Professional Documents
Culture Documents
Histoloty - DR Oday
Histoloty - DR Oday
Tissue Preparation
INTRODUCTION
o Artifacts
FIXATION
EMBEDDING
STAINING
SECTIONING
INTRODUCTION
Most fresh tissue specimens are colorless and squishy. They provide little useful information.
For scientific or diagnostic purposes, tissue specimens must undergo substantial alteration in
preparation for viewing under a microscope.
Most basic histology texts offer a minimal account of basic histological technique. For routine
examination of tissues, you probably don't need to know much more.
Artifacts. Be aware that each step of tissue preparation introduces artifacts by altering or
distorting the natural appearance of cells.
Some artifacts are unavoidable. Fixation, by its very nature, kills cells and stabilizes dynamic
cell processes. Enzyme activity is usually altered. Ions and small molecules are usually washed
away.
Some artifacts are intentional, most notably the colors added by staining. The pink and purple
colors of H&E staining can become so familiar that they appear "normal", as if they were the
natural colors of tissues.
2
Still other artifacts are accidental. Cells may shrink or swell during fixation. Extracellular
spaces may be distorted by compression or stretching. Ripples and wrinkles can be introduced
during cutting and handling of sections.
Unintended artifacts can be minimized by optimal procedures -- but optimal procedures are often
impractical, especially with human specimens. Ideal tissue preparation preserves cells in a form
that resembles the living state, but this ideal is seldom practical with clinical specimens. Often,
especially in post mortem (autopsy) material, cells have been dead and deteriorating for several
hours before fixation. Therefore, certain artifacts must be appreciated as part of the normal
appearance of tissue specimens.
The most common mode of routine tissue preparation involves fixation with buffered
formaldehyde, embedding in paraffin, sectioning into slices about 5 micrometers in thickness,
and staining with hematoxylin and eosin.
Modern cell biology uses many tools to reveal cell structures and functions that are not apparent
on routine H&E slides. Many of these involve sophisticated reagents based on the specificity of
enzymes, immunological antibodies, or gene sequences to label and localize specific proteins or
other molecules. Some textbooks present additional detail.
FIXATION
Fresh tissue samples must be preserved for future examination. This process is called fixation,
and the resulting specimen is described as fixed.
Boiling an egg and pickling a cucumber represent examples of fixation, in which heat or
chemistry stabilizes the organic materials.
A variety of chemicals can be used for fixing histological specimens. Routine fixation often uses
a solution of formaldelhyde (formalin) to react with proteins and other organic molecules to
stabilize cell structures. This solution is buffered and osmotically balanced to minimize
shrinkage, swelling, and other collateral damage.
Perfusion involves the delivery of fixative through the circulatory system of living tissue, by
direct injection into a major artery. Such a procedure is commonly used with experimental
animals but is obviously impractical for obtaining clinical specimens from patients.
3
Successful fixation by immersion requires very small samples. However, surgical removal of
very small tissue samples often entails incidental mechanical damage, especially with punch
biopsies.
These constraints on ideal fixation mean that tissue quality may vary across a specimen, with
possible distortion near edges (especially with needle or punch biopsies) and with variation in
fixation quality (and attendant staining character) in deeper areas (into which fixative diffuses
more slowly).
Frozen sections are seldom as "pretty" as well-fixed specimens, but they do have certain
advantages. Because frozen sections do not require hours for the normal schedule of fixation and
embedding, they can provide immediate diagnostic information to a surgeon in the operating
room. Frozen sections can also permit analysis of small diffusable molecules or of enzyme
activity whose presence would be lost during chemical fixation.
After fixation, tissue specimens are routinely embedded in a solid material which will support
very thin sectioning.
To embed a tissue sample, tissue water is replaced first by solvents (such as alcohol and xylene)
and then with a liquid such as melted wax (paraffin) or epoxy solution which can be
subsequently solidified by cooling or polymerization.
Sectioning is the production of very thin slices from a tissue sample. The tool used for
sectioning is called a microtome (tom = to cut, as in appendectomy). A microtome may be as
simple as razor blade, or it may be a complex machine costing several tens of thousands of
dollars (for producing the ultrathin sections needed for electron microscopy).
Sections for routine light microscopy are typically 5-10µm (micrometers, microns) in thickness.
Exceptionally thin sections may less than 2µm thick. For electron microscopy, sections are
typically 50-100 nanometers (millimicrons) in thickness.
Among the commonest artifacts, and most distracting for a beginner, are wrinkles. To
appreciate why wrinkles form, imagine trying to lay a sheet of wet tissue paper (representing the
slice from the sample) flat onto a table (representing the microscope slide). Even with great care,
wrinkles sometimes appear. Sometimes wrinkles are "forced" when the tissue section stretches
unevenly around structures of differing consistencies.
Another sectioning-related artifact is the disappearance of small structures which fall out of their
proper place on the specimen, and the occasional reappearance of such structures at other
inappropriate locations. This happens most often when the process of slicing separates a part
which is attached only outside the plane of section, such as a hair shaft within a hair follicle.
Except in the case of perfect lengthwise slices, the hair shaft will be cut into an oval slice that is
not attached to the sides of the hair follicle and may therefore come out (leaving the follicle
apparently empty) and then alight somewhere else (as an odd oval structure anywhere on the
slide).
Yet other common artifacts are scratches and "chatter". Scratches are caused by flaws or dirt
on the cutting edge, and appear as straight slashes or ragged tears across the specimen. "Chatter"
is the visible record of knife vibration. The the process of slicing sometimes induces vibrations
in the knife edge, which then cause variations in thickness (ripples) in the section. These appear
as narrow parallel bands, usually evenly spaced, across a tissue specimen. They are often most
evident in areas of smooth texture, such as the colloid in thyroid follicles.
STAINING
NOTE that all stain color is artifactual and does not represent the natural color of the tissue.
The same structures may have very different colors with different stains. For example, collagen
is pink with H&E but blue or green with trichrome. You should generally use specific aspects of
actual structure (location, size, shape, texture) to identify cells and tissues, rather than color.
Color can offer additional information if used wisely, but is unreliable by itself.
H&E stain
Hematoxylin is a basic stain with deep purple or blue color. Structures that are stained by basic
stains are described as basophilic ("base-loving"). Chromatin (i.e., cell nuclei) and ribosomes
are basophilic. With H&E staining, basophilic structures are stained purple.
Eosin is an acidic stain with a red color. Structures stained by acid stains are described as
acidophilic (or eosinophilic) and include collagen fibers, red blood cells, muscle filaments,
mitochondria. With H&E staining, acidophilic structures are stained red or pink.
Note that, basophilic cell structures are NOT necessarily acidic; they only
happen to stain with basic stains. Likewise for acidophilic structures,
which are NOT necessarily basic. Many tissue staining properties are
determined by the complex chemistry of proteins and other
macromolecules after interactions with fixatives and other processing
agents, and defy simple analysis.
Also note that absolute color intensity on H&E-stained slides can be quite
variable, with the same cell structure appearing red on one slide, pink on
another, and possibly even blue on yet another. Relative stain intensity on
the same slide is a more reliable indicator of acidophilic/basophilic quality
than is absolute color, but this can also vary, especially between edges and
center of a section.
Remember that nuclei are not really purple and collagen is not really pink. All such stain colors
are artifacts, albeit intentional ones.
If an H&E slide shows any colors other than purple/blue and red/pink -- such as yellow or brown
-- the additional color is probably due to an intrinsic pigment such as melanin.
Some cell structures do not stain well with aqueous dyes and so routinely appear clear. This is
especially so for those which are hydrophobic, containing fat. Included in this category are
adipocytes, myelin around axons, and cell membranes of the Golgi apparatus.
6
Trichrome stain
Trichrome uses three dyes (hence the name), including one that is
specific for the extracellular protein collagen. Depending on the
particular stain combination, a trichrome stain may color collagen
fibers sky-blue or bright green. The principle use for trichrome is to
differentiate collagen from other eosinophilic structures, such as muscle fibers.
Trichrome stains can be especially useful for highlighting an accumulation of scar tissue, as in
glomerulosclerosis of the kidney (see WebPath) or cirrhosis of the liver.
Other stains. Be aware that many other stain techniques exist, for special cases. Some of these
are classical procedures can yield beautiful results but depend on mysterious art and alchemy for
success. Other, more-modern techniques have been rationally designed to exploit recent
developments in molecular biology.
A variety of silver stains have been very powerful for research into the central
nervous tissue. Their only common feature is that silver grains form a dark precipitate on
selected structures, with empirical variables determining which structures are visualized.
Some cells have traditional names based on their demonstration with certain stains, such as the
"argentaffin cells" (cells with an affinity for silver) and "chromaffin cells" (cells with an affinity
for chromium) of the gastrointestinal tract.
In the "modern" category are stains based on the application of particular molecules that can be
selectively stained using radioactive labels, enzyme reactions or specific antigen binding. The
techniques of autoradiography, enzyme histochemistry and immunocytochemistry often
require sections of frozen rather than fixed tissue.
7
o
f
T
i
s
s
u
e
:
o
f
t
y
p
e
8
o
f
t
i
s
s
u
e
:
Each of the tissue-types listed in the panel on the left falls into one of the four categories
above.
However, the four "Basic Types" of animal tissues can be sub-divided further as each includes
several different sub-types of the tissue, each being specialised to meet specific needs and/or
perform particular tasks.
1. Epithelial Tissue
Epithelial tissue exists in many forms and can be classified or sub-divided in different ways.
Covering and Lining Epithelial Types of Epithelial Tissue (in this classification)
Tissue
Classification by Cell Shape:
Squamous
Cuboidal
Columnar
Transitional
Classification by Arrangement of Layers:
Simple Epithelium Simple squamous epithelium,
Simple cuboidal epithelium,
Nonciliated simple columnar epithelium,
Ciliated simple columnar epithelium.
Stratified Epithelium Stratified squamous epithelium,
Stratified cuboidal epithelium
Stratified columnar epithelium,
Transitional epithelium.
Pseudostratified Pseudostratified columnar epithelium.
columnar Epithelium
Glandular Epithelial Tissue
9
Each of the sub-divisions of epithelial tissue identified above can be described in terms of its
structure (using both text and diagrams), location, and function within the body.
2. Connective Tissue
Connective tissues serve the general purpose of supporting and connecting the tissues of the
body, and vary considerably in structure and composition. Teaching materials (incl. textbooks
and websites) sub-divide this tissue category in various different ways - hence it is useful to be
aware of variations and overlap in classifications and terminology.
Lymph
3. Muscular Tissue
There are three (3) types of muscular tissue:
Skeletal located throughout the body and under conscious (i.e. "voluntary") control, main
Muscle function movement of the structures of the body, and the body as a whole, e.g.
(Tissue): by walking, running, etc..
Cardiac which is found only in the heart and is important for effective blood-flow through
Muscle the heart.
(Tissue):
Smooth involuntary muscle tissue located around the walls of many internal structures
Muscle such as the stomach and intestines and important for aiding the passage of
(Tissue): materials/fluids through those structures.
4. Nervous Tissue
Further information about each of the basic types of animal tissue is included on the pages
indicated.
Junctions
Adhering Junctions
11
The zonula adherens junction lies below the tight junction (occluding junction). In the gap
between the two cells, there is a protein called E-cadherin - a cell membrane glycoprotein. The
cadherins from adjacent cells interact to 'zipper' up the two cells together.
Inside the cell, E-cadherin binds to catenin, which in turn binds to other proteins (vinculin, alpha
actinin) in a protein complex with actin filaments (microfilaments, shown here in red).
The actin filaments tend to be arranged circumferentially around the cell, into what is called a
'marginal' band. This marginal band can contract, and deform the shape of cells held together in
this way - this is thought to be key in the morphogenesis of epithelial cells, in forming ducts for
example.
Tight junctions
These are regions in which two cells are very tightly connected together, and they will prevent
some molecules from passing across an epithelium. The borders of two cells are fused together,
often around the whole perimeter of each cell, forming a continuous belt like junction known as
a tight junction or zonula occludens (zonula = latin for belt). Transmembrane proteins from each
cell membrane interlock across the intercellular space, all around the cell, in this belt (black lines
in the diagram).
12
The permeability of tight junctions varies from site to site, and are often can be selectively leaky.
For example, these junctions are important in the gut, in acting as a selective diffusion barrier,
preventing diffusion of water soluble molecules. They also act to restrict the localisation of
membrane bound proteins. (For more, see the section on the gut)
Desmosomes are particularly common in epithelia that need to withstand abrasion (see skin).
Desmosomes are also found in cardiac cells, but the intermediate filament in this case is desmin,
not keratin (which is found in epithelial cells).
The picture shows an EM of a desmosome formed between two cells.Notice the phase dense
material between the two cell membranes, which is mad up of transmembrane linker
glycoproteins (e.g. demosgleins and desmocollins - which are cadherin proteins). Also notice the
intermediate filaments running from the desmosome into the cytoplasm.
Other proteins run across the membrane into the intracellular space, to connect the two cells
together. These 'transmembrane linker' proteins are called desmoglein and desmocollin, which
are types of cadherin.
13
This diagram shows a desmosome. It is made up of a dense cytoplasmic plaque, to which the
intermediate filaments attach.
Hemidesmosomes
These look similar to desmosomes, but are different functionally, and in their content. The
connect the basal surface of epithelial cells via intermediate filaments to the underlying basal
lamina. The transmembrane proteins of hemidesmosomes are not cadherins, but another type of
protein called integrin.
This electron micrograph shows a Hemidesmosome (H), and two of the three layers of the
underlying basal lamina. LL - lamina densa, LD - lamina lucida. Integrins in the plasma
membrane link the cell to the extra-cellular matrix.
Gap Junctions.
14
Gap junctions are the most widespread of all cell junctions in animal tissues. Gap junctions thus
couple cells electrically and metabolocially, enabling cells to communicate with each other
directly. Gap junctions can open and close in response to changes in calcium levels, and pH. Gap
junctions form in a narrow gap of 2-4nm, between two adjacent cells.
The picture shows an EM (A) of two gap junctions between two cells, and a freeze fracture EM
(B) of the particles on the cytoplasmic face of the plasma membrane.
A group of protein molecules called connexins form a structure called a connexon (each particle
in B above is a connexon). When connexons (blue in the diagram to the right) from two adjacent
cells (red and yellow in the diagram) align, they form a continuous channel between them.
EPITHELIA
Epithelia are tissues consisting of closely apposed cells without intervening intercellular
substances. Epithelia are avascular, but all epithelia "grow" on an underlying layer of vascular
connective tissue. The connective tissue and the epithelium are separated by a basement
membrane. Epithelium covers all free surfaces of the body. Epithelium also lines the large
internal body cavities, where it is termed mesothelium. Furthermore, the internal surfaces of
blood and lymph vessels are lined by epithelium, here called endothelium.
15
Epithelia are classified on the basis of the number of cell layers and the shape of the cells in the
surface layer.
Simple Epithelia
This type is composed of a single layer of flattened, scale- or plate-like cells. It is quite
common in the body. The large body cavities and heart, blood vessels and lymph vessels
are typically lined by a simple squamous epithelium. The nuclei of the epithelial cells are
often flattened or ovoid, i.e. egg-shaped, and they are located close to the centre of the
cells.
Cells appear cuboidal in sections perpendicular to the surface of the epithelium. Viewed
from the surface of the epithelium they look rather like small polygons. Simple cuboidal
epithelium occurs in small excretory ducts of many glands, the follicles of the thyroid
gland, the tubules of the kidney and on the surface of the ovaries.
Can there be "low cuboidal" epithelia?
The cells forming a simple columnar epithelium are taller than they are wide. The nuclei
of cells within the epithelium are usually located at the same height within the cells -
often close to the base of the cells. An example is the simple columnar epithelium which
lines the internal surface of the gastrointestinal tract (GIT) from the cardia of the stomach
to the rectum.
16
? Identifying Epithelia
Suitable Slides
simple squamous epithelium: any section containing blood vessels, sections of organs
which include an outer lining (or serosa) of the organs, or sections of kidney (parietal
blades of Bowman's capsules) - H&E, trichrome
simple cuboidal epithelium: sections of ovaries (epithelium lining the surface), thyroid
gland (follicles), kidney (tubules) or large glands (e.g. parotid gland) with well preserved
small ducts
simple columnar epithelium: sections of the small intestine (duodenum, jejunum or
ileum), uterus (uterine glands), liver (large bile ducts) or gall bladder - H&E, trichrome
17
Stratified Epithelia
Stratified squamous epithelia vary in thickness depending on the number of cell layers
present. The deepest cells, which are in contact with the basement membrane, are
cuboidal or columnar in shape. This layer is usually named the basal cell layer, and the
cells are called basal cells. Basal cells are mitotically active and replace the cells of the
epithelium which are lost by "wear and tear". The basal cell layer is followed by layers of
cells with polyhedral outlines. Close to the surface of the epithelium, cells become more
19
flattened. At the surface of the epithelium, cells appear like flat scales - similar to the
epithelial cells of simple squamous epithelia.
Remember that it is the shape of the cell which form the surface of the epithelium which
gives the name to the epithelium.
are not common. A two-layered cuboidal epithelium is, for example, seen in the ducts of
the sweat glands. Stratified columnar epithelia are found in the excretory ducts of the
mammary gland and the main excretory duct of the large salivary glands.
Suitable Slides
stratified squamous epithelium: sections of the oesophagus, tongue or vagina - H&E, van
Gieson, trichrome
stratified cuboidal epithelium: skin (excretory ducts of sweat glands) - H&E
stratified columnar epithelium: sections of the parotid gland or mammary gland - H&E
Oesophagus, human - H&E
The oesophagus is lined by a
stratified squamous epithelium
consisting of many cell layers.
Basal cells often form a well
defined layer at the border of the
epithelium to the underlying
connective tissue. The underlying
connective tissue forms finger-
like extensions towards the lumen
of the oesophagus, which are
called papillae. The border
between epithelium and
connective tissue may appear
quite irregular because of the
papillae. This irregular border
aids in anchoring of the
epithelium to the connective
tissue. If these extensions are not
cut exactly along their long axis,
they may look like isolated small
islands of connective tissue and
blood vessels within the
epithelium.
Draw the stratified squamous
epithelium of the oesophagus and
label your drawing. Try to draw a
little schematic illustration which
20
These two types of epithelia are difficult to classify using the shape of the cells in the surface
layer and the number of the cell layers as criteria.
Transitional epithelium
Transitional epithelium is found exclusively in the excretory urinary passages (the renal
calyces and pelvis, the ureter, the urinary bladder, and part of the urethra).
The shape of the cells in the surface layer of a transitional epithelium varies with the
degree of distension of the organs whose lumen is lined by this type of epithelium. In the
'relaxed' state of the epithelium, it seems to be formed by many cell layers. The most
basal cells have a cuboidal or columnar shape. There are several layers of polyhedral
cells, and, finally, a layer of superficial cells, which have a convex, dome-shaped luminal
surface. In the distended state of the epithelium only one or two layers of cuboidal cells
are followed by a superficial layer of large, low cuboidal or squamous cells. In the
distended state the epithelium will resemble a stratified squamous epithelium.
Pseudostratified columnar
epithelium
Suitable Slides
transitional epithelium: sections of ureter or bladder - H&E
pseudostratified epithelium: sections of the trachea - H&E
Bladder, Monkey - H&E
At a first glance a transitional
epithelium looks like a stratified
cuboidal epithelium. Several
rows of nuclei appear to be
topped by a layer of dome-shaped
cells which bulge into the lumen
of the ureter. The shape of the
surface cells and the number of
rows change if the bladder is
distended. The number of rows
decreases. This decrease should
tell us that many of the nuclei
located in different layers of the
epithelium belong to cells which
are all in contact with the
basement membrane. With
distension, the shape of the cells
in the surface layer will become
squamous.
Draw the epithelium and label the
features you can see. Add a
simple schematic drawing of how
you expect the epithelium to look
like if the ureter is distended.
In addition to its function as support of the epithelium, the basal lamina acts as a selectively
permeable filter between epithelium and connective tissue.
Unless special stains are used, the basement membrane is rarely visible using light microscopy.
You can read more about reticular fibres and ground substance on the Connective Tissues page.
Desmosomes
are specialisations of the lateral cell membranes which mediate cell adhesion. Proteins
inserted into the cell membrane of the adjacent cells form a protein-'zipper' linking the
cells. Fibers of the cytoskeleton attach to the cytoplasmic side of the desmosome to
stabilise the area of contact. Hemi-desmosomes mediate the attachment of the epithelial
cells to the basement membrane.
A group of glycoproteins (cadherins) inserted into the opposing plasma membranes
mediate cell-to-cell adhesion at desmosomes and also at the adhesion zones or patches
mentioned below. Integrins, another group of proteins, allow the cell to attach to the
matrix proteins of the basal lamina.
The above mentioned membrane specialisations mediate cell-adhesion but are less well suited to
support one of the essential functions of epithelia - the isolation of the interior of the body from
the outside world. A tight junction (zonula occludens) between epithelial cells mediates this
aspect of epithelial function.
Proteins inserted into the cell membranes of adjacent cells 'stitch' the membranes of the
cells together and provide an effective barrier to the diffusion of substances from the
outside of the epithelium (called luminal side if the epithelium covers the surface of a
tubular structure). Several "rows of stitches" may be found. Their number depends on the
demand to reduce diffusion across the epithelium. Each of these rows reduces diffusion
by about a factor 10 of what it was 'before'.
GLANDS
Exocrine glands release the secretory product via a system of ducts that opens upon one
of the surfaces of the body which are in contact with the external world (skin,
gastrointestinal tract etc.).
Endocrine glands release their secretory product (typically hormones) into the spaces
between the secretory cells (extracellular space) from which it enters the bloodstream.
Both endocrine and exocrine glands are developmentally derived from epithelia, which form a
down-growth into the underlying connective tissue. The cells forming this down-growth then
develop the special characteristics of the mature gland. Exocrine glands maintain the connection
with the surface epithelium, whereas the connection is lost by endocrine glands.
Exocrine glands may be classified according to cell number, and/or the shape and branching
pattern of their secretory portions and ducts.
Unicellular Glands
consist of a single secretory cell. In mammals the only example of unicellular exocrine
glands are goblet cells, which occur in the epithelium of many mucous membranes.
Goblet cells secrete the glycoprotein mucin, which by the uptake of water is converted
into a slimy substance, mucus.
26
Multicellular glands
tubes in tubular
glands,
acini in acinar
glands or
alveoli in alveolar
glands.
Combinations exist - the pancreas is a tubulo-acinar gland, in which each section of the secretory
system has a specialized function.
The precursors of digestive enzymes are produced by the acinar cells. Tubular cells secrete the
alkaline bicarbonate solution which eventually neutralizes the acidic contents of the stomach that
are released into the duodenum.
Multicellular glands with an unbranched excretory duct are called simple. We talk about a
compound gland when the excretory duct is branched. Finally, the part of the gland consisting of
secretory cells is branched in a (surprise!) branched gland.
27
The classification scheme may appear somewhat elaborate - but there are many exocrine glands
around. All of them can be identified and described by this scheme, and some ideas about their
function can be derived from this description.
Suitable Slides
unicellular exocrine glands (goblet cells): sections of intestines (duodenum, jejunum,
ileum or colon) or trachea - H&E
secretory epithelial sheath: stomach - H&E
straight tubular glands: sections of stomach (principal glands) or colon (intestinal glands)
- van Gieson, H&E
coiled tubular glands: sections of skin (sweat glands) - see lab section on the
Integumentary System page.
Colon, Human - van Gieson
Straight tubular glands extend
from the surface of the colon into
the underlying connective tissue.
Although they are present
throughout the intestines they are
largest in the colon and, because
of the smooth inner surface of the
colon, they often show in good
longitudinal or transverse
sections. The lumen of the glands
is narrow and surrounded by
secretory cells of several types,
which include goblet cells. The
connective tissue beneath the
epithelium and surrounding the
glands in the colon contains more
cells than the connective tissue
beneath other epithelia that were
considered on this page. This is a
characteristic feature of the
epithelia in the digestive system.
Glands cut at slightly oblique
angles will connect to the lumen
outside of the plane of the
section.
If possible, draw both
longitudinally and transversely
sections intestinal glands. Include
part of the surrounding
connective tissue and surface
28
epithelium.
Secretory Mechanisms
The secretory cells can release their secretory products by one of three mechanisms.
Merocrine secretion
corresponds to the process of exocytosis. Vesicles open onto the surface of the
cell, and the secretory product is discharged from the cell without any further
loss of cell substance.
Apocrine secretion
designates a mechanism in which part of the apical cytoplasm of the cells is
lost together with the secretory product. The continuity of the plasma
membrane is restored by the fusion of the broken edges of the membrane, and
the cell is able to accumulate the secretory product anew. This mechanism is
used by apocrine sweat glands, the mammary glands and the prostate.
Holocrine
secretion designates the breakdown and discharge of the entire secretory cell.
It is only seen in the sebaceous glands of the skin.
There are two additional mechanisms by which secretory cells can release their products. Lipid
soluble substances may diffuse out of the secretory cell (e.g. steroid hormone-producing
endocrine cells). Transporters (membrane proteins) may actively move the secretory product
across the plasma membrane (e.g. the acid producing parietal cells of the gastric glands). These
secretory mechanisms may not involve any light microscopically visible specialisations of the
cell.
The relationship between the secretory tissue (parenchyma) of glands and the supporting
connective tissue is similar in most larger glands. Externally the entire gland is surrounded by a
layer of dense connective tissue, the capsule. Connective tissue sheets (septa) extend from the
capsule into the secretory tissue and subdivide the gland into a number of lobes. Thinner
connective tissue septa subdivide the lobes into a number of lobules. Reticular connective tissue
(hardly visible in H&E stained sections) surrounds and supports the secretory units of the glands
(alveoli, acini etc.) and the initial parts of the excretory ducts if present.
Blood and lymph vessels as well as nerves penetrate the capsule and form a delicate network
between the secretory units and the initial parts of the duct system.
29
The main excretory duct conveys the secretory product to one of the external surfaces of the
body. Other parts of the duct system are named according to their relation to the lobes and
lobules of the gland.
Lobar ducts are are large branches of the main duct which extend to the lobes of the
gland. They may be called
Interlobar ducts if they are found in the connective tissue surrounding the lobes.
Interlobar ducts branch and give rise to
Interlobular ducts, which are found in the connective tissue surrounding the individual
lobules of the gland. Branches of the interlobular ducts enter the lobules and are now
called
Intralobular ducts. The terminal branches of the duct system, which connect intralobular
ducts with the secretory units of the gland, are called
Intercalated ducts.
The appearance of the different portions of the duct system is quite variable from gland to gland
and may allow the identification of the gland. Quite often, the appearance of parts of the duct
system also permits some deductions about their functions.
? Note that lobes and lobules are defined by their relationship to each other. Many small lobules
may form one large lobe. Neither size nor the spatial relationship between different parts of the
tissue can be unequivocally determined in a single, two-dimensional section of the tissue. Lobes
and interlobar ducts may therefore be difficult to distinguish from lobules and interlobular ducts.
Suitable Slides
alveolar gland: lactating mammary gland H&E - see lab section on the Female
Reproductive System page.
serous and mucous acinar glands: sections of parotid gland, sublingual gland or tongue
(lingual salivary glands) - H&E
30
sure that the features which characterise serous and mucous acini are visible in your drawing - if
necessary use a little artistic freedom.
CONNECTIVE TISSUE
Extracellular Substance
Collagen fibres
Collagen fibres are the dominant fibre type in most connective tissues. The primary function of
collagen fibres is to add strength to the connective tissue.
The thickness of the fibres varies from ~ 1 to 10 µm. Longitudinal striations may be visible in
thicker fibres. These striations reveal that the fibres are composed of thinner collagen fibrils (0.2
to 0.5 µm in diameter). Each of these fibrils is composed of microfibrils, which are only visible
using electron microscopy.
There are many different tropocollagen types around (currently named type I to XXI). These
types differ in their content of the amino acids hydroxyproline and hydroxylysine. They also
differ in the amount of carbohydrates attached to the collagen molecules. The different types of
tropocollagen give the fibres the structural and functional features which are appropriate for the
organ in which the fibres are found. Types I, II and III are the major fibre-forming
tropocollagens. Tropocollagen type IV is an important structural component of the basal lamina.
33
A tensile force of several hundred kg/cm2 is necessary to tear human collagen fibres. The fibres
stretch by only 15-20%.
Reticular fibres
Reticular fibres are very delicate and form fine networks instead of thick bundles. They are
usually not visible in histological sections but can be demonstrated by using special stains. For
example, in silver stained sections reticular fibres look like fine, black threads - coarse collagen
fibres appear reddish brown in the same type of preparation.
Because of their different staining characteristics, reticular fibres were initially thought to be
completely different from collagen fibres. Cross-striations with the same periodicity as in coarse
collagen fibres are however visible using electron microscopy. We now know that reticular fibres
consist of collagen - although the main type of tropocollagen found in reticular fibres, type III, is
different from that of the coarse collagen fibres.
Reticular fibres give support to individual cells, for example, in muscle and adipose tissue.
Suitable Slides
sections of liver, spleen or lymph nodes - reticulin
34
Elastic fibres
Elastic fibres are coloured in fresh tissues - they are light yellow - but this colouration is only
visible if large amounts of elastic fibres are present in the tissue, for example, in the elastic
ligaments of the vertebral column. Special stains are necessary to show elastic fibres in tissue
sections.
Resorcin fuchsin is one of these stains, which gives the elastic fibres a dark violet colour.
Light microscopy does not reveal any substructure in the elastic fibres. Electron microscopy
shows that elastic fibres consist of individual microfibrils, which are embedded in an amorphous
35
matrix. The matrix accounts for about 90% of the fibre and is composed of the protein elastin.
Neither the elastin nor the microfibrils are collagens.
Elastic fibres can be stretched to about 150% of their original length. They resume their original
length if the tensile forces applied to the elastic fibres are relaxed.
Elastin is a somewhat odd protein in that its amino acid sequence does not determine a specific
three-dimensional structure of the molecule. Instead, elastin remains unfolded as a "random
coil". Elastin molecules are cross-linked to each other by desmosin and isodesmosin links, which
are only found between elastin molecules. Tensile forces straighten the cross-linked mesh of
elastin coils.
Suitable Slides
sections of blood vessels or skin - elastin
Skin, human - elastin & van Gieson or Artery, human - elastin & eosin
Like reticular fibres, elastic fibres require special stains to be visualized. Typically elastic fibres
will appear as fine, dark violet and gently undulating fibres in the tissue. Elastic fibres can form
membranes - not unlike the collagen membrane in the basal lamina of epithelia. This is the case
at some levels in the walls of blood vessels.
Collagen and elastic fibres intermingle in the dermis, i.e. the connective tissue beneath the
epithelium of the skin. Immediately beneath the epithelium both fibre types are relatively fine -
they appear much thicker in the deeper parts of the dermis. At least the internal elastic lamina
should be visible in the smaller arteries which course through the dermis.
A combination with a second stain is necessary to visualize other tissue components. Colours
visible in the sections will depend on the method used in combination with the elastin stain.
Eosin gives an even pink or red colour to many tissue components. Nuclei of cells remain
unstained without the inclusion of the haematoxylin in the staining solutions.
Identify the artery and the vein in the section. Their walls contain large amounts of elastic fibres.
Which one contains more elastic fibres - artery or vein?
Blood vessels: draw a small section of the wall of a vessel, preferably an artery, at high
magnification. Identify elastic laminae, fine and coarse elastic fibres in your drawing.
Skin: draw a small section of the dermis - preferablyof a part of the dermis where both the very
fine and the coarse fibres are visible.
36
Ground substance
Ground substance is found in all cavities and clefts between the fibres and cells of connective
tissues. Water, salts and other low molecular substances are contained within the ground
substance, but its main structural constituent are proteoglycans.
Ground substance is soluble in most of the solvents used to prepare histological sections and
therefore not visible in ordinary sections.
Proteoglycans are responsible for the highly viscous character of the ground substance.
Proteoglycans consist of proteins (~5%) and polysaccharide chains (~95%), which are covalently
linked to each other. The polysaccharide chains belong to one of the five types of
glycosaminoglycans, which form the bulk of the polysaccharides in the ground substance.
Hyaluronan (or hyaluronic acid) is the dominant glycosaminoglycan in connective tissues. The
molecular weight (MW) of hyaluronic acid is very high (~ MW 1,000,000 ). With a length of
about 2.5 µm hyaluronan is very large. Hyaluronan serves as a "backbone" for the assembly of
37
other glycosaminoglycans in connective and skeletal tissue, which results in even larger
molecule complexes (MW 30,000,000 - 200,000,000).
Hyaluronan is also a major component of the synovial fluid, which fills joint cavities, and the
vitreous body of the eye.
The remaining four major glycosaminoglycans are chondroitin sulfate, dermatan sulfate, keratan
sulfate and heparan sulfate. These glycosaminoglycans attach via core- and link-proteins to a
backbone formed by the hyaluronic acid. The coiled arrangement of the hyaluronan and other
attached glucosaminoglycans fills a roughly spherical space with a diameter of ~0.5 µm. This
space is called a domain. Neighbouring domains overlap and form a more or less continuous
three-dimensional molecular sieve in the interstitial spaces of the connective tissues.
The large polyanionic carbohydrates of the glycosaminoglycans bind large amounts of water and
cations. The bound water in the domains forms a medium for the diffusion of substances of low
molecular weight such as gases, ions and small molecules, which can take the shortest route, for
example, from capillaries to connective tissue cells. Large molecules are excluded from the
domains and have to find their way through the spaces between domains.
The restricted motility of larger molecules in the extracellular space inhibits the spread of
microorganisms through the extracellular space. A typical bacterium ( 0.5 x 1 µm) is essentially
immobilised in the meshwork formed by the domains. The pathogenicity of a bacterium is
indeed to some extent determined by its ability to find its way through the mesh, and some of the
more invasive types produce the enzyme hyaluronidase, which depolymerises hyaluronic acid.
The components of the ground substance, collagen, elastic and reticular fibres are synthesised by
cells of the connective tissues, the fibrocytes.
Connective tissue cells are usually divided into two groups based on their ability to move within
the connective tissue. Fibrocytes (or fibroblasts) and fat cells are fixed cells. Macrophages,
monocytes, lymphocytes, plasma cells, eosinophils and mast cells are wandering cells.
Fibrocytes
Fibrocytes are the most common cell type in connective tissues. They are the "true" connective
tissue cells. Usually only their oval, sometimes flattened nuclei are visible in LM sections. The
cytoplasm of a resting (i.e. inactive) fibrocyte does not contain many organelles. This situation
changes if the fibrocytes are stimulated, for example, by damage to the surrounding tissue. In this
case the fibrocyte is transformed into a fibroblast, which contains large amounts of the organelles
which are necessary for the synthesis and excretion of proteins needed to repair the tissue
damage (Which ones?). Fibrocytes do not usually leave the connective tissue. They are,
however, able to perform amoeboid movement.
38
The terms fibrocyte and fibroblast refer here to the inactive and active cells respectively - at
times you will see the two terms used as synonyms without regard for the state of activity of the
cell.
Reticular cells
Reticular cells are usually larger than an average fibrocyte. They are the "fibrocytes" of reticular
connective tissue and form a network of reticular fibres, for example, in the lymphoid organs.
Their nuclei are typically large and lightly stained (H&E) and the cytoplasm may be visible
amongst the cells which are housed within the network of reticular fibres.
Adipocytes
Fat cells or adipocytes are fixed cells in loose connective tissue. Their main function is (what
surprise!) the storage of lipids. If "well fed" the cytoplasm only forms a very narrow rim around
a large central lipid droplet. The flattened nucleus may be found in a slightly thickened part of
this cytoplasmic rim - if it is present in the section, which may not be the case since the diameter
of an adipocyte (up to 100 µm) is considerable larger than the thickness of typical histological
sections. A "starving" adipocyte may contain multiple small lipid droplets and gradually comes
to resemble a fibrocyte.
Adipocytes are very long-lived cells. Their number is determined by the number of
preadipocytes (or lipoblast) generated during foetal and early postnatal development.
Macrophages
Suitable Slides
sections of liver or lymph nodes - carbon injected animal - trichrome or H&E
sections containing irregular connective connective tissues - Alcian blue & van Gieson
In Alcian blue & van Gieson stained sections macrophages may appear as intensely
green, roundish cells with a dark nucleus.
Liver, rabbit - ink injected,
trichrome
Macrophages are usually difficult
to distinguish from other cell
types in connective tissues. One
way to visualize them is to inject
an experimental animal with very
fine carbon particles.
Macrophages which come into
contact with the circulating
particles will phagocytose some
of them. In sections the particles
will be visible as dark, black-
brown accumulations in the
cytoplasm of the macrophages.
Draw a few macrophages in situ.
Mast cells
Mast cells are - like macrophages, lymphocytes and eosinophils - in demand when something
goes wrong in the connective tissue. Quite a few of them are present in healthy connective tissue
as they stand on guard and monitor the local situation. The cytoplasm of mast cells is filled by
numerous large vesicles. Mast cells discharge the contents of these vesicles if they come in
contact with antigens, for example, proteins on the surface of an invading bacterium or, in
allergic reactions, in response to antigens found, for example, on the surface of pollen grains.
The most prominent substances contained in the vesicles are heparin and histamine. They
increase blood flow in close by vessels and the permeability of the vessel walls to plasma
constituents and other white blood cells. By facilitating access to the area, mast cells facilitate an
immune response to the antigen which triggered the release histamine and heparin.
Lymphocytes are usually small cells (6 - 8 µm). Their nuclei are round and stain very dark. The
cytoplasm forms a narrow rim around the nucleus and may be difficult to see. There are many of
them in the connective tissue underlying the epithelia of the gastrointestinal tract but usually
much fewer in other connective tissues. Again, this situation may change - in this case with
immunological reactions. Some lymphocytes may differentiate into plasma cells. Plasma cells
are lymphocytes which produce antibodies. To accommodate the necessary organelles for this
function the size of the cytoplasm increases dramatically and the cells become basophilic.
Plasma cells can occasionally be spotted in the loose connective tissue present in sections.
Like eosinophilic cells and monocytes, lymphocytes are white blood cells. More information
about these cell types can be found on the Blood page.
Eosinophilic cells
Eosinophilic cells are typically rounded or oval, large cells, which contain large amounts of
bright red granules in their cytoplasm. They originate, like the monocytes, in the bone marrow.
They enter connective tissues early in inflammatory reactions, where they phagocytose antigen-
41
antibody complexes. Their numbers in healthy connective tissue vary with location, but a few of
them can usually be found.
Mesenchymal cells
During development, mesenchymal cells give rise to other cell types of the connective tissue. A
small number of them may persist into adulthood. Mesenchymal cells are smaller than fibrocytes
and difficult to detect in histological sections. They may regenerate blood vessels or smooth
muscle which have been lost as a consequence of tissue damage.
Suitable Slides
sections of tongue, skin, mesentery or other sections containing epithelia and / or loose
connective tissue - toluidine blue, cresyl violet
These two tissues are distinguished according to the relative amounts of fibres they contain.
Dense connective tissues are completely dominated by fibres. They are subdivided according to
the spatial arrangement of the fibres in the tissue.
In dense irregular connective tissue the fibres do not show a clear orientation within the tissue
but instead form a densely woven three-dimensional network. A good example is the dermis of
the skin.
We talk about regular dense connective tissue if the fibres run parallel to each other. Good
examples of regular dense connective tissue are tendons, ligaments and the fasciae and
aponeuroses of muscles.
Loose connective tissue is relatively cell rich, soft and compliant. It is also rich in vessels and
nerves. It is best understood as a kind of generalised connective tissue in which all connective
tissue cell types may occur. Loose connective tissue may occur in some special variants: mucous
connective tissue, reticular connective tissue and adipose tissue.
Suitable Slides
sections of tendons or ligaments - van Gieson, H&E
43
Suitable Slides
sections of skin or non-lactating mammary gland - H&E, van Gieson, trichrome
44
Reticular connective tissue consists of reticular cells and the network of reticular fibres formed
by them. Most connective tissues contain reticular fibres, but only in reticular connective tissue
are they the dominant fibre type. In a number of tissues and organs, reticular connective tissue
forms the structural framework in which the cells of the organ are suspended. The open
45
meshwork of fine fibres is particularly useful in tissues and organs in which diffusion and / or
cell movements are functionally important, for example, in the liver, lymph nodes and the spleen.
Adipose tissue
Adipose tissue is essentially loose connective tissue containing large numbers of adipocytes.
There are two types of adipose tissue, which derive their names from the colour of the tissue
(white or brown) and the number of lipid droplets found in the adipocytes.
Adipocytes of white, unilocular adipose tissue contain one large lipid droplet.
Adipocytes of brown, multilocular adipose tissue contain many lipid droplets.
White adipose tissue does not only function in the storage of lipids. For example, in the palms of
the hands, on the plantar surface (sole) of the feet and in the gluteal region (buttocks) it has a
structural, cushioning function. In these regions, accumulations of adipocytes are surrounded by
strong connective tissue fibres. Also, the distribution of white adipose tissue is different in males
and females and is part of the secondary sexual characteristics. The storage and mobilisation of
lipids does require quite some metabolic activity of the tissue. Consequently, adipose tissue has a
rich supply of capillaries.
Brown adipose tissue occurs mainly during development and may account for 2 - 5 % of the
body weight in a newborn. In adult individuals most of the brown fat has further differentiated
into white fat. Adipocytes in brown fat contain plenty of mitochondria. A very rich capillary
supply and the cytochromes found in the mitochondria give the tissue its characteristic colour. A
protein (UCP-1 or thermogenin) found in these mitochondria decouples the oxidation of fatty
acids from the generation of ATP. Instead, these cells generate heat.
The location of the brown fat reflects its heat-generating function. It is located in the axilla
(armpits), between the shoulder blades, in the region of the neck and along large blood vessels.
The heat generated by the brown fat warms the blood which supplies nearby organs or which re-
enters the trunk from the limbs.
Suitable Slides
white adipose tissue: sections of skin - H&E, trichrome, van Gieson
Section are rarely prepared to show just white adipose tissue. The hypodermis, i.e.
lightest and loosest appearing layer, of skin sections will typically contain large areas of
adipose tissue. Other good candidates are bone sections which contain yellow bone
marrow or sections of lymph nodes which are often embedded in adipose tissue. Small
spots of adipose tissue should be present in many other sections.
brown adipose tissue: sections of brown adipose tissue or kidney - trichrome, H&E
Brown adipose tissue is often located in the connective tissue close to the renal hilus or in
the renal sinus of sections which contain the entire kidney of small laboratory animals.
Look for an indentation in the outline of the kidney, which corresponds to its hilus.
46
Kidney - trichrome
In the renal sinus, islands of
brown adipose tissue are often
surrounded by white adipose
tissue, which emphasises the
different appearances of the two
tissue types. In brown adipose
tissue, the nuclei of adipocytes
are round and located more or
less centrally in a cytoplasm
which, after the extraction of
lipids during tissue preparation,
looks very frothy. Cell borders
can be difficult to identify.
Capillaries are very frequent.
Sketch the appearance of the
brown adipose tissue. Contrast
the characteristic features of
white and brown adipose if both
types are present side by side.
Mesenchyme forms the undifferentiated "filling" of the early embryo. It consists of mesenchymal
cells, which interconnect by slender cell processes. Mesenchymal cells have stem cell properties,
i.e. they are able give rise to other cell and tissues types. The wide extracellular space between
the mesenchymal cells is occupied by ground substance, which can be stained with dyes that also
stain mucin - hence the alternative name of this tissue type: mucoid connective tissue. Collagen
or reticular fibres may not be visible at all or form a loose network between the cells. With fetal
development, mesenchyme forms the connective tissue between and within the developing
tissues and organs. Mucoid connective tissue also forms a compliant cushion around the vessels
of the umbilical cord, where it is also called Wharton's jelly.
In adult humans, mesenchymal connective tissue is only found in the dental pulp.
48
Suitable Slides
sections of umbilical cord, tooth (pulp), or sections of embryonic and early foetal
development - H&E, Azan or Alcian blue & van Gieson
section usable for "intramembranous ossification" during foetal development will contain
areas of mucoid connective tissue around the developing bone.
Cells which accumulate fat are often present in loose connective tissue, either
singly or in small groups. When they are present in large numbers we call such
tissue adipose (fatty) tissue. layers of fatty tissue may be present under the skin
(in what anatomically we call superficial fascia), under serous membranes
(especially the peritoneum) and even within certain organs. Groups of cells are
usually separated from each other by fine partitions of loose connective tissue.
Adipose tissue in the adult human appears white or yellowish in colour. In foetal
life and in the newborn there is another variety of fat that is brownish in colour.
The brown colour is in fact due to blood vessels. Brown fat is also present in
adult animals of species which hibernate.
When a cell accumulates fat in the cytoplasm, small "droplets" of fat grow into
a single large droplet. This pushes the remaining cytoplasm and the nucleus to
one corner of the cell. During preparation of H & E slides the fat is dissolved and
the cell appears empty, with a very thin rim of cytoplasm and the nucleus along
the edge. This appearance is described as "signet ring" appearance. (If you have
a historical or biblical movie you may have seen how the ring of a king carries
his seal which can be "impressed" on writing material by punching it!).
White adipose tissue does not only function in the storage of lipids. For example,
in the palms of the hands, on the plantar surface (sole) of the feet and in the
gluteal region (buttocks) it has a structural, cushioning function. In these
51
Brown adipose tissue occurs mainly during development and may account for 2 -
5 % of the body weight in a newborn. In adult individuals most of the brown fat
has further differentiated into white fat. Adipocytes in brown fat contain plenty
of mitochondria. A very rich capillary supply and the cytochromes found in the
mitochondria give the tissue its characteristic colour. A protein (UCP-1 or
thermogenin) found in these mitochondria decouples the oxidation of fatty acids
from the generation of ATP. Instead, these cells generate heat.
The location of the brown fat reflects its heat-generating function. It is located in
the axilla (armpits), between the shoulder blades, in the region of the neck and
along large blood vessels. The heat generated by the brown fat warms the blood
which supplies nearby organs or which re-enters the trunk from the limbs.
. A "starving" adipocyte may contain multiple small lipid droplets and gradually
comes to resemble a fibrocyte.
Adipocytes are very long-lived cells. Their number is determined by the number
of preadipocytes (or lipoblast) generated during foetal and early postnatal
development.
Suitable Slides
52
Kidney - trichrome
In the renal sinus, islands of
brown adipose tissue are
often surrounded by white
adipose tissue, which
emphasises the different
appearances of the two
tissue types. In brown
adipose tissue, the nuclei of
adipocytes are round and
located more or less
centrally in a cytoplasm
which, after the extraction of
lipids during tissue
preparation, looks very
frothy. Cell borders can be
difficult to identify.
Capillaries are very frequent.
Sketch the appearance of the
brown adipose tissue.
Contrast the characteristic
features of white and brown
adipose if both types are
present side by side.
The pictures here (at different magnifications) show both types of fat in the same
section.
55
A different kind of adipose tissue is known as brown or multilocular fat. Brown fat
contains fat droplets of varying sizes. The cells are smaller than those of white fat,
with an eccentric but not flattened nucleus. Brown fat has a very limited
distribution in adult humans, but is found in many animals. In hibernating animals,
the oxidation of brown fat warms the blood flowing through it during arousal from
hibernation. Human newborns, whose large surface to volume ratio can result in
56
heat loss, also have alot of brown fat. Most of it disappears during the first decade
of life.
Blue Histology –
Skeletal Tissues – Cartilage
CARTILAGE
Cartilage is rather rare in the adult humans, but it is very important during development
because of its firmness and its ability to grow rapidly. In developing humans, most of
the bones of the skeleton are preceded by a temporary cartilage "model". Cartilage is
57
Types of Cartilage
1. Hyaline Cartilage
Hyaline
cartilage
develops, like
other types of
connective
tissue, from
mesenchymal
cells. From
about the fifth
foetal week
precursor cells
become
rounded and
form densely
packed cellular
masses,
chondrification
centres. The
cartilage-
forming cells,
chondroblasts,
begin to secrete
the
components of
the
extracellular
matrix of
cartilage. The
extracellular
matrix consists
of, ground
substance
(hyaluronan,
chondroitin
sulfates and
keratan
sulfate) and
tropocollagen,
which
58
polymerises
extracellularly
into fine
collagen fibres.
Tropocollagen type II is the dominant form in collagen fibres of almost all types of
cartilage.
As the amount of matrix increases the chondroblasts become separated from each other
and are, from this time on, located isolated in small cavities within the matrix, the
lacunae. Concurrently the cells differentiate into mature cartilage cells, chondrocytes
.
The matrix appears structureless because the collagen fibres are too fine to be resolved
by light microscopy (~20nm), and because they have about the same refractive index as
the ground substance. Collagen accounts for ~ 40% of the dry weight of the matrix.
The matrix near the isogenous groups of chondrocytes contains larger amounts and
different types of glycosaminoglycans than the matrix further away from the
isogenous groups. This part of the matrix is also termed territorial matrix or capsule.
In H&E stained sections the territorial matrix is more basophilic, i.e. it stains darker.
The remainder of the matrix is called the interterritorial matrix. Fresh cartilage
contains about 75% water which forms a gel with the components of the ground
substance. Cartilage is nourished by diffusion of gases and nutrients through this gel.
Suitable Slides
sections of the trachea or larynx - H&E, van Gieson
Both stains are equally well suited to look at the organisation of hyaline cartilage. The
van Gieson method stains collagen red. The cartilage appears as a wide red zone
underneath the epithelium and loose connective tissue, which line the lumen of the
trachea. The staining may appear a little lighter close to the lacunae. This lighter stained
zone defines the territorial matrix surrounding the lacunae and chondrocytes. Colour
intensities appear reversed in the H&E stained section. The two compartments of the
matrix are usually better defined than in van Gieson stained sections. The interterritorial
matrix appears very light; the territorial matrix is somewhat darker. Groups of
chondrocytes surrounded by these lighter (van Gieson) or darker (H&E) staining zones
belong to the same isogenous group. A layer of dense connective tissue surrounding the
cartilage and blending with it is the perichondrium.
The isogenous groups may form small "squares" (e.g. four chondrocytes, separated by
thin cartilage membranes, in a 2x2 arrangement) or short columns (e.g. four
chondrocytes in a 1x4 arrangement).
Draw a small section of the cartilage and identify in your drawing territorial matrix,
interterritorial matrix, isogenous groups, and chondrocytes. Think about how the spatial
arrangement of chondrocytes in the isogenous group may reflect patterns of cell
divisions.
60
2. Elastic Cartilage
Suitable Slides
Sections of the epiglottis - elastin
this preparation a blue-green colour) will depend on the method used to show tissue
components other than elastic fibres. Although the matrix appears blue-green, the
typical organisation of cartilage is readily visible. Within the green matrix you can see
the fine elastic fibres which give this cartilage its elastic properties. The elastic fibres
may form dense masses in which individual fibres are difficult to distinguish. The
staining of these masses of fibres may appear more reddish than dark-violet.
A change of the colour of the stain in intensely stained tissue areas is called
"metachromatic staining".
Draw and label a small section of elastic cartilage.
3. Fibrous Cartilage
62
Suitable Slides
sections of intervertebral discs or articular discs - H&E, van Gieson
Fibrous Cartilage, Intervertebral Disc, sheep, H&E and Articular Disc, rabbit, H&E
The fibrous cartilage forming the intervertebral discs varies in appearance from the
center of the disc (the nucleus pulposus) the the periphery of the disc (the annulus
fibrosus). Centrally, the fibrous matrix is very loose. The jelly-like consistency of the
central part allows the intervertebral discs to function as a shock absorber. Towards the
periphery, the fibrous matrix is organised into layers. It is often visible that the fibres of
different layers are oriented at angles to each other - similar to the orientation of the
thread in radial tires. Chondrocytes are very flattened in the periphery and may be
difficult to find.
Midway between periphery and center of the intervertebral disc, chondrocytes are
scattered singly or in small isogenous groups in the dense fibrous matrix of the
cartilage. If you take a close look at the cells you will see that their appearance actually
resembles that of chondrocytes in other types of cartilage - their characteristic
appearance distinguishes fibrous cartilage preparations from connective tissues. The
very regular arrangement of the fibres in the articular disc may initially let you guess at
dense regular connective tissue. Isogenous groups of chondrocytes again identify the
tissue as fibrous cartilage.
Draw a small section of the fibrous cartilage, including (if possible) a group of
chondrocytes.
63
4. Articular Cartilage
Tangential layer
Chondrocytes are rather small and flattened parallel to the surface. The most
superficial part (lamina splendens) is devoid of cells. Collagen fibres in the
matrix of the tangential layer are very fine. They run parallel to the surface of
64
the cartilage.
Similar to the collagen fibres of the skin, the general orientation of collagen
fibres in articular cartilage is determined by tensile and compressive forces at
the articulating surfaces.
Transitional zone
The chondrocytes are slightly larger, are round and occur both singly and in
isogenous groups. Collagen fibres take an oblique course through the matrix of
the transitional zone.
Radial zone
Fairly large chondrocytes form radial columns, i.e. the stacks of cells are
oriented perpendicular to the articulating surface. The course of the collagen
fibres follows the orientation of the chondrocyte columns.
It rests on the underlying cortex of the bone. The matrix of the calcified
cartilage layer stains slightly darker (H&E) than the matrix of the other layers.
The main source of nourishment for articular cartilage is the synovial fluid, which
fills the joint cavity. Additional small amounts of nutrients are derived from blood
vessels that course through the calcified cartilage close to the bone.
Living chondrocytes have been found in small pieces of cartilage floating in the joint
cavity after damage to the articular cartilage.
Suitable Slides
Sections of large joints - H&E
Layers are difficult to identify in the articular cartilage of small joints.
of the cartilage close to the bone is caused by the calcification of the cartilage.
Draw the articular cartilage at low magnification. Indicate in your drawing the preferred
orientations of lacunae and isogenous groups and the expected orientation of collagen
fibres.
Due to the fairly poor access of nutrients to the chondrocytes they may atrophy in
deep parts of thick cartilage. Water content decreases and small cavities arise in the
matrix, which often leads to the calcification of the cartilage. This further
compromises nutrition. The chondrocytes may eventually die, and the cartilage is
gradually transformed into bone.
acute damage to hyaline or articular cartilage. If these cartilages are injured after the
period of active growth, the defects are usually filled by connective tissue or fibrous
cartilage. The extracellular matrix of these "repair tissues" is only poorly integrated
with the matrix of the damaged cartilage.
Fortunately, cartilage is rather well suited for transplantation - the metabolism of the
chondrocytes is rather slow, the antigenic power of cartilage is low, and it is difficult,
if not impossible, for antibodies or cells of the immune system to diffuse through the
matrix into the cartilage.
Blue Histology
Skeletal Tissues - Bone
BONE
Trabecular bone
(also called
cancellous or
spongy bone)
consists of
delicate bars and
sheets of bone,
trabeculae,
which branch
and intersect to
form a sponge
like network.
The ends of long
bones (or
epiphyses)
consist mainly of
trabecular bone.
Compact bone
does not have
any spaces or
hollows in the
bone matrix that
are visible to the
eye. Compact
bone forms the
thick-walled
tube of the shaft
(or diaphysis) of
long bones,
which surrounds
the marrow
cavity (or
medullary
cavity). A thin
layer of compact
bone also covers
the epiphyses of
long bones.
68
Compact Bone
Trabecular Bone
Suitable Slides
sections of compact bone (usually part of the diaphysis of
a long bone) - ground (unstained), Schmorl stained or
H&E
Suitable Slides
sections of part of a vertebra or an epiphysis of a long
bone - H&E, van Gieson
Sections prepared to show articular cartilage will often
also contain trabecular bone in the epiphysis below the
articular cartilage.
Bone Matrix
74
Bone Cells
3. Osteocytes.
4. Osteoclasts
75
Formation of Bone
1. Intramembranous Ossification
77
Suitable Slides
sections of the developing mandible (or some other bones
of the skull) or clavicle - H&E, van Gieson
Sections prepared to show endochondral ossification (see
below) may be an alternative if no specifically prepared
slides of intramembranous ossification are available. The
periosteal collar, i.e. the manchette of bone forming
78
2. Endochondral Ossification
Suitable Slides
sections of bones during the early stages of their
development - H&E, van Gieson
Suitable Slides
sections of the epiphyseal disc of growing bones - H&E
82
BLOOD
Blood is sometimes considered to be a fluid connective tissue because of
the mesenchymal origin of its cells and a low ratio of cells to liquid
intercellular substance, the blood plasma. In human adults about 5 liter
of blood contribute 7-8 % to the body weight of the individual. The
contribution of red blood cells (erythrocytes) to the total volume of the
blood (haematocrit) is about 43%.
Erythrocytes are the dominant (99%) but not the only type of cells in the
blood. We also find leukocytes and, in addition, blood platelets.
Erythrocytes, leukocytes and blood platelets are also being referred to as the
formed elements of the blood. Erythrocytes and blood platelets perform their functions
exclusively in the blood stream. In contrast, leukocytes reside only
temporarily in the blood. Leukocytes can leave the blood
stream through the walls of capillaries and venules and enter
either connective or lymphoid tissues.
Erythrocytes
85
Functions
Suitable Slides
blood smear - Leishman, Wright's, Giemsa or May-
Grünwald-Giemsa stains
? Where to look for cells in a
blood smear
The density of cells varies
across the smear. Cells will be
"heaped and piled" close to the
point were the drop of blood was
placed on the slide. White blood
cells appear shrunken, and
some types are difficult to
distinguish from each other.
86
Leukocytes
are quite stable. A differential leukocyte count would typically produce the
following cell frequencies (numbers in parentheses are the range of
normal frequencies reported in different texts):
Granular Leukocytes
Granular leukocytes are all approximately the same size - about 12-15 µm
in diameter. Their nuclei form lobes, and nucleoli cannot be seen. The
number of nuclear lobes varies according to cell type. All granulocytes are
motile.
primary lysosomes, although they are larger (0.4 µm) than the
"ordinary" primary lysosome. Secondary granules (or B
granules), the specific granules of the neutrophils, contain
enzymes with strong bactericidal actions. The specific
granules of neutrophils stain only weakly if they are at all
visible - they are "neutral", hence the term neutrophil.
Functions
Suitable Slides
see lab section on erythrocytes
89
Their nucleus usually has only two lobes. Almost all of the
cytoplasm appears filled with the specific granules of the
eosinophils. As the term "eosinophil" indicates, these granules are
not neutral but stain red or pink when eosin or a similar dye is used
in the staining process. Aside from the usual complement of
organelles eosinophils contain some large rounded vesicles (up to 1
µm) in their cytoplasm . These granules correspond to the
eosinophilic grains that we see in the light microscope. The specific
granules contain, in addition to enzymes that otherwise are found in
lysosomes, an electron-dense, proteinaceous crystal. This crystal is
composed of major basic protein (MBP).
Functions
Suitable Slides
see lab section on erythrocytes
Functions
Heparin and histamine are vasoactive substances. They dilate the blood
vessels, make vessel walls more permeable and prevent blood
coagulation. As a consequence, they facilitate the access of other
lymphocytes and of plasma-borne substances of importance for the
93
Non-granular leukocytes
Monocytes
Functions
Suitable Slides
see lab section on erythrocytes
Lymphocytes
These cells are very variable in size. The smallest may be smaller
than erythrocytes (down to ~5 µm in diameter) while the largest may
reach the size of large granulocytes (up to 15 µm in diameter). How
much cytoplasm is discernible depends very much on the size of the
lymphocyte. In small ones, which are the majority of lymphocytes in
95
the blood, the nucleus may appear to fill the entire cell. Large
lymphocytes have a wider rim of cytoplasm which surrounds the
nucleus. Both the nucleus and the cytoplasm stain blue (and
darker than most other cell types in the blood) . The typical
lymphocyte only contains the usual complement of cellular
organelles. The appearance of lymphocytes may change drastically
when they are activated (see below).
Functions
T-lymphocytes and B-
lymphocytes form the vast
majority of lymphocytes in
the blood stream, but they
do not add up to 100%,
and they usually are small
lymphocytes. The much
less frequent medium-
sized or large lymphocytes
may represent e.g.
natural killer
(Nk-) cells
which belong
to the group
of large
granular
96
lymphocytes,
or
haemopoietic
stem cells of
which a few
will be
circulating in
the blood
stream.
Functions
Suitable Slides
blood platelets (thrombocytes) : see lab section on
erythrocytes
megakaryoblasts and megakaryocytes: see lab section on
haemopoiesis
98
Haemopoiesis
Haemopoietic Cells
Erythrocytes
The first
identifiable stage of
erythropoiesis is the
proerythroblast - a
large, slightly
basophilic cell,
which contains a
large, lightly
stained nulceus.
Proerythroblasts
proliferate to
generate a sequence
of cells which show
a gradual decrease
in size and
condensation of
their chromatin.
They are named
after changes in
the staining
characteristic of
their cytoplasm
(basophilic
erythroblast,
polychromatophili
101
c and
orthochromic
normoblasts). The
nucleus is finally
extruded from the
normoblast. The
cell enters
circulation as a
reticulocyte, which
still contains some
organelles.
Reticulocytes
remain for a few
days in either the
bone marrow or the
spleen to mature to
erythrocytes.
In some blood
smears
reticulocytes may
be recognisable
because of a very
slight basophilic
staining - either
homogeneous or
in the form of a
basophilic
stippling.
Granulocytes
Myeloblast appear light-microscopically similar to proerythroblast.
They proliferate to generate promyelocytes. Promyelocytes begin to
accumulate nonspecific granules, but they are still able to divide.
The maturation of their progeny, the myelocytes, is characterised by
the accumulation of specific granules and changes in nuclear
morphology. Metamyelocytes have a C-shaped nucleus.
Precursors of blood cells which are usually only found in the bone marrow
can be found in peripheral blood in a variety of pathological conditions.
If a Rh-negative mother has been immunised by erythrocytes of a
Rh-positive foetus, a condition called Erythroblastosis fetalis may
develop during subsequent pregnancies. It would show itself in the
foetus or newborn by the presence of erythrocyte precursors in
peripheral blood - although other, more severe symptoms should be
obvious. Chronic myeloid leukemia is another condition - in this case
showing itself by the presence of all types of granulocyte precursors
in peripheral blood.
Suitable Slides
sections of red bone marrow - H&E or a bone marrow smear
- Leishman, Wright's, Giemsa or May-Grünwald-Giemsa
stains
If you still have some time and are desperate to get frustrated try to
hunt up a nice basophilic erythroblast - a basophilic cell with
homogeneously staining nucleus that is somewhat smaller than the
nuclei of granulocyte precursors.
MUSCLE
1. smooth muscle,
2. skeletal muscle and
3. cardiac muscle.
Smooth Muscle
Two broad types of smooth muscle can be distinguished on the basis of the
type of stimulus which results in contraction and the specificity with which
individual smooth muscle cells react to the stimulus:
cells which are often innervated by a single nerve terminal and which
never contract spontaneously (e.g. smooth muscle in the walls of blood
vessels).
2. The visceral type represents bundles of smooth muscle cells connected by
GAP junctions, which contract spontaneously if stretched beyond a
certain limit (e.g. smooth muscle in the walls of the intestines).
Suitable Slides
? The only tissues which perhaps could be confused with smooth muscle are
dense regular connective tissues and peripheral nerves. Both the number of
nuclei and their shapes clearly distinguish smooth muscle from dense
regular connective tissues. Nuclei are much more frequent and larger in
smooth muscle, and they are very elongated if cut longitudinally. Peripheral
nerves will be surrounded by a capsule of cells and connective tissue - the
perineurium. The thickness of longitudinally cut nerve fibres is constant
while smooth muscle cells are spindle shaped. Also, axon and nodes of
Ranvier should be visible in peripheral nerves
Skeletal Muscle
Skeletal muscle consists of very long tubular cells, which are also called
muscle fibres.
108
The myoblasts of all skeletal muscle fibres originate from the paraxial
mesoderm. Myoblasts undergo frequent divisions and coalesce with the
formation of a multinucleated, syncytial muscle fibre or myotube. The
nuclei of the myotube are still located centrally in the muscle fibre. In the
course of the synthesis of the myofilaments/myofibrils, the nuclei are
gradually displaced to the periphery of the cell.
Satellite cells are small cells which are closely apposed to muscle fibres
within the basal lamina which surrounds the muscle fibre. Their nuclei are
slightly darker than those of the muscle fibre. Satellite cells are believed to
represent persistent myoblasts. They may regenerate muscle fibres in case
of damage.
Suitable Slides
Tongue,
Skeletal
Muscle,
human -
H&E
Skeletal
muscle in the
tongue is
arranged in
bundles
which
typically run
at right
angles to
each other.
Both
longitudinally
and
transversely
cut skeletal
muscle fibres
are present.
In both
section
planes you
can see that
the nuclei are
located in the
periphery of
the muscle
fibre.
Myofibrils
may be
visible as
very fine dots
in some of
the
transversely
muscle fibres.
Striations
formed by the
A- and I-
Bands of the
sarcomeres
are visible in
110
longitudinally
cut fibres. Z-
lines and H-
bands can be
identified in
well-
preserved
tissue.
Details of the
sarcomeres
stand out
more clearly
if you close
the iris
diaphragm of
the
microscope.
Remember to
open the
diaphragm
after you
have seen the
striations
clearly !
In the
connective
tissue
between the
muscle fibres,
the
endomysium,
numerous
capillaries
supply the
muscle with
oxygen and
nutrients.
Draw a small
section of
longitudinal
and
transversely
cut skeletal
muscle at
high
111
magnification
.
The muscle
surrounding
the upper
one-third of
the
oesophagus is
skeletal
muscle.
Smooth
muscle
surrounds its
lower one-
third. In
section of the
middle of the
esophagus it
is usually
possible to
identify both
muscle types
and their
appearances
can be
compared.
112
The spatial relation between the filaments that make up the myofibrils
within skeletal muscle fibres is highly regular. This regular organisation of
the myofilaments gives rise to the cross-striation, which characterises
skeletal and cardiac muscle. Sets of individual "stria" correspond to the
smallest contractile units of skeletal muscle, the sarcomeres. Rows of
sarcomeres form the myofibrils (), which extend throughout the length of
the skeletal muscle fibre.
I-band -
actin
filaments,
A-band -
myosin
filaments
which may
overlap with
actin
filaments,
H-band -
zone of
myosin
filaments
only (no
overlap with
actin
filaments)
within the
A-band,
Z-line - zone
of
apposition
of actin
filaments
belonging to
two
neighbourin
g
sarcomeres
(mediated
by a protein
called alpha-
actinin),
M-line -
band of
connections
between
myosin
filaments
(mediated
by proteins,
e.g.
myomesin,
M-protein).
114
The protein titin extends from the Z-line to the M-line. It is attached to the
Z-line and the myosin filaments. Titin has an elastic part which is located
between the Z-line and the border between the I- and A-bands. Titin
contributes to keeping the filaments of the contractile apparatus in
alignment and to the passive stretch resistance of muscle fibres.
Other cytoskeletal proteins interconnect the Z-lines of neighbouring
myofibrils. Because of this connection, the A- and I-bands of neighbouring
myofibrils lie side-by-side in the muscle fibre. These cytoskeletal proteins
also connect the Z-lines of the peripheral myofibrils to the sarcolemma.
Muscle-Tendon Junction
eliptical area.
The
excitatory
transmitter at
the motor
end plate is
acetylcholine
. The space
between the
boutons and
the muscle
fibre is called
primary
synaptic
cleft.
Numerous
infoldings of
the
sarcolemma
in the area of
the motor
end plate
form
secondary
synaptic
clefts. Motor
end plates
typically
concentrate
in a narrow
zone close to
the middle of
the belly of a
muscle. The
excitable
sarcolemma
of skeletal
muscle cells
will allow
the stimulus
to spread,
from this
zone, over
the entire
muscle cell.
116
Sites of interaction between actin and myosin are in resting muscle cells
"hidden" by tropomyosin. Tropomyosin is kept in place by a complex of
proteins collectively called troponin. The binding of calcium to troponin-C
induces a conformational change in the troponin-tropomyosin complex
which permits the interaction between myosin and actin and, as a
consequence of this interaction, contraction.
Type II fibres
White
muscle
cells, which
are
predominan
tly found in
white
muscles, are
thicker and
contain less
myoglobin.
ATPase
activity of
the myosin
isoform in
white fibres
is high, and
contraction
is fast. Type
IIA fibres ctsy M. Müntener
(red)
contain
many
mitochondri
a and are
available for
both
sustained
118
activity and
short-
lasting,
intense
contractions
. Type
IIB/IIX
fibres
(white)
contain only
few
mitochondri
a. They are
recruited in
the case of
rapid
acceleration
s and short
lasting
maximal
contraction.
Type
IIB/IIX
fibres rely
on
anaerobic
glycolysis
to generate
the ATP
needed for
contraction.
nerve fibre does not match with the type of muscle. The process of
reinnervation and type adjustment may result in fibre type grouping within
the muscle, i.e. large areas of the muscle are populated by muscle fibres of
one type.
Muscle Spindles
Muscle
spindles are
sensory
specializatio
n of the
muscular
tissue. A
number of
small
specialised
intrafusal
muscle fibres
(nuclear bag
fibres and
nuclear chain
fibres) are
surrounded
by a capsule
of
connective
tissue. The
intrafusal
fibres are
innervated
by efferent
motor nerve
fibres.
Afferent
sensory
nerve fibres
surround the
intrafusal
muscle
fibres.
If the muscle
is stretched,
the muscle
fibres in the
120
muscle
spindle are
stretched,
sensory
nerves are
stimulated,
and a change
in
contraction
of the
muscle is
perceived.
Different
types of
intrafusal
fibres and
nerve
endings
allow the
perception of
position,
velocity and
acceleration
of the
contraction
of the
muscle.
The
contraction
of the
intrafusal
fibres, after
stimulation
by the
efferent
nerve fibres,
may
counteract or
magnify the
changes
imposed on
the muscle
spindle by
the
surrounding
121
muscle. The
intrafusal
fibres and
the efferent
nerves can in
this way set
the
sensitivity
for the
sensory
nerve ending
in the muscle
spindle.
Cardiac Muscle
T-tubules are typically wider than in skeletal muscle, but there is only one
T-tubule set for each sarcomere, which is located close to the Z-line. The
associated sarcoplasmatic reticulum is organised somewhat simpler than in
skeletal muscle. It does not form continuous cisternae but instead an
irregular tubular network around the sarcomere with only small isolated
dilations in association with the T-tubules.
Cardiac muscle does not contain cells equivalent to the satellite cells of
skeletal muscle. Therefore cardiac muscle cannot regenerate.
Suitable Slides
Cardiac
Muscle,
human - H&E
Use a low
magnification
to find a part
of the tissue
in which the
cardiac
muscle cells
are cut
longitudinally
. At high
magnification
you should
see striations
and the large
nuclei of the
cardiac
muscle cells.
If you follow
the course of
individual
cardiac
muscle cells
you will note
fine, darker
lines which
seem to cross
the fibres.
These are the
intercalated
discs which
connect the
individual
muscle cells
mechanically
and permit
the
conduction of
electrical
impulses
between the
cells ... how?
124
A light streak
of cytoplasm
is often
visible
extending
from the
poles of the
nucleus. This
part of the
cytoplasm
does not
contain
myofibrils,
and it appears
very light in
transversely
cut cardiac
muscle cells.
Myofibrils
are often
visible in
transversely
cut cells.
Their visible
separation
reflects the
large
numbers of
mitochondria
located
between
them. Also,
the large
number
capillaries
reflect the
need of a
good blood
supply to the
constantly
active muscle
cells.
Draw
longitudinally
125
cut cardiac
muscle cells
which show
all the
features
mentioned.
Label the
features in
your drawing,
and include
an suitable
scale.
heart rate.
Suitable Slides
The nervous system consists of all nervous tissue in the body. It is divided
anatomically into the central nervous system and the peripheral nervous
system.
The CNS consists of the brain (encephalon), which is enclosed in the skull,
and the spinal cord, which is contained within the vertebral canal. Nervous
tissue of the CNS does not contain connective tissue other than that in the
meninges and in the walls of large blood vessels. Collagenous fibers or
fibrocytes/blasts are consequently not observed, which is quite unlike other
tissues. Because of the absence of connective tissue, fresh CNS tissue has a
very soft, somewhat jelly-like consistency. The two major classes of cells
that make up the nervous tissue are nerve cells, neurones, and supporting
cells, glia.
Neurones
Neurones are generally large cells. Neural activity and its control require
the expression of many genes, which is reflected in the large and light
nuclei of most neurones. The keys to the understanding of the function of a
neurone lies in (1) the shape of the neurone and, in particular, its processes,
(2) the chemicals the neurone uses to communicate with other neurones
(neurotransmitters) and (3) the ways in which the neurone may react to the
neurotransmitters released by other neurones.
129
The shape of
the neurone
and its
processes
Neurones have
long processes,
which extend
from the part
of the cell
body around
the nucleus,
the perikaryon
or soma. The
processes can
be divided into
two
functionally
and
morphologicall
y different
groups,
dendrites and
axons.
Dendrites are
part of the
receptive
surface of the
neurone. As a
rule neurones
have one to
several
primary
dendrites,
which emerge
from the
perikaryon.
Primary
dendrites may
divide into
secondary,
tertiary etc.
dendrites.
Dendrites can
be smooth, or
130
they can be
studded with
small,
mushroom-
shaped
appendages,
which are
called spines.
Each neurone
has as a rule
one axon, and
never more
than one axon
which emerges
from the
perikaryon or
close to the
trunks of one
of the primary
dendrites. The
point of origin
of the axon
from the
perikaryon is
the axon
hillock. The
axon may, like
the dendrites,
branch as it
travels through
the nervous
tissue to its
destination(s).
The axon is the
"transmitting"
process of the
neurone.
131
The shape and orientation of the dendritic tree of the neurone determines
the amount and type of information that may reach the neurone. The course
of its axon determines to which neurones this information may be passed
on. The location of the neurone within the CNS determines to which major
system the neurone belongs.
Transmitters
Receptors
Usually there exists a multitude of receptors which are all sensitive to one
particular neurotransmitter. Different receptors have different response
properties, i.e. they allow the flux of different ions over the plasma
membrane of the neurone or they may address different second messenger
systems in the postsynaptic neurones. The precise reaction of the neurone to
the various neurotransmitters released onto its plasma membrane at the
synapses is determined by the types of receptors expressed by the neurone.
Suitable Slides
perikaryon depends on the level of activity of the neurone and the length of
the processes which the neurone has to support. An usable range for the size
of the perikaryon would be 15 - 50 µm, although much smaller and much
larger neuronal perikarya exist.
Draw the spinal cord at low magnification and indicate the distribution of
grey matter and white matter. Find a nice group of neurones in the grey
matter and draw them at a high magnification. Finally, have a look at the
white matter and identify the nuclei of glial cells. You will find similar
nuclei also in the grey matter.
Glia
Astrocytes
(or
astroglia)
are star-
shaped
cells. Their
processes
are often in
contact
with a
blood
vessel
(perivascul
ar foot
135
processes).
Astrocytes
provide
physical
and
metabolic
support to
the
neurones
of the
CNS. They
participate
in the
maintenanc
e of the
compositio
n of the
extracellul
ar fluid.
Although
not
themselves
directly
involved in
the process
of
communic
ation
between
neurones,
they may
be
involved in
the
removal of
transmitter
s from
synapses
and the
136
metabolis
m of
transmitter
s.
Astrocytes
are also the
scar-
forming
cells of the
CNS.
The
ventricl
es of
the
brain
and the
central
canal of
the
spinal
cord are
lined
with
ependy
137
mal
cells.
The
cells are
often
cilated
and
form a
simple
cuboida
l or low
column
ar
epitheli
um. The
lack of
tight
junction
s
between
ependy
mal
cells
allows a
free
exchang
e
between
cerebro
spinal
fluid
and
nervous
tissue.
Ependy
mal
cells
can
speciali
138
se into
tanycyt
es,
which
are
rarely
ciliated
and
have
long
basal
process
es.
Tanycyt
es form
the
ventricu
lar
lining
over the
few
CNS
regions
in
which
the
blood-
brain
barrier
is
incompl
ete.
They do
form
tight
junction
s and
control
the
139
exchang
e of
substan
ces
between
these
regions
and
surroun
ding
nervous
tissue
or
cerebro
spinal
fluid.
Many glial cells do express neurotransmitter receptors, but they do not form
synapses with neurones. Neuronal activity may regulate glial function by a
spillover of transmitter from synaptic sites, which are typically surrounded
by fine processes of glial cells. Glial cells may also communicate with each
other via GAP junctions.
Suitable Slides
a feeling for the structural diversity visible in the section available to you -
parts of the section that look different from others are very likely to have
different functions.
Find a spot that appears interesting (or least boring) to you and sketch its
structure at low magnification. Choose a spot for high magnification, and
draw some of the visible neurones and glial cells. Note the difference in the
size and number of glial cells and neurones.
The PNS comprises all nervous tissue outside the brain and spinal cord. It
consists of groups of neurones (ganglion cells), called ganglia, feltworks of
nerve fibres, called plexuses, and bundles of parallel nerve fibres that form
the nerves and nerve roots. Nerve fibres, which originate from neurones
within the CNS and pass out of the CNS in cranial and spinal nerves, are
called efferent or motor fibers. Nerve fibres which originate from nerve
141
cells outside the CNS but enter the CNS by way of the cranial or spinal
nerves are called afferent or sensory nerve fibres.
Peripheral Nerves
Afferent, sensory fibres enter the spinal cord via the dorsal roots, while
efferent, motor fibres leave the spinal cord via the ventral roots. Dorsal and
ventral roots merge to form the spinal nerves, which consequently contain
both sensory and motor fibres. As the spinal nerves travel into the periphery
they split into branches and the exact composition of the nerve in terms of
motor and sensory fibres is, of course, determined by the structures the
nerve will innervate.
One nerve fibre consists of an axon and its nerve sheath. Each axon in the
peripheral nervous system is surrounded by a sheath of Schwann cells. An
individual Schwann cell may surround the axon for several hundred
micrometers, and it may, in the case of unmyelinated nerve fibers, surround
up to 30 separate axons. The axons are housed within infoldings of the
Schwann cell cytoplasm and cell membrane, the mesaxon .
Suitable Slides
sheath surrounding the axon. A good impression of the different sizes of the
nerve fibres may be obtained. The axon is usually not well preserved. It
may only form a little dark spot somewhere within the dark ring which
represents the myelin sheath. Lipid droplets in fat cells, which can be found
in the connective tissue around nerves, stand out as large (much larger than
the nerve fibres), round, homogeneously stained areas.
Draw the nerve at low magnification (you may include some of the stained
lipid droplets) and a small section of it at high magnification.
lack of a myelin sheath, type C fibres are very difficult to detect in either
osmium or H&E stains.
Draw part of the longitudinally and transversely sectioned nerve at high
magnification. Include Schwann cell nuclei, myelin sheath, axons and, if
possible, nodes of Ranvier.
Ganglia
Ganglia are aggregations of nerve cells (ganglion cells) outside the CNS.
Cranial nerve and dorsal root ganglia are surrounded by a connective tissue
capsule, which is continuous with the dorsal root epi- and perineurium.
Individual ganglion cells are surrounded by a layer of flattened satellite
cells. Neurones in cranial nerve and dorsal root ganglia are pseudounipolar.
They have a T-shaped process. The arms of the T represent branches of the
neurite connecting the ganglion cell with the CNS (central branch) and the
periphery (peripheral branch). Both branches function as one actively
conducting axon, which transmits information from the periphery to the
CNS. The stem is connected to the perikaryon of the ganglion cell and is the
only process originating from it. Ganglion cells in dorsal root ganglia do not
receive synapses. Their function is the trophic support of their neurites.
145
Autonomic ganglia do contain synapses, and the ganglion cells within them
do have dendrites. They receive synapses from the first neurone of the two-
neurone chain, which characterises most of the efferent connections of the
autonomic nervous system. The second neurone is the ganglion cell itself.
Some autonomic ganglia are embedded within the walls of the organs which
they innervate (intramural ganglia - e.g. GIT and bladder).
Suitable Slides
VASCULAR SYSTEM
You have already seen blood vessels of various sizes and types
in preparations available in other lab sessions, and you should be
aware that the histological appearances of vessels of different
sizes (arterioles vs. arteries) and different types (arteries vs.
veins) are different from each other. These differences are the
result of quantitative variations of a common structural pattern
that can be seen in all blood vessels with the exception of
capillaries, i.e. the division of the walls of the blood vessels into
three layers or tunics.
delimits the
vessel wall
towards the
lumen of the
vessel and
comprises its
endothelial
lining (typically
simple,
squamous) and
associated
connective
tissue. Beneath
the connective
tissue, we find
the internal
elastic lamina,
148
which delimits
the tunica
intima from
The tunica
media is
formed by a
layer of
circumferential
smooth muscle
and variable
amounts of
connective
tissue. A
second layer of
elastic fibers,
the external
elastic lamina,
is located
beneath the
smooth muscle.
It delimits the
tunica media
from
which consist
mainly of
connective
tissue fibres.
The tunica
adventitia
blends with the
connective
tissue
surrounding the
149
vessel. The
definition of
the outer limit
of the tunica
adventitia is
therefore
somewhat
arbitrary.
Arteries
Vessels close to the heart (aorta, pulmonary trunk and the larger
arteries that originate from them) are
Elastic arteries
Suitable Slides
sections of the aorta - H&E, elastin
cells. Smooth muscle cells and collagen fibres are found between
the layers of elastic fibres. If you scan the periphery of the aorta
you may find small blood vessels, the vasa vasorum, in the
tunica adventitia and penetrating into the outer part of the tunica
media.
Draw the aorta at low magnification and label the three tunics.
Draw part of the tunica media at high magnification and identify
collagen fibres, layers of elastic fibres and smooth muscle cell
nuclei in your drawing.
Muscular arteries
The basic structure of the walls of arteries does not change much
as we come to the next type of arterial vessels. Size is used to
differentiate them from muscular arteries.
Arterioles
Suitable Slides
sections of arteries - H&E or elastin (in combination with
other stains)
Sections of small muscular arteries and arterioles are
present in many sections, and the basic features of their
structure are usually visible - even in smaller arteries.
Large muscular arteries often have their "own section" in
teaching collections.
Artery - H&E and
elastin & eosin
Identifying muscular
arteries in sections is
rather straight forward.
There are two easily
recognizable features
which distinguish these
arteries from veins. If
two vessels have a
similarly sized lumen,
the walls of arteries
will be much thicker
and more compact than
the wall of veins. At
high magnification, the
internal elastic lamina
forms a pink streak
immediately below the
endothelial cell lining
in arteries and even
arterioles, while it is
difficult to identify in
veins.
The layer of
subendothelial
connective tissue is
very thin, and the
endothelium seems to
rest on the internal
elastic lamina. Smooth
154
Capillaries
The sum of the diameters of all capillaries is significantly larger than that of the
aorta (by about three orders of magnitude), which results in decreases in blood
pressure and flow rate. Also, capillaries are very small vessels. Their
diameter ranges from 4-15 µm. The wall of a segment of capillary may be
formed by a single endothelial cell. This results in a very large surface to
volume ratio. The low rate of blood flow and large surface area facilitate the
functions of capillaries in
These functions are also facilitated by a very simple organisation of the wall
of capillaries. Only the tunica intima is present, which typically only
consists of the endothelium, its basal lamina and an incomplete layer of
cells surrounding the capillary, the pericytes. Pericytes have contractile
properties and can regulate blood flow in capillaries. In the course of
vascular remodelling and repair, they can also differentiate into endothelial
and smooth muscle cells.
Continuous capillaries
are formed by "continuous" endothelial cells and basal lamina. The endothelial cell and
basal lamina do not form openings, which would allow substances to pass the capilla
wall without passing through both the endothelial cell and the basal lamina. Both
endothelial cells and the basal lamina can act as selective filters in continuous capilla
Fenestrated capillaries
The endothelial cell body forms small openings called fenestrations, which allow compo
of the blood and interstitial fluid to bypass the endothelial cells on their way to or fro
the tissue surrounding the capillary. The fenestrations may represent or arise from
pinocytotic vesicles which open onto both the luminal and basal surfaces of the cell. T
extent of the fenestration may depend on the physiological state of the surrounding ti
i.e. fenestration may increase or decrease as a function of the need to absorb or secre
The endothelial cells are surrounded by a continuous basal lamina, which can act as
157
selective filter.
Discontinuous capillaries
are formed by fenestrated endothelial cells, which may not even form a complete layer of c
The basal lamina is also incomplete. Discontinuous capillaries form large irregularly
shaped vessels, sinusoids or sinusoid capillaries. They are found where a very free
exchange of substances or even cells between bloodstream and organ is advantageou
(e.g. in the liver, spleen, and red bone marrow).
Suitable Slides
Sections of any well preserved tissue - H&E, Whipf's polychrome
cardiac and skeletal muscle, glands or the papillary layer of the skin
contain dense capillary beds.
Cardiac Muscle, sheep -
Whipf's polychrome
Large numbers of
capillaries are present in
almost all tissues. At least
a few dozen cross sections
are present in every sqr.
mm of section of poorly
vascularised tissues. There
may be thousands in highly
vascularised tissues.
However, a "good"
capillary is not that easy to
find because of their small
size and because the
capillary walls are very
thin, which often leads to
the collapse of the capillary
during tissue preparation.
Cardiac muscle is highly
vascularised. Each muscle
cell is surrounded by one
or more capillaries. The
capillaries roughly follow
the course of the muscle
cells. To find capillaries in
transverse and longitudinal
sections it is easiest to first
find areas in which the
muscle cells have been cut
158
Veins
The walls of veins are thinner than the walls of arteries, while their diameter
is larger. In contrast to arteries, the layering in the wall of veins is not very
distinct. The tunica intima is very thin. Only the largest veins contain an
appreciable amount of subendothelial connective tissue. Internal and
external elastic laminae are absent or very thin. The tunica media appears
thinner than the tunica adventitia, and the two layers tend to blend into each
other. The appearance of the wall of veins also depends on their location.
The walls of veins in the lower parts of the body are typically thicker than those
of the upper parts of the body, and the walls of veins which are embedded in
tissues that may provide some structural support are thinner than the walls
of unsupported veins.
Venous vessels originate from the capillary network which coalesce into the
smallest venous vessels the
Venules.
They are larger than capillaries. Small venules are surrounded by pericytes.
A few smooth muscle cells may surround larger venules. The venules
merge to form
159
which contain bands of smooth muscle in the tunica media. The tunica
adventitia is well developed. In some veins (e.g. the veins of the
pampiniform plexus in the spermatic cord) the tunica adventitia contains
longitudinally oriented bundles of smooth muscle.
Aside from most veins in the head and neck, small to medium-sized veins
are also characterised by the presence of valves. The valves are formed by
loose, pocket-shaped folds of the tunica intima, which extend into the
lumen of the vein. The opening of the pocket will point into the direction
of blood flow towards the heart. One to three (usually two) pockets form
the valve. Blood flowing towards heart will pass the pockets. If the flow
reverses, blood will fill the pockets which will occlude the lumen of the
vein and prevent the return of blood into the part of the vein preceding the
valve. The ability of the valves to prevent backflow depends to some extent on
the state of contraction (tone) of the smooth muscle in the wall of the vein.
Suitable Slides
sections of veins - H&E, van Gieson or elastin (in combination with
other stains)
Like arteries, veins and venules are present in many sections. The
basic features of their structure are however more difficult to
identify because of the thin walls of veins - in particular in small
veins. It is best to resort to sections of large veins, which together
with large muscular arteries often have their "own section" in
teaching collections.
160
Suitable Slides
sections containing small to medium sized veins - H&E, van Gieson
or trichrome
Unless a specifically prepared slide is available, I would recommend
looking at skin slides, in which I have found quite a few nice valves
in the veins located at the border between dermis and hypodermis.
161
Small arteries and veins often form anastomosing networks, which provides
routes for alternative blood supply and drainage if one of the vessels should
become occluded because of pathological or normal physiological
circumstances. Some arteries are however the only supply of blood to their
target tissues. These arteries are call end arteries. Tissues which are
supplied by end arteries die if the arteries become occluded.
The segments of the kidney and the heads of the gastrocnemius muscle are
examples of tissues supplied by end arteries.
Arteries and veins may also form arteriovenous shunts, which can shunt the
blood flow that otherwise would enter the capillary network between the vessels .
These shunts usually contain specialisations of the smooth muscle in the
region of the shunt. Arteriovenous shunts are frequently seen in the blood
supply of distal parts of the limbs and the nose (thermoregulation) and in the
blood supply of endocrine organs.
Lymphatic Vessels
Parts of the blood plasma will exude from the blood vessels into the
surrounding tissues because of transport across the endothelium or because
of blood pressure and the fenestration of some capillaries (this process is
partly counteracted by the higher osmotic pressure of the blood). The fluid
entering tissues from capillaries adds to the interstitial fluid normally found
in the tissue. The surplus of liquid needs to be returned to the circulation.
Lymph vessels are dedicated to this unidirectional flow of liquid, the lymph.
Three types of lymph vessels can be distinguished based on their size and
morphology.
Lymph capillaries
which are larger and form valves but otherwise appear similar to
lymph capillaries. The lymph is moved by the compression of the
lymph vessels by surrounding tissues. The direction of lymph flow
is determined by the valves. Lymph vessels empty intermittently
into lymph nodes from which the lymph continues in efferent lymph
vessels.
Only very little lymph is returned from the limbs if they are
immobilized, which illustrates the importance of muscular action in
lymph transport. This is also the reason for immobilizing limbs that
are either infected or that have been bitten by venomous Australians.
The effect can also be observed after long intercontinental flight
when you may feel that your shoes and socks are just about one
number too small. Finally, impeded lymph drainage is one of the
problems associated with surgery which requires the removal of
lymph nodes and which thereby interrupts the lymph collecting
vessels.
Lymph ducts
which contain one or two layers of smooth muscle cells in their wall
(some textbooks call this layer the tunica media of lymph vessels).
They also form valves. The walls of the lymph ducts are less flexible
in the region of the attachment of the valves to the wall of the duct,
which may give a beaded appearance to the lymph ducts. Peristaltic
contractions of the smooth muscle contribute to the movement of
lymph towards the heart in addition to the compression of the ducts
by surrounding tissues.
The largest lymph duct of the body, the thoracic duct, drains lymph
from the lower half and upper left quadrant of the body and empties
the lymph into the circulation by merging with the vascular system
close to the junction of the left internal jugular and subclavian veins.
That it is the largest lymph duct does not mean that it is a large
vessel when compared to the large arteries and veins. It actually is
not much larger (about 5mm in diameter) than one of the superficial
forearm veins.
Suitable Slides
164
bloodvessels
The Myocardium:
Cardiac
muscle in
longitudinal
section
Figure 1 shows
cardiac muscle in
longitudinal
section. (It is taken
from slide 94.)
The striations can be seen along the length of the
muscle fibres. [The striations are easier to see
when looking through the microscope, they are
not as obvious on these scanned computer
167
Branching in
cardiac muscle
fibres
Figure 2 shows
another
longitudinal
section of cardiac
muscle (from slide
23). In this section,
several cardiac fibres are seen branching.
Figure 4 shows
Purkinje fibres in
longitudinal
section. Because
of the lower
density of
myofibrils,
Purkinje fibres
appear paler than
regular cardiac
muscle fibres (a few of which can be seen at the
bottom of the figure). A prominent perinuclear
region is seen around several nuclei, and
170
Cross section
of Purkinje fibres
Figure 5 (also
from slide 8)
shows Purkinje
fibres in cross or
oblique section
embedded in the
CT of the subendocardial layer. The wide
diameter of the fibres and the large perinuclear
region devoid of myofibrils can be seen clearly.
Regular
cardiac fibres in
cross section
Figure 6 is also
scanned from slide
8, at exactly the
same
magnification as
Figure 5. Figure 6
shows regular cardiac muscle fibres in cross
section. It can be seen that the fibres are of
smaller diameter, stain more darkly because of a
higher density of myofibrils, and have a smaller
perinuclear region than the Purkinje fibres of
Figure 5.
171
The Endocardium:
Endocardium of Endocardium of
atrium ventricle
The Epicardium:
172
Low power
view of
epicardium
Figure 9 shows a
low power view
of the epicardium
of the ventricle.
Part of the
myocardium is
also visible. In the field of view shown here, there
is a large amount of adipose tissue within the
connective tissue of the epicardium. A nerve
bundle and several blood vessels can also be seen.
The mesothelial lining (at the top) is not really
distinguishable.
Higher
power view of the
epicardium
A higher power
view of the
epicardium is
shown in Figure
10. The nerve is
the same as the
one in Figure 9 and can be used for orientation.
Blood vessels are more easily identified (brighter
red due to RBCs). The nuclei of the mesothelial
cells can be distinguished (albeit with difficulty)
at this magnification.
173
LYMPHOID TISSUES
Thymus
The thymus is situated in the upper parts of the thorax, behind the sternum and the
upper four costal cartilages, in the anterior and superior mediastina. The size of the
thymus changes in the course of life. It weighs about 10-15 g at birth and reaches its top
weight (about 30-40 g) at puberty. After puberty a progressive involution (see below)
occurs, which leaves a middle-aged person with a thymus weighing about 10 g. The
thymus consists of a right and left lobe which are joined by connective tissue.
The thymus is enclosed by a thin connective tissue capsule from which numerous septa
extend into the thymus subdividing the two lobes into numerous lobules (about 0.5 -2
mm in diameter). Blood vessels enter and leave the thymus via the connective tissue
septa. Each lobulus is divided into a darker peripheral zone, the cortex, and a lighter,
175
central zone, the medulla. Medullary tissue is continuous from lobule to lobule
throughout each lobe.
Reticular cells and macrophages are present in addition to the lymphocytes, which are
the dominant cell type within the lobules.
Reticular cells
are quite abundant. Their cytoplasm is eosinophilic, and their large, ovoid and
light nuclei may contain one or two nucleoli. The cells are branched, and their
slender processes are connected with the processes of other reticular cells to
form a cellular reticulum (or cellular network). This cellular network (reticular
fibres are scant in the thymus) provides support for other cells of the thymus.
Reticular cells sheathe the cortical capillaries; they form an epitheloid layer
which delimits the cortical tissue from the connective tissue and secrete
substances (hormones and other factors) important for thymic function. Thereby
they create and maintain the microenvironment necessary for the development
of T-lymphocytes in the cortex. Their functions thus go beyond those of
"typical" reticular cells and, to reflect this, they are also referred to as thymic
epitheliocytes.
Macrophages
occur in both cortex and medulla. They are difficult to distinguish from the
reticular cells in H&E stained sections.
Lymphocytes
are present in both cortex and medulla, but are more numerous (denser) in the
cortex. Their sizes are variable (5 - 15 µm) in the cortex but generally small in
the medulla. The vast majority of them will be developing T-lymphocytes. They
are also called thymic lymphocytes or thymocytes.
176
The thymus is necessary for the development of the recirculating pool of small, long-
lived (in humans many years) lymphocytes, the T-lymphocytes. These cells are mainly
responsible for the cell-mediated part of an immune response. Stem cells invade the
cortical regions of the thymus, where they divide to form lymphocytes. Only a small
fraction (estimates range from 10-30%) of the cells generated in the cortex leave the
thymus. They migrate via the medulla into the blood stream to populate the T-
lymphocyte areas of other lymphoid tissues and organs. Cells which do not express the
necessary receptors to recognize antigens presented to them or which react incorrectly
towards "self-antigens" die and are removed by cortical macrophages.
Since the function of the thymus is to produce lymphocytes for the other lymphoid
tissues it is a primary lymphoid organ.
After puberty much of the parenchyma of the thymus, in particular cortical lymphoid
tissue, is replaced by adipose tissue. The process, which is called involution, initially
proceeds rapidly but slows down in adulthood. Involution is under the control of steroid
hormones (both sexual hormones and stress hormones). Although most pronounced in
the thymus, involution is a common feature of all lymphoid tissues.
Another age-related phenomenon is the increase in size of the thymic (or Hassall's)
corpuscles. Thymic corpuscles are rounded eosinophilic structures, which consist of
concentrically arranged, flattened cells. Thymic corpuscles are likely to be formed by
reticular cells. Similar structures occur also in the tonsils. The size of these structures
varies from 20 µm to more than 100 µm in diameter. Thymic corpuscles may calcify,
and their core may "dissolve" leading to the formation of a cyst.
178
Lymph Nodes
Lymph nodes are small, flattened, oval or bean shaped organs, which are situated in the
course of the collecting lymph vessels. Their size is variable (from a few mm to more
than 2 cm). The capsule and trabeculae of lymph nodes are formed by connective
tissue. Afferent lymph vessels penetrate the capsule and empty into the subcapsular
space. The lymph continues thereafter through cortical and medullary sinuses towards
the efferent lymph vessels, which emerge from the hilus of the lymph node. The walls
of the sinuses can be traversed freely by all components of the lymph, which allows
lymphocytes to enter/leave the lymphoid tissue (as part of their constant circulation) or
to get in contact with antigens/antigen-presenting cells that may arrive with the lymph.
Reticular cells
which are located in the outer cortex of the lymph node are likely to represent
B-lymphocytes. They are organised into spherical masses - lymphoid nodules or
follicles. Sites within the cortex at which B-lymphocytes have been stimulated
to proliferate (by contact with an antigen) appear lighter than the surrounding
tissue and allow you to identify the centres of lymphoid nodules. The lighter
stained parts of the nodules are called germinal centres. Mature B-lymphocytes
(plasma cells) are located in cord-like extensions of the lymphoid tissue into the
medulla, the medullary cords. T-lymphocytes are located in the more diffuse
tissue between the nodules and in the paracortex, i.e. the deep part of the cortex.
Macrophages
are found scattered within the lymphoid tissue. In many preparations they are
difficult to distinguish from the reticular cells, but if an H&E stain turns out
nice, macrophages can be distinguished from the reticular cells in the sinus
system of the lymph node.
Blood Vessels
Blood vessels enter the lymph nodes through the hilus and travel initially in the
connective tissue trabeculae that extend from the hilus into the parenchyma of the
lymph node. They continue in the medullary cords towards the cortex and give off
capillaries to the surrounding tissue as they do so. High-endothelial venules (or
postcapillary venules) in the deep cortex have a characteristic low cuboidal epithelium -
quite unlike the squamous epithelium that we usually would expect to see.
Lymphocytes, which reach the lymph node via the blood stream, may migrate through
this epithelium as part of their recirculation. Larger venules accompany the arteriolar
180
INTEGUMENTARY SYSTEM
The skin or cutis covers the entire outer surface of the body. Structurally, the skin
consists of two layers which differ in function, histological
appearance and their embryological origin. The outer layer or
epidermis is formed by an epithelium and is of ectodermal origin . The
underlying thicker layer, the dermis, consists of connective tissue and
develops from the mesoderm. Beneath the two layers we find a
subcutaneous layer of loose connective tissue, the hypodermis or
subcutis, which binds the skin to underlying structures. Hair, nails
and sweat and sebaceous glands are of epithelial origin and
collectively called the appendages of the skin.
The skin and its appendages together are called the integumentary system.
Suitable Slides
sections of skin - H&E, trichrome or van Gieson
183
Epidermis
2. In the
stratum
spinosum,
the cells
become
irregularly
polygonal. The
cells are often
separated by
narrow,
translucent
clefts. These
clefts are
spanned by
spine-like
cytoplasmatic
extensions of
185
very flat and (3) the space between the cells has been filled with
lipids, which cement the cells together into a continuous
membrane. In the EM, the cell membranes appear thickened and interdigitate
with those of neighbouring cells. Closest to the surface of the epidermis, the
stratum corneum has a somewhat looser appearance. Horny cells are constantly
shed from this part of the stratum corneum.
The protection of the body by the epidermis is essentially due to
the functional features of the stratum corneum.
Keratinisation should not be used as a synonym for the formation of the stratum
corneum: other stratified squamous epithelia may become keratinised but may not form
a stratum corneum in which cells join to form a horny cell membrane.
Suitable Slides
sections of skin - H&E, trichrome or van Gieson
The red and yellow hues of the skin are due to haemoglobin in the red
blood cells, which pass through the capillaries beneath the epidermis,
and carotene, which accumulates in fat cells found in the dermis and
hypodermis.
Melanocytes
The brown colour component is due to melanin, which is produced in the skin
itself in cells called melanocytes (typically 1000-2000 / sqr. mm). These cells are
located in the epidermis and send fine processes between the other cells. In the
melanocytes, the melanin is located in membrane-bound organelles called
melanosomes. The cell bodies of melanocytes are difficult to distinguish in
ordinary LM preparations, because the melanosomes are located mainly in the
188
Melanocytes
can transfer
melanin to
keratinocytes -
mainly to the
basal cells. The
fine processes
of melanocytes
may invade
keratinocytes
and bud-off part
of the
melanocyte
cytoplasm,
including the
melanosomes,
within the
keratinocytes.
Melanin
protects the
chromosomes
of mitotically
active basal
cells against
light-induced
damage.
Pigmentation is
not just under
the control of
light. Hormones
produced by the
pituitary and the
adrenal glands
also affect
pigmentation.
Diseases of
these two
endocrine
organs often
result in
changes of
pigmentation of
189
the skin.
Although
melanocytes are
also
ectodermal
in origin, they
are derived
exclusively
from the neural
crest of the
embryo, from
where they
migrate to all
other parts of
the body.
Langerhans Cells
are another cell type found within the epidermis. Morphologically they are not
unlike melanocytes, but functionally they are more closely related
to macrophages. They are important in immune reactions of the epidermis.
Their fine processes form a network between the cells of the epidermis and
phagocytose antigens which have entered the epidermis. Langerhans cells
may only be temporary residents of the skin. If they have come into contact with
an antigen, they can migrate to regional lymph nodes, where they initiate an
immune response.
T-lymphocytes
Dermis
The dermis is the thick layer of connective tissue to which the epidermis is attached. Its
deepest part continues into the subcutaneous tissue without a sharply defined boundary.
Its thickness is for this reason difficult to determine but 1-2 mm is a good
190
guestimate for "average" skin. The dermis may be divided into two
sublayers (again without a sharp boundary):
Kraissl lines have been defined in living humans. They not always coincide with
the cleavage lines, which Langer defined (Langer's cleavage lines) about a
century before Kraissl in cadavers.
Elastic fibres are found in both the papillary (fine fibres) and reticular
(coarse fibres) layers.
They can not be identified in H&E stained sections.
Suitable Slides
sections of skin - H&E, van Gieson
Van Gieson stained sections are particularly nice if the van Gieson stain has
been combined with an elastin stain.
Skin, thin - H&E and Skin, thick - van Gieson & elastin
How easy it is to differentiate between the papillary and reticular layer of the dermis
depends on the preparation - you may have to look at several preparations. Immediately
beneath the epidermis you should see a layer which at low magnification appears rather
evenly stained. At high magnification the stain should resolve into a fine network of
collagen fibres, which blend with equally fine elastic fibres. Cells are more numerous in
the papillary layer and you should see more nuclei in this area than in the deeper
reticular layer. Also, the papillary layer contains the capillary network which supplies
the epidermis, The reticular layer contains coarse collagen and elastic fibres and the
larger vessels which feed into the capillary network of the papillary layer..
Draw part of the epidermis and the underlying dermis. Label the layers of the dermis
191
Hair
Truly hairless are only the palms of hands and soles of feet, the distal
phalanges and sides of fingers and toes and parts of the external
genitalia.
192
In those parts of the skin which we perceive as "hairy" we find terminal hairs. The
free part of each hair is called the shaft. The root of each hair is anchored in a tubular
invagination of the epidermis, the hair follicle, which extends down into the
dermis and, usually, a short distance into the hypodermis. The deepest end of the
hair follicle forms an enlargement, the bulb. Cells in the bulb are mitotically active.
Their progeny differentiates into the cell types which form the hair and the cells that
surround its root, the root sheath. Hair cells keratinise within the lower one-third of the
hair follicle. Above this level it is not possible to identify individual cells within the
hair. Each hair follicle has an associated bundle of smooth muscle, the
arrector pili muscle. This muscle inserts with one end to the papillary layer of the
dermis and with the other end to the dermal sheath of the hair follicle.
Hair growth is discontinuous. Hairs are lost and replaced by new ones. The hair follicle
goes through different stages that reflect the discontinuous hair growth.
Anagen is the phase of growth. The resting stage is called telogen. The length of
the anagen is variable in different regions of the body - lasting only a
few months for hair of the eyebrows and eyelashes but 2 to 5 years for
hair of the scalp. Hair growth is controlled by a number of hormonal
and hereditary factors and their interactions.
Suitable Slides
Sections of hairy skin or scalp - H&E
With a few exceptions (thick skin and skin covering parts of the external
genitalia), all skin sections should contain a few hair follicles.
Sebaceous Glands
Sebaceous glands empty their secretory product into the upper parts of the hair follicles.
They are therefore found in parts of the skin where hair is present. The hair follicle
and its associated sebaceous gland form a pilosebaceous unit.
Sebaceous glands are also found in some of the areas where no hair
is present, for example, lips, oral surfaces of the cheeks and external
genitalia.
Sebaceous glands are as a rule simple and branched (Remember the nomenclature of
glands!). The secretory portion consists of alveoli. Basal cells in the outermost layer of
the alveolus are flattened. Basal cells are mitotically active. Some of the new cells will
replenish the pool of basal cells, while the remaining cells are displaced towards the
194
centre of the alveolus as more cells are generated by the basal cells. The secretory cells
will gradullay accumulate lipids and grow in size. Finally their nuclei disintegrate, and
the cells rupture. The resulting secretory product of lipids and the constituents of the
disintegrating cell is a holocrine secretion.
Suitable Slides
slides of hairy skin or thin skin - H&E, trichrome, van Gieson
Sweat Glands
Apocrine sweat glands occur in, for example, the axilla. They are
stimulated by sexual hormones and are not fully developed or
functional before puberty. Apocrine sweat is a milky, proteinaceous
and odourless secretion. The odour is a result of bacterial
decomposition and is, at least in mammals other than humans, of
importance for sexual attraction.
The histological structure of apocrine sweat glands is similar to that of merocrine sweat
glands, but the lumen of the secretory tubulus is much larger and the secretory
epithelium consists of only one major cell type, which looks cuboidal or
low columnar. Apocrine sweat glands as such are also much larger
than merocrine sweat glands.
The excretory duct of apocrine sweat glands does not open directly onto the surface of
the skin. Instead, the excretory duct empties the sweat into the upper part of the hair
follicle. Apocrine sweat glands are therefore part of the pilosebaceous unit.
Some texts argue that the apocrine sweat glands use a merocrine or a combined
merocrine / apocrine secretory mechanism.
Suitable Slides
merocrine sweat glands: sections of thick skin or thin skin - H&E
apocrine sweat glands: sections of skin from the areolae (pigmented skin
surrounding the nipples), the axilla (arm pit) or skin covering the external
genitalia - H&E
preparations. The different cell types in the secretory epithelium of merocrine sweat
glands are only visible in well preserved glands. The red rim around the secretory
tubulus is formed by the cytoplasm of myoepithelial cells. Their small, dark nuclei may
be visible close to the periphery of the tubulus.
Draw a small schematic illustrating the relative position of the sweat gland in the skin.
Identify and draw the secretory tubulus and excretory duct. Label as many features as
can be identified.
199
Nipple - H&E
Like merocrine sweat
gland, the secretory
tubulus of apocrine
sweat glands will coil
close to the border
between the dermis and
the hypodermis. Only
one type of secretory
cell is present, and the
lining of the secretory
tubulus looks more
uniform than that of
merocrine sweat
glands. The key feature
though is the very wide
lumen of the secretory
tubulus. The secretory
cells are surrounded by
a layer of myoepithelial
cells. Their cytoplasm
forms the slightly
darker outline of the
secretory tubulus.
Draw the secretory
tubulus of an apocrine
seat gland - preferably
next to your drawing of
the merocrine sweat
gland. Label the
structures included in
your drawing.
ORAL CAVITY
The oral cavity is formed by a bewildering array of tissues which function in or are
associated with the processes that are performed with what we typically refer to as
our mouth. Lecture and lab focus on the one organ found within the oral cavity, the
tongue, and the glands which empty their secretory products into the oral cavity, the
salivary glands. In the lab you will also have the opportunity the examine one other
specialized epithelial area, the lip. The oesophagus is the first part of the alimentary
canal. Its organisation is also typical for all parts of the gastrointestinal tract (GIT).
The oral cavity is divided in a vestibule, the area "outside" the teeth, and an oral cavity
proper. The entire oral cavity is lined by a stratified squamous epithelium. The
epithelial lining is divided into two broad types:
1. Masticatory epithelium covers the surfaces involved in the processing of food (tongue,
gingivae and hard palate). The epithelium is keratinized to different degrees
depending on the extent of physical forces exerted on it.
2. Lining epithelium, i.e. non-keratinised stratified squamous epithelium, covers the
remaining surfaces of the oral cavity.
The Tongue
The dorsal surface of the tongue is divided by the sulcus terminalis into an oral part, the
anterior two-thirds, and a pharyngeal part, the posterior one-third. The dorsal surface of
the oral part has a characteristic appearance due to the presence of a large number of
small projections, the lingual papillae. The epithelium of the pharyngeal part forms a
somewhat irregular surface which covers the lingual tonsils.
Filiform papillae
201
are the smallest and most numerous papillae. By providing the tongue with a
rough surface they aid in the manipulation and processing of foods.
Prof. Oxnard brought another function to my attention, i.e. the cleaning of the
surfaces of the mouth, in particular the teeth.
Fungiform papillae
occur singly and are fairly evenly spaced between the filiform
papillae. Their connective tissue core is richly vascularised.
The epithelium is slightly thinner than on the remaining
surface of the tongue.
Circumvallate papillae
are the largest and least numerous papillae - in humans there are between 8 and
12 of them. They occur in depressions of the surface of the tongue and are
surrounded with a trench formed by the infolding of the epithelium. Taste buds
are particularly numerous on the lateral surfaces of these papillae. The excretory
ducts of serous glands open into the trenches surrounding the papillae ("rinsing
glands" or glands of von Ebner).
Foliate papilla
are not well developed in humans and may be absent in aged individuals. If
present, they form lamellae along the posterior and lateral border of the
202
tongue.
The epithelium of the dorsal surface of the tongue rests on a fairly dense layer of
connective tissue, which connects the epithelium firmly with the underlying muscular
and connective tissues.
The muscles of the tongue (skeletal muscle) are organized into strands oriented more or
less perpendicular to each other. Their actions provide the tongue with the necessary
motility to participate in the formation of speech and to aid in the initial processing of
foods. Control of the movement of the tongue muscles and the collection of sensory
information necessitate a profuse innervation of the tongue in which a number of the
cranial nerves participate (V, trigeminal nerve - sensory - anterior two-thirds; VII, facial
nerve - taste; IX, glossopharyngeal nerve - sensory/taste - posterior one-third; XII,
hypoglossal nerve - motor).
Suitable Slides
Taste Buds
203
Taste buds are most numerous in the fungiform, circumvallate and foliate papillae. In
addition, taste buds are found in the palate, palatoglossal and palatopharyngeal arches
and in the pharynx and larynx.
In histological sections they appear as ovoid lightly stained bodies, which extend
perpendicular from the basement membrane to a little opening formed in the
epithelium, the taste pore. The elongated cells that form the taste bud can functionally
be divided into three groups: sensory cells, supporting (or sustentacular) cells, and basal
cells. Sensory cells extend microvilli into the taste pore. These microvilli contain the
receptors for the different basic taste modalities (sweet, salty, bitter and acid). Basal
cells regenerate the two other cell types.
Cell turnover is quite high, and it is thought that the cells of the taste buds are
replaced (on average) every 10th day.
Suitable Slides
Circumvallate Papilla and Taste Buds, sheep - Alcian blue & van Gieson, H&E
Find and inspect the taste buds embedded in the epithelium of the lateral walls of the
circumvallate papillae. The taste pore may not always be visible (outside the plane of
section). Now look at the bottom of the trench surrounding the circumvallate papillae.
Sometimes it is possible to find a duct opening into the trench. If the actual opening is
outside the plane of section it is usually possible to find a section of the duct in the
underlying connective tissue. Slightly deeper in the connective tissue you may be able
to identify the serous glands, which rinse the trenches surrounding the circumvallate
papillae.
Draw a part of the tissue in which these structures (as many as possible) are visible.
204
Salivary Glands
Saliva is a mixed secretion, which is derived from numerous large and small salivary
glands that all open into the oral cavity. Small salivary glands are situated in the
connective tissue beneath the epithelia lining the oral cavity, and, in the case of the
tongue, they may also be found between the muscular tissue. Depending on the
localisation they are grouped into lingual, labial, buccal, molar and palatine glands.
1. the sublingual glands, which are positioned beneath the tongue and embedded deeply
in the connective tissue of the oral cavity,
2. the submandibular glands and
3. the parotid glands, which lie outside the oral cavity.
All of these glands are tubuloacinar glands, i.e. they have secretory acini but the first
part of the duct system originating from the acini also participates in the secretory
process. The salivary glands are divided by connective tissue septa into lobes, which are
further subdivided into lobules.
Functionally the secretory acini can be divided into two groups: those that secrete a
205
rather liquid product - serous acini, and those that secrete a very viscous product -
mucous acini. This functional differentiation is reflected in the appearance of these
acini in histological sections.
The cells forming the serous acini contain a round or slightly ovoid nucleus which is
placed basally in the cell. In an H&E stain, the apical cytoplasm may appear
pinkish/red or, in well-preserved tissue, contain reddish granules. The granules
represent the vesicles which contain the secretory products of the cell. The digestive
enzyme α-amylase is also secreted by the acinar cells.
The cells forming the mucous acini usually contain flattened nuclei which appear to be
"pressed" against the basal surface of the cell. Secretory vesicles fill much of the apical
cytoplasm. The secretory product has either been dissolved during the staining
process or remains unstained. Small amounts of cytoplasm which remain between the
vesicles gives the apical part of the cell a distinct "spongy" appearance.
Glands located close to the oral cavity have mainly mucous secretions, whereas glands
located further away from the oral cavity have mainly serous secretions. Following this
general rule, the parotid glands contain almost exclusively serous acini, the
submandibular glands contain both serous and mucous acini, and the sublingual glands
contain mainly mucous acini or mucous acini with serous demilunes.
The ducts of the salivary glands can, according to their position in relation to the lobes
and lobules of the glands, be divided into two parts. Interlobular and interlobar ducts
are embedded in the connective tissue surrounding the lobes and lobules of the
glands. Intralobular ducts are located in between the secretory acini within the lobules
and, consequently, only surrounded by scant, if any visible connective tissue.
Interlobar and interlobular ducts function mainly in the conduit of the saliva and are
formed by a stratified cuboidal or stratified columnar epithelium. The epithelium is
replaced by the stratified squamous epithelium as they approach the opening into the
oral cavity.
The product of serous glands is extensively modified by the initial part of the duct
system. Intralobular ducts can on the basis of their function be divided into intercalated
ducts and striated ducts. The secretory acini empty into intercalated ducts which merge
into the striated ducts.
Cells forming the intercalated ducts add bicarbonate ions to the saliva (buffering
function) and absorb chloride from the saliva. They are typically formed by cuboidal
epithelium.
Striated ducts are formed by columnar cells. In contrast to many other columnar
epithelia, the nucleus of these cells is located approximately midways between the
apical and basal cell surfaces. The striations of the striated duct are found in the basal
part of the cytoplasm of the cells where numerous mitochondria are found between
infoldings of the basal cell membrane. This specialisation provides the cell with the
necessary energy and surface area to perform its task in the modification of the saliva
- the secretion of potassium and the absorption of sodium. Cells of the striated ducts
also take up a secretable form of antibodies and release them into the saliva.
Suitable Slides
The Oesophagus
Throughout the remainder of the digestive system, the histological composition of the
alimentary canal can be described by the following blue-print:
The lumen is lined by an epithelium, which rests on a vascular connective tissue, the
lamina propria. The lamina propria is in turn surrounded by a narrow band of smooth
muscle (muscularis mucosae). These three tissues are collectively referred to as the
mucosa of the alimentary canal.
canal.
The alimentary canal is delimited from other tissues by a layer of loose connective
tissue, the adventitia. In the case of the intraperitoneal parts of the alimentary canal,
i.e. those parts which are suspended in the peritoneal cavity, a simple squamous
epithelium, the serosa, delimits the adventitia from the peritoneal cavity.
Glands may be present in some parts of the wall of the alimentary canal canal. These
glands are called mucosal glands if they are located luminal (or superficial) to the
muscularis mucosae. If the glands extend into the submucosa they are called
submucosal glands.
Oesophageal glands are located in the submucosa. These submucosal glands produce a
mucous secretion, which lubricates the epithelium and aids the passage of food. In the
part of the oesophagus closest to the stomach there may be mucosal mucus-producing
glands, which resemble the glands in the adjacent mucosa of the stomach.
The mucous glands in the part of the oesophagus closest to the stomach protect the
oesophageal mucosa from acidic reflux from the stomach. Mucous glands in the
adjacent mucosa of the stomach are called cardiac glands, and this name is also used
for submucosal mucosal glands in the the part of the oesophagus closest to the stomach.
The muscularis externa is somewhat unusual in that it contains striated muscle in its
upper one third, a mixture of striated muscle and smooth muscle in its middle one-third
and smooth muscle in its lower one-third.
210
The adventitia consists only of a layer of loose connective tissue. Only the lowest part
of the oesophagus (approx. the lowest 2 cm) enters the peritoneal cavity. A serosa
forms the outermost part of the adventitia of this short intraperitoneal segment of the
oesophagus.
Suitable Slides
The Lips
When we think of lips we usually only think of a small part, the vermilion border (or
prolabium), of the "anatomical" lips, which comprise the entire fleshy fold surrounding
the oral orifice. The outside and inside of the lips are lined by skin and oral mucosa
respectively. Between the two, we find labial vessels, nerves, the orbicularis oris
muscle (striated), which shapes the lips, and labial salivary glands.
The vermilion border is the area of transition from the skin to the oral mucosa. The
epithelium is somewhat thicker than in other parts of the facial skin. Connective tissue
papilla extend deep into the epithelium and are heavily vascularized. It is the proximity
211
of these vessels to the surface of the epithelium which gives the prolabium it's red
appearance.
Suitable Slides
GASTROINTESTINAL TRACT
The gastrointestinal tract (GIT) comprises the stomach,
duodenum, jejunum, ileum, colon, rectum and anal canal. The
GIT and oesophagus form the alimentary canal. The basic
structure of the walls of the alimentary canal has been described
on the "Oral Cavity and Oesophagus" page.
The Stomach
a cardiac part,
fundus,
body or corpus, and
a pyloric part (pyloric antrum and pyloric canal)
Histologically, most of the layers of the wall of the stomach
appear similar in its different parts. Regional differences are
mainly restricted to the appearance of the gastric mucosa.
213
Cardiac glands
214
Note that so far only one type of bacteria has found which
can live happily in the stomach - Helicobacter pylori.
Unfortunately these bacteria are involved in the
pathogenesis of gastritis and gastric ulcers.
Endocrine cells
Endocrine cells are scattered, usually solitary, throughout
the epithelium of the gastrointestinal tract. They are part of
the gastro-entero-pancreatic (GEP) endocrine system. The
best characterized endocrine cells in the gastric mucosa are
gastrin-producing cells (G cells) and somatostatin-
producing cells (D cells). G cells are most frequent in the
middle third of the glands. They stimulate the secretion of
acid and pepsinogen. G cell function is stimulated by
nervous input, the distension of the stomach or
secretagogues. D cells are found mainly in glands of the
pyloric antrum. They inhibit G cells and thereby acid
production. D cell function is stimulated by acid in the
lumen of the stomach and duodenum.
decarboxylation cells.
Pyloric glands
Pyloric glands are more coiled than principal glands, and they
may be branched. Endocrine cells, in particular gastrin-producing
cells, are more frequent than in principal glands. A few parietal
cells may be present but chief cells are usually absent.
Large blood vessels, lymph vessels and nerves are located in the
submucosa which consists of loose connective tissue.
Suitable Slides
slides of stomach - H&E (with PAS), van Gieson (with
Alcian Blue)
Small Intestine
The small intestine is divided into duodenum (25-30 cm),
jejunum (about first two-fifths of the rest) and ileum. The three
segments merge imperceptibly and have the same basic
histological organization.
The Mucosa
The mucosa of the small intestine has various structural features
which considerably increase the luminal surface area and
consequently support the main function of the small intestine -
the absorption of the degraded components of the food.
Plicae circulares
The muscularis mucosae has two layers and extends into the
intestinal villi, where the smooth muscle cells form a longitudinal
bundle in the centre of the villi.
The Submucosa
221
Suitable Slides
slides of the duodenum - H&E, van Gieson, trichrome
Duodenum - H&E
Take a close look at the epithelium lining the villi and crypts of
the duodenum, and note the absence of plicae circulares. Where
in the duodenum are we? You will see this tall columnar
epithelium composed of enterocytes, goblet cells and endocrine
cells throughout the remainder of the GIT. Now identify the
lamina propria and muscularis mucosae and the "packages" of
glandular tissue (Brunner's glands) in the connective tissue
between the muscularis mucosae and muscularis externa, i.e. in
the submucosa. Occasionally you will see ducts of Brunner's
gland which penetrate the muscularis mucosae and ascend
through the lamina propria. Note that goblet cells are absent from
these ducts.
You will not be able to identify endocrine cells in the H&E
stained sections.
Draw a section of the duodenal wall in which these structures are
visible, and identify them in your drawing.
222
Suitable Slides
slides of jejunum or ileum - H&E, van Gieson
Jejunum - H&E
Look at the slide without the microscope and see if you can
identify plicae circulares, muscularis externa and villi. Next
identify surface epithelium (simple columnar with goblet cells),
crypts, muscularis mucosae, submucosa and muscularis externa.
Crypts will probably be smaller than you expect them from
schematic drawings. They are fairly short and narrow. The
connection of the lumen of the crypt with the lumen of the
intestine will not always be visible in the plane of the section.
Accumulations of lymphocytes are common in the mucosa of the
GIT, and they occur frequently in the small intestine. They can
form very large aggregates in particular in the ileum, where they
may be covered by a specialised form of epithelium which
facilitates their function in the immune-defence of the body
223
Large Intestine
The large intestine constitutes the terminal part of the digestive
system. It is divided into three main sections: cecum including the
appendix, colon, and rectum with the anal canal. The primary
function of the large intestine is the reabsorption of water and
inorganic salts. The only secretion of any importance is mucus,
224
Suitable Slides
slides of the colon - H&E, van Gieson, trichrome
Colon - H&E
Again look at the slide without the aid of the microscope.
Bundles of longitudinal muscle should be clearly visible on the
outside of the colon. Plicae circulares are absent from the luminal
side of the colon.
Draw a small schematic figure of these features.
Now have a closer look at the components of the wall of the
colon. Villi are absent and the crypts appear deeper than the ones
225
Suitable Slides
227
Junctions
Junctions between the major parts of the alimentary canal share a
rapid transition from tissues characteristic of one part to those
characteristic for the next part, e.g. the transition from the
stratified squamous epithelium of the oesophagus to the glandular
epithelium of the stomach. Many junctions are in addition
accompanied by morphological specializations, e.g. the pylorus at
the gastro-duodenal junctions or the ileo-caecal valve at the ileo-
caecal junction.
Suitable Slides
sections containing the gastro-oesophageal, gastro-
duodenal, ileo-caecal or ano-rectal junction - van Gieson,
H&E, trichrome
229
The Pancreas
The main pancreatic duct opens into the summit of the major duodenal
papilla, usually in common with the bile duct. A duct draining the lower
parts of the head of the pancreas, the accessory pancreatic duct (of
Santorini), is very variable. If present, it may open into the minor duodenal
papilla ~2 cm above the major papilla in the duodenum.
acids;
pancreatic amylase hydrolyses starch and glycogen to glucose and
small saccharides;
pancreatic lipase hydrolyses triglycerides into fatty acids and
monoglycerides;
cholesterol esterase breaks down cholesterol esters into cholesterol
and a fatty acid.
While the enzymes are secreted by the secretory cells of the pancreatic
acini, the bulk of fluid and bicarbonate ions of the pancreatic juice are
secreted by the cells which form the intercalated ducts of the pancreas.
Bicarbonate ions in the pancreatic juice neutralize the acidic contents
which the stomach empties into the duodenum.
Now have a closer look at the secretory tissue within the lobules. At low
magnification most of the tissue appears to be composed of small reddish
packages, the secretory acini. Intercalated ducts are difficult to find and so
are the initial segments of the (non-secretory) intralobular ducts (cuboidal
epithelium). You may try to find them and include them in your drawing,
but don't get upset if you or the demonstrators have difficulties locating
them.
Draw at the largest magnification a number of exocrine secretory acini. If
possible, include in your drawing some centroacinar cells.
233
The Liver
lobules.
functions as an exocrine gland because it secretes bile.
The portal vein, hepatic artery and bile duct enter the liver through the
porta hepatis. These three vessels travel together through the liver
parenchyma. If one of these vessels gives off a branch it is usually
accompanied by branches of the other two vessels. Terminal branches of
one of the vessels will consequently be accompanied by terminal branches
of the other two vessels.
These groups of three tubes - a branch of the portal vein, a branch of the
hepatic artery and a branch of the bile duct - are called portal triads. Portal
triads are a key feature of the organization of the liver. Portal triads are
embedded in interlobular connective tissue.
An idealized representation of
the "classical" liver lobule is a
six-sided prism about 2 mm long
and 1 mm in diameter. It is
delimited by interlobular
connective tissue (only little, if
any, visible in humans; plentiful
in e.g. pigs). In its corners we
find the portal triads. In cross
sections, the lobule is filled by
cords of hepatic parenchymal
cells, hepatocytes, which radiate
from the central vein and are
separated by vascular sinusoids.
There are other ways of dividing the parenchyma of the liver into units.
Two common ways are divisions into portal lobules and liver acini. Portal
lobules emphasize the afferent blood supply and bile drainage by the
vessels of the portal triads. The secretory function of the liver is
emphasized by the term liver acinus. Acini are smaller units than portal or
"classical" liver lobules and relate structural units to terminal branches
formed by the vessels of the portal triad but not the portal triad itself.
Representations of portal lobules and liver acini vary in different
236
textbooks.
The liver lobule is drained by the central vein, which open into the
intercalated or sublobular veins of the liver. These in turn coalesce to form
the hepatic veins. They run alone through the tissue, are usually covered by
connective tissue and eventually empty into the inferior vena cava.
Adjoining liver cells form the walls of the bile canaliculi , which form
a three dimensional network within the sheets of hepatocytes. Bile
canaliculi connect via very short canals (of Hering; formed by both
hepatocytes and cells similar to those in the epithelium of bile ducts) to
terminal bile ducts (cholangioles) which empty into the interlobular bile
ducts found in the portal triads.
stained section.
Draw a small part of sheets of
liver cells and their supporting
reticular connective tissue.
Include in your drawing some of
the hepatocyte nuclei.
Hepatocytes
Biliary System
Terminal bile ducts are lined by a cuboidal epithelium. All other parts of
the bilary system are lined by a tall columnar epithelium. In the gall
bladder the epithelium is often folded and "caved". The gall bladder
functions in the storage and concentration of bile. Microvilli on the apical
surface of the epithelial cells facilitate the resorption of water from the bile.
The epithelium lining the biliary system does not contain mucus-producing
cells and a muscularis mucosae is absent. These features allow you to
distinguish the gall bladder from other parts of the gastrointestinal tract.
RESPIRATORY SYSTEM
241
The complex of organs and tissue which are necessary to exchange blood
carbon dioxide (CO2) with air oxygen (O2) is called the respiratory system. It
consists of
1. structures, which function as ducts, and which together are called the
conductive portion of the respiratory system
2. structures which form the respiratory portion of the respiratory system, in
which the exchange of CO2 and O2 is occurring and
3. the parts of the thoracic musculo-skeletal apparatus and specialisations
of the lung which allow the movement of air through the respiratory
system - the ventilating mechanism.
Nasal Cavity
The Nasal cavity is divided into three structurally and functionally different parts.
1. The vestibules (the first ~1.5 cm of the conductive portion following the
nostrils) are lined with a keratinised stratified squamous epithelium.
Hairs, which filter large particulate matter out of the airstream, and
sebaceous glands are also present.
2. At the transition from the vestibule to the respiratory region of the nasal
cavity the epithelium becomes first stratified squamous and
then pseudostratified columnar and ciliated. This type of epithelium is
characteristic for all conductive passages dedicated to the respiratory
system and therefore also called respiratory epithelium. Mucus
producing goblet cells are present in the epithelium.
The surface of the lateral parts of the nasal cavity is thrown into folds by
bony projections called conchae. These folds increase the surface area
of the nasal cavity and create turbulence in the stream of passing air,
both of which facilitate the conditioning (warming, cooling and filtration)
of the air. Mucous and serous glands in the connective tissue underlying
the epithelium, the lamina propria, supplement the secretion of the goblet
cells. Veins in the lamina propria form thin-walled, cavernous sinusoids,
242
3. Tissues on the superior concha and the nasal septum form the olfactory
region of the nasal cavity. Cilia in the epithelium of the olfactory region
arise from olfactory cells. Although their internal structure resembles
largely that of normal cilia they do not move, because they lack dynein
arms which are necessary for cilial motility. The cell membrane covering
the surface of the cilia contains olfactory receptors which respond to
odour-producing substances, odorants, dissolved in the serous covering
the epithelium. The axons of the olfactory cells collect into bundles in the
lamina propria. The olfactory cells and their processes receive
mechanical and metabolic support from supporting cells (or
sustentacular cells). Basal cells can divide and differentiate into either
olfactory or supporting cells.
The supporting cells and the secretion of the serous glands contain lipofuscin
granules, which give a yellow-brown colour to the surface of the olfactory
region.
Pharynx
The pharynx connects the nasal cavity with the larynx. Depending on the
extent of abrasive forces on the epithelium, the pharynx is either lined
with respiratory epithelium (nasopharynx or epipharynx) or with a
stratified squamous epithelium (oropharynx or meso- and hypopharynx),
which also covers the surfaces of the oral cavity and the oesophagus.
Lymphocytes frequently accumulate beneath the epithelium of the
pharynx.
The nasal cavity and pharynx form the upper respiratory passages.
Suitable Slides
243
sections of the respiratory region of the nasal cavity - H&E, van Gieson
Suitable Slides
sections of the olfactory region of the nasal cavity - H&E, van Gieson
Nasal Cavity, Olfactory Region, rat - Alcian blue & van Gieson
In humans, olfactory epithelium lines the superior concha and parts of the nasal septum.
The bony structures beneath the epithelium form an irregular surface, which increases
244
turbulence in the air passing them and thereby the chances that odorants come into
contact with the olfactory epithelium. In macrosmatic animals, like the rat, the olfactory
epithelium also covers the middle conchae and the surface is considerably more
irregular than in humans.
The olfactory epithelium is formed by olfactory cells, sustentacular cells and basal
cells. Basal cells can be identified by their location. Sustentacular cells are
preferentially located in the superficial cell tier of the epithelium but are difficult to
distinguish from olfactory cells in this preparation. Cilia are not visible and goblet cells
are absent from the olfactory epithelium. Lightly stained rounded areas in the lamina
propria represent bundles of olfactory axons in the lamina propria. Small mucous
glands, olfactory glands or Bowman's glands, in the lamina propria moisturise the
epithelium.
Draw the olfactory epithelium and underlying lamina propria at high magnification.
Label the features included in your drawing.
Larynx, trachea, bronchi and bronchioles form the lower respiratory passages.
Larynx
The larynx connects the pharynx and trachea. The vocal folds of the larynx
245
control airflow and allow the production of sound. The vocal folds are lined by
stratified squamous epithelium and contain the muscle (striated, skeletal) and
ligaments needed to control the tension of the vocal folds. The larynx is
supported by a set of complexly shaped cartilages.
Trachea
The trachea is a fairly short tube (10-12 cm) with a diameter of ~2 cm.
Epithelium and underlying lamina propria are called the mucosa. The
lamina propria consists of loose connective tissue with many elastic
fibres, which condense at the deep border of the lamina propria to form
an elastic membrane. This elastic membrane forms the border between
the mucosa and the connective tissue below it, which is called the
submucosa. Muco-serous glands in the submucosa (submucosal glands)
supplement the secretions of cells in the epithelium. The submucosa
ends with the perichondrium of the tracheal cartilages.
Tracheal cartilages
The trachea bifurcates to give rise to the main bronchi. Their histological
structure corresponds largely to that of the trachea.
Suitable Slides
sections of the trachea - H&E, van Gieson
Trachea, human - H&E
In the trachea you should be able to
identify the following structures:
respiratory epithelium, basement
membrane, submucosal glands (both
serous and mucous parts),
perichondrium, tracheal cartilage and
trachealis muscle (smooth muscle).
One can perceive different appearances
of the connective tissue immediately
below the epithelium and the
connective tissue surrounding the
submucosal glands, but the elastic
lamina forming the border between the
mucosa and submucosa is not visible
in H&E stained slides. Accumulations
of very dark small dots represent
lymphocytes (not illustrated). If
present, you are likely to see them
close to the glandular tissue.
Draw a composite, i.e. assemble all the
main features of the trachea in one
drawing, of a segment of the trachea.
Label the main features.
Bronchi
In the lungs we find the last segments of the conductive portion of the
respiratory system. The main bronchi divide into lobar bronchi which in
turn give rise to segmental bronchi. The latter supply the bronchopulmonary
segments of the lungs. Bronchial branches are accompanied by branches of
the pulmonary artery, nerves and lymph vessels. These structures usually
247
Bronchioles
Bronchioles divide into respiratory bronchioles, which are the first structures
that belong to the respiratory portion of the respiratory system. Small out
pouchings of the walls of the respiratory bronchioles form alveoli, the site of gas
exchange. The number of alveoli increases as the respiratory bronchioles
continue to divide. They terminate in alveolar ducts. The "walls" of alveolar
ducts consists of entirely of alveoli.
Cilia are absent from the alveolar epithelium and cannot help to remove
particulate matter which continuously enters the alveoli with the inspired air.
Alveolar macrophages take care of this job.
They migrate freely over the alveolar epithelium and ingest particulate matter.
Towards the end of their life span, they migrate either towards the bronchioles,
where they enter the mucus lining the epithelium to be finally discharged into
the pharynx, or they enter the connective tissue septa of the lung.
Suitable Slides
sections of lung - H&E, elastin, reticulin
Sections may not contain bronchi.
249
The formation of the lower respiratory passages begins in the fourth foetal
week. An outpouching of the foregut gives rise to the laryngotracheal tube. The
lining of this tube will eventually give rise to the epithelia covering the surfaces
of the larynx, trachea, bronchi, bronchioles and alveoli. Most of the other
tissues of the lower respiratory passages are derived from splanchnic
mesoderm. The laryngotracheal tube divides distally to form two lung buds.
1. The bronchi grow and branch during the glandular period, which last until
approx. the 17th foetal week. Alveoli are not present at this time.
2. Bronchi and bronchioles expand and branch during the canalicular
period. The lung tissue is vascularised during the canalicular period.
Bronchi and bronchioli begin to form terminal sacs (developing primitive
alveoli), in which cuboidal and squamous cells become associated with
vessels. Respiration becomes possible towards the end of this period
around the 25th foetal week.
3. The number of terminal sacs increases during the intial part of the
alveolar period (sometimes also considered a separate period of lung
development and called terminal sac period). The capillary network is
developing between the terminal sacs. The late alveolar period is
marked by the development of mature alveoli from the terminal sacs.
The period begins shortly before birth, but the first mature alveoli appear
only after birth. Alveolar sacs continue to be formed during early
childhood (up to year 8) and mature into alveoli. Alveolar maturation and
growth continue for another decade, but their numbers do not increase
further.
Suitable Slides
Sections of developing lung - H&E
Draw a small section of developing lung including terminal sacs and connective tissue
at high magnification.
URINARY SYSTEM
253
The kidneys, ureters, urinary bladder and urethra are the main components
of the urinary system. A function of the urinary system that immediately
comes to mind is the excretion of waste products from the body. This is
only one of many functions of the system. Others are
Kidney
Glomeruli and the tubular system are both part of the basic functional unit
of the kidney, the nephron.
Mesangial cells in the glomerulus form the connective tissue that gives
structural support to podocytes and vessels.
Kidney - H&E
Locate the cortex of the kidney and scan over the tissue at low
magnification. Note the presence of numerous glomeruli and the apparent
absence of any preferred orientation of the tubules visible between the
glomeruli (convoluted parts of proximal and distal tubuli). You should be
able to identify the vascular pole of a good glomerulus by the attachment
of the capillary tuft to the wall of the glomerulus. What would make your
glomerulus VERY good would be the presence of a tubulus which contains
a dense row of nuclei in the part of its wall closest to the vascular pole of
the glomerulus, the macula densa. The nuclei are located side by side or
may even overlap. It should be possible to find this structure in all slides. It
is also very likely that it may take you a few minutes of carefully scanning
the tissue at high magnification before you will find it. Proximal tubules
are characterised by their eosinophilic (pink) low, columnar cells and by
large amounts of fuzzy material, which may fill the entire lumen of the
tubulus. This fuzzy material represents the remains of the brush border of
the cells of the proximal tubules, which is difficult to preserve during the
preparation of the tissue.
Draw a glomerulus and label its components: the anatomical glomerulus,
255
The tubular system can be divided into proximal and distal tubules, which
in turn have convoluted and straight portions. Intermediate tubules connect
the proximal and distal tubules. Running from the cortex of the kidney
towards the medulla (descending), then turning and running back towards
the cortex (ascending), the tubules form the loop of Henle.
256
The thin segment of Henle's loop leads into the straight part of the
distal tubule, which is formed by low cuboidal cells without a
brush border. A few short microvilli are present, but they are
difficult to see in the light microscope. The diameter of the tubule
expands to ~35 µm. Epithelial cells in the ascending parts of the
intermediate and straight distal tubules cells transport chloride
258
(active) and sodium ions (passive) out of the tubular lumen into
the surrounding peritubular space. The epithelium can not be
penetrated by water. Consequently, the transport of ions over the
epithelium sets up a gradient in osmotic pressure, which serves as
driving force in the further concentration of the urine.
the lumen of the tubulus, diameter of the duct is small) and distal
tubule (cuboidal epithelium, cells stain weakly pink). Both
transversely or longitudinally cut tubules are suitable. Note that it
will be difficult to identify ALL tubules that are visible.
In most of our sections only little medulla is present - try to scan
along the margins of the tissue and see if you can find some
medulla. If that should not be possible take a look at medullary
rays instead, although they will contain few, if any, good thin
tubules.
Draw the tubules you could identify and label them. Also include
some tubules which could not be identified in your drawing,
which will give you a better impression of how the medulla of the
kidney looks than just three circles on a white background.
Excretory Passages
ENDOCRINE GLANDS
264
Endocrine (or internally secreting) glands are also named ductless glands, since they
lack excretory ducts. Instead, the secretory cells release their products, hormones, into
the extracellular space. From the extracellular space, the hormones may enter the
blood stream, by which they reach their target organs. Alternatively, the hormones may
affect nearby cells (paracrine acting hormones).
The major endocrine glands are the pituitary gland, the pineal body, the thyroid gland,
the parathyroid gland, the pancreas, the adrenal glands, the ovaries and the testes. In
some of these glands/organs the endocrine tissue constitutes only part of the
parenchyma of the organ (Which ones?). Small groups or individual endocrine cells are
also found in a variety of other organs, e.g. the GIT and the kidneys.
Pituitary Gland
The pituitary gland (or hypophysis) is attached to the inferior surface of the
brain by an extension of the nervous tissue of the tuber cinereum /eminentia
mediana of the hypothalamus, the infundibulum. The infundibulum and small
amounts of non-neural secretory tissue surrounding it form the hypophyseal
stalk. The pituitary gland is located in the sella turica, the hypophyseal fossa of
the sphenoid bone. The pituitary gland is surrounded by a thin connective
tissue capsule. The loose connective tissue between the capsule and the
periosteum of the sphenoid bone contains a dense plexus of thin-walled veins,
which surround the entire pituitary gland.
Any pituitary slide which contains both the adenohypophysis and neurohypophysis is
suitable to look at the general organization of the pituitary. Try to identify the portal
venules. Several of them can usually be seen in the pars tuberalis, where they descend
towards the pars distalis of the adenohypophysis.
Draw the pituitary at low magnification and identify its divisions (those visible in the
slide) and portal venules in your drawing.
Adenohypophysis
The pars distalis of the adenohypophysis accounts for about 75% of the hypophyseal
tissue. The glandular cells are arranged in irregular clumps or cords between a network
of capillaries with large and irregular lumina. Connective tissue, which supports the
glandular cells, is scant. Traditionally, glandular cells are subdivided into chromophobe
cells and acidophil and basophil (chromophil) cells. This division into three cell types
is based on their differential staining with H&E. Cocktails of other dyes, some of which
are mentioned below, also allow a differentiation between these cell types.
The contents of the secretory vesicles are responsible for the staining characteristics of
the chromophil cells.
Acidophils are rounded cells and typically smaller than basophil cells. Acidophils
account for roughly 65% of the cells in the adenohypophysis.
Based on their hormone products basophils are divided into three subtypes.
When stained with the PAS reaction all three types of basophils appear reddish.
Morphological criteria may be used, aside from the secretory products, to distinguish
between the cells, but differences are so subtle that it is hopeless to try to tell them
apart in the available preparations.
Chromophobe cells
The blood supply to the pituitary gland is extraordinary complex. At this stage it is
important to know that the primary capillary network in the neural part of the
hypophyseal stalk drains into 20 or more portal venules, which form a secondary
capillary network in the pars distalis of the pituitary gland.
The release of hormones from the adenohypophysis is under the control of hormones
which are produced by nerve cells in the hypothalamus.
factors into the extracellular space associated with the primary capillary plexus. They
are transported towards the adenohypophysis within the portal venules and reach their
target cells via the secondary capillary plexus.
Neurohypophysis
neurohypophysis consists of
Usually only the oval or round nuclei of the pituicytes are visible. Hypothalamic nerve
fibres typically terminate close to capillaries. Scattered, large, and bluish-violet (in
PAAB/PAS/Orange G stained sections) masses represent dilations of these nerve fibres.
The dilations are named Herring bodies. They are filled by small vesicles which contain
the neurosecretory products of the hypothalamic cells.
Pituitary, sheep -
PAAB/PAS/Orange G
Identify nuclei of pituicytes and
Herring bodies. Herring bodies are
not quite as well-defined structures
as e.g. Hassall bodies, but rather
areas of darker staining in the tissue.
You may also want to take a quick
look at neurones of the
hypothalamus, part of which should
be visible on the slides.
Staining in adenohypophysis of the
PAAB/PAS/Orange G section
corresponds largely to that in the
other PAS/ORANGE G section, but
the colours are somewhat subdued
because of the additional PAAB
stain.
Sketch the appearance of the tissue
of the neurohypophysis at high
magnification and label the
structures you can identify.
PINEAL BODY
The pineal body is a flattened, cone-shaped organ attached to the roof of the third
ventricle, where it occupies a depression between the superior colliculi.
The pineal body is surrounded by pia mater, which functions as its capsule and which
sends connective tissue septa into the pineal body, subdividing it into lobules.
In the pineal we find two cell types: pinealocytes (about 95% of the cells;
large, light and round nuclei) and astrocytes (glial cells; dark, elongated
nuclei).
Aside from the cells the pineal gland also contains ..... sand - well - brain sand (or
acervuli cerebri or - just for good measure - corpora arenacea). These are calcium-
containing concretions in the pineal parenchyma, which increase in size and number
with age. These concretions are radioopaque, and, since the pineal is located in the
midline of the brain, they provide a good midline-marker. They have no other known
function.
The most prominent secretory product of the pineal body is melatonin. The
cocktail of substances released by the pinealocytes can have several functions:
270
they may decrease secretory activity in most other endocrine glands (in part
indirectly, by way of influencing hypothalamic neurones), and they may "delay"
puberty through antigonadotrophic effects.
THYROID GLAND
The thyroid gland is situated on the lateral sides of the lower part of the larynx and
upper part of the trachea. The two lateral lobes of the thyroid are connected by a narrow
isthmus in front of the trachea. The size is quite variable but typically ranges around
20g (slightly larger in females than in males).
271
The thyroid gland consists almost entirely of rounded cysts, follicles, which are
separated by scant interfollicular connective tissue. The follicle is the structural and
functional building block of the thyroid gland. It consists of a simple cuboidal
epithelium (variable - depending on the functional state) which surrounds a lumen
filled with a viscous substance, colloid. The size of the follicles is variable ranging from
about 50 µm to about 1 mm.
The colloid is the secretory product of the follicular cell (extracellular storage!). Its
main component, thyroglobulin, consists of triiodothyronine and tetraiodothyronine
(or thyroxine).
C cells (or parafollicular cells) are part of the follicles. There are only few of them, and
they are typically situated basally in the epithelium, without direct contact with the
follicular lumen. They are always situated within the basement membrane, which
surrounds the entire follicle.
Arterial supply is abundant with a dense network of capillaries between the follicles.
Sympathetic fibres (from the superior, middle and inferior cervical ganglia) are mainly
vasomotor (there is some evidence that sympathetic input may have a stimulatory effect
on secretory activity).
The main secretory products of the thyroid gland are thyroxine and
triiodothyronine. TSH stimulates the endocytosis of thyroglobulin from the
follicular lumen and the subsequent release of its components into the blood stream.
TSH also stimulates their synthesis and release into the follicular lumen.
C cells produce the hormone calcitonin, which decreases blood calcium concentration
by inhibiting the resorption of bone (primarily by inhibiting osteoclast activity).
PARATHYROID GLANDS
The parathyroid glands are four small oval bodies located at the posterior surface of
the thyroid gland (close to the middle and inferior ends of the lateral thyroid lobes - but
a bit variable; the inferior pair may actually be located in the mediastinum).
These glands are small (average total weight is about 130 mg - that's 130
milligrams) but essential for life.
1. Chief cells are the most numerous type. They are rather small, a round, light and
centrally placed nucleus and a very weakly acidophilic cytoplasm. They
273
There are plenty of transitional cells, i.e. cells that morphologically represent
transitions between chief cells and oxyphilic cells.
Both the release of calcitonin by C cells in the thyroid gland and the release of
parathyroid hormone are regulated by negative feedback from blood calcium
concentrations.
ADRENAL GLANDS
The adrenal (or suprarenal) glands consist of an outer cortex (the main part of
the adrenal glands) and an inner medulla (which accounts for about 10% of the
adrenal glands). The gland is surrounded by a thick connective tissue capsule.
Vessels and nerves reach the medulla by way of connective tissue trabeculae
which extend from the capsule towards the medulla. Cortex and medulla are
two distinct endocrine organs(in lower vertebrates they may actually form two
entirely separate organs).
Cortex
The cortex is divided into three concentric zones which, from the surface
inwards, are termed the zona glomerulosa (accounting for about 15% of the
cortical thickness), the zona fasciculata (about 75%) and the zona
reticularis(about 10%). Transitions are usually gradual.
Both the zona fasciculata and zona reticularis depend on ACTH to sustain their
function and survival.
Hormones produced in the cortex are all steroids. Consequently, cortical cells contain
large amounts of smooth endoplasmatic reticulum and lipid droplets. Since the
hormones are synthesised in the cortex they are more precisely termed corticosteroids.
Corticosteroids are further subdivided into mineralocorticoids and glucocorticoids .
The most important mineralocorticoid is aldosterone, which regulates the resorption of
sodium and excretion of potassium in the tubules of the kidney. The most important
glucocorticoids is cortisol, which has a wide range of effects on most cells of the body.
Cortisol effects protein catabolism in almost all cells aside from liver
cells, gluconeogenesis, glycogen storage, mobilisation of fat from
adipocytes, anti-inflammatory effects, inhibition of allergic reactions) .
Medulla
The medulla is not sharply delimited from the cortex. Cells are arranged in strands or
small clusters. Capillaries and venules in the intervening spaces. The cytoplasm of the
cells is weakly basophilic. They are called chromaffin cells because the granules of
these cells can be stained with potassium bichromate. Chromaffin cells correspond to
the adrenaline- (80%) and noradrenaline-producing cells of the medulla. The two
groups cannot be distinguished using routine histology. Chromaffin cells are innervated
by preganglionic sympathetic fibres and correspond functionally to postganglionic
neurones. The correspondence is not only functional - chromaffin cells are, like
ganglion cells of the PNS, derived from neural crest cells.
The internal male genitalia consist of the testes with the adjoining epididymis, the vas
deferens and the accessory sex glands, namely the seminal vesicles, the prostrate and
278
the bulbourethral glands (the latter sometimes are included in the external genitalia).
Testes
The testes have, like the ovaries, two functions: they produce the male gametes or
spermatozoa, and they produce male sexual hormone, testosterone, which stimulates the
accessory male sexual organs and causes the development of the masculine extragenital
sex characteristics.
The testis is surrounded by a thick capsule, the tunica albuginea, from which a conical
mass of connective tissue, the mediastinum testis, projects into the testis. The tunica
albuginea is covered externally by a serosa.
From the mediastinum, delicate fibrous septa radiate towards the tunica albuginea and
divide the parenchyma of the testis into about 300 lobuli testis, which communicate
peripherally. Each lobule contains 1-4 convoluted seminiferous tubules (about 150-300 µm
in diameter, 30-80 cm long).
Interstitial tissue between the convoluted tubules is continuous with a layer of loose
vascular connective tissue, the tunica vasculosa testis, which is found beneath the tunica
albuginea.
Each seminiferous tubule continues near the mediastinum into a straight tubule, a
tubulus rectus. The straight tubules continue into the rete testis, a labyrinthine system of
cavities in the mediastinum.
Suitable Slides
-
279
These tubules are enclosed by a thick basal lamina and surrounded by 3-4 layers of
smooth muscle cells (or myoid cells). The insides of the tubules are lined with
seminiferous epithelium, which consists of two general types of cells: spermatogenic
cells and Sertoli cells.
Spermatogenic cells:
Spermatogonia
are the first cells of spermatogenesis. They originate in the 4th week of foetal
development in the endodermal walls of the yolk sac and migrate to the
primordium of the testis, where they differentiate into spermatogonia.
Spermatogonia remain dormant until puberty. They are always in contact with
the basal lamina of the tubule.
280
Primary spermatocytes
which lie in the cell layer luminal to the spermatogonia. They appear larger than
spermatogonia. They immediately enter the prophase of the first meiotic
division, which is extremely prolonged (about 22 days!). A large number of primary
spermatocytes is always visible in cross-sections through seminiferous tubules.
Cell divisions, from the formation of primary spermatocytes and onwards, to the
production of the spermatocytes, are incomplete. The cells remain connected by
bridges of cytoplasm. The completion of the first meiotic division results in the
formation of
Secondary spermatocytes,
which are smaller than primary spermatocytes. They rapidly enter and complete
the second meiotic division and are therefore seldom seen in histological
preparations. Their division results in the formation of
Spermatids,
which lie in the luminal part of the seminiferous epithelium. They are small
(about 10 µm in diameter) with an initially very light (often eccentric) nucleus. The
chromatin condenses during the maturation of the spermatids into spermatozoa,
and the nucleus becomes smaller and stains darker.
Spermatozoa
It takes about 48 days from the time cells enter meiosis until morphologically mature
spermatozoa are formed. Depending on the length of reproduction of spermatogonia
(which is not precisely determined) it takes approximately 64 days to complete
spermatogenesis.
Sertoli cells
are far less numerous than the spermatogenic cells and are evenly distributed
between them. Their shape is highly irregular - columnar is the best
approximation. Sertoli cells extend from the basement membrane to the luminal
surface of the seminiferous epithelium. Processes of the Sertoli cells extend in
between the spermatogenic cells (cell limits are therefore not clearly visible in
283
the LM). The nucleus of Sertoli cells is ovoid or angular, large and lightly
stained and often contains a large nucleolus. The long axis of the nucleus is
oriented perpendicular to wall of the tubule. A fold in the nuclear membrane is
characteristic for Sertoli cells but not always visible in the LM (well ... actually
... it's not that difficult to find, but not that easy either ....).
Lateral processes of Sertoli cells are interconnected by tight junctions, which are
likely to be the structural basis for the blood-testis barrier. Spermatogonia and
primary spermatocytes are located in the basal compartment, other cellular
stages of spermatogenesis are located in the adluminal compartment. Tight
junctions may temporarily open to permit the passage of spermatogenic cells
from the basal into the adluminal compartment. Sertoli cells provide mechanical
and nutritive support for the spermatogenic cells. Sertoli cells also secrete two
hormones - inhibin and activin - which provide positive and negative feedback
on FSH secretion from the pituitary.
Interstitial tissue
Leydig cells (15-20 µm), located in the interstitial tissue between the convoluted
seminiferous tubules, constitute the endocrine component of the testis. They synthesise
and secrete testosterone. Ledig cells occur in clusters , which are variable in size and
richly supplied by capillaries. The cytoplasm is strongly acidophilic and finely granular.
The nucleus is large, round and often located eccentric in the cell.
Suitable Slides
-
284
Spermatozoa pass via the tubuli recti (low columnar epithelium) and the rete testis
(flattened or cuboidal epithelium) into numerous ductuli efferentes, which are lined by a
columnar epithelium, which consists of both absorptive and ciliated cells. The height of
the two cells types which form the epithelium of the ductuli efferentes is variable which
gives the lumen a characteristic wavy outline.
The ductuli efferentes leave the testis and open into a common duct, the ductus
epididymidis (about 6 m long!). It is lined by a very tall pseudostratified columnar
epithelium. Most cells of the epithelium, also called principal cells, have long
stereocilia. Stereocilia are non-motile structures, which in the EM resemble large
microvilli. Towards the basal lamina we see a number of small nuclei, which belong to
the basal cells of the ductus epididymidis. These cells regenerate the epithelium.
move the spermatozoa towards the middle segment of the duct, which is the site of final
functional maturation of the spermatozoa - now they are motile. The terminal segment
of the ductus epididymidis is the site of storage of the mature spermatozoa. Smooth
muscle fibres of the terminal part of the ductus epididymidis do not contract
spontaneously. They contract during sexual stimulation concurrently with the
contraction of the musculature of the duct into which it opens, the vas deferens.
Suitable Slides
sections of the epididymis or testes sections which include the epididymis - H&E, van Gieson
Note that ductuli efferentes are located mainly in the head of the epididymis, whereas the ductus
epididymidis forms the body and tail of the epididymis. Sections of epididymis may therefore not
contain both types of ducts.
The mucosa of the vas deferens forms low longitudinal folds. It is lined by a
pseudostratified columnar epithelium. Similar to the epididymis, cells have long
stereocilia. The lamina propria is unusually rich in elastic fibres. The muscularis is well
developed (up to 1.5 mm thick) and consists of a thick circular layer of smooth muscle
between thinner inner and outer longitudinal layers. The muscularis is the structure
which makes the vas deferens palpable in the spermatic cord. The vas deferens is
surrounded by an adventitia, which is slightly denser than usual.
Suitable Slides
The accessory (or secondary) male sex glands consist of the seminal vesicles, the
prostrate and the bulbourethral glands.
Prostate
The prostate is the largest accessory sex gland in men (about 2 × 3 × 4 cm). It contains
30 - 50 tubuloalveolar glands, which empty into 15 - 25 independent excretory ducts.
These ducts open into the urethra. The glands are embedded into a fibromuscular
stroma, which mainly consists of smooth muscle separated by strands of connective
tissue rich in collagenous and elastic fibres. The muscle forms a dense mass around the
urethra and beneath the fairly thin capsule of the prostrate.
The secretory alveoli of the prostate are very irregularly shaped because of papillary
projections of the mucosa into the lumen of the gland. The epithelium is cuboidal or
columnar. Basal cells are again present, and the epithelium may look pseudostratified
where they are found. The secretory cells are slightly acidophilic and secretory granules
may be visible in the cytoplasm. Small extensions of the apical cytoplasm into the
lumen of the alveoli may represent cells which release their secretory products
(secretion is apocrine/merocine). The secretion of the prostate contains citric acid, the
289
The secretory ducts of the prostate are lined by a simple columnar epithelium, which
changes to a transitional epithelium near the openings of the ducts into the urethra.
Macroscopically the prostrate can be divided into lobes, but they are inconspicuous in
histological sections. In good histological sections it is possible to distinguish three
concentric zones, which surround the prostatic part of the urethra.
The peripheral zone contains large, so-called main glands, whose ducts run
posteriorly to open into the urethra.
The internal zone consists of the so-called submucosal glands, whereas
the innermost zone contains mucosal glands.
This subdivision of the prostate is of clinical importance. With age the prostate
becomes enlarged due to benign nodular hyperplasia. The onset age of these
hyperplastic changes is 45. About 3/4 of the males above 60 are affected of which half
will be symptomatic. This condition affects the mucosal glands. Cancer of the prostate,
which is the second most common malignant tumor in western males, involves the
peripheral zone.
Suitable Slides
-
290
Seminal Vesicles
The seminal vesicles develop from the vas deferens. Their histological organisation
resembles to some extent that of the vas deferens. They are elongated sacs (about 4 cm long
and 2 cm wide), which taper where they unite with the vas deferens. Each seminal vesicle
consists of one coiling tube (about 15cm long). All the lumina visible in sections of the
seminal vesicle are in continuity in the intact organ.
The mucosa shows thin, branched, anastomosing folds. The structure of the epithelium
is variable appearing columnar or pseudostratified columnar (columnar cells and basal
cells). The lamina propria of the mucosa is fairly thin and loose. The muscularis
consists of inner circular and outer longitudinal layers of smooth muscle.
Seminal vesicles were thought to store semen - hence there name. This turned out to be
wrong. They are glands, whose secretion constitutes 60-70 % of the ejaculate. The
secretory product of the columnar cell, which may be seen in the lumen of the seminal
vesicles, is strongly acidophilic. It contains large amounts of fructose which the
spermatozoa utilise as a source of energy. Furthermore, the secretion contains
prostaglandins, flavins (yellow fluorescing pigment - of use in forensic medicine to detect
semen stains) and several other proteins and enzymes.
The secretion of the seminal vesicles is the third fraction of the ejaculate (the
spermatozoa are released with the second fraction - the contents of the vas deferens).
Suitable Slides
This section focusses on the internal female reproductive organs: the ovaries, oviducts,
uterus and vagina. We will also have a look at the mammary gland, an accessory
reproductive gland. The external female genitalia include the labia minora and majora,
clitoris and vestibule.
The Ovaries
The ovaries have two functions - "production" and ovulation of oocytes and the
production and secretion of hormones. The ovary is attached to the broad ligament by a
short fold of peritoneum, called the mesovarium (or ligament of the ovary), through
which vessels and nerves pass to the ovary and enter it at the hilus of the ovary.
The surface of the ovary is covered by a single layer of cuboidal epithelium, also called
germinal epithelium. It is continuous with the peritoneal mesothelium. Fibrous
connective tissue forms a thin capsule, the tunica albuginea, immediately beneath the
epithelium.
Like so many other organs the ovary is divided into an outer cortex and an inner
medulla. The cortex consists of a very cellular connective tissue stroma in which the
ovarian follicles are embedded. The medulla is composed of loose connective tissue,
which contains blood vessels and nerves.
Ovarian Follicles
Ovarian follicles consist of one oocyte and surrounding follicular cells. Follicular
development can be divided into a number of stages.
Primordial follicles
are located in the cortex just beneath tunica albuginea. One layer of flattened
follicular cells surround the oocyte (about 30 µm in diameter). The nucleus of
the oocyte is positioned eccentric in the cell. It appears very light and contains a
prominent nucleolus.
Most organelles of the oocyte aggregate in the centre of the cell, where they
form the vitelline body (probably not visible in any of the available
295
preparations).
is the first morphological stage that marks the onset of follicular maturation
(Which hormone stimulates follicular maturation and where is this hormone
produced?). The previously flattened cell surrounding the oocyte now form a
cuboidal or columnar epithelium surrounding the oocyte. Their cytoplasm may
have a granular appearance, and they are for this reason also called granulosa
cells. The continued proliferation of these cells will result in the formation of a
stratified epithelium (with a distinct basement membrane) surrounding the
oocyte. The zona pellucida (glycoproteins between interdigitating processes of
oocyte and granulosa cells) becomes visible. Parenchymal cells of the ovary
surrounding the growing follicle become organised in concentric sheaths, the
theca folliculi.
Secondary follicle
Small fluid-filled spaces become visible between the granulosa cells as the
follicle reaches a diameter of about 400 µm. These spaces enlarge and fuse to
form the follicular antrum, which is the defining feature of the secondary
follicle. The oocyte is now located eccentric in the follicle in the cumulus
oophorus, where it is surrounded by granulosa cells. The theca folliculi
differentiates with the continued growth of the follicle into a theca interna and a
theca externa. Vascularization of the theca interna improves, and the spindle-
shaped or polyhedral cells in this layer start to produce oestrogens. The theca
externa retains the characteristics of a highly cellular connective tissue with
smooth muscle cells. The oocyte of the secondary follicle reaches a diameter of
about 125 µm. The follicle itself reaches a diameter of about 10-15 mm.
increases further in size (in particular in the last 12h before ovulation). The
Graafian follicle forms a small "bump" on the surface of the ovary, the stigma
(or macula pellucida). The stigma is characterised by a thinning of the capsule
and a progressive restriction of the blood flow to it. Prior to ovulation the
cumulus oophorus separates from the follicular wall. The oocyte is now floating
freely in the follicular antrum. It is still surrounded by granulosa cells which
form the corona radiata. The follicle finally ruptures at the stigma and the
oocyte is released from the ovary.
magnification. Identify the cuboidal epithelium covering the ovary and the underlying
tunica albuginea. Find a part of the cortex where you can observe primordial, primary
and secondary follicles.
Draw this section of the cortex with its follicles, the surrounding theca (if present),
connective tissue stroma, tunica albuginea and epithelium.
Atresia
Atresia is the name for the degenerative process by which oocytes (and follicles) perish
without having been expelled by ovulation. Only about 400 oocytes ovulate - about
99.9 % of the oocytes that where present at the time of puberty undergo atresia. Atresia
may effect oocytes at all stages of their "life" - both prenatally and postnatally. By the
sixth month of gestation about 7 million oocytes and oogonia are present in the ovaries.
By the time of birth this number is reduced to about 2 million. Of these only about
400.000 survive until puberty.
Atresia is also the mode of destruction of follicles whose maturation is initiated during
the cyclus (10-15) but which do not ovulate. Atresia is operating before puberty to
remove follicles which begin to mature during this period (none of which are ovulated).
Given that atresia affects follicles at various stages of their development it is obvious
that the process may take on quite a variety of histological appearances.
297
The corpus luteum is formed by both granulosa cells and thecal cells after ovulation has
occurred. The wall of the follicle collapses into a folded structure, which is
characteristic for the corpus luteum. Vascularization increases and a connective tissue
network is formed. Theca interna cells and granulosa cells triple in size and start
accumulating lutein (Which hormone stimulates this process? Where is this hormone
produced?) within a few hours after ovulation. They are now called granulosa lutein
cells and theca lutein cells and produce progesterone and oestrogens.
Hormone secretion in the corpus luteum ceases within 14 days after ovulation if the
oocyte is not fertilised. In this case, the corpus luteum degenerates into a corpus
albicans - whitish scar tissue within the ovaries.
Hormone secretion continues for 2-3 month after ovulation if fertilisation occurs.
The Oviduct
The oviduct functions as a conduit for the oocyte, from the ovaries to the uterus.
Histologically, the oviduct consists of a mucosa and a muscularis. The peritoneal
surface of the oviduct is lined by a serosa and subjacent connective tissue.
The mucosa
The muscularis
Oviduct is a nice descriptive term, but (sigh) not the only one commonly used for these
structures - you will also find the terms Fallopian tubes or uterine tubes. The term
salpinx (Greek, trumpet) seems to have passed its "use-by-date" in many histology text
but (sigh) not in pathology, where salpingitis refers to chronic or acute inflammation of
the oviduct. Let's see how "tubal inflammation" will fare in the future.
The Uterus
The uterus is divided into body (upper two-thirds) and cervix. The walls of the uterus
are composed of a mucosal layer, the endometrium, and a fibromuscular layer, the
myometrium. The peritoneal surface of the uterus is covered by a serosa.
Myometrium
The muscle fibres of the uterus form layers with preferred orientations of fibres
(actually 4), but this is very difficult to see in most preparations. The muscular
tissue hypertrophies during pregnancy, and GAP-junctions between cells
become more frequent.
Endometrium
The endometrium can be divided into two zones based on their involvement in the
changes during the menstrual cycle: the basalis and the functionalis.
Vagina
302
The vagina is a fibromuscular tube with a wall consisting of three layers: the mucosa,
muscularis and adventitia of the vagina
Mucosa
Muscularis
Inner circular and outer longitudinal layers of smooth muscle are present.
Inferiorly, the striated, voluntary bulbospongiosus muscle forms a sphincter
around the vagina.
Adventitia
The part of the adventitia bordering the muscularis is fairly dense and contains
many elastic fibres. Loose connective tissue with a prominent venous plexus
forms the outer part of the adventitia.
The mammary glands are modified glands of the skin. Their development resembles
that of sweat glands. They are compound branched alveolar glands, which consist of
15-25 lobes separated by dense interlobar connective tissue and fat. Each lobe contains
an individual gland. The excretory duct of each lobe, also called lactiferous duct, has its
own opening on the nipple.
The lactiferous duct has a two layered epithelium - basal cells are cuboidal whereas the
superficial cells are columnar. Beneath the nipple, the dilated lactiferous duct forms a
lactiferous sinus , which functions as a reservoir for the milk. Branches of the
lactiferous duct are lined with a simple cuboidal epithelium. The secretory units are
alveoli, which are lined by a cuboidal or columnar epithelium. A layer of myoepithelial
cells is always present between the epithelium and the basement membrane of the
branches of the lactiferous duct and the alveoli.
The above description corresponds basically to the appearance of the resting mammary
gland. Pregnancy induces a considerable growth of the epithelial parenchyma leading to
the formation of new terminal branches of ducts and of alveoli in the first half of
pregnancy. Growth is initiated by the elevated levels of oestrogen and progesterone
produced in the ovaries and placenta. Concurrently, a reduction in the amount of intra-
and interlobular connective tissue takes place. The continued growth of the mammary
glands during the second half of pregnancy is due to increases in the height of epithelial
cells and an expansion of the lumen of the alveoli. They contain a protein-rich (large
amounts of immunoglobulins) eosinophilic secretion - the colostrum or foremilk).
mammary gland. Do all parts of the secretory tissue look similar? Why/why not?
Draw and label part of the secretory tissue.
The Eye
The eye is formed by three layers, or tunics. Each of these three layers contributes with
parts that have structural / nutritive functions and parts that form the optic and
305
photoreceptive apparatus of the eye. From the outside to the inside of the eyeball the
three tunics are the
1. fibrous tunic, which forms a capsule enclosing and protecting the other
components of the eye. It is subdivided into the sclera, with primarily structural
functions, and the cornea, which is part of the optic apparatus.
2. vascular tunic, which forms the choroid, ciliary body and iris. This tunic is also
called the uveal tract. The choroid has primarily nutritive functions. The ciliary
body generates the aqueous humor of the eye, but the ciliary muscle also
functions in the optic apparatus. The iris is part of the optic apparatus in which it
functions a contractile diaphragm, i.e. the aperture of the eye.
3. neural tunic consists of the retina. The retina proper forms the photoreceptive
layer of the eye. As a double-layered epithelium, the retina also covers the
ciliary process and the posterior surface of the iris, where it has both nutritive
and structural functions.
The ciliary and iridial parts of the retina are described together with the ciliary
process and iris.
Cornea
The cornea forms the anterior surface of the eye in an area largely corresponding to the
pigmented iris, which is visible behind the cornea. The diameter of the cornea is ~11
mm; the thickness ranges from ~0.5 mm centrally to ~1mm along the margins of the
cornea. The cornea is formed by three cellular layers, which are separated from each
other by two thin, acellular layers. Blood vessels are not normally found in the cornea,
and the cells are not pigmented.
The anterior surface of the cornea is lined by a stratified squamous epithelium. The
basement membrane of this anterior corneal epithelium rests on the first acellular layer,
the anterior limiting lamina or Bowman's membrane. It separates the epithelium from
the corneal stroma and consists of densely packed collagen fibrils embedded in ground
substance.
The corneal stroma consists of 200 - 250 layers of regularly organized collagen fibrils
(mainly tropcollagen type I, but also types III, V and VI). Collagen fibres within each
layer will run parrallel to each other but at large angles to collagen fibres in the next
layer. Flattened fibrocytes, referred to as keratocytes, are located between the layers of
collagen fibres. The regular arrangement of the collagen fibres and their small diameter
(20 - 60 nm) acount for the transparency of the cornea.
306
The lateral margins of the cornea are continuous with the conjunctiva (anterior corneal
epithelium) and sclera (corneal stroma).
Sclera
The sclera is a tough layer of dense connective tissue consisting of collagenous fibres
and networks of elastic fibres. Melanocytes are present in deep parts of the sclera in
addition to the usual complement of connective tissue cells. Distended by the
intraocular pressure, the sclera maintains the shape of the eyeball. It is also the site of
attachment of the ocular muscles.
Anteriorly, the sclera forms a slight protrusion into the eyebal before it merges with the
cornea - the scleral spur, which provides a point of insertion for part of the ciliary
musle. The sclerocorneal junction houses the canal of Schlemm, through which the
aqueous humor is drained into ciliary veins.
Suitable Slides
sections of the eye - H&E, van Gieson
It is difficult to prepare good sections of the eye. The sclera is quite tough, while
the hyaline body is very soft and contains a high proportion of water.
Differential shrinkage and hardness typically give rise to a number of artefacts.
Detachment of the outer retina from the pigment epithelium and distortions of
the lens and cornea are the most common ones. The preparation of only parts of
the eye is one way to overcome at least some of the problems. You may have
access to sections of just 'retina' or 'anterior eye'.
307
Choroid
The coroid consists of loose connective tissue, which houses a dense network of blood
vessels. Connective tissue cells and melanocytes are numerous. The latter give the
choroid its dark colour. Small blood vessels are especially frequent in the innermost
part of the choroid, which is called the choriocapillary layer. This layer supplies the
retina with nutrients. Bruch's membrane is located between the choroid and the retina. It
consists of two layers of collagen fibres and a network of elastic fibres between them.
Ciliary body
The ciliary body is an inward extension of the choroidea at the level of the lens. Ciliary
processes are short extensions of the ciliary body towards the lens. A small amount of
loose connective tissue similar to that of the choroid is located between smooth muscle
cells which form the bulk of the ciliary body. They form three bundles, the ciliary
muscle.
The inner surface of the ciliary body and its processes are lined by two layers of
308
columnar cells which belong to the retina - the ciliary epithelium formed by the pars
ciliaris of the retina. The outer cell layer is pigmented, whereas the inner cell layer, i.e.
the layer that faces the posterior chamber of the eye, is nonpigmented.
The ciliary processes contain a dense network of capillaries. The cells of the inner layer
of the ciliary epithelium generate the aqueous humor of the eye. , i.e. they transport the
plasma filtrate generated by the capillaries in the ciliary processes into the posterior
chamber of the eye. Thight junctions between the cells form the blood - aqueous humor
barrier.
Fibers, which consist of fibrillin, extend from the ciliary processes towards the lens and
form the suspensory ligament of the lens. These fibres are also called zonule fibres.
Two of the bundles of the ciliary muscles attach to the sclera and strech the ciliary body
when they contract, thereby regulating the tension of the zonule fibres. The reduced
tension will result in a thickening of the lens which focusses the lens on close objects -
a process called accomodation.
Iris
The posterior surface of the iris is covered by the retina. The inner layer of the retina,
i.e. the layer facing the posterior chamber, is called the posterior epithelium of the iris.
Both layers of the retina are pigmented, but pigmentation is heavier in the inner layer.
In the region of the central opening of the iris, the pupil, the retina extends for a very
short distance onto the anterior surface of the iris. The iridial stroma consists of a
vascularized loose connective tissue rich in melanocytes in addition to macrophages
and fibrocytes, which are all surrounded by a loose meshwork of fine collagen fibers.
The anterior surface of the iris is not covered by an epithelium - instead of we find a
condensation of fibrocytes and melanocytes, the anterior border layer of the iris.
The iris forms the aperture of the eye. Myoepithelial cells in the outer (or anterior) layer
of the retina, i.e. the layer adjacent to the stroma of the iris, have radially oriented
muscular extensions. These extensions form a flat sheet immediately beneath the
anterior layer of the retina, the dilator pupillae muscle. Embedded in the central portion
of the iridial stroma are smooth muscle cells which form the annular sphincter pupillae
muscle. In humans, this muscle surrounds the pupil as a less than 1 mm wide and only
0.2 mm thick band. The two muscles regulate the size of the pupil.
Pupillary constriction, which is mediated by the sphincter pupillae muscle, is clinically
refered to as miosis - dilation, mediated by the dilator pupillae muscle, as mydriasis.
The pigmentation of cells in the stroma and anterior border layer of the iris determines
to color of the eyes. If cells are heavily pigmented the eyes appear brown. If
pigmentation is low the eyes appear blue. Intermediate levels create shades of green and
grey.
Suitable Slides
309
sections of the
Lens
The lens consists of a lens capsule, the subcapsular epithelium and lens fibres. It does
not contain blood vessels or nerves.
The lens capsule is generated by the cells of the subcapsular epithelium and
corresponds to a thick, elastic basal lamina. The zonule fibres insert into the lens
capsule.
Cells of the subcapsular epithelium (or anterior lens cells) are mitotically active. In
adult individuals they only cover the anterior "hemisphere" of the lens. As they divide,
310
cells gradually move towards the equator of the lens where they tranform into lens
fibres. The apical part of the gradually elongating cell extends between the subcapsular
epithelium and adjacent lens fibres towards the anterior pole of the lens. The basal part
extends towards the posterior pole. The nucleus remains close to the equatorial plane of
the lens.
The optical properties of the lens change from periphery to central parts because of
differences in the amounts of crystallins contained in lens fibres. These difference
correct for distortions of colours and shapes (called spherical and chromatic
aberrations) which commonly occur at the margins of glass lenses. These aberrations
are easy to observe when you look through a loupe - or even in not-so-good
microscopes at the margins of the field of view, where they are easy to detect when to
slide is moved
Suitable Slides
sections of eye - H&E, van Gieson
Similar to the retinal lining of the iris and ciliary body, the outer layer of the light
sensitive retina forms a single layer of cuboidal cells - the pigment epithelium. The
inner layer of the retina contains the photoreceptors, the first neurones which process
the sensory information, and the neurones which convey the pre-processed sensory
information to the central nervous system. Receptors, neurones, supporting cells and
their processes are segregated into nine layers:
1. The layer of rods and cones contains the outer, rod- or cone-shaped light
sensitive segements of the photoreceptive cells. The lights sensitive part and the
perikayon of the rods and cones are connected by a narrowed bridge of
cytoplasm. At the level of this connection the rods and cones are surrounded by
the processes of a specialised type of glial cells, Müller cells, which form the
2. outer limiting membrane.
3. The outer nuclear layer contains the nuclei and perikarya of the rods and cones.
Their processes form part of the
4. outer plexiform layer, where they form synapses with the processes of neurones
whose cell bodies are located in the
5. inner nuclear layer. The cells of the inner nuclear layer are concerned with the
312
initial processing of the sensory input. The three major neurone types are
horizontal, bipolar and amacrine cells. The inner nuclear layer also houses the
perikarya of the Müller cells.
6. The inner plexiform layer contains the processes of the inner nuclear layer
neurones which convey the sensory input to the
7. ganglion cell layer. Ganglion cells are not evenly distributed. There are few of
them towards the periphery of the retina. Close to the fovea, ganglion cells form
a densely packed layer. Both ganglion cells and the cell bodies located in the
inner nuclear layer which contact the rods and cones of the fovea are displaced
towards the margins of the fovea.
8. Layer of optic nerve fibres. The axons of the ganglion cells travel in this layer
towards the optic disc. Towards the optic disc, the thickness of this layer
increases as more and more axons are added to it.
9. The inner limiting membrane corresponds to a basal lamina formed by the
Müller cells.
Suitable Slides
The eyes of most mammals are suitable to look at the general organization of
the retina. However, the retina of some mammals does not contain cones, and,
as mentioned before, cell layers which would be pigmented in normal humans
will not be so if the eye has been collected from an albino (many laboratory
strains of small ammals are albinoid).
Optic Nerves
Because of the origin of the retinae and optic nerves from the developing forebrain, the
optic nerves (cranial nerves II) corresponds to fibre tracts connecting parts of the CNS -
in this case the ganglion cells of the retina with neurones in the lateral geniculate
nucleus of the thalamus and neurones in the superior colliculus and pretectum of the
midbrain.
Ganglions cell axons run towards the optic disc where they turn towards the sclera.
Numerous bundles (or fascicles) of axons pass through the choroid and openings in the
sclera, the lamina cribosa. The axons become myelinated in this region. Collectively,
the bundles form the optic nerve. Like other parts of the CNS, the optic nerve is
surrounded by the three meninges - the outer dura mater, the middle archnoid and the
inner pia mater, which are separated from each other by subdural and subarachnoid
spaces. At the eyeball, the dura fuses with the sclera while the arachnoid and pia mater
merge with the choroid. Connective tissue septa, which arise from the pia mater,
separate the fibre bundles in the optic nerve. The axons in the optic nerve are supported
by astrocytes and oligodendrocytes. Microglia is also present.
314
Suitable Slides
sections of the optic nerve - H&E, trichrome
Optic nerve, monkey, van Gieson
The organisation of the optivc nerve
corresponds at a first glance to that of
peripheral nerves. Differences relate to
the connective tissue surrounding the
nerve and the fascicles of nerve fibres.
In the optic nerve, the dura mater,
arachnoid and pia mater take the roles
of the epi-, peri- and endoneurium of
peripheral nerves. Narrow cleft-like
spaces are found between the
meninges, forming the subdural space
(between dura and arachnoid) and
subarachmoid space (between
arachnoid and pia).
These spaces may expand during tissue
preparation due to differential
shrinkage of the tissues and appear
much wider than they are in the living
organism. This applies also to the optic
nerve illustrated.
Draw the optic nerve at low
magnification and label its
components.
Eyelid
The posterior (facing the eyeball) and anterior (facing the world) surfaces of the eyelid
are also called conjunctival and cutaneous parts. The cutaneous part is covered by skin
and contains sweat glands, sebaceous glands and, along the margins of the lid, 3-4 rows
of hairs - the eyelashes. Modified apokrine sweat glands, the glands of Moll, empty into
the follicles of the eyelashes. The eyelashes lack arrector pili muscles.
The inner, conjunctival part of the lids is lined by conjunctiva. Beneath the conjunctiva,
large sebaceous glands are embedded in a plate of dense connective tisssue containing
many elastic fibres. The plate and the sebaceous glands within it are called the tarsal
plate the tarsal glands (also Meibomian glands). Extensive skeletal muscle bundles
between the tarsal plate and the skin belong to the orbicularis oculi muscle.
Conjunctiva
315
The margins of the cornea merge with the conjunctiva. The conjunctiva extends over
the the 'white of the eye', which corresponds to the anterior part of the sclera, folds back
and continues over the posterior part of the eyelid. At the opening formed by the
eyelids, the conjunctive merges with the skin which covers the anterior surface of the
eyelids.
The epithelium of the conjunctiva varies from stratified squamous (most of it) to
stratified columnar (at the reflection from the sclera to the eyelid). It contains goblet
cells. The conjunctival epithelium rest on the loose connective tissue of its lamina
propria.
Ear
316
R
e
g
i
o
n
s
o
f
t
h
e
e
a
r
.
Images
o Cochlea (overview)
o Cochlea (low magnification)
o Organ of Corti (high magnification)
o Spiral ganglion (high magnification)
o Saccule and ampulla (low magnification)
o Macula of saccule (high magnification)
o Crista of ampulla (high magnification)
317
The ear has three distinct regions -- outer ear, middle ear, and
inner ear.
The outer ear includes the pinna (the visible ear, consisting
mostly of skin and cartilage) and the ear canal. The latter
is lined by keratinized stratified squamous epithelium. This
lining differs from skin by the presence of specialized
ceruminous (ear-wax) glands.
The
inner
ear,
locat
ed
withi
n the
bony
labyr
inth,
cont
ains Image courtesy Alec Salt, Cochlear Fluids
sens
Laboratory, Washington University; used with
e
orga permission.
ns
serving both balance and hearing.
All of these several senses of the inner ear utilize the same
319
The oval
window
forms a
potential
opening
from the
middle ear Image courtesy Alec Salt, Cochlear Fluids
into the Laboratory, Washington University; used with
bony permission.
labyrinth.
o The
stapes of the middle ear plugs this opening; but . . .
o The stapes is flexibly attached and can vibrate to
transmit pressure waves to the fluid that fills the
bony labyrinth. (Sound is carried from the eardrum
across the middle ear by the three middle ear
ossicles, ending with the stapes at the oval window.)
HAIR CELLS
Hair cells, the specialized mechanoreceptor cells of the auditory
and vestibular systems, are found in several positions along the
chambers and passageways of the membranous labyrinth.
SEMICIRCULAR CANALS
COCHLEA
The cochlea houses an elaborate
configuration of membranous
labyrinth and hair cells, called the
organ of Corti, designed for
auditory reception.
The
scala
medi
a,
also
calle
d the
cochl
ear
duct,
lies
alon Image courtesy Alec Salt, Cochlear Fluids
g the
Laboratory, Washington University; used with
lengt
h of permission.
spiral
cochlea, in a "medial" position between the scala vestibuli
and scala tympani.
o The scala media contains endolymph.
o The organ of Corti lies within the scala media.
o The scala media is separated from the scala vestibuli
by the very thin Reissner's membrane.
o The scala media and the scala tympani are separated
by the basilar membrane.
ORGAN OF CORTI