Zoology 115 Animal Histology Laboratory Exercises

Exercise 9
THE CARDIOVASCULAR SYSTEM AND THE BLOOD
A merry heart doeth good like a medicine. Proverbs XVII:22
THE CARDIOVASCULAR SYSTEM
The cardiovascular system is concerned with the transport of blood and lymph through the
body. It can be anatomically subdivided into the heart and the blood vessels, and the latter category is
further subdivided into different vessel types.
Arteries are blood vessels which conduct blood away from the heart; veins are blood vessels
which conduct blood towards the heart. These two major categories are broken down further, and there
are subcategories of arteries and veins based on size, construction of the vessel wall, etc. However, the
direction of blood flow is the criterion which separates arteries from veins, and which defines a vessel
as being in one or the other major class. You may have heard the hoary old chestnut that "Arteries carry
oxygenated blood, and veins carry deoxygenated blood." THIS IS NOT TRUE. Some arteries carry
deoxygenated, and some veins carry oxygenated blood. The direction of flow is the only criterion for
classification.
OBJECTIVES: After completing the exercises on the cardiovascular system, you should be able to:
Identify cardiac muscle in longitudinal and cross section in light microscopic preparations.
Identify the Purkinje fibers of the heart.
Identify microscopic sections of different classes of arteries and of veins of various sizes.
Identify venous and cardiac valves in light microscopic sections.
General Structure of Blood Vessels
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.
The tunica intima
The tunica intima 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, which delimits the tunica intima from
The tunica media.
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
The tunica adventitia,
The tunica adventitia consist mainly of connective tissue fibres. The tunica adventitia blends with
the connective tissue surrounding the vessel. The definition of the outer limit of the tunica
adventitia is therefore somewhat arbitrary.

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Zoology 115 Animal Histology Laboratory Exercises

Locate and label specific structures mentioned in bold face.

1. The Heart, Entire, H&E
Examine an entire heart from some small animal (most likely a rat) which has been sectioned
from the apex to the cranial end of the atria. Start by holding it up to the light. After orienting yourself,
place it on the microscope under low power. You will easily be able to make out at least two, and
probably three of the chambers; if you have a favorable section, you may be able to see all four. The
atria and ventricles are separated from each other. Running from the apex to the cranial end you will
see the septum dividing the right and left sides. There should be at least one of the heart valves in
your section, connecting the atria and ventricles. External to the heart proper, you should have a cross
section of one of the great vessels (the pulmonary artery) and some adipose connective tissue as well.
The heart is a large mass of cardiac muscle. observe the pattern of the muscle cells of the
myocardium. Myocardial cells (cardiac myocytes) are much smaller than the myofibers of skeletal
muscle, and they don't form long cylindrical structures the way myofibers do. Rather than forming neat
bundles of parallel fibers with well-defined striations, myocardium forms an anastomosing network
with a sort of "spongy" appearance. The contacts between myocytes are such that a branching network
with blood vessels in the spaces between them is the result. While both atria and ventricles are
composed of the same type of specialized muscle cells, those of the atria tend to be less numerous,
thinner, and more elongated than those of the ventricles. The ventricles of the heart are more stoutly
constructed than the atria because they have more work to do. All the atria do is pass blood to the
ventricles below: the right ventricle sends blood to the lungs against the resistance of the pulmonary
circulation, and the the left ventricle has to deal with the entire system circulation's resistance to flow.
While the resistance of the lung capillaries to blood flow is considerable, that of the entire peripheral
circulation is much higher.
The most prominent feature of myocardium, and the one that's absolutely diagnostic for it, is the
intercalated disk. The ID is a specialized cell-to-cell adhesion/communications site. It demarcates the
beginning of one myocyte and the end of the next, and information is pass across it from cell to cell.
These structures are found only in cardiac muscle.
Examine the lining of the chambers. The chambers of the heart is lined with a simple squamous
epithelial covering termed endocardium.If you traverse the thickness of the myocardium and examine
the outer surface of the wall, you will see a similar (though less well defined) layer of simple squamous
epithelium termed epicardium, which translates as "upon the heart." Anatomically, this is the visceral
layer of the pericardial sac. The epicardium is the inner portion of the CT envelope that surrounds the
heart. It corresponds to the peritoneum of the abdominal cavity in origin (from the mesoderm lining the
embryonic coelom) and function (to allow the heart to move freely in its cavity without adhesion to
surrounding structures). Here you see it as a thin serous membrane overlying the outer surface of the
organ. A stain for connective tissue would reveal a very delicate sub-epicardial CT layer, mainly
reticular fibers.
Arteries
Arteries may be subclassified by type. Conducting or elastic arteries are large ones, with very
strong and relatively elastic walls, whose function is to "conduct" the bulk of the blood to regions of
the body where it's to be distributed. Examples include the aorta, subclavian, and pulmonary arteries.
Once the blood has reached the region of distributionsay, the limbsit will be handled by smaller
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Zoology 115 Animal Histology Laboratory Exercises

(but still fairly large) distributing or muscular arteries, which send it to sub-regions. As the distribution
area gets more and more limited the arteries become smaller. In very local areas you will see small
arterioles, essentially mini-arteries with a wall considerably less muscular than the larger ones
"upstream."
1. Elastic Arteries: Aorta, human H&E, elastin & vanGieson
The lumen is lined with a thin squamous epithelial layer, endothelium, (the tunica intima)
which may be likened to the rubber bore of the hose; like the rubber, it offers a smooth and unimpeded
passage for the flow of blood. The thin endothelial lining of the aorta corresponds to that of other
vessels. The flattened cells are easily damaged during preparation and it may be difficult to identify the
endothelium. The subendothelial layer of connective tissue is characterised by a lower density of cells,
i.e. fewer nuclei, a fibrous appearance of the tissue and the absence of well-defined elastic layers.
Because the lamellae of elastic fibers diffract light differently from the remaining tissues they should
also be visible in H&E stained sections.
The tunica media is a region of elastic and collagen fibers. Elastic arteries (the aorta most of
all) must withstand an enormous head of pressure to pump against the peripheral systemic resistance:
consequently the wall is heavily reinforced to prevent bursting, just as the wall of a fire hose has
reinforcing cords in it. The elastic fibers allow some stretching and "springiness" in response to the
pressure, and the collagen fibers limit the degree of stretch permitted. (In some disease or deficient
nutritional statesfor example lathyrism, a copper deficiency caused by certain plantsthe wall may
be weakened, resulting in an aneurysm which may leak, or burst with fatal effects. Elastic lamellae
become visible in the tunica media. The elastic laminae is revealed as red-staining or black-staining
material by the elastin stain. The majority of cells in the tunica media are smooth muscle 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.
The tunica adventitia of the aorta is the fabric covering on the outside of the vessel. The tunica
adventitia of conducting arteries is scanty, and collagenous in nature.
2. Muscular arteries: Artery - H&E and elastin & eosin
The proportion of elastic fibers decreases, and the proportion of smooth muscle increases. Some
elastic fibers and collagen fibers will always be present, but eventually the great bulk of the tunica
media will be smooth muscle, and at that point we're dealing with muscular or distributing arteries,
whose function is to "distribute" blood supply to their regions of responsibility, such as a limb.
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. The internal
elastic lamina is most easily seen in smaller muscular arteries. In these it usually stands out as a bright
pink undulating band just below the lining epithelial cells and their supporting CT (termed tunica
intima). 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 muscle cell nuclei are frequent in the tunica media. The external
elastic lamina stains similar to the internal elastic lamina, but it is thicker and appears fibrous instead
of forming a continuous band. Collagen fibres and a few connective tissue cell nuclei are visible in the
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Zoology 115 Animal Histology Laboratory Exercises

tunica adventitia.
NOTE: If you close the iris diaphragm of the microscope, the elastic layers will stand out very
clearly, but remember to open the diaphragm once you have seen them.
In addition to the inner and outer elastic laminae, elastin stains will show fine elastic fibres in
the tunica media and coarse elastic fibres between the collagen fibres of the tunica adventitia. The
appearance of other structures will depend on the stain used together with the elastin stain. Eosin, the E
in H&E, gives a pink colour to both collagen fibres and the cytoplasm of cells. Nuclei are not stained if
the H is omitted from the H&E.
3. Arterioles
These are arterial vessels with a diameter below 0.1 - 0.5 mm (different values in different
textbooks). Endothelial cells are smaller than in larger arteries, and the nucleus and surrounding
cytoplasm may 'bulge' slightly into the lumen of the arteriole. The endothelium still rests on a internal
elastic lamina, which may be incomplete and which is not always well-defined in histological sections.
The tunica media consists of 1-3 concentric layers of smooth muscle cells. It is difficult to identify an
external elastic lamina or to distinguish the tunica adventitia from the connective tissue surrounding the
vessel.
The smooth muscle of arterioles and, to some extent, the smooth muscle of small muscular
arteries regulate the blood flow to their target tissues. Arterioles receive both sympathetic and
parasympathetic innervation. The final branching of the arterioles finally gives rise to the capillary
network (microcirculation).
Veins
Veins are those vessels leading blood back towards the heart. As a rule, they have much thinner
walls than arteries do, though in cross sectional area they're usually larger than the corresponding
artery, because they have to carry the same volume of blood at a lower pressure. Since veins are on the
post-capillary side of the circulatory loop, operating at much lower pressures, there's less need for burst
resistance. Thin walls are also important because much of the pressure that drives blood through veins
is generated not by the heart, but by contraction of the muscles in the region of the vein. This
"squishes" the blood back through the vein. Because they have low pressures, some veins have venous
valves in them to prevent back flow. This is especially true of medium sized veins in the extremities, as
they have to lift blood against gravity.
1. Vein, human H&E
The tunica intima is very narrow and the internal elastic lamina is difficult to identify - even in
elastin stained sections. A few elastic fibres below the endothelium form only a very thin and
incomplete internal elastic lamina. Smooth muscle is present in the tunica media, but it is organised
less regular than in the artery. The tunica media is, again as compared to the artery, very thin and there
is no sharp border between the tunica media and the tunica adventitia. The tunica adventitia of the
largest veins contains coarse collagen fibres, elastic fibres and longitudinal bundles of smooth
muscle. Small and medium sized veins will not contain smooth muscle in the tunica adventitia.
2. Small to medium sized vein: Vein Valve - H&E
If a vein with valves is present in the section, it should be easy to identify. Valves are only
found in small to medium-sized veins. You should see one or two bands of tissue in the lumen of the
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Zoology 115 Animal Histology Laboratory Exercises

vein. Each band is formed by two apposing layers of tunica intima. The bands may share their origin
from the inner aspect of the wall of the vein or they may have separate origins. Folding of the tissue
bands forming the valves is variable.
Note: You can observe the function of vein valves. Put your finger firmly on one of the veins
which are visible on the back of your hand. Use another finger to stroke over the vein in the direction
of the wrist. The vein will empty and not backfill (closed valves!) until you release the first finger.
Capillaries
Capillaries are small vessels, whose walls are thin enough to allow the diffusion of nutrients,
oxygen and carbon dioxide across them. They are "where the action is" in gas and nutrient exchange:
with a few exceptions, no cell of the body is very far from one, because access to the blood is an
absolute requirement: cell death would result from anoxia or the loss of nutrient and waste transport.
1. Capillaries: Cardiac Muscle, , sheep - Whipf's polychrome
Large numbers of capillaries are present in almost all 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 in these planes. Only one or two red blood cells fit side by side in the capillary. A single
endothelial cell forms the wall around the entire circumference of a segment of the capillary.
Endothelial cell nuclei are therefore not always visible, and some red blood cells are only surrounded
by a fine line representing the capillary wall.
THE 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.
OBJECTIVES: After completing the laboratory exercises of the blood, you should be able to:
Identify all the cellular elements in normal blood smears and in tissue sections
Identify the platelets of mammals
1. Blood smear, Wright's stain
To study the cell types in your preparations first scan the smear with the low power objective
and select a region where the red cells do not overlie one another and where they are stained pink. After
finding an area that is well smeared and well stained, examine the blood cells, and in particular the
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Zoology 115 Animal Histology Laboratory Exercises

leukocytes, at highest magnification (Find and study under the oil-immersion lens the formed
elements). Identify neutrophils, lymphocytes, eosinophils, and monocytes; basophils also will be
present but they account for less than one percent of the total number of leukocytes. Platelets will also
be found.
RBC: RED BLOOD CELL (ERYTHROCYTE)
The most common formed element is the erythrocyte, or "red blood cell" (RBC). The RBC is
the mature stage of development of a cell line in which the nucleus (present in earlier forms) has been
lost. Erythrocytes take the shape of biconcave discs. They are essentially containers full of hemoglobin
without any nucleus or evidence of internal organelles.Due to the biconcave shape of the erythrocytes,
their center will look lighter than their periphery. One should note any abnormalities of the
erythrocytes. Megaloblastic anemia will result in large RBCs, while iron deficiency will result in
erythrocytes that are too small. A significant variation in shape of the erythrocytes should also be
noted; this might include spherocytic changes, stippling or irregularly shaped cells (including sickle
cells). When the area of central pallor occupies more than two-thirds of the cell, hypochromia is likely,
and abnormalities in hemoglobin production should be suspected (such as iron deficiency). You will
become acquainted with these and other less frequent abnormalities of erythrocytes when you study
hematology.
Question: Why is the shape of mammalian RBC biconcave? What is the significance?
WBC: WHITE BLOOD CELLS (WBCs, LEUKOCYTES):
neutrophils (polymorphonuclear leukocytes, PMNs or polys)
eosinophils
monocytes
lymphocytes
basophils (rare)
As you study the various kinds of white blood cells, compare them in size to the surrounding
red blood cells. Normal, undistorted, adult human red blood cells will be in the 7-8 m diameter range.
(In histological sections of paraffin embedded tissues the diameter of RBCs is in the range of 6-7 m
due to some shrinkage artifact.) The primary criteria for identifying the various kinds of white blood
cells are: 1) the size of the cell, 2) the size and shape of the nucleus and the distribution of chromatin
within it, and 3) the amount of cytoplasm and the number and staining reactions of the granules present
within it.
Question: Which type of white blood cell is the largest? Which is the smallest?
Neutrophil granulocytes are easy to find. They are the most frequent type of white blood cells,
and the complex shape of their nucleus identifies them unequivocally. In darkly stained smears it is
possible to see some faintly purple, very small granules in the cytoplasm. These granules represent the
primary, lysosom-like granules of neutrophils. Be sure to identify neutrophils with their multi-lobed
nuclei and perhaps also a band (stab) cell, which is an immature neutrophil with a horseshoeshaped nucleus.
Eosinophils are easily identifiable because of their obvious red cytoplasmic granules and bilobed nuclei. The two lobes of the nucleus of eosinophils are usually well-defined and of about equal
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Zoology 115 Animal Histology Laboratory Exercises

size. 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.
Basophils are so rare that you will probably not encounter one, but, if you do, it will have very
dark cytoplasmic granules, obscuring the nucleus. The 2-3 nuclear lobes of basophils are not as well
defined as those of eosinophils, granules are not as numerous as in eosinophils, and pretty much all of
them can be identified "as individuals" rather than the dense mass they form in eosinophils. The
specific granules of basophils are stained deeply bluish or reddish-violet. In some smears, their colour
corresponds closely to the colour of the nucleus which sometimes is difficult to distinguish amongst or
behind the granules.
Monocytes are the largest white cells often with bean-shaped nuclei and agranular
cytoplasm.Their cytoplasm stains usually somewhat stronger than that of granulocytes, but it does not
contain any structures which would be visible in the light microscope using most traditional stains.
Monocytes and lymphocytes definitely look much prettier in darker stained smears than in lighter ones
mainly because of a clearer distinction between cytoplasm and nucleus. The C-shaped nucleus of a
textbook monocyte may not be easy to find. The nuclei will vary from a peanut- to a "fat" S-shape in
smears. Whatever the shape of the nucleus, it is usually not lobed and not round. The nucleus is bound,
at least on its concave side, by a wide rim of non-granular cytoplasm. Note also the light area of
monocyte cytoplasm, which is often visible close to the concave surface of the nucleus.
Finally, lymphocytes are the smallest WBC, slightly larger than the RBCs, and tend to be round
with round nuclei that fill up almost the entire cell leaving scant cytoplasm. The cell is very likely to be
a lymphocyte if the nucleus is round and surrounded by a narrow rim of cytoplasm.These cells are very
variable in size. The smallest lymphocytes may be smaller than erythrocytes (down to ~ 5 m in
diameter) while the largest may reach the size of large granulocytes (up to 15 m 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 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 in most other cell types in the blood).
After you are satisfied with your ability to identify the various kinds of white blood cells,
compare the percentages of the leukocytes in your smear to the expected differential values that you
should have committed to memory. Abnormally high or low percentages of white blood cell types often
accompany certain disorders and may be diagnostic.
PLATELETS, (or thrombocytes)
Finally, the number of platelets per oil immersion field should be noted. These are the smaller
particles, also without a nucleus. These may be individual or clumped together. In lightly stained
smears, blood platelets will appear like light blue, fairly ill-defined specks between the other blood
cells. In darker smears, you will sometimes be able to see that the blue specks are formed by an
accumulation of small bluish grains, the granules of the blood platelets.
When successive high power fields contain fewer than four platelets, and they are not present
elsewhere on the slide in clumps, platelets are said to be decreased. This will be confirmed with platelet
counts.

ATES-CAMINO, Fritzie B. (AY 2011-2012), BSES-CSM, UPMindanao