HISTOLOGY FULL-TEXT

CONTENTS

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'

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

INTRODUCTION

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

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

            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

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

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

• (a) Made of unit membrane roughly 7.5 nm wide; in the EM picture with appropriate fixation it has a trilaminar appearance of two dark bands with a light layer between them; the light layer is mostly non-polar lipid, the dark layers mostly the charged ends of the lipid molecules with attached protein. In LM the membrane is often unseen.
• (b) A coat of glycoproteins (sugars+protein) - glycocalyx - adheres to the outside of most cells.
• (c) The plasma membrane is flexible, semi-permeable, and experiences active transport and potential differences across it.
• (d) By the cell membrane's fusing with the intracellular membrane around stored material, then breaking open at a point along the line of fusion the material can be released to outside the cell - exocytosis/emiocytosis. By bringing extracellular things into an invagination, followed by selective membrane fusion and separation, materials are brought into the cell - endocytosis: phagocytosis for solids, pinocytosis for fluids; endocytosis includes these bulk movements, but also signifies the internalization of membrane receptors and bound ligands on a smaller scale.
• (e) Specializations of form shown by the plasma membrane include: (i) microvilli, (ii) cilia with basal bodies, (iii) pinocytotic vesicles/pinosomes/caveolae intracellulares, (iv) infoldings or plications, (v) desmosomes, (vi) gap junctions/nexuses, (vii) occluding junctions, (viii) stereocilia (long microvilli).
• (f) Some vesicles involved in transport into the cell have prominent coats of clathrin attached to the membrane.

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:

• (i) Fat droplets appearing as vacuoles in ordinary LM preparation, but usually preserved by EM procedures as dark rounded bodies.
• (ii) Lamellar bodies contain lipids to be secreted.
• (iii) Glycogen granules visible as dark l5-30 nm wide granules in EM, e.g., in cardiac muscle and liver cells.
• (iv) Secretion granules, e.g. zymogen, in pancreatic cells.
• (v) Pigments, e.g., melanin (skin cells), lipofuscin (old neurons), and haemosiderin (natural), carbon (exogenous - from outside the body).
• (vi) Crystals, e.g., in testicular interstitial cells.
• (vii) Bacteria and viral inclusion bodies (pathological).

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.

• (a) The basophilic reaction is a chemical one and different basic stains can be used to show cytoplasm in various colours, red, blue, green, etc. The same principle applies to an acidophil reaction of the cytoplasm.
• (b) Because proteins are amphoteric, a structure reacting basophilically at one pH may change to acidophilia, if another stain of a markedly different pH is used.
• (c) Two stains are used one after the other in the H and E treatment. It is not difficult to get the balance wrong so that one swamps the other. For example, an excess of eosin can mask a basophilic reaction of the cytoplasm.
  

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

A CELL MEMBRANE OR PLASMALEMMA

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.
   

D NUCLEAR-CYTOPLASMIC SYSTEM

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:
   • Nuclear structure is seen in TEM.
   • Radioautography for rates of cell division uses tritium-labelled precursors of nucleotides, e.g., thymidine and cytidine. Bromodeoxyuridine (BrdU) is incorporated in place of thymidine, so that cells synthesizing DNA prior to division can be revealed by a labelled antibody to the BrdU.
   • Chromosomes and their banding patterns are studied in cytogenetics; see Chapter 30.
   • Fluorescent in-situ hybridisation/FISH uses 'coloured' nucleotide probes to identify a particular chromosome and, on it, the gene of clinical interest - normal, mutated, translocated, deleted, duplicated, truncated, etc.
   • Silver staining reveals the nucleolar organizer regions (NORs) as intranuclear black dots, because it marks the acidic proteins binding to the genes coding for ribosomal RNA located on certain chromosomes. An increase in the number of NORs so shown (AgNORs) correlates with dysplastic (abnormal) growth, and may indicate malignant tendencies in epithelial cells.

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

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.

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

C MORPHOLOGICAL VARIETIES OF 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
   • (a) Cell shape is indicated approximately by the name; most epithelial cells are really polyhedral with many sides or faces.
   • (b) Cells stand one cell high, although their nuclei may lie at slightly different levels.
   • (c) Cells are fastened and sealed at the top of their sides by encircling junctional complexes.
   • (d) Cells have three surfaces: free/luminal, lateral and basal; each may have membrane specializations, e.g., cilia at the free, occluding junctions and desmosomes at the lateral, and infoldings at the basal surfaces.
   • (e) Note the position and shape of the nucleus, and special locations of organelles and inclusions that also indicate the cell's polarization.
     

• (a) Very flattened cells presenting a minimal barrier to the passage of materials, e.g., oxygen, through them.
• (b) Cytoplasm is very hard to see with LM.
• (c) The very similar endothelium and mesothelium line blood and lymph vessels, and serous cavities, respectively.

• (a) Nuclei lie at different levels suggesting stratification, but all cells are in contact with the BL.
• (b) Two or more cell types are present: short basal, tall columnar.
• (c) More details: Chapters l2.B.5. and 22.B.2.

7 Stratified squamous

• (a) Many cells thick.
• (b) Surface cells are flat plates and flake off as squames.
• (c) Basal-most cells are cuboidal or columnar and divide.
• (d) Cells above the base become polyhedral and are held together by many desmosomes to resist the abrasive forces on this protective epithelium.
• (e) Underside of the epithelium is indented by vascular papillae of connective tissue, except in the cornea.

8 Keratinized/cornified stratified squamous

• (a) Similar in its basal and middle layers to 7, but the uppermost epithelium has granular cells concerned with forming special, dead cells solidly packed together as a surface keratin layer for greater protection.
• (b) More details: Chapter 21.A.

9 Transitional/urinary

• (a) Several cells thick, but the surface cells are large, rounded, alive and sometimes binucleate, with spare cell membrane in vesicles.
• (b) No connective tissue papillae indent the epithelium.
• (c) More details: Chapter 23.B.I.

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

2 Attachments: function and observation
l Attachments provide for:

• mechanical strength;
• barriers to the indiscriminate passage of materials, and thus the possibility of selective transport;
• formation of intercellular compartments, e.g., bile canaliculus;
• communication for migration, differentiation, function and exfoliation.
  But, for repair and normal cell replacement, cells move relative to one another and must break their attachments. Also, white blood cells can pass between them.
  

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

• (a) a change from pseudostratified columnar to stratified squamous after repeated chemical or thermal insult, say, smoke in the larynx and trachea;
• (b) from oesophageal stratified squamous to gastric or intestinal simple columnar (Barrett's oesophagus), after repeated acid reflux;
• (c) from columnar, cuboidal or transitional to cornified stratified squamous in severe vitamin A deficiency.
• (d) from transitional to stratified squamous in bladders with a stone, or infested with worms. Image - R Lindquist.

F SPECIAL ELEMENTS IN EPITHELIA

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

A CELLS OF CONNECTIVE TISSUES

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

• (a) Fibres are long, wavy or straight, and colourless.
• (b) They have great tensile strength and resistance to stretching, whilst retaining considerable flexibility.
• (c) Fibres are made up of finer fibrils packed together.

• (a) Fibrils show a characteristic, complex cross-banding pattern regularly along their length with a periodicity of 67 nm (64 nm when shrunken).
• (b) Chemical dissolution of collagen followed by chemical manipulation results in a reforming of fibrils displaying a longer periodicity - a long-spacing collagen, believed to comprise rearranged collagen molecules.
• (c) The fundamental unit is a sequence of 234 amino acids, 67 nm long. These units in differently staggered arrays account for the various banding patterns of artificially and naturally occurring collagens, including the main natural collagen molecule with a length of 300 nm.
• (d) In the natural shorter-spacing fibril, these 300 nm-long units lie end to end, side by side, but staggered in such a way as to produce regions of greater and lesser density with the 67 nm periodicity.

• (a) The collagen molecule is made up of three intertwined and cross-linked helical chains of amino acids, with glycine, proline and hydroxyproline prominent amongst them. Dependent on the amino-acids, a chain may be a 1, types I or II, or a 2, etc. Triplet combinations of these and other chains furnish around 19 types of collagen.
• (b) Procollagen is made in the GER of the fibroblast, osteoblast, etc. and cleaved to 300 nm collagen at its release from the cell.
• (c) Fibril-formation takes place immediately outside the cell, by self-assembly followed by a crosslinking crucial to strength.
• (d) Fibrils farther from the cells are thicker (30-300 nm), so presumably the initially thin fibrils can thicken by additions from soluble collagen to the orderly crystalline array.
• (e) Various enzymes, e.g., collagenase, break down collagen molecules.
• (f) Man can achieve man-made fibrous strength by repeated spinning, e.g., for hawsers to tow ships: cells can only spin inside themselves at the molecular level of trimers, thereafter strength comes solely from crosslinking the molecules and the diverse glycoprotein and FACIT-collagen binders.

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       

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

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:

• (a) basophilic with basic stains, e.g. in hyaline cartilage;
• (b) positive with Alcian blue and Hale's iron;
• (c) metachromatic, e.g., with toluidine blue, where the blue molecules, in binding to the closely spaced anions, interfere with each other, so that their effect on light becomes a red one, e.g., in mast cell granules.

7 Physical properties - the high negative charge:

• (a) attracts cations, and restricts the movement of water, ions and molecules (and bacteria), thus controlling transport through the CT to the cells and other tissues;
• (b) structures the matrix, making it gel-like, or even firmer in cartilage, where the proteoglycans are themselves strung along a hyaluronic acid backbone;
• (c) binds CT fibers to one another, influencing their strength and functioning;
• (d) acts as a reservoir for growth factors and other agents controlling cell behaviour.

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

• (a) Forms of the glycoprotein, fibronectin, occur in CT matrices, basal laminae and blood plasma.
• (b) Fibronectin is a multiple adhesive, since various domains of the molecule bind glycosaminoglycans, collagen, fibrin, and some cells.
• (c) Made by fibroblasts and available from blood, it helps in the scaffold-building, and cellular migrations and attachments, which give tissues their microarchitecture during embryogenesis and wound repair.
• (d) Tenascin shares some structure with fibronectin, but plays its part more during development, e.g., at sites of epithelial-mesenchymal interaction. It reappears in malignant tumours.
  
• Anti-adhesive macromolecules provide another control on cell interactions with the ECM. Tenascin and decorin are respectively glycoprotein and proteoglycan examples.

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

D TYPES OF CONNECTIVE TISSUES

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

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

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

• (a) Ground substances rich in soluble collagens and the proteoglycan - aggrecan: a core protein with chondroitin and keratan sulphates side chains, and a link protein for attachment to hyaluronic acid. The aggregated proteoglycans impart basophilic and metachromatic staining properties, and bind water and confer resilience.
• (b) Collagen fibrils visible in EM; and LM after silver impregnation, digestion of the ground substance, or in polarized light. The reinforcing fibrils are oriented in some relation to stresses experienced by the cartilage.

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:

• (a) Appositional/perichondral by the recruitment of fresh cells, chondroblasts, from perichondral stem cells, and the addition of new matrix to the surface;
• (b) Interstitial by the mitotic division of, and deposition of more matrix around, chondrocytes already established in the cartilage.

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

C FIBROCARTILAGE

D DISTRIBUTION OF THE THREE CARTILAGE VARIETIES

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

B CLASSIFICATIONS OF BONE

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

Chapter 8 BONE FORMATION

A INTRAMEMBRANOUS OSSIFICATION

B PREPUBERTAL LONG BONE

C ENDOCHONDRAL/INTRACARTILAGINOUS OSSIFICATION

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

• (a) Cartilage cells hypertrophy.
• (b) Matrix around them becomes basophilic, then calcifies.
• (c) Perichondral cells close by become osteoblasts; i.e., the perichondrium becomes a periosteum.
• (d) Periosteal bone deposition forms a subperiosteal bony collar or ring around the centre of the shaft.
• (e) At the same time, through gaps in the bone, osteogenic/vascular buds invade the calcified cartilage matrix to form the primary ossification centre of bone forming in, and on the calcified walls of, spaces eroded in the matrix.

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.

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

• (a) Reserve/quiescent zone of hyaline cartilage with small cells and few cell divisions.
• (b Proliferative zone where chondrocytes multiply and arrange themselves in ordered parallel columns (palisades) of disc-shaped cells. Growth, interstitial, in the long axis of the bone occurs mainly here.
• (c) Hypertrophic/maturing zone has the chondrocytes no longer dividing, but enlarging at the expense of their matrix.
• (d) Calcification zone, where the matrix stains basophilic and is impregnated by crystals of calcium salts. Mineralization may be triggered by the seeding action of calcium-rich matrix vesicles released from chondrocytes. Whether or not these chondrocytes then all die is disputed.
• (e) Erosion zone encroaches into the calcification zone. Vascular bud elements destroy some cartilage, but leave longitudinal spicules as a scaffold on which bone is laid down. In HE preparations the calcified cartilage cores are blue, the superficial bone red.

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

E PHYSIOLOGICAL FACTORS AFFECTING CONNECTIVE TISSUES

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

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

5  (a) Skeletal/striated/voluntary;              |   are the various
   (b) Cardiac/heart/(striated);                 |-  names, of the
   (c) Smooth/unstriated/involuntary/visceral;   |   three kinds.

B SKELETAL MUSCLE

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

                             _________________ A __________________
                            |                                      |
                 ____ I  ___                                         ____ I ____
                |           |                                       |           |

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

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

5 Some events and chemistry of contraction

• (a) Excitation. The ion permeability of the sarcolemma is altered by acetylcholine to the point where it propagates an action potential. This depolarization extends down the T-tubules and influences adjacent cisternae of the sarcoplasmic reticulum (SR), which extend feet to receive the stimulation.
• (b) The agent coupling excitation to contraction is ionic calcium, released by the stimulated SR to interact with one of two regulator proteins attached to the contractile protein - actin. The binding of Ca²⁺ to troponin causes tropomyosin to stop interfering with the link between actin and myosin.
• (c) Contraction occurs because the myosin head, linked to the actin, somehow generates the force moving the thin filaments deeper between the thick ones. At the same time, the actomyosin complex acts enzymatically to release energy from ATP bound to the myosin. The actomyosin bond is between actin and heavy meromyosin that protrudes as many cross-bridges, or lateral projections (seen in EM), from the thick filament of light meromyosin.
• (d) Relaxation happens when Ca²⁺ is actively taken up by the SR. Troponin then permits tropomyosin again to inhibit actomyosin binding, thus unfastening the cross-bridges.

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

F MYOGENESIS (skeletal muscle)

G TENDON

• (a) many bundles of dense, regular, collagen fibres with
• (b) flattened tendon cells (fibroblasts) between them;
• (c) each bundle is loosely bound in a CT sheet - endotendineum;
• (d) peritendinial CT, bearing vessels and nerves, encloses several primary units as one fasciculus; and
• (e) a thick sheath - epitendineum - wraps around the whole tendon of several fasciculi.

Chapter 10 NERVOUS ELEMENTS

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

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:

• (a) Nissl bodies/granules - basophilic, cytoplasmic structures are concentrations of granular ER.
• (b) Neurofilaments - a variety of intermediate filament - are aggregated into neurofibrils visible in the cytoplasm after silver impregnations.
• (c) Surrounding the nucleus are elements of the Golgi apparatus, mitochondria, lysosomes, and microtubules. Actin filaments move vesicles in the zone directly under the neuron's plasmalemma.
• (d) Pigment is sometimes present, e.g., melanin in substantia nigra neurons, and lipofuscin in old neurons.
• (e) Cell membrane has specialized receptive areas, the subsynaptic membranes of synapses.

2 Dendrites

• (a) Contain mitochondria, microtubules, and granular ER.
• (b) Membranes have receptive subsynaptic membrane areas.
• (c) Some dendrites have spine-like side processes, also receptive,
• (d) Dendrites integrate the excitatory influences along them, and modify their responses and morphology in learning.

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

• (a) Contains axoplasm flowing centrifugally from the somatic starting-point of the axon - the axon hillock.
• (b) Has mitochondria, neurofilaments, microtubules, travelling vesicles, and, in some neurons, secretion droplets, in the axoplasm.
• (c) Membrane of the tube is the axolemma, swelling out into a bag at its terminal/synapse which holds vesicles/microvesicles. (The axon is also termed the axis cylinder.)
• (d) Myelin sheath of lipoprotein around the axolemma is interrupted at regular intervals to leave the axolemma `bare' at nodes of Ranvier. The membrane propagates the action potential.
• (e) EM reveals myelin to have lamellae with alternate dark (major dense) and light (intraperiod) lines, apparently concentric around the axon.
• (f) Closer examination reveals a mesaxon from the innermost lamella by the axolemma, and an outer mesaxon from the outermost lamella to join the enclosing Schwann or glial cells' plasmalemma.
• (g) To control the neuron's ionic and transmitter environment, enclosure of the axon (and myelin) by Schwann or glial cells is complete, including nodes of Ranvier and the terminal bags (synapses). At intervals along the myelin, Schwann cell cytoplasm penetrates into conically oriented separations of the myelin lamellae, constituting the incisures of Schmidt-Lantermann, perhaps giving the myelin some flexibility and aiding molecular turnover.

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

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

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)

• (a) Cerebellar cortex: Purkinje cells, granule cells, stellate cells, basket cells.
• (b) Olfactory bulb: mitral cells, granule cells* and tufted cells.
• (c) Cerebral neocortex: pyramidal cells, fusiform and stellate cells of many kinds.
• (d) Neural retina: ganglion cells, bipolar cells, horizontal cells, amacrine cells* and photoreceptors (modified neurons).

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

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

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

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

• (a) Anatomical, based on the lobes, and the pattern of sulci and gyri.
• (b) Functional, founded on functional specializations revealed by extirpation, clinical lesions, stimulation of certain regions, on gross and microrecording of the neurons' electrical activity, or PET-detected patterns of glucose use, in response to specific stimuli, e.g., visual.
• (c) Histological, based on cell- or cytoarchitecture, and fibroarchitecture. Histologically defined areas often coincide with functional areas. A conservative view of the histological parcellation of the cortex is now taken, that commonly adopts the numbering used by Brodmann, but does not acknowledge all his subdivisions in the frontal, parietal and temporal association areas.
• (d) Projection areas demonstrated by histological studies of degeneration or transport (F.4 below) to be connected with particular brainstem nuclei.

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

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

• (a) Make an experimental destruction (lesion), confined as far as is possible to the particular nucleus, tract or cortical region of interest.
• (b) Allow the animal to stay alive for one or two weeks after recovery from anaesthesia. During this post-operative survival period, the injured fibres will degenerate in the Wallerian manner, whilst the nerve cell bodies where the damaged fibres originated may develop a retrograde cell reaction.
• (c) Kill the anaesthetized animal by perfusing fixative through its vascular system, thus fixing the brain in situ.
• (d) Remove and section the brain and spinal cord.
• (e) Stain the brain or cord sections:
  ... (i) with a degeneration-specific method, e.g., Nauta, for the degenerating fibres.
  ... (ii) with a Nissl method to find neurons experiencing the retrograde cell reaction.
  ... (f) Electron microscopy of a suspected site of termination of a tract can confirm the presence of degenerating fibres and terminals, and may reveal the existence of surviving intact terminals/synapses.

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

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

A PERIPHERAL NERVE

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

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

• (a) A spindle-shaped C.T. capsule encloses several thin specialized intrafusal skeletal muscle fibres. Away from the muscle fibre's central region with clustered or linear nuclei (nuclear-bag and nuclear-chain fibres) are plate or trail motor endings served by mostly gamma (fusimotor) nerve fibres.
• (b) Two types of sensory ending on the fibres - primary annulospiral and secondary flower-spray - increase the combinations of innervations.
• (c) The larger extrafusal muscle fibres stimulate the attached spindle by their movements, while its sensitivity is varied by its own alpha and gamma motor control.
• (d) The spindle informs on the complexities of muscle contraction, movement and stretch, and gives a `muscle sense'.

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)

• (a) Three cell kinds are in the pseudostratified, columnar neuro-epithelium:
  1. (i) Bipolar sensory cell, with an axon going directly through the ethmoid bone to the olfactory bulb and, at the apical end, several, long, cilium-like processes (olfactory hairs) extending into the surface coat of mucus and lipid.
  2. (ii) Sustentacular cell has pigment in its apical cytoplasm and is equipped with microvilli.
  3. (iii) Basal cells to divide and replace sustentacular and sensory cells.
• (b) All cell types rest on a BL penetrated by axons that become grouped as unmyelinated nerves in the lamina propria.
• (c) Bowman's compound serous glands also contribute secretion to the surface. Unlike other serous cells, they have much smooth ER.

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

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

• (a) The sarcolemma is depressed into a trough.
• (b) The swollen ending of the axon lies in the trough.
• (c) The sarcolemma lining the trough invaginates into many small junctional folds/secondary synaptic clefts. It has ACh receptors.
• (d) The space between the sarcolemma and axolemma is filled by a basal lamina-like material.
• (e) The axon terminal has mitochondria, and vesicles containing the chemical transmitter substance, acetylcholine, which, when released by electrical activity in the axon, indirectly causes the muscle fibre to contract.
• (f) Schwann cells cover the axon and its terminal bag.

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)

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)

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

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.

A ANTERIOR EYE

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

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

3 Retinal layers details:

• (a) Pigmented epithelium, simple cuboidal, lying on Bruch's wide membrane of basal laminae reinforced by collagen and elastic fibrils. Pigment cells absorb light, and destroy the used-up tips of the rods.
• (b) Photoreceptors
  
  • (i) An outer segment of stacked, infolded cell membrane, incorporating the light-sensitive chemical, is connected via a cilium-like neck to
  • (ii) an inner segment with mitochondria, Golgi body and ER for the continual replacement of outer segment materials.
  • (iii) Then comes a dilation with the nucleus, and further inward the cell narrows to become an inner fibre before synapsing with the dendrites of bipolar nerve cells.
  • (iv) The nerve and photoreceptor cells are separated by processes of the special glial cells, Müller cells.
  • (v) Photoreceptors are classified by shape as: (a) rods (rhodopsin as the visual pigment and converging neural connections give them high sensitivity, but poor acuity and no colour discrimination); or (b) cones (varieties of iodopsin and less convergence in connections provide for colour vision and fine acuity).
• External limiting 'membrane' is formed of outer terminal processes of Müller cells and lies at the level of the photoreceptors' inner segments, to which the Müller cells tightly attach by junctional complexes.
• (d) Outer nuclear layer - nuclei of photoreceptors.
• (e) Outer plexiform layer - synapsing processes: photoreceptors' spherules or pedicles with bipolar neuron dendrites.
• (f) Inner nuclear layer - nuclei and bodies of bipolar neurons; Müller cells, horizontal neurons and amacrine cells.
• (g) Inner plexiform layer - axons of bipolar neurons synapsing with dendrites of ganglion neurons.
• (h) Ganglion cell layer - somas of ganglion neurons, whose axons pass over the internal surface of the retina as the
• (i) nerve fibre layer to converge on the optic papilla, where they pass out unmyelinated through the eye's other two coats to form the optic nerve.
• (j) Inner limiting membrane - a basal lamina separates the inner processes of Müller cells from the vitreous.

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)

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.

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

Chapter 14 AUDITORY AND VESTIBULAR ORGANS

A SITUATION AND EXTERNAL RELATIONS

• (a) The oval window/fenestra ovalis occupied by the moveable stapes bone acting as a plunger.
• (b) The round window/fenestra rotunda covered with fibrous tissue and epithelium, and moving passively as a pressure release for pressures generated within the fluid system by movement at the oval window.

B DIVISIONS OF THE EAR

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

• (a) Macula, flat with:
  1. Basal lamina penetrated by
  2. nerve fibres from bipolar neurons of Scarpa's vestibular ganglion passing to synapse with
  3. sensory hair cells, of two types, with their companion
  4. sustentacular cells, making a pseudostratified neuroepithelium.
  5. The 25 µm-long hairs (long microvilli and one cilium per cell) of the hair cells project into
  6. the gelatinous otolithic membrane, in which are imbedded
  7. small crystalline otoconia/otoliths (weights subject to gravity).
• (b) Crista - rounded, crest-like prominence similar to a macula in its components, but the gelatinous mass is called the cupola, and is without otoliths. The crista detects the start and end of movement in the plane of its duct.

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

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

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.     

B SYSTEMIC BLOOD VESSELS

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)

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

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

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.)

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:
   • (a) diffusion or carrier-mediated transport through and between endothelial cells;
   • (b) active transcytotic transport through endothelial cells; then passage through the basal lamina.
   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

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]

3 Heart wall`s three layers

1. Endocardium (innermost)
   • (a) Lined by endothelium on a basal lamina.
   • (b) Subendothelial layer of collagenous and elastic fibres, fibroblasts and some smooth muscle cells.
   • (c) Subendocardial layer of CT with blood and lymphatic vessels, nerve fibres, and Purkinje fibres of the heart's conducting system. A layer worth calling a subendocardium is not everywhere present.
2. Myocardium
   • (a) Cardiac muscle fibres, bundled and wound in spiralling sheets, thickest in the left ventricle, thinnest in the atria.
   • (b) Blood vessels and lymphatics and fine CT.
3. Epicardium (visceral pericardium) and subepicardium
   • (a) Outer mesothelial sheet and BL on
   • (b) loose subepicardial CT of fat cells and collagen fibres with
   • (c) blood vessels (coronary), lymphatics and nerves to the heart nodes.
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.

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

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

• (a) Intima of endothelium, BL, CT, some longitudinal smooth muscle and an elastic lamina.
• (b) Thick media of mixed longitudinal and circular smooth muscle.
• (c) Thin adventitia of collagen and a little longitudinal smooth muscle, vasa vasorum and nerve fibres.
• (d) A valve is at the venous exit.

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

Chapter l6 GLANDS

A VARIETIES OF GLAND

• (a) Simple, with one duct have secretory units (end-pieces) of a form either:
  .. (i) tubular (straight, coiled, branched), or
  .. (ii) acinar/alveolar (dilated acini may be termed saccules).
• (b) Compound with a branching duct system and secretory units of three forms:
  .. (i) tubular,
  .. (ii) acinar/alveolar,
  .. (iii) mixed tubulo-alveolar.

11 B STRUCTURE OF A COMPOUND EXOCRINE GLAND

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.

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

• (a) Mucous cells, in the majority, surround the lumen.
• (b) Serous cells lie at one end as a serous crescent/demilune between the mucous cells and the BL.
• (c) Serous secretion may pass in fine intercellular canaliculi between the mucous cells to reach the lumen.

C CYTOLOGY OF SECRETION

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

                         _
            Plasma      |  Red blood corpuscles/Erythrocytes (RBCs)
              +         |
      formed, visible   |
         elements    - -|  White blood cells/Leucocytes (WBCs)
     (46% by volume)    |
                        |
                        |_ Platelets

A MICROSCOPIC TECHNIQUES FOR BLOOD

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:
   • (a) Oxygen binds to ferrous iron of haemoglobin (RBC) for transport:
     air --> lungs -- > blood --> tissues
   • (b) Carbon dioxide leaves bicarbonate of the plasma and carbaminohaemoglobin (RBC) for transport:
     tissues --> blood --> lungs --> air
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)

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

• (a) Nucleus has coarse, clumped, deeply staining chromatin, usually in two or more lobes or segments connected by thin chromatin strands. Unlobated band nuclei are in immature cells; older nuclei have several lobes.
• (b) Cytoplasm is granular from many, small, weakly staining (neutrophil) granules of two kinds:
  .. (i) non-specific azurophil granules that are lysosomes with destructive enzymes; and
  .. (ii) numerous specific non-lysosomal granules holding a selectin-type glycoprotein for adhesion to endothelium and ECM, and lysozyme, and other bactericidal substances.
• (c) This motile cell is attracted out of vessels into the tissues, where it attacks bacteria and phagocytoses them and immune complexes. The attack on bacteria is two-pronged:
  .. (i) with a respiratory burst that generates free radicals; also myeloperoxidase catalyzes the production of hypochlorous acid; and
  .. (ii) by proteins, e.g., defensins, and bactericidal permeability-inducing protein (BPI), that damage bacterial cell walls.
• (d) PMNs make up 55-65 per cent of the total leucocytes.

• (a) Nucleus is darkly staining and bilobed.
• (b) Cytoplasm has many large, eosinophil granules 0.5-l µm diameter, and some smaller core-less granules.
• (c) Specific granules are a form of lysosome, which in EM have a crystalline core and a fine granular region. Defensive basic/cationic proteins, e.g., major basic protein, give the acidophil reaction.
  The enzymes differ somewhat from the neutrophil's, e.g., generating antimicrobial O₂ metabolites differently.
• (d) Motile, and enter inflamed tissues, especially at sites of allergies and parasitic infestations. They attack helminths using the basic proteins and oxygen derivatives, and also may dampen mast cell-dependent reactions, e.g., by phagocytosing mast-cell granules.
• (e) They comprise 2-3 per cent of leucocytes (but rising for (d)).
• (f) In EM, the large granules, like a skunk, have a dark lengthwise central stripe, which helps in identifying the eosinophil.

• (a) Nucleus is bilobed and sometimes twisted, but palely staining and often obscured by
• (b) basophilic cytoplasmic granules, containing sulphated proteoglycans, heparin and the vasodilator, histamine.
• (c) Basophils are reluctant to enter CTs, where there are mast cells holding the same materials. Thus, the function of basophils is in doubt, but they bind IgE and participate in various hypersensitivities.
• (d) They are rare; 0.5 per cent of the leucocytes.

• (a) Small spheroid cell about 5-8 µm in diameter.
• (b) Large, spheroid, darkly staining nucleus leaves only a
• (c) narrow rim of cytoplasm with a few small azurophil granules.
• (d) Motile to enter CT and epithelial tissue, but is not phagocytic.
• (e) Larger lymphocytes up to l2 µm diameter, with more abundant cytoplasm, may be seen in small numbers. The large granular lymphocyte is the natural killer cell.
• (f) Unlike granular leucocytes, small lymphocytes can be stimulated to enlarge and divide by antigens, cytokines, and some plant lectins.
• (g) The complex role of lymphocytes in immune defences is outlined in Chapter l9 and Fig. 10.
• (h) Lymphocytes circulate in blood and lymph systems and migrate to CT and mucous membranes. Some lymphocytes have a lifespan of months or years.
• (i) They amount to 25-35 per cent of the leucocytes.

• (a) Large, spheroid cell about 12-20 µm in diameter.
• (b) Nucleus has fine chromatin not densely stained, and is an indented sphere.
• (c) Golgi body and centrioles lie by the nuclear indentation.
• (d) Cytoplasm is abundant, with a few granules that are precursors of many larger lysosomes seen in EM when the cell is actively phagocytic.
• (e) Motile, to leave the vessels after only a day or so to become the phagocytic macrophages/histiocytes of CT, or other derivatives.
• (f) Macrophages/MØs spend months in CTs cooperating with lymphocytes in defensive responses (Chapter l9). Macrophages, by releasing cytokines after activation, coordinate inflammatory and defensive reactions.
• (g) They comprise 3-l0 per cent of the leucocytes.

D PLATELETS (clotting and vessel-sealing)

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

Chapter 18 HAEMOCYTOPOIESIS

A DIVISIONS OF HAEMOPOIESIS

B SITES OF MYELOPOIESIS

• (a) blood islands in the yolk sac;
• (b) fetal liver haemopoietic tissue;
• (c) bone marrow in cavities of the developing bones;
• (d) marrow in the spleen and lymph nodes.

• (a) Red/myeloid marrow remains mostly in the axial skeleton - skull diploë, ribs, sternum, parts of the vertebrae and pelvis.
• (b) Ectopic/extramedullary myelopoiesis may be seen in adults, usually in disease, e.g., fibrosis of marrow, and at the sites of myelopoiesis in the fetus.

C THEORETICAL CONSIDERATIONS OF HAEMOPOIESIS

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

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

2 Red marrow has many elements, see Powerpoint:

• (a) Blood sinusoids are lined by endothelial cells on an incomplete BL. Collagen fibrils (reticular fibres) support these, and
• (b) adventitial stromal/reticular cells, similar to fibroblasts, but extending processes between, and greatly influencing, the haemopoietic cells.
• (c) Macrophages cleanse blood, and detect and destroy worn-out RBCs and other elements. The iron recovered is stored, combined with protein as ferritin granules, before release to the labile pool and reuse.
• (d) Blood cells develop extravascularly, are stored, then released through the sinusoidal wall into the circulation.
• (e) Megakaryocytes form and release platelets.
• (f) Fat cells are present, large and empty of fat in embedded sections.
• (g) Bone surface cells act as an enclosing sac for the marrow.

F CELL COMPARTMENTS, POPULATIONS AND KINETICS

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

 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

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

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

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.

• some of its cells have short lives and their remains must be disposed of;
• foreign non-living matter may enter, e.g., dust and grit, and has to be eliminated or made harmless;
• foreign living matter may gain entrance, carrying the additional hazard that the intruder may poison or proliferate and overwhelm its host.

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____________

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

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.

• (a) B lymphocytes develop in the bone marrow from stock originating perhaps in the fetal liver, then populate germinal centres, and can proliferate and differentiate to become the sessile (non-moving) antibody-forming plasma cells;
• (b) T lymphocytes originate in marrow, develop and survive elimination in the thymus, are more mobile, and play many roles.
  

3 Roles of the T lymphocyte

• (a) By gene rearrangement and selective expression, to offer a very broad range of T-cell receptors (one specific TCR per clone) for the diverse antigens that might be met.
• (b) Patrolling the body as a naïve or virgin lymphocyte able to be stimulated by a new antigen taken up by macrophages or dendritic cells.
• (c) For some antigens, T lymphocytes help the humoral response of B lymphocytes.
• (d) Less often, they have a suppressor action on the B cell's response.
• (e) For certain antigens, e.g., virally-infected cells or foreign transplanted tissue, the stimulated lymphocyte proliferates, sending its progeny via the circulation as cytolytic/cytotoxic lymphocytes (CTLs) to attack the target cells at close range - the cell-mediated response.

• (f) Cytolytic lymphocytes /CTLs release substances that:
  • (i) lyse the target cells or organisms, e.g., perforin, which inserts uncontrollable pores into the target cell's membrane;
  • (ii) Fas ligand presented on the surface binds to the Fas/death receptor triggering apoptosis;
  • (iii) as cytokines, attract other leucocytes to the site of antigen,
  • (iv) activate naive lymphocytes and cause their proliferation,
  • (v) enhance leucocytes' phagocytic activity;
  • (vi) and trigger the release of histamine by basophils.

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

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

• (a) pathologically, at any site of chronic infection;
• (b) regularly in the lamina propria of tracts exposed to antigens - respiratory, GI and genito-urinary, and the ocular conjunctiva; lymphocytes also migrate into the epithelia;
• (c) in dedicated secondary lymphoid organs - spleen and lymph nodes.

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:

• (a) APCs/reticular cells and macrophages to activate lymphocytes;
• (b) many lymphocytes to respond to a major antigenic challenge coming via the blood (spleen) or lymph (nodes);
• (c) lymphocytes to propagate the immune response further, say, to recruit other nodes;
• (d) a cleansing action by macrophages to remove undesirable materials from blood and lymph undesirable.

B MUCOSO-LYMPHOID ORGANS

• (i) Over the nodules, special low columnar epithelial cells - M cells - develop in order to pass antigens to the underlying antigen-presenting cells in the lamina propria. The APC and lymphocytes sometimes lie in a pocket in the M cell. ('M' for microfolds on the M cell surface.)
• (ii) The antibodies subsequently made by the plasma cells are immunoglobulins of a kind that the typical epithelial cells can take up basally, and secrete apically into the lumen needing protection.
• (iii) It is also necessary for certain types of lymphocyte to enter the epithelium.

C LYMPH NODES

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

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.

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

2 Thymic finer structure
l Cells are:

• (a) packed lymphocytes (thymocytes), less densely packed in the medulla, making it paler, supported by
• (b) stellate epithelio-reticular cells of pharyngeal-pouch endodermal origin, not phagocytic, and with their processes attached by desmosomes (note that the main thymic stromal cell is thus an epithelial cell);
• (c) pale interdigitating dendritic/reticulum cells in the medulla;
• (d) a few macrophages in cortex and medulla;
• (e) some myoid cells, resembling dystrophic skeletal muscle fibres;

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

A EPIDERMIS (epithelium)

2 Cytological details of the layers
l Stratum germinativum/basale

• (a) Keratinocyte precursor cells, cuboidal or columnar in form, lie on a BL.
• (b) Cells project down many small basal processes.
• (c) The whole underside of the epithelium is indented by CT dermal papillae for effective attachment, nutrition, and sensation.
• (d) Cells proliferate to replace lost surface cells.

• (a) Keratinocytes/prickle cells
  ... (i) Principal cell kind; ectodermal in origin; move upwards in the layer and continue to proliferate, despite the many desmosomes holding them together (which, with processing shrinkage, lead to the cells' spiny, prickly appearance).
  ... (ii) Cytoplasm is rich in keratin filaments, bundled into tonofilaments and increasing in number towards the keratin layer, and formed from prekeratin monomers.
  
• (b) Melanocytes
  ... (i) Ectodermal; but migrated neural crest cells.
  ... (ii) Constitute l in 4 to l in l0 of basal epithelial cells.
  ... (iii) Deficient in tonofilaments and desmosomes.
  ... (iv) Synthesize melanin and transfer it via their long dendritic processes to neighbouring keratinocytes.
  ... (v) EM shows that the Golgi apparatus participates in forming the melanosome granules.
  ... (vi) Melanin is formed from tyrosine by the enzyme tyrosinase. Cells with melanin-forming ability can be revealed in a section by treating it with dihydroxphenylalanine (DOPA), which is oxidized to melanin.
  ... (vii) Albinos have an inborn error of metabolism, making them unable to synthesize melanin in the skin and eye.
  ... (viii) UV light causes greater melanin formation and a thickening of the keratin layer. Pituitary and adrenal hormones also increase pigmentation, which is a useful sign for diagnosis.
• (c) Langerhans cells are poorly phagocytic, marrow-derived, specialized macrophages, with long dendrites. They are antigen-presenting cells, accessory to T-cell immunity.
• (d) Merkel cells are sensory cells with vesicles and a polylobulated nucleus. They attach to disc-shaped endings of some of the axons that penetrate the epithelium.

B DERMIS (Corium)

C SWEAT GLANDS (Glandulae sudoriparae)

D SEBACEOUS GLANDS

E HAIR

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:

• (a) Medulla, as the central core of soft keratin and sometimes air spaces. The medulla may be absent.
• (b) Cortex of closely packed, elongated, hard-keratinized cells, formed without any intermediary kerato-hyaline granules developing. Melanin and other pigments may be incorporated in the cells during keratinization.
• (c) Cuticle - outermost coat of shingled/imbricated cells, with their free edges projecting upwards.

• (a) Outer CT sheath and inner basal lamina (hyaline membrane).
• (b) Vascular papilla lies directly under the synthesizing epithelial area, responsible for the upward growth of the hair and its inner root sheath.
• (c) External root sheath is a continuation of the epidermal living layer, expanding to form the basal hair bulb.
• (d) Internal root sheath forms a cuticle layer from which the other cuticle, on the hair, can separate at the level of entry of the sebaceous gland's duct. The internal root sheath thus comprises: (a) innermost cuticle cells, (b) Huxley's layer of cells with trichohyaline granules, (c) Henle's single, outer layer of clear cells.
• (e) Epidermal germinal matrix, above the papilla, forms the hair's cuticle, cortex and medulla. NB - the appearance of cross-sections varies with the level in the hair follicle at which they are taken.
• (f) Arrector pili of smooth muscle fastens the lower hair bulb's CT sheath to the upper dermis nearby.

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

G SKIN FUNCTIONS

Chapter 22 RESPIRATORY TRACT

A RESPIRATORY TRACT TO LUNGS

9 Nasal functions:

• (a) air-filtering, material trapped in mucus is swept by the cilia towards the pharynx,
• (b) air-warming,
• (c) air-humidifying,
• (d) olfaction,
• (e) sensitivity for nasal reflexes such as sneezing,
• (f) resonating the voice.

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

• (a) the lungs are covered by a slippery membrane and are enclosed in another membrane, adherent to the inner chest wall, with a potential space between;
• (b) the lungs are stretched out against their considerable elasticity, so that this space remains only a potential one;
• (c) the larger conducting tubes of the lung need firm cartilages in their walls to prevent their collapsing during the inspiratory sucking in of air.

2 Mucosa of the lower airway

1. Cell types in the epithelium:
   • (a) ciliated columnar cells, with lysosomes and some microvilli;
   • (b) mucus-secreting goblet cells;
   • (c) basal 'undifferentiated' cells to replace the specialized kinds;
   • (d) Clara's non-ciliated bronchiolar secretory cells with granules and GER;
   • (e) neuroendocrine cells;
   • (f) lymphocytes migrated from the lamina propria.
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.

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

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

A KIDNEY

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

• (a) Blood is fed, via an afferent arteriole, under pressure into groups of capillaries, tufting out as loops from the vascular pole, and ensheathed in visceral squamous epithelium.
• (b) Glomerular wall of
  ... (i) fenestrated endothelium,
  ... (ii) thick basal lamina (two laminae fused together),
  ... (iii) podocytes' pedicels (visceral epithelial cells' feet), separated by filtration slits of controllable width, permit
• (c) the filtration of water and solutes, with a molecular mass less than 30 kDa, into a capsular space between
• (d) glomerular/visceral epithelium and the parietal squamous epithelium and BL of Bowman's capsule.
• (e) The altered blood is collected from the capillary tufts, and passes out via the narrower efferent arteriole.
• (f) Between the capillaries at their base lie mesangial cells, synthesizing and maintaining the glomerular basal lamina, and also probably phagocytic and contractile.
  Mesangial cells are significantly involved in renal disease, e.g., in diabetes and glomerular nephritis.

• (a) Most common of those tubules seen in the sectioned cortex, since it is longer than the distal tubule.
• (b) Simple, acidophilic, cuboidal, epithelial lining cells with: large round nuclei;
• (c) very many microvilli (brush border), and a surface glycoprotein coat containing peptidases to reduce polypeptides;
• (d) vesicles and lysosomes just below the microvilli, and involved in endocytotic protein uptake and breakdown to amino acids;
• (e) marked lateral membrane infoldings and interdigitation with adjacent cells,
• (f) to which they attach with junctional complexes.
• (g) The basal region has many membrane infoldings and long mitochondria (basal striation) for the provision of energy for active transport of Na⁺, and with it glucose and amino acids, through the basolateral membrane,
• (h) basal lamina, and thence into adjacent capillaries, with their fenestrated endothelium.

• (a) Squamous epithelial lining on a BL.
• (b) Cells are pale, tightly fastened, with small, short microvilli, and a few mitochondria scattered randomly.
• (c) The lack of red blood corpuscles in the lumen, and plumper nuclei, distinguish thin segments from capillaries.

• (a) Weakly acidophilic, cuboidal epithelial cells enclose large lumens.
• (b) No brush border is seen because only a few short microvilli are present.
• (c) Basal infoldings and interdigitations, with very many long mitochondria, give a basal striation.
• (d) Cells lie on a BL, also supporting fenestrated endothelial cells of the surrounding capillaries.
• (e) Macula densa is a specialized, more nucleated region of the epithelium, where it attaches to the arterioles of the glomerulus to form part of the juxtaglomerular apparatus. It senses the [Cl^-] locally in the distal tubule and signals, via mesangial cells, for renin release, and arteriolar and mesangial contraction.

• (a) Afferent arteriole, nearing the JGA, loses its elastica interna.
• (b) Smooth muscle cells change to epithelioid with
• (c) secretory granules and some GER.
• (d) The juxtaglomerular secretory cells are in contact with the endothelium of the arteriole and, indirectly, with the macula densa of the distal tubule: for sensing (i) renal tubular chemistry, and (ii) stretch, indicating blood pressure.
  The cells' sympathetic innervation is another element in the control matrix.
• (e) Granules are the enzyme renin for release into the blood, where it cleaves a potentially hypertensive polypeptide (angiotensin I) from angiotensinogen.
• (f) A juxtaglomerular interaction with the adrenal cortex and Na⁺ excretion also occurs.
• (g) Polkissen/Goormatigh/lacis cells lie in the angle between the afferent and efferent vessels and the attached distal tubule.

• (a) Pale cuboidal cells, with the lateral cell membranes prominent because lateral interdigitation is lacking, are of three kinds:
• (b) principal collecting-duct cells, and, set between them, alpha/A and beta/B intercalated cells, all differing in their ion-transport roles.
• (c) Principal cells have few microvilli, and few mitochondria, but are tightly connected by occluding junctions. Aquaporin 2 constructs the channels making the luminal cell membrane permeable to water in the presence of vasopressin/ADH, so that the cells reabsorb water. Basolaterally, a membrane Na,K-ATPase lets the cells secrete potassium, while absorbing sodium.
• (d) Intercalated cells have darker cytoplasm, and more and darker mitochondria, than principal cells. The number of vesicles is highly variable, because they function to insert or remove ion pumps into the cell membrane, in a similar way to the gastric parietal cell.
• (e) Type A intercalated cells bear a luminal-membrane H,K-ATPase to secrete hydrogen ions and reabsorb potassium; type B cells have a luminal Cl/HCO₃^- countertransporter to secrete bicarbonate and recover chloride.
• (f) A simple columnar epithelium lines the final papillary ducts of Bellini, and covers the papillae.

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

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

A ORAL STRUCTURES

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:

• (a) Filiform - most numerous, spiky, with a partly keratinized tip which is shed.
• (b) Fungiform - less numerous, larger, with some taste buds in their smooth tops.
• (c) Circumvallate - least numerous, largest, lie along the terminal sulcus, each surrounded by a trench, and with taste buds in its wall.
• (d) (Foliate - small ridges on the sides of the tongue, prominent in rabbit, vestigial in man; also with taste buds in the walls.)

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

• (a) Enamel: covers the crown of the tooth; tall tightly packed prisms/rods; 96 per cent mineral crystals, 4 per cent organic content; but completely acellular.
• (b) Dentine: supports enamel and acts as the skeleton of the tooth; hard material of collagen fibrils, impregnated with crystals of calcium salts; penetrated from the pulp side by dentinal tubules enclosing long, thin processes of odontoblasts, whose bodies lie outside the dentine at the pulp.
• (c) Cementum: a thin layer of bone-like material, with cementocytes (like osteocytes), but no Haversian systems, covers the root. Sharpey's collagen fibres of the periodontal ligament insert into cementum, and also into the bone of the alveolus, thus attaching the tooth to the jaw.
• (d) Pulp: jelly-like ground substance, with CT cells, blood and lymphatic vessels and nerves, on a network of fine collagenous fibres; dentine's pulp-surface is covered with the columnar odontoblasts.

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:

• (i) Migrating neural crest cells become 'mesectoderm,'
• (ii) and induce overlying ectoderm to thicken and grow down producing a
• (iii) dental lamina, under which 'mesectoderm' cells group,
• (iv) These induce the ectodermal dental lamina above to separate into tooth germs, and to provide for each an enamel/dental organ, with its stellate reticulum and inner and outer epithelia. The inner epithelium of the enamel organ, in its turn, induces
• (v) 'mesectodermal' cells of the dental papilla to become odontoblasts, which form dentine.
• (vi) The dentine acts as a stimulus to inner epithelium to become functional ameloblasts and deposit enamel on the dentine. Ameloblasts and odontoblasts are tall, columnar, secretory cells.
• (vii) The enamel organ's epithelium extends down as Hertwig's root sheath, determining the form, size, and number of the roots.
• (viii) The root sheath perforates, and, through the holes, cells of the surrounding mesectodermal dental sac approach root dentine and lay down cementum. Other sac cells become fibroblasts forming the periodontal ligament.

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

• (a) Of epithelium, lamina propria and smooth muscle muscularis mucosae. GI Powerpoint. . and GI Function Powerpoint
  
• (b) The epithelium in most places takes a glandular form, with simple tubular glands and a secreting surface epithelium.
• (c) Some parts have discrete compound glands lying in the mucosa.
• (d) Single lymphoid nodules can occur anywhere.

• (a) Of fairly dense CT, with blood and lymphatic vessels, and having a plexus of unmyelinated autonomic nerve fibres - Meissner's submucosal plexus.
• (b) Glands are present in a few places.

• (a) Two or more helical layers of smooth muscle: the inner, tight 'circular'; the outer, loosely coiled 'longitudinal'.
• (b) Served by a nerve fibre plexus - Auerbach's myenteric plexus, whose parasympathetic ganglion cells lie between the muscle layers.
• (c) Circular coat is more developed at sphincters and valves.

• (a) Of loose CT, with collagen and elastic fibres, nerves and vessels.
• (b) The serosa has a smooth mesothelial covering, and that part of the tract is suspended on a mesothelium-covered tissue fold - omentum or mesentery.
• (c) Mesothelial cells bear microvilli, are well attached, and secrete lubricants to allow viscera to move freely.
  (If mesothelium is lost during inflammation or operations, and is replaced by the fibrous scar tissue of an adhesion, function is lost, e.g., uterine tubal adhesions can cause infertility.)
  To avoid knots and obstruction, the plan for the GI tract is fasten, loosen, fasten, and so forth, so that only the small intestine and transverse colon have long stretches of mobile tube: fastening requires an adventitia, mobility, a serosa. [ Powerpoint story on when a serosa rather than an adventitia is found.]

3 Stomach
l General structure

• (a) Anatomical regions - cardia, fundus, corpus, pyloric antrum and pyloric canal: the regions are histologically distinct.
• (b) Outer covering is a serosa, from which hang omenta.
• (c) Muscular coat of three smooth muscle layers - outer, longitudinal; middle, circular; inner, oblique. The middle layer is more developed to form a sphincter at the pylorus. The muscle churns the contents (chyme), and passes them periodically in regulated amounts to the duodenum.
• (d) Submucosa - no glands; CT carries vessels and the nerve plexus.
• (e) Muscularis mucosae - two layers, with the inner circular one sending a few muscle fibres up towards the lumen.
• (f) Mucosa is deep and glandular, with only a little lamina propria tissue; produces acid and enzymes for digestion, and undertakes some absorption, e.g., of water and alcohol.

• (a) Empty stomach's lining is folded in ridges - rugae.
• (b) Surface is pitted by recesses - gastric pits/foveolae gastricae.
• (c) Long tubular glands extend from the muscularis mucosae up to empty into the pits. A gland has a base, neck and isthmus.
• (d) The surface of the stomach and the pits are lined by simple, columnar, special mucous epithelial cells.
• (e) Gastric glands throughout the body and fundus of the stomach are simple, branched tubules with these cells:
  • (i) Chief/zymogenic/peptic serous cells: in the majority; basophilic, with 'zymogen' granules and rich granular ER.
  • (ii) Parietal/oxyntic cells: occur peripherally and singly; large and eosinophil; packed with mitochondria and smooth ER; have long secretory canaliculi, lined by microvilli, and opening to the gland's lumen.
  • (iii) Mucous neck cells: concentrated near the neck of the gland.
  • (iv ) Endocrine/enteroendocrine/argentaffin/enterochromaffin/ Kultschitsky cells: few in number, seen with EM, silver methods, or cytochemistry, but may be recognized from their empty look with H & E, and their rarity.
• (f) In the narrow cardiac region lie cardiac glands - compound tubular, with mucous and a few parietal cells.
• (g) In the pylorus, pits are much deeper, and glandular tubules are wider and more branching. The main kind of glandular cell present is pale and resembles fundic mucous neck cells.

4 Gastric protective mechanisms

• (a) Digestive secretions (survived by typhoid and other bacilli, and eggs of parasites, which do their damage in the gut, and by Helicobacter pylori). H. pylori, resident in many stomachs, may cause intestinal metaplasia - a pre-malignant state - or peptic/gastric ulcers, in some people.
• (b) Mucous and bicarbonate outer coating of the epithelium.
• (c) A film of surfactant-like lipid secreted by the epithelium.
• (c) Regenerative power of the epithelium, by cell proliferation and migration (normally renewed every few days).
• (d) Lymphoid nodules and lymphocytes, and other leucocytes, in the mucosa and submucosa.
• (e) Tight junctions between the epithelial cells.
• (f) Vomiting.

• (a) Three regions - duodenum, jejunum and ileum, anatomically and histologically distinguishable.
• (b) Serous coat over all except part of the duodenum and the terminal ileum, which are fixed to the abdominal wall.
• (c) Suspended on a mesentery carrying blood and lymphatic vessels, lymph nodes and nerves.
• (d) Muscularis externa has two complete layers.
• (e) Submucosa - occupied by Brunner's mucous, compound tubular glands in the duodenum; elsewhere is CT as for the rest of the tract.
• (f) Muscularis mucosae - inner, circular, and outer, longitudinal smooth muscle.
• (g) Mucosa has:
  .. (i) Villi - finger- or leaf-like projections.
  .. (ii) Crypts of Lieberkühn - simple tubular glands.
  .. (iii) Lamina propria forming the core of each villus and lying between the gland tubules.
  .. (iv) Covering of simple columnar epithelium.

• (a) Enterocytes are columnar absorptive epithelial cells on the villi; with a brush border (many microvilli); are held apically by junctional complexes; the many vesicles at the base of the microvilli communicate with agranular ER.
• (b) Goblet cells, with the nucleus, GER and Golgi apparatus basally, stored mucigen droplets apically.
• (c) Paneth cells, with eosinophil granules holding defensin and enzymes; remain at the base of the crypts.
• (d) Enteroendocrine cells (other names 3.2.e.iv. above), with hormone- and serotonin-containing basal granules.
• (e) Undifferentiated columnar crypt stem cells: few microvilli; able to divide, migrate, differentiate into the other kinds, function, and be extruded at the villus tip, over approximately four days.
• (f) Villus core has the basal lamina for the epithelium, a central lymphatic capillary (lacteal), blood vessels, smooth muscle fibres. The loose stroma of reticular and elastic fibres is heavily infiltrated by WBCs, e.g., CD4+ helper-inducer lymphocytes and eosinophils, and plasma cells.
• (g) Ileum has Peyer's patches of extensive lymphoid tissue, erasing villi, breaking into the epithelium, and interrupting the muscularis mucosae to invade the submucosa. Elsewhere, only solitary lymphoid nodules are to be seen. The epithelium domed over the Peyer's-patch follicles is specialized, with M cells, which transport antigen and otherwise assist immune functions.

• (a) Secretory
  .. (i) Goblet cells give mucus.
  .. (ii) Columnar cells make disaccharidases and other digestive enzymes which, as ecto-enzymes, remain tethered in the microvillous membrane, so constituting one component of the glycocalyx.
  .. (iii) Paneth cells form defensins, etc, for defence.
  .. (iv) Endocrine cells produce hormones to coordinate the functions of the gut, liver and pancreas.
  .. (v) Simple tubular intestinal glands/glands of Lieberkühn also contribute to the enteric juice.
  These secretions are additional to those already present from:
  .. (vi) Salivary and oesophageal glands.
  .. (vii) Stomach mucosa.
  .. (viii) Pancreas and liver, introduced into the duodenum.
  .. (ix) Brunner's duodenal glands (alkaline mucus led into the bottom of crypts).
  
• (b) Gut mucosal absorption of materials degraded by the secretions.
  
  • (i) Membrane transports, active and passive, of many kinds, with appropriate pumps, channels, and transporters. The tricky part is to get lipid in across a barrier based on lipids - the cell membrane, and back out again.
  • (ii) Pathway, through the absorptive cell, from the lumen to the lacteal capillary lumen for lipid:
    .. (a) hydrolysis, by mostly pancreatic lipase, of the dietary triacylglycerols;
    .. (b) interaction of the resulting free fatty acids and monoacylglycerol with bile, to form micelles for solubilization;
    .. (c) in this form, the lipids can be transported through the enterocyte's apical membrane;
    .. (d) in the apical smooth ER (SER), the lipids are re-acylated to triacylglycerol, and bound to a protein for intracellular transport.
    .. (e) Meanwhile, the GER is producing proteins to which some lipid is added - apolipoproteins - which meet up with the apically reacylated lipid at the SER, where
    .. (f) the apoplipoprotein is used as a kind of cage, into the core/interior of which increasing amounts of lipid are introduced, as the lipid droplet - the chylomicron - is assembled.
    .. (g) The Golgi complex is the accumulation centre for the chylomicrons before their basolateral secretion by exocytosis into the baso-lateral intercellular space.
    .. (g) The chylomicrons and similar smaller lipid bodies pass through the basal lamina to enter the lacteal lymph capillary, giving the gut and mesenteric lymphatic vessels their white colour, and constituting chyle.
• (iv) Devices for increasing the effective gut surface area for absorption:
  .. (a) the long length of the gut;
  .. (b) villi;
  .. (c) microvilli on absorbing cells;
  .. (d) plicae circulares/valves of Kerckring (high folds of mucosa and submucosa)
  .. (e) contractions of villus muscle, muscularis mucosae, and two main muscle coats; (microvilli can slowly elongate, but not contract and relax.)

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:

• (a) alkaline mucus of Brunner's glands;
• (b) lubricating and protective goblet-cell mucus;
• (c) immune responses by APCs, lymphocytes and plasma cells;
• (d) rapid reactions of eosinophils, macrophages, and neutrophils
• (e) lysozyme and other antimicrobial contributions of Paneth cells;
• (f) barrier of tight junctions between the enterocytes;
• (g) diarrhoea;
• (h) rapid regeneration by the epithelium.

5 Large intestine
l General features

• (a) Crypts, but no villi or plicae circulares.
• (b) Columnar epithelial cells are: (i) undifferentiated; (ii) goblet (numerous); (iii) colonocytes, absorbing, with microvilli, for water, and some products of bacterial metabolism of the faeces; (some excretion occurs). Endocrine cells are also present.
• (c) Dehydrating faeces need lubrication, hence many goblet cells are present in the simple columnar epithelium.

• (a) Colon and caecum: outer longitudinal muscle coat is gathered into three bands - taeniae coli - which pucker or sacculate the tube, forming haustrations.
• (b) Appendix: continuous muscle coats; few crypts; the mucosa is mainly occupied by lymphoid tissue; the muscularis mucosae may be deficient and lymphoid tissue seen in the submucosa. The wall may be thick. With age the lumen may be blocked off/occluded by fibrosis.
• (c) Rectum: outer longitudinal muscle is one continuous sheet.
• (d) Anal canal
  .. (i) Morgagni's anal columns are 6-10 vertical mucosal folds.
  .. (ii) Dentate line lies at the level of the bases of the columns, where there are tiny flaps and pockets - anal valves and sinuses.
  .. (iii) The histological epithelial anal transitional zone (ATZ) lies between unbroken simple columnar colo-rectal epithelium and lower stratified squamous epithelium.
  .. (iv) The ATZ - the common site of anal cancers - is very variable in its extent and outline, in its kinds of epithelia, and the number of crypts.
  .. (v) Submucosal veins display periodic dilations. Deterioration of their supporting connective tissue permits enlargement and prolapse - haemorrhoids.
  .. (vi) The complex anal musculature includes external skeletal-muscle and internal smooth-muscle sphincters. (The muscles and their innervation are particularly at risk of stretching and damage in women giving birth.)

Chapter 25 PANCREAS, LIVER AND GALLBLADDER

A PANCREAS

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:

• (a) Alpha cells, 20 per cent, and large - produce the hormone, glucagon, which raises the blood's glucose level.
• (b) Beta cells, 75 per cent, smaller - produce insulin, which promotes the intracellular movement of glucose and glycogen storage, thereby lowering the glucose level of the blood.
• (c) Delta cells, 5 per cent, with large argyrophil granules; form somatostatin, which inhibits insulin and glucagon release.
• (d) F cells/PP cells, in islets and among exocrine cells, making pancreatic polypeptide (PP), acting centrally on the brainstem to influence the vagal control of GI functions, and on the liver.

B LIVER AND GALLBLADDER

• (a) Portal vein bringing food-rich blood from the gut.
• (b) Hepatic artery bringing arterial blood.
• (c) Hepatic veins taking away processed blood into the vena cava.
• (d) Lymphatics taking away some lymph.
• (e) Hepatic ducts removing bile to the gallbladder and gut.

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

• (a) Central vein/terminal hepatic venule - very thin wall; lies in the centre of a lobule, with sinusoids converging towards and opening into it.
• (b) Sublobular/intercalated vein - thicker wall; lies alone at the periphery of the lobule.
• (c) Branch of portal vein - again at the periphery of the lobule, but accompanied by one or more small hepatic arteries/arterioles, one or more bile ducts/ductules lined by cuboidal epithelium, and lymphatics.
  Vein, artery, and bile duct constitute a portal triad; the area in which they lie is a portal area/canal. (The lymphatics are ignored for this naming).
  

7 Hepatic lobular blood flow is:

• (a) from branches of the portal vein and hepatic artery; from the periphery towards the centre;
• (b) in the sinusoids, between the cell plates.
• (c) Blood collected in central veins goes to sublobular veins, thence to collecting veins, and then hepatic veins leaving the liver.

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:

• large, spheroid nucleus (sometimes two), with membrane pores, and ribosomes on the outer membrane;
• extensive granular ER (protein synthesis for enzymes, plasma proteins, etc.);
• smooth ER (steroid hormone and cholesterol metabolism; lipids are taken in, processed, and secreted in a way very like the enterocyte's; SER carries enzymes for detoxifications);
• mitochondria (oxidative and other enzymes);
• actin and other filaments, near the bile canaliculi and elsewhere.
• cell membrane projecting microvilli into the space of Disse, and held firmly to adjacent cells, especially around the channel, the bile canaliculus, formed by the separation of two or three cells' membranes and equipped with a few microvilli;
• Golgi body lying near the canaliculus, as do the
• lysosomes; both appear to help form bile;
• peroxisomes with other enzymes, e.g., catalase;
• glycogen granules stored in association with smooth ER (an association seen elsewhere);
• fat droplets occurring briefly after meals;
• lipofuscin or aging pigment, as another normal inclusion; and sometimes brown haemosiderin, with its iron, may be seen.

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

B CHEMISTRY OF HORMONES

• (a) Amino-acid derivatives
  .. (i) iodinated, e.g., tetraiodothyronine
  .. (ii) catecholamines, e.g., norepinephrine
  .. (iii) melatonin
  
• (b) Polypeptides
  .. (i) oxytocin, and vasopressin (ADH)
  .. (ii) glucagon and insulin
  .. (iii) neuroendocrine hormones, e.g. gastrin, motilin
  .. (iv) parathormone and calcitonin
  .. (v) adrenocorticotrophic hormone (ACTH)
  .. (vi) melanocyte-stimulating hormone (MSH)
  .. (vii) hypothalamic releasing factors and somatostatin
  ..(viii) relaxin
  .. (ix) atrial natriuretic factor (ANF)
  .. (x) pancreatic polypeptide (PP), and leptin
  
• (c) Proteins and glycoproteins
  .. (i) thyrotrophic hormone (TH/TSH)
  .. (ii) growth hormone (GH/STH) and prolactin (PRL/MTH)
  .. (iii) gonadotrophins; follicle stimulating hormone (FSH)
  and luteinizing hormone (LH/ICSH); chorionic gonadotrophin (hCG)
  .. (iv) renin
  .. (v) erythropoietin (EPO)
  .. (vi) inhibin
  
• (d) Steroids
  .. (i) mineralocorticoids
  .. (ii) glucocorticoids
  .. (iii) sex hormones
  .. (iv) vitamin D and its metabolites.
  
• (e) Various agents act rather like hormones, but are not classified as such, e.g., kinins, prostaglandins. Some peptide hormones have local paracrine, rather than distant, endocrine, effects.
  

• (a) Bioassay - a known volume of fluid is injected into an animal lacking the hormone, and showing some measurable deficiency. The extent to which the injected fluid restores the animal to a normal condition gives a measure of its hormone content. (The method is old, but still useful.)
• (b) Radioimmunoassay - using antibody to the hormone, a radioactive label, a method of separating immunologically bound from free hormone, a scintillation counter for determining the concentration of radioactive material, and previously established curves.

C CYTOLOGY OF HORMONE SECRETION

D QUESTIONS TO CHARACTERISE A HORMONE

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).

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:
   • (a) Removing the target tissue, when it itself forms a hormone?
   • (b) Giving an excess of the material, e.g., water or glucose, whose homeostasis is being controlled?
   • (c) Removing from the animal's diet the substance, e.g., Na⁺, whose homeostasis is being controlled?
   • (d) Interfering with possible links in the control, e.g., by cutting the pituitary stalk, giving an antibody to neutralize a releasing factor, sectioning nerves, e.g., vagus, or transplanting the endocrine cells?

Chapter 27 ENDOCRINE SYSTEM

A HYPOPHYSIS/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

• (a) Adenohypophysis develops from the ectodermal Rathke's pouch above the oral cavity.
  
• (b) Rostral wall of Rathke's pouch becomes the anterior lobe; caudal wall gives the intermediate lobe; the cleft between the intermediate and anterior lobes occludes to a line of cysts; and the dorsolateral corners of the pouch give the pars tuberalis.
• (c) Neurohypophysis comes as a downgrowth of the floor of the diencephalon. The brain connection is maintained.

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

• (a) Thick, branching cords and plates of cells, supported on basal laminae and reticular fibres. Between the cords run wide sinusoidal capillaries of fenestrated endothelial cells on their own BLs.
• (b) Classical division of the cells was into acidophils (40 per cent), basophils (l0 per cent), and chromophobes (50 per cent).
• (c) Chromophobes are sparsely granular, small, pale, and often clustered together. They are thought to be less active forms of the five secretory, granular, chromophil cell kinds.
• (d) Chromophils can be distinguished by various stains, since some form peptide hormones, others glycoproteins; by EM, from the size, density and shape of the granules; and by immunostaining, for LM and EM.
  • (i) ACIDOPHILS
    Somatotroph - makes growth hormone (GH)/somatotrophin (STH); stained by orange-G
    Lactotroph/Mammotroph - makes prolactin/mammotrophin (MTH); stained by erythrosin
    
  • (ii) BASOPHILS, staining also with PAS and aniline blue
    Thyrotroph gives thyrotrophic hormone (TSH/TH)
    Gonadotroph gives luteinizing hormone (LH) and follicle-stimulating hormone (FSH)/interstitial cell-stimulating hormone (ICSH)
    Corticotroph makes adrenocorticotrophin (ACTH) by cleaving pro-opiomelanocortin (POMC) appropriately
• (e) Hypothalamic regulation of the adenohypophysis is via the hypothalamo-hypophyseal portal circulation, and for gonadotrophins, ACTH, and TSH, functions by negative feedback thus:
  1. Hypothalamic neurons are specialized to be sensitive to a blood deficiency of the target gland's hormone, e.g. thyroxine.
  2. From the sensitive neuron's terminal, a neurosecretory, chemical peptide releasing factor, e.g. TH-RH/TH-RF, passes into
  3. blood capillaries of the median eminence, whence it drains down
  4. via the portal circulation to the pars distalis.
  5. The releasing factor passes out of the blood to activate the appropriate
  6. chromophil cell, which produces more trophic hormone, e.g., TH.
  7. The trophic hormone passing in the blood to the target gland, thyroid,
  8. promotes an increased output of target gland hormone, thyroxine, whose raised blood level
  9. then reduces the activity of 1. the sensitive hypothalamic neurons, i.e., the system uses a negative feedback.
  This simplification ignores the inhibitory factors, such as hypothalamic somatostatin preventing the release of growth hormone.
• (f) The hypothalamus thus acts as an endocrine organ.

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:

• (a) oxytocin/pitocin: makes mammary gland myoepithelial cells and uterine smooth muscle contract;
  
• (b) vasopressin/pitressin/antidiuretic hormone (ADH): makes the kidney collecting tubule permeable to water, and influences vascular and gut smooth muscle.

B PINEAL GLAND/EPIPHYSIS CEREBRI

• (a) Pinealocytes: basophilic, with secretory inclusions and lipid droplets; nuclei indented; many ribosomes and smooth ER; innervated by sympathetic fibres; and release melatonin.
• (b) Interstitial glial cells: 5 per cent; stellate with long processes.

C THYROID GLAND

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

• (a) Are APUD cells of neural crest origin,
• (b) and produce the polypeptide calcitonin for the reduction of high plasma Ca²⁺ and phosphate levels.
• (c) Although diffuse, in sum they form a gland antagonistic to the action of the parathyroids.

• (a) Are stimulated by pituitary thyrotrophic hormone (TSH) to produce and release two iodinated amino-acid hormones - tetraiodo-thyronine (thyroxine/T4) and 3,5,3-triodo-L-thyronine(T3),
• (b) which are stored in the colloid, as component amino acids of the glycoprotein - thyroglobulin.
• (c) The hormones accelerate general and specific metabolic processes of the body.
• (d) Electron radioautography has shown the sites in the sequence of hormone production by the follicular cells:
  • (i) Iodide concentration - basal part of the follicular cell.
  • (ii) Iodide oxidation - throughout the cell.
  • (iii) Synthesis of thyroglobulin - basal cell, granular ER, Golgi body, by vesicle to the lumen.
  • (iv) In the luminal thyroglobulin, tyrosine residues are iodinated, then pairs condense.
  • (v) Cellular retrieval of thyroglobulin from colloid storage - cell's apical region by endocytosis.
  • (vi) Transport to lysosomes, where cathepsins degrade the large modified molecule.
  • (vii) Release of freed iodothyronines - out of the base of the cells into the blood.

D PARATHYROID GLANDS

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

• (a) Chief cells: small, 7-l0 µm diameter; some dark, some light: contain glycogen, a Golgi complex, lipofuscin pigment, and argyrophil secretory granules; form occasional small follicles.
• (b) Oxyphil cells; larger, acidophilic, and often occur in clumps; cytoplasm is packed with mitochondria; no secretory granules; serve no known role. More oxyphil cells are seen in older individuals.

E ADRENAL/SUPRARENAL GLAND

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

• (a) Zona glomerulosa - under the capsule, rounded balls or groups of columnar cells with dark nuclei.
• (b) Zona fasciculata - long, straight cords of large cells, swollen with lipid droplets.
• (c) Zona reticularis - network made up of cells, small and often lipid-free; lies nearest to the medulla.

• (a) Aldosterone (mineralo-corticoid) helps control water and electrolyte balance, e.g., by promoting renal Na⁺ reabsorption, and having repercussions on blood pressure; secreted in the Z. glomerulosa, and released in response to angiotensin II.
• (b) Cortisol (gluco-corticoid) helps control carbohydrate metabolism, e.g., facilitates protein catabolism and gluconeogenesis (thus interfering with processes requiring a high rate of protein synthesis, such as wound repair and antibody responses): formed in Z. fasciculata and reticularis in response to pituitary ACTH, itself released under hypothalamic control; glucocorticoids affect the cells and ground substances of connective tissues.
• (c) Other glucocorticoids, and significant amounts of sex hormones, in Z. Fasciculata and reticularis.

• (a) Sparse ganglion nerve cells, probably serving vascular smooth muscle in arterioles and the central vein.
• (b) Chromaffin cells: large, granular, and arranged around venules, with their other pole by blood capillaries; by far the major cell type.
• (c) Schwann cells to accompany the nerve fibers.

• (a) Norepinephrine (transmitter substance for sympathetic, postganglionic fibres).
• (b) Epinephrine (increases cell respiration, cardiac output, and glucose mobilization, for the great muscular effort needed in fighting or fleeing).

F KIDNEY

G APUD NEUROENDOCRINE AND PEPTIDE SYSTEMS

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

Chapter 28 MALE REPRODUCTIVE SYSTEM

A TESTIS

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:

• (a) spermatogenic cells, quiescent or in the various phases of development;
• (b) Sertoli supporting cells; well attached, tall with an irregular columnar form, and a pale ovoid nucleus with a prominent nucleolus; taking up testosterone; and controlling spermatogenesis.

• (a) spermatogonium, spheroid cell lying basally, divides mitotically for several generations, then become a
• (b) primary spermatocyte, larger, divides by the first meiotic division (to halve the chromosome number to haploid 23 and introduce genetic variety), to produce
• (c) secondary spermatocytes, small, soon undergoing the second meiotic division, maintaining the chromosome number at 23, to give
• (d) spermatids, smaller and incompletely separated, which, without dividing, metamorphose by the process - spermiogenesis - into
• (e) spermatozoa, released into the tubule's lumen.

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:

• (a) acrosomal head cap, with an enzyme - proacrosin - to aid binding to, and penetration of, the zona pellucida of the oocyte;
• (b) nucleus, streamlined in shape, with dense chromatin;
• (c) neck joining the head (nucleus and head cap) to the flagellar tail, which has the:
  • (i) middle piece, with an axial axonemal core of microtubules in a cilium-like array, nine dense longitudinal fibres and, outermost, a sheath of mitochondria ending at the annulus;
  • (ii) principal piece, with both longitudinal and circumferential fibres around the axoneme;
  • (iii) end piece, with microtubules like a cilium, but no dense fibres.

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

• (a) construction of the acrosome by the Golgi complex;
• (b) the nucleus, thus polarized at one end, condenses and elongates;
• (c) at the other end, one of the centrioles initiates formation of the flagellar tail;
• (d) mitochondria migrate to form a sheath in the tail;
• (e) excess cytoplasm is shed as a residual body;
• (f) the head of the spermatid throughout spermiogenesis stays held in a recess in a Sertoli cell.

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

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

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

5 Corpus spongiosum

• (a) is erectile, but less turgid than the corpora cavernosa;
• (b) has less smooth muscle in the CT trabeculae;
• (c) originates proximally as the bulbus urethrae and
• (d) extends distally to form the bulbous glans, occupied by many veins and nervous receptors, and covered by stratified squamous epithelium, variably keratinized;
• (e) ensheaths the cavernous/penile urethra, lined by stratified columnar and finally stratified squamous epithelium.

E MALE & FEMALE REPRODUCTIVE 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

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

                     ___|___            ___.___
                    |   |   |          |   .   |
                    |   |   |        ------.-------->
                    |___|___|          |___.___|
                        |
                        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.            |

Chapter 29 FEMALE REPRODUCTIVE SYSTEM

A OVARY

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

• (a) Oocyte increases in size.
• (b) Golgi complex and other organelles become more dispersed in the cytoplasm, and lipid droplets appear.
• (c) Zona pellucida of glycoprotein forms between the oocyte and surrounding follicular cells; both extend processes into it. The zona pellucida may protect the ovulated and fertilized oocyte from phagocytosis and immune rejection.

2 Development of follicular/granulosa cells and follicle

• (a) Follicular cells are present as a single squamous layer, encircling the dormant oocyte (stage of primordial follicle).
• (b) The primary follicle arises by enlargement of the follicular cells - they become cuboidal - and of the oocyte.
• (c) Follicular cells proliferate to a multilayered state (secondary/ preantral follicle).
• (d) Primary oocyte moves to an eccentric position. Fluid forms, separating follicular cells and collecting in antra (spaces). Further cell multiplication, and fluid coalescence, lead to a large follicle, with liquor folliculi filling a single antrum (antral/vesicular/ tertiary/Graafian follicle).
• (e) In the follicular lining of granulosa cells, a hillock - cumulus oöphorus - encloses the oocyte.
• (f) The granulosa cells synthesize materials for the oocyte, and also oestrogens, and inhibin to reduce FSH release from the pituitary.

3 Changes in stroma around maturing follicle

• (a) Stromal fibroblast cells build a capsular theca, which
• (b) differentiates into:
  .. (i) an inner theca interna: ovoid secretory cells, with lipid droplets; vascular;
  .. (ii) an outer theca externa: fusiform fibroblastic cells packed densely.
• (c) The growing theca interna secretes androgenic precursors of oestradiol-l7b for aromatase-mediated conversion by the granulosa cells.
• (d) A glassy basal lamina develops between the theca cells and the membrana granulosa lining the follicle.

4 Ovulation

• (a) A sudden surge in LH, coupled with an increase in FSH and a peaking oestrogen level, triggers ovulation, after the completion of meiosis I by the oocyte.
• (b) Graafian/antral follicle, grown huge (l5 mm diameter), extends to and protrudes from the ovarian surface.
• (c) Protruding apical tissue weakens at the stigma, by apoptosis, and enzymatic action on its matrix, and ruptures, helped by thecal cellular contractions; the fluid flows out.
• (d) The fluid takes with it the already floating secondary oocyte (a first maturation division having recently occurred), and some attached granulosa cells as a corona radiata.

5 Corpus luteum: formation, function and fate

• (a) Burst follicle's wall collapses, becoming folded/plicated.
• (b) Lining granulosa cells become secretory granulosa lutein cells - the main component of the corpus luteum of menstruation (CLM), or of pregnancy (CLP);
  theca interna cells become secretory theca lutein cells (found as small nests of darker cells at the periphery of the main mass of granulosa lutein cells, and accompanying vascular septa into the CLM).
• (c) Lutein cells become enlarged, with many lipid droplets (vacuoles, in H&E preparation) and much smooth ER, and secrete the steroid hormone - progesterone,
• (d) which is collected in capillaries that grow in from the theca interna.
• (e) Progesterone makes the uterine mucosa secretory; and inhibits menstruation and uterine muscle contraction, if implantation occurs.
• (f) The centre of the collapsed follicle fills with clotted blood, which is reorganized by ingrowing fibroblasts and capillaries to form a pale, central core of CT.
• (g) Late in pregnancy, or late in the menstrual cycle (if the shed oocyte is not fertilized), the glandular lutein cells degenerate; the corpus luteum shrinks, and is replaced by a small pale mass of hyalinized CT - corpus albicans (white to the naked eye in the fresh, unstained ovary).

B FALLOPIAN/UTERINE TUBE (oviduct)

C UTERUS

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

E EXTERNAL GENITALIA/VULVA

F MAMMARY GLAND/BREAST/MAMMA

4 In each lobe are:

• (a) a stroma of CT - loose collagenous and adipose tissue, with many lymph and blood vessels;
• (b) parenchymal tissue of alveoli and ducts, lined with secretory, cuboidal and columnar epithelia. Alveoli and ducts also have myoepithelial cells between epithelium and basal lamina.

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

• (a) Prolactin/mammotrophic hormone of the anterior pituitary and placenta promotes an 'apocrine' secretory activity in alveolar cells; first of protein-rich colostrum, then milk.
• (b) Milk comprises:
  .. (i) proteins, e.g., casein (seen as granules in EM);
  .. (ii) lactose;
  .. (iii) minerals;
  .. (iv) fat (seen as osmiophilic droplets in EM) extruded apically as large, membrane-bound bodies;
  .. (v) water.
• (c) Secretion needs adequate nutrition, and proper levels of thyroid, adrenal, and other hormones.

6 Post-lactational regression and post-menopausal involution

• (a) If the milk is not suckled, it accumulates and secretion slows down.
• (b) Alveolar cells die by apoptosis. The secretions and degenerating alveolar cells are resorbed, leaving the gland with ducts, but fewer alveoli.
• (c) After the menopause or surgical oophorectomy, the alveoli and ducts regress further, cysts may form, and the CT tends to become pale, acellular and hyalinized.

G PLACENTATION

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:

• (a) outer syncytiotrophoblast layer of fused-together, basophilic, hormone-secreting, and absorptive 'cells', with smooth and granular ER, and a brush border (microvilli) on their maternal blood-facing surface;
• (b) cytotrophoblast layer of distinct Langhans' cells, paler and reducing in number as time goes on; on a
• (c) basal lamina: the outer boundary of a
• (d) loose stroma of reticular fibres and ground substances, with large Hofbauer vacuolated macrophages, and fibroblasts;
• (e) fetal blood vessels and capillaries with their BL and unfenestrated endothelium; capillaries move nearer to the wall of the villus, as gestation proceeds, and the stroma acquires more collagen.
• (f) The nature of the placental barrier between maternal and fetal bloods thus changes somewhat during pregnancy.

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

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

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 (HPO₄^2-) 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

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).

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:

• (a) By using special fixatives, e.g., glutaraldehyde for EM.
• (b) By cutting unfixed sections in a freezing environment in a cryostat.
• (c) Freeze-drying involves the rapid freezing of fresh tissue in fluid cooled to - l500 C. Subjecting the frozen tissue to vacuum removes the water (ice), which can be replaced straightaway by an imbedding medium, or by a fixative (freeze-substitution).

• (a) The presence of the enzyme, phosphatase, in a section can be demonstrated by:
  • (i) Incubating the fresh section with a natural substrate, e.g., glycero-phosphate.
  • (ii) Liberated phosphate ions combine with calcium ions, also present in the medium, to form an insoluble calcium phosphate precipitate at the site of enzymatic reaction.
  • (iii) Treatment of the section with a cobalt or lead salt results in a replacement of the calcium with the new metal, in the phosphate precipitate. (The lead phosphate is electron-dense; electron histochemistry is therefore possible for some enzymes.)
  • (iv) Treating the section with ammonium sulphide now transforms the lead salt to black, lead sulphide visible for LM.
• (b) Another method for phosphatase involves:
  • (i) Incubation of the fresh section with an artificial substrate, b- naphthol-phosphate.
  • (ii) The enzyme's action liberates naphthol.
  • (iii) Treatment with a diazonium salt gives a coloured azo product, forming at the reaction site.
• (c) Enzymes of the dehydrogenase class can be revealed in their location because they convert tetrazolium salts in solution to coloured, insoluble formazans.

4 Electron-microscopic control for cell fractions

• (a) Is an adjunct to electron histochemistry in situ. Thus, if an enzyme is visualized in mitochondria, confirmation of its presence there can be had by obtaining cell fractions by biochemical density-gradient fractionating techniques.
• (b) Examine samples of each fraction by EM to establish that some are nearly homogeneous.
• (c) Biochemical studies should then show that only the mitochondrial fractions have the enzyme's activity.
• (d) Conversely, some enzymes are present in only one of kind of organelle. Thus a cytochemical method for the enzyme can also confirm the nature of an organelle seen in EM, e.g., acid phosphatase as the marker for lysosomes.

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

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

F RADIOAUTOGRAPHY AND ELECTRON RADIOAUTOGRAPHY

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

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

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

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:

• (a) Polyethylene glycol or Sendai virus promotes unusual cell fusions, and thus can make hybrid cells, e.g., chick-mouse, for use in many problems, e.g., how genes are expressed, and what makes cells malignant.
• (b) Lectins, e.g., concanavalin A, bind to sugar groups of cell-surface glycoproteins, and thus interfere with the many cell membrane functions (Chapter 3.A.5), e.g., inhibiting phagocytosis, but stimulating lymphocytes to divide.
  (The older lectin name, phytohaemagglutinin, referred to plant proteins named for causing red blood cells to stick to one another.)
• (c) Other tools for interfering selectively, but maybe not specifically, with cell components are: colchicine for disrupting microtubules, and cytochalasin B to make actin filaments fall apart; monensin or cooling to 20º C perturbs transport through the Golgi complex.

• (a) The release of free oxygen radicals by macrophages, in response to cytokines, can be measured by the extent to which the radicals turn nitrotetrazolium-blue in the medium into an optically dense precipitate around the cells.
• (b) Change in the intracellular calcium ion concentration affects probes such as fura-2, 'Calcium Green', etc.
  • (i) A loading medium or carrier is needed to get the probe into the cells.
  • (ii) The probe allows microspectrophotometric measurement of changes in [Ca²⁺] of individual cells reacting to a stimulus.
  • (iii) The probe, upon chelation with cytoplasmic Ca²⁺, alters the intensity and wavelength of the fluorescent light that it emits, when excited by a source.

• (a) Phase-contrast microscope
  • (i) Different structures diffract and retard the waves of light to varying extents, putting those coming through the various structures of the tissue more or less out of phase with one another.
  • (ii) A recombination of these variously retarded waves with other light that has passed diffracted, but slowed, through the condenser and specimen permits summation and cancelling out (visible as light and dark intensity differences), as the two sine waves meeting correspond or cancel out.
• (b) Interference microscope
  • (i) Is likewise based on a variety of phase differences resulting from the various refractive indices of structures in the specimen.
  • (ii) However, the variously phased waves are recombined with a separate, reference, beam sent around the specimen, and not through it.
  • (iii) Since the reference beam passes by the tissue uncontaminated, it provides a stable factor, allowing quantitative measurements of the optical densities of structures in the tissue to be determined, from which the concentrations of their constituent materials can be estimated.
• (c) Interference-contrast microscope
  Nomarski added the interference principle to heighten the clarity and relief of the phase-contrast image - Nomarski optics.
• (d) Dark-field microscopy
  • (i) Relied on the same phenomenon that makes dust particles 'visible' in a shaft of sunlight.
  • (ii) Small particles, e.g. bacteria, are illuminated by a strong light beam not itself directed into the objective, hence the field is dark.
  • (iii) The particles diffract the light, some of which goes into the objective, enabling the particles to be seen as light 'sources' against the background, even when their size is below the limit of optical resolution for the usual, brightfield, microscopy.
• (e) Lapsed-time cinéphotomicrography/cinémicroscopy
  • (i) Through the phase or Nomarski microscope, pictures of living cells were taken on ciné film.
  • (ii) A time-lapse device arranged for one frame to be taken every few seconds.
  • (iii) When the film was projected at, say, l5 frames per second, movement of the magnified cells was seen on the screen. The movement is, of course, many times speeded up over the natural rate.
  • (iv) Thus, visibly dynamic cell activities could be recorded, e.g., mitosis, phagocytosis, contact inhibition of movement.
    Ciliary and flagellate movement, and heart muscle contraction needed slow motion filming.
  • (v) Digital storage of video-camera or digitized-microscopy data now permits much more scope for image replay, analysis, and manipulation.
• (f) Confocal laser scanning microscopy
  1 At any one time, the laser light-source illuminates one small spot within the width and depth of the specimen. The detector is likewise restricted to observing the same spot.
  2 The illumination beam and detector are scanned, in synchrony with the fine focus, to furnish a series of optical sections down through the specimen, whose images can be stored in the computer.
  3 The laser beam can penetrate up to 1 mm into the specimen, making the ability to offer 3-D information even more useful.
  4 The microscope can work also in fluorescence and phase-contrast modes to provide clear images and exact localisation of probes.

J MEDICAL CYTOGENETICS

l Techniques
l Chromosomes of cultured cells

• (a) Cells, e.g., lymphocytes from the child's peripheral blood or amniotic foetal fibroblasts, are taken for culture.
• (b) A growth factor or lectin added to the medium causes some cells to divide.
• (c) Dividing cells are arrested at metaphase by adding colchicine to disrupt the microtubules of the spindle apparatus.
• (d) Metaphase chromosomes are brought, by a squashing procedure, into one visual plane for examination, and stained.
• (e) Karyotype is the name for all the chromosomes presenting flattened in one plane (can be photographed, cut out, and arranged in order, or digitally rearranged, for convenience).
• (f) Chromosomes are counted to see if their number is diploid (46), hyperdiploid (more than 46), or hypodiploid (less than 46); and individual chromosomes are studied for malformations, and changes in their Giemsa-stained banding patterns. In situ hybridization (3 below) allows a particular gene to be mapped to an individual band of a known chromosome.

• (a) In interphase cells, the sex chromatin is a clump of undispersed and inactivated chromatin.
• (b) One is seen per nucleus in normal females, since it is derived from one of her X chromosomes; the other X chromosome uncoils and loosens and is not seen at interphase.
• (c) The sex chromatin or Barr body was first seen in female cat neurons, attached to the nucleolus, later observed in oral mucosa and epidermal cells attached to the nuclear membrane, and in neutrophil leucocytes, as a drumstick extension to the nucleus.
• (d) Take a smear of buccal mucosa cells, stain well for chromatin, and count the proportion of cells without a sex chromatin, and note whether any have more than one sex chromatin body.

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

• (i) to identify - paint - a particular chromosome, by using a library-derived cocktail of DNAs from multiple sites, specific to that chromosome.
• (ii) to locate the specific gene of interest (o, in Fig.) at a region along the marked chromosome.

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)

• (a) Philadelphia chromosome - a 22 with abnormally short long arms - is observed in 90 % of cases, and is an acquired abnormality, specific for the disease.
• (b) It is seen on long arms of chromosomes of pairs 22 and 9, because of a partial translocation - tr(9:22)(q34;q11).
• (c) The translocation of part of the proto-oncogene c-bcr from 9 creates a hybrid gene, bcr-abl, on 22, which is transcribed and translated into an abnormal kinase: this is read by the molecular circuits controlling cell division as requiring ceaseless myeloid cell proliferation.

4 Mongolism (Down's syndrome)

• (a) Characterized by defective brain development, and severe mental retardation.
• (b) A gamete is deranged - a nondisjunction - so that an extra chromosome appears, usually additional to pair 21 making it trisomic. Other trisomies are Edward's at l8, and Patau's at pair l3.

5 Intersex states (genetic)

• (a) Can sometimes be revealed as genetic by examining the sex chromatin of an interphase epithelial-cell preparation, but are checked in chromosome preparations.
• (b) Turner's syndrome of a female appearance (phenotype), but with 45 chromosomes (an X is missing), and no sex chromatin; and Klinefelter's with male genitalia, but 47 chromosomes (two X and one Y), and having sex chromatin. These are abnormal people, but extra X's in women and Y's in men may not be significant.
• (c) X-linked androgen-insensitivity: the genetically XY man has a woman's external genital appearance (phenotype), because, although testes are present, the gene for the androgen receptor on his one X chromosome is missing, shortened, or mutated. Internally, the Wolffian ducts are undeveloped. Some mutations result in only partial androgen insensitivity, and ambiguous phenotypes.

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

• (a) In transplantation research, when cells behave differently after a transplantation of foreign cells, it is of vital theoretical interest to know whether the cells behaving in the new way are derived from the host, or came in as donor cells with the transplant.
• (b) If mouse cells are transplanted to the chick chorio-allantois, or Japanese quail cells into a chick, the chromosomes (and chromatin) of chick and mouse (or quail and chick) are sufficiently different to indicate the source of the cells in which they are seen. 1950s haematologists used strains of mice with spontaneously altered and distinctive chromosomes.
• (c) The prior insertion into the cells' DNA of retroviral markers (e.g., driving expression of beta-galactosidase) is a more recent way to trace the fate of introduced cells.

K ELECTRON MICROSCOPY

• (a) Solutions of salts of heavy metals, e.g., lead, manganese, uranium, may act as stains and make structures more electron-dense, i.e., improve their contrast.
• (b) Variations in the surface structure of certain things, e.g., collagen fibre, can be made visible by 'shadowing' the structure by applying the vapour of a heavy metal from one side. The metal will deposit more on the exposed side of the surface elevations.
• (c) Staining and shadowing can be applied to spreads of materials, e.g., large proteoglycans and chromatin.
• (d) Negative staining involves filling empty interstices of a structure with a heavy metal, thus outlining the constituent elements, e.g., protein capsomeres in the case of an adenovirus particle.
• (e) Electron histochemistry, e.g., of enzymes, is possible using lead compounds as the substrate or in the medium, since the lead precipitate is electron-dense.
• (f) Electron radioautography uses in the usual way a section having a very thin coating of photographic emulsion. The silver grains are seen as black threads in the EM. For example, this method has shown the secretory sequence in pancreatic exocrine cells using tritiated leucine.

               

            ________| |Electron-emitting, heated FILAMENT
           |        $$$        |
           |         .         | 
           |   ~~~~~~.~~~~~~   | Electron-accelerating ANODE
           |   __    .    __   |
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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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           |       .       |
Anode +    | ~~~~~ . ~~~~~ |                                    Cathode ray tube/CRT
           |       .       |                                           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|__|__________        ___________          | /    .      |
           |     s .   / _____________|------|___________|----------/     .      |
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       ____|      |a** /   |                                       |_____$$$_____|
Pump <-____       | g  "   |                                             | |
           |      | |e  "  |
           |______|_|___"__|
                  | |   "
                  W |   " " " " X-ray detector
         Stage tilt W      
         & rotate           

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

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

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

                    - 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 +

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