HISTOLOGY FULL-TEXT

William A Beresford MA, D Phil ©
Professor of Anatomy
Anatomy Department, West Virginia University, Morgantown, USA
With a home modem, this online book may take up to five minutes to be fully on-screen, but it arrives without registration or fee. It weighs in at only 605 Kilobytes, so that you can copy it onto a floppy disc, or HD, in about half a minute.

CONTENTS

Introduction and Preface

Chapter
The first listing of chapters keeps you within the one large file. The second listing of chapters (see below) links you to individual chapters, so that if you wish to print, you print just that one chapter, not the whole book.

  1. Histology: Method and Microscopy
  2. Cytology I
  3. Cytology II
  4. Epithelia
  5. Connective Tissues
  6. Cartilage
  7. Bone
  8. Bone formation
  9. Muscle
  10. Nervous Elements
  11. Central Nervous System
  12. Peripheral Nervous System
  13. Eye and its Adnexa
  14. Auditory and Vestibular Organs
  15. Circulatory System
  16. Glands
  17. Blood
  18. Haemocytopoiesis
  19. Defence and Immunity
  20. Lymphoid Organs
  21. Skin
  22. Respiratory Tract
  23. Urinary System
  24. Alimentary System
  25. Pancreas, Liver and Gallbladder
  26. Hormones
  27. Endocrine System
  28. Male Reproductive System
  29. Female Reproductive System
  30. Techniques of Experimental Morphology
  31. Regeneration
  32. Molecular Mechanisms of Cellular Identity
  33. Eponymous Structures and Methods of Histology
  34. 'Preface'
Individual Chapters
The following list of chapters links you to individual chapters, so that if you wish to print, you print just that one chapter, not the whole book.
  1. Histology: Method and Microscopy
  2. Cytology I
  3. Cytology II
  4. Epithelia
  5. Connective Tissues
  6. Cartilage
  7. Bone
  8. Bone Formation
  9. Muscle
  10. Nervous Elements
  11. Central Nervous System
  12. Peripheral Nervous System
  13. Eye and its Adnexa
  14. Auditory and Vestibular Organs
  15. Circulatory System
  16. Glands
  17. Blood
  18. Haemocytopoiesis
  19. Defence and Immunity
  20. Lymphoid Organs
  21. Skin
  22. Respiratory Tract
  23. Urinary System
  24. Alimentary System
  25. Pancreas, Liver and Gallbladder
  26. Hormones
  27. Endocrine System
  28. Male Reproductive System
  29. Female Reproductive System
  30. Techniques of Experimental Morphology
  31. Regeneration
  32. Molecular Mechanisms of Cellular Identity
  33. Eponymous Structures and Methods of Histology
  34. Preface
More detailed contents

INTRODUCTION

How this book comes to be on the Web, and the thinking behind it are in the 'Preface', at the end.
. Here, it should be said that this is a potential aid to thought and confidence in what matters - working with the microscope, or its reproduced images: looking, thinking, looking again, relating the detail and its implications to the patient. (If it makes some things clear, it might also help with exams.)

How does one illustrate the descriptions and ideas of histology? The solution chosen here is to develop links to freely accessible (but copyrighted) Powerpoint diagrams and lists, most of which have now been done (June, 2001). Powerpoints are at the mercy of browser settings and projector light intensity, so that I am now making the backgrounds as black as possible. You can adjust via 'Format', 'Custom Background', 'Down arrow', 'Custom Background', your choice, 'Apply to the one slide/Apply to all slides', 'OK' (Then, reverse arrow, if you do not like the result)

Some of the Powerpoints are 'busy'. For projection, the content would be spread over two or more slides. They are in the condensed form so that they can be printed out six-to-a-page, then enlarged 120% for handouts.

For those seeking images of actual sections, here are links to some Histology Websites with illustrations :Bergman, Afifi, & Heidger: U Iowa --- Heidger & U Iowa's histology slides - - - JayDoc HistoWeb - - - Vanderbilt Histology Lessons
Dr Alex Imholtz has more useful sites linked from his own site at Imholtz links

Histology is important for the medical student: it introduces her or him to the broad range of cell types and molecules that let man live, and at which therapy is aimed.
. Beyond this meet-the-family role, histology reveals time and again how function is reflected in microscopically visible structural patterns of organization: patterns that really mean something for detecting and curing man's ailments and injuries.
. With many sources available for the actual visual images of microscopic anatomy, I have used the space here to show the patterns of knowledge as elements, structure, and sequence. For curricula where formal lectures have been cut back, the layout preserves for the independent student what the lecture offers by way of organization. (It does not yet offer much of the stories, analogies, props, and jest, customarily used to bring the subject alive.)
. The following storyline is sometimes helpful, and can be recognized later:
context - structures - tasks - means - mechanisms - molecules - malfunctions


Chapter 1 HISTOLOGY: METHOD AND MICROSCOPY

A NATURE OF HISTOLOGY

Medical histology applies microscopy to the human body, seeking to discover the nature of its smaller structures, how they relate to each other, and what they do. Thinking in histology runs along these lines. How does one prepare living and dead tissues for microscopy to best keep their images faithful to their true nature? What kinds of microscopy can be applied? How does one analyse and describe the images yielded at different orders of magnification by the various microscopes? Does the microscopic appearance of the tissue or cell suggest something of how it works, its chemical nature, and what may go wrong in disease? What experiments can one do to test ideas on how structure relates to function?

The answers comprise a large body of knowledge graced in several ways. First, histology is colourful. Secondly, almost everything seen is actually there; which is not to say that what is not seen is absent. Third, one handles and views actual slides - the source material for most of histology, not just someone else's selected images. Fourth, the structures can be interpreted as parts in developmental and functional sequences, and be fitted together by satisfying accounts, for example, of how cells defend the body. So much is now known of the roles of cells and structures that histology is both descriptive microanatomy, and an introduction to function for the whole body. Powerpoint
.

B PREPARATION OF THE MATERIAL

1 A major distinction can be drawn between dead and living preparations
            Dead                          Living
(a)Section - a thin slice of           Such preparations may be out of the
   tissue or organ - on a glass slide  body in a tissue culture system, or
   or metal grid.                      living within the body but in an 
(b)Smear on a glass slide -            observable situation, e.g., a  
   suitable for suspensions, e.g.,     transparent chamber inserted into 
   blood, urine, mucus, cyst fluid,    the ear or skin. The  first need is 
   bone marrow, etc.                   to keep the preparation alive. This  
(c)Spread sheet of tissue              seriously limits the facilities for
   stretched thin, e.g., areolar       observation. For example, staining
   connective tissue.                  is usually impracticable. Thus,   
(d)Teased apart fibrous                phase-contrast or interference-contrast
   elements, e.g., muscle.             microscopy has to be used in order
                                       to overcome the poor contrast
                                       between natural structures.

2 Steps needed to make and study a histological section

  1. Fixation to prevent post-mortem decomposition, preserve structure, and intensify subsequent staining.
  2. (a) Steps involved in imbedding the tissue in a block of wax or plastic, or (b) freezing of the material to a firm mass, for
  3. cutting into thin sections on a microtome; 1-l50 microns (µm) thick for light microscopy (LM); 30-60 nanometres (nm) for electron microscopy (EM).
  4. Units: based on the metre (m): micron/micrometre (µm) = 10-6m; nanometre (nm)/ millimicron (mµ) = l0-9m; Ångström (Å) = l0-10m; l0Å=1nm.
  5. Mounting of the section on a glass slide or metal grid. Staining of the section with one or more reagents, e.g., solutions of metallic salts, in one or more stages.
  6. For light microscopy, the removal of surplus stain and water, and steps involved in holding a thin glass coverslip to the section with a mounting medium having a refractive index close to that of glass.
  7. Observation and recording by means of the microscope, and notes, photomicrography, projection drawing, labelled sketches, counting and reconstructions, digital and videorecording. A drawback to using our eyes as part of the observing instrument is that the visual system does not record accurately. Memory is unreliable.

C MICROSCOPY

1 Microscopy in general The main distinction is between light microscopy and electron microscopy. The usual light microscope uses a visible light source with a system of condenser lenses to send the light through the object to be examined. The image of this object is then magnified by two sets of lenses, the objective and the eyepiece. Total magnification is then the product of these two lens systems, e.g., 40 X 10 = 400. The resolution or resolving power - how close two structures can be and still be seen as separate - is a measure of the detail that can be seen, and for the light microscope is about 0.25 µm. This limit to resolution is determined mostly by the wavelength of the light; and, however powerful the lens, 0.25 µm cannot be improved upon.

The only way to improve resolving power is to reduce substantially the wavelength of the light. This is achieved by the electromagnetic beam of the electron microscope. The beam is focused through the object suspended on its metal grid, and is magnified before striking a fluorescent screen to be transformed into a visible image (Chapter 30.K). [Caution! the link takes you there , but 'back' brings you only to the start of the last Chapter that you linked to.]
The resolutions so far achieved in biology with transmission electron microscopy (TEM/EM) are of the order of 1 nm at a magnification of X 2 000 000. The other forms of microscopy - phase-contrast, interference, fluorescence, confocal scanning, atomic-force (and X-ray diffraction) - will be discussed in Chapter 30. in relation to the problems for which they are suited.

2 Microscopy for the student (may not apply in toto to the reader's use)

  1. The usual class microscope has eyepieces/oculars magnifying X 10, and an objective nosepiece carrying X 4, X 10, X 44, and X 95 (oil immersion) lenses. Normally the three lower-power lenses are kept mounted on the nosepiece, whilst the oil immersion objective may be mounted or kept separately.
  2. Every time it is used, the microscope should be set up to the best optical advantage. How to do this is described briefly below.
  3. Keep in mind the limit to resolution. In practical terms, make special note of those structures that need an oil immersion lens to be seen or are visible only in electron microscopy.
  4. The section has some thickness, so that the fine-focusing adjustment should be used continually during observation to bring out fine detail, e.g., cilia on cells. Essentially, though, we are getting a two-dimensional picture from an originally three-dimensional piece of material. For what the structure looked like in the third dimension, the student can try to reconstruct mentally what is going on in the missing dimension, and look up views of the structure in scanning electron microscopy.
  5. Artefacts (appearances not due to the original nature of the material as obtained from the body) can arise at all stages in the treatment of the section. Gross examples arise from: (l) clumsy excision from the body; (2) poor or inappropriate fixation; (3) shrinkage and, worse, uneven shrinkage, leading to artificial spaces and distorted relations; (4) cutting scores from a bad microtome knife; (5) the section not flat on the slide; (6) water, dirt or bubbles on or in the section; (7) dirt on the microscope lenses; (8) patchy or faded staining; unbalanced staining when more than one stain has been applied; (9) precipitate from fixative or stain; (l0) tears and folds in the section.
  6. Setting up the microscope
    (a) Ask for and read the instruction booklet for the 'scope.
    (b) Familarize yourself with the parts and controls of the microscope, in particular with the: (your 'scope may not have all these)
    ... (i) light source and switch,
    ... (ii) two-sided mirror, if present,
    ... (iii) iris diaphragm lever on the condenser assembly,
    ... (iv) condenser focusing knob,
    ... (v) stage slide clips and controls for a mechanically movable stage,
    ... (vi) objective selection on the three- or four-way objective nose-piece
    ... (vii) coarse focusing adjustment,
    ... (viii) fine focusing adjustment,
    ... (ix) pointer in the eyepiece lens,
    ... (x) any control for eyepiece focusing.

(c) Before plugging in the microscope, check to feel how the switch and rheostat work. Plug in, switch on, and adjust the rheostat up one third of its range to start.
(d) Otherwise, use artificial light provided by an electric bulb behind a ground-glass screen to furnish a constant and reliable source. Light intensity can be increased by bringing the lamp nearer to the mirror, if the lamp is not built-in.
(e) If the condenser in use (nearly always), use the plane side of the mirror, if the lamp is not built-in.
(f) Raise the condenser to very near the underside of the stage, and open the iris diaphragm.
(g) Place a clean, stained slide on the stage and using the coarse and fine focusing controls bring it into focus with X l0 objective.
(h) With the condenser racking knob focus the light source on the specimen. This has happened when the specimen itself is in focus and some aspects of the light source is also seen sharply defined, e.g., the bulb filament or scratches on the frosted glass screen. If this feature of the light source is obtrusive, now place the condenser very slightly out of focus. Do not lower the condenser way out of focus as a means to reduce the light intensity.
(i) The iris diaphragm should now be closed just to the point where glare is eliminated. Further closure will make the field too dark and reduce resolving power.
(j) The microscope is now set up for use, but the requirements change for each objective. Higher power objectives require more light thus the iris will need to be opened and perhaps the lamp brought nearer to the mirror and the condenser refocused.
(k) Note that the objective lenses are of different lengths, and they are not always parfocal. Be careful when switching in a higher power lens that it does not hit the slide because of its greater length. Clean the lenses only with lens paper.
(l) If the X 44 objective will not focus to a clear image, check first that the slide is not upside down on the stage.

(m) Use of the 'oil immersion' lens:
... (i) Select field of interest with the high dry lens (X40); centre precisely the cell or object in the microscopic field; if X 95 lens is already mounted go to (v).
... (ii) Raise the objective lens assembly and remove the low power (X4) lens.
... (iii) Place it in the container to be found on the door of the microscope cabinet from which you have taken the oil immersion (X 95) lens.
... (iv) Screw the oil lens into the now vacant place on the objective nosepiece.
... (v) Place carefully one drop of immersion oil from the small bottle issued on the area of the slide to be studied.
... (vi) Switch round the objective nosepiece to bring the oil immersion lens into play.
... (vii) Very carefully lower the objective assembly with the coarse focusing, until the tip of the oil lens touches the drop of oil. This operation must be controlled by observing the descending lens from the side. Do not yet look down through the eyepiece. Once the lens has touched the oil raise it slightly, but not so far that the drop breaks away.
... (viii) Look in the eyepiece and focus down with the fine focusing control very slowly and gently until the specimen comes into focus. If you seem to have gone down a very long way without a clear image, again check from the side that you have not overshot and the lens is not nearly on the glass of the coverslip. If this has happened raise the lens slowly, while looking for a focused image.
... (ix) The oil objective lens needs much light so that the iris diaphragm may have to be opened.
... (x) As soon as you have finished using the oil lens, raise (remove) and clean it. (Replace X 4 lens on the nosepiece and the oil lens in its box.) Clean the slide of oil with lens or tissue paper. Do not allow oil to get on to the other, dry, lenses.

(n) Other controls the class microscope may have include eyepiece focusing, filter-holders, centring screws for the condenser, and a rheostat, lens and light-stop for the light source. Ask for instructions in their use and for help with any mechanical problem.

(o) Take care of the microscope, carry it only by its arm, protect it from dust by keeping it locked in its case, and do not stand it or boxes of slides near the edge of the bench. If lens paper alone is insufficient to clean a lens, use no solvents but consult a demonstrator. On no account exchange the lenses of your microscope for those of any other microscope.

Spectacle-wearers need not use their glasses in microscopy; if they do, they should beware of damaging their glasses, while trying to compensate for the narrowed field of view.

3 Differences between light and electron microscopy
l Chapters 2 and 3 deal with microscopic details of cells - cytology, for which EM is better suited than LM. Table l gives some differences between the two approaches. The detailed morphology revealed by EM may be called fine or submicroscopic structure/ultrastructure.
2 The direct comparison of LM and EM images of a structure requires that the magnifications be of the same order. Noting the magnification, on the 'scope or in the figure legend, allows one to adjust one's expectations of what may be seen, and should always be done.
3 A growing practice in histology and pathology is to fix and prepare the tissue by EM standards, imbed in plastic and cut semi-thin (l µm) sections for staining by modified LM methods. LM then reveals good cellular detail and fewer artefacts.
4 Two other techniques yield anatomical images - fibre-optic endoscopy and scanning EM, and are being digested by the anatomical texts. Endoscopy from its low magnification is marginal to histology, but related in that endoscopy is used to obtain biopsy specimens for histopathology.
SEM strengthens one's conception of microscopic structures, e.g., cilia, renal podocytes, bone under resorption, and effectively counters the unavoidable impression of structures existing only in two dimensions. (From hereon, EM is standard transmission electron microscopy.)

Table 2. Some differences between light and electron microscopy.

Light microscopy                        Electron microscopy
--------------------------------------------------------------------------

Image is presented directly to the     Image is in shades of green on
eye. Image keeps the colours given     the screen; photographically,
the specimen by staining.              only in black and white.                                       

Modest magnification to X 1500;        High magnification, up to X 2,000,000
but a wider field of view and easier   thus the range of magnification
orientation                                        is greater
                           

Resolving power to 0.25 µm.            Resolving power to 1 nm (0.001µm.)

Frozen sections can yield an image     Processing of tissue takes a day at
within 20 minutes.                     least.

Crude techniques of preparation        High resolution and magnification
introduce many artefacts.              demand good fixation (e.g. by
(Histochemical methods are better.)    vascular perfusion), cleanliness
                                       and careful cutting, adding up to
                                       fewer artefacts.

Section thickness (1-30 µm) gives      Very thin sections provide no
a little depth for focus for           depth of focus, but 3-D information
appreciation of the third dimension.   can be had from: (a) thicker sections
Serial sections can be cut, viewed     by high-voltage EM; (b) shadowed
and used to build a composite image    replicas of fractured surfaces; (c)
or representation.                     scanning electron microscopy (SEM).

Most materials and structures cannot   Heavy metal staining gives a more
be stained and viewed at the same      comprehensive picture of membranes,
time; stains are used selectively to   granules, filaments, crystals, etc.;
give a partial picture, e.g. a stain   but this view is incomplete and even
for mucus counterstained to show       visible bodies can be improved by
cell nuclei.                           varying the technique.

Specimen can be large and              Specimen is in vacuo. Its small size
even alive.                            creates more problems with sampling
                                       and orientation.                 
 
Light microscopy                       Electron microscopy

Chapter 2 CYTOLOGY l

l Body components

These are cells, extracellular substances, and body fluids. Fluids can have their suspended solid constituents viewed microscopically as smear preparations (see Blood; Chapter l7.A), but are otherwise of limited histological interest. Extracellular substances are important for the cells whose environment they form: they reflect and help control cellular activities, aside from their critical structural mechanical properties. Individual materials can be seen and localised by histochemistry.

2 Cells: chemical constitution and fixation

  1. Composition: much water; proteins, nucleic acids, lipids, carbohydrates, amino acids, minerals, hormones, vitamins, etc.
  2. Fixation stabilizes mainly proteins, and protein conjugates. These substances are used as building materials for the firmer structures of the cell. Lipids, minerals, glucose, and smaller molecules are usually lost from the section during processing. What is left is a skeleton of structures - membranes, granules, filaments - made up of proteins, polypeptides, polysaccharides and some other macromolecular materials. The special steps of histo- and cytochemistry (Chapter 30.C) preserve and reveal some smaller molecules.

3 Cells: living properties and specialization
1 Properties of cells: (a) general - communication, respiration and energy storage and release, synthesis, excretion, growth, differentiation, reproduction; (b) specialized - irritability to stimuli (excitability), motility, contractility, conductivity, absorption, phagocytosis, secretion.
2 During development from the fertilized oocyte, a great variety of cells is formed in the mammal, each kind specializing in a certain function, e.g., secretion, but many activities, such as energy production, are common to all cells. The cells of the four primary tissues - epithelial, connective, nervous and muscular - are divided along lines of specialized function, e.g., muscle for contractility and excitability.

4 Cell morphology
Powerpoint
1 Cells performing a given function have a characteristic size, form and fine structure adapted to that task. However it may help at this stage to think in terms of a composite cell having all the features the various cells of the body display.
2 The cell is defined as a distinct entity by having a thin skin or plasmalemma/cell membrane separating off from the outside a soft, viscous, almost fluid cytoplasm, in which are suspended a number of firmer, recognizable structures - organelles and inclusions - and one or more nuclei. The nucleus, likewise, is a mass of material enclosed in nuclear membranes.

5 Cell components
1 Cytoplasm: the so-called soluble phase of the cell, consisting mostly of water, dissolved solutes, and larger molecules in suspension tending to link repetitively with covalent bonds giving the cytoplasm a dense, viscous colloidal sol or gel consistency.

2 Cell or plasma membrane/plasmalemma

3 Nuclear membrane
(a) Is doubled with a 20 to 25 nm perinuclear space between the two membranes. Each is of unit membrane with a 'trilaminar' nature similar to that of the plasma membrane.
(b) Many of the apparent interruptions or pores/fenestrations through the doubled membrane are covered by a very thin 'diaphragm', actually granular and fibrillar in nature.
(c) Transport between nucleus and cytoplasm takes place at assemblies of proteins at the pore complexes.
(d) The outer nuclear membrane sometimes leads on out to a membrane system existing in the cytoplasm.

4 Organelles
Structures 1-8, below, are organelles - actively participating cytoplasmic bodies of characteristic structure and behaviour.
Points to note: (a) their morphology in light microscopy; (b) morphology in EM; (c) multiple or single; (d) any special location in the cell; and (e) functions.

1. Endoplasmic reticulum
(a) Is the name given to the cytoplasmic membrane system of many parallel membranes and tubules in communication with one another. This system of closed channels (cisternae) leads towards the Golgi complex.
(b) Two varieties of endoplasmic reticulum (ER) are seen.
....(i) Granular/rough/GER has fine granules, ribosomes of ribonucleo-
... protein (RNP), l5 nm in diameter, in clusters studding the outer surface
... of the parallel membranes.
...(ii) Agranular/smooth lacks the ribosomes and is more tubular. This
... kind is associated with cholesterol metabolism among other things, whereas
... the granular variety is related to protein synthesis, e.g., enzyme
... formation.

2. Ribosome particles: may lie free in the cytoplasm in small clusters (polyribosomes/polysomes) unrelated to membranes. This is noted particularly in growing cells.

3. Golgi body/complex/apparatus
(a) This usually takes one area near to the nucleus and often in a specific place, e.g., supra-nuclear in cuboidal epithelial cells.
(b) It consists of a complex of stacked smooth cisternae-enclosing lamellae, tubules, and vesicles of various sizes.
(c) It is more disorderly in appearance than the smooth endoplasmic reticulum and has more closed sacs or vesicles, often with dark staining material within them.
(d) In LM after special silver staining, the Golgi apparatus may be seen as a tangled network. With routine haematoxylin staining in certain cells, e.g., active osteoblasts, the juxta-nuclear vacuole reveals the site of the Golgi structure as a pale negative image.
(e) Its tasks are the concentration and preparation for storage of proteins, and completing the synthesis of complex sugars.

4. Microtubules: with a diameter of 20-25 nm; seen lying in the cytoplasm only with EM or fluorescent tagging; they give form to the cell, and are responsible for much intracellular transport and some movements.

5. Filaments: fine threads visible in EM, but may be aggregated into thicker fibrils visible in LM; in muscle cells they are a very important contractile element; in all cells they furnish a flexible skeleton articulated by the cell itself.

6. Mitochondria
(a) The many to be found in almost all cells may or may not have a special location within the cell.
(b) In LM they can be stained with special methods and appear as coloured rods or granules. In EM their shape tends to be tubular or spheroid.
(c) They are hollow bodies enclosed in two unit membranes; the inner membrane projects inwards as plates or tubules called cristae, studded with small 9 nm wide elementary particles - rounded bodies on stalks. In the matrix of the intercristal space inside the mitochondrion, granules may be found.
(d) Mitochondria are very rich in the enzymes associated with the storage and release of energy, and with respiration, and fatty acid metabolism, etc. They may also store calcium.

7. Lysosomes
(a) Are round, single-membrane-limited, darkly staining bodies without cristae and containing another class of enzyme, hydrolytic or digestive.
(b) The material that they digest may be:
... expended organelles of the cell itself;
... extracellular matter engulfed by phagocytosis and membrane-enclosed in a phagosome;
... endocytotic vesicles.
Material resistant to digestion may persist as a residual body.

8. Centrosome/diplosome/division body/centriolar complex
(a) A body which lies alongside the nucleus at the cell centre or cytocentrum and is just visible as dots with LM. EM reveals that it consists of two centrioles lying at right-angles to one another. Each is an open-ended cylindrical body with a wall composed of nine bundles, each of three microtubules. These tubules help to initiate and control the aster, also microtubular, during cell division.
(b) A similar cylindrical structure is seen at the base of each cilium and is called a basal body/kinetosome. One way basal bodies and cilia develop is by a multiplication of centrioles.

5 Cell nucleus
(a) Nuclear membranes have been discussed in 5.3.
(b) Chromatin granules/karyosomes seen in the sap during the interphase period are chromosomes composed of deoxyribose-nucleic acid molecules (DNA) not fully uncoiled. The sex chromatin of female cells is an extreme example of not uncoiling, where one of the female's two X chromosomes continues in a condensed heterochromatin, rather than the dispersed euchromatin, form, although a few genes on that chromosome escape inactivation.
(c) The nucleolus seen as a dense body in most cells' nuclei with LM. is a condensation of protein and RNA and DNA, comprising granules supported by microfilaments. The dense branching strand is the nucleolonema.
(d) There is great variety in the size of the nucleus, its shape, the densities of staining of chromatin and nucleolus, and in the position of the sex chromatin, if present.
(e) In general, a large nucleus, with much pale euchromatin and a prominent nucleolus, is very active in the control of protein synthesis by transcribing mRNA from chromatin DNA. The nucleolus is mostly pre-ribosomal RNA. Nucleoli are prominent in nerve and Sertoli cells.

6 Cell inclusions
(a) Non-living, non-participating, poorly structured cell elements, very rarely seen in an intra-nuclear position; usually cytoplasmic.
(b) Examples:

7 Dynamic nature of the cell
The cell is not a static entity in life. Its chemical constitution and morphology are in continuous flux. Its complement of organelles is altering, with wearing-out and replacement, i.e., the cell is having to synthesize its own material. The cell itself represents a system of activities isolated to partial extent from an extracellular environment. Within the cell things are constantly being altered, moved around and joined up within the membranes. The membranes define temporary compartments separated from the cytoplasm, where particular activities can be confined and controlled by enzymes attached to the extensive membrane surfaces. Dynamic aspects of the cell's existence are partly deduced from a study of its morphology in specimens fixed in various states, partly from microscopical observations of living cells, and from histophysiological experiments outlined in Chapter 30.

8 Cell staining
l Cellular material contains a lot of water and has little natural colour. It was discovered that various dyes intended for textiles would selectively stain different structures in the cell. These stains may be discussed for how their chemical nature relates to the particular things in the cell with which they react and stain: for more details see Chapter 30.B.3.
2 Routine staining of human material usually employs haematoxylin, which can be made to react preferentially with nucleic acids and acidic groups of proteins. The nucleic acids are concentrated in the nucleus and the rough endoplasmic reticulum. The colour is usually some shade of blue or brown depending on the particular method of haematoxylin staining used.
3 A few cells have a lot of granular ER in their cytoplasm, which gives a strong staining reaction, and the cell is said to be basophilic/ basophil - liking basic stains and haematoxylin. This term depends on how the cytoplasm reacts: it is taken for granted that the nucleus will react positively with basic stains.
4 Most cells do not have such a concentration of GER that their cytoplasm stains significantly with haematoxylin. Consequently, another cytoplasmic stain is needed. Such a general stain is eosin, acidic in nature, which stains the cytoplasm of most cells red or pink. Some structures stain bright red. These are said to be strongly eosinophilic or acidophilic/acidophil - liking an acidic stain.
5 Three points should be mentioned.

6 Haematoxylin and eosin, then, are used as a routine procedure to stain the tissues - cells, and some extracellular materials, such as hyaline cartilage matrix (by haematoxylin), and collagen (by eosin) - and make things in general easier to see. When the interest is in specific cell features, special methods are used: for example, methods for glycogen granules, fibrils, etc. After performing the special staining, a counterstain is often used to reveal the nuclei or some other aspects of the general background to the special feature.

9 Cytological description of an individual cell
In light microscopy involves: (l) relative and absolute size; (2) shape; (3) number of nuclei; (4) shape and size of nucleus/nuclei; (5) intensity of nuclear staining; (6) amount of cytoplasm; (7) staining affinity of cytoplasm, e.g., basophilic, acidophilic (eosin), argentophilic (silver stains), or chromophobe (liking no stain); (8) granular cytoplasm; (9) nature of any inclusions, for instance, melanin pigment, fat, carbon, bacteria, zymogen granules, glycogen, mucus; (l0) specializations of the cell membrane, e.g., cilia, a brush/striated border (many microvilli); (ll) distinctive organelles in cytoplasm and their position, e.g., prominent Golgi complex, many fibrils, numerous orderly mitochondria giving another striated effect, Nissl substance (GER) in nerve cells; (l2) whether the cell is in some phase of mitosis or meiosis; (l3) the cell's surroundings; (l4) manifest properties of the living cell, e.g., motility, phagocytosis, contractility.

10 Aspiration cytology
The above cellular detail discriminates pathological, e.g., malignant, change in cells. These can be obtained, single and clumped, from any tissue or organ of the body, where a needle can be introduced to suck out cells to make a smear for fixing and staining - fine-needle aspiration cytology (FNA).

11 Cell division
l Review from biology what happens to (a) the centrioles and spindle, (b) the nuclear chromatin and nucleolus, (c) the nuclear membrane, (d) the cell membrane, and (e) the cytoplasm and its organelles and inclusions,
2 during the various phases: pro-, meta-, ana-, telo-phase and interphase.
3 During interphase the chromatids duplicate themselves by an exact replication, when DNA has to be synthesized.
4 Pursue biochemists and molecular biologists for their accounts of the molecular controls on cell division, progress through the cell cycle Powerpoint, and the continue working-proliferate-die decisions, and the medical relevance.

12 Apoptosis
1 The orderly or programmed death of cells is needed to balance cell proliferation in mature renewing tissues, such as blood and epithelia. Also, one strategy of development is to overproduce cells, then select, e.g., for the survival of correctly connected neurons, or of lymphocytes reactive to non-self antigens. Thirdly, if an organ cannot work properly, e.g., a gland has a blocked main duct, or its hormonal drive stops, many cells die by apoptosis. Apoptosis

2 For apoptosis, endonucleases are activated which break up the chromatin, chopping up the DNA, transcription slows and stops, organelles clump, and the GER dilates. Caspases digest relevant cellular materials and structures. The cell and internal membranes bleb out. Finally the shrunken cell is phagocytosed by macrophages.[Caspases - cysteinyl aspartate-specific proteinases]
3 With LM, only the increasing nuclear density and cell shrinkage are noticeable, unless special cytochemical methods to detect apoptotic events (2 above) are used.
4 Apoptosis is intentionally unobtrusive, to remove single cells without provoking inflammation or upsetting tissue function.
5 Quite often, the inclination of cells is to die, and they need survival signals not to undergo apoptosis.

13 Stem cells
For a stable population, the corollary to cell death is cell renewal. This requires:
..(i) the proliferation of cells;
..(ii) an enduring population of stem cells;
..(iii) controls (+ & -) that promote division of stem cells to maintain their numbers - self-replication;
..(iv) controls that cause differentiation of certain of the stem cells to become the determined/committed precursors of the mature cells of the tissue;
..(v) factors to promote division of the precursors/progenitors and their further differentiation. The controlling factors include cytokines (Chapter 8.F).

More is known about the ensuing progenitor cells than about the stem cells. Although not essential to the concept of stem cells, at step (iv) above, stem cells usually give rise to more than one lineage of differentiated cells, in order to furnish the needed diversity of cell types in blood and most epithelia. One mechanism for this is the asymmetric cell division, wherein the daughter cells of a mitosis differ.

The next chapter (Cytology II) reviews in more detail the structures of the cell, emphasizing their functions.


Chapter 3 CYTOLOGY ll

This account deals, by custom, with a generic cell. For a similar story based on specific working cell types - the poster-cell approach - try the Medical Cytology Module.

A CELL MEMBRANE OR PLASMALEMMA

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1 Although looking trilaminar in routine EM, it behaves as if it comprises a double layer of lipid molecules, in which proteins are distributed asymmetrically in a mobile mosaic pattern. Thus, some proteins span the width of the membrane, and may vary rates of transport by changes in their conformation. Others, as enzymes, receptors, or adhesion molecules, etc. have active domains at the surface held in correct position by intramembranous domains imbedded in the lipid layer, and intracellular domains to engage in events inside the cell.
A sometimes fuzzy-looking coat of glycoprotein - glycocalyx - sticks to the external face of the membrane.

2 Functions of the membrane are:

  1. Firm attachment to other cells or a basal lamina; membrane specializations for this are: (a) junctional complexes, (b) gap junctions/ nexuses, (c) desmosomes, (d) hemi-desmosomes, (e) intercalated discs, and (f) membrane interdigitations; more details Chapter 4.D.l.
  2. Movement of the cell itself by pseudopodial, filipodial, or lamellipodial extensions (think karate: fist, finger, or side of the hand) and the release of any firm attachments, or by flagellate activity, e.g., by sperm. (Microspikes and ruffles are alternative names for filopodia and lamellipodia, respectively.)
  3. Movement of materials outside the cell by the activity of cilia, e.g., ciliated epithelia of the respiratory tract and uterine tube. The wide-spread occurrence of solitary cilia (flagella), e.g., on neurons, adrenal cells, smooth muscle, may involve a vestigial body or one still functional. The stereocilia of the male reproductive tract are non-motile, clumped, long microvilli, probably absorptive.
  4. Transport of materials in and out of the cell served by: (a) permeability (selective) of the membrane, (b) active transport through the membrane, (c) endocytosis, and its more scaled-up forms - pinocytosis and phagocytosis, (d) exocytosis; and increased exchange surface area by (e) microvilli (thousands on a cell), and (f) infoldings of membrane.
  5. Communication and transduction. Each cell collaborates with both adjacent cells, and those of the whole body, for development, growth, homeostasis, regeneration, and its own particular task. The importance of the cell membrane in receiving and sending the necessary signals is stressed by the number of examples given:
    (a) The binding of hormones to receptors on the membrane.
    (b) The binding of the lymphocyte's membrane receptor to an antigen.
    (c) Transmitter chemicals depolarize neurons and muscle cells.
    (d) Excitable tissues propagate action potentials along the membranes.
    (e) Schwann cells wrap their membranes many times round an axon's to make myelin sheath segments for faster signalling.
    (f) Chemical stimuli are transduced into nerve impulses in chemoreceptors; mechanical stimuli in mechanoreceptors.
    (g) Gap junctions permit ions and excitation to spread from cell to cell, and unify and synchronise actions of many cells/cell assemblies.
    (h) In development, epithelial and mesenchymal cells interact in sequence to induce cell differentiations, e.g., in tooth and glands.
    (i) Cells attract and fuse with one another to form multinucleated cells, e.g., skeletal muscle and osteoclasts.
    (j) Chemotactic agents act on phagocytic cells to attract them to their targets.
    (k) Keratinocytes of the skin phagocytose melanin pigment offered to them in the processes of melanocytes.
    (l) Macrophages detect spent or abnormal red blood cells, and hold and engulf either the whole cell, or the part holding an unwanted body.

3 Molecules Wherever such actions are described, special molecules are acting, by binding to each other, changing their conformation, or some other means. Examples are:
.. (a) Spectrin/fodrin provides a subplasmalemmal skeleton attached to the cell membrane by ankyrin, and to actin of the cytoskeleton, to permit control of the membrane's shape and movement.
.. (b) Cell adhesion molecules (CAMs) allow cells to attach to only certain cell types or substrates.
.. (c) Integrins are cell-surface-membrane dimeric molecules (an alpha with a beta), by which cells choose to which extracellular matrix (ECM) components they wish to fasten, e.g., laminin.
.. (d) Connexins are proteins that combine as hexamers to form connexons - the gap-junction channels, allowing ions and small molecules to pass between cells. Connexins and the transports allowed vary among liver cells, neurons, etc.
.. (e) Occludins are responsible for the seal preventing passage of materials past inter-epithelial tight junctions.

B INTERNAL (CYTO-) MEMBRANE SYSTEMS

l Nuclear membrane is a doubled membrane with pores that separates off genetic material and influences its degree and kind of interaction with the cytoplasm. The membrane sometimes has deep infoldings. (The rare annulate lamellae are parallel membranes with pores, and closely resemble nuclear membranes, but are stacked in the cytoplasm.)

2 Granular endoplasmic reticulum (ergastoplasm) is a membrane system providing some communication with the nucleus via the latter's outer membrane. Both have ribosome particles studding their outer sufaces, i.e., those facing outwards towards the cytoplasm. The membranes and ribosomes, in association with the nucleus, are concerned with protein synthesis. Some proteins pass into the sacs or cisternae, formed between the double membrane layers, for folding and processing. Membrane vesicles holding the protein bud off and travel along microtubules to the cis (receiving) face of the Golgi complex.

. Endosomes are the natural transport vesicles moving from the donor membrane compartment to the acceptor compartment. Endosomes require active mechanisms for: membrane budding, separation, transport, targetting & sorting, docking, and fusion. The donor and acceptor compartments may be the plasmalemma (via endocytotic vesicles), the Golgi complex, lysosomes, or the endoplasmic reticulum.
Some materials, e.g., receptors, are in a constant cycle between cell membrane, early endosomes, and late endosomes, with branch points for materials to come or go to the Golgi complex, lysosomes, or elsewhere in the cell.

[Note that microsomes are small bodies formed during separation by biochemical cell fractionation techniques: morphologically they are artefacts. They are derived from broken parts of the smooth or granular ER whose membranes 'heal' to form small vesicles, with or without ribosomes.]

3 Golgi apparatus serves as a hub for traffic out of, into, and around the cell. It is a complex transit region (there may be more than one in large cells) occupied by smooth-surfaced tubules, sacs and flatter chambers varying considerably in size. It concentrates, modifies and packages certain secretory products to await transport to the cell membrane for release, or application to some intracellular purpose. Vesicles depart from the trans or releasing face. Also, the Golgi is where glycoconjugates are finished by adding the remaining sugars (using glycosyltransferases), e.g., in cartilage cells and mucus-secreting goblet cells. Glyco

4 Many inclusions - structures not actively participating in the metabolism of the cell at the time of observation - are products bound in membranes by the Golgi apparatus, e.g., melanosomes, zymogen granules. Other inclusions, e.g., glycogen granules, form in the cytoplasm without any enclosing membrane.

5 Smooth endoplasmic reticulum resembles the granular form in sometimes having systems of parallel membranes following the curvature of nearby structures, but it usually exists in tubular and vesicular forms. Its functions are varied and include:

  1. Acting as a source or reserve for membranes of new structures, e.g., Golgi body, GER; and cell membrane, as for the gastric oxyntic cell's canaliculi, and in the megakaryocyte's formation of platelets.
  2. It is prominent in cells involved in cholesterol metabolism, e.g., liver cells and cells making steroid hormones. It seems to interact with lipid droplets, also present, in the synthesis of the steroids.
  3. Smooth ER in liver cells is the site of enzymes detoxifying phenobarbital and other foreign chemical materials. It is also concerned there with the formation of plasma lipoprotein as a normal responsibility.
  4. A smooth membrane system is found in muscle fibres as the sarcoplasmic reticulum. It synchronizes contractions and relaxations throughout the fibre, by moving calcium ions to and from the sarcoplasm.

6 Mitochondria
l These are complex bodies with a double membrane, the inner membrane extending inwards as sheets or tubules called cristae.
2 The inside of the mitochondrion is occupied by a matrix in which dense bodies may sometimes be found.
3 Enzymes of oxidation and energy-release, and for some syntheses, are present; some associated with the crista membranes or with the external/ outer limiting membrane, others and coenzymes are in the matrix.
4 Mitochondria of steroid cells are distinctive in having tubular cristae.
5 Mitochondria are able to reproduce themselves. Also, they contain circular DNA for 13 respiratory-chain proteins. One rare mitochondrial genetic disorder of this DNA causes, for example, a muscle disease with mitochondrial inclusion bodies.
6 Apoptosis involves mitochondria. Increased permeability of the outer mitochondrial membrane allows the release of inter-membrane factors, e.g., cytochrome c, that help start the caspase enzymatic cascade.

7 Lysosomes
l They are roughly spherical with a single enclosing membrane.
2 The storage/primary form is derived from the Golgi apparatus and contains hydrolytic enzymes,
3 whose access to other intracellular materials is controlled by the enclosing membrane and processes of membrane fusion. The stability of the membrane can be influenced by vitamin A and glucocorticoid hormones.
4 Lysosomes fuse with endosomes, phagosomes, surplus secretory granules or expended organelles, which they destroy.
Multivesicular bodies contain distinct vesicles, inside a limiting membrane. The vesicles seem to be endosomes on their way to meet lysosomes, or for storage as a way to keep membranes and membrane proteins intact for redeployment.
5 The form of lysosomes changes from round and fairly homogeneous to varied secondary kinds including myelin figures and residual bodies. Some residual bodies become yellow or brown lipofuscin/lipochrome granules.
6 In autophagy, the lysosome fuses with the autophagosome, consisting of a double membrane wrapped around the target organelles and cytoplasm to be broken down. . Autophagy PowerPoint
7 Lysosomal enzymes are also used in the turnover of extra- and intra-cellular materials. In lysosomal deficiency diseases, the inherited absence of an enzyme causes the massive accumulation of the material, e.g., glycogen, normally broken down. (Excess storage, hence "storage disease/disorder", e.g., Glycogen storage disorder II from a lack of a-glucosidase.)
8 A devastating storage disease is Hurler's, where a deficiency in lysosomal a-L-iduronidase causes intra- and extra-cellular excess accumulations of dermatan-sulphate and heparan-sulphate glycosaminoglycans. Aside from the dwarfism and mental retardation, the many cardiovascular defects bring about early death.

8 Peroxisomes/Microbodies are widespread, but particularly in hepatocytes and renal proximal tubule cells. They have a dense matrix enclosed by a single membrane, and hold enzymes involved in the beta and alpha oxidations of certain fatty acids, and some phospholipid synthesis. Catalase is a useful marker enzyme for peroxisomes. The congenital lack of peroxisomes - deficient 'biogenesis' of the organelle - causes fatal syndromes with brain, liver and kidney dysfunctions. Zellweger's syndrome is the best known. If the peroxisomes have formed, but only one enzyme is genetically at fault the damage is less severe and may permit life.

C CYTOSKELETAL (FILAMENTO-TUBULAR) SYSTEM

The three components of the cytoskeleton - actin-myosin, microtubules, and intermediate filaments - are functionally linked as the dynamic organizer of the cellular domain, controlling cell shape, cell locomotion, where materials move in the cell, and hence cell polarity.

l Filaments

  1. Several kinds of filaments, derived from unpolymerized precursors, attach to the plasmalemma, and to structures within the cell in order to change their form and position as the cell works.
  2. Subdivision of filaments is by: (a) size - thick myosin (l5 nm diameter) and thin actin (6 nm) versus intermediate (l0 nm), roughly contractile versus noncontractile (also labile versus insoluble); and (b) by distribution - general versus tissue-specific.
  3. Relatively tissue-specific intermediate filaments are: keratin in epithelia; desmin in muscle cells; neurofilaments in neurons; and glial filaments of acidic protein (GFAP) in certain glia. Immunostaining for these filament types gives some idea of the cellular origin of, and prognosis for, tumours.
  4. Nestin is a protein closely associated with intermediate filaments in stem and progenitor cells of tissues generally, and serves as a stem-cell marker.
  5. More generally distributed filaments are: (a) vimentin (intermediate) in connective tissue and other cells of mesenchymal origin, and in muscle, glia, and sometimes epithelia; (b) actin (thin); and (c) myosin, often not easily demonstrable as filaments.
  6. In motile cells, e.g., white blood cells/leucocytes, the peripheral zone of cytoplasm clear of organelles and inclusions in phase-contrast microscopy, and used to extend pseudopodia, is the ectoplasm. This zone is now known as the actin cortex because of the actin filaments attached to the cell membrane for locomotion and changes in cell shape.
  7. In absorptive and secretory epithelia, actin filaments are a major component of the apical terminal web inserting into junctional complexes and running up inside the microvilli. The actin is responsible for the movement involved in endocytosis and exocytosis.
  8. Isoforms of actin (at least seven) have roles in striated muscle, smooth muscle, and non-muscle cells; and several proteins interact with these actins to achieve their controlled rapid reorganization for movement, e.g., profilin, a-actinin, filamin, severin, villin, etc.

2 Microtubules Each microtubule is built of 13 tubulin filaments. The dimers constructing the filaments confer a repeated polarisation along the tubule so that one end is 'plus', the other 'minus' - a difference that tells the attached microtubule motor proteins which end to head for.

  1. Microtubules, not in a doublet or triplet formation, are supporting or cytoskeletal elements used, for instance: (a) to give some shape to platelets, (b) to cause and orient the elongation of cells, e.g., ameloblasts, (c) to allow neurons and glia to grow long processes and keep them patent; (d) microtubules are responsible for axoplasmic flow. (e) Microtubules direct certain materials to regions of the cell membrane, e.g., basolateral, creating cell polarity.
    Microtubules are dynamic structures in themselves and their construction and disassembly from tubulin dimers are under constant control.
  2. Centrioles are cylinders open at one end with a wall composed of microtubules disposed longitudinally as nine triplets. The centrioles and additional microtubules direct and move the chromosomes along the mitotic spindle in cell division.
  3. Cilia have, at their base, basal bodies, each identical with a centriole and often developing from one. Of the triplet, two microtubules extend up inside the cilium which thus has a core of nine peripheral doublets, plus an additional central pair. The doublets, by a sliding-tubule action using dynein arms, move the cilium to beat strongly in one direction by a bending-wave action.
  4. Flagellum (tail) of the spermatozoon uses a cilium-like array of microtubules, aided by very thick fibres, for powerful swimming.
  5. Intracellular movement of vesicles by microtubules is achieved by a repeating detachment and re-attachment of motor proteins - kinesin and/or cytoplasmic dynein - to the microtubule, energized by an ATPase. Two different motor proteins can achieve movement in opposing directions, as in axons, although the microtubules are polarised (+ & -) only one way.
  6. Sensory cilia containing microtubules occur on cells in the special sense organs, e.g., olfactory, and play some role in sensory transduction.
In conclusion, filaments and microtubules give support, and both participate in various kinds of movement.

D NUCLEAR-CYTOPLASMIC SYSTEM


l Access: Through the nuclear pores: the complex of glycoproteins at the pore - the pore complex - permits the diffusion of materials of Mr greater than 40-60 kDa and actively transports macromolecules.

2 Protein synthesis is the primary cell activity influenced by the nucleus. Proteins, as enzymes, transcription factors, receptors, and so on, are the key to the particular cell's character (Chapter 32).

3 Nuclear constituents are:

  1. Chromatin - mainly DNA and dispersed at interphase (except for one female X chromosome); has a fine granular appearance in routine EM, but is fibrillar; some RNA is present.
  2. Nucleolus/nucleoli - dense clumped granules; nucleic acid is mainly RNA, but some DNA is there. Peri-nucleolar/nucleolus-associated chromatin has a special relation to the nucleolus. A range in the number of nucleoli present usually exists, e.g., in the liver cells with one nucleus, l to 6, with 2 the modal value. Removal of DNA by DNase allows one to see nucleoli in the very dense nuclei of lymphocytes and some other cells.
  3. Nuclear proteins - histones and non-histone proteins are associated with the DNA and RNA. A nucleosome comprises a length of DNA wrapped around a core of histones. The proteins mostly leave the nucleus during cell division.
  4. Nuclear skeleton and karyoplasm/nuclear sap - A fibrous lamina is attached to the inner nuclear membrane, and supports the DNA periodically, creating intervening loops of DNA. The sap contains the more labile biochemical materials.
  5. Histological methods for the nucleus:

4 Protein synthesis (also Chapter 32)

  1. Is controlled by messenger RNA, synthesized by transcription from the chromatin DNA, and leaving via the nuclear pores.
  2. The nucleolus is a staging post for ribosomal RNA,
  3. before it passes out to the cytoplasmic ribosomes, free or attached to membranes. Ribosomes are held in clusters by mRNA.
  4. Transfer RNA brings to the ribosomes
  5. activated amino acids for linking together according to the messenger RNA`s molecule (the translation step),
  6. thus producing specific chains - a polypeptide or protein, with a sequence and length specified in the first instance by the transcribed nuclear DNA.

5
Cell fusion techniques, using the Sendai virus or chemicals, to produce hybrid cells have revealed that the cytoplasmic environment can alter the activity of the nucleus. Thus, a dense and inaccessible chromatin in a nucleus may be transformed by new cytoplasmic surroundings to one that is looser, paler and more reactive, as is seen in the small lymphocyte after antigenic stimulation. Cell fusion can also be between a nucleated cell and one made nucleus-free - a cytoplast.

E CYTOPLASM (cytosol/cell sap)

Structureless at the EM level, but of vital biochemical and physiological importance with its water, electrolytes, soluble proteins, amino acids, sugars, enzymes, etc. It is in continuous interaction with the filamentous and membrane-enclosed systems. Unwanted or defective proteins to be destroyed are conjugated with ubiquitin , which takes them to chemically structured concentrations - proteasomes - of proteolytic cytoplasmic enzymes Powerpoint.


Chapter 4 EPITHELIA

A INTRODUCTION

1 In the early development of the embryo the rapidly multiplying cells lie in layers. From each of these three germ layers a class of tissue develops that persists to maturity as a tightly packed layerof cells, contrasting with the connective tissues where the cells are spaced out in an extensive extracellular matrix. The class is the epithelial kind of tissue. Epithelia cover the outside of the body and line the spaces and tubes within the body. Powerpoint
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2 Epithelium is more than an inert covering or lining: it works. Examples of its activities are:

  1. The tongue has receptors for touch and taste in its protective epithelium.
  2. In the gut, digested foods, but not unwanted and toxic materials have to be absorbed into the blood.
  3. In the skin, heat is transferred from underlying blood vessels to the air, and skin is a surface on which sweat spreads and evaporates: mechanisms for cooling the body.
  4. In the eye, epithelia serve both transparency and light screening.
  5. In many epithelia the cells are secretory. They take raw materials from blood and build up complex materials for release from the cell as a secretion. Epithelial secretions protect, lubricate, digest, or, in the case of hormones, control. For instance, in the GI tract and airway, epithelial cells make anti-bacterial defensive chemicals.
This list indicates some of the many epithelial functions, for which there are several types of epithelium. The last function listed, secretion, often entails a gland.

3 A gland is a structure formed to increase greatly the epithelial working surface, without occupying too much space in the body. The several ways of doing this are presented as a separate topic - Glands (Chapter l6). Here, the interest is the epithelia that lie more stretched out in a covering or lining attitude.

4 The embryological origin, in terms of the three germ layers, of epithelial and other tissues does not correspond with the morphological divisions set out in Section C, and is clinically significant only as a basis for certain terms such as 'mesenchymal tumour'. Should you need them, details of the origins are tabulated in older histology textbooks.

B EPITHELIUM: SUPPORT AND NUTRITION

l Support of the cells of a simple epithelium or of the bottom layer of a stratified epithelium is by their lying on, and attachment to, a glycoprotein sheet reinforced by fine filaments - the basal lamina (BL). This is anchored by collagen fibrils to the denser fibres of a supporting connective tissue lamina propria.
2 The basement membrane (BM) seen in LM is the basal lamina, fibrils and connective-tissue ground substances.(The term 'basement membrane' is often used for just the basal lamina.) Of principally epithelial origin, the basal lamina comprises interacting macromolecules: special glycoproteins, e.g., laminin, nidogen (a sulphated glycoprotein), and collagen type IV and others, and also heparan sulphate proteoglycan.
The principal epithelial grip is by cell-membrane integrin to laminin.
Seen with TEM, the basal lamina is subdivided into two or three layers - a pale lamina lucida next to the epithelium, a lamina densa, then the deeper lamina fibroreticularis (less consistently visible).
BMs differ by location, and experience various pathological changes - thickening, breaks, duplication, autoimmune attack, etc.
3 The lamina propria has collagenous and elastic fibres, other matrix materials, fibroblast cells, blood and lymphatic vessels, and wandering defensive cells to protect it and the epithelium.
4 The nutrition of epithelial cells is by indirect exchange through the BL and matrix substances with blood in the capillaries of the lamina propria.
5 Tunica mucosa (abbreviated to mucosa)/mucous membrane comprises an epithelium, its BL, and the lamina propria, including structures such as glands lying in it. The exceptions are the skin (epidermis on a dermis), the mesothelium-covered serous membranes where tunica serosa is applied, and the endothelium-lined tunica intima of blood vessels.
6 In glands, the working epithelial cells constitute the parenchyma . The supporting connective tissue and other elements make up the stroma.

C MORPHOLOGICAL VARIETIES OF EPITHELIA

l Simple and compound epithelia
  1. The primary classification is based upon the layering: one cell thick is simple, two or more cell layers thick constitutes stratified/ compound. Cell shapes give the secondary classes.
  2. Simple epithelia, in general, are adapted to absorptive and secretory roles, while compound epithelia protect against damaging mechanical and chemical actions.
  3. Compound epithelia frequently, and simple sometimes, have several types of cell present. Cells lying basally on the BL are mitotically active and migrate upwards, differentiating to replace cells lost from the surface, or cells that have destroyed themselves by apoptosis.
  4. Epithelia shed cells continually. Such cast-off or desquamated cells may be examined in smears of the appropriate fluid - sputum, gastric, uterine cervical - for signs of malignant change and/or chromosomal abnormality in their epithelium of origin: the technique of exfoliative cytology.

2 Simple epithelia

  1. Cuboidal and
  2. Columnar
3 Squamous/pavement
4 Pseudostratified columnar
3 Stratified/compound/layered epithelia
5 Stratified cuboidal, and 6 Stratified columnar
(a) Surface cells of 5 and 6 resemble those of 2.1 and 2, but between them and the BL is a layer of cuboidal basal cells.

7 Stratified squamous

8 Keratinized/cornified stratified squamous

9 Transitional/urinary

4 Sites of occurrence, examples
(a) Simple - l, cuboidal, kidney tubules; 2, columnar, gall-bladder, gut, uterus (ciliated); 3, squamous, Bowman`s capsule in kidney, lining of lung alveoli; 4, pseudostratified columnar, epididymis, trachea (ciliated);
(b) Stratified - 1, cuboidal, sweat gland's duct; 2, columnar, penile urethra; 3, squamous, oesophagus, vagina; 4, keratinized squamous, skin; 5, transitional, urinary tract.

D CELL ATTACHMENT

l Devices for attachment
These are used to attach not only epithelial cells but, with some modification , those of the other tissues, e.g. muscle, osteocytes, neurons. To be seen clearly or at all, EM is needed.
l Junctional complex of: the girdle-like zonula occludens and zonula adhaerens/belt desmosome, below which is a ring of maculae adhaerentes/ spot desmosomes. Filaments of the terminal web in each cell's apical cytoplasm fasten to the complex. Something of the complex was seen as the terminal bar of LM.
2 Desmosome (the macula/spot/punctate kind of adhaerens attachment): disc-like structures scattered on cell's surface; each is contributed to by membranes of two cells; cytoplasmic tonofilaments (keratin intermediate filaments) converge on and insert into dense subplasmalemmal plaques. There are distinct plaque and desmosomal membrane proteins.
3 Hemi-desmosome: for better adhesion of the basal cell membrane to the basal lamina; includes a plaque and tonofilaments.
4 Gap junction/nexus: where two cells' membranes come closely together with only a 2 nm gap bridged by 'connexons' allowing ions, nucleotides, and amino acids to pass from cell to cell for coupling and coordination of many cells' activities.
5 Tight junction (resembles a zonula occludens but is not always belt-like): outer parts of two cells' membranes are fused together thereby occluding the intercellular cleft.
6 Plication/folding and interdigitation of the adjoining cells' folded membranes.
7 Glycocalyx in the usual 20 nm cleft existing between membranes where specialized attachment are absent.
8 Cell bridges with true cytoplasmic continuity: seen only rarely, e.g., between spermatids.
9 Fascia adhaerens: at intercalated discs of cardiac muscle.

2 Attachments: function and observation
l Attachments provide for:

2 Something (glycocalyx + ?) appears as a black line between cells treated with silver nitrate and sunlight. This outlines well the individual cells, e.g., in a stretched mesothelial sheet. Otherwise, cell membranes are not easily seen in LM except in the kidney collecting tubules. Elsewhere, the nuclei and their spacing are often the only guides to the number and shape of the cells and their layering. Even so, the pseudostratified epithelia show that this guide is fallible.

E METAPLASIA

l Metaplasia in any tissue is a change (usually abnormal) from one distinctive tissue to another, at a definite site after development is over. It implies a change in cell type - a transdifferentiation. Metaplasia is noted in epithelia, for example: 2 The term is applied neither to normal differentiation, e.g., from basal to ciliated cell, nor to a change to an abnormal tumour cell for which 'neoplasia' is used. Although, having become neoplastic, a cancer cell may then change its identity in a metaplastic manner.
3 Epithelioid is a term for a non-metaplastic epithelium-like appearance of non-epithelial osteoblasts as a layer on bone, or secretory cells of muscular origin as in the kidney's juxta-glomerular apparatus.
4 Oncocytes are large, eosinophilic, mitochondrion-rich epithelial cells with small dark nuclei, occurring with increasing age in glandular and lining epithelia, and constituting a kind of metaplasia.

F SPECIAL ELEMENTS IN EPITHELIA

Non-epithelial structures sometimes occur within an epithelium:
  1. Capillaries - very rarely; only in cochlear stria vascularis.
  2. Nerve axons - common in skin, oral mucosa; less common elsewhere.
  3. Neural crest derivatives - as melanocytes, and accessory glial-type cells associated with receptors.
  4. Lymphocytes - common in gut and airway; less common elsewhere.
  5. Langerhans cells - contributors to immune defence in stratified squamous epithelia.
  6. Globular leucocytes - a special granular leucocyte of some epithelia.

Chapter 5 CONNECTIVE TISSUES

Connective tissues (including cartilage and bone: Chapters 6, 7 and 8) are derived from mesoderm or mesectoderm (for the head) of the embryo, via an intermediate stage called mesenchyme. Mesenchyme consists of pale cells, with extended processes, lying in jelly-like matrix. In later development, the cells and extracellular matrix (ECM) become specialized for various tasks, and the matrix comprises amorphous 'ground substance' reinforced to greater or lesser extent by specialized fibres. The various cells, fibres, and ground substances will be discussed, followed by a treatment of the tissues that they combine to build. Connective tissue from hereon may be abbreviated to CT. (Caution! This abbreviation usually signifies computed tomography.)Powerpoint

A CELLS OF CONNECTIVE TISSUES

l Fibroblast
l Occurs in young active, and adult quiescent/less active forms.
2 Young has abundant, basophilic cytoplasm, with a well-developed Golgi complex and GER for protein and proteoglycan synthesis.
3 Nucleus is ovoid, with weakly staining chromatin granules.
4 The cell is elongated, and often sends out processes to take on a more elongated or stellate form.
5 Adult fibroblasts (fibrocytes) have smaller, darker nuclei, and very little cytoplasm. They remain fixed and squashed into a spindle/cigar form amongst the fibres that they formed.
6 Function - forming and remodelling collagen, reticular and elastic fibres, and the ground substances. The remodelling requires the production of destructive enzymes, and inhibitors to help restrain their action. TIMPs - Tissue Inhibitors of MetalloProteinases - are an example.
In some sites, e.g., the periodontal ligament holding the teeth in place, the fibroblasts more aggressively destroy fibres, in the process of matrix turnover.
7 Young fibroblasts, aside from making fibres, may in some circumstances (e.g., wound repair) take on some smooth-muscle characteristics, and become contractile myofibroblasts, which contribute to the disabling contractures of some scar tissue.

2 Mesenchymal cell
l Has a similar appearance to a small, young fibroblast, but is far more multipotential in what cell types it can turn into.
2 In adult tissues, two views are:
...(a) a few are present and can explain such findings as the formation of ectopic (out of its expected place) bone in soft CT, otherwise difficult to account for unless differentiated cells such as fibroblasts can dedifferentiate and change their role;
...(b) mesenchymal cells all differentiate early in life and thereafter are not present, and fibroblasts or other cells can de- and redifferentiate and become osteoblasts.

3 Macrophage/histiocyte
l An ovoid or spheroid cell, which may change its shape while lying alongside fibres, or when extending pseudopodia to move and ingest materials.
2 Phagocytoses dead cells, cell debris, live and inert foreign bodies.
3 Coordinates the inflammatory response and healing by means of signalling peptides and proteins - cytokines, e.g., IL-1, TGF-b (Chapter 8.F).
4 Nucleus is smaller and more condensed than that of the active fibroblast.
5 Cytoplasm is pale with little GER, but has many lysosomes, when digesting phagocytosed material.
6 Macrophages may fuse to become foreign-body giant cells with many nuclei, when faced with a large object for digestion. More on macrophages.

4 Macrophage/reticuloendothelial/mononuclear phagocyte system (MPS)

l Comprises cells related directly to blood monocytes, or derived from the same precursor in marrow.

2 A tentative division of the macrophage-system cells recognizes:
...Phagocytic antigen-presenters (Chapter l9.B.l)
... (a) Macrophages of connective tissues and serous cavities.
... (b) Alveolar macrophages/lung dust cells.
... (c) Macrophages of lymph nodes, spleen and bone marrow.
... (d) Kupffer sinusoid-lining cells of liver.
.........................................
... Weakly phagocytic antigen-presenters
... (e) Dendritic and interdigitating reticulum cells of lymphoid tissues.
... (f) Langerhans cells of epidermis and other epithelia.
.........................................
... Specialized (Some not phagocytic? Some not antigen-presenters?)
... (g) Foreign-body giant cells.
... (h) Microglia cells of CNS.
... (i) Synovial A cells lining joints.
... (j) Osteoclasts resorbing bone.

3 The phagocytic group (i.e., the original reticulo-endothelial series) can be revealed by vital injection (into the living animal) of colloidal or particulate coloured matter, e.g., Trypan blue or India ink, which the phagocytic cells of the system preferentially accumulate in their cytoplasm, thereby identifying themselves. Nowadays, MPS cells are distinguished by their cell-surface glycoprotein profiles, e.g., CD antigens.

5 Mast cell
l A `watchdog' cell starting the inflammatory response to noxious intruders.
2 From the German verb, mästen, it meant a `fattened' cell.
3 Spheroid or ovoid with a small central nucleus, and its cytoplasm packed with dense basophilic granules.
4 Granules give a metachromatic staining reaction with thionine or toluidine blue, i.e., a reddish-purple colour, because they contain a sulphated polysaccharide - heparin.
5 Heparin is an anticoagulant for blood, first obtained from the liver (hepar), but it also inhibits vascular smooth muscle proliferation and some immune complement reactions. As a polyanion, it can complex materials, e.g., the trypsin-like enzyme, tryptase, in the granules.
6 Histamine, increasing capillary permeability, is also present in the granules. The chemokines also released can then more easily attract white blood cells out of the vessels.
7 Many stimuli (e.g., antigens and agents released by lymphocytes during an immune response) activate a release of the granule contents, from this `mobile-pharmacy' cell, with its many chemical mediators.
8 Mast cells favour positions in CT close to veins (MCt subtype), and at dermal and mucosal interfaces with the hostile environments of the skin, airway, and gut (MCtc subtype).
9 The mast cell subtypes in man differ in the proteases that they contain:
...MCt cells have mast-cell tryptase and are involved directly with defence.
...MCtc cells contain chymase, cathepsin G, and other proteases, in addition to mast-cell tryptase, and are more concerned with adaptive and remodelling responses of blood vessels and CT.

6 Fat cell/adipocyte
l A genuinely fattened cell, initially resembling a fibroblast with a few droplets in the cytoplasm.
2 For the white or yellow unilocular fat seen in adult man, the droplets (mainly glycerides of fatty acids) coalesce and more fat is added,
3 until the nucleus is bulged to one side of a spheroid cell up to 200 µm in diameter, distended by a huge droplet.
4 Cytoplasm, with a Golgi complex, ER and mitochondria, is present as an attenuated peripheral shell.
5 The cell is static, but its content is not. The stored fat is participating in the body's carbohydrate and fat metabolism.
6 Fat in the usual wax-imbedded section is dissolved out, but with osmium tetroxide fixation it remains and is black. Some dyes will colour it, if it is preserved by frozen sectioning.
7 Besides a number of adipocyte-specific enzymes for fat metabolism, fat cells secrete leptin, which helps control energy balance and body fat mass.

7 Melanophore/CT pigment cell/CT melanocyte
l A process-bearing cell with melanin pigment granules in its cytoplasm.
2 Found in the skin`s dermis, brain's pia matter and the scleral and choroid coats of the eye.

8 Plasma cell
l Many tissues, particularly those lining tracts open to outside the body, are not immunologically virgin, but have been exposed to foreign organisms that have provoked immune responses by local CT plasma cells and lymphocytes. A lamina propria may have many of both and some eosinophils, e.g., in the gut.
2 Plasma cells are ovoid, roughly l0 µm in length, with an eccentrically placed nucleus having its denser chromatin granules clumped regularly around the nuclear membrane (clock-face appearance).
3 Cytoplasm is deeply basophilic from the rich GER, except for a pale central region where the Golgi complex lies.
4 Proteins synthesized by plasma cells in lymphoid organs reach the plasma as immunoglobulins/ antibodies, inactivating foreign invaders, e.g., viruses.
5 Plasma cells in CT make antibodies for local use, e.g., in the airway or gut, to counter toxins and control microbial populations.

9 Reticular/reticulum cells
l Immunocytochemistry, EM, and enzymatic analysis distinguish at least three kinds of reticular cell: fibroblastic, and two phagocytic kinds - interdigitating (T-zone:) and dendritic (B-zone: antigen-presenting).
2 The supporting reticular fibres of lymphoid tissues and bone marrow are presumed to be produced by the fibroblastic variety.
3 Caution! The principal reticular cell in the thymus is an epithelial kind, although extending cell processes to build a reticulum.
[Any time you hear 'reticular cell', ask for the type meant.]

B FIBRES OF CONNECTIVE TISSUES

l Collagen fibres
l Fibres 2 Fibrils 3 Collagen molecule and fibril-formation 4 Collagen types: distribution and use
1 All collagen molecules are trimers of helical alpha chains (with two-number designations) intertwined together, mostly as a robust super-helix, e.g., [a-1(II)]3 in cartilage, [a1(IV)2a2(IV)] in basement membrane.
2 Collagen molecules differ in their amounts of helical versus globular shapes along the molecule. The ones (fibrillar) that are cleaved to be only helical assemble into fibrils, the others (non-fibrillar) attach to and space the fibrils, in scaffolds of various patterns, fibril-widths, densities and strengths, appropriate to the mechanics of the tissue. Some of the scaffold-glueing ones, e.g., types IX, XII, and XIV, are termed Fibril-Associated Collagens with InterrupTed helices - FACIT.
3 The types are relatively tissue-specific, but not absolutely so as once was thought.
4 Of the twenty plus types, some important ones are:
Type I in bone, fibrocartilage, and established soft connective tissues
Type III in these same tissues as embryonic or reparative forerunners (and as a minor mature component)
Type II in hyaline cartilage
Type IV in basement membranes
Type VII to anchor BMs, and
Type VIII from endothelium lining vessels.

5 Collagen staining
(a) Collagen (type I) often is present in bulk, and is stained selectively by: aniline blue in Mallory's method, light green in Masson's, or red acid fuchsin in van Gieson's. (Eosin stains it orange.)
(b) Mallory's, Masson's and van Gieson's trichrome methods distinguish collagen from muscle, and also react with the nuclei and cytoplasm of other cells.

6 Caution for rat CT spreads. Preparations of rat subcutaneous tissues may be contaminated by hairs. The segmentation of the medulla of a hair gives a crossbanding effect in LM. Collagen fibril crossbanding is visible only in EM.

7 Collagen types: extended from the classification of Prockop DJ & Kivirikko KI. Ann Rev Biochem 1995;64:403-434

Fibril-forming     I  II  III    V                   XI
Network-forming               IV          VIII     X
Beaded filament-forming            VI
Anchoring filament-forming            VII
FACIT                                          IX       XII       XIV       XVI             XIX
Nonsecreted transmembrane                                   XIII                XVII*            XXIII  XXV                                                         XV             XVIII
Basement membrane zone                                                XV             XVIII       
* transmembrane collagen XVII is a component of hemidesmosomes. An autoimmune reaction to it can cause poor epidermal adhesion and hence skin blistering in humans.
The above Table looks confusing enough, but with 27! collagen types now known, any textbook table is only a distraction, and is already out of date at publication.

2 Reticular fibres
l Collagen fibres, running parallel to one another, do not join up with others running differently. Such an arrangement is seen, however, with reticular fibres, which form a network or reticulum.
2 Reticular fibres stain black with reduced silver methods, hence their other names - argyrophil or argentophil. H and E and some trichrome stains leave them unstained.
3 X-ray diffraction and EM show them to be like fine collagen fibres, having the same 67 nm-repeating crossbanding. Furthermore, they appear first at many sites, as in mesenchyme and healing wounds, where collagen fibres will later form. Thus reticular fibres are an immature, fine kind of collagen fibre, mostly of type III collagen.
4 They persist into the adult in several organs, where a fine fibrous support is needed that does not interfere with a close relation between fixed cells and blood or lymph, e.g., in endocrine glands.
5 Reticular fibres fasten to the underside of basal laminae of epithelia and endothelium, and bind and secure muscle and nerve fibres, using their external laminae.

3 Elastic fibres
l May be fine, single and branching in areolar CT, or thick and parallel in elastic ligaments. Walls of blood vessels have incomplete elastic membranes.
2 The elastic nature of the fibres is shown by the spiralling and kinking of their recoiled broken ends, in spread preparations.
3 Elastic fibres and membranes, if thick, stain pink with eosin, or red with Masson's method; otherwise, they remain unseen, unless elastic stains, e.g., orcein or Verhoeff's, are used.
4 In bulk, unstained, they appear yellow to the naked eye.
5 Formation and nature - fibroblasts and vascular smooth muscle cells form and release two components: (a) fine protein microfibrils thought to orient (b) tropoelastin as it polymerizes into amorphous elastin. With little structure in EM, elastin is a network of long protein chains held in a springy arrangement crosslinked by desmosines, each derived from four lysines of the protein amino-acid chains.

C GROUND SUBSTANCES

l Location - in interstitial/tissue spaces, cartilage and bone matrices, under basal laminae, on and between CT fibres. Ground substance(s) is the extracellular matrix, less the fibrous and fibrillar elements.

2 Nature - large negatively charged proteoglycan molecules (polyanionic macromolecules) bind to a varying degree water, electrolytes, and other macromolecules, such as collagen, and the glycoproteins, fibronectin and tenascin.

3 Proteoglycan chemistry - from a long protein backbone molecule, many long sugar side chains stick out, because negative charges along each chain repel adjacent chains and each other. The chains are composed of repeating pairs of sugar/saccharide units. Each pair has an hexosamine and a uronic acid. The loss of hydrogen ions from the many acids in the chain of glycosaminoglycans (GAGs) leaves negative charges, only some of which are neutralized by counterions such as Na+.

4 Nomenclature - the many linked sugars of the side-chains are polysaccharides, hence with the protein backbone the general name - 'protein-polysaccharide'. However, this also describes glyoproteins, for example, mucoproteins and mucopolysaccarides. Proteoglycans differ from glycoproteins in: their core proteins; the use of fewer species of sugar; lack of branching of the sugar chains; and usually their longer sugar chains, and more acidic/negative character

    O-LINKED GLYCOPROTEIN an example
                                             
 |Core protein
 AA                    Fu    Side chain is short (1-20 sugars) & branching
 |                     |
 AA        Ga - Gln - Ga     Wide variety of sugars
 |       /  ^
 T - Gln                     Uses one of several sugar core^ types
 |    ^   \                   for attachment to the protein
 AA        Gun - Gln - Na                 
 |          ^                Na - sialic acid,  Fu - fucose



    TYPICAL STRAIGHT PROTEOGLYCAN SUGAR SIDE-CHAIN
                                                            -------->
 |                                        Repeating disaccharide pair 
 AA                                       ___|___
 |                                       |       |
 S - Xy - Ga - Ga - Ua - Gln - Ua - Gln - Ua - Gln - Ua - Gln
 |                              -     -          - Unsatisfied negative
 AA                                                             charges
 |Core protein, with serines (S) & threonines (T)      

5 Proteoglycan varieties - dependent on the specific sugars, and the sites of sulphation, if any:
... Hyaluronate - soft connective tissues; synovial fluid; vitreous humour;
... Dermatan sulphate (chondroitin sulphate B) - skin and corneal CT;
... Keratan suphate - cartilage matrix;
... Chondroitin-4-sulphate (A) - cartilage matrix;
... Chondroitin-6-sulphate (C) - cartilage matrix:
... Heparin (also sulphated) - granules of mast cell and basophil.

6 Staining - the failure of counterions to neutralize all anions leaves regions of high negative charge density. If the proteoglycan is prevented from dissolving out, its reactions are:

7 Physical properties - the high negative charge:

8 Overview of proteoglycans (PGs) and glycoproteins in connective tissues

1 The large PG monomer molecules may be aggregated by being strung along a hyaluronate backbone, by means of a link protein for the core protein-HA attachment.
PG aggregation produces huge molecules extending over micrometres, and visible with conventional TEM. Proteoglycans amenable to such assembly are aggrecans, susceptible to breakdown by aggrecanase.
However, the chemical nature and heterogeneity of monomers and their aggregates make study of these important matrix constituents difficult.
Note that proteoglycans are also kept within some cells to work with other molecules.

2 The glycosaminoglycan side chains of proteoglycans vary in number, nature and length. Combinations of sulphated and non-sulphated hexosamines, and relatively tissue-specific core proteins, yield a diversity of PGs, crudely classifiable by molecular size into large and small:

LARGE
Chondroitin-6-sulphate, skeletal keratan sulphates - Cartilage
Versican/Fibroblast PG - Soft CTs
Cell-surface-associated, e.g., the membrane-attached PGs syndecans, with heparan-sulphate and chondroitin-sulphate chains, and the HSPGs - glypicans - on epithelial and other cells
Basement-membrane heparan-sulphate PGs - basement membranes, e.g., perlecan

SMALL
Decorin/PGII (chondroitin/dermatan sulphates) - extracellular matrix
Biglycan/PG-S1 ( " ) - associated with a variety of cells including non-CT ones
Fibromodulin (keratan sulphate)
Dermatan sulphate
Small bone proteoglycans I & II

3 Non-collagenous glycoproteins of connective tissues include: Fibronectin, Tenascin, Thrombospondin, Bone sialoprotein/BSPII, Osteopontin/BSPI, Osteonectin/Bone Gla protein, Cartilage-matrix protein, Alkaline phosphatase, Chondronectin, and Fibrillin.
They interact with other macromolecules and influence cell behaviour.

One clinical aspect is their use as urinary or serum markers of excessive turnover, e.g., Gla protein for bone disease.
Fibrillin is a crucial component of elastic fibres and other structures in CTs; and genetic defects in its formation result in the weak arterial walls, poorly suspended eye lens, lax ligaments, etc. of Marfan's syndrome.

4 Fibronectin and Tenascin

5 For more on vulnerabilities from the cellular and ECM-molecular interactions see ECM <>

D TYPES OF CONNECTIVE TISSUES

Based upon: (a) the density and order of fibre packing; and (b) the predominant cell and fibre types.

l Areolar tissue
l Loose textured with a mixture of all cell and fibre types (but seldom pigmented cells).
2 Rich in ground substances which fill the spaces or areolae, and confer physical properties and control transport.
3 Locations - the lamina propria of the gut, under the skin, around joints, muscles and some viscera, and other sites needing some freedom of movement; the eye's choroid coat serving a more nutritive role also has pigment cells.
4 Areolar tissue merges with the somewhat denser CT of D.6. Both types may be regarded as belonging in one broad loose category.
5 Serous membranes are similar to areolar tissue but also have a layer of simple squamous mesothelium (sometimes two layers).
6 Milky spots on serous membranes are dense accumulations of the macrophages and lymphocytes present to protect serous body cavities.

2 White adipose tissue
l Comprises primarily fat cells enclosed in basal lamina, and held on a framework of reticular fibres in association with many blood capillaries.
2 Fibrous CT encloses the tissue, subdividing it with septa.
3 Found subcutaneously in the hypodermis (in the child, a panniculosus adiposus), and in the mesentery, omentum, and retroperitoneal area.
4 Padding fat in palmar, plantar and intraorbital sites is not so freely available as an energy store, and can survive starvation.
5 Adipose deposits in the hips, buttocks, and breasts are especially under the control of female sex hormones, but many hormones control fat metabolism.
6 Functions - energy store; insulation; padding; steroid conversions.

3 Brown adipose tissue
l Cells have many separate (multilocular) fat droplets, relatively more cytoplasm, and are smaller than white fat cells.
2 Found around the thorax and kidneys of animals naturally exposed to severe cold, particularly hibernators.
3 Brown fat is a thermogenic organ providing a prompt and direct source of heat to maintain the temperature of vital organs. Uncoupling protein 1 lets mitochondria divert energy in this otherwise unwanted thermal way by uncoupling respiration from ATP formation.
4 Seen in the human newborn; in adults BAT is detectable after adrenergic stimulation. Brown fat might dissipate surplus energy from overeating.

4 Reticular tissue
l Has the reticular fibre as the supporting fibre, and phagocytic fixed macrophages.
2 The fibres are made by some of the stellate reticular cells acting as fibroblasts.
3 Reticular tissue also contains parenchymal cells (the main working cells) held by the fibres, e.g., hepatocytes or lymphocytes.

5 Elastic tissue
l Elastic fibres or membranes are the predominant element.
2 The fibres may be:
(a) thick or very thick (l0-l5 µm) and orderly as in the elastic ligaments, e.g., ligamentum nuchae (in the neck of heavy-headed grazing animals), vertebral ligamentum flavum, penile suspensory ligament, and in the vocal chords; or
(b) finer and mixed with membranes in elastic arteries. The lung and airway also have many elastic fibres.
3 In the ligaments, elastic fibres are formed by fibroblasts and held together by reticular fibres, proteoglycan, and glycoproteins.

6 Dense fibrous (collagenous) tissue
Two kinds:
...(a) Regular, e.g., tendon, ligament, aponeurosis, fascia, with collagen fibres oriented to take stress principally in one direction. (The dense corneal stroma has very orderly collagen for transparency as well as strength.)
...(b) Irregular, e.g., dermis, organ capsules, periosteum, perichondrium, epitendineum, with irregular, interwoven bundles of collagen.

7 Loose fibrous (collagenous) tissue
l Although 6(b) and 7 have fibroblasts and collagen fibres as the principal elements, reticular and elastic fibres and other cells are present to a lesser degree, together with blood and lymphatic vessels and nerves.
2 An example of a loose fibrous tissue is the lamina propria of the urinary bladder, looser than dermis, denser than that of the gut. Indeed, the gut's lamina propria is so given over to defence and defensive cells that it is hardly recognizable as a CT.
. However, fibrous CTs form a continuum from dense, regular to areolar, making implausible any assignment to rigid categories.

8 Mucous/mucoid/primitive connective tissue
l Very rich in proteoglycans and water, has some fine collagen fibres and widely separated young fibroblasts.
2 As Wharton's jelly of the umbilical cord it encloses and cushions the vessels; the ocular vitreous and young dental pulp also fit tolerably well in this class.

E FUNCTIONS OF CONNECTIVE TISSUES

1 Mechanical and protective - supporting, restraining, binding, separating, directing and padding.
2 Transport of nutrients, metabolites, and signalling factors.
3 Storage of energy-rich lipids, water and electrolytes.
4 Defence against pathogenic organisms.
5 Repair of damage to itself, and organs supported or enclosed, by fibrosis - the formation of irregular collagenous scar tissue.
6 Thermogenesis (brown fat) and insulation (white fat).

Physiological factors controlling connective tissues are listed in Chapter 8.E.


Chapter 6 CARTILAGE

A specialized CT to resist compression, and provide modest rigidity with flexibility, by having its cells, chondrocytes, produce a firm resilient matrix of ground substances, and fibres or fibrils. The rapid growth of cartilage is used to assist the growth of bones and the repair of fractures. Based on the composition of the matrix, three kinds are distinguished: hyaline, elastic, and fibrocartilage. Powerpoint.

A HYALINE CARTILAGE

l Occurs fused with bone or as discrete pieces, looking hyaline/translucent (glass-like) to the unaided eye. Most surfaces, except joint/articular ones, are covered by a nutritive CT perichondrium/capsule with collagen and elastic fibres, fibroblasts and blood vessels. It merges gradually via a chondrogenic zone with the cartilage proper.

2 Matrix, apparently amorphous with HE staining in LM, contains:

3 Chondrocytes or cartilage cells are large and rounded, each lying in a space - lacuna - enclosed by matrix. Cells often are grouped in nests of 2, 4, or 6 as a result of mitoses and restricted cellular movement. EM reveals cells to have short stubby processes, fat droplets, glycogen and the GER and Golgi complex appropriate for secretion of the matrix components: proteoglycans, type II collagen [with the homotrimeric molecule a1(II)3], and glycoproteins.

4 Growth occurs in two ways:

Growth is vulnerable to X-rays, poor nutrition, and disturbed blood supply, for example, from fractures at the growth plate.

5 Territories Most noticeably in articular cartilage there are:
.. (i) the chondron - the chondrocyte and the pericellular matrix immediately around it;
.. (ii) proteoglycan-rich territorial matrix outside the chondron;
.. (iii) interterritorial matrix, lying between the territorial matrices.
The matrix of the chondron has its own profile of special collagens, proteoglycans, and cartilage glycoproteins, whereas the differences between territorial and interterritorial matrices are more quantitative, and related to collagen fibril thickness and orientation.

6 Nutrition - cartilage is avascular and no blood vessels serve the matrix directly, but cartilage canals may carry vessels through the matrix to non-cartilaginous regions, e.g., secondary ossification centres. Therefore, nutriment and wastes must diffuse through the matrix for the cells to stay alive and perform their slow turnover of the matrix macromolecules. The diffusion may break down and various degenerations then occur, e.g., calcification. This last is prompted, organized and made use of in the process of endochondral ossification.

B ELASTIC/YELLOW CARTILAGE

l Is more opaque and flexible than the hyaline kind, but the cells are similar in appearance and distribution; and it occurs as separate pieces with a perichondrium.
2 Matrix is permeated by many elastic fibres that can be selectively stained by stains such as orcein or Verhoeff's. The matrix is not prone to degeneration and calcification.

C FIBROCARTILAGE

l In the intervertebral (IV) disc, fibrocartilage at first appears to have a rather disorderly matrix with many thick collagen fibres, amongst which are dispersed only a few chondrocytes in lacunae. However, the fibres are orderly in their alternating orientations and layering, like the burst-resisting fibres of an old-style bias-ply car tyre.
2 The matrix gives the staining reaction of collagen, mostly type I, except for close around the cells where proteoglycans are abundant.
3 Lacks a perichondrium and is not seen as discrete pieces; rather it is a strong tension-resistant, but flexible transitional tissue located between tendon and bone, bone and bone, hyaline cartilage and hyaline cartilage.
4 In the IV disc, the enclosed central nucleus pulposus is not cartilage, but nevertheless has collagen type II, which diminishes in the innermost layers of the annulus fibrosus as it is replaced by type I.

D DISTRIBUTION OF THE THREE CARTILAGE VARIETIES

l Hyaline - articular surface of most synovial joints; costal cartilages; nasal and respiratory tract cartilages; basis of most of the fetal skeleton; fracture callus, Chapter 31.E.1
2 Elastic - external ear, pharyngotympanic tube, epiglottis, and some laryngeal and bronchiolar cartilages.
3 Fibrocartilage - intervertebral disc's annulus fibrosus (around a nucleus pulposus of notochordal origin, present until late in life); pubic symphysis; femoral ligamentum teres; many tendon insertions into bone; and the articular surface of some joints, e.g., temporomandibular.

Chapter 7 BONE

Bone is a hard CT with cells, osteocytes, in much matrix, and serves for support, attachment, leverage, protection and mineral storage.

l To obtain great strength and rigidity with some elasticity, the matrix is composed of densely packed collagen fibrils infiltrated with bone mineral as fine crystals of calcium salts resembling hydroxyapatite crystals. Mineral constitutes about 65 per cent of the dry weight of bone. The densely packed collagen fibrils are primarily type I. There are small amounts of distinctive non-collagenous proteins, e.g., calcium-binding osteocalcin and bone sialoproteins (Chapter 5.C.8).

2 Matrix is strong but dense, thus nutritive fluids cannot diffuse freely through it. Osteocytes therefore have to differ from chondrocytes in having many long processes extending through canaliculi (narrow passages) and making contact with one another and, indirectly, with blood vessels. The cell body lies in a cavity, a lacuna, in the matrix.

3 Throughout life, for mineral homeostasis, and for its special problems of growth, bone is subject to an unending turnover, with selective destruction and replacement - the remodelling process. Powerpoint

A PARTS OF A BONE

See Chapter 8.B for terms, e.g., diaphysis, epiphysis, etc.

B CLASSIFICATIONS OF BONE

l Based on the size of the spaces within the bone, and its trabecular (lattice-like) or dense nature:
.. (a) Cancellous/spongy/trabecular
.. (b) Compact/dense

2 Based on the presence or absence of lamellae (layers) and osteons/Haversian systems:
.. (a) Woven/primitive
.. (b) Lamellar/Haversian

Woven bone's matrix has disorderly fibrils, whereas in lamellar bone the fibrils of a lamella share a predominant orientation. Note that a particular bone will have areas of woven and lamellar bone, depending on how far remodelling has involved all regions.

C HAVERSIAN BONE

l An Haversian system is roughly cylindrical and arranged around one or two small vessels in a central Haversian canal.
2 Osteocytes and bone lamellae making up the system are disposed in 4-20 concentric rings centred on the canal.
3 A lamella is the territory formed and maintained by the osteocytes lying in a ring when seen in a cross-section. From the orderliness of the fibrils, lamellae can be distinguished in polarized light, but it is only in a smaller unit, the domain, that SEM reveals the fibrils to be aligned in the same direction.
4 Haversian canals branch and join up with others. Their vessels originally entered the bone from the periosteum or marrow via Volkmann's canals, around which osteocytes are not especially ordered.

D MATURE HUMAN BONE

Studied from the outside working inwards has:
l Periosteum of dense CT divisible into:

2 Dense cortical bone. Where wide, e.g., femoral shaft, this layering is often present:

In practice, some areas of dense bone remain woven or primary and are not replaced by this classic lamellar architecture.

3 Cancellous medullary bone whose trabeculae are lined by a thin cellular endosteum and have some lamellae, but can be sustained by marrow blood vessels without the need for Haversian canals.

4 Marrow cavities lie between trabeculae, inside the tubular shaft, or in the diploic spaces of flat skull bones.

E BONE CELLS

l Osteoblast
l Lies on the surfaces of bone, in a one-cell thick layer, as most of the endosteum and inner periosteum.
2 May be in two states: 3 Forms the collagen, glycoproteins, and proteoglycans of the matrix, and controls the deposition of mineral crystals on the fibrils.

2 Osteocyte
l Osteoblast becomes an osteocyte by forming matrix around itself and becoming buried or immured.
2 Young osteocyte thus resembles an active osteoblast; older ones have smaller, flattened bodies.
3 Processes extending from the body down the canaliculi are not visible by LM; but EM shows that osteocytes, like osteoblasts, remain connected by gap junctions.
4 The mature osteocyte is involved in maintaining the matrix of its territory. SEM evidence puts into doubt the proposal that osteocytes can resorb bone by osteolysis. Lacunae empty of osteocytes indicate dead bone.

3 Osteoclast
l Large, multinucleated cell, with a pale acidophilic cytoplasm.
2 Lies on the surface of bone, often in an eaten-out hollow - Howship`s lacuna.
3 Cell surface is attached to the bone by podosomes to create a sealed compartment against the bone, in which the moving long cell processes of the ruffled border can agitate the resorbing - bone-destroying - materials.
4 Cytoplasm has vacuoles and lysosomes, since the mechanism of bone resorption is partly an enzymatic digestion, by cathepsins and collagenase, and also from acid made by an osteoclastic proton pump.
5 In dense bone, many osteoclasts act together to erode resorption tunnels, which are later partially filled in with lamellar bone to become osteons.

4 Bone cell dynamics
l Skeletal growth, changes of shape, and the physiological responses of bone need changes in the populations of 'blasts and 'clasts.
These rely on a proliferation of osteoblasts or a precursor, while osteoclasts come from the fusion of blood-derived monocytes, which also partipate indirectly as macrophages in the bone resorption.
2 The osteoprogenitor cell is a small, organelle-poor cell on the surface or lying just behind the osteoblasts. It might be just an inactive osteoblast: that it is more of a stem cell is shown by its occasionally becoming chondroblastic, e.g., in tumours and fracture repair.

F HISTOLOGICAL METHODS FOR BONE

Special techniques are needed because of the difficulty of cutting such hard material into sections thin enough for microscopy.
1 Ground sections with the mineral present are made by sawing out a slice of bone (or tooth) and grinding it thinner. They show osteons, lacunae and canaliculi, but these hold air or debris and no longer cells.
2 Decalcified sections are cut from bone imbedded in the usual way after removal of the mineral by dilute acids or chelating agents. Cells and the organic matrix remain. Eosin and selective collagen stains reveal the dense collagenous matrix, but individual fibrils and canaliculi are not seen unless special stains are used.
3 Mineral density can be studied by the magnified X-ray image of microradiography in ground sections or microtome-cut sections of plastic-imbedded undecalcified bone obtained by biopsy.
4 Electron microscopy of such plastic sections gives a comprehensive view of mineral, collagen, and cells, and their interactions. Scanning EM reveals bone's trabecular architecture, and how bone is formed and destroyed.
5 Vital labelling, with the fluorescing tetracyclines, alizarin red (in madder), or the radioactive isotopes, 45Ca or 31P, given at known times, permits the amount and sites of new bone formation, and its patterns of deposition and resorption to be identified, and related to bone diseases or experimental manipulations.

G JOINTS

1 Synarthroses (poorly movable)
1 Syndesmosis. Bones linked by dense fibrous CT, e.g., a skull suture, which may be replaced by bone with increasing age to become a synostosis.
2 Synchondrosis. Bones linked by cartilage, e.g., pubic symphysis.

2 Diarthrosis (movable)
1 Articular cartilage, usually hyaline, covers the moving bone ends, and is nourished and lubricated by synovial fluid.
2 Joint capsule of dense irregular fibrous CT, continuous with the periostea, encloses a joint space for synovial fluid.
3 Nervous joint receptors for proprioception are in the capsule.
4 Synovial membrane: lines the capsule; a cellular layer, with macrophage (A/M) and fibroblastic (B/F) cells, lies on a loose vascular CT, sometimes thrown up into folds, synovial villi. The cells make lubricating hyaluronic acid and glycoproteins, and determine the nature of the cartilage-sustaining synovial fluid.
5 Articular cartilage layers: although the cartilage is not thick, variation in the amounts and arrangements of proteoglycans and collagen with depth distinguishes these layers:
... superficial/tangential
... intermediate/transitional
... deep/radial
... calcified/mineralized, attached to the 'subchondral' bone.
A lamina splendens is at the free surface of the superficial layer: below it the collagen fibrils are better organized, in a packed series of 'leaves' that curve up from the radial layer, run parallel with the surface superficially, then descend to the radial layer.
6 In arthritis, inflamed synovium threatens articular cartilage. Synovial cytokines stimulate chondrocytes to emphasize cartilage breakdown over renewal.


Chapter 8 BONE FORMATION

Introduction
l Occurs in only one way - by the appositional or surface-depository action of osteoblasts; soon accompanied by the selective destructive action of osteoclasts in a remodelling process, continuously adapting the growing bone to developing soft tissues and dynamic mechanical forces, whilst meeting metabolic mineral demands.
2 Growth by remodelling is necessary because no interstitial growth is possible (except in growth cartilages).
3 Bone formed in the fetus is woven: only later is it mostly replaced by lamellar bone.
4 Dependent on whether bone is formed de novo in a soft tissue area, or in a site already taken by an established cartilaginous model, two situations of bone formation are noted - intramembranous and endochondral.

A INTRAMEMBRANOUS OSSIFICATION

l Seen in the skull vault, facial skeleton, and parts of the clavicle.
2 In one or more ossification centres for a given bone, mesenchymal cells become osteoblasts and start to lay skeletal claim to territory by forming branching trabeculae/struts of bone. The initial thin struts may be called spicules.
3 This trabecular bone becomes denser by widening of the trabeculae, and is then remodelled externally and internally, e.g., in the skull vault to two denser plates, tables, with spongy bone - diploë - between them.
4 The remodelling plates expand from their centres, but during growth remain separated by CT sutures for better adjustment to the enlarging brain, eyes, nasal cavities, etc. Skull bones grow by complex interactions and remodelling patterns that must cope also with, for instance, more teeth in the older child's jaws and the need for articulating cartilages on the mandible .

B PREPUBERTAL LONG BONE

l Diaphysis is the long tubular shaft containing marrow. The dense bone is the cortex, the marrow constitutes the medulla.
2 Epiphyses lie at each end of the bone. Each has:
... (a) a cap of hyaline articular cartilage over a
... (b) cushioning lattice of secondary-ossification-centre bone;
... (c) this bone on its deeper aspect is fused with an epiphyseal plate/growth disc of hyaline cartilage.
3 Metaphysis is a lattice of bone trabeculae (primary ossification bone) with cross-struts, that joins each end of the shaft to an epiphyseal plate.
4 Endosteum lines all internal bony surfaces.
5 Periosteum ensheaths the bone, except for a small circumferential perichondrium around the epiphyseal plates, and where tendons and ligaments fasten to the bone. The articulating surfaces are bare.
6 Longitudinal growth, while the bone is under the stresses of use, is provided for by the interstitial growth of cartilage in the growth plates.

C ENDOCHONDRAL/INTRACARTILAGINOUS OSSIFICATION

l Mesenchymal cells retracting their processes round up to become chondroblasts, which form a minute hyaline cartilage precursor having roughly the shape of the eventual bone, e.g., the femur. Other mesenchymal cells differentiate and make a perichondrium.

2 In the central, shaft, region of the cartilage:

3  Cells and functions of an osteogenic bud are:
   (a) macrophages               |
   (b) chondroclasts             |- for selective cartilage erosion
   (c) endothelial cells?        |
   (d) progenitor cells of osteoblasts and osteoclasts/chondroclasts;
   (f) marrow cells - to populate intertrabecular spaces;
   (g) endothelial cells - to form capillaries and sinusoids.
4 Primary ossification zone establishes itself across the width of the shaft and starts extending in both directions towards the epiphyses, resulting in two transverse fronts of ossification across the diaphysis. At each front is the cartilaginous growth plate.

5 Epiphyseal plate. (This only becomes plate-like after secondary ossification has started within the epiphysis.) Starting farthest from the front, the zones are:

6 Within the cartilage of the young epiphysis, a secondary ossification centre develops, again by processes of cartilage cell hypertrophy, matrix calcification, and its erosion by vascular elements penetrating from the perichondrium. However, orderly columns of chondrocytes and a defined marrow cavity are lacking.

7 The epiphyseal, secondary, ossification centre spreads to occupy much of the epiphysis and forms the bony border to the cartilaginous epiphyseal plate. The cartilage grows (thus lengthening the whole bone) keeping pace with the front of ossification invading it from the metaphyseal side, until puberty. Then resorption and ossification slowly overtake halting chondrocyte proliferation, until the primary ossification front fuses with the secondary epiphyseal bone - epiphyseal fusion/closure. The growth plate is obliterated, but an irregularity in the trabecular bone pattern marks its site.

8 Hyaline cartilage remains as a thin cap over the epiphysis to be the articular surface.

9 Growth in width of the shaft is by a periosteal deposition on the outside surface, coordinated with an osteoclastic resorption on the inner, marrow, aspect. These patterns may be reversed at sites of change in shape or drift. At the same time, shaft bone is remodelled internally to be more lamellar and have the layers of Chapter 7.D.

D OSTEOID

l The osteoid seam is a very poorly mineralized, narrow zone of organic matrix seen sometimes with LM between the true bone and active osteoblasts.
2 It results from a definite lag between the formation of collagen fibrils and the later deposition of mineral crystals.
3 The presence of osteoid can be determined for sure by methods, e.g., von Kossa's silver, microradiography, EM, which are able to show the absence of mineral, but certain stains for decalcified sections are reliable.
4 The seam widens significantly in osteomalacia and rickets when too little Ca2+ is available, e.g., in kidney disease.

E PHYSIOLOGICAL FACTORS AFFECTING CONNECTIVE TISSUES

l Hormones
  1. Growth: for matrix synthesis, particularly in epiphyseal cartilage; lack causes dwarfism, excess gigantism, or in adult-onset, acromegaly.
  2. Parathyroid: acts on osteoblasts to cause osteoclasts to resorb bone, thus raising blood Ca2+; lack of hormone results in death by tetany; large eroded spaces from an excess fill with fibrous CT in 'osteitis fibrosa' - hyperparathyroidism. (There is also the paradox that small doses of PTH stimulate bone formation.)
  3. Calcitonin: acts on the 'clast to block bone resorption and lower blood Ca2+, but its role seems to be minor (except in treating Paget's disease - uncoordinated 'clasts and 'blasts).
  4. Thyroid: acts indirectly on all cell activities by controlling metabolic rate; lack thus slows growth; an excess favours resorption.
  5. Sex: affect genital tract CT, e.g., endometrial stroma; also the timing of secondary ossifications and epiphyseal closure (premature fusion and dwarfing follow an excess of gonadal hormone in childhood).
  6. Glucocorticoid: excess impairs bone and CT matrix synthesis; used clinically to reduce inflammation, perhaps by reducing prostaglandins.
  7. Insulin, Norepinephrine, etc: control fat cell metabolism.
  8. Parathyroid hormone-related protein/PTHrP is a PTH-like peptide released from various tumours that causes a cancer-linked destruction of bone, and hypercalcaemia. (PTHrP acts more in a local - paracrine - manner than the endocrine PTH.)

2 Agents
l Vitamin D: in its active form is needed for Ca2+ to be absorbed in the gut; low blood Ca2+ from a lack of D prevents mineralization of growth cartilage matrix, resulting in rickets, and causes the failure of osteoid to mineralize in osteomalacia; excess D may raise blood Ca2+ to the point where soft tissue calcifications occur.
2 Prostaglandins: stimulate osteoclastic bone resorption
3 Peptides: although thought of originally in the contexts of immunity and haemopoiesis, the cytokines (see F below) influence matrix formation and destruction, and the numbers and activities of all connective tissue cells, e.g., macrophages stimulate fibrogenesis, and osteoblasts interact with osteoclasts, etc.

3 Diet
l Calcium, phosphorus: see 2.l above for Ca2+ deficiency.
2 Vitamin A: excess and deficiency disturb ossification and remodelling in different ways.
3 Vitamin C: deficiency (scurvy) impairs collagen synthesis in all CTs.
4 Copper: needed for making elastin.
5 Toxic elements, e.g., Pb, 90Sr, F, may substitute for the natural elements and ions in the mineral crystals of bone and teeth.

4 Use
CTs respond to more use by making a matrix better able to withstand the greater forces, e.g., osteoblasts build more and wider bone trabeculae; conversely, disuse leads to the few thin and frail trabeculae of osteoporosis.

F CYTOKINES

1 The name does not indicate the only source or action; all cell types use cytokines for signalling, including neural and epithelial cells.
2 The materials are protein or glycoprotein and express sub-types, e.g., acidic and basic fibroblast growth factors (aFGF & bFGF), and control cells by binding to receptors, but comprise a system separate from hormones, neurotransmitters, and the eicosanoids (derivatives of arachidonic acid, e.g., prostaglandins and leukotrienes).
3 Cytokines' actions are diverse, and not consistently stimulatory or inhibitory, but depend on the target cell type and the action of other agents.
4 Factors that could qualify, but are already known as hormones, such as erythropoietin & insulin, are not listed.
5 Cytokines are important for the control of renewing cell populations, in inflammation and healing (wound and fracture), and the immune responses, and are used clinically to influence disease or tardiness in these processes.
6 A few cytokines and their actions are listed below, but which cytokine does what can wait.
Epidermal growth factor  EGF            Insulin-like growth factors IGFs            
Platelet-derived growth factor PDGF     Fibroblast growth factors  FGFs             
Transforming growth factors  TGF-1      Interleukins 1,2,3-- IL-1,2, --IL-10   
Tumor necrosis factor-alpha TNF-a      Colony-stimulating factors CSFs
Interferons  IFs                        Stem-cell factor  SCF

Help to start and/or complete cell proliferation;
Promote or inhibit differentiation;
Activate white blood cells, osteoclasts, etc.;
Raise or lower the rate of synthesis of ECM;
Alter the release of proteases or their inhibitors;
Induce chemotaxis, motility & change of cell shape;
Change sensitivity to other cytokines or hormones;
Cause fever;
Cause vasoconstriction

Chapter 9 MUSCLE

A INTRODUCTION

l Most muscular tissue is derived from mesoderm, by a modification of the cells into elongated muscle fibres.
2 The muscle fibre is itself a cell, and muscle has relatively little extracellular tissue.
3 Each fibre has a special cytoplasm (sarcoplasm), in which lie contractile filaments, which can contract the fibre or cell along its long axis.
4 The three kinds of muscle found are adaptations to the requirements of:
... (a) strong contractions of short duration (skeletal muscle);
... (b) rhythmic, strong unfaltering contractions (cardiac);
... (c) contractions of longer duration and greater cell shortening (smooth).
These varieties differ also in their innervation and the way in which their forces are applied. Powerpoint
5  (a) Skeletal/striated/voluntary;              |   are the various
   (b) Cardiac/heart/(striated);                 |-  names, of the
   (c) Smooth/unstriated/involuntary/visceral;   |   three kinds.
6 Sarcoplasm stains pink with eosin, and selectively purple with Masson's trichrome stain, or yellow with van Gieson's.

B SKELETAL MUSCLE

l Connective tissue (CT) sheaths and subdivisions
... CT epimysium encloses the whole muscle;
... CT perimysium encloses each fasciculus (bundle) of fibres;
... CT endomysium encloses each muscle fibre.

Connective tissue carries blood vessels, lymphatics and nerves, and serves to harness and direct to the attached tendons the force developed by contraction.

2 Individual skeletal muscle fibre
l Outside lies a connective tissue endomysium with some fibroblasts, collagen fibrils, and capillaries.
2 Cell membrane is the sarcolemma.
3 Directly under the sarcolemma, i.e., peripherally, lie elongated nuclei. The cell, as another product of cell fusion, is multinucleated.
4 In one place, the sarcolemma is modified to take a nerve fibre's terminal motor-end-plate/ myoneural junction (Chapter l2.C.l.2.).
5 The interior of the fibre has sarcoplasm with orderly myofibrils.
6 Fibre is large and cylindrical, with a diameter between l0 and l00 µm and a length between l and 40 mm.
7 Regularly along the length of the fibre a cross-banding of light and dark lines is seen.
8 Fainter longitudinal lines, the myofibrils, are also visible.

3 Myofibril
The fibre is cross-banded because the many constituent myofibrils are banded, and lie side by side with their dark areas in register. High power light microscopy reveals the repetitive sequence (see Fig. l) along the myofibril.

   Light I band            Dark A Band             Light I band
    0.8µm wide              1.5µm wide               0.8µm wide
  with central dark                                with central dark
        Z line                                         Z line
          |                                               |
          |                                               |
          |            Sarcomere extends                  |  
          |                                               |  
          |-------- from one Z line ---- to the next -----|                               
          |                                               |
                      Resting fibre has a paler
                      zone H (Hensen's) in the
                      centre of the A band. It 
                      disappears in contraction.
                      The A band stays the same 
                      during contraction.

The I bands narrow in contraction, and thus the sarcomere (Z to Z) shortens

4 Red and white fibres
Groups of only one kind of fibre can be identified by colour with the naked eye in some fresh, unstained muscles. They differ physiologically with red, richer in myoglobin and mitochondria, providing slower responses, but being less prone to fatigue. Histochemistry reveals further subtypes: white, intermediate, red (fast-twitch), and red (slow).
(The classification of muscle fibre types is used in assessing muscular disease, but the classification by roman numeral can be based on the profiles of contractile proteins, or on the metabolic behaviour of the fibre, so one needs to ask the particular criteria for any types encountered.)

C CARDIAC MUSCLE COMPARED WITH SKELETAL

  1. Cross-banded, with the same repetitive sequence as in Figs. 1 and 2, but the banding is weaker.
  2. Sometimes a Z line's place is taken by a dark line across the width of the fibre - intercalated disc.
  3. Intercalated discs mark a strong end-to-end cell connection. The muscle thus pulls upon itself during contraction.
  4. Each cell has only one or two nuclei lying centrally, elongated, but with blunt ends.
  5. Fibres are narrower, around 9-22 µm in diameter.
  6. Fibres branch and anastomose and, until intercalated discs were discerned using EM, the muscle was believed to be syncytial - one huge cell.
  7. EM shows the intercalated discs to be extensive, interdigitated cell junctions with gap junctions, fasciae adhaerentes, where the myofibrils attach, and desmosomes.
  8. Mitochondria are more numerous.
  9. There is less CT.
  10. Cardiac myofilaments are not clearly aggregated into myofibrils.

D ELECTRON MICROSCOPY OF SKELETAL AND CARDIAC MUSCLE

1 Sarcolemma is the plasmalemma, outside which is a basal lamina.
2 Sarcoplasm contains glycogen granules, lipid droplets, many mitochondria and soluble proteins. A Golgi complex lies by some nuclei.
3 T-system and sarcoplasmic reticulum/SR (smooth)
The sarcolemma extends down, at the junction between A and I bands (or at the Z lines in cardiac muscle), into the fibre as centrotubules of the T-system. Cisternae of junctional SR lie immediately adjacent to the T-system, and from these a tubular and vesicular system of free SR extends on either side along, and wrapping around, the myofibrils. A centro-tubule and the two lots of cisternae on either side constitute a triad of separate compartments, but ones joined by densities (feet).
4 Myofilaments (contractile elements). Thick (of myosin) and thin (of actin protein) lie densely packed in orderly array in each myofibril (Fig. 2). Cross-sections revealing sites with only thin (at I band), only thick (at H), and thick and thin filaments (A band outside the H zone), and differences between the resting and contracted states, led Huxley to propose a `sliding filament' theory of contraction. In contraction, the thin filaments are induced to slide in further between the static thick ones, with their many heads/lateral projections of heavy meromyosin.
                             _________________ A __________________
                            |                                      |
                 ____ I  ___                                         ____ I ____
                |           |                                       |           |

                                               M
                   N     N                     |
          |-----      |      ------------------|---------------------     |
          |-----------|-------------------     |     ---------------------|--------------
Relaxed  _|-----      |      ------------------|---------------------     |
myofibril |-----------|-------------------     |     ---------------------|--------------
          |-----      |                        |                          |
                      |                   |_________|                     |
                      Z                      H zone                       Z

                      <--------------------- Sa ------------------------->

Fig. 2 Key
A .. Anisotropic band (bright in polarized-light microscopy; dark in bright-field)
I .. Isotropic band (dark in polarized light, light in bright-field microscopy)
Z .. Zwischenscheibe (between disc): the Z line or band to which the thin actin myofilaments attach
Sa .. Sarcomere is the contractile unit of the fibril extending longitudinally between one Z line and the next Z line
M .. Mittelscheibe (middle disc): the M line or band bisecting the A band
H .. Hensen's pale zone or band devoid of actin myofilaments
N .. Nebenscheiben (accessory discs): the N bands on either side of the Z line

5 Some events and chemistry of contraction

6 Some other muscle molecules
(a) a-actinin is a non-contractile Z-line actin-attachment protein.
(b) Nebulin is a giant ruler-like molecule to align, and establish the length of, the actin filaments.
(c) The ryanodine receptor - a calcium channel - is the principal protein of the SR feet, linking the T-tubule to the sarcoplasmic reticulum. Other proteins are at the complex.
(d) Desmin is the intermediate filament of muscle.
(e) Dystrophin lies just under the sarcolemma to help attach it to the cytoskeleton. Its lack causes one type of muscular dystrophy.
(f) Titin is another huge, long molecule, connecting the Z disc with the material of the M-line. It provides an elastic framework for filament movement, and helps actinin anchor the actin filaments in the Z disc.
(g) The contractile and regulatory proteins exist as isoforms, characteristic of slow, fast, and cardiac muscles. The isoforms change during development and in disease, e.g., muscular dystrophy, cardiac hypertrophy.
(h) Caveolin is associated with the inwardly budding sarcolemmal protrusions - caveolae - of cardiac and smooth muscle that are part of the signal transduction machinery for contraction.

E SMOOTH MUSCLE

l The fibres are spindle-shaped (fusiform) with one, central, cigar-shaped nucleus, and are usually 200 µm or less long, but in the hypertrophic uterus they may reach 0.5 mm. Width is around 6 µm.
2 Fibres show no cross-banding, but have many fine filaments.
3 Cells are firmly attached by gap junctions, and elsewhere by glycoprotein external laminae (like basal lamina). Diverse patterns of attachment and contraction occur in gut, vessel walls, genital organs, etc.
4 Fibres are usually packed to form a sheet or bundle. Reticular fibres enfold the muscle fibres, assist in holding them together and carry blood vessels, and fine autonomic nerve fibres going to inconspicuous myoneural junctions (Chapter l2.C.2).
5 The nuclei may be wrinkled in the contracted state.
6 EM shows thin and thick filaments, but the thick are labile and not easily preserved. These filaments connect with Z line-like densities in the cytoplasm, or at the cell membrane. Desmin intermediate filaments help to structure the filamentous arrays.
By the nucleus lie mitochondria and the Golgi body.
7 Peripheral vesicles are part of a vesicular and tubular Ca2+-holding sarcoplasmic reticulum. These organelles, and inward protrusions of cell membrane - caveolae - function similarly to the better-defined SR and T-tubules of striated muscle.
8 Contraction is triggered by a Ca2+-dependent phosphorylation of myosin light chain by smooth-muscle MLC kinase. This is the primary control, fine-tuned by the calcium-mediated binding of caldesmon and calponin to actin in ways which interfere with actomyosin force-generation and ATPase activities.
9 Myoepithelial cells, wrapped around glandular secretory or duct cells, have contractile processes resembling smooth muscle cells.
10 Vascular smooth muscle cells also can make elastin and collagen during development.

F MYOGENESIS (skeletal muscle)

l Mesodermal cells of the myotome become elongated premyoblasts.
2 These multiply, acquire more cytoplasm and elongate further, becoming granular, with many mitochondria and ribosomes.
3 Filaments and microtubules appear in the cytoplasm of the myoblasts. New myoblasts fuse with the more mature ones accumulating myofilaments to build long, multinucleated cells.
4 Filaments aggregate into myofibrils near the sarcolemma, leaving a paler central core, with a row of nuclei (myotube stage).
5 The fibrils develop prominent striations; nuclei move to the periphery of the fibre; and mitochondria and SR order themselves in relation to the myofibrils.
6 Some cells stay in a peripheral position to lie within the basal lamina as a regenerative reserve of satellite cells.

G TENDON

l Musculo-tendinous junction entails no continuity between myofibrils and collagen fibres: the sarcolemma intervenes.
2 Myofibrils pull on the tapering sarcolemma at the muscle fibre's extremity, and its contraction is conveyed by the muscle's CT to the tendon with which the muscle CT merges.
3 Tendon is composed of: 4 Freedom of movement is provided for some tendons by enclosing them in lubricated synovial sheaths, or interposing a synovial bursa between the tendon and a bony prominence or ligament.
5 Tendons and skeletal muscle have nervous proprioceptors - Golgi tendon organs and muscle spindles.

Chapter 10 NERVOUS ELEMENTS

The nervous system provides for the control and coordination of all the body's activities. It spreads out widely to all organs from central nervous organs of a complex and discriminating nature, permitting a multitude of finely graded responses to changes in the external and internal states. It makes use of millions of nerve cells having especially the properties of excitability and conductivity. Information is conducted along long nerve cell processes as an electrical excitation generated across the cell membrane.
The nervous system comprises the central parts (CNS) of brain and spinal cord, and neural parts of the eye, and the peripheral (PNS) of nerves, ganglia, receptors, and neural endings on effector structures - muscle and glands. Powerpoint

A ELEMENTS OF NERVOUS SYSTEM

l  Nerve cells/neurons
                  _
                 | Oligodendrocytes, Astrocytes, Ependymal cells,
                 | Microglia, Special glial cells - (CNS)
2 Glial cells ---|
                 | Schwann cells, Satellite/Capsule cells,
                 |_Enteric (gut) glia - (PNS)
                 
3  Blood vessels
4  Connective tissue enclosing sheaths

B NEURONS

l Shape
Neurons are characterized by having long processes extending from a cell body/soma. One of these is the axon transmitting information; the others are receptive dendrites.
l Unipolar have one process, e.g., neuroblast.
2 Pseudounipolar have one process branching into two a short way from the cell soma, e.g., dorsal-root ganglion cell.
3 Bipolar have two processes, e.g., bipolar cell of the retina.
4 Multipolar have many processes. Shapes include:
... (a) stellate or star-like,
... (b) pyramidal with apical and basal dendrites, or
... (c) Purkinje with a plump body tapering to an espalier-oriented dendritic tree.

Note that the dendrites branch repeatedly, becoming finer. The axon retains its diameter along most of its length. The axon, though, may give off side branches or collaterals, and will usually divide into many fine branches, telodendria or the preterminal axonal arborization, near to its terminal structures.
The Golgi method which delineates only a few (roughly l in 70) of the neurons allows the full dendritic spread and the axon to be seen in a thick (l50 µm) section. Thus the shape of the neuron could be determined and classified, e.g., Golgi type l cells with long axons (projecting neurons) distinguished from type II cells with short axons (non-projecting neurons). No intracellular detail was seen.
Current methods yielding Golgi-like detail are to fill the neuron with Lucifer Yellow which can be lighted up with fluorescence microscopy, or with horseradish peroxidase which produces a visible product by acting on a substrate.

2 Nerve cell structure
l Soma contains a large central nucleus with much sap, but little visible chromatin. The nucleolus is prominent because the neuron has to synthesize organelles and much cytoplasm to fill its long processes.
2 Around the nucleus is the perikaryon with:

2 Dendrites

3 Nerve fibre (includes the axon and its myelin sheath, if present).

Fig. 3 Staining methods for CNS neurons and glia(Stain #s in Table 3 below)

               \/   Full extent of dendritic tree
          \    /    shown by 3 GOLGI
           \  /
            \/
            /                                 5  GLIAL
         \ /                          \    /  for glial cell 
          \                            \  /   processes
           \                       ____ OO____
         / /                            OO
 \ \    / /                            /  \
  \ \  / /                            /    \
    \ / /           OO
     | |            OO             _
     | |           Glial nuclei     |
     | |                            |
     |*|Base of dendrites           |____ 1  NISSL
     |*|                            |
    /   \ Soma with Nissl granules* |
   /  *  \    and the nucleus      _|
 /*   * * \
/ *  ___   \     
* * | 0 | * \______########################_ _###################_________
 *  |___|    _______________________________|___________________________axon
*  *  *  *  /      ######################## | ################### myelin   /
\  NEURON  /                                |          |                  /
 \ *   *  /                                 |          |                 /
  \ *  * /                                  |          |                /
   \  * /                                   |      4  MYELIN           /
    \ */                                    |                         /
    |* |                                    |                        /
    |* |                                    |                       /
    |  |                                   Axon                    /
   / /\ \                                   |        Synapses O---/
 / /    \ \                                 |                    /
/ /      \ \                                |             &     O
          \ \ --------First dendrites-- 2 SILVER    terminal axonal 
                                       for neurofibrils    branchings

3 Neuron staining
l Fundamental to an understanding of nerve cell histology is the knowledge:
(a) that most neurons' processes are so extensive that only part of the cell is present in a 8 µm-thick section;
(b) that different parts of the neuron contain different elements, and staining for one of these elements reveals only the part of the cell containing it.
For example, a basic stain like toluidine blue will stain only nuclei of nerve and glial cells and Nissl bodies of nerve cells, leaving the large areas of surrounding tissue pale and apparently structureless, although other stains reveal that these areas of neuropil are packed with dendrites, axons, and processes of glial cells.
2 The staining methods for normal neural tissue, numbered l to 5 in Table 3, just below, reveal correspondingly numbered elements in Fig. 3 showing details of a CNS neuron and glia cell. Table 4 later lists the kind of information obtained by applying these techniques and degeneration-specific ones to the normal and pathological CNS.

Table 3 (a) and (b). Histological methods for the central nervous system.

Staining or
impregnation                                  Elements of nervous tissue
method              Nature of reagent          revealed


1  Nissl      Basic, e.g. methylene blue,  Nuclei of nerve cells, glia
              cresyl violet, thionine.     and blood vessels. Nissl
              Haematoxylin.                granules in nerve cell bodies (blue)

2  Silver     Reduced silver nitrate       Nerve cell bodies and larger
              methods of Cajal, Biel-      dendrites, axons and synapses,
              schowsky and Glees           because of their neurofibrillar
                                           content. (Soma, yellow: axons
                                           and synapses, black.)

3  Golgi      Silver nitrate.              Complete outline of only a 
                                           few (1/70) nerve cells - soma,
                                           dendrites and axon (black).

4  Myelin     Mordanting followed by       Myelin sheaths (blue).
              haematoxylin - Weigert-
              Pal technique. Luxol blue.

5  Neuroglia  (1) Cajal's gold-sublimate.  Astrocytes, oligodendroglia;
              (2) Hortega's silver         Microglia
                carbonate.

Degeneration-specific methods

6  Nauta,     Reduced silver nitrate,      Nerve fibres experiencing
   Fink-      after suppressive pre-       Wallerian degeneration
   Heimer     treatment.                   (black); pale, but identifiable
                                           background of normal nerve
                                           cells and fibres (yellowish-
                                           brown).

Staining or impregnation Used in combination Elements of nervous method with other methods tissue not revealed 1 Nissl Yes. Myelin Nerve fibres (axon and myelin) and synapses 2 Silver No Myelin of nerve fibre. Synapses without neuro- fibrils. Glial cell processes. 3 Golgi No Most nerve and glial cells. Intracellular structures of the few cells revealed. 4 Myelin Yes. Nissl Axons, synapses and nerve cell somas. Glia. 5 Neuroglia No Most nerve cells and processes. 6 Nauta No Glial cell processes.

C GLIAL CELLS AND MYELINATION

Consult Chapter 11.C. . . . . . . . . . . . . 11.C

D CELL LINEAGES OF THE NERVOUS SYSTEM

1. For the PNS, the problems are: (i) the large number of neural-crest-derived cell types, including many non-neural ones (mostly because the crest is the major constructor of the head); (ii) that additions are still being made to the list; (iii) the evidence is chiefly from birds; and (iv) some head structures, e.g., receptors and ganglion neurons for hearing and balance come from ectodermal placodes.

2. In the CNS, the story is coming to resemble that for haematopoiesis, with a multipotent neural stem cell giving rise to a self-propagating progenitor pool. From this pool, self-sustaining populations of neuroblasts and glioblasts derive. Further specifications, under the control of neural 'growth factors', are for transmitter type, shape, and axon length, and for glioblast derivatives, whether to be type 1 or 2 astrocytes, or oligodendrocytes. Microglia are regarded as invaders of haematopoietic origin, but is this true for all of them, always? Other questions are: do neural progenitors live on in the adult CNS? (They are present in olfactory mucosa.) And how well does the astrocyte 2 correspond to the fibrous astrocyte, and the 1 to the protoplasmic?

Neural and other cell derivatives of neural tube and crest

NEURAL TUBE
CNS: Neurons, Astrocytes, Oligodendrocytes, Ependymal cells, Special central glia

NEURAL CREST
PNS: Sensory- & autonomic-ganglion neurons, Adrenal neurons, Satellite cells, Schwann cells, Enteric glia
OTHERS: Chromaffin cells, C-cells, Melanocytes, some Cardiac (outflow tract) & Carotid-body cells

NEURAL CREST via Mesectoderm
ANTERIOR CRANIAL SKELETAL TISSUES: Osteoblasts, Chondroblasts
DENTAL TISSUES: Odontoblasts, Cementoblasts, Ligament fibroblasts
HEAD MUSCLES & CONNECTIVE TISSUES: Smooth & skeletal muscle cells, Fibroblasts, Adipocytes, Meningeal cells


Chapter 11 CENTRAL NERVOUS SYSTEM

A BRAIN BOUNDARIES

The brain, spinal cord and optic nerves are enclosed in vascular connective tissue sheaths - the meninges - and protected by bone. From the inner meninges, the leptomeninges, blood vessels pass into the substance of the brain to vascularize it extensively and to supply the CSF-forming choroid plexus. CSF dilutes and carries away metabolites and excess neurotransmitters, and drains to form a cushion around the brain.

l Meninges

l Dura mater - (pachymeninx) - dense fibrous CT; osteoblastic outside (skull), or mesothelial facing the epidural space (spine); specialized layer of dural fibroblasts* attaches dura to arachnoid.

2 Arachnoid complex - apposed to the dura is a layer of well attached cells, several cells thick; between this layer and the pia are open subarachnoid spaces, crossed by trabeculae of collagen, clad in other arachnoid cells, and supporting the vessels.

3 Pia mater - thin cellular, vascular and collagenous layer, adherent to the BL of the nervous tissue.
(Arachnoid and pia comprise the leptomeninges.)

* The idea that the arachnoid was merely a membrane led to the mistaken notion that it had to be separated from the dura by a 'sub-dural space'. Such a space only arises by a forcible cleaving between the fibroblasts of the inner dura, as occurs in 'sub-dural' haematomas.

2 Ependyma and choroid plexus
Ependymal epithelium lining the ventricular cavities and canals of the CNS is simple, columnar or cuboidal. In regions of each ventricle, tufts of blood vessels (mainly fenestrated capillaries) project out from the pia, and are covered by a loose CT coat, then a layer of cuboidal ependymal cells on a BL. This choroid plexus forms cerebrospinal fluid (CSF) secreted into the ventricles.
These plexus ependymal cells have ion pumps, deep basal infoldings, and luminal microvilli.

3 Cerebrospinal fluid's return to blood
The subarachnoid space, which dilates into chambers, cisterns, fills with CSF spilled out of the ventricular system via the foramina of Lushka and Magendie in the fourth ventricle. Some CSF may come out of the brain tissue via spaces between blood vessels and the pia. CSF returns to the dural sinus blood through the thin walls of the arachnoid villi and granulations.

4 Blood-brain barrier
The blood capillaries serving the brain tissue have a characteristic structure of unfenestrated endothelial cells held together by tight/occluding junctions on a thick basal lamina, whose outer surface is enclosed by glial cell processes (astrocytes' pedicles). The endothelium has few transcytotic vesicles and is very selective in what it transports. In most regions of the brain the endothelium blocks the passage of most materials from the blood into the neural tissue, and a blood-brain barrier (BBB) is said to exist for such substances.

5 CNS elements
The two cells specific to neural tissues are the neuron/nerve cell and the glia cell, for the latter of which several varieties exist. Some of the glial cells are used to form a layer - glia limitans - separating neurons from the numerous blood vessels and the enclosing pia matter.

B NEURON STRUCTURE

                                              _      
                                             |  dendrites
                                             |  (receptive)
l Cell with soma and extended processes -----|
                                             |  axon
                                             |_ (transmissive)
2 Axon may or may not be myelinated. It may or may not give off collaterals, sometimes recurrent back to near the soma.
3 Final part of the axon branches to give preterminal fibres, often serving very many nerve cells.
4 Axon synapses in various ways, discussed later in D, with the cell body, dendrites, and axons or synapses of other neurons.
5 Soma contains granular ER as Nissl granules and the characteristic vesicular neuron nucleus, plus a Golgi apparatus, mitochondria, microtubules, filaments, etc.
6 Some of the dendrites can be seen with silver methods for the neurofibrils. Also, axons appear black and some synapses are seen as little rings on the surface of other nerve cells.
7 All around the processes of the nerve cells, the space is almost fully taken by the glial cells' processes, unseen except in EM or after special staining.
8 When the full extent of the dendritic and axonal ramifications is seen in Golgi preparations, nerve cells can be categorized by their size and shape and the course of the axon. Thus the kinds of nerve cell in any brain area can be described. For example: [* These cells lack axons.]

9 When a neural area has such a variety of cells, the Golgi and other methods to be described later are used to discover:
(a) how the various cells are interconnected (intrinsic connections);
(b) how projections of nerve fibres from outside and to outside the nucleus or brain area terminate or originate (extrinsic connections).

C GLIAL CELLS

l Glial cell types
l Protoplasmic astrocytes: large, star-shaped with many processes, some of which attach pedicels/pedicles/sucker-feet to blood vessels or the basal lamina under the pia mater; have cytoplasmic filaments and microtubules; are common in grey matter.
2 Fibrous astrocytes: similar to protoplasmic astrocytes, but have more filaments and glycogen, and lie in the white matter.
3 Oligodendrocytes/oligodendroglia: plump cell body with fairly dense cytoplasm and a darker nucleus and fewer, shorter processes than an astrocyte; common in white matter, but some are perineuronal.
4 Microglia: - (a) derived from mesenchyme via bone marrow; (b) potentially phagocytic; (c) dispersed throughout the brain; (d) a small elongated cell with many short processes and a dark nucleus.
This is the ramified or resting microglial cell, which becomes round and phagocytic as a reactive microglial cell (Gitter cell), when responding to damage.
5 Ependymal cells: lining ventricles, and covering the choroid plexus.
6 Peripheral glia: satellite cells and Schwann cells may be roughly equated with oligodendrocytes by function. Peripheral glia in the gut autonomic system - enteric glia - are more like astrocytes. Olfactory ensheathing cells enwrap the unmyelinated axons of the olfactory nerve bundles, and may provide favourable cues for axonal regeneration.
7 Specialized central glia: Müller astrocytes of the retina, pituitary-gland pituicytes, and periventricular tanycytes extending away from the ventricles.
Because of the readily measured electrical activity, much is known of the neuron's physiology, but glial activities are less easily studied. Certain functions are special to the various types of glia.

2 Glial functions
l Myelination of myelinated axons (oligodendrocytes).
2 Augmenting the extracellular space, e.g., being an active compartment for ionic buffering by taking up and redistributing K+, and metabolizing transmitters (astrocytes). The CNS has little true tissue space and no lymphatics.
3 Helping to induce endothelial cells to create the blood-brain barrier (astrocytes).
4 Insulating chemical and electrical events from nearby sensitive structures (astrocytes and oligodendrocytes).
5 Storing glycogen and passing on raw materials for the energetic and synthetic processes of the neuron (astrocytes).
6 Acting as macrophages to remove degenerating nerve cell components (microglia).
7 Protecting neurons by metabolising excess ammonia from liver disease (astrocytes).
8 Mechanically supporting the neuronal elements and keeping them properly spaced (astrocytes and oligodendrocytes).
9 Transient radial glia guide the migration of developing neurons.

3 Some evidence for cell types performing these functions
l Oligodendroglia contain myelin basic protein. Their membranes are connected with myelin lamella that they form.
2 Excluding myelin, insulation is a task of astrocytes whose processes enfold synapses and neural membranes.
3 Astrocyte cytoplasm also could serve as a nutritive pathway via its pedicles and processes from the blood capillary wall to the neuron, and can transfer ions and inactivated transmitters in the reverse direction.
4 Fibrous astrocytes have long processes, firm connections with one another and very little in their cytoplasm but filaments and glycogen. They would seem to be fitted for the role of mechanical support.

4 Myelination process
l Many axons remain unmyelinated throughout their existence. However, for rapid saltatory (jumping) nerve conduction a myelin sheath interrupted by nodes is necessary. This sheath is a modified lipoprotein membrane, rich in cerebrosides and other special lipids and proteins.
2 The process of myelination in peripheral fibres is by an apparent 'rotation' of the Schwann cell in relation to the axon that it has enfolded, thus enclosing the axon in many layers of Schwann-cell membrane. These membranes fuse together, but the lamellar structure remains visible in EM, and an outer mesaxon connects the last wrapping to the Schwann cell's own plasmalemma.
One Schwann cell myelinates a given length of axon, which is separated by an unmyelinated node of Ranvier from the next myelinated segment. Outside the Schwann-cell or neurolemmal sheath lies a basal lamina, beyond which are found the collagen fibrils and fibroblasts of the endoneurium.
3 In the CNS, the oligodendrocyte incrementally adds membranes to several axons, and to more than one segment per axon. This myelin configuration is compatible with 'spiralling' membrane synthesis, but not actual rotation. Nodes are present, but not as distinct as in the PNS.
4 Myelination takes place in different tracts of the brain at different times during development. The time of myelination correlates fairly well with the development of the ability to function in that system.
5 Remyelination (successful or attempted) is involved in the mature nervous system in two circumstances - the regeneration of peripheral nerve fibres, and demyelinating diseases in the CNS and peripheral NS.

D SYNAPSES

Synapses are specialized neuron-to-neuron cell contacts, firmly attached and functionally polarized to transfer excitation one way (except for 7).

l Types of synapse
l Axosomatic: to the neuron's body.
2 Axodendritic: e.g., from climbing fibres to Purkinje cells' dendrites.
3 Axodendritic to spines, e.g., from parallel fibres to Purkinje cells' dendritic spines. (The presence of spines on dendrites is used to subclassify neurons in many brain regions.)
4 Glomerular: a rounded structure serving several dendrites, e.g., from mossy fibres to cerebellar granule neurons.
5 En passant: made 'in passing' on the way to other synapses.
6 Axo-axonic: synapse onto another synapse or the axon's initial segment (for presynaptic inhibition).
7 Reciprocal dendro-dendritic: e.g., in retina and olfactory bulb.

2 Synapses also differ in the number, size and density of their vesicles, in the transmitter and neuromodulator substances that these hold, in the organelles present, and in the cleft material and membrane densities.

3 Chemical neuroanatomy involves mapping which connections of the CNS employ particular neurotransmitters, e.g., serotonin, acetylcholine, dopamine, etc.

E SPECIAL FEATURES OF BRAIN REGIONS

l Spinal cord
l Enclosed in CT meninges with pia extending in at the ventral fissure with the anterior spinal artery.
2 The ependyma-lined central canal lies centrally.
3 Surrounding the canal in a butterfly shape is grey matter (grey to the naked eye when fresh and unstained).
4 Horns of grey matter partly separate three columns of
5 white matter: dorsal (posterior), lateral, and ventral (anterior) columns.
6 White matter is composed of nerve fibres, many thickly myelinated, running mainly up or down the cord. Generally, fibres projecting to or from a particular brain region run together in a tract.
7 Grey matter has groups of multipolar nerve cell bodies, nerve fibres entering and leaving the grey matter, and preterminal fibre branches (poorly myelinated, hence the grey colour in the fresh, unstained cord).
8 Glial cells and blood vessels are in both white and grey matter. Grey matter is more vascular. The oligodendrocyte is the principal glial cell of white matter.
9 Roots of nerve fibres enter the cord on the dorsal sides; other roots leave on the ventral sides.
l0 Substantia gelatinosa lies at the extreme margin of the dorsal horn of grey matter.
ll The multipolar neurons include: motoneurons, whose axons pass out of the cord to join peripheral nerves and serve skeletal muscles; and short-axoned interneuron/ Renshaw cells.

2 Cerebellar and cerebral cortices
Differ from the spinal cord in these ways: (a) grey matter lies to the exterior with white underlying it; (b) tissue of both kinds of cortex is folded: into gyri for the cerebral cortex and folia in the cerebellum; (c) nerve cells are of various types and are disposed in layers parallel to the pial surface, thus
l Cerebellar cortex (Pia). l Molecular layer (cell processes, but few cells). 2 Purkinje cell layer. 3 Granule cell layer (densely packed small neurons) (underlying white matter).
2 Cerebral neocortex (Pia). l Molecular layer. Layers 2, 3, 4, 5, 6 with varying proportions of stellate, fusiform and small, medium, and large pyramidal cells (white matter).
The number of layers to be clearly seen depends on the particular area of the cerebral cortex and the criteria of the investigator. Thus Cajal worked with an 8-layered scheme, whereas Brodmann adopted 6 - today's choice. Even so, in the motor region only 5 are to be easily made out.

3 Divisions of the cerebral cortex

3 Brain stem
(a) Resembles the spinal cord in having nerve cell bodies grouped in nuclei and nerve fibres in tracts.
(b) Some special nuclei of the brain stem and hypothalamus are:
... (i) The reticular formation is an extensive system of groups of neurons serving many vital tasks, but whose nuclear organization is hard to discern.
... (ii) Neurons of the substantia nigra contain melanin pigment and dopamine.
... (iii) Certain hypothalamic nuclei have neurosecretory neurons.

F NEURAL DEGENERATION AND REGENERATION

l Degeneration
l There is a marked contrast between the successes of regenerations in the central and peripheral nervous systems, although their degenerations are similar.
2 Because of the extended nature of the nerve cell, its axon can be injured without direct damage being inflicted on the soma. It is unclear how much damage the dendrites can repair.
3 For axonal injuries, because of the steady production of axoplasm in the cell body and its flow down the flexible axolemmal tube, mere traumatic distortion is soon corrected.
4 Injury resulting in a total cessation of flow down a section of axon leads to total loss by Wallerian degeneration of that deprived part of the axon (Fig. 4).
5 This involves axonal beading and break-up, and fragmentation of the myelin, the lipids of which alter their chemical nature, thus permitting degeneration-specific staining techniques to be applied. The use of neural degeneration for pathway tracing in the CNS is noted below.

Fig. 4 Neuronal degeneration (with changes used earlier in tracing neural pathways)


        /                                                                                   
       /                                                                                0\/ Next neuron in the chain
      |                              SEVERANCE                                         .   \  may display a trans-
      |                                 or                                           .       \  neuronal atrophy
     / \                             CRUSHING                                      .         /\
    / * \                               !                                        .         / * \
__ / OO *\                              !                                     .           /* NN \ _______
   \*OO  /------------------------------!  -  -  -  -  -  -  -  -  -  -  -  -  . . . . . 0\* NN*/
    \ * /    Proximal fibre stump                           |                              \* */
     \ /     remains relatively intact      Entire distal segment                          0\/                        
      |      for a while                    experiences a Wallerian                        |                
      |                                     degeneration:                                  | 
      |                                                                                    |
      |                                     1. Fibre breaks up into fragments             Terminal changes occur in 
Nerve cell soma exhibits                    2. which become osmiophilic (Marchi           the synapses - bouton
a loss of Nissl substance* -                   reaction) & argyrophilic (Fink-Heimer      changes - before they too
retrograde cell reaction                       reaction)                                  are resorbed
with swelling & recovery,                   3. and are later removed by glial 
if this distance........................!       phagocytosis. Absence of the fibres
is not too short; otherwise,                   can then be revealed by silver or
the reaction becomes a                         myelin methods for normal fibres.
progressive atrophy, and the
cell disappears to be replaced
by glia


(i)   Retrograde cell reaction shows from where the damaged fibres have come
(ii)  Wallerian degeneration of the fibres shows their course through the CNs
(iii) Bouton changes 0 and degeneration in the fine preterminal branches . . .
      indicate whereabouts the fibres of the tract terminate

2 Regeneration in the peripheral nervous system .
l This requires a two-sided effort, but not symmetrically two-sided as in other tissues' healing.
2 On the distal side of a cut through a nerve, macrophages and Schwann cells remove the degenerating axons and myelin, and Schwann cells proliferate and organize themselves to keep open the endoneurial tubes.
3 On the proximal side the axon degenerates back a little way and forms a retraction bulb, from which many fine axonal branches sprout.
4 The energy and synthesizing capacity for this new axonal material reside in the intact nerve cell body.
5 The soma to do this has to disperse its Nissl substance in order to form proteins; the cell swells and the nucleus may move off centre.
6 This change in the Nissl substance is termed chromatolysis, part of the retrograde cell reaction.
7 Some of the axonal sprouts find their way down the endoneurial tubes, aided by the Schwann cells' keeping out fibroblasts,
8 and may eventually re-innervate old end-organs or develop new ones.
9 Some new axons will be myelinated by their Schwann cells.
10 The pace of peripheral fibre regrowth is that of slow axonal transport - about 2 mm per day.

3 Regeneration in the CNS
l The lack of endoneurial tubes and a different kind of glial cell responsible for the fibres lead to the formation of a 'scar' of glial cells, leucocytes, and extracellular matrix, blocking any effective regeneration by the axonal sprouts.
2 The glial cells release factors inhibiting axonal growth and guidance.
3 Also, the axonal lesion results in a greater degree of 'shock' to the neuron soma, which may undergo a progressive atrophy to the point of disappearance, i.e., a loss of neurons may follow.
4 Their place is taken by numerous glial cells, constituting a gliosis.

4 Pathway tracing and neural degeneration
l Some fibre tracts can be seen to originate from or to enter particular brain nuclei. However, it was usually impossible to decide from a histological examination of a normal tract where its fibres have come from, and where they are going. This pathway information was learned by taking advantage of the special degenerations seen in the nerve cell and its fibre, when they are severely injured (Fig. 4). (Nerve cell is often used loosely to refer to the nerve cell's soma.)

2 Pathway-tracing procedure

3 Recent pathway-tracing methods are based on axoplasmic transport (which takes place in both directions).
(a) Radioactively labelled leucine injected near the soma is carried by orthograde transport to the axon terminals of the neuron, where it can be revealed by radioautography (Chapter 30.E).
(b) Horseradish peroxidase injected in the vicinity of the axon terminals is transported retrogradely back into the neuron's soma. The HRP accumulates in, and can be used to mark, those cells projecting to the site of injection.

Table 4. Applications of histological staining methods for central nervous system

For the normal CNS

  1. Nissl. Shows grouping of nerve cell bodies into nuclei or layers - cytoarchitecture: density of neuron packing; size of neuron somas; state of Nissl substance.
  2. Silver. Reveals cytoarchitecture and fibroarchitecture; size of axons and their distribution in tracts; some synapses. So many fibres seen that interpretation is difficult.
  3. Golgi. Sampling method showing types of neuron present; shape, number and extent of dendrites; length of axon, and its relations with other cells.
  4. Myelin. Myeloarchitecture; tracts or bundles of nerve fibres and their course; size of myelin sheaths; number of myelinated axons.
    This is the stain often used for brain atlases.
  5. Glial. Kinds of glia present, and their relations with nerve cells and fibres, and with blood vessels
Histological methods for the pathological CNS
  1. Nissl. Shows atrophic or degenerated neuron bodies; absence of neurons; increase in glial cell nuclei - a gliosis; white blood cells.
  2. Silver. Reduction in axon size, i.e., an atrophy of fibres; degeneration and loss of axons and terminals.
  3. Golgi. Changes in the shape of neurons, e.g., fewer dendrites.
  4. Myelin. Loss of myelinated fibres after Wallerian degeneration; demyelination of fibres with little damage to the axons, e.g., in multiple sclerosis.
  5. Neuroglia. Constituent cells of glial tumours; reactions of glia to degeneration and trauma.
  6. Nauta. Fibre degeneration in tracts and in fine preterminal fibres at the end of tracts.

Chapter 12 PERIPHERAL NERVOUS SYSTEM

Connected to the brain by cranial nerves or to the cord by roots combining to form nerves are sensory, relay and effector structures, which send raw sensory data to the central nervous system and receive from it and carry out its instructions.

A PERIPHERAL NERVE

l Nerves fibres present may be:
l centripetal sensory fibres,
2 centrifugal motor fibres to skeletal muscle,
3 centrifugal autonomic fibres to glands, and smooth muscles.

2 Connective tissue wrappings
l Epineurium around the whole nerve trunk with blood and lymphatic vessels (vasa nervorum), collagen and fibroblasts, and fat cells.
2 Perineurium around each fasciculus of nerve fibres: the site of the blood-nerve barrier. Perineurial cells are tightly attached.
3 Endoneurium around each individual myelinated nerve fibre, but separated from its Schwann cells by a basal lamina.

3 Cross-section of nerve in LM shows:
l Close-to-round shape with no lumen; CT coat and divisions.
2 Nuclei of Schwann cells, fibroblasts and a few capillaries.
3 Axons and some remnant of myelin (so-called neurokeratin) around them (with H & E staining); or
4 brownish-black rings (myelin with an unstained axon within each) (osmium tetroxide treatment).
5 The eosin of H & E shows the collagen of epi- and perineurium, which remain very pale yellow with osmium. Osmium tetroxide will, however, show intensely black the fat in the adipocytes, usually present in epineurium.

4 Single nerve fibre
Single fibres that have branched off from nerves to pass to and enter some kind of end-organ remain unseen unless special techniques are used, although the CT capsule and supporting cells of the end-organ are usually discernible with HE staining. The fibre-revealing techniques are EM, silver impregnation, or histochemical ones for cholinesterase, neuropeptides, and catecholamines (Chapter 30.D.2.).

B RECEPTORS AND SENSORY FIBRES

Sensory fibres (except for those of cranial nerves) are derived from the dorsal root/spinal ganglion cells lying just outside the spinal cord. The fibre is T-shaped with one branch entering the cord as an element of the dorsal root, and the other coming from a sensory receptor of one of the following kinds *.

l Skin and some mucous membranes (exteroceptors)
l *Meissner's corpuscles - common in dermal papillae of fingers, palms, nipple, etc.
2 *Krause's end-bulbs and Ruffini's end-organs - in external genitalia, dermis, tongue, joints, etc.
3 *Pacinian corpuscles - large, lamellated bodies, in external genitalia, also lie more deeply under the skin, in tendons, mesentery, joints, etc.
... Receptors/endings l to 3 are definitely encapsulated.
4 *Merkel`s discs - intra-epithelial in lower layers of the epidermis and oral epithelium.
5 *Free nerve endings - also intra-epithelial.
6 *Palisade/peritrichal endings around a hair follicle.

How far the morphology of a receptor can be related to a special sensitivity to a particular modality, e.g., pain, touch, cold, is disputed.

All receptor axon terminals lack myelin and contain vesicles, mitochondria, and filaments.

2 Muscle, joint and tendon (proprioceptors)
l *Golgi tendon organ - branching nerve fibres with thickenings between a tendon's collagen fibres. Joint and ligament receptors are similar, but some take more specialized forms, e.g., Pacinian.
2 *Muscle spindle

3 (Mechano-receptors of the vestibular apparatus (Chapter 14.C.) inform the CNS independently of the results of the muscles' actions in terms of changed position and movement of the head. Skin receptors likewise contribute to 'proprioception' in the lax sense.

3 Viscera (interoceptors)
l Carotid body - chemoreceptor for blood O2 tension; has sinusoids with blood passing in close relation to glomus/Type I cells. Clusters of these cells, with their cored vesicles, are innervated by axons of the glossopharyngeal nerve. The intermixed sustentacular/Type II cells are glial, and have no known role in signal transduction. The aortic body is similar in structure and function, and connects with the vagus nerve.
2 Carotid sinus and aortic arch - pressoreceptors/baroceptors (for blood pressure) set within the vessel's wall.
3 *In lung, gut, bladder, and other viscera - measuring distension, motility and chemical irritation.

4 Brain (other intero-chemoreceptors)
l In hypothalamus: for blood osmolarity, glucose, hormones (and for temperature).
2 In medulla: for CO2 tension of the blood.

5 Special senses (extero-chemoreceptors)
1 Olfactory mucosa (smell)

2 Taste bud (taste)
(a) Barrel-shaped; lying within the stratified squamous epithelium of the tongue's circumvallate and fungiform papillae
(b) At the apex towards the opening of the taste pore, project processes of two fusiform cell kinds: (i) thin, neuro-epithelial receptor cells (dark, light, and intermediate), and (ii) paler sustentacular or supporting cells.
(c) Receptor cells have axons from the facial and glossopharyngeal nerves terminating synaptically upon them. (Taste buds in the pharynx, epiglottis and oesophagus are served by the vagus nerve.)
(d) Towards the bottom of the taste bud are basal cells, proliferating slowly to replace receptor cells.
(e) Von Ebner's glands in the lamina propria send a serous secretion into the trench around the vallate papilla, in whose walls the taste buds lie.

C MOTOR ENDINGS AND MOTOR FIBRES

l Skeletal-muscle motor fibres are derived from motor neurons/motoneurons within the CNS, either in the ventral-horn grey matter of the spinal cord or in motor nuclei of cranial nerves.
l Light microscopic view (after-gold-chloride impregnation). Nerve fibre branches to serve several skeletal muscle fibres, terminating on each as an irregular branching net lying on a small area of the muscle cell, its sole plate.

2 End-plate or neuromuscular/myoneural junction (EM morphology)

3 A motor unit comprises a motoneuron and all muscle fibres on which it has end-plates. .

2 Postganglionic autonomic nerve fibre terminals
l Control smooth muscle contraction and exocrine glandular secretion, or go to the heart muscle and adrenal medullary cells.
2 Axons lie against, or sometimes within, invaginations of the muscle fibres or glandular cells, making mostly en passant contacts,
3 but specialized sarcolemmal structures comparable with a motor end-plate's are not present.
4 The nerve fibres are, however, widely dispersed as a plexus between the smooth muscle fibres, and contain many vesicles concentrated periodically.
5 These vesicles may contain one of the two principal transmitter substances - acetylcholine (ACh) and norepinephrine (Ne)/noradrenaline, along with other chemicals, e.g., peptides. Some neurons and fibres are neither cholinergic nor adrenergic. A chemical mapping of the PNS (crucial to pharmacology) is under way, including the sensory pathways to autonomic ganglia.

3 Origin of autonomic nerve fibres Powerpoint
l Parasympathetic

Cranial nerves: III,VII,IX,X   |______  have parasympathic
Sacral pelvic nerves           |        preganglionic fibres (ACh)
These run near or into the organ to be controlled before synapsing with local parasympathetic ganglion neurons (e.g., of Auerbach`s plexus), whose own short post-ganglionic fibres (ACh) innervate the muscle or glandular tissue.

2 Sympathetic
Thoraco-lumbar outflow has sympathetic preganglionic fibres (ACh) synapsing with neurons of the sympathetic ganglion chain along the vertebral bodies or going farther to ganglia, e.g., coeliac, serving a visceral or cranial region. Sympathetic post-ganglionic fibres (usually Ne) thence pass to the muscle or gland to be controlled. (Ac - acetylcholine is the transmitter substance; the fibre is called cholinergic. Ne - norepinephrine is the transmitter substance; the fibre is called adrenergic.)
Beware. There can be more than one transmitter, for example, ATP can be a cotransmitter for both sympathetic and parasympathetic neurons, making these also purinergic.

3 Adrenal medulla
Receives direct, cholinergic, preganglionic, sympathetic fibres whose activity causes the release of norepinephrine (and epinephrine/adrenaline) into the blood, thus contributing to a widespread sympathetic tone.

D GANGLIA (relay structures)

l Spinal/dorsal root ganglion (no synapse involved)
l Has a collagenous connective tissue investment.
2 Many bundles of thick, myelinated, nerve fibres separate
3 groups of large, round-bodied nerve cells.
4 Each neuron has a thin CT capsule like an endoneurium.
5 Between capsule and neuron is a layer of small satellite cells of a glial nature.
6 Neuron has only one process (not a dendrite) branching into two near to the soma. The thinner axon runs centrally via a dorsal root into the spinal cord, the thicker runs peripherally to a nervous receptor.

2 Autonomic ganglion (compared with a spinal ganglion)
l Fewer myelinated fibres are present.
2 Neurons and fibres are interspersed.
3 Neurons are smaller and have dendrites, with preganglionic fibres synapsing upon them.
4 Many of the neurons' own axons (post-ganglionic fibres) are unmyelinated.

In a cross-sectional view, several unmyelinated fibres share one Schwann cell, lying in many deep invaginations of its membrane. In the gut, enteric glia take the place of Schwann cells.


Chapter 13 EYE AND ITS ADNEXA

The eyeball is one of a pair of roughly spherical, rigid structures sensitive to precise light stimuli and movable in coordination with its fellow. The camera performs a similar task, and the camera and the eye have in common:
  1. a rigid supporting structure,
  2. a light-excluding lining,
  3. a moveable control or stop for the light allowed to enter,
  4. a lens to focus that light on
  5. a light-sensitive sheet, and
  6. protective devices.
Before the histology is considered the overall anatomy should be briefly reviewed. Then the various structures will be taken in order as they are met on the light path. After that the accessory structures or adnexa will be dealt with, to lead to a final classification of all the structures along functional lines. . Eye Powerpoint

A ANTERIOR EYE

l Cornea
l Stratified squamous epithelium roughly five cells thick. Cells are held together by desmosomes, and supported on
2 Bowman's membrane: collagen fibrils in an amorphous matrix, viewed as a limiting condensation of the wide
3 Corneal stroma: orderly lamellae of collagen fibrils of uniform diameter, and keratocytes/fibroblasts with plenty of chondroitin and keratan sulphates; no blood vessels or lymphatics; takes up to 90 per cent of the corneal thickness.
4 Descemet's membrane: thick, distinct basal lamina with collagen fibrils in orderly array.
5 Endothelium: single layer of pavement/squamous cells, working to control the water content of the cornea.

Corneal functions: refraction, transparency, protection, and sensitivity (from intraepithelial free axons) for protective reflexes.

2 Anterior chamber
Limited by the posterior surface of the cornea and anterior surfaces of the iris and lens. It is filled to turgor with aqueous humour resembling serum, but very low in protein, and produced in the posterior chamber. To define this, some structures off the optical axis must be discussed.

3 Angle of the iris/anterior-chamber angle
Limbus forms the boundary between the cornea and the sclera which, although collagenous, is not transparent because of the disorder of its collagen fibres, its deficiency of sulphated ground substances, and its greater water content than the cornea.
Where Descemet's membrane terminates is a corneo-scleral trabecular meshwork/pectinate ligament enclosing the spaces of Fontana. These drain the aqueous humour towards Schlemm's canal, from which it passes to the episcleral or aqueous veins for venous return. The meshwork lies in the drainage angle between the sclera and the scleral spur.

4 Iris
l Rings the pupil and controls, by dilation or constriction, the light entering and the depth of focus.
2 Stroma: loose vascular CT with a variable proportion of pigment cells/melanophores.
3 Posterior surface is covered by a pigmented cuboidal epithelium forming the inner layer of the iridial retina.
4 Sphincter smooth muscle near the pupillary margin receives parasympathetic fibres, eliciting a contraction in response to increased light intensity.
5 Dilator muscle is a less substantial myoepithelial structure lying peripherally and posteriorly as the outer layer of the iridial retina, with fibres oriented radially and under sympathetic autonomic control.

5 Lens

  1. Is a biconvex, elastic, transparent, protein structure with:
  2. thick `elastic' glycoprotein outer capsule tending to give it a round form, under which lies
  3. an inner layer of cuboidal epithelial cells which peel off, elongate and insinuate themselves into the inner substance as lens fibres at the lens bow, thereby adding to lens crystallins - the main lens proteins.
  4. The lens is held out of the rounder shape of its own inclination by its attaching zonule/suspensory ligament running to the smooth muscle ciliary body, which is itself firmly attached to the CT sclera. Lens nutrition is indirect by the aqueous humour.

6 Ciliary body
l Circular smooth muscle (Müller's muscle): innervated by para-sympathetic fibres from the ciliary ganglion to contract, reducing tension in the zonule thus allowing the lens to become rounder and accommodate to near vision.
3 Radial and meridional muscle fibres (Brücke's muscle): function and innervation are uncertain.
3 Covered by a double layer of cuboidal epithelial cells (ciliary retina), with the outer ones heavily pigmented.
4 Gives off a number of projections, ciliary processes, covered by the two-layered epithelium and enclosing fenestrated blood capillaries, which produce the aqueous humour in a manner similar to the production of CSF by the choroid plexus.

7 Posterior chamber
l Is limited by the posterior surface of the iris, the zonule and parts of the lens and ciliary body;
2 from the last of which comes the aqueous humour that fills it and passes out via the pupil to the anterior chamber.

B POSTERIOR EYE

Here the three tunics of the wall - sclera, uvea, retina - are most clearly recognized.

l Vitreous body
l Viscid and transparent fluid which, although mainly water, contains proteoglycans, hyaluronic acid, and collagen.
2 It fills the space bounded by the lens, zonule, pars plana and neural retina.
3 The hyaloid canal extends anteroposteriorly through it.

2 Neural retina

  1. Curved membrane terminating its receptor function as an irregular line at the ora serrata/ora terminalis.
  2. Contains a pigment cell layer, light-sensitive photoreceptors, and nerve cells arranged in layers to partially integrate the nervous information and transmit it out of the eye to the brain.
  3. In most regions of the retina, light has to pass through the inner structures to reach the outer ones that are actually photosensitive.
  4. Retinal layers in brief:
    .. (i) Pigment-cell layer
    .. (ii) Photoreceptors
    .. (iii) External limiting membrane
    .. (iv) Outer nuclear layer
    .. (v) Outer plexiform layer
    .. (vi) Inner nuclear layer
    .. (vii) Inner plexiform layer
    .. (viii) Ganglion cell layer
    .. (ix) Nerve-fibre layer
    .. (x) Inner limiting membrane
When looking at slides of the posterior eye, resist the temptation to view the neural retina as an epithelium facing a lumen. The reference point for 'inner' and 'outer' is the unseen vitreous, not the BL on which the pigment cells sit.

3 Retinal layers details:

4 Retinal modifications
l Macula lutea with fovea centralis - on the visual axis is a yellow-ringed depression, from which the inner layers have been displaced to a peripheral hump so that: (a) the light can fall directly on the photoreceptors, that (b) are all tightly packed cones with straight-through neural connections, for high acuity.
2 Optic papilla/nerve head, where optic nerve fibres leave the eye (no receptors, therefore a blind spot), and where retinal blood vessels leave and enter for widespread retinal distribution.
The condition of these vessels is a crucial part of the ophthalmoscopic examination.
3 Optic nerve - the ganglion cells' fibres acquire myelin sheaths, then run centrally with accompanying glial cells and a meningeal sheath as a CNS tract. The retinal artery and vein run centrally in the intraorbital section of the nerve.

5 Choroid
Posterior part of the uvea - the eyeball's middle tunic - acts as a light-dense, nutritive backing for the retina with:

  1. Bruch's membrane supporting the retina.
  2. Choriocapillaris - a plexus of large capillaries.
  3. Choroid and outermost epichoroid/suprachoroid - highly vascular, loose stroma of collagen and elastic fibres, fibroblasts and pigmented melanophores. (The pigment is static inside mammalian melanophores, which are thus unlike those of lower vertebrates.)

6 Sclera
Dense, tough outer tunic of collagenous fibrous tissue. It has some regional variations:
l At the lamina cribrosa, where its fibres interweave with bundles of optic nerve fibres leaving the eye.
2 At the limbus, where it is more vascular, related to Schlemm's canal and the ciliary body.
3 Near to the limbus are the insertions for the oculomotor skeletal muscles moving the eye.
4 Throughout, its innermost layer (lamina fusca) also has melanophores and elastic fibres.

C ACCESSORY STRUCTURES (ADNEXA)

l Eyelids protect and lubricate the eye's anterior surface.
  1. Fine skin with a loose dermis faces outward.
  2. Palpebral conjunctiva (stratified columnar epithelium with goblet cells on a lamina propria) faces inward.
  3. Orbicularis oculi skeletal muscles (served by VIIth nerve) close lids.
  4. Levator palpebrae muscle raises the upper lids.
  5. Tarsal plates of dense CT have imbedded in them
  6. Meibomian sebaceous glands to make protective secretions.
  7. Eyelash hair follicles are separated by the
  8. sweat glands of Moll and sebaceous glands of Zeiss.
2 Conjunctiva
l Palpebral conjunctiva lines the eyelids, and bulbar covers the eyeball's sclera, with the fornices as the angle of reflection.
2 Stratified columnar epithelium has goblet and Langerhans cells, with many lymphocytes in the loose lamina propria.
3 Epithelium changes at the limbus (to corneal) and at the lid margin (to skin). Conjunctival epithelium is a source of cells to repair damaged corneal epithelium.
4 Plica semilunaris is a small conjunctival fold in the medial margin of the eye above the
5 caruncle, with its sebaceous glands.

3 Lachrymal glands
l In upper, lateral orbit, opening via ducts to the conjunctiva.
2 Compound, tubulo-acinar, serous gland with many myoepithelial cells. Mucous cells also are present.
3 Tears drain through the lachrymal punctum via lachrymal canaliculi into the lachrymal sac. Then they pass via the nasolachrymal duct to the lateral side of the inferior meatus of the nose.
4 Tear fluid is chemically complex. Tears have water, salts, glycoproteins, and bactericidal factors, e.g., lysozyme.

4 Other orbital structures
l Tenon's CT capsule.
2 Extraocular skeletal muscles (fine-fibred).
3 Adipose tissue.
4 Ciliary ganglion.

D FUNCTIONAL CLASSIFICATION OF THE EYE AND ADNEXA

  1. Optical refractive agents: cornea, lens, aqueous humour, and vitreous humour; form a small, inverted, real image.
  2. Receptor and neural tissues: retina and optic nerve.
  3. Sustaining and light-excluding tissue: vascular uvea comprising the pigmented choroid coat, ciliary body and iris.
  4. Form- and rigidity-endowing tissues: cornea, sclera and intraocular fluid.
  5. Oculomotor system: sclera and three pairs of muscles.
  6. Protective tissues: lids, conjunctiva, cornea, lachrymal, Meibomian and other glands, and the orbital bone.

E DEVELOPMENT OF THE EYE

l Forebrain grows out as the hollow optic vesicle, whose
2 proximal part constricts to become an optic stalk, later the optic nerve.
3 Superficial ectoderm over the optic vesicle thickens, then separates to become the lens vesicle.
4 Meanwhile, the anterior wall of the optic vesicle invaginates into the posterior producing a two-layered cup that becomes the retina with its posterior pigment epithelium.
5 Mesectoderm gives the corneal stroma, uvea and sclera.
6 Ectoderm provides the corneal and conjunctival epithelia.

Chapter 14 AUDITORY AND VESTIBULAR ORGANS

A SITUATION AND EXTERNAL RELATIONS

l Organs - cochlea and vestibular apparatus - sensitive respectively to air vibrations (sound), and movement of the head and its position relative to the gravitational field (balance), are combined in the inner ear within communicating spaces - the bony labyrinth - of the temporal bone. . Ear PowerPoint
2 Actually the receptors are enclosed in membranous tubes forming a membranous labyrinth that lies within, but does not fill the bony labyrinth.
3 The two separate systems contain different fluids. The membranous labyrinth is filled with endolymph and is a closed system, although it extends a ductus endolymphaticus through the bone to end blindly by the brain as an intradural sac involved in metabolic functions. This sac can be drained surgically to relieve damaging excess endolymphatic pressure - endolymphatic hydrops.
4 The space between the tubules of the membranous labyrinth and the bone is occupied by perilymph, which is in communication via the aqueductus cochleae and aqueductus vestibularis with the meninges and with the CSF of the brain's subarachnoid space.
5 The fluid in the bony labyrinth can interact with the middle ear (and indirectly with the external environment) by means of two soft areas in its bony walls: 6 The stimulus in the environment that causes movement of the oval window and pressure changes in the fluids is movement of air/sound, allowed a little way into the head via the external ear.
7 The fluids of the labyrinth are also subject to the gravitational force and that accompanying movement of the head.

B DIVISIONS OF THE EAR

l External ear
l Auricle: core of elastic cartilage; lobule of adipose tissue; skin-covered.
2 External auditory meatus: lined with skin and stratified squamous epithelium; has ceruminous (modified apocrine sweat) and sebaceous glands; supported by cartilage and, further in, by bone.
3 Tympanic membrane/eardrum: inner limit of the external ear, core of atypical collagen with thin epidermis externally, and a mostly simple squamous epithelium internally; the manubrium of the malleus bone inserts into the collagen. Elastin is present in the flaccid region.

2 Middle ear
l Epithelium-lined, air-filled, bony spaces of the tympanic cavity.
2 Communicates with the nasopharynx via the Eustachian/auditory/ pharyngotympanic tube, allowing equalization of air pressures on either side of the tympanic membrane. The mucosa of the tube and middle ear has several kinds of cell, and defensive systems.
3 Auditory ossicles articulate with one another - malleus, incus and stapes. The malleus is vibrated by air moving the tympanic membrane. This movement is then transmitted via the incus to the stapes with its foot held in the oval window by the annular ligament.
4 Elastic membrane of the round window transforms the fluid pressures generated in the inner ear into other forms of energy, thus acting as a pressure-release.
5 Fine skeletal muscles, (a) stapedius and (b) tensor tympani, inserting into the stapes and malleus are protective, and influence sound discrimination. Fine nerves pass to them.

3 Inner ear
The outer and middle ear thus have an exteroceptor function, transmitting air vibrations (20-20000 cycles per second is the perceptible range) to the perilymph fluid in the bone of the inner ear. Although the resulting pressure changes involve all perilymph, the receptors sensitive to the changes are localized in only one part of the labyrinth, the cochlea, and lie in the inner endolymph-filled system. Elsewhere in this inner system lie the intero- or proprioceptors for balance and movement, located in the vestibular apparatus.

C VESTIBULAR APPARATUS

l Bony vestibule houses the membranous utricle and saccule.
2 The vestibule extends into three semicircular tubes or canals distributed in three planes perpendicular to one another and containing the membranous semicircular ducts/canals, each swelling out at one end into an ampulla.
3 Movement of endolymph within the connecting membranous chambers stimulates receptors in maculae and cristae - modified, small, neuroepithelial areas of the lining membrane.

4 Utricle (with a macula) is oriented in the plane of the base of the skull, and the saccule (macula) in the sagittal plane. Both are responsive to gravity and linear acceleration, thus giving information on how the head is positioned.

5 Ampulla (with a crista) oriented in each of the horizontal, sagittal and transverse planes; responsive to movement of the head in the plane of that canal, thus furnishing information on the rate of angular acceleration.

7 The insensitive remainder of the vestibular membranous labyrinth is lined by a simple squamous epithelium on CT, which is supported by collagen and fibroblasts passing to the periosteum of the bony labyrinth, except on the side it fastens to the bony wall.

D COCHLEA

l Structures and elements
The tube - the cochlear duct - containing the cochlear endolymph is not surrounded by perilymph, but has it on two of its triangular sides. Thus, three chambers are contained within the bony cochlea which spirals for 2 1/2 turns around an axis of spongy bone, the modiolus. The spiralling unit comprises:
  1. Scala vestibuli with perilymph and mesothelium-lined.
  2. Reissner's membrane (membrana vestibularis), thin;
  3. Cochlear duct/scala media with K+-rich endolymph made in the stria vascularis; epithelium-lined, and containing the
  4. Organ of Corti (the actual receptor) on the
  5. basilar membrane, stretched from the tympanic lip of the bony spiral lamina to the spiral ligament.
  6. Scala tympani with perilymph and mesothelium-lined, separated by bone from the scala vestibuli adjacent in the spiral.
  7. Scalae vestibuli and tympani connect by a helicotrema at the apex of the cochlea, whilst the cochlear duct ends blindly as the caecum cupulare.
    The cochlear duct at its base communicates with the saccule via the ductus reuniens.
 Fig. 5 Turn of the cochlea
                      # # # # # bone
                    #  (  (  #
                  # (        #
               #  (          #
             #  (    SCALA   #
           #  (* ~ VESTIBULI # b
          # (*     ~r        # o      
         # (*  SCALA ~m      # n
        # (*   MEDIA   ~   # # e   
        # (___________!! ~_# Modiolus
        # (Basilar membrane  #      r~m Reissner's membrane
         # (                #        
          # (   SCALA     #         !! Organ of Corti
           # ( TYMPANI  #
            # (       #             *** Stria vascularis
             # (    #
              # # #   SCALA
                # (   VESTIBULI of the next turn
2 Organ of Corti
l Rests on the tympanic lip and basilar membrane.
2 Internal border cells and internal hair cells (receptive).
3 Internal pillar cells lean outwards towards inwardly leaning
4 external pillar cells, thereby enclosing an inner tunnel.
5 Phalangeal cells/Dieter's supporting cells support
6 external hair cells, (50-l00 hairs per cell); contractile to amplify the response of the mechano-sensory system; in three rows; damaged by loud sounds, streptomycin, cisplatin, etc.
7 Hairs (stereocilia of graded lengths) of outer cells go through a reticular plate to attach to the overlying
8 tectorial membrane - a gelatinous body attached at the vestibular lip to the CT limbus spiralis.
9 Nerve fibres derived from bipolar neurons of the spiral ganglion/ganglion of Corti in the bony spiral lamina, passing through the bone, serve the inner and outer hair cells.
(Centrifugal fibres also run from the brain stem to the outer hair cells, to enhance the response.)
l0 The centripetal fibres of Scarpa's vestibular and Corti's cochlear ganglia join to form the auditory/VIIth cranial nerve.

3 Organ of Corti's transducer function
Inner hair cells convert into neuronal discharges fluid pressure changes, transmitted through the basilar membrane to the cochlear endolymph, from the perilymph of the scala tympani. These changes originated at the oval window in response to vibration of the auditory ossicles caused by air moving the tympanic drum.
Discrimination of pitch (sound frequency) is based on different cochlear regions responding preferentially to particular tones, with high frequency received at the basal cochlea and lower ones apically where the basilar membrane is broader.

4 Fluids and gelatinous bodies
Although these are lost or grossly distorted in the histological processing, they are very important. The fluids transmit forces, and provide a metabolic pathway and favourable ionic environment for the receptor and other cells of the membranous labyrinth. In life, the gelatinous cupola and tectorial and otolithic membranes are large, filling or almost filling their respective membranous chambers.


Chapter l5 CIRCULATORY SYSTEM

A GENERAL FUNCTIONS AND ASPECTS OF THE CIRCULATORY SYSTEM.

Fig. 6               |    GAS EXCHANGE    |
                     |      (Lungs)       |
                     |                    |
                     |                    |
                     |      PUMPING       |
                   /  --------------------- \  
                  /        (Heart)           \         
                 /                            \
                /                              \ 
 HORMONES  __  /                                \ __ FOOD & (Gut)                                           
(Endocrine    |                                  |   WATER
 Glands)      |                                  |
              |                                  |
              |                                  |   
   WATER      |                                  |
              |                                  |
WASTE & HEAT /\                                  |__ STORAGE &     
( Kidneys, Gut \                                 /   PROCESSING
 Lungs, Skin)   \                               /     (Liver)
                 \                             /
                  \                           /
      BLOOD CELLS  \ _______________________ /                       
           &       /          | |       ~     \ CLEANING (Spleen, Liver,
      ANTIBODIES              | |         ~                       Marrow)
      (Red Marrow             | |           ~           
       Lymphoid Organs)       | |             ~
                              | |               ~
                              | |              Lymphatic drainage
                              | |                   ~
                 DETERMINING CELL ENVIRONMENTS        ~
                                                        ~
        1. By forming special fluids,      2. Forming tissue fluids in
           e.g., CSF, aqueous                 extracellular space by
           humour, synovial fluid             more widespread diffusion
                                              and transport, serving, e.g.,
                                              CT, epithelia, muscles.     

l Closed system of tubes, through which blood is forced by the pumping action of the four-chambered, contractile heart.
2 Tubular walls are permeable so that exchange of materials can take place between the system of small blood vessels and their environment - cells, or tissue spaces.
3 Lymphatic system collects fluid and colloids and crystalloids from the tissue spaces and returns them to the bloodstream.
4 There is a balance whereby materials are lost, e.g., from kidneys, lungs, skin, and replenished by the intake of foodstuffs, air and water.
Vessels Powerpoint

B SYSTEMIC BLOOD VESSELS

l Blood capillaries
l Very numerous, anastomosing, delicate tubes of diameter 7-9 µm.
2 Total cross-sectional area of the capillary bed is very great, thus blood flows slowly under low pressure.
3 Wall is made up of curved, thin, plate-like endothelial cells lying on a BL and oriented with the tube's long axis.
4 Type l unfenestrated capillaries have complete endothelial cells, e.g., in muscle and skin: type ll capillaries have endothelial cells with fenestrations/pores through them (not between them), e.g., in kidney and choroid plexus.
5 Endothelial cells have serrated margins where they attach by adhaerens and tight junctions to each other, tight/occluding junctions predominate where more of a barrier is needed, e.g., in the brain. Continuous capillaries have no gaps between the endothelial cells, in contrast to discontinuous capillaries.
6 Transport is controlled by the cells, with diffusion and facilitated transport for small molecules, and transcytotic vesicles or passage through the pores for larger materials.
7 Some capillaries have the occasional pericapillary cell - pericyte - imbedded within the BL, perhaps playing a contractile role.
8 Show transitions at both ends: to arterioles (by acquiring smooth muscle cells), or venules (by widening and taking on more collagen fibrils).
9 Endothelial cells secrete vasoconstrictor, vasodilator, and mitotic agents, and their own BL; they interact with blood, leucocytes and platelets, vary their permeability, and proliferate. Despite their lack of presence in routine light microscopy, they keep very busy, and are specialised for each organ that they serve.
10 Selectins are molecules expressed on the endothelial cells of small vessels, and on white blood cells. They bond intermittently with the sugars of a glycoprotein on the corresponding cell to cause the WBC to roll to a stop attached to the endothelium, before squeezing through the vessel wall into the connective tissues for defence. Sometimes the selectin is on endothelium, the ligand on the WBC, at other times the reverse achieves a similar result.
11 von Willebrand factor (vWF) also has a dual distribution, being present in Weibel-Palade storage granules of endothelial cells and alpha granules of platelets. Vascular injury releases vWF from endothelium to cause platelet activation, aggregation, binding to subendothelial collagen, and blood clotting - processes of haemostasis.

2 Sinusoids
l Have wider, more irregular lumens than capillaries.
2 Some of the lining cells are phagocytic.
3 Basal lamina may deficient or absent so that lining endothelial and phagocytic cells lie directly on reticular fibres and other cells, as in the liver.

3 Sinusoidal capillaries
l Have wide irregular lumens and a continuous, but fenestrated, non-phagocytic lining;
2 are the usual smallest vessel in endocrine tissue.

4 Arteries
l Have three main layers composed of several tissues:

Tunica intima
... (a) Endothelial lining on a BL
... (b) Subendothelial CT
... (c) Internal elastic lamina (fenestrated)

Tunica media
... (d) Smooth muscle cells (tightly spiralling or 'circular') 
... (e) Sparse reticular and elastic fibres

Tunica adventitia
... (f) External elastic lamina (interrupted)
... (g) Collagenous and elastic CT (mostly longitudinal)
2 Arterioles, less than 0.5 mm wide, have (a),(c),(d),(e) and (g) of the above.
3 Small and medium-sized arteries (muscular/distributing) have all elements.

4 Large arteries (elastic/conducting) differ significantly:

Tunica intima
... (a) Endothelium on a BL
... (b) Subendothelial CT
... (c) Innermost fenestrated elastic lamina

Tunica media
... (d) Many fenestrated elastic laminae interspersed with
... (e) smooth muscle cells and collagen fibres

Tunica adventitia
... (f) Collagenous CT with vessels and nerves
The larger arteries and veins have nutrient vessels and nerves (of vessels) in the adventitia - vasa vasorum and nervi vasorum.

In atherosclerosis, the arterial smooth muscle cell (SMC) changes its phenotype from static and contractile to proliferative, migratory, and synthetic. The converted SMC is further delinquent in invading the territory of the intima, where it lays down matrix and encourages the deposition of lipid, which, aside from narrowing the lumen, attracts platelets and macrophages. Their activation carries worse implications for blood flow, clotting, and deterioration of the vessel wall.

5 Veins
l Venules have an endothelial lining, BL and a collagenous outer sheath. Pericytes are numerous. The wall is thin enough to permit transport through it. White blood cells can squeeze between endothelial cells (transmigration/ diapedesis) and escape into the tissues. Lymphocytes may migrate actually through the interior of the endothelial cell.
[Emperipolesis is the migration of a cell into (and out of) another cell, while remaining intact: high endothelial cells, megakaryocytes, and thymic epithelio-reticular cells are hosts for such activity.]
2 Small veins acquire an additional thin media of smooth muscle and a thicker adventitia of collagen and elastic fibres.
3 No distinct elastic laminae are seen, but sparse elastic networks are found throughout the wall.
4 Many veins have valves - leaf-like projections of the intima, usually in a bicuspid form.
5 Large veins (e.g., vena cava) have bundled longitudinal smooth muscle in the CT adventitia and intima, whilst the media is thin or absent.

6 Comparison between a vein and its companion muscular artery
Both are tubes lined by endothelium and may contain RBCs.

Artery                          Vein
 (a) Shape less deformed       (a) Flattened
 (b) Thick wall                (b) Thin wall
 (c) Intima crinkled           (c) Intima smooth
 (d) Three distinct layers     (d) Layering indistinct
     (media prominent)             (media weak)
 (e) Internal elastic lamina   (e) No internal elastic lamina
7 Exceptional vascular structures
l Cerebral, retinal and osseous veins have no valves and no media. Veins in general are very variable in their structure.
2 Cerebral arteries are thin walled and have no external elastic layer.
3 Umbilical vein is very muscular; and the umbilical arteries have little elastic, and a media with distinct longitudinal and circular muscle layers.
4 Arterial intimal cushions are present in arteries to erectile tissue, kidneys, etc.
5 Some vessels have a high protruding endothelium, e.g., fetal stem arteries.

8 Exceptions to the vascular pattern of arteries, arterioles, capillary bed, venules, veins, heart

  1. Arteriovenous anastomoses - bypassing the bed (e.g., in the skin and gut) with thick muscle to close the bypass.
  2. Arterial anastomoses, e.g., circle of Willis to the brain.
  3. In the periphery, arteries and veins run together with nerves bound in CT as a neurovascular bundle. In the brain, arterial and venous distributions are separate.
  4. Vasa vasorum are blood vessels serving the adventitia and media of larger arteries and veins.
  5. Portal systems exist to the liver and pituitary gland, where venous blood drained from one organ is fed as a supply to the sinusoids or capillaries of another.
  6. Sinusoids may take the place of a capillary bed. Thus, for instance, sinusoidal capillaries permit blood to pass slowly by and influence chemosensitive epithelioid cells in the carotid body/glomus caroticum.
  7. Venous sinuses are endothelium-lined CT spaces where blood can collect for purposes other than metabolic exchange, thus, as part of the venous collecting system, e.g, coronary and dural sinuses, and in erectile tissue. (Caution: sinus is also a term for the pocket behind a venous valve - a site causing problems, when veins are grafted to substitute for arteries.)
9 Morphology in relation to physical factors in various vessels of the system
  1. Large elastic or conducting arteries - collagen fibres and elastic laminae predominate for strength, and elastic distensibility provides for elastic recoil during diastole thus damping the pulsatile flow resulting from the intermittent contractions of the heart. Endothelium provides a smooth lining to facilitate flow and prevent clotting.
  2. Muscular distributing arteries - lumens of a controllable size are narrowed by smooth muscular contraction to direct blood flow appropriately for the needs of various regions; mainly muscular media, strong CT adventitia with vasa vasorum, and autonomic nerve fibres to the muscle; elastic tissue limits distension of the lumen, and acts with the muscle.
  3. Arterioles - smooth muscle provides for a great reduction, by vasoconstriction, of the blood flow to a region; they maintain an adequate arterial pressure, but reduce blood pressure to an acceptable level for:
  4. Capillaries - pressure is low so can be thin-walled to permit exchange of gases, minerals, carbohydrates and small molecules + water by: The wall serves to keep back in the capillary most of the colloidal proteins of the plasma; the presence of these then encourages the return of fluids at the venous end of the capillary.
  5. Veins - low, even pressure so have large lumens, thin collagenous walls, and valves to prevent backflow; larger veins acquire some muscle, circular and longitudinal, in the media and adventitia. In general, elastic is not needed for recoil, for variations in pressure between diastole and systole are insignificant, but the vena cava has significant numbers of elastic fibres.

C HEART

1 Thick-walled, hollow, muscular pumping, and endocrine, organ.

2 Heart structures

       |                                                |
Systemic Veins                                    Pulmonary Veins
       |        Coronary sinus                          |
       |       /                                        |
       |      /                                         |
 RIGHT ATRIUM/                                     LEFT ATRIUM
       |                 . . . . . . . .  .             |
Tricuspid Valve . . . .  . Annuli fibrosi . . . . . Mitral Valve
       |                 .                .             |
       |                 . Trigona fibrosa.             |
       |                 .                .             |
       |                 .                .             |
RIGHT VENTRICLE          .    Septum      .        LEFT VENTRICLE
       |                 .  membranaceum  .             |
       |                 .                .             |
Pulmonary Valve . . . .  . Annuli fibrosi . . . .  Aortic Valve
       |                 . . . . . . . .  .             |
       |                         .                      |
Pulmonary artery                 .                    Aorta
       |                         .                      |
                         [Cardiac Skeleton]

Fig. 7 Heart structures.

3 Heart wall`s three layers

  1. Endocardium (innermost)
  2. Myocardium
  3. Epicardium (visceral pericardium) and subepicardium
  4. Pericardium (parietal)
    CT membrane of fibres supporting a mesothelium. This faces the epicardium across the pericardial cavity containing a small amount of lubricating fluid.
4 Cardiac skeleton of dense fibrous CT, with a tendency to turn into fibrocartilage. Elements are listed above in Fig. 7.

5 Heart valves
1 Atrio-ventricular valves
... (a) Leaflets are covered with endothelium on a
... (b) core of dense CT fused to the supporting annulus.
... (c) Cords of CT (chordae tendineae) connect the valve to
... (d) the papillary muscles in the ventricular wall.
2 Semi-lunar valves
... (a) Deploy three leaflets.
... (b) Thinner than the atrio-ventricular valves.
... (c) Lack chordae tendineae.
... (d) Fibrous core enlarges to the nodule of Arantius at the free margin.

6 Impulse-conducting system (coordinates myocardial contractions)
l Sino-atrial node of thin, modified, cardiac muscle fibres, influenced by parasympathetic (ganglionic neurons are found in the heart) and sympathetic autonomic nerve fibres, initiates contraction (pacemaker).
2 Contraction spreads through the atrial myocardium to the
3 atrio-ventricular node (Tawara's) consisting of a tangled plexus of modified cardiac fibres in the medial wall of the right atrium.
4 These fibres enlarge into Purkinje fibres and continue through the septal CT as the bundle of His, which then branches.
5 Purkinje fibres are rich in sarcoplasm and glycogen, but poor in myofilaments. They lack T-tubules, and are connected by intermediary transitional cells with ordinary myocardial fibres, whose contraction they can thus evoke in many regions of the ventricles.
7 In ungulates, Purkinje fibres are very large, pale and easily recognized: in man, the system is less obvious.

7 Endocrine role of heart
Atrial myocytes synthesize atrial natriuretic factor (ANF), which relaxes blood vessels and increases the excretion of sodium and water by the kidney. ANF is thus a partial counterweight to the renin-angiotensin system.

D LYMPHATIC VESSELS

l Lymphatic capillaries
l Network of blindly ending or anastomosing tubes, 5-50 µm wide.
2 The wall is made of an endothelial tube, with a discontinuous basal lamina and fine anchoring fibrils.
3 The wall permits the capillary to collect water, solutes and macromolecules from the tissue spaces.
4 Capillaries (i.e., a lymphatic drainage) are absent from the CNS, bone marrow, eye, and parts of the spleen.

2 Collecting vessels
l Lymph passes from capillaries into larger lymphatic vessels with very thin walls of endothelium, basal lamina and collagen, and numerous valves.
2 Lymph is led to small protective ovoid bodies - lymph nodes - through whose tissues it must filter before going further.
3 Lymph collects in the thoracic duct before entering the circulating blood at the left innominate vein; the right lymphatic duct also collects lymph for return to the bloodstream.

4 Thoracic duct

3 Lymph
l Adds to the blood proteins leaked from blood capillaries, new and recirculated lymphocytes, and antibodies, fat droplets (chylomicrons), etc.
2 Fat is collected from the gut in blind lymphatic capillaries lying centrally in intestinal villi. The fat-whitened lymph (chyle) gives these vessels a milky colour, hence their name lacteal.

4 Oedema and its causes
Oedema is an excessive accumulation of tissue fluid, involving mainly the extracellular space (except in CNS), and making the tissue swollen and puffy. It is caused by:

  1. Venous obstruction, e.g., from cardiac incompetence, which
    .. (a) raises intracapillary hydrostatic pressure, thus forcing more fluid into the tissues;
    .. (b) reduces the volume of blood collected from the capillaries.
  2. Injured capillary walls, e.g., from heat, become permeable permitting greater egress of fluid, solutes and colloids.
  3. Resulting reduction in intracapillary colloid at the venous end of the blood capillary lessens the osmotic attraction for tissue fluid to come back into the capillary.
  4. Lowering of systemic plasma colloids (proteins), from
    .. (a) proteinuria (excretion of protein in the urine),
    .. (b) protein starvation, or
    .. (c) exudation from burnt skin surfaces,
    will likewise reduce the osmotic return of extracellular fluid to capillary blood.
  5. Obstruction of lymphatic vessels receiving the lymph drained from tissue fluid by lymph capillaries, for instance, blockage by metastatic cancer cells. The tropical filaria parasites often block the lymphatics of their host causing gross swelling (elephantiasis) of affected extremities.

E VESSEL DEVELOPMENT

l Blood and lymphatic vessels (except sinusoids) form initially as simple endothelial tubes developed from mesenchymal cells - angioblasts.
2 Larger vessels and systems start independently of one another.
3 Their tunics with muscle and CT are added from mesenchymal condensations around the endothelium.
4 Capillaries of the adult can multiply or regenerate by extending cords of endothelial cells, which arrange themselves into a tubule. Cords can fuse with one another to build an anastomosing network.
5 Various cytokines promote or inhibit angiogenesis, e.g., vascular endothelial growth factor (VEGF).

Chapter l6 GLANDS

Glands are composed of secretory epithelial cells and their supporting connective tissue, nerves and blood vessels. Powerpoint
.

A VARIETIES OF GLAND

l Epithelial secretory layer, e.g., lining stomach and uterus.
2 Single cells amongst others in an epithelium - goblet cells, secreting glycoproteins, which with water, make mucus. Mucus is vital for the protection and lubrication of epithelial surfaces.
3 Intra-epithelial clusters of glandular cells, e.g., in urethra.
Glands as structures distinct from an epithelium can hold more synthesizing cells, but remain related to the surface epithelium by a duct - exocrine type of gland.
Other glands originate in an epithelial layer, but lose their duct and send their secretion instead into blood capillaries - endocrine or ductless glands.
4 Exocrine glands, which may be: 5 Endocrine glands making hormones: details in Chapters 26 and 27.
6 Mixed exocrine and endocrine glands, e.g., pancreas.
7 Mixed germinal exocrine/cytogenic (forming reproductive cells) and endocrine - testis and ovary.
8 Neurosecretory nerve cells and their axons constituting a neurofibrous gland are an exception to glands' being epithelial.
This classification takes on more meaning when all glands in all the organs have been studied.

11 B STRUCTURE OF A COMPOUND EXOCRINE GLAND

l Encapsulated in fibrous CT which sends in partitions around lobes.
2 Septa (sheets of CT) divide the glandular tissue further into lobules. Septa carry ducts, blood and lymphatic vessels, and autonomic nerves and neurons.
3 Each lobule contains:
  1. Many epithelial, parenchymal cells grouped as tubules or alveoli, cut at a variety of angles to the plane of section.
  2. In each tubular or alveolar secretory unit, the cells lie on a BL and face inwards towards a very small lumen.
  3. The lumens lead to ducts, also seen in the lobule.
  4. Outside the BLs, in the spaces between alveoli are the blood capillaries, CT cells and autonomic nerve fibres of the supporting stroma.
  5. A duct system runs through and out of the lobule and the gland, converging and widening as shown in Table 5.
Table 5. Secretory passages of a compound exocrine gland.
Structure and site                                Lined by

Intercellular canaliculi (alveolus)      Alveolar secretory cells
Alveolar lumen (alveolus)                Alveolar secretory cells
Intercalated duct (intralobular)         Squamous or cuboidal epithelium
Intralobular duct (intralobular)         Cuboidal epithelium
Interlobular duct (interlobular septum)  Columnar epithelium
Lobar duct (interlobar septum)           Pseudo-stratified columnar epithelium
Final duct (lamina propria of tract)     Stratified columnar epithelium
4 Compound exocrine glands were classified by their secretory product as serous (water+enzymes), mucous (glycoproteins), or mixed serous and mucous.
l Serous acini have pyramidal darkly basophilic cells, with spheroid nuclei and apical zymogen (pro-enzyme) granules.
2 Mucous acini are made up of pale cells, with the nuclei flattened towards their bases, and a cytoplasm crowded with mucus/mucin droplets, which can be stained to reveal the presence of the sulphated or neuraminic-acid/sialic-acid moieties that confer viscosity on mucus.
3 Mixed acini/alveoli: Mixed glands may also form two products by having pure mucous and pure serous alveoli.

C CYTOLOGY OF SECRETION

l Serous secretion (e.g., in pancreatic exocrine acini)
l Enzymes formed are proteins, and the path of synthesis can be revealed by following radioautographically the fate of tritium-labelled amino acids, e.g., leucine.
Other serous products include antimicrobial proteins.
2 In the basal region of the cell, amino acids are chain-linked at the ribosomes attached to the GER, in sequences determined by mRNA from the nucleus. The energy needed is released by plentiful mitochondria.
3 The protein passes into the cisternae of the GER and
4 travels in the cisternal space to near the Golgi complex.
5 The protein is shuttled to the cis/forming/proximal face of the supra-nuclear Golgi complex by transporting vesicles.
6 Condensing vacuoles concentrate the secretion before its dispatch from the concave trans/secretory/maturing/distal face of the Golgi to become
7 membrane-bound, apical, zymogen storage granules.
8 With appropriate stimulation, the granules pass to the cell's luminal membrane for release by exocytosis, whereby the granule's enclosing membrane fuses with the cell's, which then breaks allowing the granular content to spill out into the acinar lumen.

2 Mucous secretion (e.g., by goblet cell)
l Oligosaccharides are completed by the Golgi complex, sulphated, if necessary, and linked with a protein to form
2 mucin, stored as droplets dilating the apical cytoplasm.
3 Granular ER - for synthesis of the core protein of the glycoprotein and of sugar-attaching (glycosylating) enzymes - is well developed in the narrow basal stem of the cell.
4 After one cycle of activity, the gut goblet cell is normally shed to be replaced from a pool of undifferentiated cells.
5 Mucous cells of salivary glands are not shed. They have GER and, when they are immature, or in the early secretory phase with little mucin accumulated, they are basophilic and may resemble serous cells.
6 The mucin type of glycoprotein has its hundreds of chains of sugar moieties attached to the peptide core - the apomucin - by hydroxyls of serine or threonine - the O-linkage. In contrast, serum-type glycoproteins are N-linked, since their sugars attach via amido groups of asparagine. The O- and N-linked classes differ in their affinities for lectins, what agents block sugar-chain biosynthesis, and in whether the first glycosylation is in the Golgi complex or GER.
7 The mucin molecules are further classified as neutral or acidic, based in part on the amount of sialic acid present. The molecules join end-to-end, and then tangle up for bulk and high viscosity.

3 Liberation of secretion

  1. Merocrine/epicrine/eccrine manner involves exocytosis, or the discharge of only secretory material without any loss of cytoplasm, as in a serous gland. The cell then returns to the synthesizing phase of its secretory cycle.
  2. Holocrine secretion requires that the cell fill itself up with secretion which is liberated by the cell's breaking open and dying, e.g., in a sebaceous gland. Precursor cells must multiply to replace those lost, for the gland to continue secreting.
  3. Apocrine way was thought to involve a significant loss of apical cytoplasm along with the secretion, but not cell death. EM suggests that this occurs rarely, if at all, and the classic apocrine-merocrine distinction is invalid. However, apocrine is now applied to a release of secretion where the product, milk fat, departs from the mammary cell enclosed in a membrane.

4 Myoepithelial cells (basket cells)
These lie between glandular and duct cells and the BL, and clasp those cells in long branching processes filled with filaments. They closely resemble smooth muscle cells, and contract to help squeeze the secretion out of large exocrine glands (breast and salivary) or the long, tortuous sweat gland.

5 Duct-lining cells
Ducts are not usually passive tubes for conveying secretions. Their lining cells often are cuboidal or columnar, and acidophilic, with many basal mitochondria serving active transport mechanisms to modify the secretion's concentration and electrolyte composition, by actions similar to those of kidney tubules. Such ducts may be called secretory or striated (from the many parallel mitochondria); they lead to less active excretory (drain pipe) ducts.
(Secretory ducts are usually intralobular, but not all intralobular ducts are secretory.)


Chapter l7 BLOOD

Blood might be classed as a specialized connective tissue because its cells are mesodermal in origin and are separated by plasma.
Also available in colour as a series of Powerpoint slides.
                         _
            Plasma      |  Red blood corpuscles/Erythrocytes (RBCs)
              +         |
      formed, visible   |
         elements    - -|  White blood cells/Leucocytes (WBCs)
     (46% by volume)    |
                        |
                        |_ Platelets

A MICROSCOPIC TECHNIQUES FOR BLOOD

l A blood drop is smeared across a slide and
2 stained with a Romanowsky-type combined stain - a neutral combination of acidic (eosin) and basic (azure) stains.
3 In the stained smear, a differential count by eye or automated counter gives the proportions of the different varieties of leucocyte.
4 Absolute counts of blood, diluted by a known amount, in a counting chamber give the numbers of the formed elements: RBCs - about 5.2 million mm3 (man), 4-5 million (woman); WBCs - 5000-9000 mm3 (healthy adult); platelets 200000-400000 mm3.
5 EM study of WBCs and platelets in the buffy coat after its centrifugal separation from the RBCs.
6 Light and EM examination of cells in the lumens of blood vessels in sections of imbedded tissues.
7 Phase-microscopy and videorecording of leucocytes alive in fresh blood on a warmed slide under a sealed coverslip.
8 Tagged monoclonal antibodies to recognise cell-surface glycoproteins characteristic for particular subtypes of blood cell. This approach allows a specific cell population to be sorted for culture and study using automated flow cytometry.

B ERYTHROCYTES (gas transport)

  1. Biconcave discs; close to 7.5 µm diameter in a smear.
  2. Comprise a flexible membrane enclosing haemoglobin (iron-porphyrin-protein) in a closely packed state which, with membrane-spectrin-actin interactions, maintains the RBC's optimal shape for gas exchanges involving the haemoglobin.
  3. Osmolarity of the plasma affects the shape of an RBC. Hypertonic solutions in vitro cause crenation and shrinkage; hypotonic, swelling and haemolysis.
  4. Globin is acidophilic, and RBCs stain orange with eosin.
  5. Mature RBCs have no nucleus, Golgi body, ER, ribosomes or mitochondria.
  6. RBCs do have glycolytic enzymes and substrates, and methaemoglobin reductase and carbonic anhydrase for their respiratory function:
  7. Reticulocyte/polychromatophil erythrocyte. An immature RBC, when stained supravitally with cresyl blue, has a blue condensed network of clumped, residual ribonucleoprotein not yet used for protein (globin) synthesis.
  8. Life in circulation is estimated by 51Cr labelling at around l20 days, then the RBC is sequestered in the spleen, liver or bone marrow to be phagocytosed by macrophages. The spleen is most responsible.
  9. The volume of RBCs as a percentage of centrifuged whole blood - the haematocrit - is a quick, crude measure of the O2-carrying quality.

C LEUCOCYTES (defence)

These are true cells, divided according to the granularity of their cytoplasm into two groups - granular and agranular.

l Granular leucocytes
All kinds appear round in a smear with a diameter l0-l4 µm.

l Polymorphonuclear neutrophil (neutrophil/PMN/polymorph, for short).
Defence Powerpoint

2 Eosinophil 3 Basophil 2 Agranular leucocytes
l Lymphocyte 2 Monocyte

D PLATELETS (clotting and vessel-sealing)

Clotting Powerpoint
l Rounded or ovoid parts of cells, 2-5 µm diameter.
2 Consist of cytoplasm, organelles and inclusions, bounded by a cell membrane, reflecting their formation as pseudopodia breaking away from extravascular cells - megakaryocytes.
3 The dense central granulomere (organelle zone) has mitochondria, dense bodies and alpha granules; the pale peripheral hyalomere (sol/gel region) is cytoplasm deficient in organelles, except for contractile filaments and a shape-giving ring of microtubules.
4 Platelets adhere to collagen, neutrophils and monocytes, and especially to each other; this platelet aggregation/agglutination is used to seal defects in blood-vessel walls.
5 Apart from several molecules for adhesion, the membrane supplies a phospholipoprotein: one of many factors in the cascade causing blood fibrinogen to form fibrin fibres in clotting. Platelets contract and cause a compacting of the fibrin to which they adhere - clot retraction.
They also release from their granules several factors, e.g., serotonin and cytokines, having vasoconstrictive and other actions.

E BLOOD IN BRIEF

Liquid PLASMA + visible FORMED ELEMENTS - supplement, control, & extend
                                          what goes on in the plasma

A GAS TRANSPORT - Erythrocytes/Red blood cells/RBCs |
                                                    |
B DEFENCE against the - Leucocytes/WBCs             |___ Describe:
  bad & the dead                                    |    appearances,
                                                    |    roles, &
C DEFENCE for vessel integrity - Platelets          |    means

Description includes Measurements:

ABSOLUTE COUNT numbers/cubic mm  RBCs - 6 X 106, Platelets - 2 X 105
                                 WBCs - 6 X 103

DIFFERENTIAL COUNT  individual % proportion of 5 kinds of leucocyte

HAEMATOCRIT   Centrifuge tube
            __________________________________
             |///////////////}................\
            _____________________________|____/
                Plasma       |           |
                           Buffy         | RBCs - 45% by volume
                           coat          a measure of O2-carrying
                           (WBCs)        capacity

ERYTHROCYTE     <--- 7.5µm --->
                Biconvave disc  -  high surface/volume ratio

 Membrane with subplasmalemmal cytoskeleton: holds shape, but is
   deformable
 No organelles; no nucleus; haemoglobin binds O2

NEUTROPHIL/polymorphonuclear leucocyte/PMN [granular] 55%  lobed nucleus
t   phagocytosis of bacteria & debris   m  destructive acid hydrolases
a   bacterial killing                   e  generation of free radicals
s   adhesion to & migration through     a  proteins, e.g., defensins
k    venule & capillary walls*          n  adhesion molecules* to stick
s                                       s   to 'infected' endothelium
     
EOSINOPHIL [granular] 2%, bilobed nucleus, large granules
t                                m  lysosomal enzymes         } similar 
a  anti-parasites                e  oxygen-radical generation } to PMN's
s  role in allergies             a  anti-parasite proteins, e.g. ECP
k                                n   eosinophil cationic protein
s  adhesion & migration*         n  cytokines
                                 s  adhesion molecules*

LYMPHOCYTE [agranular] 30% small cell, dense round nucleus
t  attack foreign cells & microrganisms    m  immune responses
a  attack foreign materials                e  cytokines
s  instruct other cells - come, stay,      a  cell-surface contacts
k   be quiet, be active, proliferate       n
s  proliferates itself                     s  *
   *

MONOCYTE [agranular] 8%, large cell, with indented nucleus
t  general phagocytosis, e.g. of      m  becoming activated macrophage
a   leftovers of damaged tissues      e  lysosomal enzymes
s  coordination of defence and        a  cytokines
k   repair                            n  antigen presentation
s  *                                  a  *

BASOPHIL [granular] ½% lobed, obscured nucleus
t  back up mast cells' responses     m  histamine
a   in inflammation                  e  leukotrienes
s  affects capillaries & speed &     a  prostaglandins
k   intensity of immune responses    m  cytokines
s   e.g. hypersensitivity            s  proteoglycans
   *                                    *

PLATELETS small cast-off pieces of giant marrow cell - megakaryocyte
t  stick together, and to endothelium   m  von Willebrand factor
a   and vessel collagen                 e  other clotting factors
s  start blood clotting                 a  5-hydroxytryptamine
k  control blood flow                   n  thrombospondin
s                                       s  adhesion molecules
* adhesion to endothelium and the cell-specific means thereto
Note that some lymphocytes , e.g., NK cells, have azurophil lysosomal granules

Chapter 18 HAEMOCYTOPOIESIS

Continuous formation of the cells, corpuscles, and platelets of the blood is necessary to keep their numbers relatively constant as they wear out or are lost from the body. The formation is called haemocytopoiesis or haemopoiesis for short.

A DIVISIONS OF HAEMOPOIESIS

l Myelopoiesis - formation of granular leucocytes (granulopoiesis), monocytes (monopoiesis), erythrocytes (erythropoiesis), and platelets (thrombopoiesis).
2 Lymphopoiesis - formation of lymphocytes and plasma cells. (Plasma cells are not normally seen in the blood.)

B SITES OF MYELOPOIESIS

l Embryonic: mesenchyme gives rise to: 2 Adult

C THEORETICAL CONSIDERATIONS OF HAEMOPOIESIS

l Granular leucocytes and RBCs are specialized end products in being unable to divide, and living for only a few weeks. Since their numbers in the blood stay constant, new cells must be forming from less specialized ones.
2 Bone marrow, stained as for a blood smear, has cells, construed from their granularity, eosinophilia, nuclear morphology, etc, as members of developmental sequences, apparently starting with a large, undistinguished weakly basophil, primitive cell, and ending as one of the clear-cut specialized kinds.
3 If all the primitive marrow cells multiplied and then turned into blood cells, when the blood cells were spent, no primitive ones would exist to replace them. Thus, the primitive cells must act as stem cells able to divide, and with two possible fates: some to stay as primitive stem cells, others to differentiate into special forms.
4 Since there are several specialized blood cells, are there separate, but histologically indistinguishable, stem cells: one for each blood cell type? - The polyphyletic theory of committed progenitors for each lineage. Yes, but the monophyletic theory also survives, because rare multipotent/pluripotent stem cells exist, and can replenish the restricted stem cells, e.g., those for erythropoiesis.
5 CFU-S denotes the pluripotent cell in mouse, and forms the basis for naming progenitor cells in humans. Colony-forming unit - spleen/CFU-S was the cell that could give rise to an island/colony of complete haemopoiesis in the spleen of the mouse, after splenic and other sites of haemopoiesis had been totally destroyed by irradiation. Where, then, did the rescuing cell come from to form the colony? The CFU-S was obtained from infant mice and injected just after the irradiation. (A convenient human source for equivalent stem cells is blood from the umbilical cord.)
6 All cell divisions and differentiations need controlling growth factors (cytokines), not only to maintain the stem cell population, but to persuade some of them to fill precisely the ranks of the various blood cells.
7 After a stem cell becomes a committed precursor/progenitor for a certain cell line, a period elapses when histology, without immunostaining, cannot identify the line. Later, perceptible morphological changes make the cell a recognizable precursor, say a pro-erythroblast. Thereafter, the development of the cell is divided into named stages, each based on a significant change in appearance from the previous stage.
The potential for confusion exists, since workers have differed in the number of stages chosen, e.g., omitting pre-stages, and their names for a given cell type, e.g., rubriblast/normoblast for erythroblast.
8 The ability of the few stem cells to divide does not preclude proliferation by committed precursors, and by cells at later, recognizable, stages of development, for continued amplification of cell numbers.
9 Fig. 8 omits: how the early elements shown match the committed precursor-recognizable precursor classification; typical population figures for each cell kind; how recognizable the kinds are; and the controlling factors for proliferation and differentiation (G below).

Fig. 8 Pathways of blood cell differentiation - Stained marrow-smear view

                                                                                
                           . Haemocytoblast/Pluripotent stem cell                     
                        .~     /     |        \                 \                      
H                    .~      /       |          \                 \                       
A                 .~       /         |            \                 \                 
E              .~        /           |              \                 \               
M   Lymphoblast     Monoblast     Myeloblast       Pro-erythroblast   Megakaryoblast  
O        |             |              |                   |                 |         
P        |             |              |                   |                 |         
O        |             |              |                   |                 |         
I        |             |              |                Basophil             |         
E        |             |         Pro-myelocyte        Erythroblast          |         
T        |             |              |                   |                 |         
T        |             |              |                   |                 |         
I        |             |              |                   |                 |  
C        |             |              |              Polychromatic          | 
T        |             |          Myelocyte          Erythroblast           |  
         |             |              |                   |                 |        
T        |             |              |                   |                 |
I        |             |              |                   |                 |
S        |             |              |                   |                 |
S        |             |         Metamyelocyte       Orthochromatic         |
U        |             |              |               Erythroblast          |
E        |             |              |                   |                 |
S        |             |       Band granulocyte      Reticulocyte     Megakaryocyte
- - - - -| - - - - - - | - - - - - - -| - - - - - - - - - | - - - - - - - - | - - - - -
B        |             |              |                   |                 |
L        |             |              |                   |                 |
O   Lymphocyte      Monocyte     Granulocyte         Erythrocyte        Platelets
O        |             |              |
D        |             |              |
- - - - -| - - - - - - | - - - - - - -| - - - - - - - - - - -  - - -  - - - - - - - -
C        |             |           Tissue
T   Plasma cell    Macrophage    Granulocyte

D CHANGES IN DEVELOPING BLOOD CELLS

l Erythrocytes
l Large, weakly basophilic pro-erythroblast increases the free ribosomes in its cytoplasm to become a basophil erythroblast.
2 Cell size decreases, and organelles are lost.
3 Nucleus, initially large and pale, with nucleoli, gets smaller and stains more darkly.
4 Cytoplasm acquires haemoglobin at the expense of ribosomal ribonucleprotein (RNP) - thus its staining affinity changes from basophilia to acidophilia; the mixed-hued halfway stage is the polychromic/polychromatophil erythroblast.
5 Small cell, with orange cytoplasm and a round dark nucleus, is the orthochromic erythroblast/normoblast.
6 Nucleus, in a little cytoplasm, is extruded for phagocytosis.
7 Reticulocyte/polychromatophil erythrocyte is an RBC that is released into the blood still with RNP in its cytoplasm. Supravital staining with brilliant cresyl blue causes this material to clump as a blue network (reticulum) in around 2 per cent of the RBCs of normal blood.

2 Granulocyte
l Myeloblast/granuloblast develops into a
2 promyelocyte synthesises non-specific azurophil granules (lysosomes) in the cytoplasm, and with its nucleus getting smaller and darker.
3 Myelocyte, after a pause, then makes additional granules specific for one of the three kinds of granulocyte in their staining affinity.
4 Nucleus elongates and indents, and chromatin becomes coarser, giving the metamyelocyte (now unable to divide).
5 More granules form and the nucleus becomes sausage-shaped - band/juvenile granulocyte. Then the nucleus starts segmenting, as the cell becomes the mature granulocyte.

3 Platelets
l Haemocytoblast enlarges to become a megakaryoblast.
2 The nucleus experiences several rounds of DNA replication, but each time with reassembly of a single nuclear envelope and no segregation into separate nuclei. Thus the nucleus takes on a distinctive lumpy, polyploid form. (The single, large, lumpy nucleus is the criterion for distinguishing megakaryocytes from nearby osteoclasts in bone sections.)
3 Fine cytoplasmic azurophil granules accumulate as the cell becomes a very large granular megakaryocyte.
4 Many paired membranes of smooth ER (demarcation membranes) appear and contribute plasmalemma to the formation of
5 pseudopodia, which are extended into the lumen of a sinusoid, where they cast off in the blood as platelets.
6 Megakaryocyte cytoplasm might also serve as a transcellular migration pathway for some new leucocytes passing from the marrow into the blood.

4 Agranular leucocytes
l In developing, they do not become so strikingly different from their stem cells as do granulocytes and RBCs.
2 Monocytes form from monoblast/pre-monocyte precursors in bone marrow.
3 Lymphocytes develop from lymphoblasts in bone marrow and lymphoid organs.
4 Some circulating lymphocytes appropriately stimulated can also become lymphoblasts.

E BONE MARROW


l The naked-eye appearance of fresh, unstained marrow may be red from many developing RBCs, or yellow from mainly fat cells.

2 Red marrow has many elements, see Powerpoint:

3 Microscopic methods for marrow include sections, and smears of aspirated sternal marrow stained with a blood stain.

F CELL COMPARTMENTS, POPULATIONS AND KINETICS

l Although the marrow smear picture appears static, it represents part of a dynamic system of cell populations. For example an RBC may be: (a) in the marrow developing, (b) in the marrow being stored, or (c) circulating in the blood. The population of each compartment at any time represents the balance between the numbers entering and leaving.
2 Most cells of the RBC sequence/erythroid complex/erythron are circulating; whereas the granular leucocyte system has most of the cells developing or being stored in the marrow, and only a minority in circulation. Many of these leucocytes in the vessels hug the wall as a barely moving, marginated reserve.

3 Some factors affecting blood cell formation
l Bacterial infection increases the number of circulating granulocytes (a leucocytosis) and their rate of formation.
2 Erythropoietin is a humoral factor, released from the kidney in response to hypoxia, that increases RBC production. Thrombopoietin controls platelet formation, but has multiple sources, including liver and kidney.
3 RBC formation requires adequate dietary elements, e.g., folic acid, iron, vitamin B12.
4 Androgenic steroid hormones stimulate erythropoiesis.
5 Stromal cells release cytokines and, with the matrix, create a microenvironment favourable for haemopoiesis.

G . A LITTLE CLINICAL HAEMATOPOIESIS

The scheme below depicts the cell types and early events in haemopoiesis, detected mostly by cell-surface antigens. The usual histological chart telescopes these events and, instead, concentrates on later visible changes, which lie on the right side of this Figure, and are under the control of the last-acting specific factors such as EPO and IL-5. (Growth factors causing progress in a stage of a lineage are in italics or a symbol, e.g., *.)

 Fig. 9 Haematopoiesis

                   LP - - - - - - - - - - - - - - - -  T lymphocytes
                  /
               LPC
              /   \
            /      BP - - - - - - - - - - - - - - - -  B lymphocytes
          /         
PSC----CSC                  Meg-CSF            TPO
^  |    #\      CFU-Meg - - - - - - MegaK - - - - - - Platelets
|__|     @\       /
          *\   **/              **              EPO
            \   / - - - BFU-E - - - - - CFU-E - - - - - Red blood cells
             MPC    **
              ~ \  
               ~ \                             M-CSF
                ~ \                  CFU-M - - - - - - Monocytes
                 ~  \**             /
                  ~   \         ** /
                    ~   \         /   **          G-CSF
                      ~   \CFU-GM - - - - CFU-G - - - - - - Neutrophils       
                       ~          \  
                        ~          \
                     IL-3~       ** \
Key                        ~         \          IL-5
#  SCF                        ~       \ CFU-Eo - - - - - Eosinophils
@  IL-6                         ~
*  IL-1                            ~
** IL-3 &                             ~                IL-3
   GM-CSF                                ~ CFU-Mast - - - - - -Basophils
EPO Erythropoietin
TPO Thrombopoietin
PSC Pluripotent stem cell
CSC Committed stem cell
MPC Myeloid progenitor cell; LPC Lymphoid progenitor cell
CFU Colony-forming unit; BFU-E Burst-forming unit-erythroid
CFU-GM Colony-forming unit-Granulocytes & Monocytes;
CFU-M Colony-forming unit-Monocyte, etc.

The above diagram is modified from Kenneth Kaushansky's Fig. 1 in Proteins1992;12:1-9, with the kind permission of John Wiley & Sons, Inc, New York, holders of the 1992 copyright ©, and of the author

[ The ideas and terminology of the diagram are up-to-date. A full 1998 version would include only more growth factors and separate lineages for natural killer and dendritic cells.]

1 Tasks & solutions

  1. To keep the process going - stem cells
  2. To arrive at precise numbers of special cell types - proceed in steps that can be separately controlled, and give precursor cells some options of what they are going to turn into.
  3. To control the necessary events of cell division, choice, specialization /differentiation, and migration into the blood - cytokines and certain hormones - collectively called haemopoietic growth factors
  4. To make the cells responsive to needs, e.g., bacterial infection (more neutrophils), low O2 (more RBCs) - use feedback from defensive reactions or in hormonal loops

2 Ideas of haemopoiesis
.. The controlling microenvironment, with stromal cells;
.. self-renewal of a cell population;
.. cell differentiation;
.. restricted versus wide-ranging potential/competence for differentiation;
.. progression through stages of differentiation;
.. lineage or sequence of precursors, as the ancestry of a particular cell type;
.. early versus late events and controls;
.. cascades and combinations of factors (signals);
.. colonies of cells grown in culture, thought to mimic clusters/nests in marrow;
.. the clonal colony derived from one original cell.

3 Abbreviations
Lots of them. Welcome to medicine! Here the difficulties are that:

  1. The same letter means more than one thing, e.g., M - myeloid, monocyte; C - cell, committed, colony; P - pluripotent, progenitor/precursor.
  2. Where a factor causes selective differentiation, the convention is to put the abbreviation of the target cell type first, before the one specifying that it is a growth factor, e.g., M-CSF.
  3. Cell-type abbreviations are combined, where the factor acts on less specialized, i.e., multipotent progenitors, e.g., GM-CSF for a factor stimulating differentiation to a precursor of neutrophils (and eosinophils) and monocytes.
  4. 'Unit' (U) is a term for 'cell'.

4 Feasible clinical uses for growth factors
Erythropoietin - to combat anaemia from renal disease
Various CSFs:
.. to boost marrow performance after chemotherapy
.. to help injected stem cells or grafted marrow 'take' and perform
.. to restore PMN numbers in AIDS, certain anaemias, and neutropenia
.. as a differentiation therapy for leukaemia

5 Histological accounts
1 Focus on the very late events, discernible with conventional stains of smears, (nuclear and cell-size changes, acquiring granules, etc).
2 Can be wrong, e.g., too closely relating basophil formation to that of other granulocytes.
3 Leave vague the early events that clinicians need to know for untangling types of leukaemia, stimulating greater numbers of a deficient cell type, and finding stem cells to transplant, instead of marrow.
However, haematologists and pathologists use extensively the knowledge and terms, e.g., reticulocyte, band cell, etc, derived from the simple and available techniques of histology.


CHAPTER l9 DEFENCE AND IMMUNITY

A PROBLEMS AND DEFINITIONS

1 The problem is 'bugs in me'; specifically, in my connective tissues.

The                              ANSWERS
problem
            Early, loosely targetted*             Later, precisely 
     |b  Innate non-specific defences       targetted immune defences
B    |l
U<.a.|o.......MACROPHAGES
G  t |o                           <.a........CYTOLYTIC    cell-mediated
S<.t.|d.......NEUTROPHILS & EOs     t        T LYMPHOCYTES
   a |                              t
I .c.|v.......COMPLEMENT PROTEINS   a                  
N  k |e                           <.c........ANTIBODIES    humoral
  ...|s.......NATURAL KILLER CELLS  k        from plasma cells
M    |s                                      (originally B lymphocytes)
Y    |e
     |l
C    |_wall & other BARRIERS to the chemical & cellular attack on the   
T          bugs are overcome by inflammation and its events, 
           such as the release of MAST-CELL and other mediators.
     
OUTCOMES                    MEANS

Me intact: recognised as self and not to be attacked.
           Chemical protections: on cell surface; neutralisers for
           destructive enzymes; antioxidant mechanisms

Bugs gone: cells phagocytose & digest bugs; antibodies counter their
           toxicity & make them more eatable; superoxide & other O2-based
           radicals attack them; defensive cells release other antimicrobial
           chemicals, e.g, defensins, major basic protein, cytokines; 
           liver hepatocytes make acute-phase proteins to circulate for
           more chemical defence

Infected cells: pores created in cell membrane; apoptosis/suicide
    gone                                                 triggered
    
Bugs made unwelcome
  next time: circulating memory B & T lymphocytes with specific surface
              antibodies or T-cell receptors (TCR) for that kind of bug
Precise targetting is made possible by the prior proliferation of billions of B & T lymphocytes, accompanied by the generation of diversity in the antibody or TCR. The diversity is immense, covering all the possible molecular forms that might show up and injure one. Controlled mutation and rejoining of DNA (V & J regions) of the Ab or TCR genes produce the variety.

The initially crude targetting* of the innate or primitive system is refined and made more effective by the evolutionarily more recent lymphocyte-based immune system, which, in its turn, receives directions from the innate system.
Primitive animals had too few cells for the strategy of winning the anti-microbial lottery by buying all the tickets - making an Ab and TCR for every possible Ag.
Why keep two systems? The specific immune takes days to get going after a new antigenic encounter, because of the need to recruit cells and greatly amplify their number.
The above Fig. and text are in colour at Powerpoint.


2 A multicellular organism has to contend with three related problems:

3 The macrophage system (Chapter 5.A.4) can recognize and phagocytose decrepit and dead cells, and cell debris, and tries to cope with inert foreign matter. Material that cannot be digested can be held in cells, or surrounded by giant cells enclosed in a collagenous capsule.
In the lungs, the collagenous fibrosis impedes elasticity and is harmful.
The macrophage system, in dealing with foreign living intruders, tries routine phagocytosis, but it also calls upon several kinds of defensive cell working together to combat the intruder and its harmful products, toxins, and its various strategies, e.g., encystment, viral commandeering of host cells, mimicry of self materials, etc.
3 Against living things the defence has to be prompt, coordinated and successful, but also selective enough to cause little harm to the tissues of the host. The selectivity and coordination call for special cells to recognise the intruder for what it is - a foreign/non-self entity. The macrophages and other antigen-presenting cells recognize the foreign nature of such materials of living organisms as their surface proteins and carbohydrates. After phagocytosis, fragments of the foreign materials are presented as antigens, to which lymphocytes respond with specifically targetted immune responses - cell-mediated and humoral.

Fig. 10 Immune responses: cell-mediated & humoral

                                                               _
Antigen-presenting   presents Ag to the  T                      |
 cell  APC-Ag                        Lymphocyte                 | LYMPH
                                      /        .                | NODES
                           activation/           .              |
                         recruitment/         helper            | SPLEEN
                                   /                 .          |
                                  /             action.         | TONSILS
                                 T                     B        |
                             Lymphocyte            Lymphocyte   | GUT
                                 |                     |        |
                                 |                     |        |
                                 |                 Plasma cell  |
                                 |                     |       _|
                                 |                     |
                                 |                     |
- - -  - - - - - - - - - - - -   |    - - - - - - -    |    - - -      
B                                |                     |
L                                |                     |  CM
O                                |                     |   |Complement
O      |                         |                     |   |activation
D      |                         |                     |   |
 - - - | - - - - - - - - - - - - | - - - - - - - - - - | - | - .- -
       | mast cell activation    |                     |   |  . Mast-cell
T      |                   .     |                     |. .| . activation
I      | chemotaxis          .   |                     |   |
S      |. . . . . . . . . .    . |                     Ig CM
S      |                    .    T                      Ag 
U  Macrophage. . . . . . . . Lymphocyte           IMMUNE COMPLEX      
E     Ag      activation         .                           
S                               .    
                  direct lysis .                  Ig also boosts NEUTROPHIL
                       Ag.  .                     migration & phagocytosis
                                                  of immune complexes

   |________CELL-MEDIATED RESPONSE_________|    |______HUMORAL RESPONSE____________

4 Immunologically competent cells - T & B lymphocytes and plasma cells - show an exquisite specificity to an individual kind of alien body, e.g., polio virus rather than smallpox, in binding themselves (by the T-cell receptor), or in their humoral product - immunoglobulins/ antibodies.
The accessory cells of the immune system do not have this specificity, but their activities are guided and enhanced by lymphocytes and antibodies, and they in turn contribute, by presenting antigen, under histocompatibility restriction, to the specificity of the lymphocytes' responses. An important aspect of this restriction is that the immune system does not attack one's own cells and materials.

5 Sources of antigen, actual or potential, are:
.. (a) viruses and microorganisms;
.. (b) venoms;
.. (c) inspired particles, e.g., fungi, pollen, dander;
.. (d) foods;
.. (e) semen;
.. (f) the embryo;
.. (g) transplanted tissues, e.g., skin;
.. (h) altered autologous (own) cells, e.g., tumour products
.. (i) some medicaments, e.g., penicillin.

B DEFENSIVE CELLS AND MECHANISMS

The details belong in other disciplines, but the main actions of the competent and accessory cells are listed below and in Fig. 10 to show the defensive cells, met individually in Chapters 5 and l7, and the lymphoid organs of the next chapter, as participants in a highly integrated system.

1 Plasma cells (immunologically competent)
l Develop from B lymphocytes (see 2.l below for B and T lymphocyte definitions) via a transitional cell involved in rearranging its immunoglobulin genes for expression, first for the cell-surface, then for secretion.
2 Synthesize and release specific humoral antibodies (immunoglobulins), after engagement with the presented antigens, and stimulations from helper T lymphocytes.
3 Immunoglobulins:
.. (a) bind and inactive the antigenic bodies;
.. (b) neutralize toxins;
.. (c) enhance phagocytosis;
.. (d) trigger the activation of special blood proteins - complement factors - which amplify the immune response.
4 Complement also binds to the antigen, potentiating the action of the bound antibody, and itself has lytic, signalling, and other effects. The three-part entity - antigen, antibody and complement - is an immune complex.

2 Lymphocytes (competent)
l Start as stem cells of fetal haemopoietic tissue, but fall into two classes differing in where they were conditioned for distinct tasks.

2 Both B and T lymphocytes seed out to populate the secondary lymphoid organs: spleen, nodes, and major mucosal lymphoid structures, and some lymphocytes then circulate. (Thymus, bone marrow, and fetal liver are primary lymphoid organs.)

3 Roles of the T lymphocyte

4 Natural killer/NK cells are marrow-derived lymphocytes that act early and independently of antigen presentation to attack tumour cells and infected cells, using membrane-damaging perforin, Fas ligand, and other agents.

5 Lymphocytes are classified by the reaction of certain of their surface glycoproteins to monoclonal antibodies. Thus, inducer/helpers are CD4+; cytolytic lymphocytes are CD8+; natural killer cells are CD3-, CD16+, CD56+; B lymphocytes are CD19+, etc.
CD means Cluster-of-Differentiation antigens, and stems from the patterns of response of differentiating leucocytes to a great variety of monoclonal antibodies. It turns out that many kinds of cell aside from leucocytes express one or more of the antigens that the CD antibodies mark. These antigens only incidentally help characterize cells (e.g., marrow stem cells are CD34+), since they are working molecules - in adhesion and signalling, as enzymes, protective agents, etc.

6 Some T and B cells, having participated in an immune response to a certain antigen, patrol the body as long-lived memory cells ready to initiate an early and stronger secondary response, should the same antigen intrude again - the basis of vaccination.

7 The distinction between self- and non-self-recognition, and the acquisition of memory by lymphocytes, may be confounded by presentation of the antigen in high doses, by unusual routes, or in immaturity just after birth. The confused lymphocytes that result remember to tolerate an antigen, to which they should react. This tolerance is believed to be a byproduct of a normal mechanism, whereby all normal cells are telling circulating T lymphocytes, with receptors for the normal cells' materials, not to react, but to die.

3 Dendritic antigen-presenting cells (APCs) and Macrophages (accessory)
1 APCs and macrophages/MØs concentrate some antigenic fragments on their surface, presenting them in a form more potent for stimulating lymphocytes.
2 What is presented on the surface is a small peptide, derived by degradation from the antigen, bound to a histocompatibility protein (MHC class I or II depending on whether the antigen is of intracellular (self) or foreign/exogenous origin). Intracellular antigens presented in this way include materials that viruses have forced the cell to make.
A non-sequitur: antigen-presentation is not limited to MØs and antigen-presenting cells. For example, B lymphocytes present antigen to T lymphocytes.
3 Once activated by a particular antigen, lymphocytes and macrophages exchange cytokine messages to:
.. (a) recruit more macrophages from the circulating monocytes;
.. (b) inhibit macrophage migration to keep macrophages at hand;
.. (c) activate macrophages to attack more vigorously the antigen by which the lymphocyte is activated, e.g., tuberculosis bacilli.
(These cytokines convey simple 'doggy' orders: Come! Stay! Attack!)
4 Macrophages phagocytose toxins and cells killed by other immune actions, and make cytokine factors, e.g., chemotactic for neutrophils.
5 Macrophages and other phagocytes liberate destructive enzymes and oxygen metabolites to lyse cells. They also digest matrix, e.g., by MØ elastase, so that they themselves may move more freely. Enzymes may also be regurgitated in phagocytosis, or be spilled after death of the cell.
To reduce the damage to surrounding tissues, extra-cellular degradative enzymes normally are neutralized by protease inhibitors in the plasma and tissues, such as alpha l-antitrypsin.
6 'Tingible-body' macrophages are in germinal centres. Their darkly stained (tingible) inclusion material is nuclear debris of apoptotic B lymphocytes that were selected against for not improving their affinity for antigen fast enough.

4 Granular leucocytes
l Neutrophils respond in strength to certain bacterial and fungal infections, avidly ingesting, say, streptococci, dying, and often accumulating to become pus.
2 Neutrophils and eosinophils are attracted to immune complexes which they phagocytose, but the materials that they use to attack microbes and parasites also damage tissues, e.g., airway epithelium in allergies.

5 Mast cells
l One kind of immunoglobulin (Ig) is already bound to their surface. Antigen entering the tissue bridges these IgE molecules, triggering the release of
2 histamine, which dilates vessels, increases their permeability and facilitates the exit of granular leucocytes, monocytes, antibodies, etc.
3 Heparin may hold histamine and other factors ready for discharge; if released itself, it might, as a polyanion, bind and neutralize toxins. Among the many other mediators are bradykinin and factors attracting granulocytes - chemokines.
4 The mast cell's reaction is an immediate hypersensitive one: the basis of allergies. An anaphylactic hypersensitive response in the airway lining is life-threatening, by overconstricting smooth muscle, and other effects.

C TRANSPLANTATION

Of the many intriguing manifestations of immunity, such as anaphylaxis, autoimmunity, graft rejection, graft-versus-host reaction, and immunodeficiency syndromes, only autoimmunity and transplantation will be considered further.

Transplantation has wide use in the experimental approaches of Chapter 30.
l Most tissues can be grafted autologously to another site in the same individual, where they will live, if they can soon gain a new blood supply by revascularization by, or anastomosis with, the vessels of the host bed.
2 Transplants between two individuals will take - not be rejected - if they are isogeneic/syngeneic, and thus have identical tissue proteins synthesized according to the same DNA, e.g., in identical twins, or animals of the same sex whose forebears have been many times inbred.
3 Transplants between genetically different individuals can be:
.. (a) allogeneic/homologous between members of the same species;
.. (b) xenogeneic/heterologous between members of different species or orders.
The grafted tissue is antigenic and evokes the delayed T cell-mediated immune response.
4 An allogeneic graft made to a neonatal host can induce a permanent tolerance for that graft and subsequent grafts of the same tissue. The host, now composed of tissues differing genetically, has been made a chimaera.
5 Certain sites for allogeneic grafts slow down or prevent the antigen from draining to lymphoid tissue and eliciting an immune response. Such immunologically privileged sites are the cornea and brain.
6 Immunity depends on the proliferation and synthesis by cells. To help a graft to take, the response could be inhibited for a while by provoking apoptosis in the competent cells, or hindering their proliferation, with irradiation with X-rays, or with cytotoxic drugs or glucocorticoids. Transplant surgeons can also use agents, e.g., cyclosporin, to block the activation of T cells.

Autoimmunity
Sometimes the mechanisms of restraint against attacking one's own materials go awry. Clinically significant autoimmune targets include: gastric parietal cells, renal mesangial cells, pancreatic beta cells, thyroid follicular cells, skeletal muscle, myelin components, and basement membranes.


Chapter 20 LYMPHOID ORGANS

A INTRODUCTION

l Lymphoid cells mediating the immune response - lymphocytes, plasma cells, dendritic cells (APCs) and macrophages - occur: 2 Lymphocyte and APC traffic Lymphocytes circulate between tissue sites and blood and lymph flows; and APCs, such as the Langherhans cell, travel to nodes as the lymph-borne dendritic antigen-presenting cell - the veiled cell. Lymphocytes travel locally in a lamina propria of a mucosa.

3 The lymphoid cells are densely packed in rounded nodules/follicles in parts of the spleen and nodes. Aggregates of nodules occur in the tonsils, appendix and ileal Peyer's patches of the GI tract; whereas solitary nodules may exist anywhere in the mucosae of all 'open' tracts.
Wherever nodules may be found, close by are lymphoid cells dispersed more diffusely.

4 Most nodules have paler central regions - germinal centres, but these are not essential for cell proliferation. Germinal centres recruit virgin B cells, and follicular dendritic cells then present them with antigen. The B cells progressively refine their response to the antigen, in terms of Ig class, affinity, cell numbers, and whether to be plasma cells or memory cells.

5 The primary lymphoid organs - thymus and fetal bone marrow - store, release and confer competence on the lymphocytes that populate the secondary organs and CTs, but do not participate directly in defence. Primary-secondary Powerpoint.

6 Lymphocytes migrate in the blood and lymphatic flows for:
.. (a) the initial colonization of spleen, etc;
.. (b) a constant vigilant patrol by recirculation around the body, as memory or naïve cells;
.. (c) the propagation of an active immune response, as activated cells.

7 The secondary lymphoid organs provide:

B MUCOSO-LYMPHOID ORGANS

1 Aggregates of nodules occur in the tonsils, appendix and ileal Peyer's patches of the GI tract; whereas solitary nodules may exist anywhere in the mucosae of all tubular systems open to the outside.
2 Wherever nodules may be found, close by are lymphoid cells dispersed more diffusely.
3 The gut- and bronchus-associated diffuse lymphoid tissues (GALT, BALT) are notable. MALT (mucosa-associated lymphoid tissue) usually refers to the unorganized lymphoid tissue of the gut.
4 Having an epithelium between the microörganisms and the connective tissue, where most of the lymphoid cells reside, poses problems:

C LYMPH NODES

l Situation
Nodes are small bodies placed at intervals along the lymphatic vessels, and structured so that the lymph has to pass through them. Afferent lymphatics bring lymph from a drainage area. The node is responsible for combating intruders and confining infection to that area, by sending out antibodies and cells via efferent lymphatics.

2 Lymph-node structure
l A CT capsule, with some smooth muscle cells, sends in thin CT trabeculae, supporting a network of reticular fibres, and reticular cells of fibroblastic and the accessory dendritic kinds.
2 A denser outer cortex and a looser, inner medulla are present.
3 Efferent lymphatics leave at a hilus: the point of entry for blood vessels, serving a mostly cortical microvasculature.
4 Afferent lymphatics open through the capsule at several places to feed a system of 'sinus' channels running so:
subcapsular/marginal sinus --> cortical/intermediate sinuses --> medullary sinuses --> efferent lymphatics.
Sinuses are lined by reticular cells, accompanied by macrophages.
5 Denser masses of lymphoid tissue, extensive and follicular/nodular in the cortex, and continuing into the medulla as widely spaced medullary cords, have packed cells: lymphocytes, lymphoblasts and antigen-trapping dendritic reticular cells with processes. Lymphoblasts/centroblasts occur in the paler germinal centres of the cortical follicles.
The follicular zone contains B lymphocytes separated by follicular dendritic cells (FDCs).
6 The deeper lying paracortical region has mostly T lymphocytes, and dendritic APCs wrapping so intimately around lymphocytes that they received the name interdigitating reticular cells (IPCs).

3 Lymph-node functions

  1. Mechanical filtration of lymph in the sinuses, trapping, for instance, soot carbon particles. Tumour cells are not so easily stopped.
  2. Phagocytosis of materials in lymph by macrophages along the sinuses or lodged across them. The materials taken up include antigens, e.g., on bacteria. APCs and MØs process antigens for lymphocyte activation.
  3. Proliferation of sensitized lymphocytes to become lymphoblasts, large, with little GER, but many ribosomes, stainable with pyronin. These lymphoblasts are the source of:
    .. (a) plasma cells, and hence humoral antibodies, or
    .. (b) cytolytic lymphocytes, which set out for their distant target - antigen in the drainage area - in the sinus lymph.
  4. Recirculation of mature lymphocytes from venule blood to sinus lymph by migration through the cuboidal endothelium of the venules (high-endothelial venules - HEVs).

D SPLEEN

l Situation
Lies in the upper left of the abdomen, but there may also be small accessory spleens. It receives blood from the splenic artery for a treatment similar to that given the lymph by the node.

2 Splenic structure
l Thick fibro-elastic CT capsule has some myofibroblasts and a covering mesothelium.
2 Internally, thick CT trabeculae bear branches of the splenic artery and veins, entering and leaving at the hilum.
3 To the naked eye, most of the freshly cut organ is red pulp with white spots - white pulp.
4 Red pulp consists of a loose reticular tissue infiltrated with blood cells, and arranged in the so-called cords of Billroth around sinusoidal channels/sinuses - a Swiss-cheese situation of red-pulp cheese and sinusoidal holes.
The outermost white pulp, abutting the red pulp, is a boundary zone - the marginal zone, not to be confused with the mantle zone of densely packed mature lymphocytes around germinal centres.
(A mantle zone is not usually symmetrical; it is concentrated to one side of its germinal centre.)
5 Cord tissue has dendritic and fibroblastic reticular cells, and collagen fibrils supporting macrophages, and white and red blood cells.
6 Sinusoids/sinuses are lined by non-phagocytic endothelial/littoral cells, separated by slits and oriented longitudinally on a fenestrated BL. Blood cells thus can pass from sinusoid to cord and back, and cordal macrophages can extend pseudopodia into the sinusoidal lumen.
7 White pulp is a dense lymphoid tissue ensheathing branches of the arteries, once these have left the trabeculae. The sheath (PALS) dilates into follicles/nodules, some with germinal centres.
8 Lymphocytes are predominantly B in the nodules, and T in the periarterial lymphoid sheath (PALS). To match, reticular antigen-presenting cells are follicular/dendritic in the B-zone, interdigitating (IDCs) in the T-zone.
However, PALS and nodules/follicles work together, in that, the outer PALS is where B lymphocytes are initially selected for population-expansion in the nodules.

3 Splenic blood flow

  1. Fed by the splenic artery, a trabecular artery branches out away from the CT as a
  2. central artery (arteriole) of the white-pulp lymphoid sheath, which it supplies by small branches. The artery is not central in the nodules.
  3. The arteriolar branches of the central artery turn towards the red pulp, as several very straight branches - penicilli/pulp arterioles.
  4. The vessels become smaller, and some have discontinuities in the BL, and gain a sheath of macrophages - sheathed capillaries - before
  5. the terminal capillaries open into a cord (Open Circulation Theory) or a sinusoid (Closed/Fast Circulation). Probably both kinds of termination exist.
  6. Sinusoids and cords both contain blood.
  7. Pulp venules collect the blood and carry it to
  8. trabecular veins for return to the hilum,
  9. and exit via the splenic vein.
Note that the spleen displays substantial species differences: the dog spleen has very muscular trabeculae; rodent spleens have a significant marginal sinus, along the white-red border; MØ-sheathed capillaries are not prominent in man, and lie in a perifollicular zone of red pulp, special to man.

4 Splenic functions
l Until birth, the spleen takes part in myelopoiesis, as do lymph nodes.
2 White pulp serves for:
.. (a) recirculation of lymphocytes;
.. (b) formation of new lymphocytes and plasma cells for immune responses to blood-borne antigens, met first at the marginal zone.
3 Red pulp provides:
.. (a) blood cleansing by the sequestration and phagocytic destruction by macrophages of unfit blood cells and platelets, and bacteria;
.. (b) metabolic breakdown of RBCs so that their iron can be reused;
.. (c) a place to accumulate platelets;
.. (d) sites by the marginal zone for plasma cells after antigenic stimulation, analogous to the cords and medulla of the active lymph node.

E THYMUS

l Situation and basic structure
l Lies in the upper midline of the thorax.
2 Markedly lobulated, with thin partitioning septa of fibrous CT, and adipose tissue which increases greatly with age.
3 In each lobule, a cortex surrounds a more palely staining medulla.
4 However, the medullary tissue is continuous from lobule to lobule as an axial cord.

2 Thymic finer structure
l Cells are:

2 Absent are afferent lymphatics, germinal centres, and significant numbers of reticular fibres.
3 Epithelio-reticular cells form concentrically lamellated, rounded, keratinizing, eosinophilic bodies - thymic/Hassall's corpuscles - in the older medulla.
4 Blood capillaries have intact basal laminae, few fenestrations in the endothelium, and an outside sheath of epithelio-reticular cells: all comprising the basis for a barrier hindering cells, e.g., B cells, and perhaps blood-borne antigens, from reaching the thymic cortical lymphocytes.

3 Thymic function
l Neonatal removal of the thymus causes the secondary lymphoid organs - nodes, spleen, tonsils, etc - to develop only partially and be unable to respond to many antigens.
2 Before birth, the thymus - a primary lymphoid organ - receives stem cells from the marrow that proliferate and undergo selection and maturation (by interacting with epithelial-reticular cells and APC reticular cells), before seeding out via the blood to populate the secondary organs with T or thymus-dependent immunologically competent lymphocytes.
Self-reactive lymphocytes are selected against, die, and are phagocytosed, while the surviving T lymphocytes migrate from subcapsular cortex towards the medulla.
3 At puberty the thymus starts a slow involution and replacement by adipose tissue, accelerated by severe stresses.
4 Despite the involution, the adult thymus maintains a low level of T-cell development from immature precursors that have not yet rearranged their TCR genes.
5 The thymus was assigned the status of an endocrine organ because the epithelio-reticular cells produce thymosin. It turns out that members of this family are not made solely by the thymus; and they act intracellularly as actin-binders and locally as cytokines.

4 More details of T-lymphocyte development based on the mouse
1 T progenitor cells arrive in the subcapsular region, where they multiply and express each its own pre-T cell receptor type.
2 This expression is used to select thymocytes to become cells that are double positive (CD4+8+) and expressing low levels of TCR-alpha/beta.
3 From encounters with peptide-MHC on the membranes of cortical thymic epithelial cells, some double-positive thymocytes - those with the appropriate TCR - are positively selected to become active and to downregulate either CD4 or CD8 expression to become mature CD8+ or CD4+ thymocytes.
4 Meanwhile, in both inner cortex and medulla, dendritic antigen-presenting cells negatively select, by the MHC-complexed presentation of self peptides, those thymocytes with TCRs for self antigens. Autoreactive thymocytes undergo apoptosis and removal by macrophages.
5 Thus, the thymocytes leaving the medulla as T lymphocytes have experienced positive selection and survived negative selection, and now await any further peripheral instruction on tolerance - how not to react with one's own tissues.
6 The story is similar, but more complicated for lymphocytes with gamma/delta TCRs.


Chapter 21 SKIN

Skin/integument covers the body and serves many functions. It consists of a thick, protective, cornified, stratified squamous epithelium (epidermis), on a firm, dense CT lamina propria (dermis), and has special appendages, hair and nails, and accessory glands, sweat, sebaceous, and mammary glands (Chapter 29.D). Powerpoint

A EPIDERMIS (epithelium)

l Layers
l Stratum corneum of keratinized cells (outermost).
2 Stratum lucidum, a thin pale layer of keratin seen when the stratum corneum is very thick.
3 Stratum granulosum of cells with basophilic granules.
4 Stratum spinosum of keratinocytes/prickle epithelial cells.
5 Stratum germinativum, bordering on the BL.

2 Cytological details of the layers
l Stratum germinativum/basale

2 Stratum spinosum
3 Strata granulosum and corneum.
(a) Stratum granulosum cells form a kerato-hyaline matrix from their basophil granules, binding together packed tonofilaments within the cells to convert the cells to soft keratin. Other organelles and the nucleus vanish, while the plasmalemma thickens and toughens, to build a cornified envelope.
(b) Flattened, dead, keratinized, surface cells desquamate.
(c) Only with EM is keratin seen to be cellular. In the usual HE preparation it is eosinophilic, and often splits and breaks.
(d) Epidermis is thrown up into ridges - cristae cutis - on the palmar and plantar surfaces of the hands and feet: the basis of finger and palm prints.
(e) At the top of the ridges, spiralling holes open through the keratin to let out the sweat.
(f) Keratin layer may be very thick, for instance on the soles and palms. Such thick skin is hairless, and lacks sebaceous glands.
(g) The molecular epidermis:- Filaggrin is the protein of keratohyaline granules, and aggregates 'keratinocyte' keratins Nos. 5/14. These acidic-basic combinations of keratins (numbered indirectly according to Mr) are characteristic for particular classes of epithelia, e.g., simple versus squamous, and help in interpreting pathological changes. Directly under the plasmalemma is a complex of proteins that are made dense and insoluble - to constitute the "envelope" - by transglutaminase-mediated cross-linking. One protein of the cornified cell envelope is involucrin. Ceramide and other extracellular lipids surround the envelope to boost the barrier function.

B DERMIS (Corium)

l Divided into layers: papillary, fine-textured CT adjacent to the epidermis, and a deeper reticular layer.
2 Reticular layer is thick collagenous CT of a variable thickness, not always related to that of the overlying epidermis.
3 Elastic fibres of the dermis give skin its elasticity, but cause wounds to gape. Ruptured dermis often heals as a white line visible through the epidermis, e.g., a mother's stretch marks.
4 Has the usual cells of CT - fibroblasts, macrophages and other defensive cells, and sometimes pigment-bearing chromatophores/dermal melanocytes.
5 Smooth muscle of arrectores pilorum, nipples and scrotal dartos, and skeletal muscle in the scalp and face, are attached in the dermis.
6 Blood vessels are derived from arterial plexuses: a deep cutaneous plexus/rete, and a subpapillary plexus sending capillary loops up into dermal papillae. Lymphatics accompany blood vessels. Blood flow is varied greatly by shunts through glomi (coiled arteriovenous anastomoses), and by the constriction or relaxation of arterioles.
7 Nervous receptors (Chapter 12.B), with sensory nerve fibres are present; and autonomic nerve fibres:
.. vasomotor to vascular smooth muscle,
.. pilomotor to hair arrector muscles,
.. sudomotor to sweat glands.
8 Hair follicles and glands lie mostly in the dermis.

C SWEAT GLANDS (Glandulae sudoriparae)

l Single coiled tubules, lined by simple cuboidal light and dark cells; distributed over the body except for the lips, glans penis and inner prepuce.
2 Secretory part lies in the lower dermis, or subcutaneously in the hypodermis/superficial fascia. One tubule is cut through many times in one section.
3 The secretion, mainly water and electrolytes plus some lipids, is led to the epidermis through a duct, lined by stratified cuboidal epithelium, then through the living/Malpighian layer and a spiralling hole in the keratin. The gland's chloride channel is one that is impaired in cystic fibrosis.
4 Myoepithelial cells are seen within the basal lamina of the secretory tubule. Their contraction is under autonomic control.
5 The larger variety of gland seen in the axillary, perianal and perigenitalial regions is termed apocrine, in contrast to the eccrine glands in the majority. Apocrine glands become active with pubertal development of the ambosexual hair, and may be related to animals' scent glands.
6 The ceruminous glands of the external auditory meatus seem to be enlarged sweat glands, producing a secretion of pigmented lipids.

D SEBACEOUS GLANDS

l Pear-shaped, simple, branched alveolar, with large cells, usually looking vacuolated because their fatty content is dissolved out.
2 Several glands are clustered by the side of a hair follicle, into which they discharge the secretion - sebum. Their short duct is lined by stratified squamous epithelium.
3 Sebum, formed in a holocrine manner by the total breakdown of the cells, may lubricate the hair shaft, protect the skin from drying and moisture, and be bacteriostatic.
4 Lie independently of hairs on the labia minora, glans penis, in the oral mucosa by the red margin of the lips, and as the Meibomian glands of the eyelid. They are absent from the palms and soles.

E HAIR

l Varieties and sites
1 Lanugo - fine, fetal, hairy covering, shed at birth.
2 Replaced by the vellus - fine body hairs.
3 Scalp, eyebrow and eyelash hairs are thicker.
4 Ambosexual hair - pubic and axillary.
5 Masculine hair - face (beard), chest and extremities.

2 Hair development
l Hair is a hard keratin derivative of the epithelium of a hair follicle.
2 In development, an epithelial bud grows down from the young epidermis; a vascular CT dermal papilla invaginates the bud; in the bud a germinal matrix develops, forming the special keratin; and side buds form sebaceous glands.

3 Hair shaft comprises:

4 Hair follicle

5 Epithelial replacement and hair growth are cyclical, not constant activities. The hair stops growing, via a relatively short catagen period of regression or involution, to enter a long non-growing telogen phase of being a club hair, which eventually falls out. It is replaced during an anagen/growth phase by a new hair from the reactivated deep region of the follicle.

3 Pilomotor activity
Hairs are raised from their relaxed, inclined attitude by contraction of their arrectores pilorum muscles in response to cold, so that more insulating air is trapped near to the skin. Hairs also 'stand up' in fear and other emotional reactions.

F NAIL

l The horny plate of hard beta keratin is synthesized by
2 the proximal, germinal, part of the nail bed.
3 The nail bed comprises the living layers of the epidermis, ridged longitudinally, and lacking glands and follicles. Part of its germinal region is seen by the naked eye as the
4 lunule, the pale half-moon area just distal to the eponychium - an extension of the stratum corneum of the dorsal skin.

G SKIN FUNCTIONS

l Protection against water, bacteria, sunlight, mechanical forces, dehydration, cold, etc.
2 Retaining body fluids, i.e., protection against dehydration.
3 Temperature regulation by: (a) varying peripheral blood flow, (b) sweating, (c) hair elevation, and (d) insulation by adipose tissue under the skin. (Note that heavy sweating defeats 2 above.)
4 Food storage and fat metabolism in the subcutaneous hypodermis.
5 Vitamin D formation by the action of ultraviolet light.
6 Sensory appreciation of the environment by nervous receptors: Chapter l2.B.L.
7 Friction surface for motor tasks involving grasping, rubbing, scratching, etc.
8 Display and communication: social, sexual, and diagnostic. Many diseases distinctively affect the skin and its hair and nails.

Chapter 22 RESPIRATORY TRACT

The tract rhythmically expels spent air and takes in fresh through conditioning passages, conducting it to the respiratory portion of the lungs, where the walls of the air-filled chambers are thin enough to permit an exchange of gases between blood and air. The respiratory movements involve chemoreceptors, brain centres, the thoracic cage, and various muscles: these structures belong, together with the respiratory tract, in the respiratory system Powerpoint. The lungs also have important metabolic functions not directly related to gas exchange, e.g., the activation of circulating angiotensin I, and the inactivation of some other vasoactive agents.

A RESPIRATORY TRACT TO LUNGS

l Nasal cavity
l Divided by a hyaline-cartilage nasal septum in the midline.
2 Stratified squamous epithelium (hairy) of the nares changes to
3 a lining nasal mucosa of:
.. (a) pseudostratified, columnar, ciliated epithelium with mucus-secreting goblet cells, on
.. (b) a loose lamina propria, with many leucocytes, blood vessels, and mixed muco-serous glands.
4 Venous plexuses, to warm the air, underlie the epithelium.
5 Turbinate bones in the conchae support the mucosa.
6 A small part of the mucosa is olfactory, with a neuroepithelium (Chapter l2.B.5.la) and Bowman's glands.
7 Paranasal air sinuses open off the main cavity.
8 The folded pharyngeal tonsil, covered by pseudostratified, columnar, ciliated epithelium, lies posteriorly in the pharynx.

9 Nasal functions:

2 Larynx
l Hollow chamber, whose walls are supported by cartilages, connected by ligaments and membranes, and moved by skeletal muscles.
2 The extrinsic and intrinsic muscles move the larynx up and under the epiglottis in swallowing, and move the cartilages and tense the vocal cords during phonation and breathing.
3 The cartilages are hyaline tending to calcification, or elastic for the epiglottis, cuneiforms, corniculates, and the apices and vocal processes of the arytenoids.
4 Mucosa is mostly pseudostratified, columnar, ciliated epithelium with goblet cells, on a loose lamina propria rich in elastic fibres, mucous and mixed glands, leucocytes and sometimes lymphoid nodules.
5 Two constrictions occur: the false vocal cords/ventricular folds; and the lower, true, cords. The true vocal chords are elastic ligaments tensed by the adjacent vocalis muscle, and are covered with stratified squamous epithelium. There are no glands in their lamina propria.
6 The epiglottis, too, has stratified squamous epithelium on its exposed tip and upper surface.

3 Trachea
l Flexible, extensible tube, with an always-patent lumen.
2 Mucosa as for the larynx, and the cilia sweep towards the pharynx, but the elastic fibres run longitudinally as a layer between mucosa and submucosa.
3 Supporting C-shaped pieces of hyaline cartilage are incomplete on their oesophageal side.
4 The gap in the C is crossed by trachealis smooth muscle and CT.
5 Outer adventitia is fibro-elastic CT.

B LUNGS

The structure of the lungs reflects the way in which the air is moved: l Bronchial tree serving the lungs
l Primary bronchi branch to form the
2 intrapulmonary lobar bronchi, branching to form segmental bronchi, then lobular bronchioles. After about 9-l2 generations of branching, bronchioles replace bronchi.
3 Terminal bronchioles lead to respiratory bronchioles, off which open the respiratory exchange units, and not just at the end, but along the bronchiole. [For efficiency, the branching, tubular architecture of air conductance overlaps slightly the honeycomb architecture of gas exchange.]
4 Bronchi resemble the trachea in structure, except that the cartilage pieces in the wall have very irregular shapes, and the smooth muscle forms a nearly complete layer - muscularis mucosae - between the cartilages and the lumen.
5 Bronchioles are smaller than bronchi:
.. they have no cartilages;
.. their elastic fibres merge with those of the surrounding lung tissue;
.. the epithelium changes to simple, low ciliated columnar with a few goblet cells;
.. no mucous glands are present in the lamina propria, where the smooth muscle is relatively substantial.
6 Sharing the connective tissue of the branching bronchi are blood vessels, nerves and lymphatic vessels, entering or leaving at the hilum or lung root.
7 Hilar structures include arteries (bronchial and pulmonary), veins, lymphatics (from two systems), bronchi, lymph nodes, ganglia, nerves (to bronchial, bronchiolar, and vascular smooth muscles; and sensory), and adipose and other CT.
The carotid body-like glomus pulmonale in the pulmonary artery's adventitia is of uncertain function.

2 Mucosa of the lower airway

  1. Cell types in the epithelium:
  2. A sheet of sticky mucus is moved by ciliary action over the mucosa to catch and remove particles - the mucociliary escalator.
  3. The basal lamina typically is thick.
  4. Muco-serous mixed glands, where present in the lamina propria, are small, compound tubular, and respond under nervous control to irritant stimuli, e.g., smoke.
3 Respiratory chambers
l Respiratory bronchiole has simple, low columnar or cuboidal bronchiolar and ciliated cells; elastic fibres and smooth muscle support the epithelium's BL.
2 Opening out along the respiratory bronchiole are alveoli, whose openings are ringed by smooth muscle.
3 At the end of the respiratory bronchiole are one or more long alveolar ducts.
4 Alveolar ducts can be viewed as being three to six atria, vestibules, leading to alveolar sacs, made up of varying numbers of alveoli.
Processing distortions in lung slides often make the atria and sacs hard to make out.
5 One alveolus or cubicle shares an alveolar wall with the ones adjacent and backing on to it. The wall is thus interalveolar and carries the many capillaries, whose blood is to receive oxygen and give up carbon dioxide.
6 Angiotensin converting enzyme in pulmonary capillaries cleaves angiotensin I to make it the potent angiotensin II.

4 Interalveolar wall
l Air side - continuous alveolar epithelium with:
.. (a) type I pneumocytes/squamous cells; and
.. (b) pneumocytes type II/septal or great alveolar cells, with prominent lipid cytosomes/ multilamellar bodies in their cytoplasm.
2 Surfactant is a stabilizing fluid film of lipids (90%) and proteins (10%), covering the epithelium and lowering surface tension. The principal surface-active agent is the lipid, dipalmitoyl phosphatidylcholine (DPPC). The type II cells synthesize this film, but also are the stem cell to replace themselves and Type I cells. Cytosomes are stored surfactant.
3 Alveolar macrophages/dust cells lie free in the alveoli.
4 Alveolar epithelium lies on a basal lamina sometimes merging with, and sometimes separated from, the
5 basal lamina of a blood capillary, on which lies an
6 unfenestrated endothelium on the blood side.
7 Where the two basal laminae are separated, the space - zona diffusa - is taken by elastic and reticular fibres, fibroblasts, macrophages and other CT cells.
8 The pulmonary blood-air barrier can therefore be as thin as 300 nm, and has a very extensive area.
9 Communication between adjacent alveolar sacs is through holes in the wall - alveolar pores.
l0 Basal laminae, fibres, and surfactant maintain the shape and patency of alveoli during respiration.

5 Pleurae are fibro-elastic vascular membranes with mesothelial coverings. From the visceral pleura, CT septa run in to subdivide the lung into lobules and carry lymphatic and venous vessels.

C RESPIRATORY TRACT

l Development
l From an endodermal bulge on the foregut, which gives the trachea, then two buds for the bronchi and lungs.
2 Continued budding and branching, and enclosure of the hollow buds by mesenchyme, produce a system of cuboidal epithelium-lined tubules with surrounding differentiating CT and vessels.
3 Early development thus is analogous to that of a compound exocrine gland, until the later phase, when the pulmonary alveoli form. Inadequately developed alveoli, with no surfactant, are a major hazard of premature birth.
4 Surfactant comprises mainly lipids, with surfactant-associated glycoproteins SP-A, -B, -C, & -D, which variously cause the lamellar material to become a monolayer, enhance the lowering of surface tension, stabilise the lipids and counteract their oxidation, and modify host defences.
5 For the development of glands and the lung, complex mesenchymal-epithelial inductive (instructional) interactions occur, and recur during repair and tumour development.

2 Respiratory protective mechanisms

  1. Secretion of entrapping mucus by goblet cells and mixed glands,
  2. which is swept pharynx-wards by the ciliary beating action.
  3. Solitary lymphoid nodules and tonsils, and their lymphocyte progeny, for immune defence.
  4. Phagocytic alveolar macrophages/dust cells.
  5. Reflex coughing, sneezing, and constriction of bronchioles.
  6. Secretion of serous bacteriolytic materials, e.g., defensins and lysozyme.
  7. Upper airway recovers water and heat, preventing too much loss in the expired air.

Some protection is hazardous in that enzymes from WBCs can break down elastin; and activated lung macrophages stimulate fibroblasts to lay down movement-restricting collagen - an interstitial fibrosis.

Various defects in the arms and microtubules of cilia (primary ciliary dyskinesia) can prevent proper clearance and cause recurrent lung infection. Affected men are often infertile from an accompanying paralysis of sperm.


Chapter 23 URINARY SYSTEM

The kidneys eliminate waste metabolic products using water, but conserve water, electrolytes and other materials to maintain the body homeostatically in fluid, pH and electrolyte balance. The urine produced is evacuated periodically via urinary passages.

A KIDNEY

This separates from the blood large quantities of ultra-filtered fluid in more than a million small, tubular units, nephrons/uriniferous tubules. Most needed materials are then recovered to the bloodstream, and some secretion of other substances occurs, to give a solution of unwanted materials -the excretion - to be collected as urine from the tubules. The kidney is a compound, tubular, excretory gland, and an endocrine gland. Kidney Powerpoint.

l Kidney's general architecture
l Outside are perirenal fat, and nearby suprarenal glands.
2 Thin, fibrous capsule.
3 Reniform (kidney-shaped!), around a hilum and sinus for the
4 renal artery, renal vein, and ureter.
5 Ureter opens from a renal pelvis, for which
6 major and minor calyces* collect the urine from
7 bluntly pointed apical papillae of pyramids.
8 Pyramid + overlying tissue constitute a lobe.
9 The human kidney is multilobar, with 8-l8 lobes.
l0 Pyramidal tissue has a pale striated appearance from many parallel tubules and blood vessels. It is the medulla.
ll The outer cortex of the kidney is darker, with many round structures - renal corpuscles/Malpighian corpuscles, and coiled tubules cut in cross and oblique section.
l2 Cortical tissue - columns of Bertin - runs inward to partly separate the pyramids.
l3 Medullary tissue extends rays up from the medulla into the cortex. A medullary ray defines the centre of a lobule, but the lateral limits of the lobule remain undefined in the cortical tissue.
(* The minor calyces act as a curved row of funnels with cut-off stems, dripping into a second row of stem-less major funnels, delivering into the single pelvis. This arrangement and the concept of the sinus cannot be made out in a single section.)

2 Form of nephron and relations with cortex and medulla
Cortex
l Renal corpuscle (round, l50-240 µm diameter) - glomerulus of epithelium-invested capillaries, and enclosed in a Bowman's capsule, opening out at the urinary pole into the
2 proximal convoluted tubule, which leads to the

Medulla
3 descending limb of the hairpin loop of Henle,
4 then the ascending limb of Henle's loop.

Cortex
5 Distal convoluted tubule follows, attached at one point to the renal corpuscle of origin; thence leading to an
6 arched collecting/junctional tubule joining a

Medulla
7 straight collecting tubule, receiving many branches and running down from a medullary ray through the medulla to a
8 papillary duct of Bellini, opening at the papilla of the pyramid. The papilla is cribriform from the many openings.

Alternative terms for these parts are:
.. 2 may be termed the pars convoluta of the proximal tubule;
.. 5 may be termed the pars convoluta of the distal tubule;
.. the loop of Henle comprises the pars recta of the proximal tubule, the thin segment, and the pars recta of the distal tubule.
Thin segments and loops vary in length dependent on the position in the cortex of their glomeruli of origin. The appearance of the kidneys is dominated by the nephrons, since the connective tissue element (reticular fibres) is slight, and the very many small blood vessels follow the pattern of the nephrons, because the two work together.

3 Functional unit of the kidney
Consists of (a) nephron, (b) blood vessels, (c) interstitium, and (d) collecting tubule.
The functions of the various parts of the unit are given briefly, so that all aspects of the finer structure can be presented together.
l Renal corpuscle, with vascular glomerulus - ultrafiltration of arterial blood.
2 Proximal convoluted tubule - from the ultra-filtrate received from the corpuscle, the prompt massive recovery (reabsorption), by active transport, cotransport, facilitated and downhill diffusion, of sodium, chloride, glucose, amino acids, etc, and of small proteins by endocytosis.
3 Loop of Henle - urine concentration by active and passive functions in a complicated counter-current osmotic multiplier interaction of loops of Henle, blood vessels, interstitium, and collecting tubules.
4 Distal tubule (partly in the loop) - continued active reabsorption of Na+ under the control of aldosterone, and the secretion of potassium.
5 Collecting tubule - passive reabsorption of water to the blood, making the urine hypertonic, under the influence of pituitary antidiuretic hormone (ADH); and a variety of fine adjustments to electrolytes and acidity.
6 The nephron is controlled by hormones from other endocrine glands, but the kidney itself produces hormones that affect non-renal tissues (Chapter 27.F).

4 Nephron cytology
1 Glomerulus

2 Proximal tubule (40-50 µm diameter)
3 Thin segment (l5 µm diameter) 4 Distal tubule (20-50 µm diameter) 5 Juxtaglomerular apparatus 6 Collecting duct (40-200 µm diameter)

5 Renal interstitium
1 lies between the kidney tubules and vessels.
2 It comprises: (a) reticular fibres, (b) a little ground substance, and (c) interstitial fibroblasts, looking after the matrix and secreting erythropoietin.
3 The interstitial elements are more prominent in the medulla than the cortex.

6 Renal blood vessels
l Renal artery branches to form
2 interlobar arteries (interpyramidal), extending to the cortico-medullary junction, where they branch and turn as arching
3 arcuate arteries, giving off outward branches called
4 interlobular arteries; from which
5 intralobular arteries provide
6 afferent arterioles to
7 glomeruli; from the capillaries of which the blood is taken via
8 efferent arterioles to serve one or both of
9 two capillary beds - around the convoluted tubules, and between the straight medullary tubules.
l0 The blood collected in stellate, deep cortical, and interlobular veins, traces back the arterial path to the renal vein.
ll The sympathetic nervous supply to the kidney goes mainly to the renal vasculature, including the juxtaglomerular cells.
l2 Vasa recta is a collective name for arteriolar, capillary, and venous straight blood vessels in the medulla. They participate in the counter-current exchange.

B URINARY PASSAGES

. The kidney's calyces and pelvis, and the passages to the urethra are lined by transitional epithelium. Powerpoint.

l Transitional epithelium/urothelium
l Multilayered, with large surface/umbrella cells, intermediate cells and basal cuboidal cells on a thin BL.
2 The surface cells have unique properties of:
.. (a) making a barrier impermeable to urine;
.. (b) changing their shape and extent during bladder distension.
3 For 2 (a), the luminal umbrella cell membrane is asymmetrically thickened (to l2 nm) and has unusual lipids and proteins, including uroplakins
4 For 2(b), the Golgi complex forms fusiform vacuoles, bounded by thick membranes. During bladder dilation, the vesicles attach to the thick luminal membrane and become part of it, thus increasing its extent and allowing the cell to flatten. No cell-over-cell sliding occurs, the cells being joined by tight and adhaerens junctions and desmosomes.
5 Large lysosomes destroy defective membrane.
6 The rate of cell turnover is very low for an epithelium.

2 Ureter
l Transitional epithelium lies on a collagenous lamina propria.
2 Mucosa has several longitudinal folds, giving the lumen a stellate shape in the cross-section.
3 Two smooth muscle coats: outer, circular; inner, longitudinal; (the terminal ureter has an extra, outer longitudinal one).
4 CT adventitia, rich in vessels and nerves.

3 Urinary bladder
l Transitional epithelium, on a wide collagenous lamina propria without glands, constitutes the mucosa.
2 Three smooth muscle tunics interweave in the muscularis, in a pattern to squeeze the bladder empty. Retention of urine invites infection.
3 A CT adventitia has blood and lymphatic vessels, nerve fibres and ganglion cells. The part of the bladder facing the pelvic cavity has a serosa.
4 The ureters enter obliquely, with mucosal flaps to prevent reflux; smooth muscle forms a sphincter at the urethral outlet.

. Urethra (male)
l Epithelium lies on a very loose, elastic, vascular, distensible lamina propria. The lumen is stellate in cross-section.
2 Epithelium is transitional changing to pseudostratified columnar, stratified columnar, and finally stratified squamous, as it traverses the three sections: prostatic, membranous (short) and penile/cavernous (long).
3 Branching out in the penile mucosa are Littré's small tubular mucous glands.
4 There is a meagre smooth muscle muscularis, except at
5 the smooth and skeletal muscle sphincters
6 Female urethra is much shorter than the male; structurally it is similar, but, ending in the pelvic floor, has a skeletal muscle sphincter at its terminus.


Chapter 24 ALIMENTARY SYSTEM

Long, muscular, tubular structure for ingesting food and water, separating them from the intake of air, breaking the food down mechanically and chemically (digestion) for absorption, while propelling it anally. Ancillary glands, liver and pancreas (Chapter 25), are included, since they produce materials used for digestion or to be excreted via the tube, and they participate metabolically and in the control systems. . Oral Powerpoint

A ORAL STRUCTURES

l Salivary glands
l Generally compound tubulo-alveolar, with intralobular intercalated ducts and secretory ducts (with basal striations), leading to interlobular excretory ducts.
2 Parenchyma is divided by CT septa into lobes and lobules.
3 Saliva is water, salts, and organic materials (mainly mucin and salivary amylase/ptyalin and maltase), with suspended lymphocytes (salivary corpuscles), epithelial cells, and bacteria.
4 Mucin is formed by mucous cells (pale in HE staining).
5 Enzymes are formed by serous cells (basophil, with zymogen granules).
6 Parotid gland is serous; submandibular/submaxillary has serous alveoli, and mixed tubules with serous demilunes/crescents; and the sublingual gland has mucous and mixed branched tubules, but lacks intercalated and secretory ducts.( The tubules are long enough to reach the excretory ducts.)
7 Smaller mucous and mixed glands are in lingual, labial, buccal, pharyngeal and palatine sites.
8 Chapter 11 gave more details of salivary glandular structure.

2 Lip
l Core of fibro-elastic CT and skeletal muscle.
2 Outside is thin skin with hairs and glands.
3 Transition zone is the red margin/vermilion border, where the skin's cornified layer thins out; a rich capillary plexus is responsible for the colour. Glands are absent.
4 Inside is a thick stratified squamous epithelium, with mucous glands in its lamina propria.
5 The cheek is similar, but has more adipose tissue, and no red margin.

3 Gingiva/gum and raphe of hard palate
Stratified squamous epithelium (partly keratinized) on a dense CT lamina propria, with deeply penetrating papillae, and fastened tightly to tooth or bone.

4 Soft palate
l Fibrous and skeletal muscle core, with mucous glands;
2 pseudostratified, columnar, ciliated epithelium covers the pharyngeal side, and stratified squamous the oral surface.
3 Functions in deglutition (swallowing), speech, blowing, coughing, and sneezing.

5 Tongue
l Core is interlaced skeletal muscle bundles oriented in three directions, with attendant nerves and blood vessels.
2 Covered by stratified squamous epithelium, modified over the anterior dorsum by being thrown up with the dense lamina propria into projections called
3 papillae of various kinds, with special distributions:

4 Lingual glands - (a) posterior mucous; (b) posterior serous of von Ebner, opening into the trenches; (c) anterior mixed sero-mucous.
5 Lingual tonsils are stratified squamous epithelium-covered aggregations of lymphoid nodules, with shallow crypts flushed out by mucous secretions of the posterior lingual glands.

6 Palatine/faucal tonsils
l Covering is stratified squamous epithelium.
2 Deep, branching, epithelium-lined pits or crypts run down from the surface into the tonsils, but the epithelium is infiltrated by
3 lymphocytes produced in germinal centres of lymphoid nodules (often confluent) in the lamina propria, and by macrophages.
4 Immunoglobulins and lysozyme are present.
5 Glands and skeletal muscle lie nearby, outside the underlying CT capsule.
6 The palatine tonsils have substantial depth; the lingual are a narrow region interposed between the epithelium and the muscular core of the tongue.

7 Tooth
l Anatomical features: crown, cervix/neck, root, apical foramen, pulp cavity, bony alveolus/socket, attaching periodontal ligament and the gingiva.

2 Tooth components

3 Histological details of tooth
(a) Decalcification for sectioning destroys mature enamel. It can be studied in the ground section.
(b) Enamel prisms have a spiral curvature to better withstand masticatory forces.
(c) Bands/striae of Retzius are growth/incremental lines across the enamel; Owen's contour lines are analogous features in dentine.
(d) Interglobular areas are poorly mineralized regions in the dentine.
(e) Dentinal tubules branch, and may penetrate a little way into the enamel as enamel spindles.
(f) Von Korff's 'fibres' seen in the pulp by young odontoblasts are either collagen awaiting incorporation into the matrix of the dentine, or are an artefact of silver impregnation.
(g) Secondary dentine (sometimes reparative) may be formed later to increase the thickness of the dentine.
(h) Epithelial attachment is a cuff-like extension of the gingival epithelium, attached to the neck of the tooth by glycoprotein.
(i) Acellular cementum lacks cementocytes.

4 Tooth development
(a) Two stages with (i) 20 deciduous/milk teeth, (ii) followed by 20 successional teeth and l2 permanent or accessional molars, totalling 32. Powerpoint
(b) Involves complex inductive processes:

8 Functions of oral structures
l Obtaining, approving, masticating, and swallowing food and water.
2 General exploration of the environment.
3 Vocalization and communication. (Many systemic diseases have oral signs).
4 Preening, mating, fighting, etc, where chewing, licking, grasping or biting is needed.
5 Oral glands contribute to digestion, and the lymphoid tissues to protection.
6 Breathing in exercising, and when the nose is blocked; coughing, and blowing.

B GASTROINTESTINAL TRACT

l General plan
l Mucosa (innermost) 2 GI submucosa 3 GI muscularis externa 4 GI serosa or adventitia/fibrosa (outermost) 2 Oesophagus
l Mucosa has stratified squamous epithelium ending sharply, but along a jagged line, at the gastric junction, creating a white-red distinction between proximal and distal sides of the Z-line in endoscopy. Here, abnormalities of the oesophageal epithelium and the position of the epithelial junction are quite common - Barrett's oesophagus, where the stratified squamous epithelium is replaced metaplastically by simple columnar epithelium with some or all of the small-intestinal cell types.
2 Muscularis mucosae - longitudinal smooth muscle.
3 Cardiac glands - make neutral mucus and are branched tubular, in the mucosa near the gastric cardia, and in mucosa of the upper oesophagus; inconsistently present.
4 Oesophageal glands - acidic mucous, compound, tubulo-alveolar, and lying in the submucosa, less numerous in the middle segment of the oesophagus.
5 Circular and longitudinal external muscle coats of skeletal muscle in the upper fifth or so give way progressively to only smooth muscle in the lower half.
6 Outermost coat is CT adventitia, except on a small piece below the diaphragm.
7 Function - rapid passage of food to (and from) the stomach.

3 Stomach
l General structure

2 Stomach mucosa 3 Gastric secretions and cell types responsible
(a) Surface mucous cells - mucus, to prevent auto-digestion of the mucosa, and bicarbonate ions held in the mucus.
(b) Chief/zymogenic cells - enzymes, e.g., pepsin, rennin, gastric lipase.
(c) Oxyntic/parietal cells - Cl-/HCO3- is exchanged basolaterally to balance the apical Na+/H+ proton pump used to form the hydrochloric acid of the digestive juice.
(The stimulated active parietal cell has greatly extended canaliculi.)
(d) Mucous neck cells - mucus and enzymes, e.g., dipeptidases.
(e) Endocrine cells - hormones and amines; e.g., a hormone - gastrin - produced by the pyloric antral G cells controls the release and formation of acid from parietal cells, and of digestive enzymes from chief cells.
(f) Parietal cells - intrinsic factor - to assist in the absorption of vitamin B12: this role is upset when the parietal cells' proton pump is an autoimmune target in pernicious anaemia, leading to the cells' destruction.

4 Gastric protective mechanisms

4 Small intestine
l General structure 2 Cytology of small-intestinal mucosa 3 Functions of small-intestinal mucosa

4 Changes within small intestine during descent:
(a) Goblet cells increase in number.
(b) Villi become more finger-like.
(c) Lymphoid tissue increases.
(d) Plicae circulares diminish.

5 Protective mechanisms of the gut:

5 Large intestine
l General features

2 Regional details of large intestine

Chapter 25 PANCREAS, LIVER AND GALLBLADDER

A PANCREAS

This gland combines exocrine and endocrine functions. The exocrine secretion passes via the duct of Wirsung (and any accessory duct) into the duodenum for digestive and neutralizing purposes. Powerpoint

l General structure
l Elongated, lobulated, compound, acinar gland, with a very thin CT capsule and septa.
2 Long duct system and its CT provide support.
3 Exocrine part is major with very many serous acini and some ducts.
4 Endocrine part is minor: many small clusters of cells staining palely (with HE) - islets of Langerhans.

Exocrine pancreas

2 Acinar structure
l Pyramidal epithelial cells line the acini; are rich in basal granular ER (deeply basophil); have a prominent supranuclear Golgi complex and apical zymogen granules (precursors of several digestive enzymes).
2 Electron-radioautography with labelled leucine showed the secretory pathway through the cell and its time aspects; see Chapter l6.C.l.
3 A pale duct cell (or a pair) may be seen intruded into the centre of the acinus as a centroacinar cell.

3 Ducts
1 Commence as narrow intercalated ducts within the acini, although vagaries of section plane result in one finding centroacinar cells in only some acini.
2 Beyond the intercalated ducts, ducts have pale cuboidal cells, with few organelles and some microvilli, changing to columnar epithelial cells in the larger ducts.
3 Ducts are less often seen than in the serous parotid gland, and probably actively change the secretions only in the smaller, early ducts.
4 Ducts are accompanied by less connective tissue than in the salivary glands, which are exposed to masticatory forces.

4 Exocrine function
l Formation of alkaline secretions, which counter the gastric fluid's acidity, thereby activating pancreatric pro-enzymes for digestion.
2 The release of alkaline and enzymatic secretions is under the hormonal control of secretin, and cholecystokinin/CCK, respectively.

Endocrine pancreas

5 Islet structure and functions
l No ducts, but rich in capillaries with a fenestrated endothelium.
2 Pale cells contain granules differing in alcohol-solubility and staining characteristics (distinguishable also in EM and immunocytochemically) for the differentiation of:

3 Blood drained from the pancreas and bearing the polypeptide hormones passes, via the portal flow, to the liver.

B LIVER AND GALLBLADDER

l Liver's general features
l Large, lobated exocrine and blood-processing gland, with
2 vessels and ducts entering and leaving at the porta.
3 Enclosed by a thin CT capsule, mostly covered by mesothelium.
4 CT of the branching vascular system provides gross support.
5 Parenchymal cells are supported by fine reticular fibres.
6 The internal structure is understandable in terms of the several vessels entering or leaving the organ;

2 Liver lobule
l First impression is of a uniform mass of large glandular cells throughout the liver substance.
2 Closer examination shows that the cells are arranged in perforated plates, one cell wide. Between the plates are sinusoidal blood channels 9-l2 µm wide, lined by endothelial cells.
3 Scattered in the glandular mass are blood vessels, alone and accompanied by other vessels.
4 The distribution of these vessels defines or marks out the classic hepatic lobules.

5 Varieties of liver vessel

6 In pig and camel, the lobules are separated from one another by CT and thus much more easily identified.

7 Hepatic lobular blood flow is:

8 Intralobular bile flow is from the lobule's centre towards the peripheral bile ducts, and runs, within any one cell plate, between the liver cells in bile canaliculi.

9 Rappaport's liver acinus was a functional unit comprising parts of three or so lobules. It sought to account for differences in exposure to the blood supply among various parts of lobules. Such differences are reflected in varied functional activities and degrees of susceptibility to toxic agents - a metabolic zonation (Gebhardt R. Pharmacol Therapeut 1992;53:275-354; Cell Biol Toxicol 1997;13:263-272).
The territory of an acinus has, as its axis, one final branch of the portal vein, and is subdivided into: 1 periportal, 2 intermediate, and 3 perivenous (close to the central vein) zones, with the initial periportal zone being roughly spheroid, and isolated from periportal zones of adjacent acini.
The concept is not easy for students to follow, nor, it seems, for hepatocytes, which, for many processes, heed different patterns. To best fit events to the architecture, hepatologists are now more likely to employ the simpler concept of separately continuous periportal and perivenous/pericentral zones, than that of discrete acini.

                    pp  pp  pp            pv .
                pp _ _ _ _ _ _ _ _ _ _        . pv
                 /  pp  pp   pp             pv .
              pp/                               . pv
portal veins   / pp     pv    pv    pv     pv    .
- - - - - - - -       . . . . . . . . . . . . . . . . . .central veins
 pp   pp    pp \          pv      pv    pv     pv
                \   
              pp \_pp_ _ pp_ _ _pp_ _ _
                    pp   pp  pp     pp

3 Liver sinusoids
l Are lined by fenestrated endothelial cells, loosely attached, and hold
2 phagocytic Kupffer cells (larger, stellate, with a pale oval nucleus), demonstrated by the vital intravascular injection of trypan blue or carbon particles, or latex particles for microscopy in vivo.
3 Fenestrated lining cells are not tightly attached and rest on microvilli of underlying hepatic cells, without a BL intervening.
4 Plasma can thus pass through the sieve plate, formed by the lining cells, out into the perisinusoidal space of Disse to interact with the hepatocytes. Some of this fluid may pass to the periphery of the lobule to be collected as lymph.
5 Disse's 'space' contains ECM materials, but not a visible basal lamina.
6 Scarce, fat-storing, stellate cells of Ito lie outside the endothelial cells. They store vitamin A. They respond to a variety of insults by making collagen and causing cirrhosis (fibrosis).
7 The sinusoidal wall provides for:
.. (a) blood cleansing, e.g., of gut bacterial toxins;
.. (b) haemopoiesis in the embryo;
.. (c) bringing plasma into intimate contact with the hepatic cell for its many metabolic functions of storage, transformations, syntheses, regulation of plasma concentrations, detoxifications, the production of bile, and assisting defence by producing acute-phase proteins.

4 Hepatocyte/hepatic cell
l Large, polyhedral, 30 µm x 20 µm cell with:

5 Bile pathways
l System of canaliculi (seen easily only with EM or special impregnation) between the hepatic cells leads to
2 canals of Hering/cholangioles, with both hepatocytes and pale duct cells in their walls. Next come, in the portal areas,
3 bile ductules with only small, pale cuboidal cells, firmly held by membrane interdigitations and junctional complexes, and having a few luminal microvilli.
4 Bile ducts' epithelium changes to columnar mucous cells and, extrahepatically, the ducts acquire smooth muscle as well as CT.
5 Cystic duct allows reflux into the gallbladder, when Boyden's sphincter choledochus at the duodenal outlet of the common bile duct is closed.

Bile production starts with inward bile-acid pumping across the sinusoidal region of the hepatocyte membrane. Then, transporters in the canalicular membrane send bile salts, phosphatidylcholine, and toxic metabolites out into the bile.

6 Gallbladder
l Extensively folded mucosa of tall, simple, columnar epithelial cells with many microvilli, lying on a loose lamina propria.
2 Goblet cells are absent, but in the neck there may be small glands of uncertain function.
3 The middle layer has variously disposed (mainly circular) smooth muscle bundles.
4 Outermost is a serosa of mesothelium-covered areolar CT with vessels and nerves, except where the gallbladder attaches to the liver.
5 Function - stores and concentrates the bile by actively absorbing sodium, coupled with water and anions. The hormone - cholecystokinin - released from gut endocrine cells in response to fat or amino acids causes contraction of the muscle to expel the bile.


Chapter 26 HORMONES

A GENERAL CHARACTER

l These are potent chemical substances travelling via the bloodstream from one cell to another. They work in conjunction with the nervous system to control the homeostasis of the body, and to anticipate future events such as birth, lactation, fighting or fleeing. . Powerpoint
2 The hormone reaches many cells but, except for hormones affecting growth and some general metabolic processes, only certain target or end-organ cells respond. The response is often a start or increase in a cell's activity, e.g., contraction, release of a secretion, growth by proliferation (hyperplasia) or by an increase in size (hypertrophy). However, a hormone may sometimes inhibit a cell's activity, e.g., calcitonin inhibits osteoclasts' resorption of bone.
3 The hormone may stimulate its target cell either by binding with a membrane receptor in the plasmalemma that starts a signal transduction sequence, say, to alter the level of a second, internal, messenger, within the cell, e.g., cyclic AMP or GMP; or by penetrating the cell membrane and binding with a cytoplasmic receptor. Once inside the cell the bound hormone itself, the second messenger, or downstream effectors such as Ca2+, can trigger the release of secretion, an increase in nucleus-controlled synthesis, a contraction, or some other useful task.
4 In its usual concentrations, a hormone's action is called physiological. Pharmacological effects are seen when abnormally high quantities are injected.
Pathological effects are observed when: too little or too much hormone is present; the target organ is insensitive to the hormone; or the hormone molecule is defective.

B CHEMISTRY OF HORMONES

l Hormones differ chemically and may be classified thus: 2 Hormones may be bound to proteins for storage, e.g., to thyroglobulin in thyroid colloid; or for transport, down a neurosecretory cell's axon, or in the blood.
3 The small amounts of hormone normally circulating in the blood, or present in a tissue, can be measured by two principal methods:

C CYTOLOGY OF HORMONE SECRETION

l Hormones are formed in:
.. (a) pure endocrine glands, e.g., thyroid;
.. (b) mixed exocrine and endocrine glands, e.g., pancreas and testis;
.. (c) some of the cells in organs having other functions, e.g., placenta, kidney, GI tract.
2 The hormone is a product synthesized and released by glandular cells, mostly epithelial, but some are modified neurons or muscle cells.
3 Cells have organelles associated with synthesis, e.g., granular or smooth ER, Golgi complex, and may store the hormone or prohormone as membrane-bound inclusion granules. Lysosomes may be used to destroy excess hormone. Actin filaments are used to discharge the granules by exocytosis. The chemical nature of the hormone is reflected in the cytology, e.g., steroid cells store the lipid precursor, but not the hormone, and have much smooth ER.
4 The secretory granules may stain selectively because of the chemical nature of the hormone, e.g., glycoprotein with the PAS reaction, and have a distinctive size, shape and density in EM.
5 The stored hormone can be demonstrated in its cell by immunostaining, using an antibody that binds specifically with that hormone, coupled with a visually demonstrable tag, e.g., a fluorescent compound (for LM) or a peroxidase (for EM and LM).
Catecholamines can be seen with fluorescence microscopy after treatment with an aldehyde. The mRNA for the hormone or its precursor, or for enzymes necessary to its synthesis, can be seen by using in situ hybridization.
6 The synthesis of a hormone can be followed within the cell, moving progressively between organelles and inclusions, by sequential radioautographic following of a radioactively labelled precursor, e.g., an amino acid, or 125I.
7 The stimulus for the release of hormone, and the synthesis of more hormone, may be:
.. nervous by synaptic action, e.g., adrenal medulla;
.. another hormone, e.g., TSH for thyroid follicular cells; or
.. the blood level of a non-hormonal chemical, e.g., Ca2+ for parathyroid chief cells.
8 To facilitate the blood-endocrine cell interactions, cords and small clusters of endocrine cells are supported by reticular fibres, close to numerous wide capillaries (sinusoidal capillaries), lined by fenestrated but non-phagocytic endothelial cells.

D QUESTIONS TO CHARACTERISE A HORMONE

l For each hormone - questions for the physiological state:
  1. Its chemical nature?
  2. What cell, in what part, of which organ, forms it?
  3. What cell specializations are associated with its synthesis, storage and release?
  4. What are other morphological details of the cell? e.g., size.
  5. What controls hormone release?
  6. Is the pathway for the control of release simple and direct, or complex and indirect?
  7. What are the target tissues?
  8. What actions does the hormone have on those tissues?
  9. Do some effects appear more important than others?
  10. Do other hormones affect the same tissue? If so, how do the hormones interact?
  11. Do the endocrine cells and the effects of the hormone on the target change with time? e.g., the timescales of menstruation, gestation, childhood growth.
  12. Are the hormone's effects the same in the two genders? e.g., the role of prolactin in the male?
  13. What pre-secretion proteolytic processsing is required to release active hormone? For example, the corticotrophs of the anterior pituitary gland employ a serine endoprotease to release ACTH and small amounts of beta-endorphin from a 30 kDa peptide precursor - Pro-OpioMelanoCortin (POMC). In the intermediate lobe, extra convertase enzymes increase the output of endorphin and process ACTH to alpha-Melanocyte Stimulating Hormone (MSH).
2 For each hormone - questions for abnormal states, pathological and induced:
  1. What are the effects of its loss?
  2. Is it essential for life?
  3. What happens when there is an excess from a tumour, or by experiment?
  4. To find out the pathways and mechanisms controlling hormone release: What are the effects on the endocrine cells of:

Chapter 27 ENDOCRINE SYSTEM

A HYPOPHYSIS/PITUITARY GLAND

l General morphology and development . . Endocrine Glands Powerpoint
l Linked by a stalk to the base of the brain, and lies surrounded by dural membrane (capsule) in the bony sella turcica.
2 Stalk extends through the dural diaphragma sellae. Pituitary weighs 0.5-l.0 g.
3 Divisions of the pituitary gland
     Primary                      Secondary
                     _
                    |  Pars distalis (anterior lobe)
                    |
Adenohypophysis ----|  Pars intermedia (intermediate lobe)
                    |
                    |_ Pars tuberalis*

                     _
                    |  Pars nervosa/infundibular process (posterior lobe)
                    |                   _
Neurohypophysis ----|                  | infundibular stem*
                    |                  |
                    |_ Infundibulum ---|
                                       |
                                       |_median eminence (of tuber
                                                             cinereum)
  (* together form hypophyseal stalk)

4 Embryological origins

2 Adenohypophysis (histology and function)
l Pars tuberalis - wrapped around the neural stalk are cords of basophilic cells containing gonadotrophic hormones.
2 Pars intermedia - rudimentary in man; variable in width; several colloid-filled cysts; glandular cells - chromophobe or basophil; basophilic cells may extend into the neural lobe; function - unknown in man, but in fish and amphibia the melanocyte stimulating hormone (MSH) formed varies skin pigmentation.

3 Pars distalis

3 Neurohypophysis
May be viewed as a downward extension of the hypothalamus, allowing for hormone storage and a complete breach of the blood-brain barrier for hormone release. Its structure follows:
l The neural stalk and posterior lobe consist of the unmyelinated axons (grouped as the hypothalamo-hypophyseal tract)
2 of neurosecretory neurons of the hypothalamic supraoptic and paraventricular nuclei.
3 The neurosecretion collects, and dilates some axons and their terminals into Herring bodies. Gomori staining or EM shows the presence of granules in these axons, but not in the
4 pituicytes - a neuroglial kind of cell.
5 The secretion collects in terminals arranged as a palisade around blood vessels. Its release may involve electrical discharge in the axon and chemical factors in the 'synaptic' vesicles also present.
6 Two polypeptide hormones in the secretion are:

7 The neural lobe has a direct arterial supply from the inferior hypophyseal arteries to its fenestrated capillaries.

B PINEAL GLAND/EPIPHYSIS CEREBRI

l Originates as a dorsal outgrowth at the caudal end of the diencephalon. Unlike the pituitary, it is not connected directly by nerve fibres with the CNS.
2 The capsule of pia extends in septa to lobulate the organ, and carry in extensive blood vessels.
3 There is a regulatory autonomic nerve supply via the superior cervical ganglia.
4 Constituent cells 5 Increasing in number throughout life are mineral concretions - so-called brain sand (acervuli cerebri/corpora arenacea).
6 The pineal is responsive to changes in environmental light, initially mediated via the accessory optic tract and the suprachiasmatic nucleus.
Darkness raises the production of the enzyme hydroxyindole-O-methyl transferase (HIOMT), which methylates N-acetyl-serotonin to give melatonin.
7 Melatonin is part of the internal clock, matching the rhythm of alertness, and gonadal and other endocrine functions, to external light-based circadian and seasonal cycles. (In amphibia, melatonin also reduces the dispersal of pigment within melanocytes, hence the name.)

C THYROID GLAND

l General morphology
l Develops from an endodermal downgrowth at the base of the tongue. The thyroglossal duct, connecting it with its point of origin, later disappears. Two lateral lobes, an isthmus (and sometimes a pyramidal lobe) are established.
2 The inner, true, CT capsule sends in septa to partially enclose lobules.
3 In the lobules are rounded or elongated bodies - follicles, in a loose stroma of CT, with many blood vessels.

2 Thyroid follicle
l In man, they vary between 0.02 and 0.9 mm in diameter. A gland has several million follicles.
2 Filled with viscous fluid - thyroid colloid - variably acidophil or basophil, and often shrunken and showing knife chatters.
3 Lined by basophilic cuboidal follicular cells, varying in height as a simple epithelium on
4 a basal lamina, outside which is an extensive plexus of blood capillaries, and reticular fibres and fibroblasts.
5 Follicular cells are polarized with respect to the follicle lumen; the nucleus is central, the Golgi complex supranuclear; EM shows plenty of granular ER, some luminal microvilli, endocytotic vesicles, and lysosomes.
6 Between the follicular cells and the BL, and sometimes outside the BLs, lie occasional C cells (clear/parafollicular cells), having no direct access to the lumen, and no colloid droplets, but with small argyrophil, secretory granules.

3 Thyroid histophysiology
l C Cells

2 Follicular cells

D PARATHYROID GLANDS

l General morphology
l Derived embryologically from the 3rd and 4th pharyngeal pouches.
2 Adherent to the true capsule of the thyroid.
3 Each of the four or more rounded or ovoid bodies has a fine CT capsule and delicate, incomplete septa.
4 These septa carry vessels, nerves and many fat cells.

2 Histophysiology
l Supported on fine reticular fibres are many fenestrated blood capillaries and sheets and cords of
2 glandular cells:

3 Functions
(a) Secretory granules of chief cells are the polypeptide hormone, parathormone/PTH, released in response to low blood Ca2+, and acting on osteoclasts and macrophages to increase bone resorption.
(b) In the kidney, PTH: promotes the tubular reabsorption of calcium, and the 1, activation of vitamin D; and inhibits the renal tubular reabsorption of phosphate - a phosphaturic action.
(c) Unlike most other endocrine glands, no specific pituitary trophic hormone is involved in its control.

E ADRENAL/SUPRARENAL GLAND

l General morphology and development
l Elongated glands of cocked-hat or crescentic shape.
2 Composite of medullary and cortical tissues, linked by blood supply, but embryologically and functionally distinct.
3 Mesodermal cells of coelomic mesothelium differentiate into:
(i) inner, provisional or fetal cortex (involutes at birth); and
(ii) outer, permanent cortex.
4 Neural crest ectodermal cells migrate: (i) to coeliac ganglion; and (ii) then some go beyond to invade the adrenal cortical tissue and form the medulla.
5 Mature adrenal has a thick CT capsule, bringing arteries to serve radial capillaries draining down towards the venules and central vein of the medulla. Arterioles also penetrate the cortex to serve a medullary capillary bed.
6 The medulla is a long, thin strip of basophilic cells, which can be made outstanding by the chromaffin reaction - a darkening produced by dichromate ions.
7 The supporting element throughout is the reticular fibre.

2 Cortex
l Polyhedral glandular cells, in cords usually two cells wide, run roughly radially, along with sinusoidal capillaries.
2 Three layers are visible:

3 Lipid droplets (Sudanophilic and osmiophilic) contain cholesterol and cholesterol esters, used in conjunction with the Golgi body, smooth ER and special mitochondria, to produce two kinds of
4 steroid hormones: mineralo- and gluco-corticoids. Examples: 3 Medulla
l Two cell kinds: 2 Release is controlled by a direct, 'preganglionic', sympathetic innervation, terminating synaptically on the glandular cells.
3 The hormones released are: 4 The hormones are stored in characteristic membrane-bound granules, visible in EM. The granules form in relation to the Golgi body, but a dense GER is not required. They also contain enkephalins and chromogranin.
5 Both principal hormones are catecholamines, which can be converted by oxidizing agents, e.g., dichromate or ferric salts, to brown-coloured polymers - adrenochromes: this is the chromaffin reaction.

F KIDNEY

The kidney is not only the target for hormones, but it also makes several.
l Renin is an enzyme, formed in the juxtaglomerular modified muscle cells, that acts on a blood protein to form the potentially hypertensive angiotensin l. One triggering stimulus is the chloride concentration in the distal tubule detected by the macula densa cells.
2 l,25-hydroxycholecaliferol - the active form of vitamin D, needed for the intestinal absorption of Ca2+ and some direct actions on bone cells, is made in the kidney. Vitamin D from synthesis in the skin, or from the diet, is changed to 25-HCC in the liver, but the final 1,25 step is a renal task.
3 Erythropoietin is a protein growth factor, made by predominantly medullary renal fibroblasts, that stimulates the production of erythrocytes by marrow, e.g., when the atmospheric O2 falls at high altitude.

G APUD NEUROENDOCRINE AND PEPTIDE SYSTEMS

In the 1970s, the focus was on the amine metabolism that gave a unifying aspect to rather perplexing cells, scattered in many organs, which had been noticed and considered on an individual basis as clear (empty looking), or having granules reacting with silver salts. It turned out that most of these cell types made and released non-cytokine peptide mediators, to act locally or at a distance. The peptide story has now overwhelmed the amine or APUD idea, because these peptide factors are many, and are made and used for signalling in every part of the body, including the brain. The basis of the APUD classification is outlined below, because it helps explain aspects of pathology.

l APUD Within some endocrine glands, chemoreceptors, the brain, and dispersed in epithelia, are cells that form amine compounds. After an Amine Precursor has been taken Up, the cell Decarboxylates it to form serotonin (5-HT) from 5-hydroxytryptophane, or a catecholamine from dihydroxyphenylalanine (hence APUD).
Noticing that many of these cells secrete polypeptide hormones, Professor Pearse proposed a far-flung 'APUD' neuroendocrine system, secreting peptide mediators. The amines and peptides function variously as neurotransmitters, hormones, and modulators of neural action. Some vary their role by site. Some cells come from neural crest; for others, their origin is disputed.

2 Established APUD members
Peripheral
l Pancreatic islet cells -> insulin, glucagon, and somatostatin
2 Thyroid C cells -> calcitonin
3 Parathyroid chief cells -> parathormone
4 Gastrointestinal endocrine cells -> gastrin, secretin, pancreozymin/ cholecystokinin, glucagon, motilin, somatostatin, and many other active peptides. (Cells have a designating letter, if the hormone is known).
5 Other endocrine/neuroendocrine cells in respiratory and genito-urinary tract epithelia hold granules, reacting with silver salts in the argyrophilic and argentaffin ways of the GI-tract endocrine cells, and produce a variety of peptides, e.g., vasoactive intestinal polypeptide/VIP.

Tumours of these neuroendocrine cells often draw attention because of symptoms resulting from an excess of ectopic (out of place) polypeptide hormone, e.g., ACTH from the bronchial neuroendocrine cell, and/or an excess of serotonin, resulting in the flushing, bronchoconstriction, diarrhoea, etc. of the carcinoid syndrome.

Central
5 Pituitary
.. somatotrophs -> growth howmone (GH)
.. mammotrophs -> prolactin (PRL/MTH)
.. corticotrophs -> adrenocorticotrophic hormone (ACTH)
.. melanotrophs -> melanocyte-stimulating hormone (MSH)
6 Hypothalamic large neurosecretory cells -> oxytocin, vasopressin
7 Hypothalamic small neurosecretory cells -> releasing factors/hormones, e.g., LH.RF; and somatostatin (SRIF) inhibiting GH release from pituitary somatotrophs.
8 Pinealocytes -> melatonin

3 APUD members with an uncertain peptide role
The peptide substance normally formed, if any, has not yet been identified, or its role is unclear.
l Carotid-body type l cell and similar cells in the aortic and other chemoreceptive bodies contain norepinephrine and/or dopamine.
2 Chromaffin-system cells, in the adrenal medulla and abdominal paraganglia, contain catecholamines and enkephalins.
(The GI tract cells of 2.4 above, despite their old 'enterochromaffin' name do not form catecholamines.)
4 Melanocytes of skin, and dermal and ocular CT cells using amines to form melanin, come from the neural crest.

4 Neuroendocrine cells
The granular cells of the GI tract, airway, and genitourinary system produce a variety of peptide factors, some acting locally in a paracrine mode, others maybe having more distant effects. A common denominator is the presence along with the peptide(s) of certain materials in the dense-cored granules, e.g., chromogranin A or B, which provide markers for histopathologists seeking to find these relatively rare and dispersed cells.

H HEART, PANCREATIC ISLETS, TESTIS, OVARY and PLACENTA

Chapters 15.C.7, 25.A.5; 28.A.3; and 29.G.4 respectively.

Chapter 28 MALE REPRODUCTIVE SYSTEM

Male reproductive organs form spermatozoa, suspend them in secretions produced by accessory glands, and conduct them, via seminal pathways, to the female reproductive tract by mating behaviour. These activities are influenced by hormones, including ones formed by the testes. . Powerpoint

A TESTIS

l General morphology
l Very dense CT capsule - tunica albuginea, with an outer mesothelium-covered visceral tunica vaginalis propria.
2 Septa/septula extend from the capsule to the CT mediastinum.
3 In the partitions thus formed (lobuli testis), lie looped, coiled seminiferous tubules, lined by germinal epithelium, and feeding via straight
4 tubuli recti into cuboidal epithelium-lined ducts of the
5 rete testis, which lead through the mediastinum to roughly 6-l2
6 ductuli efferentes. These take the spermatozoa to a
7 single, coiled, tubular epididymis lying behind the testis.
8 Between, and outside, the coils of a seminiferous tubule lie blood and lymph capillaries, cells and fibres of CT, and hormone-secreting Leydig interstitial cells.
9 The testis is a mixed endocrine and compound, tubular, cytogenic exocrine gland.

2 Seminiferous tubule and spermatogenesis
l The tubule has a substantial support of the basal lamina, plus two or more alternating layers of collagen fibres and muscle-like/myoid cells, with adherent external lamina.
2 The stratified germinal epithelium has cells of two kinds:

3 Spermatogenesis in the epithelium is initiated by the pituitary hormone FSH, and passes through these stages: The stages are not all seen at any one place in the germinal epithelium; various combinations exist and are distributed as a mosaic in the tubule's wall.

4 Spermatogenesis is vulnerable to heat, X-rays, dietary deficiencies, pesticides, and other poisons. Conventional microscopy reveals defects in sperm shape and motility, leading to infertility. FISH and other molecular techniques are needed to assess genetic damage, sometimes arising during meiosis.
Spermatogenesis is protected to a degree by the tight attachments between the capillary endothelial cells and, separately, between the Sertoli cells, creating a two-tiered blood-testis barrier, for example, against immune attack. The inner protected compartment of the seminiferous tubule is the 'adluminal' compartment.

5 The spermatozoon is a very elongated motile cell, with a cell membrane enclosing the:

6 Spermiogenesis - whereby the spermatid, a typical cell (except for its chromosomes) becomes a spermatozoon - involves:

7 Sertoli cell functions: to protect, nourish, and release the spermatids; to phagocytose residual bodies; and to make androgen-binding protein, fluid, and inhibin to influence pituitary FSH release.

3 Endocrine testis
l Leydig cells, eosinophilic, with much smooth ER, lipid droplets, and crystals of Reinke, lie outside the tubules' BLs, constituting a diffuse, steroid-secreting endocrine gland.
2 Leydig interstitial cells are controlled by gonadotrophic interstitial cell-stimulating hormone (ICSH/LH) of the anterior pituitary, and produce the androgenic hormone - testosterone, responsible for:
3 (a) spermatogenesis; (b) development and maintenance of reproductive ducts and accessory glands; (c) secondary sexual characteristics; (d) male mating behaviour; (e) general anabolic effects on metabolism.

B PATHS TRAVERSED BY SPERMATOZOA

l Efferent ducts/Ductuli efferentes
l Unevenly lined by simple, columnar, epithelial cells, in groups of tall ciliated and short secretory; the wall has circular smooth muscle;
2 functions - reabsorption of the fluid used to move sperm out of the testis; maturation of the sperm.

2 Epididymis/ductus epididymidis
l Regularly lined by tall, absorptive, columnar cells with non-motile stereocilia, and smaller basal cells, together forming a pseudostratified epithelium;
2 outside the BL is a little smooth muscle and, between the coils, is a stroma of dense CT with capillaries;
3 functions - as for ductuli efferentes.

3 Ductus deferens/vas deferens
l Lined by an epithelium similar to that of the epididymis, on a lamina propria; in the ampulla, this mucosa has many folds;
2 most of the very thick wall is smooth muscle: inner, longitudinal; middle, circular; outer, longitudinal;
3 adventitia of CT binds it to nerves, blood and lymphatic vessels, and the skeletal cremaster muscle, to comprise the spermatic cord;
4 function - rapid transport of sperm during ejaculation, under sympathetic control.

4 Ejaculatory ducts
l Each occurs after a dilation of the ductus d. - the ampulla;
2 lined by pseudostratified or simple columnar epithelium on CT, without smooth muscle.
3 Ducts open into the prostatic urethra through a hillock on the posterior urethral wall - verumontanum/colliculus seminalis, with its blind recess - utriculus masculinus.

5 Urethra
l Three portions; prostatic, membranous, and cavernous;
2 more details - Chapter 23.B.4

C MALE ACCESSORY GLANDS

l Prostate gland
l Lobulated by septa of CT, with much smooth muscle.
2 Divisible, with histology and rectal-probe ultrasound, into several zones:
.. peripheral (prone to cancer),
.. transitional,
.. central,
.. peri-urethral (subject to benign prostatic hypertrophy), and
.. an anterior non-glandular fibromuscular zone.
3 Large-lumened secretory acini are lined by pale columnar or cuboidal epithelial cells, on a BL. Epithelium is patchily pseudostratified, i.e., bearing some small basal cells.
4 Acini open into many ducts, entering the urethra individually, thus the prostate is a collection of compound tubuloacinar glands.
5 Laminated, rounded, prostatic concretions (originally glycoprotein, but later calcifying) - corpora amylacea - develop in some acini as age increases.
6 Functions - secretion of a watery fluid to dilute the semen; the protease - prostate-specific antigen (PSA) - liquifies the gel from the seminal vesicles to free the sperm; the roles of the citrate (the anionic counterpart to Na+) and acid phosphatase are uncertain.
7 PSA serves as a serum marker of prostatic cancer, if excessive for the man's age.
8 The stroma has abundant smooth muscle to make the prostate a self-squeezing gland, without the need for myoepithelial cells. Stroma interacts with the epithelium in the control of growth and secretion, and is a major player in benign prostatic hypertrophy.

2 Seminal vesicles
l Coiled, convoluted, tubular structures; with a
2 very extensively folded mucosa, having
3 a pseudostratified, columnar, secretory epithelium.
4 The wall has circular and longitudinal smooth muscle, and a thin, outer, fibro-elastic adventitia.
5 Functions - secretion of a viscid gel composed of seminogelin, with fructose to provide energy for the sperm, and prostaglandins that may alter contractions in the female tract.

3 Cowper's bulbo-urethral glands
l Compound, tubulo-alveolar gland making special mucus, thought to
2 lubricate and prepare the urethra for ejaculation.

D PENIS

l The thin, elastic skin of the shaft is loosely attached.
2 Connective tissue capsules or tunicae albugineae enclose
3 three roughly cylindrical erectile bodies - two corpora cavernosa penis, and one corpus spongiosum/cavernosum urethrae.
4 The two corpora cavernosa are incompletely separated by a sagittal pectiniform septum. Their endothelium-lined venous sinuses, between a meshwork of dense trabeculae of muscular CT, can be engorged with blood from helicine (coiled) arteries causing erection.

5 Corpus spongiosum

6 Erection and detumescence are controlled by autonomic nerve fibres to the arteries and trabecular smooth muscle. Erection results from parasympathetically directed trabecular and arterial relaxation, and passive occlusion of the veins draining the corpora.
Sensory nerves serve the glans, skin and deep receptors.
7 Functions - urination/micturition; copulation.

E MALE & FEMALE REPRODUCTIVE DEVELOPMENT

1 The primordial germ cells (prospective gametes) migrate to the gonadal ridges, then a system of dual paired tubules develops, to be either the male or female reproductive tract. Why dual sets of tubules?
2 The para-mesonephric/Müllerian ducts provide the default pathway to turn into female organs.
The mesonephric/Wolffian ducts furnish the driven pathway to a male tubular system.
3 How is the choice made?
The male is male by virtue of the Y chromosome, bearing the SRY gene for the human testis-determining factor (SRY - Sex-determining Region on Y).
The sequelae of the protein expression of SRY are:
.. (i) The indifferent gonad becomes a testis, with Sertoli and Leydig cells. Products of these cells act, gardening-style, as weed-killer and fertilizer.
.. (ii) Sertoli cells make Müllerian-inhibiting factor (MIF), which causes the apoptosis and degeneration of almost all the Müllerian duct (MD).
.. (iii) Leydig cells' testosterone boosts the growth and differentiation of the mesonephric/ Wolffian duct (WD), to make the male tubules - efferent ducts to ejaculatory ducts, and the seminal vesicles.
.. (iv) Testosterone, as 5a-dihydrotestosterone (DHT), also:
(a) converts the urogenital sinus into the male urethra and prostate;
(b) drives the external genitalia into male forms: larger phallus, urethra through the phallus, scrotal halves fused, etc. (The female-male homologues from embryology are needed to understand and correct inter-sex pathologies, seen in the newborn.)
.. (v) In the foetal girl, the Wolffian duct, left without testosterone, withers, while the Müllerian structures continue development.

4 Outcomes of successful sexual development


(OVARY)          UTERINE TUBE           UTERUS        VAGINA      - MD
epoöphoron, paraoöphron                         Gärtner's cyst    - WD


appendix testis                                  prostatic utricle - MD
(TESTIS) EFFERENT DUCTS  EPIDIDYMIS  VAS DEFERENS EJACULATORY DUCT - WD
                                                  SEMINAL VESICLES
In lower case, are the epithelial-lined vestiges of the opposite sex's unneeded duct system. Note that the paradidymis is a remnant of male tubules in the male: surplus efferent ducts. . Sexual Development Powerpoint

5 Problems of sexual development can arise at several points, thus:
.. (i) Absent or faulty SRY gene in the male;
.. (ii) Failure of testis cells to respond to the gene's product;
.. (iii) Absent or defective MIF gene; or problems in the MD's response;
.. (iv) Leydig-cell failure to make and deploy the enzymes to produce testosterone;
.. (v) Defective or absent androgen receptor in the Wolffian-duct and external-genital targets for testosterone (J.2)

Meiosis provides an opportunity for such genetic defects to arise.

F MEIOSIS

Ma & Pa meiosis
Aim: from one primary spermatocyte to produce four spermatids, each with:
.. (i) 23 chromosomes (haploid number);
.. (ii) each chromosome derived from either ma or pa - random assortment;
.. (iii) but, with bits of pa's chromosome replacing some of ma's, and vice versa - crossing over.
.. (iv) genetic diversity is the goal, with crossing over (genetic recombination) providing far more diversity than the random assortment of m & p chromosomes
.. (v) Also, think perpendicular quartering cuts for how the cells 'divide' in relation to their chromosomes:
                     ___|___            ___.___
                    |   |   |          |   .   |
                    |   |   |        ------.-------->
                    |___|___|          |___.___|
                        |
                        v
                                                                                                                 m
1º Spermatocyte                                              m                               3-----------------M }
                                         3-----------------M }                             p                     |
3-----------------M       DNA            3-----------------M }    maternal-paternal        {3-----------------M  |
                       ------>                               p     --------------------->  {3*****************P  |
3*****************P     replication      3*****************P }    homologue pairing                              |
                                         3*****************P }                         /     3*****************P }
maternal &                                                                          /
paternal #3 chromosome                   each chromosome now                     /         bivalent for
                                         a pair of chromatids         }       /            crossing-over of
                                         held together by a centromere}    /               aligned chromatids
                                                                        /                  -- for ** 
                                                             DNA excision
                                                             & ligation  
             p                                            <            8 
             { 3---**************M                  <
             { 3-------------****P              <
                                    m       <
               3***----------****M }                                            
               3*****************P }
                                                                                 3***--------------M
                Meiotic division I

   2º spermatocyte           2º spermatocyte                                     3-------------****P

p                                              m     Meiotic division II        
{ 3---**************M     3***----------****M }     --------------------->         4 spermatids
{ 3-------------****P     3*****************P }     centromere splitting
                                                                                 3***----------****M 
                 /          \
                /            \
          random assignment# of maternal                                         3*****************P
          & paternal chromosomes                                                 
          (disjunction), e.g.,

         1 p                   1 m
         2 m                   2 p
         3 p                   3 m
         4 p                   4 m
         5 m                   5 p
         etc                   etc


Sources of trouble . #                                     8
wrong assignment of chromosomes (nondisjunction), e.g.,   |imprecise DNA exchange
2 #21's to one 2º spermatocyte results in one spermatid   |at recombination
having 2 #21's. If, as a sperm, this is the unlucky       |disrupts or cuts out genes
fertilizer, its 2, combined with the 1 from the oocyte,   |
= trisomy 21 in the zygote and eventual child.            |
The Fig. cannot do justice to the intimacy, tight spacing, and connections of synapsis and crossing over, but does convey the outcomes and vulnerabilities. It owes the chromatid recombination pattern, after the reductional and equational divisions, to GS Roeder Genes & Devel 1997;11:2600-2621

Chapter 29 FEMALE REPRODUCTIVE SYSTEM

This is a tubular system for the production of ova, and the reception of spermatozoa, their transport and union. It accommodates the fertilized oocyte and ensuing fetus, then expels the fetus at term. The ovary and placenta also have hormone-secreting functions, for instance, to prepare the uterine mucosa to receive, accept, and sustain the fertilized oocyte. Mammae are modifications of the skin for feeding the infant. Powerpoint
.

A OVARY

l General structure
l Covered by mostly simple epithelium (variably columnar, cuboidal, or squamous),
2 under which is a loose CT, a nominal capsule - tunica albuginea.
3 Has a stroma of atypical fibroblasts; collagen, as reticular fibres, is present, but not a dominant element; and stromal cells secrete hormones.
4 A fold of peritoneum, the mesovarium, connects the ovary at its hilum to the broad ligament, and sends many blood vessels to the fibrous, central, medullary, region of the ovary.
5 Peripheral, cortical, regions have many primordial and primary follicles, maturing Graafian follicles, which shed the ova (to be fertilized in the upper third of the Fallopian tube), and glandular masses.
6 Certain vestigial structures remain after development has ceased. These take the form of blind epithelium-lined tubules - epoöphoron and paroöphoron - lying in the broad ligament by the ovary.
7 Hilar stromal cells may include hormone-secreting hilus cells, resembling testicular Leydig cells, which occasionally give rise to tumours causing a hyperandrogenic syndrome in the woman.

2 Ovarian events and structures Fig. 11

Primordial follicle with primordial germ calls migrated from the yolk 
            |                   .                                sac
            |                   .
  Primary follicle of           .        At any time during development
    oocyte surrounded by        ........ a follicle can degenerate, and 
    follicular cells            .        most do, becoming an
            |                   .                        |
            |                   .                        |
develops under stimulus of      .                 Atresic follicle
FSH of anterior pituitary       .                        |
            |                   .                        |
  Graafian follicle             .                        |
large, fluid-filled, cyst-like  .            followed by invasion by
            |                   .            vessels and CT; if a theca
            |                   .            interna was present, it
grows, extends to the surface of             forms a temporary glandular
ovary, and bursts at mid-cycle               tissue - the
         /                |                              |
        /                 |                        Interstitial gland
shedding ovum       Empty cavity collapses        more obvious in rodents
to be caught        Surrounding cells grow        than woman; other names are
by the nearby       large and glandular,          corpus atreticum &
   /                forming, under the influence  pseudolutein body
  /                 of pituitary LH, a
Fallopian tube            |
                          |
                      Corpus luteum
                       /             \
                      /               \
          grows considerably         grows for 12 days
          under the influence of     only, if the oocyte
          placental hCG, if the      is not fertilised
          oocyte is fertilised              |
                   |                        |
                   |                        |
                   |                        |
            Corpus luteum             Corpus luteum 
            of pregnancy              of menstruation      
                    \                     /
                     \                   /
                      \                 /
                    Glandular cells degenerate, and
                    fibroblasts form a pale scar-like
                          \         /
                           \       /
                            \     /
                       Corpus albicans
                      formed while the cycle starts again 

3 Hormonal background
l Dealing with changing structures, either developing or degenerating; with marked changes in events and appearances at the menarche, when ovarian cycles begin, and the menopause, when they end.
2 The constant physiological change makes difficult recognizing pathological changes, e.g., uterine bleeding. Female reproduction is a considerable burden in its energy demands, e.g., for fat storage and lactation, which can only be met on an intermittent, i.e., cyclic, basis.
2 FSH and LH/ICSH are pituitary gonadotrophins - hormones with the gonads as their target organ.
3 Corpus luteum is also influenced by hormones produced by the placenta, if fertilization has occurred - chorionic gonadotrophins.
4 Distinguish between hormones acting on the gonads, and those produced by the gonads and acting on other organs, e.g., uterus.

4 Ovarian/menstrual cycle (with details not included in Fig. 11)
l Maturation of oöcyte

2 Development of follicular/granulosa cells and follicle

(A caution for the primary-secondary-tertiary staging of follicles: ask in your own setting how these terms and 'Graafian' are to be applied.)

3 Changes in stroma around maturing follicle

4 Ovulation

5 Corpus luteum: formation, function and fate

6 Signs of follicular atresia (aborted development)
.. (a) Granulosa lining breaks up and sheds apoptotic cells into the antrum.
.. (b) Follicle's wall collapses; vessels and CT cells invade.
.. (c) Basal lamina thickens to become a 'glassy membrane'.
.. (d) Oocyte's nucleus shrinks and becomes pyknotic.
.. (e) Zona pellucida folds in, as the oocyte degenerates.
.. (f) Theca interna cells enlarge, becoming more glandular to form a temporary interstitial gland.

B FALLOPIAN/UTERINE TUBE (oviduct)

l Four parts: (a) infundibulum with the fimbria - a fringe of processes, engorgeable with blood and moved by smooth muscle to catch the oocyte, (b) wide ampulla, with a cell-ensnaring labyrinth of protruding mucosal processes, (c) narrow isthmus down to the uterus, and (d) an intramural/ interstitial section through the uterine wall.
2 Lined by a highly folded mucosa, comprising a cellular lamina propria covered by a simple columnar epithelium of
3 columnar ciliated cells, and secretory cells, varying in height and secretory activity during the menstrual cycle. Secretion is more in the late oestrogen phase around ovulation than in the post-ovulatory progesterone phase. Cilia beat toward the uterus.
4 Muscularis of inner, circular, smooth muscle, and a few outer, longitudinal bundles.
5 Covered outside by a serosa, with nerves and blood vessels.
6 Functions - meeting place for sperm and oocyte; helps 'capacitation' of sperm to their most energetic and zona pellucida-penetrating state; nourishes and transports the zygote.

C UTERUS

l Outer serous coat (perimetrium), with vessels, nerves, and ganglia.

2 Myometrium of interwoven smooth muscle, capable of a great hypertrophy during pregnancy, with many blood vessels in the middle stratum vasculare.

3 Mucosa/endometrium with:
l simple, columnar, epithelial lining (some cells ciliated);
2 simple, tubular mucous glands;
3 loose vascular stroma of special fibroblasts, reticular fibres and much ground substance; some stromal cells can become decidual around the implantation site;
4 helicine/coiled spiral arteries, a capillary bed, and veins.

4 Mucosa of the sexually mature woman experiences cyclic menstrual changes, involving all elements and considerable changes in mucosal thickness, and driven hormonally by the ovary:
l Oestrogens, e.g., oestradiol, from the growing follicle cause cell proliferation, and an increase in endometrial height.
2 Progesterone, formed by the corpus luteum, then increases cell secretion and glycogen accumulation, and the stroma dilates with fluid. The glands coil and sacculate. Spiral arteries continue to grow up towards the surface.
3 Helicine arteries rhythmically constrict, then dilate, inducing menstruation or breakdown of the endometrium, altered in the last few days of the secretory phase by a reduction in progesterone level, and by cytokine signals for cellular apoptosis. This sloughing of the functional layer of the endometrium is unaccompanied by blood clotting.
4 Regeneration (physiological) takes place from the basal layer of the endometrium, where the epithelium survives at the bottom of the glands.
5 The mucosa may experience these cyclic changes minimally, even though no oocyte was shed from the Graafian follicle - an anovulatory cycle.

5 Uterine cervix differs from the corpus thus:

  1. It has more collagen and elastic in the wall than muscle.
  2. Mucosa is furrowed by complex clefts - plicae palmatae; and does not participate in menstruation.
  3. Lining columnar epithelial cells produce a mucus, richly hydrated and penetrable at mid-cycle.
  4. Epithelium changes to stratified squamous on the portio vaginalis.
    The boundary between simple columnar and stratified squamous epithelia is unstable, and shifts position by a process of columnar-to-squamous conversion. This transformation zone is prone to dysplasia, then malignant change, which can be detected early by examining 'Pap' smears.

D VAGINA

l Adventitia of CT, with abundant nerves and blood vessels, merges with some longitudinal and a few circular smooth muscle bundles, around a wide collagenous lamina propria. All these layers loosen in gestation.
2 Epithelium is stratified squamous, rich in glycogen (to promote the growth of benign lactobacilli in the lumen), and influenced by gonadal hormones, but not to the degree seen in rodents.
3 Mucosa has transverse folds or rugae, and may have lymphoid nodules, but is without glands.

E EXTERNAL GENITALIA/VULVA

l Labia majora and minora, vestibule and hymen - skin, or stratified squamous epithelium on a loose, fatty or vascular lamina propria.
2 Clitoris and vestibular bulbs - erectile tissue.
3 Sensory receptors are distributed widely in the clitoris, vestibule and labia.
4 Bartholin's glands - mucus-secreting, compound, tubulo-alveolar - are homologues of the male Cowper's glands. Other, minor, vestibular, mucous glands lie near the urethra and clitoris.

F MAMMARY GLAND/BREAST/MAMMA

l Structure
l A collection of compound, tubular (tubulo-alveolar, when active) glands grouped around the
2 nipple, where the lactiferous duct of each gland opens.
3 Glands are in lobes, separated by dense interlobar CT.

4 In each lobe are:

5 Lactiferous ducts are lined successively by cuboidal, columnar, stratifed columnar, and stratified squamous epithelia. Each duct widens below the nipple into a sinus.

2 Nipple
l Cornified stratified squamous epithelium covers a stroma of elastic fibres, smooth muscle, and collagen, through which pass the lactiferous ducts.
2 Epithelium is continuous with the somewhat pigmented, glabrous (hairless) epidermis of the surrounded areola, with its sebaceous glands and high dermal papillae.
3 The many autonomic nerve fibres to the nipple's smooth muscle control its rigidity for suckling, and the relaxation of the milk sinuses.
4 Numerous sensory receptors and nerve fibres are present.

3 Histophysiology
l Prepubertal period
.. (a) Nipple remains small and weakly pigmented.
.. (b) Glands stay rudimentary as multiple, branched, tubular units in a CT stroma.
2 Puberty
.. (a) Oestrogen promotes ductal growth, and the formation of stromal adipose tissue.
.. (b) Increasing levels of progesterone cause some alveoli to bud out from the duct ends.
3 Early pregnancy
Progesterone and oestrogen cause a marked epithelial proliferation, with increased branching of ducts, which bud out and form many alveoli; these form at the expense of stromal tissue.
4 Late pregnancy and post-parturition

5 Lactation
(a) Numerous white blood cells infiltrate the stroma; some of which
(b) on gaining access to alveolar lumens, phagocytose the secretion and become colostrum bodies, seen in the first few days after parturition.
(c) The actual release of milk depends on the stimulus of suckling, acting on receptors in the nipple, which inform the brain to liberate pitocin (let-down hormone) from the pituitary's posterior lobe. This hormone makes the myoepithelial cells of ducts and alveoli contract.

6 Post-lactational regression and post-menopausal involution

G PLACENTATION

The placenta, with the umbilical cord and uterine mucosa, provides for the physiological exchange of oxygen, nutrients, and waste materials between the fetal and maternal circulations across the placental barrier, which protects the fetus from some infections. The placenta also performs metabolic transformations and synthesizes chorionic hormones: gonadotrophin, prolactin, oestrogen, progesterone, etc. . Placenta Powerpoint

l Fertilization and blastocyst formation
l Oocyte, when penetrated by a spermatozoon (by enzymatic action), completes its second meiotic/maturation division, with the formation of another polar body.
2 After one entry, the zona pellucida reacts, becoming impenetrable by other sperm.
3 The condensed sperm head turns itself into the male pronucleus, with reconstitution of the nuclear membrane and lamina, and of the chromatin.
4 After DNA replication in male and female pronuclei, these fuse, and male and female chromosomes pair up to give the diploid 46.
5 Over roughly four days, the zygote passes down the uterine tube, dividing to form a solid mass of cells - the morula.
6 Fluid accumulates amongst the cells resulting in a blastocyst.
7 Blastocyst remains free in the uterine lumen for another two or so days.
8 Blastocyst has an:
.. (a) outer shell of trophoblastic cells;
.. (b) inner cell mass to become the embryo;
.. (c) outermost zona pellucida.
9 Blastocyst sloughs off the zona pellucida, and implants in the glandular uterine mucosa.
10 Occasional ectopic sites of implantation are the Fallopian tube, peritoneal cavity, and ovary.

2 Implantation/nidation
l Trophoblastic cells, coming into contact with the uterine epithelium, attach, proliferate, and invade into the stroma.
2 The blastocystic structure sinks in deeper to become covered by mucosa (uterine mucosa is henceforth termed decidua).
Mucosa under the blastocyst is decidua basalis; overlying it is decidua capsularis; opposite, across the uterine lumen, is the decidua parietalis/vera.
3 Trophoblast encircles the embryonic germ disc, with its amniotic cavity, yolk sac, and exocoelom.
4 Trophoblast has an inner layer of distinct cells - cytotrophoblast - and an outer layer of fused cells - syncytiotrophoblast.
5 Syncytiotrophoblast extends out, interrupting maternal blood vessels, thereby spilling blood into lacunar spaces within its own mass.

3 Formation of the placenta
l Trophoblastic layer proliferates, and takes on an additional innermost mesenchymal layer, to constitute the chorion.
2 From the chorion, cords of trophoblasts extend out as primary chorionic villi.
3 Mesenchyme of the extraembryonic mesoblast grows down inside these, converting them to secondary villi.
4 The villi extend into spaces (intervillous) filled with maternal blood, replenished via the uterine arteries and veins.
5 Peripherally, the distal tips of anchoring villi, composed of cytotrophoblasts, unite to form a layer - trophoblastic shell - covering the uterine decidua cells (except at the openings of maternal blood vessels).
6 Fetal blood vessels then appear in the cores of the villi, making them tertiary, or definitive, placental villi.
7 These fetal vessels connect with the body stalk that will form the umbilical cord to the embryonic vascular system.
8 Later, the villi that have grown all over the chorion: (a) grow and branch basally to form the chorion frondosum, and eventually the discoidal placenta;
(b) over the rest of the surface towards the uterine cavity, villi shrink and disappear (followed by the decidua capsularis) leaving the smooth chorion laeve.
9 Think of the placenta as two frisbies - chorionic plate and basal plate - set against each other, face-to-face, creating a space for the maternal blood and foetal villi.
On slides, the chorionic plate resembles umbilical cord, but on the uterine side the thin basal plate has separated from the myometrium.

4 Placental villi
l Free villi branch out extensively from each stem villus. The whole branching unit constitutes one of the fetal cotyledons.

2 Each tertiary villus has an:

3 Syncytiotrophoblast is unique in combining these talents:
.. (a) being invasive;
.. (b) forming both steroid and peptide hormones;
.. (c) performing metabolic transformations;
.. (d) participating in a barrier;
.. (e) absorbing and transporting materials.

5 Maternal-fetal junction
l Trophoblastic shell becomes the foetal part of the basal plate, in contact with the maternal decidua basalis.
2 Plate subdivides into units - maternal cotyledons - separated by septa, with perforations allowing some lateral passage of blood.
3 Blood spurts up out of the basal plate from spiral arteries into the intervillous space, and drops down into venous outlets.
4 The chorionic plate faces the basal plate as the other boundary to the space for maternal blood. Just below the chorionic plate, where the septa do not reach, is the open space of the subchorial lake.
5 Cytotrophoblasts persist in the basal plate, as cell islands, and in the septa, and are often embedded in an eosinophilic, non-fibrous, intercellular material - fibrinoid.
Basal fibrinoid provides a cleavage plane for separation of the placenta at term.
6 Maternal decidua cells are large, with lipid and glycogen, lie in a rich ground substance with reticular fibres, and perform steroid conversions.
7 Granulated endometrial-gland cells (K/Körnchenzellen) are an endometrial kind of leucocyte, with acidophil granules. Present in the late secretory phase, their numbers increase in the first-trimester decidua, so they may somehow assist implantation.

6 Umbilical cord
l Enveloped in amnion and covered by simple, cuboidal, amniotic epithelium, it consists of mucous CT - Wharton's jelly: a gelatinous ground substance, with sparse collagen bundles and stellate fibroblasts.
2 Located in the jelly are three umbilical blood vessels:
.. (a) One vein, with much muscle, but without valves and vasa vasorum.
.. (b) Two arteries, with thick, inner, longitudinal, and outer circular muscle coats, no internal elastic lamina, and an insignificant adventitia.
3 The jelly, the thick vessel walls, and their spiral course prevent kinking and occlusion of the vessels.
4 Remnants of the (a) allantoic endoderm, and (b) yolk sac's stalk, with vitelline vessels, may persist until quite late in pregnancy.


Chapter 30 TECHNIQUES OF EXPERIMENTAL MORPHOLOGY

The histology so far has given details of the appearance of dead cells, tissues and organs, and of their functions, but this functional knowledge cannot be derived from the microscopic examination of a single fixed specimen. Cell function is learned by applying microscopy is such experimental situations as follow.
. Going over methods of morphology conveys the idea of histology as an active science, helping solve basic and clinical problems. Unfortunately, a factual, note-style format does a disservice in not contributing to another aspect. Outlining techniques gives no feel for the worth of the results of their application as evidence for the descriptions and stories of histology.
Plausible though they may sound, hypotheses of function must, above all, satisfy the evidence, old and new, and be appraised with an ever alert and sceptical mind.

A COMPARATIVE CYTOLOGY

l Interspecies comparisons
Examples:
l The many brown fat cells in hibernating mammals suggested that brown fat is related to the hibernating state, or to exposure to cold.
2 The all-cone retinas of diurnal mammals with sharp vision, e.g., squirrels, implied that cones provide for high acuity.

2 Changes in the number of cells
Examples:
l Normal and physiological
An increase in the number of anterior pituitary alpha cells in pregnancy suggested that they secrete a substance needed by the pregnant or post-partum woman - prolactin hormone.
2 Pathological
A decrease in the number of anterior-pituitary acidophil (epsilon) cells in one kind of dwarfism, relative to those seen normally, implied that they secrete something stimulating growth - somatotrophin.

3 Changes in individual cell morphology
Examples:
l Normal and physiological
The cross-banding patterns of contracted and relaxed skeletal muscle fibres were evidence for the 'sliding-filament' theory of contraction.
2 Experimentally manipulated
Numbers of granules in rat pancreatic-islet beta cells, before and after the administration of diabetes-inducing alloxan, indicated a secretion and storage of the hormone, insulin, by these cells.

B STAINS AND METHODS FOR SPECIAL STRUCTURES

Before the influence of physiological variables on certain structures can be recognized, ways must be found of demonstrating the structures clearly.

Further detailed information on histotechnique (including TEM methods and images) can be found under "Histopathology' at E Klatt's WebPath - U. Utah

l Special staining techniques exist for many structures and materials.
l Methods have been given for blood (Chapter l7.A), bone (Chapter 7.D), and nervous tissues (Chapters 10.C.3 and 11.F.l.).
2 A need for special stains has been mentioned for epithelial cell outlines, basal laminae, ground substances, elastic and reticular fibres, osteoid, melanocytes, bile canaliculi, neurosecretion and other stored secretions, Golgi body, mitochondria, etc.
3 By and large, transmission EM has superseded the LM staining of cell organelles, but many other special stains, including ones for bacteria and other pathological things, e.g., amyloid, are still used.

2 Blood vessels within an organ can be revealed by:
l Injection of the vessels with red carmine-gelatin, which is allowed to set, before thick sections are cut for microscopy.
2 Injection of the vessels with a coloured resin that sets in, maintains, and reveals the vascular pattern, after the organic tissue is destroyed - corrosion cast method. The cast can also be viewed by SEM.
3 Injection of the vessels with a radio-opaque suspension, which makes them visible with magnified roentgenography - microangiography.
4 A capillary bed can be demontrated by immunostaining for endothelium (e.g., for CD31), and viewing the thick section with laser confocal scanning microscopy.

3 Chemical basis for structural staining
It is the chemical nature of a structure that permits it to be stained by a particular stain. Its protein and other materials contain groups which bind the staining chemical.
l Thus a protein may have active carboxyl (COO-) or phosphoric (HPO42-) groups, able to bind covalently with the basic chromophoric ions, of, for example, methylene blue (tetramethylthionine chlorhydrate).
2 The same protein, at a lower pH, may have active amine (NH3+) groups which can bind acidic stains, e.g., potassium eosinate.
3 Because of this amphoteric character of proteins, pH thus determines which stain reacts with a particular protein, and also the intensity of the staining. For instance, raising the pH increases the staining by basic stains.
4 At a pH of around 6, some proteins are acidophilic, others and the nucleic acids are basophilic. Hence the use of combined stains, with two or more ingredients to reveal more structures.
5 Even though the staining pH may be increased above 6, certain materials, e.g., haemoglobin of red blood cells and granules of eosinophil white blood cells, continue to give an acidophil reaction. Oxyphil, oxyntic, eosinophilic, and acidophilic are used synonymously for cells or components behaving in this way.
6 Mordants are used as an intermediary for some stains to effect an indirect union between tissue groups and radicals of the dye, e.g., for haematoxylin's active derivative haematein.

4 Orthochromatic and metachromatic staining
l Orthochromatic staining is usual. The structure stains with the colour of the stain employed, e.g., collagen, green with light green.
2 Metachromatic staining (Chapter 5.C.6) is seen with some materials, e.g., cartilage matrix. The dye, say toluidine blue, combines with the sulphated proteoglycan in such a way that the dye molecules aggregate, causing a colour change from blue to reddish-purple.

5 Progressive and regressive staining
l Progressive staining leaves the section in the dye until it is adequately coloured.
2 Regressive staining overstains the tissue, then the excess stain is removed or differentiated out of the section by a solvent or oxidizing agent.

Specific and selective staining
l Specific staining shows just one structure or material.
2 Selective staining preferentially stains one structure or material; others are stained, but less strongly.

7 Vital staining involves the injection of materials into a living animal to reveal, say, macrophages (Chapter 5.A.4), or newly formed bone matrix (Chapter 7.F.5). Supra-vital staining is applied to live cells held briefly in culture, see below (l.3.l).

C STAINS FOR SPECIAL MATERIALS: HISTOCHEMISTRY

The chemical properties of materials, proteins in particular, have been used to reveal structures more clearly by selective staining. Another interest lies in the distribution and concentrations of chemicals, as materials per se, being used, stored, transported, altered, and secreted by cells. The histochemical and cytochemical approaches permit the visual localization of certain substances within tissues and cells, respectively.

l Fat
l Sudan dyes dissolve in fat, preserved by frozen sectioning, and colour it. This does not involve ionic combination.
2 Osmium tetroxide forms a black complex with unsaturated fat and, at the same time, acts as a fixative.

2 Glycogen, glycoproteins and proteoglycans
l These have

              H     H                    H     H
              |     |                    |     |
            - C - - C-       or        - C - - C -    linkages that
              |     |                    |     |
              OH    OH                   OH    NH2

                                         H
                                        /
2  are oxidized by Periodic Acid to  - C    (aldehyde) groups 
                                        \\   
                                         O  

3  which restore colour to Schiff's reagent (hence PAS technique).
4 Colourless Schiff's reagent/leucofuchsin is prepared by bleaching basic fuchsin with sulphurous acid.
5 Glycogen may be distinguished from glycoconjugates by a pretreatment with saliva, whose enzyme, amylase, will destroy only glycogen.
6 PAS method also shows the glycoproteins of BLs and the glycocalyx, and some mucins.
7 For EM, ruthenium red makes some glycoconjugates electron-dense (not red) and visible.

3 Deoxyribose-nucleic acid
l DNA of the nucleus could be shown by the Feulgen reaction:
2 mild acid hydrolysis unmasks aldehyde groups of the DNA, but leaves the RNA unchanged.
3 Schiff's reagent then reveals these free aldehyde groups.
4 A control is provided by repeating the procedure after a pretreatment with deoxyribonuclease, the enzyme known to remove specifically DNA.

4 Enzymes
l Enzymes require careful preservation by various methods:

2 An enzyme acts on another material, its substrate, to cause the separation or addition of groups, e.g., phosphate ions may be separated from a phosphoric ester of glycerol by a phosphatase. 3 Localization may be imprecise because of diffusion of the enzyme or coloured marker from the site of enzymatic activity. Thus, it is better to use more than one method to see if two give the same distribution pattern for the enzyme.

4 Electron-microscopic control for cell fractions

5 Inorganic materials
l To show some of the ferric iron present, e.g., in liver, by its formation of Prussian blue with ferrocyanide in solution.
2 For calcium, see I.2.7 below.

6 Actin
l When EM shows cytoplasmic filaments 4-7 nm thick, treat a section with a solution of heavy meromyosin (HMM).
2 If the filaments are F-actin, they should bind HMM at regular intervals, and oriented away from any Z lines or densities. The filaments thus become 'decorated' with arrowheads in EM.
3 Current methods of choice are immunostaining, or the use of phalloidin which binds actin, and can be conjugated to rhodamine for fluorescent visualisation.

D FLUORESCENT TECHNIQUES

Certain substances, when exposed to ultraviolet (UV) light, emit light of longer and visible wavelengths, i.e., they fluoresce. The fluorescence microscope illuminates the section with UV light, and its areas of fluorescence are viewed through eyepieces incorporating a UV filter to protect the eyes.

l Autofluorescence
Porphyrins and vitamin A are naturally occurring autofluorescent materials of interest.

2 Induced fluorescence
. Formaldehyde converts the catecholamines to fluorescent quinoline compounds. The UV microscopy of formaldehyde-fixed sections shows the distribution of norepinephrine, for example, in the sympathetic, post-ganglionic, nerve fibres and adrenal medulla. An APUD cell (Chapter 27.G), if given a suitable precursor, should form an amine, in which a formaldehyde-induced fluorescence can be shown.

Immunofluorescent visualization involves:
l The preparation of a pure sample of the material (peptide or polysaccharide), whose distribution in the tissues to be studied.
2 Injection of this substance into a rabbit, whose plasma cells will treat it as an antigen and produce antibodies against it. Testing the rabbit serum for antibody activity. Conjugation of the serum antibody with fluorescein isothiocyanate to make its position traceable, when viewed in UV light.
3 A better way is to fuse antibody-forming and malignant mouse lymphocytes in vitro and clone them. Kohler and Milstein's procedure thus taps, in combination, the potentials of antibody specificity and tumour growth, in the making of a monoclonal antibody (MoAb).
4 Treating, with the fluorescein-labelled antibody, a section from the animal or person in which the protein of interest may be present. The antigen will combine with and hold the antibody.
5 UV microscopy of the section, after washing out the uncombined antibody, reveals the location of the antigen, i.e., the material of interest, for instance, to show that a particular peptide hormone is in only a certain type of cell, already categorized by its staining properties and EM morphology, e.g., prolactin in acidophil anterior-pituitary cells.
In practice, for stronger binding and better visualization, the method employing a tagged secondary antibody is more frequently used than just one antibody.
6 The very strong bond between avidin and biotin is the basis for other very effective means of tagging reagents for immunohistology.
7 Immunostaining, with its high specificity and sensitivity, is used in electron microscopy by conjugating the antibody not with fluorescein, but with:
.. (a) ferritin, recognizable as granules; or
.. (b) a peroxidase that, when incubated with a substrate, gives a visible reaction product;
.. (b) gold particles, which do not react, are visible, and are of standardized sizes, so that two materials can be tested for at the same time, for any co-localization, or separate distributions.

E TRACERS FOR BARRIERS

l Around the body, several structures, in and around blood capillaries, combine to act physiologically as selective barriers, e.g., the blood-brain, blood-testis, blood-aqueous humour barriers, and the renal glomerular filtration barrier.
2 To find out which of the various structures holds back which macromolecules, probe or tracer molecules of known size and molecular weight, e.g., ferritin, horseradish peroxidase, etc., are injected into the blood.
3 Ultrathin sections of the organ are viewed by EM, after a pre-treatment to form a reaction product if the tracer is enzymatic, to see at which structure, say the BL, the tracer was hindered and had to accumulate. If gold particles of known size are used, they can be seen without this reaction step.
4 Tracer studies are applied to other routes of selective transport, e.g., in glands, and sites of absorption such as the gut.

F RADIOAUTOGRAPHY AND ELECTRON RADIOAUTOGRAPHY

Early histochemistry showed the distribution of certain materials in relation to cells and tissues. Electron histochemistry localizes certain compounds to organelles, and other structures within and outside the cell. But a knowledge of distribution alone is limited in its significance. It is desirable to know what is happening over a period of time to a substance: where it is being produced, from what, by what, where it is being transported, and how it is to be used.
One method for gaining this information is radioautography/autoradiography. It uses radioactive isotopes, e.g., 14C, 3H, 35S, 125I, combined in materials that will be processed, e.g., used in synthesis, by the cells, as they perform their activities. Examples:

l Fibroblasts and collagen
l Fibroblasts use the amino acid proline to form collagen.
2 If an animal is injected with proline, having some hydrogen replaced by tritium, this labelled or tagged proline is used by the fibroblast as if it were normal proline.
3 However, the tritium emits beta radiation, i.e., electrons. The presence of this radioactive emission can be shown by its action on the silver halide of a photographic emulsion, coating the histological section as a film.
4 The emulsion is exposed for several weeks to the radioactivity, before being developed and fixed.
5 Sites of concentration of radioactive material are marked by black grains, seen as black coiled threads in the EM. (The grains lying over particular structures may be counted.)
6 Thus proline, for example, can be followed into the fibroblast and its organelles, and then later into the collagen fibres themselves, by taking tissue, e.g., wound tissue, from animals at various times after the injection of the tagged material.

2 DNA-synthesis
l Another example, with very widespread application, employs an injection of tritiated thymidine.
2 This material is used in the synthesis of the deoxyribose-nucleic acid (DNA) of the nucleus that occurs during interphase, prior to cell division.
3 Tissue specimens taken after injection show radioactivity in only the cells experiencing DNA synthesis, while the tagged thymidine was circulating in the body.
4 The cells may migrate, which is shown by a change from their previous position.
5 This method also shows how mitotically active a particular tissue is, for instance, the gut epithelial cells are very active and move up from the crypts on to the villi, before being shed in only four days.
The technique also yields valuable data on the migrations and cell kinetics involved in the development (histogenesis) of tissues.
6 This radioactive approach is being supplanted by letting cells incorporate, as the thymidine analogue, bromodeoxyuridine, which can be recognized by a monoclonal antibody (MoAb).
In either case, the method is limited, if a continuation of cell division dilutes the marker until it is indistinguishable from background.

3 RNA-synthesis
Labelled uridine is injected for RNA; inadvertently labelled DNA can be removed by DNase before putting on the emulsion.

4 Rate of utilization of material
Persistence of a labelled material at a site indicates a slow rate of turnover (the label should have a long half-life).

G SURGICAL EXTIRPATION AND TRANSPLANTATION

Followed by histological and physiological examination at the cell and tissue levels, at intervals after operation, either by serial biopsies on one animal, or by killing many operated animals in groups, at intervals of time. The kinds of information obtained after various procedures are indicated by the queries given below.

l Total extirpation of an organ
What loss of function? What recovery of function? Associated with what compensatory changes in other organs? e.g., castration results in a degranulation, then an increased activity of the pituitary gonadotroph cells.

2 Partial extirpation of an organ or tissue
Extent of regeneration of the remaining tissue? What recovery of function? e.g., regeneration of liver. What follows extirpation of the nervous supply to an organ, or the ligaturing of its arterial supply or venous drainage? e.g., study of the re-innervation of denervated muscle.

3 Transplantation of tissue or organ
Can the transplanted tissue survive in the new site? Functional value of the transplanted tissue? e.g., in endocrine research, clinical organ replacement of blood vessels, cornea, kidney, lung, etc. Reaction of the site to the transplanted tissue? e.g., problems of immunity, induction phenomena.

4 Implantation of substitute materials
Reaction of the site to the implant? e.g., the use of implants to strengthen weak or broken bones; joint replacements; plastic heart valves; and pacemakers.

5 Parabiotically paired animals
The vascular systems of two live animals are connected so that the same blood passes through both. What material or cells mediating a response to a stimulus in one animal is borne in the blood, via the connection, to evoke a response in the other?

H MICROMANIPULATIVE METHODS

l Micromanipulative techniques are used blindly:
l To puncture single nerve cells or axons with microelectrodes, recording their electrical activity.
2 To eject pharmacologically active substances in the vicinity of nerve cells, whose activity is being recorded.
3 To puncture the nephron by micropipette at various points to extract fluid for analysis.
These methods are followed by a histological check on the position of the microelectrode or pipette in relation to the cells.

2 Micromanipulative techniques are used under direct microscopic control : e.g.,
l To dissect out individual cells, e.g., for tissue culture, transplantation of nuclei, biochemical analysis, physiological measurement; to irradiate parts of a cell in tissue culture, e.g., individual chromosomes at anaphase.
2 To effect surgical repairs, e.g. microsurgery of the eye, auditory ossicles, nerves, and blood vessels.

I IN VIVO AND IN VITRO TECHNIQUES

These allow living cells, tissues, and organs to be observed by microscopy, often providing confirmation of functions deduced from the histological examination of dead specimens. For some of the sites listed, incident rather transmitted light is used.

l In vivo (in the living organism)
l Perspex observation chambers set in the rabbit's ear, the skin of the mouse's back, or in the skull.
2 Frog's foot web, amphibian tail, human nail bed, and the human skin window.
3 Visible transplantation sites, such as the anterior chamber of the eye, and the chorio-allantoic membrane of the hen's egg with a window set in the shell.
4 Natural and surgically made fistulae between a viscus and the skin.
5 Exenteration of an organ, e.g., exposing the living spleen for trans-illumination on a microscope stage, and the study of its blood flow.

2 Tissue and organ culture
(In vitro - separated from the whole organism and its many interacting factors for control.)
l Careful and aseptic extirpation of the living organ or tissue (perhaps followed by an enzymatic dissociation of the cells, if only a certain kind of cell is desired).
Cell sorters then allow one to separate cells according to cell-surface markers; or cells may be 'panned for', by coating a dish with a material to which only one type sticks.
2 The tissue to be cultured is placed on a raft or adheres to the side of a tube at or near the gas-medium interface, in an incubator held at body temperature.
3 The gas phase provides oxygen for aerobic processes and CO2 for pH regulation; the liquid medium has nutrients to maintain the cells' activities.
4 Agents added to the medium may promote cell proliferation, e.g., insulin or a growth factor.
5 A cell response to other single variables (e.g., Ca2+ in the case of parathyroid cells, male sex hormone for prostatic cells, vitamin A for bone or cartilage) may be investigated by:
.. (a) observing the live tissue,
.. (b) studying the cells by EM and LM after fixation,
.. (c) measuring biochemically what the cells themselves are contributing to the medium.
6 The reaction of the cells to bacteria, viruses, chemotherapeutic and toxic agents, and to embryonic growth and inducing factors may be examined by these methods. Examples:

7 Measurement of the cells' reactions in vitro to stimuli is possible:
3 Observation methods
l Supra-vital staining is used to reveal certain structures. Methylene blue stains nerve fibres of neurons in culture.
2 Unstained living cells can be examined undisturbed by using modified light microscopes designed to enhance the small differences in contrast between biological structures.

J MEDICAL CYTOGENETICS

Histological medical genetics uses LM to study genes, chromosomes, and the sex chromatin, revealing relations between various disease states and visually detectable chromosomal and genetic change.

l Techniques
l Chromosomes of cultured cells

2 Sex chromatin 3 FISH - fluorescence in situ hybridization
To explore the molecular basis for disease by seeing, in individual cells, whether genes, or parts of genes, have been deleted, added, or altered
Locate the particular DNA sequence o (gene) in:

(i) nuclear DNA of interphase cells
            .   .                      (ii) DNA of metaphase chromosomes
         .         .                                  .  .  .      
 DAPI-stained __     .                             .            .
      .     / o  \    .                          .               .
  nucleus->/  ^   \    .                       . \./     \./      .
      .    \    o /   .                       .  / \     / \       .
       .    \ __^/   .                        . /   \   o   o      .
         .         .                           .       /     \     .
           .     .                              .                  .
              .                                  .       \./      .
        INTERPHASE                                 .     / \    .
                                                       .   .  .  
                                                   ARRESTED METAPHASE

STEPS

1 Separately label probe DNAs
DNA - biotin - yellow FLUORESCEIN (FITC) } colours seen in DNA - digoxigenin - antibody to digox - red RHODAMINE } UV light
2 Separately denature the target DNA and probe DNAs to make them single-stranded, and able to hybridize.
3 Put probe solution onto target cells/tissue on a slide; coverslip; incubate warm for hybridization.
4 Wash off unbound probe; process slide for probe/signal detection.
5 In the fluorescence microscope, count the number of hybridization signals (coloured sites) per nucleus, or per chromosome, noting more than 2, less than two, and translocations (e.g., red probe + yellow probe, combined at a site, show as an orange spot).
The number of probes is limited only by technical constraints and colour clashes: two-colour FISH is common.
6 For metaphase chromosomes, two probes can be used simultaneously thus: 7 The regions along the short (p) and long (q) arms of each chromosome already have numbers, from the banding patterns seen with Giemsa staining after a trypsin pretreatment - G-banding. But, mutations and small deletions cannot be seen as banding changes.

2 Diseases (examples)
1 The DiGeorge syndrome results from a one-copy submicroscopic deletion on the long arm of 22 (22q11). The variable picture includes defects in thoracic, neck, and facial development. The abnormal heart and aorta call early attention to the infant. The thymus and/or parathyroids may be absent, causing an immune deficiency (infections) and/or hypocalcaemia.

2 Leukaemia (chronic myeloid/granulocytic)

3 In chronic B-lymphocytic leukaemia, some patients have a deletion of one of the alleles (13q14) of the retinoblastoma 1 gene, coding for a tumour-suppressor protein.

4 Mongolism (Down's syndrome)

5 Intersex states (genetic)

Note the general lines of analysis:
chromosome - gene - gene product - product's role - normal phenotype
(Chromosomal defect) - gene defect - absent/disruptive product - abnormal phenotype

3 Basic research
l Chromosome damage
Metaphase chromosome preparations are used to determine what doses of noxious agents, e.g., radiation and drugs, given previously to the tissue, cause breakages and other structural damage to chromosomes.

2 Chromosome markers

K ELECTRON MICROSCOPY

l Preparation of material for transmission EM
l Fixation: of small pieces of tissue, or centrifuged pellets of cellular material, by immersion (may result in a marked gradient of fixation); or (b) by intravascular perfusion of fixative (better fixation, but the pressure may cause distortions).
2 Imbed the dehydrated material in a plastic or an epoxy resin.
3 Cut the block into very thin sections, 0.03 µm/30 nm thick, on an ultramicrotome under microscopic observation.
4 The cut sections float out on water in a small trough, and are picked up on a thin carbon or plastic film, itself supported upon a copper grid, or are put directly on the grid.
5 This grid can later be placed in the specimen-holder of the microscope (Fig. 12).
6 The tissue can be treated in several ways before observation: 2 Transmission electron microscope (Fig. 12)
               

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           |  |XX    .    XX|  |
           |         .         | 
Shielded   |         .         |
air-tight  |      ------>      | Object in SPECIMEN-HOLDER able
COLUMN     |   __     .   __   | to be manipulated from outside
           |  |XX    .    XX|  |
           |  |XX   .     XX|  | Electromagnetic OBJECTIVE coil
           |       .           |
           |      .            |
           |     .             |
        ___|   < . . . . . .   |
        ___    __ .       __   |
To PUMP to |  |XX   .     XX|  | Electromagnetic PROJECTIVE coil
maintain   |  |XX     .   XX|  |
vacuum     |            .      |
           |              .    |
           |                .  | 
           |___:::::::::::::>__| Image on FLUORESCENT SCREEN (direct)
                                    or PHOTOGRAPHIC PLATE (indirect)

3 Scanning electron microscope
l The tissue is fixed, carefully dehydrated, and coated under vacuum with very thin conducting layers of carbon and/or gold.
2 In the SEM (Fig. l3) the electron beam, l0 nm wide, scans the coated specimen, causing the emission of secondary electrons, the quantity of which can yield information about the nature of that area of the specimen's surface.
3 The secondary electrons pass to a charged scintillator, where their energy is changed to light, then converted to an electrical potential for more amplification, before being applied as the signal controlling the beam intensity in a cathode ray tube (CRT).
4 The electron beam of the CRT scans its fluorescent screen in synchrony with the 'scope beam, and builds up a picture of the surface of the specimen, which can be viewed on the screen or photographed.
5 The image has much greater depth than in LM, and yields a strong 3-D impression, when stereo pairs are photographed and viewed. The magnification range is wide, l0-l00 000, with l0 nm resolution. But the image is of the surface, unless the tissue was fractured, and is influenced greatly by the tilt of the specimen to the beam.
6 The beam also makes the specimen emit X-rays of wavelengths characteristic of the chemical elements in that part of the specimen. Thus, with an X-ray detector and analyser, the SEM acts as an electron-probe microanalyser, e.g., revealing the nature of inhaled particles in specific parts of the lung.

           
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           |       .       |
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           |       .       |                                           Screen
           |   __  .  __   |                                     \:::::::::::::::::/
First lens |  |XX  .  XX|  |                                      \           .   /
           |  |XX  .  XX|  |                                       \         .   /
           |   __  .  __   |                                       |        .    |
Second lens|  |XX  .  XX|  |             ___________               |       .     |
           |  |XX  .  XX|  |            |   Scan    |              |      .      |
           |       .       |            |_generator_|              |      .      |
           | @     .    @..|.................::....................|..@   .   @..|
Scanning   | @ @   .  @ @..|.......................................|..@   .   @..|
 coils     | @ @   .  @ @. | Electron beams . . . in the 'scope and|  @   .   @  |
           | @ @   .  @ @. | CRT synchronously scan the specimen **|  @   .   @  |
           | @     .    @  | and the fluorescent CRT screen        |      .      |
           |   __  .  __   |                                       |      .      |
Final lens |  |XX  .  XX|  |                                       |   _ _._ _   |
           |  |XX  .  XX|__|__________        ___________          | /    .      |
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           |      | |e  "  |
           |______|_|___"__|
                  | |   "
                  W |   " " " " X-ray detector
         Stage tilt W      
         & rotate           

Fig. 13 Scanning electron microscope

3 Atomic-force microscopy
1 Electron microscopy subjects the specimen to harsh processing. Atomic-force microscopy (AFM) yields 0.2 nm resolutions of unfixed, uncoated, and partly hydrated biological specimens, without the need for a vacuum.
Chromatin and collagen fibrils are two of many structures examined raw, and with mild treatments to enhance contrast.
2 AFM relies on the near-field interactions between a charged, sharp tip/probe and the specimen surface, very close by.

Fig. 14 Atomic-force microscopy in constant-force mode

                         visual image
                              ^
                        image | processing
                              |
          --------------  COMPUTER -----------------
         |                                          |    feedback
         |                                          |---controller--
     scan|control                                   |               |
         |                        mirror            |               |
         |     laser                ~  ~  ~  ~ photodetector        |
         |          ~             ~               array             |
         |            ~         ~                                 piezo
         |              ~     ~                                  ceramic
         |            ____~_~________ force-sensing cantilever   to keep
probe v scans           v                           <............force 
        specimen___Oo0ooÖoOOooO0..0_____                        constant
This mode works with the repulsive force.. between the scanning probe v and the surface of the specimen. The reflecting spring cantilever holding the probe is kept still, as the piezo ceramic maintains the repulsive force constant, by adjusting sample height. The piezo ceramic also separates the variable voltage information that is turned into visual contrast for that point on the specimen. Correlated with the scan-control record, the contrast data offer an image of the surface topography.

Alternative modes to constant-force are: constant-height, for the sample, while the deflections of the cantilever are recorded; and tapping, where the cantilever oscillates.

L VISUAL ANALYSIS OF SUBMICROSCOPIC ELEMENTS

l Polarizing microscope l Natural light vibrating in all planes is passed through a Nicol prism polarizer, from which it leaves displaying only one plane of vibration.
2 Such plane-polarized light can pass through many biological structures with its plane unaltered. These are called isotropic.
3 Others, having an internal submicroscopic structure, (i) with elements having one dimension longer than the others, and (ii) these elements in an orderly arrangement, alter the plane of light vibration, and are called birefringent or anisotropic.
4 The light leaving such anisotropic objects can be shown to have had its polarization plane altered by passing it through another polarizing filter, used as an analyser.
5 The plane of the analyser is set perpendicular to the plane of the light transmitted through the polarizer to the object. Thus all altered light resulting from the double refraction/anisotropy of the object, except one plane (the analyser's), is blocked. The anisotropic parts appear light against a dark background.
6 Orderly biological structures, showing anisotropy in polarized light, are collagen fibres, muscle fibres, bone matrix, cell membranes, crystals, etc.
Stains, e.g., picrosirius red for collagen, can be used to enhance the effect, which is useful for assessing fibrosis in liver, lung, and kidney.

2 X-ray diffraction
l Used to determine the molecular structure of crystallizable materials.
2 Bone mineral's diffraction pattern was compared to find the best match with the patterns of various calcium salts of known composition.
3 Proteins and nucleic acids that can be crystallized, e.g., from haemoglobin and DNA to histones and DNA-histone complexes, have been very fruitfully studied to relate changes in their molecular conformation with the tasks that they do. Enough is known to make sound predictions of protein molecular shape and active sites, from the amino-acid (and nucleotide) sequences. Gorgeous, fascinating, and revealing, describe the images of the anatomy of these biological molecules.

M QUANTITATIVE METHODS

Absolute numerical values, for such quantities as the number of red blood corpuscles and kidney glomeruli, the extent of the pulmonary alveolar surface, the number of fibres of a certain size in a nerve, are needed for the better understanding of physiological data, and their correlation with the elements seen histologically. How are measurements to be made of microscopically small biological structures?

l Morphometry
l Direct: e.g., maceration in acid and fixative of the testis, teasing out of the epididymis and measurement of its length; similar studies on individual smooth muscle fibres.
2 Direct by sampling: e.g., maceration of the kidney to free the glomeruli in their corpuscles, homogenization, dilution by a known factor, and counting of the number of glomeruli in samples in a counting chamber; counting of blood cells and corpuscles (Chapter l7.A).
3 Indirect by reconstruction or casts: e.g., from serial histological sections, construct a model of lung alveoli and measure their surface area; make a plastic cast of blood vessels or lung alveoli for measurement.

To have most structures sufficiently clearly visible for measurement means examination in thin histological sections, which bring in their train three difficulties:
.. (a) artefacts, such as shrinkage and compression distort absolute and relative values;
.. (b) they offer only a sample of the whole structure;
... (c) knowledge of the shape and extent of the structures in their third dimension is lost.
A mathematical analysis and specific formulae can be applied to data measured from sections to deduce values for structures, as three dimensional entities, such as volumes, surface areas, mean widths of trabeculae, etc.

2 Stereology
Is such a mathematical treatment, leading to formulae, of the relations between:

  1. three-dimensional structures, e.g., spheres of different sizes, tubes, curved membranes, and trabeculae;
  2. the images seen with various random probes into the structures, e.g., point, linear and planar (a histological section approaches a planar probe for structures, compared with whose dimensions its own thickness is small);
  3. and the various probabilities of encountering specific images with one kind of random probe.
The formulae devised can be applied to biological structures, after making some initial assumptions about their nature, e.g., that the gut is tubular, and kidney corpuscles spheroid; and also to structures that have departed from isotropy in a known, regular way.

3 Aids to measurement and quantitative analysis
l Eyepiece grids and graticules, counting chambers, stage micrometers: for direct measurements at the microscope.
2 The section's microscopic image may be projected, enlarged, on a screen for tracing on graph paper, or for tracing outlines, e.g., of cells, to be cut out and weighed.
3 The image may be recorded permanently as a light or electron micrograph, for measurements and tracings.
4 Features of observed images, micrographs or tracings can be entered, by means of a scanner or digitizing tablet, into an image-analysing computer, programmed to calculate such quantities as areas, number of intercepts, etc. - data that can, via the stereological formulae, give the 3-D quantities of interest.
5 Another permanent record is when a computer memory holds a complete microscopic image, built up from digitized picture elements. Such stored images save the specimen further EM-beam or ultraviolet exposure, and can be processed for image enhancement and reformatting, comparisons with stored images, measurements, and the incorporation of graphics.


Chapter 3l REGENERATION

Regeneration is the regrowth of a tissue or part of an organ after its destruction or loss. The ability to restore structural and functional integrity after injury is essential for survival, and genetic selection has left man with much of the regenerative ability of lower vertebrates.
In medicine, many of the injuries seen are complicated by such factors as infection, chemical, heat and radiation damage, extensive haemorrhage, delayed treatment, shock, multiple injury, old age, malnutrition and metabolic disease.
Nevertheless, it is helpful to know how well individual tissues and organs can mend after injury, under optimal conditions of diet, age, treatment and its timing. In man, such conditions may prevail in elective surgery. The discussion of regeneration below applies to aseptic, experimental injury in mammals.

A GENERAL CONCEPTS

l Regrowth of a tissue and its organization for function in many ways recapitulate the initial embryonic formation of the tissue. The formation of new tissue needs the development of new cells as shown below.


SOURCE CELLS     (a) Surviving differentiated cells (may de-differentiate)
     |           (b) Surviving undifferentiated stem cells
     |           (c) Circulating cells in the blood
     |
proliferation   1 Stimulated by: reduced density of cell packing?
     |             Physiological overload? Growth factors? Loss of growth
     |             inhibitors?
     |          2 Cell membranes 'feel' that tissue is missing, and are
     |             prompted to migrate? and proliferate?
     |
     |
     V                      Specialisation
GROWING CELL POPULATION -------------------------> DIFFERENTIATED CELLS
                            brought about by
                                                      
                       1 Maintained regulatory programmes
                       2 Continuity of regenerating part with mature,
                          specialized part, e.g., in skeletal muscle
                          fibre
                       3 Inducer substances/factors
                       4 Interactions with extracellular matrix
                       5 Cell contacts & gap junctions
                       6 Mechanically and electrically
                          polarized fields

2 New tissue requires new cells, derived by cell proliferation
The extent to which cell division is taking place can be determined by radioautographic study using tritiated thymidine, or BrdU-based methods. Radioautography has shown, for example, that smooth muscle has more regenerative ability than the none credited to it previously. Tissue with cells incapable of division, e.g., neurons, is unable to restore the lost cells, although individual neurons can repair some damage to their processes.

3 Organization of the differentiating cells to repair an organ
l Coordination of regenerations of more than one tissue is needed, e.g., (a) glandular cells, blood vessels, and stromal cells, ECM and later reticular elements, to build new lobules in the liver; (b) skeletal muscle fibres, connective tissues, and nerve fibres, to regenerate muscle and restore musculo-skeletal function. Tissues interact inductively and trophically.
2 Regenerations of tissues may compete, with a functionally unfavourable outcome, e.g.,
.. (a) collagenous connective tissue may outgrow the regenerating cartilage and bone in a skeletal fracture, and fill the fracture gap with a fibrous tissue not rigid enough for support;
.. (b) in the injured brain, glial cells multiply, and actively inhibit the growth of axons.

4 Requirements for regeneration
l Space: the growth of tissue requires space and may, in itself, be a response acknowledging that a spatial defect exists. Where tissue has been damaged, phagocytosis and lysis of the necrotic tissue make room for the new cells. When the defect has been filled, the cell proliferation is reduced or stopped. This inhibition happens even when the 'wrong' tissue, e.g., fibrous CT, fills the gap.
2 Communication between the participating cells by means of cytokine and other agents.
3 Adequate hormone levels, e.g., thyroid.
4 Adequate stores or intake of amino acids, vitamins, etc., for the synthesis of new protein and other materials.
5 Freedom from infection.
6 An intact blood supply and drainage for the area. Sometimes, too, a continuing innervation is a necessary coordinating and trophic factor.

5 Hyperplasia and hypertrophy
l Hyperplasia is a response by the tissue involving mitosis and the formation of new cells, increasing cell number to meet a demand for greater output, e.g., of glandular secretion.
2 Hypertrophy again tries to meet a requirement for increased effort or output, not by cell proliferation, but rather by the cells in their original number increasing their size and hence content of productive organelles and materials, e.g., uterine smooth muscle cells in pregnancy.

6 Regeneration and physiological regeneration
l Physiological regeneration is a normal process going on continuously, e.g., in gut epithelium, or continually, e.g., in hair follicle epithelium, involving cell division for the replacement of cells lost naturally.
Blood cells, epithelial, bone and connective tissue cells show the phenomenon. Muscle, cartilage and nerve cells, on the other hand, are stable and static cells, in maturity.
2 Tissues regenerating physiologically without any injury are better able to regenerate to repair damage than those with stable, long-living cells.

E EPITHELIAL AND CONNECTIVE TISSUES

l Initial events
A sterile cut into epithelium that involves its lamina propria will:
.. (a) kill some epithelial cells,
.. (b) cut CT fibres allowing the wound to gape open;
.. (c) sever small blood vessels which
.. (d) will spill blood into the gap.
Powerpoint

2 Below the epithelium
l Blood forms a clot with fibrin fibres, platelets, and fibronectin.
2 Leucocytes migrate through intact vessel walls; polymorphs attacking the few bacteria and monocytes removing cell debris and fibrin. (The inflammatory response and agents involved in it, e.g., affecting capillary permeability and the migration of leucocytes, will be studied in pathology and pharmacology.)
3 Fibroblasts in CT become active, proliferate, migrate into the clot, and lay down new collagen fibres, glycoproteins, and proteoglycans, in the construction of granulation tissue.
4 Endothelial cells of cut capillaries concurrently proliferate and move into the clot, rebuilding the capillary network.
5 Continued activity along these several lines results in the rapid formation of a new lamina propria. Meanwhile,

3 In the epithelium
l Epithelial cells at the cut margins migrate out as a thin layer, penetrating the fibrin, and replacing the temporary substrate of fibronectin and tenascin with basal lamina as a robust and permanent support.
2 While the epithelial surface is being restored as a thin sheet, its cells start to multiply and differentiate, to restore the original thickness and variety of specialized cells of the epithelium.
3 Since the new epithelium has to grow in from the edges, the degree of gaping of the cut determines the distance over which the cells must migrate, and hence the time needed for healing. The cut margins of the wound should therefore be drawn into apposition by sutures. The sooner the epithelium is restored, the earlier is the underlying tissue protected from invasion by pathogenic organisms.
The above describes the smooth, progressive sequence of healing by first intention.
4 The surviving deep part of exocrine glands can provide a source of regenerative epithelial cells to replace the surface epithelium, in addition to the lost part of the gland. Significant examples are:
(i) in the physiological restitution of the uterine lining from the basal layer of the endometrium after menstruation;
(ii) the replacement of epidermis from the sweat glands (and hair follicles), after a second-degree burn has killed the more superficial epithelial cells.

Although the regenerations of epithelium and CT have been treated separately, in life these processes, and, indeed, their normal day-to-day working, are tightly coordinated by cytokines and cell-matrix interactions.

In general, repair can restore both epithelium and its lamina propria of connective tissue to almost as good a condition as before. When there is pathological delay, cytokines are being tried in order to speed up epithelial events and to boost construction of the lamina propria.

C GLANDULAR REPAIR

Because they are composed of epithelial cells, glands can show considerable regenerative ability. For example:
l Liver, after aseptic, surgical removal of half of its substance, can make good the deficit with organized hyperplasia and some hypertrophy of the remainder.
2 Pancreas. Ligaturing the pancreatic duct causes the enzymatic destruction of most of the acinar tissue. If the ligature is released before the duct epithelium is killed, regrowth of glandular acini and islets can take place from the duct epithelium.
3 Kidney can replace tubular epithelium injured, for example, by toxins or ischaemia, but lost or damaged glomeruli are not restored.

D MUSCULAR REGENERATION

l Skeletal muscle
l Some regeneration occurs at the cut ends of fibres. (A cut is insufficient injury to kill the cell throughout its length.)
2 The end-piece reverts to the narrow myotube stage, seen in embryonic growth.
3 Just outside the sarcolemma of intact muscle fibres lie satellite cells that act as residual, peripheral myoblasts, able to respond to injury by becoming active myoblasts.
4 The end grows out a little way into the defect, then increases in thickness. If the cut is not wide, myotubes regenerating from each side may fuse and restore the fibres.
5 A deep cut may sever nerves disturbing regeneration in two ways:
.. (a) Denervation of muscle fibres reduces their regenerative response.
.. (b) Dense fibrous CT then fills the gap and obstructs reinervation of the muscle.

2 Smooth and cardiac muscle
l Radioautographic studies indicate that smooth muscle cells, e.g., in the gut, are capable of some proliferation to replace damaged cells and partially restore continuity in a muscular tunic.
2 Cardiac muscle is at a disadvantage, because it cannot relax and rest for a period to permit cell division and muscle reorganization; and there may be an early and unfavourable response from its CT.
Lung, likewise, is prevented by its elasticity and motion, and other factors, from effective regeneration, despite its epithelial content.
3 Cuts into cardiac muscle fill quickly with collagenous CT, but muscle fibres injured by infections can regenerate.
In general then, a large lesion in muscle will be filled with C.T. Only a little new muscle tissue forms to replace that lost or to fill gaps. Surviving muscle fibres may hypertrophy in an attempt to restore the power of the muscle as a whole.

E BONE AND CARTILAGE

If a long bone of an extremity breaks, the animal's ability to forage, and to escape from predators is seriously curtailed. Most animals, including man, are able to repair such broken bones, and use them again for locomotion and other tasks. .

l Long-bone fracture (involving the shaft)
l Initial phase

2 Early repair 3 Later repair 4 Union and non-union 5 Consolidation 2 Some terms used clinically
Remembering that the diaphysis of a long bone has a long axis: 3 Skull vault (compared with long bone's shaft)
l Initial phase is essentially similar.
2 Early repair. The difference is that the surviving bony surfaces (periosteal/pericranial, outside; dural, inside the vault) produce only a little new bone, and only very rarely any cartilage. The fibroblasts meet little to obstruct them from filling the gap with CT, which is too soft to protect the brain.

4 Cartilage
l As on bone, restitution of tissue is performed by the surface covering - the perichondrium.
2 In youth, when it is still active in appositional growth, the perichondrium can restore significant defects.
3 In mature cartilage, defects are likely to be filled with fibrous CT, or the lesion may precipitate a degeneration of adjacent cartilage.
Lacking a perichondrium, articular surfaces are especially unable to repair damage. In end-stage osteoarthrosis, the cartilage is completely worn away, leaving painful, grinding bone ends.

F ASSORTED TISSUES

l Tooth. The enamel, deprived of its forming cells at eruption, is incapable of repair. Additional dentine can be laid down by the odontoblast layer on the pulp-chamber surface of the dentine - again an example of restitution from the surface.
2 Tendon. Fibroblasts of the cut tendon's sheath and other sources proliferate, become active, and lay down orderly collagen fibres, which can restore most of the original strength of the tendon.
3 Myeloid and lymphoid tissues
l The phagocytic filtering action can be performed in other organs, if only one member of the system is removed. Thus, splenectomy leaves the bone marrow and liver's macrophage cells with the task of treating blood, e.g., removing old RBCs, but creates a vulnerability to certain pathogens. Therefore, surgeons try to repair ruptured spleens.
2 Removal of myeloid tissues from the sternum and calvarium leaves much marrow in other bones. Surviving marrow becomes more active, and can repopulate (by passage through the bloodstream) sites denuded of haemopoietic tissue.
A drastic demand for new RBCs and granulocytes may be met by the resumption of myelopoiesis in such ectopic sites as the liver.
3 After the killing of all the blood cell-producing elements by whole-body X-irradiation, activity can be restored by the injection into the circulation of isogenous bone marrow cells.

G NERVOUS TISSUE

See Chapter
11.F.2

Chapter 32 MOLECULAR MECHANISMS OF CELLULAR IDENTITY

This Chapter is still as written in 1992, in order not to delay putting the book online. The amount of recent primary literature to read and try to turn into teaching topics was too much for now. However, an update is in the works, in the form of Powerpoint slides Powerpoint
.
The identity of cells, i.e., the character or phenotype that each cell has, expresses itself in histological appearance and specific functions, e.g., organelle-free acidophilic cytoplasm and oxygen transport for the RBC. Compiling these characterisations has been the the meat of what has been outlined so far.
Molecular species, e.g., involucrin, uroplakin, etc. have been mentioned only as materials enabling particular tasks. It is time to tackle differential gene expression, or how a cell comes by its unique profile of specialized molecules.
Such molecular explanations are becoming a necessary part of cardiology, gastroenterology, immunology, oncology, surgery, and so forth. For example, cancer cells rearrange their genes, causing unusual, disruptive, and fatal expressions of materials; heart-muscle molecules change in disease; and lives are made difficult, if not miserable, by genes defective from conception. By starting now, one should be able to keep up with the changing complexity of molecular analyses of what cells are up to, and how molecular diagnosis and intervention will aid medical practice.

Histology stays in the picture because in situ hybridization and immunocytochemistry let one see some of the molecular action in relation to individual, identifiable cells and organelles.
In experimental testing for gene function with mutations, deletions, knock-outs, and excess gene dosage, histology reveals the altered phenotype at cell, tissue, and organ level, if there are phenotypic consequences.

A PROTEINS AS THE KEY TO CELL IDENTITY

1 Protein species provide the key to a given cell's nature and repertoire of activities - its phenotype. The proteins may be very abundant, e.g., keratin intermediate filaments in terminal keratinocytes, or minor in amount, but potent, as in the case of enzymes involved in the synthesis of a hormone or neurotransmitter.

2 Proteins are large molecules, distinctively shaped to offer regions or domains for interaction with other molecules. They achieve their eventual size, shape, and ability to act chemically, initially by the linear joining of specific amino acids in set sequences, based upon the informational content of DNA and RNA nucleotide sequences.

3 Proteins can be cell type-specific (CTS) in three ways:

  1. The protein is present in only one cell type - absolute specificity.
  2. The protein occurs in a few kinds of cell, but in each type is slightly modified to constitute an isoform of the protein - isoform specificity.
  3. The protein is found in several cell types, but its abundance is much greater in one or two types than in the others - quantitative 'specificity'.
'Tissue-specific' substitutes for CTS where one cell type predominates, e.g., in cardiac muscle.

4 In practice, there are hundreds of proteins that meet one or more of these criteria. For instance, alkaline phosphatase has separate isoforms in gut and placenta, a third isoform that is plentiful in bone, liver and kidney - the B/L/K isoform, and a fourth, similar to the placental, but occurring in thymus and testis.

5 The theoretical position is that a cell's molecular identity is represented by the constellation of special cell type-specific/luxury proteins, underpinned by a pattern of levels of housekeeping (basic-function) proteins, common to most cells of the species.

B THE ANATOMY OF A GENE

                    - bps   IIIIIIIIIIIIIII*IIIIIIIIIIIIIIII + bps
                                upstream      downstream

           gene's regulatory region        start site of transcription                    
                                           --->
  ___ _______________ _____________________|coding region --->____________________________________________________
5'__//_____ENHANCER_//______P R O M O T E R__________EXON1 INTRON1 EXON2 INTRON2 EXON3____________________________3'
*   $        #   distal         %     proximal           @                                                                 *

                                                    >start of translation                 cleavage & poly-A site
                                           |________|
                                           5' untranslated                            > 3 ' untranslated region
Fig. 14 Regulation of a gene               region of RNA +
* 5' and 3' refer to carbon positions in nucleotides, and hence to nt attachment and DNA orientation
# Enhancers and repressors may be distant or close
$ The breaks // in the DNA keep the enhancer in view
% There may be more than one promoter
@ Number of exons and introns varies by gene, including no introns. Introns are transcribed, but spliced out to create the mRNA
+ The 5' UTR may influence translational efficiency

C TRANSCRIPTIONAL REGULATION

Moving now to the general cell-type-specifying mechanism of differential gene activation: how is a gene chosen for expression?
1 DNA accessibility to transcription: DNA undergoes localised changes in its binding to histones of the nucleosomes, and in the methylation of C-G cytosines.
2 The aim is for a transcription complex centred on RNA polymerase II to bind to the DNA of the gene to be transcribed, to initiate transcription, and to continue it, until a stop codon is met. Why does the polymerase bind here? And what else is needed?
3 RNA polymerase II binds and starts because: 4 Why is the transcription complex at the 5' end of this gene, and not of another (Fig. 14)? 5 The activating power of individual TFs is usually weak, and may be + or -. Several TFs in combination must be bound, and fall exactly into place, to create a transcriptional complex that transcribes.

6 In sum, the phenotypes of cells reflect the varied activities performed, special proteins subserve the functions, and selective gene control furnishes the proteins; hence the spectrum of cell types derives from the repertoire of combinations of transcription factors.
Because of the high informational content and synergistic/antagonistic possibilities of TF combinations, far fewer regulatory factors are needed than there are genes to be controlled.
Also, a restricted number of factors makes it easier to bring the production of the phenotype's many CTS proteins into play at roughly the same time - coordinated regulation. But, there is still a need for 'master' TFs to take the lead.

7 A consensus sequence in DNA is detected: either by the high number of nucleotides held in common with another sequence that is known to bind a TF; or the TF binds to a newly studied region of DNA, which sequencing then reveals to have most of the known binding sequence. As these lines of inquiry proceed, the idea gains power as: (i) other similar (homologous) sequences are found to bind the TF; (ii) it becomes evident that, even where binding-region nucleotides differ, there are restrictions on the differences, e.g., only purine substitutions are seen. A sample consensus sequence is GTTAATNATTAAC for hepatocyte nuclear factor 1, where N stands for any nucleotide.

D TRANSCRIPTION FACTORS

TFs have devices to stabilize their shape to present an alpha helix to bind the DNA in a sequence-specific way, domains for pairing with other TFs as dimers, and domains for activating transcription by other protein-protein interactions. The classification of TFs is currently based on the structures concerned with DNA-binding and making dimers, rather than the transcription-activating or -silencing domains.

1 Types
1 Leucine-zipper - aligned ridges of leucine-rich regions on two such TFs (the same or different) join to create the 'zipper' union. The leucines are lined up so, because they occur every seventh residue along each coil. Nearby, is a basic region in the TF to bind to the DNA. The dimerization of TFs so created: (i) multiplies their instructional power, with 'allowed' and 'non-allowed' combinations; and (ii) presents the DNA-binding domains to match the DNA's shape.
2 Helix-loop-helix (HLH) - A basic DNA-binding domain lies adjacent to two alpha helices (13 & 15 amino acids (AA) long), separated by a loop (5-20 AA). The HLH region mediates oligomer formation between TFs, which can change the DNA-binding preferences. Several bHLH TFs recognize the sequence CANNTG.
3 Homeodomain is around 60 AA, arranged in a helix-turn-helix DNA-binding conformation. It came to notice through genetic-molecular studies of the products of homeotic genes controlling insect development.
POU domain comprises a 75-82 AA POU-specific domain, a variable link, and a 60 AA POU homeodomain: all involved in binding to DNA.
Why POU? The first TFs where the domain was noticed were Pit-1 (in pituitary cells), Oct-1 (general) and Oct-2 (B lymphocytes), and a TF controlling the nematode's gene unc-86. [Genes' names are in italics; their protein products in roman.] The octamer TFs bind to the 8-nt sequence ATTTGCAT.
4 Zinc-finger, C2-H2 - a zinc ion, tetrahedrally linked to pairs of appropriately spaced cysteines and histidines, creates short loops of amino acids (the fingers) to interact with the DNA.
5 Zinc-finger, C2-C2 , is a different (cysteine only), zinc-centred structure used to construct two fingers, which fold together and help orient the alpha-helical 'DNA-recognition' domains. The steroid/thyroid/retinoid receptors employ this motif. Attachment of the hormone ligand brings about the receptors' dissociation from heat shock protein 90, and movement into the nucleus, where they bind as dimers.

2 What controls TFs?
Positive and selective regulation
1 As proteins, their regulation can be at typical places in the general sequence of protein synthesis, e.g., transcription, alternative splicing of mRNA, protein stability, etc.
2 Auto-regulation, by the TF activating transcription of its own gene, e.g., for MyoD 1, Pit-1, which helps maintain and stabilize the phenotype specified by the TF, and renders the cell less dependent on the outside stimuli that evoked the phenotype.
3 Dimerization: homo- and heterodimerization of TFs.
4 Ligand activation, e.g., the binding of steroid and thyroid hormones and retinoids causes their receptors to be moved into the nucleus, and to activate transcription. The DNA sequence to which the receptor-ligand complex attaches is a 'something' response element, e.g., oestrogen RE (ERE); thyroid RE (TRE); and the CRE allows genes to be controlled by the CREB TFs stimulated by cyclic AMP.
5 Phosphorylation of TFs can induce DNA-binding, e.g., by CREB, or transcriptional activation, e.g., by Oct-2.

Negative regulation
6 Heterodimerization, e.g., the Id factor has a HLH, but no basic region to bind DNA. When Id forms heterodimers with bHLH TFs, e.g., MyoD, binding to DNA is blocked.
7 Competitors for DNA binding - competitive inhibition, e.g., NF-kappaB binds to the CCAAT box of the foetal g-globin gene, obstructing CP1's activation of the gene.
8 Inactivation by bound protein factors that do not prevent DNA binding - quenching. NF-kB's control of an Ig light chain gene in B cells is prevented by a cytoplasmic protein IkappaB, which detains NF-kB in the cytoplasm, until the IkB is phosphorylated.
9 Non-translation of TF mRNA, e.g., Pit-1 mRNA is made, but not translated, in corticotrophs and gonadotrophs.
10 A great excess of one factor in solution may so tie up its normal binding partner, another TF, that the latter is unavailable for participating in the transcription complex - squelching.
11 The TF itself inhibits transcription as a silencer TF [negative regulation by, not of the TF], e.g., thyroid hormone receptor alone (without ligand) can bind to the TRE, causing a repression of transcription.

E STEPS IN PROTEIN SYNTHESIS

The following steps provide levels of possible regulation: decision points in the overall choice of how much of what kind of protein is to be formed by the cell.
1 Extra- and intracellular signalling, with signalling molecules, receptors, signal-transduction machinery, binding proteins, and transport into the nucleus.
2 DNA accessibility to signals, regulatory factors, and the polymerization apparatus.
3 Transcription: pre-initiation, initiation, elongation, and termination.
4 RNA processing of the primary transcripts to make mRNA.
5 Stabilization of the mRNA.
6 Transport of the mRNA to the ribosomes in the cytoplasm.
7 Use and re-use of the mRNA in translation to protein sequences.
8 Direction of the protein to sites for post-translational modification, e.g., cleavage, glycosylation, phosphorylation, addition of prosthetic groups, e.g., haeme to globin.
9 The use of chaperones for the stability and folding of the protein.
10 Intracellular storage or degradation of the product.

F LEVELS OF REGULATION

1 The above progression creates a hierarchy of control points: if no primary RNA is transcribed, post-transcriptional controls are redundant; if a mRNA is made unstable, post-translational influences are superfluous.
2 For most CTS proteins, the prime control is at transcription.
3 The mechanisms can act in concert, thus as transcription is increased, the mRNA produced may be made more stable, and translational and post-translational efficiencies improved.
4 Signals from outside the cell act not only on transcription, but on the other steps, and upon the intracellular signalling pathways, which include feedback loops and network interactions.
5 Many cell type-specific products are constructed by means other than differential transcription: one gene yields more than one protein or polypeptide.

6 Significant examples whereby one gene results in different products are:

7 Variants of a protein can derive from multiple genes. These can differ slightly in their coding region, but markedly in how and when they are regulated, and may be scattered over different chromosomes, e.g., non-muscle myosin heavy chains A & B on 22 & 17 respectively. On the other hand, a family of genes can be close together on the same chromosome, may share some controls, and be in a developmentally meaningful order 5' to 3', e.g., the complex of beta globin genes on chromosome 11 is under the control of a distant upstream 'locus control region'. But genes do not have to be on the same chromosome to be regulated coordinately.

G CELL PHENOTYPE: UNTIDINESS OF THE CONCEPT

1 Although it is possible to pick out several abundant luxury proteins on a two-dimensional electrophoresis gel, the regulation of a protein's synthesis has to be studied one protein at a time. The underlying assumption is that far fewer than a hundred proteins can illustrate the general principles of regulation; and that by looking at eight or so CTS proteins in hepatocytes or skeletal muscle cells, one can conclude that since five, say, proteins are synthesized in coordination (they appear at the same time in development, and are extinguished together in de-differentiation), and three proteins are not, one can conclude that coordinate regulation occurs, but is not obligatory; and one has to go on examining proteins case by case.
2 What goes on in humans may not be exactly what transpires in animal cells, and transformed human ones that are not above living and multiplying in plastic dishes, but is close.
3 Cells acquire their identity in stages, controlled by sequences of signals and cell-cell interactions. Cells continue to respond to their environment as their activities are controlled to fit in. Where is the line between control of ongoing activities, and regulation of the phenotype to be maintained as the means to execute the activities?
4 What is known about cells is patchy, and varies in amount: much for hepatocytes, far less for pericytes.
5 In considering differentiation, the properties common to cells also have significance, but attention is seized by the differences. Likewise, quantitative differences are less inspiring then qualitative ones, although probably not that much further from the truth of cell differentiation.
6 Ubiquitous cells - fibroblasts, endothelial and smooth muscle cells, and macrophages - are adapted to the local needs of each organ that they serve in: there is no single hard-and-fast cell phenotype.

H EXAMPLES OF THE MOLECULAR CONTROL OF CELLULAR IDENTITY

Questions for a given cell type are: What are the CTS proteins? And in what sense: absolute, isoform, quantitative?
For each, at what stage of synthesis is the primary control? When is it transcriptional? What are the cis aspects - the regulatory regions and sequences of DNA? And what are the corresponding trans-acting factors - the TFs - in terms of: their class (e.g., bHLH vs. homeodomain, specific versus general), dimerization, regulation, and what is special about the circumstances, e.g., the role of growth factors.
These questions form the basis for Table 6 presenting a few results for some cell types. The point is to have a small armamentarium of informed molecular questions with which to confront issues of cell phenotype.
Viewed in total, there is a daunting jungle of interactions among a host of sometimes cryptically abbreviated entities. In practice, investigators take them on one cell type and one gene at a time, and then look for evidence of coordination.
Table 6 MOLECULAR REGULATION OF PHENOTYPE IN PARTICULAR CELLS

Cell type          DNA: position & Sequence         Cell type-specific       
& gene                                              transcription factors

Skeletal muscle
fast skeletal    Enhancer internal regulatory       MyoD, myogenin, Myf-5
troponin I       element (IRE) in intron 1, with    bind the MRF
                 25 bps muscle reg.factor-binding
                 sequence (MRF)


Thyroid follicular cell
thyroglobulin    Sites A, B, C, & K in promoter     TTF-1 bind A,B, & C
                 (-168 to -42); a consensus         TTF-2 binds to K
                 sequence for TTF-1


Pituitary lactotroph
prolactin       Proximal enhancer has four sites    Pit-1
                (-200 to -38); distal enhancer
                also has 4 sites (-1718 to -1386)

Erythrocyte
 ? globin       Proximal promoter (+12 to -60)      NF-E1
                including CCAAT; distal
                promoter (-252 to -226)


Hepatocyte
albumin         Promoter (-185 to -74) with CCAAT   HNF-1
                proximal element (PE) -62 to -45
                distal element II (-123 to -110)

a-          Promoter regions I through V        NF-1 binds IA; 
fetoprotein     (-1 to -839); enhancers at -2.5,    C/EBP - IB & V
                -50, & -6.5 kb;                     HNF-1 & C/EBP to II
                repressor at -250 to -836           NP-III binds III
                                                    NP-IV  binds IV

ApoB-100        Proximal promoter sequences         C/EBP to more distal
lipoprotein     -169 to -152 & -86 to -61           AF1 to more proximal
The full 1992 table included the necessary: animal species for the protein; type of the CTS TF ; general/ubiquitous TFs participating; and references. All will be given in the coming version.

More points on transcription-factor action

  1. One gene can have multiple binding sites for one TF.
  2. One CTS T factor can be used in the control of many CTS genes, e.g., hepatatocyte NF-1 for albumin, fibrinogens, a1-antitrypsin, a-fetoprotein, & transthyretin.
  3. There can be several different CTS TFs for the activation of one gene; and one factor can be used before another during development, e.g., MyoD precedes myogenin.
  4. Negative regulation by TFs, rather than at the chromatin level, is not uncommon. One use is to repress expression in the adult cell of an embryonically active gene, e.g. a-fetoprotein.
  5. A so-called cell-type-specific TF can be used by closely related cells, e.g., in erythrocytes and megakaryocytes.

I FINAL COMMENTS

1 Cell-specific gene regulation goes on under the influence of hormones, extracellular-matrix components, growth factors, etc. Such factors affect phenotype, and are not just physiological modulators of levels of activities, whose nature is specified once and for all when the cell first becomes terminally differentiated.
2 The regulation of one cell phenotype is very complicated, given the many CTS genes to be set, the numerous TFs, and the many levels of control for each protein, including the TFs. It is a little early to recognize the integrating mechanisms that make the task manageable for the cell, but they are starting to take shape as temporal and spatial patterns of homeodomain gene expression.
3 Is this all too high-flown for clinicians? More elaborate versions of the above table are appearing in the journals of clinical research. For example, the table in Eckert RL et al. The epidermis: genes on - genes off. J Invest Dermatol 1997;109:501-509. It covers many genes and transcription factors, for just keratinocytes of the epidermis, and paves the way for strategies of diagnosis and treatment, just a few years off.
4 The goal is to target therapy at the molecular controls on the activity of particular cells. Histology, with its approaches and methods, is there to show one whether the molecularly corrected cell is also now working properly in its cell-to-cell and organ contexts.

Chapter 33 EPONYMOUS STRUCTURES AND METHODS OF HISTOLOGY

Some terms deserve to be here, but only here.

Ångström unit of linear measurement for electron microscopy
Arantius nodule of CT at margin of ventricular-arterial valves
Askanazy's (Hürthle) mitochondria-rich thyroid cells
Auerbach's autonomic myenteric nerve plexus of alimentary tract
Baillarger's bands - horizontal layers of myelinated fibres in cortical grey matter
Balbiani's vitelline body of several organelles grouped by nucleus of oocyte
Barrett's distal oesophageal metaplasia to gut or gastric epithelium
Barr's sex chromatin body in female cells, e.g., neurons
Bartholin's compound mucous glands of female vulva
Bartholin's main excretory duct of sublingual mucous salivary gland
Bellini's urinary ducts opening into renal minor calyx
Bergmann's astroglial cells in molecular layer of cerebellar cortex
Bertin's cortical columns between pyramids of kidney
Best's carmine staining method for glycogen
Betz giant pyramidal neurons of motor region of cerebral cortex
Bielschowsky's silver impregnation methods for neurofibrils
Billroth's 'cords' of blood and lymphoid cells in splenic red pulp
Biondi's filamentous inclusions in aging choroid plexus epithelium
Birbeck's granules in Langerhans' dendritic cells of skin
Blandin's (Nuhn's) anterior lingual mucous salivary gland
Böttcher's small cells on basilar membrane of cochlea
Bowman's capsule around each kidney glomerulus
Bowman's compound serous gland of olfactory mucosa
Bowman's (exterior) membrane supporting corneal epithelium
Brodmann's numbered cytoarchitectonic areas of the cerebral cortex
Bruch's membrane supporting retina's pigment epithelium
Brücke's radial smooth muscle of ciliary body of eye
Brunner's compound mucous submucosal gland of duodenum
Bunger's cords of Schwann cells in regenerating nerve
Buniña's acidophil cytoplasmic inclusion bodies in spinal motoneurons of amyotrophic-lateral-sclerosis patients
Cabot's ring - a remnant of nucleus not extruded from normoblast
Cajal's accessory body - argyrophil granule in the nucleus of some neurons (accessory to the nucleolus; have marker protein - coilin; and make small nuclear ribonucleoproteins)
Cajal's interstitial pacemaker cells in autonomic myenteric plexus of gut
Call-Exner's dark bodies amongst follicular cells of Graafian follicle
Charcot-Bottcher's 'crystalloid' in cytoplasm of testicular Sertoli cells
Charcot-Leyden crystals - a lysophospholipase - in granules of eosinophil and basophil leucocytes
Chievitz' neuroepithelial juxtaoral bodies within cheek
Clara's non-ciliated secretory bronchiolar epithelial cell
Claudius' cuboidal cells of cochlear organ of Corti
Cloquet's hyaloid canal through vitreous of eyeball
Cohnheim's fields or groups of skeletal muscle myofibrils
Coon's fluorescein conjugation method for antibody visualization
Cooper's suspensory ligaments attaching mammary gland to dermis
Corti's auditory organ of cochlea and its ganglion
Cowper's compound mucous gland of male urethral bulb
Crooke's cells - keratin filament-filled pituitary corticotrophs suppressed by excess glucocorticoid
Deiter's phalangeal supporting cells in organ of Corti
Descemet's (interior) membrane of the corneal endothelium
Disse's perisinusoidal space between hepatocytes and sinusoid-lining cells of liver
Donne's large vacuolated phagocytic cells (bodies) of mammary colostrum
Ebner's compound serous gland (taste) in posterior tongue
Eustachian or pharyngotympanic tube
Fallopian uterine tube or oviduct to uterus
Fanañas cells - a deeper-lying variant of cerebellar Bergmann cells
Farquar's folliculo-stellate 'glial ' cells of pituitary pars distalis
Ferrein's medullary rays projecting into kidney's cortex
Feulgen reaction for revealing DNA
Feyrter's neuroendocrine APUD argentaffin cell of airway epithelium
Flemming's intermediate body briefly linking two daughter cells of a mitotic division
Fontana's spaces in trabecular meshwork of anterior eye chamber
Fontana's spiral banding on relaxed nerves
Fordyce's yellow spots - ectopic sebaceous glands in cheek mucosa
Gärtner's cyst - a blind tubular remnant of mesonephric duct by vagina
Gennari's white (myelinated) stria in grey matter of visual cortex
Gerlach's lymphoid tonsils at pharyngeal end of Eustachian tube
Gianuzzi's serous crescents in alveoli of mixed salivary glands
Giralde's body or paradidymis - vestigial mesonephric tubules in CT at head of epididymis
Glisson's connective tissue capsule ensheaths liver's vessels and ducts
Golgi's apparatus of the cell, particularly secretory cells
Golgi's long- and short-axoned types of nerve cell
Golgi's silver encrustation staining method for neurons
Golgi's tension-sensitive tendon receptor organ
Golgi's thorns/spines projecting from neurons' dendrites
Golgi-Mazzoni's lamellated sensory corpuscles of dermis
Gomori's staining method for neurosecretory material
Goormaghtigh's group of lacis/polkissen cells behind macula densa of kidney
Graafian follicle of an ovary stimulated by FSH
Hassall's eosinophil keratinizing epithelial corpuscles of thymic medulla
Hassall-Henle's protruding bodies at periphery of Descemet's corneal membrane
Haversian vascular canals and systems of lamellar bone
Heidenhain's (Gianuzzi's) serous demilunes of salivary alveoli
Heinz bodies of abnormal haemoglobin seen occasionally in RBCs supravitally stained with crystal violet
Heister's spiral mucosal valve in cystic duct of gall-bladder
Henle's endoneurial CT sheath of peripheral nerve fibres
Henle's layer of epithelial inner root sheath of developing hair
Henle's loop (descending and ascending) of kidney tubule
Hensen's columnar cells on basilar membrane of organ of Corti
Hensen's paler zone across middle of A band of striated muscle
Hensen's bodies - assemblies of vesicles in upper region of cochlear outer hair cells and related to ion transport
Hering's canals (preductular) of liver biliary system
Herring's neurosecretory bodies of the neural or posterior pituitary
Hertwig's epithelial root sheath of developing tooth
Highmore's corpus or body - mediastinal CT of testis
Hirano bodies - rare weakly eosinophil filamentous inclusion bodies in CNS
His' atrio-ventricular conducting bundle of heart
Hofbauer's fetal macrophage cells in stroma of placental villus
Hortega's microglial or 'mesoglial' cells of brain
Howell-Jolly bodies - intracellular inclusions in RBCs indicating impaired splenic function
Howship's resorption lacunae made by osteoclasts on bone
Hoyer-Grosser's digital arteriovenous anastomoses
Hürthle cells - see thyroid Askanazy cells
Huschka's auditory teeth - projections of cochlear spiral limbus
Huxley's layer of inner root sheath of developing hair
Ito's perisinusoidal fat-storing stellate liver cells
Jacobson's vestigial (olfactory) vomero-nasal recess in nasal septal mucosa
Janus green B for supra-vital mitochondrial staining
Jensen's ring or annulus in the spermatozoon's tail
Keith and Flack's sinu-atrial pace-maker node of heart
Kent's accessory muscular (conducting) bridge connecting right atrium and ventricle in animal and human infant hearts
Kerckring's transverse valves (plicae circulares) in lining of small intestine
Key-Retzius CT endoneurial sheath, i.e. Henle's sheath
Kierman's spaces very weakly demarcating liver lobules in man
Kohn's (Henle's) pores between adjacent lung alveoli
Kolmer's epiplexus macrophages on the surface of choroid plexus
Korff's fibres inserting into dentine between odontoblasts (artefacts?)
Kossa's histochemical staining method for calcium
Krause's accessory lachrymal gland at eyelid's fornix
Krause's sensory end-bulb or receptor in skin
Kultschitsky's argentaffin (APUD) cells in glands of stomach and gut
Kupffer's phagocytic lining cells of liver sinusoids
Kurloff's azurophil cytoplasmic inclusion bodies of guinea-pig's agranular leucocytes
Lambert's epithelium-lined tubular interalveolar channels in the lung
Langendorff's colloid-filled thyroid glandular cells
Langerhan's pale endocrine islets in pancreas
Langerhan's antigen-presenting dendritic cells of epidermis and stratified squamous epithelia
Langhan's cytotrophoblast cells of placental villi
Leydig's endocrine interstitial cells of testis
Lieberkühn's crypts or simple tubular glands of the gut
Lissauer's zone or dorsolateral fasciculus in spinal cord's white matter
Littré's tubular mucous glands of urethral mucosa, especially male
Lubarsch's cytoplasmic 'crystalloid' of some spermatogonia - a rod-like bundle of microtubules
Ludwig's arterioles directly connecting renal intralobular arterioles with arteriolae rectae verae
Lugaro's horizontal neurons of outer cerebellar granular layer
Luschka's aberrant blind ducts in the neck of gall-bladder
Luschka's foramina in lateral recesses of 4th ventricle
Magendie's medial foramen between 4th ventricule and cisterna magna
Malassez' rests or remnants of tooth's epithelial root sheath of Hertwig
Mall's periportal tissue space in liver
Malpighian corpuscle (glomerulus+Bowman's capsule) of kidney cortex
Malpighian germinal lymphoid corpuscles (white pulp) of spleen
Malpighian layer of skin's epidermis/epithelium
Malpighian pyramids of multilobar human kidney
Marchi's osmium tetroxide method for degenerating nerve fibres
Marinesco's acidophil intranuclear paranucleolar body in substantia nigra and locus coeruleus neurons
Martinotti's cerebral cortical neurons with ascending axons
Meckel's mandibular cartilage from embryonic first branchial arch
Meibomian 'sebaceous' gland of eyelid tarsal plate
Meissner's autonomic submucosal nerve plexus of alimentary tract
Meissner's sensory corpuscle of skin's dermal papillae
Merkel's intra-epithelial sensory transducer cells in skin
Meynert's giant stellate neurons of cerebral cortical striate area
Moll's large 'sweat' glands of eyelid margin
Mongolian spot - concentration of dermal melanocytes in sacral skin
Monro's interventricular (lateral to 3rd) foramina
Montgomery's areolar tubercles/glands - miniature nipples
Morgagni's hydatid (in male: appendix of testis) - epithelium-lined cystic remnants of Muller's duct
Morgagni's lacunae - outpouchings in mucosa lining urethra
Morgagni's longitudinal columns in lining of anal canal
Müller's circular ciliary smooth muscle of eye for accommodation
Müller's ducts - embryonic origin of uterine tubes, uterus, and vagina?
Müller's radial fibres - glial cells of neural retina
Müller's smooth-muscle levator palpebrae of eyelid's tarsal plate
Nabothian follicles or retention cysts of uterine cervical mucosal glands
Nissl's basophil rough-endoplasmic granules of neuron cytoplasm
Nitabuch's outer layer of fibrinoid in the placental basal plate
Nuel's outer tunnel by outer hair cells in organ of Corti
Oddi's duodenal sphincter (including Boyden's sphincter around bile duct)
Odland's granular cytoplasmic bodies in keratinocytes of skin
Oort's bundle of centrifugal nerve fibres in the cochlear division of cranial nerve VIII
Owen's contour/incremental growth lines in dentine
Pacchionian arachnoid villar granulations of the brain's dura mater
Pacinian onion-like sensory corpuscle in hypodermis, muscles, etc
Palade's ribosomal granules, free and membrane-attached
Paneth's acidophil granular cells in crypts of Lieberkühn of gut
Perl's Prussian blue method for revealing iron
Peyer's lymphoid patches in mucosa and submucosa of ileum
Pflüger's egg-tubes - cords of germinal cells growing into ovarian stroma
Philadelphia chromosome acquired in certain leudaemias
Prussian blue staining for ferric iron
Purkinje's large output nerve cells of cerebellar cortex
Purkinje's pale conducting muscle fibres of heart
Ranvier's nodes regularly interrupting myelin sheath of nerve fibre
Rathke's pouch of pharyngeal ectoderm - origin of adenohypophysis
Regnaud's residual body of excess cytoplasm cast off in spermiogenesis
Reichert's embryonic cartilage of the second (hyoid) pharyngeal arch
Reinke's crystals of testicular interstitial cells
Reissner's membrane in cochlea separating scalae vestibuli and media
Reissner's fibre - an stringy thread of glycoprotein from the subcommissural organ extending down into the spinal canal
Remak's unmyelinated peripheral nerve fibres (often autonomic)
Renaut's hyaline whorled cellular bodies in endoneurial CT
Renshaw's internuncial neurons of spinal cord's grey matter
Retzius contour or incremental growth lines in dental enamel
Retzius fibre cells (sensory) in cristae of semicircular canals
Riolan's ciliary part of cyelid's skeletal orbicularis muscle
Rivinian minor excretory ducts of sublingual mucous salivary glands
Rohr's layer of fibrinoid at base of inter-villous space
Rokitansky-Aschoff sinuses - mucosal outpouchings herniating out through muscular wall of gall-bladder
Rolando's substantia gelatinosa of spinal cord's dorsal horn
Romanowsky's stains for blood cells
Rosenmüller's vestigial Wolffian organ (epoöphoron) representing male's epididymis in female
Rosenthal's fibres - elongated inclusions in reactive or neoplastic astrocytes and composed of crystallins
Rouget cell or pericyte sometimes wrapped around capillary
Ruffini's encapsulated sensory mechanoreceptor II of skin's dermis
Russell's acidophil inclusion bodies of some plasma cells - mutated immunoglobulin that cannot move out of the endoplasmic reticulum
Santorini's accessory pancreatic duct
Scarpa's elastic fascia of anterior abdominal wall
Scarpa's vestibular ganglion with bipolar neurons
Schlemm's annular canal draining anterior chamber of eye
Schmidt-Lantermann incisures or clefts in myelin sheath
Schneiderian membrane or mucosa lining most of nasal cavity
Schrapnell's flaccid part of ear's tympanic membrane
Schwann's neurolemmal cells investing peripheral nerve fibres
Schweigger-Seidel phagocytic sheath around splenic pulp capillaries
Sertoli's columnar supporting cells in testis seminiferous tubule
Sharpey's imbedded periosteal fibres of bone and cementum
Skene's paraurethral ducts representing prostate in female
Stensen's duct of compound serous parotid gland
Sudan staining of fat
Suquet-Hoyer's (Hoyer-Grosser's) arteriovenous anastomosis/glomus of plantar, palmar and facial skin
Sylvian aqueduct of brain linking 3rd and 4th ventricles
Tawara's atrio-ventricular node of heart's conducting system
Tenon's orbital CT capsule within which eyeball rotates
Thebesian veins (venae minimae cordis) draining directly into heart's lumen
Timofeew's Pacinian-like corpuscles of prostate of human infant
Tomes' granular dentine layer adjacent to cementum of tooth root
Tomes' odontoblast fibres (cell processes) in tubules of dentine
Tomes' process of ameloblast - distal compartment actively forming enamel matrix
Tyson's 'sebaceous' glands of penile glans
Vater's duodenal ampulla of fused bile and pancreatic ducts
Virchow-Robin perivascular spaces of brain's pia
Volkmann's canals entering bone from periosteum or medullary cavity
von Willebrand clotting factor (vWF) in endothelial Weibel-Palade storage granules and platelets
Wallerian degeneration of severed or poisoned nerve fibres
Weibel-Palade body of membrane-enclosed vWF, microtubular and other materials in endothelial cells
Welsh's oxyphil mitochondria-rich parathyroid cells
Wharton's duct of mixed sero-mucous submandibular gland
Wharton's mucoid jelly of umbilical cord
Willis' arterial anastomosing circle at the base of brain
Wolffian duct - embryonic origin of ductus deferens and epididymis
Wolfring's tubulo-alveolar glands (mucous?) serving eyelid's conjuctiva
Wrisberg's elastic cuneiform cartilage of larynx
X cell - dark cell of corpus luteum of pregnancy
X cell - modified decidual cell of uterus
Zeiss' sebaceous glands of eyelash follicles
Zinn's suspensory zonule of the eyeball lens
Zuckerkandl's paraganglionic bodies (by abdominal aorta) of chromaffin system


Preface

Background - Students expect plentiful coloured illustration in textbooks, and this is essential for learning so visual a subject as Histology. This unillustrated note-style book had a supplementary role when times, time, and prices allowed, but a fourth print edition for the 1990s was not worthwhile. The publishers - Blackwell Science - kindly returned the copyright © to me, where it remains, so that this revised Web version is to be downloaded and printed out for fair personal use, but not for profit.

Print-to-Web adaptation - I saved the 1992 WordPerfect version (thank you, Margaret Beresford) in DOS text, and updated and edited it on a 286, using elementary HTML tags. On a 12-inch monitor screen, the tags give a format very like that of the pocket-sized print edition, which students had earlier found useful.
The aim thereby is not to hamper anyone with a small monitor and a basic Web browser. On a larger screen, the book format is achieved by a 640 X 480 pixels selection. (Some of the figures need a 600 X 800 display to be seen in their entirety.) Also, the style of indentation is varied to make the layout less tedious to the eye.

The order of chapters originally reflected a curriculum of basic histology, and then histology integrated with physiology and biochemistry, taught respectively in two semesters. Hence, there was some duplication of chapter topics. Here, like has been brought together with like, e.g., all neural topics together.

History - I wrote the first edition for the "Lecture Notes on - -" series in 1967-'68, after a few years of teaching medical histology. Then, it was a brief preview-review pocket book, in an era of heavy texts which students had to use. Now the large textbooks, where they still exist, are seldom mentioned or recommended. (The related weighty texts on cellular and molecular biology are premedical fare.)
The result is that what earlier was minimal detail sometimes goes beyond that nowadays presented in class, and expected for testing. However, the difference is minor, and lies mostly in the inclusion of topics and details of protective, clinical, and molecular interest, which should connect with what students engaged in problem-based-learning are coming across.
A sizeable component of histology is the light-microscopical classifications of the nineteenth century. The dated terms and synonyms have been jettisoned to make room for current matters. Electron microscopy has proven to have a place in pathological practice, and still contributes much to the understanding of function, but details that are not significant in either context, e.g., the size of granules in endocrine cells, are excluded.
There is more emphasis on clinical aspects of histology than in the past. The student's experience of basic medical sciences often is of having been dealt a deck of cards - facts and ideas, but without any indication of what plays high or low: as much weight being given to the muscularis layer of the vagina, as to the clinically important transformation zone of the cervix.

Relevance to your histology course? - The effort to separate, structure, and explain should have resulted in a layout that is sometimes helpful, but lets you skip what seems to be totally unfamiliar. An index is not provided, since, if you can see the text on a monitor, you can search by typing in.
Some topics may not be in your histology text, e.g., the 'modern' (1978) zonation of the prostate. The student should use this edition cautiously, paying attention to the particular context of his or her own course and examinations. Still, it is free, and offers another viewpoint.

Death by List - List-management is a basic skill of medicine, but the meal only starts with the grocery list. What is then done with the ingredients decides whether the experience will be enjoyable and memorable, or the patient is properly diagnosed and treated.
One problem is that almost everywhere one can say that a cell does this or that, many of the molecular species used to perform the tasks are known. These materials help define the cell's identity, make function more understandable, and are the basis for disease, e.g., by mutation or autoimmune reaction, but there is just too much of everything - too many types of collagen, cytokines, transcription factors, isoforms of aquaporin, subtypes of T lymphocytes, etc. The brain's expression of 'perplexin' surges.
Some lists have been introduced to this edition, not for memorization, but so that some of the basis for subtyping can be seen, e.g., that proteoglycans are viewed fruitfully as being large or small, aggregated and non-aggregated, and matrix or cellular.
Ideally, the material of medical basic sciences would be in explanatory narrative form. The very format of a book such as this saturates with lists, at the expense of the wealth of intriguing stories possible, although I have tried to keep a narrative running in some of the lists. However, it may not be all bad, since learning and thinking by list prepares one for the many questions on professional exams that use a list format.

Illustrations - With many sources - slides, atlases, textbooks, videodiscs, 35mm transparencies, CD-ROMs, & the Web (see Introduction) - available for the actual visual images of microscopic anatomy, I have used the space here to show the structure of histological knowledge by a condensed, numbered note form, almost unbroken by illustrations.
I thought of replacing the crude figures by 'Photoshop' ones, but these would not allow the book to fit on a I.4 Mb disc, i.e., to stay shirt-pocket-portable. Individual links will eventually be provided to crisp colour figures, as these become freely accessible.
I have started (March, 1999) the introduction of links in the text to Powerpoint slides, e.g., on blood and marrow. Download and use these as you like, with verbal acknowledgement at the time, if projected. [A guide to projection on one make of projector is at Powerpoint.] The Powerpoint slides, as linked, are busy summary ones, designed to be printed out six-to-a-page for review. For projection, they can be copied more than once, and items systematically deleted to provide simpler sequences for more complicated topics.

How does one illustrate the descriptions and ideas of histology? The solution chosen here is to develop links to freely accessible (but copyrighted) Powerpoint diagrams and lists, half of which have now been done (November, 1999). Some are based on lectures for pharmacy students and may not give all the detail expected of medical students, but nevertheless help the beginner.
For those seeking images of actual sections, here are links to some Histology Websites with illustrations: Bergman, Afifi, & Heidger: U Iowa - - JayDoc HistoWeb --- Vanderbilt Histology Lessons

Molecular histology
The discipline has evolved since the last printed edition in several ways. Cells signal and work by means of special chemicals, and each cell type has a recognizable biochemical identity, significant for the cell's own purposes and potentially in diagnosis and treatment. Mention of key functional and marker chemicals belongs in histology, but here these are mostly introduced unobtrusively at the end of sections. Also, the molecular mechanisms of cellular identity, or how cells come by their distinctive materials, make up a new final chapter.
One way for today's student to prepare for the era of molecular medicine - for diagnosis, and targetting therapy at the cell's controls on particular molecules - is in three early courses: biochemistry for general molecular mechanisms; microbiology for molecular specifics of immunology; and histology for the characteristic molecular species for the full range of cell types, and the mechanisms of how the cells make them. This edition makes a start on the histological side of the endeavour.

Why methods? In Chapter 30 and scattered about, there is more than the usual amount about histological techniques. These approaches have given histology its present substance and form, as molecular thinking will contribute to shaping histology in the future. Both aspects are included in order to aid in comprehending what histology means to medicine. The book looks towards what will probably be useful to medicine, as well as to what has served in the past.

Reliability In a time of specialists and sub-sub-disciplines, is a single-authored book reliable? My longstanding scientific interest is in metaplasia and transdifferentiation, which can involve any cell type and organ, and requires knowing normal phenotypes, and how cells achieve them. Hunting the clinical and basic-science journals for these phenomena also helps to satisfy my curiosity about all the dangling ends from the structures and events of general histology.
Many thanks to all who have sent me reprints.

I would appreciate your calling to my attention by e-mail, fax, or letter:

Servers are more ephemeral than bookshops: URLs for this book are
http://www.geocities.com/Athens/Academy/1575/ a refined, faster version, but needs (May, 2000) to be updated
http://wberesford.hsc.wvu.edu/histol.htm
http://wvnvm.wvnet.edu/~beresfo
Copy it onto a disc, and you only need to come back to see if changes have been made.

William A Beresford, Anatomy Department, School of Medicine, West Virginia University, Morgantown, WV 26506-9128, USA - - e-mail: -- wberesfo@wvu.edu -- wberesfo@hotmail.com -- beresfo@wvnvm.wvnet.edu -- fax: 304-293-8159