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