A MECEGFCQ‘FEC FURVEY CF FHE. FEMOEAL AEYEEY AME YEN 63F FEE EGG Thesis Fax? fan Seam a? M. 5.. MECHEGAN STRTE BNWERSI'FY Dadrid M. Ediefit 3%9 THESIS A MICROSCOPIC SURVEY OF THE FEMORAL ARTERY AND VEIN OF THE DOG BY David M. Elliott A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Anatomy 1969 DEDICATED TO: LITTLE PAL ii ACKNOWLEDGMENTS The author wishes to express sincere thanks to Dr. A1 W. Stinson for his guidance and encouragement during the course of this study. His timely suggestions and advice, both technical and literary, have been of great value. Thanks is also due to Drs. M. Lois Calhoun and Rexford E. Carrow for serving on the guidance committee and giving constructive criticisms of this manuscript. The author also wishes to thank the Department of Physiology and the Department of Veterinary Surgery and Medicine for their fine cooPeration in supplying animals for this study. Special thanks is extended to all fellow graduate students and technicians of the Department of Anatomy for their many suggestions and technical advice and to Mrs. Judy Vargo for typing this manuscript. The author is deeply indebted to his family and friends, and especially his wife, Lois, for their constant encouragement, under- standing, and complete faith throughout the course of this study. iii TAB LE OF CONT ENTS Page INTRODUCTION. . . . . . . . . . . . . . . . . . . 1 LITERATURE REVIEW . . . . . . . . . . . . . . . . 3 Femoral Artery. . . . . . . . . . . . . . . . . . 3 Femoral Vein. . . . . . . . . . . . . . . . . . . 9 Innervation of Blood Vessels . . . . . . . . . . . . 12 MATERIALSANDMETHODS............. 15 Perfusion . . . . . . . . . . . . . . . . . . . . 18 Valves in Veins. . . . . . . . . . . . . . . . . . 19 Collateral Studies. . . . . . . . . . . . . . . . . 19 Optical and Photographic Equipment . . . . . . . . 19 OBSERVATIONS AND CONCLUSIONS. . . . . . . . . . 23 Femoral Artery . . . . . . . . . . . . . . . . . 23 Intima . . . . . . . . . . . . . . . . . . . . 2 3 Media . . . . . . . . . . . . . . . . . . . . 24 Adventitia . . . . . . . . . . . . . . . . . . 2 5 Va sa Va 3 orum . . . . . . . . . . . . . . . . 2 6 Nerves . . . . . . . . . . . . . . . . . . . . 2 6 Mea sur ement s . . . . . . . . . . . . . . . . 2 7 Femoral Vein . . . . . . . . . . . . . . . . . . 28 Intima and Media . . . . . . . . . . . . . . . 2 8 Adventitia . . . . . . . . . . . . . . . . . . 2 9 Va sa Va scrum . . . . . . . . . . . . . . . . 2 9 Nerves . . . . . . . . . . . . . . . . . . . . 30 Mea sur ement s . . . . . . . . . . . . . . . . 3 0 Valve 8 . . . . . . . . . . . . . . . . . . . . 31 SUMMARY.....................32 LITERATURECITED................34 APPENDICES....................75 iv Figure 10. 12. 13. 14. LIST OF FIGURES Aluminum holder for vessels . . . Longitudinal section of femoral artery demon- strating elastic fibers and circular folds of the internal elastic membrane . . Cross section of normal post-mortem femoral artery................ Cross section of femoral artery after perfusion athOmm. Mercury. . . . . . . . . . Splitting of internal elastic membrane of femoral artery................ Fenestration in the internal elastic membrane Closure of the fenestration seen in Figure 6 Intima and media of relaxed femoral artery . . Intima and media of perfused femoral artery . Longitudinal section of perfused femoral artery demonstrating elastic fibers . . . . . . . Longitudinal section of the femoral artery demon- strating reticular fibers . . . . . . . Cross section of femoral artery showing nutrient vessels Cross section of femoral vein showing nutrient vessels and elastic fibers . . . . . . . Longitudinal section of a femoral artery demon- strating the origin of a nutrient vessel . . . . V Page 21 39 41 41 43 45 45 47 47 49 49 51 51 53 Figure Page 15. Longitudinal section of a femoral artery showing nutrient vessel in the media . . . . . . . . . 53 16. Longitudinal section of femoral artery showing a nutrient vessel proceeding obliquely through themedia................. 55 17. Longitudinal section of femoral artery showing nutrient vessel as it approaches the adventitia . 55 18. Cross section of femoral vein showing elastic fibers . . . . . . . . . . . . . . . . . . 57 19. Cross section of femoral vein . . . . . . . . . 57 20. Cross section of femoral vein showing a nutrient vessel . . . . . . . . . . . . . . . . . . 59 21. Cross section of a femoral vein showing smooth muscle in the media . . . . . . . . . . . . 59 22. Femoral vein and valve . . . . . . . . . . . . 61 23. Origin of a valve in a femoral vein . . . . . . . 61 24. Level of section for Figure 22 . . . . . . . . . 63 25. Thickness of tunica media of femoral artery - upperthird................ 65 26. Thickness of tunica media of femoral artery - rniddlethird................ 66 27. Thickness of tunica media of femoral vein - lowerthird 67 28. Thickness of adventitia of femoral artery - upper third............ 68 29. Thickness of adventitia of femoral artery - middle third 69 30. Thickness of adventitia of femoral artery lower third 70 Figure Page 31. Thickness of the femoral vein - upper third . . . 71 32. Thickness of the femoral vein - middle third . . 72 33. Thickness of the femoral vein - lower third . . . 73 34. Distribution of valves in the femoral vein . . . . 74 LIS T OF APPENDICES Appendix 1. Measurements of Arterial Walls . 2. Measurements of Vein Walls 3. Animals Used and Measurements of the Walls of Perfused Arteries . 4. Animals Used and Measurements of the Walls ofPerfusedVeins . . . . . . . . . . 5. Animals Used and Number of Valves in the Veins viii Page 75 77 79 80 81 INTRODUCTION Blood vessels, arteries in particular, are a hard working group of organs which have specific characteristics that set them apart from any other organ system. They have unique susceptibilities to disease; vary greatly in structure, and age at different rates. As was pointed out by Hare (1929), impr0per functioning blood vessels jeopardize the efficiency of the heart. According to Lansing (1959), two out of three adult deaths are caused either directly or indirectly by cardiovascular disease. It is, therefore, reasonable to assume that a complete understanding of the normal structure of the arteries and veins is essential to our under- standing of altered structure and function. A great amount of work has been done on the histology of the vascular system of humans, but that of laboratory animals has been neglected. If it can be shown that the vessels of the dog are similar to those of the human, it would make cardiovascular research on this animal more meaningful in its translation to human disease. Some vascular diseases, such as varicose veins and thromboses, are limited exclusively to humans. It is, therefore, a reasonable assumption that lower animals may have some morphological charac- teristic which helps prevent these problems. With this in mind, a microsc0pic survey of the femoral artery and vein of the dog was con- ducted in an effort to demonstrate some of the morphological structures of these vessels. LITERATURE REVIEW FEMORAL ARTERY Very little work has been done on the histology of the blood vessels of domestic animals, therefore, unless otherwise stated, the following account concerns human material. The femoral artery is described by most authors as a medium sized muscular artery consisting of the usual three layers, tunica intima, tunica media, and tunica adventitia (Clark, 1958, Greep, 1966), although Trautmann and Febiger (1957) describe the femoral artery of domestic animals as an elastic artery. Three definite layers have been ascribed to the intima: endothelium, intermediate (subendothelial), and internal elastic membrane (C0penhaver, 1964). Henle, in 1841, was the first to describe the internal elastic membrane (Has 5, 1939), and also the first to de- scribe it as having a fenestrated appearance (Wright, 1963). As the size of the vessel increases in caliber, the number of fibers in the membrane also increases (Hass, 1939). It appears as a thick membrane with holes in it, but actually it consists of two or more layers of thick and thin fibers arranged in a network with overlapping openings, of which the larger fibers run parallel to the axis of the vessel (Huber, 1916, Gross, 1949, Hall, gt__a;_l_., 1955, Wright, 1963). The media of the muscular artery is the predominant layer. Its main constituent is 25 - 40 layers of concentrically arranged smooth muscle (C0penhaver, 1964, Arey, 1968). It has been shown that the media is not arranged in circular rings of smooth muscle, as it at first might appear, but it actually consists of obliquely arranged fusiform muscle cells, which do not separate into distinct rings but rather is a continuous compact spiral structure (Strong, 1938, Clark, 1958). The thickness of the muscle coat seems to be somewhat pr0portional to the size of the vessel, but there may be considerable difference in arteries of the same size (C0penhaver, 1964). Between the muscle layers are fibers of elastic, collagenous and reticular connective tissue, the amount varying with the caliber of the vessel. In the larger muscular arteries, the elastic tissue exists as fenestrated lamellae, while in some of the smaller muscular arteries, elastic fibers may be almost absent in the media. The collagenous fibers correspond to the reticular fibers seen surrounding individual muscle fibers (Bloom and Fawcett, 1968, Greep, 1966). The adventitia consists of thick collagenous and elastic fibers which course predominately in a longitudinal direction and sometimes appear as groups of smaller fibers in transverse sections (Wright, 1963, C0penhaver, 1964, Greep, 1966). The portion next to the media abounds with prominent elastic fibers and is known as the elastica externa. In this type of artery, the adventitia varies greatly in thickness. In some it is much thinner than the media, while in others it may be as thick or thicker than the media. The collagenous fibers of the adventitia extends into the surrounding connective tissue without a clear boundary (Maximow and Bloom, 1938). All elastic tissue has essentially the same structure. It consists of dense ill-defined elastic fibers intermingled with fibers of collagen. These fibers have a tendency to Split into fibrils which lie parallel to one another and in many cases cross (Gross, 1949). The content of elastic and collagen fibers of the femoral artery was found to be 34. 3% :1- 10 (Harkness §t_a_l. , 1955). The elastic fiber content was assessed as 1—5% in the media and 10-30% in the adven- titia and appeared constant at different ages for any one site (Wright, 1963). With age, profound changes occur in the elastic tissue of blood vessels. Primary among these changes is fragmentation of the fibers in which the coarser plexuses break up into fibrils. Another change is an increase in thickness of the intima and atr0phy of the media with the replacement of muscle and elastic tissue with collagen (Dock, 1950). The basic age change, however, seems to be calcifi— cation of the media which apparently occurs after the second decade of life (Blumenthal £1331. , 1950, Lansing, 1961). Correspondingly, there is an increasing resistance to stretch with age. This seems to have some bearing on atherosclerosis, because vessels not affected by this disease, show a significant increase in elasticity in later age (Roy, 1880, Roach and Burton, 1959). For a vascular disorder to occur, the structure of the vessel must in some way be impaired, such as calcification of the elastic tissue or the dissolution of the integrity of the intima or media (Duff, 1954). Differences in the prevalence of certain vascular diseases between man and dog have been noted. It was shown that arterioscle- rosis of the intramural coronaries is more frequent in the dog, while atherosclerosis is much less prevalent (Detweiler, 1963). It would appear that there could be some morphological reason for this, although it has been shown that with increasing age the arterial wall of the canine takes on a truly sclerotic appearance. This is less common in animals of limited life span and is almost nonexistent in wild animals (Dahme, 1962). Carnivores and birds show the greatest degree of atherosclerosis and come close to the type seen in humans, however, they do have distinctive morphological differences. In contrast to human arteriosclerosis, cholesterol deposition seems to play a subordinate role in this disease in animals (Davies and Reinert, 1965). Immediately after death, the structure of blood vessels is modified more greatly than perhaps any other organ in the body. The internal elastic membrane appears highly convoluted, and the lumen decreases markedly in size, sometimes varying as much as 25-100% from the living state (MacWilliam and McKie, 1908, Bunce, 1965). The thickened wall contains loosely arranged contracted smooth muscle and tightly convoluted elastic fibers. Early speculation was that this phenomenon was caused by mechanical stimulation, cooling, and exposure to air (MacWilliam, 1902). Later, it was thought to be caused by the intrinsic elastic qualities of the vessel (Strong, 1938), but it is now held that this change is caused by a contraction of the smooth muscle component of the vessel and also by a decrease in length (Finerty and Cowdry, 1960, Galloway, 1936). In young persons, this decrease in length may be as much as 40% (Hesse, 1926). It has been shown that by certain mechanical means the post- mortem vessel can be relaxed. MacWilliam (1902) accomplished this in several ways including freezing, sulphocyanide, ammonia vapors, heating at 38°C. and kneading or stretching. The waviness of the internal elastic membrane almost com- pletely disappears when exposed to an intralurninal pressure equal to the systolic blood pressure however, in some cases, the folds do not entirely disappear and contrary to expectation, the vessel wall was identical at diastolic pressure to that of systolic pressure (MacWilliam and McKie, 1908, Galloway, 1936, Bunce, 1965). With the increase in pressure, the wall of the vessel becomes thin and compact: the relaxed smooth muscle cells are elongated and the elastic tissue becomes straightened. Methods of experimentally distending vessels have been de- scribed as early as 1875 by Tait. The amount of change in thickness appears to be related to the size and structure of the vessel with the small muscular arteries showing a greater proportionate increase in thickness when collapsed than did the larger elastic type arteries. The width of the distended media of the arteries varied from 36-70% less than the collapsed media. An intima was not found in the dis- tended arteries. The endothelium was found pressed tightly against the internal elastic lamina. In the wall of the collapsed vessels, tissue of the media seemed to be forced through the fenestrations of the internal elastic membrane into the subendothelial layer and appeared to form an intima (Bunce, 1965). This same appearance was described in sections of vessels in a state of chemically induced vasoconstriction. The endothelial cells were quite irregular and bulged into the lumen, the smooth muscle cells next to the internal elastic lamina became deformed with the nuclei much shorter and the lamina itself was highly convoluted. The wall thickenss to lumen ratio in the convluted vessel was 1:5, while in the control (with intra- luminal pressure) the ratio was 1:30, and no convolutions in the internal elastic membrane were evident (Wagner, 1962, Hayes, 1967). Very sparse information on actual measurements of the thick- ness of blood vessels was found. Klingelh'offer and Meyer (1962) did a study on 30 humans in the third, fifth, and seventh decades of life. He found that the relationship between the inner surface and the vascular volume is more favorable for nutrition of the vascular wall of the arteries of the arm than it is for the femoral artery. The preportions of the femoral artery, a thicker wall and larger radius, indicate a greater mechanical stress to the wall. He found the vessel wall to be 510 microns thick in the normal artery of those in their third decade and 750 microns when contracted. In the seventh decade, this had increased to l, 080 microns and 1, 430 microns respectively. The tunica media of these same arteries during the third decade, was 472 microns at normal pressure and 699 microns when contracted, while in the seventh decade it measured 555 microns and 608 microns. It can be seen from these figures that the tunica media of the younger individual is the predominant layer and with ageing increases very little, yet the vessel wall more than doubles in thickness. These peculiarities of the femoral artery may be important in the vessel becoming sclerose later in life. In a study of the arteries of the bat wing, the total cross sectional area of the vessel at different portions of the arterial bed showed a linear relationship from artery to capillary. These values varied greatly from those on fixed material (Wiedmann, 1962). The vasa vasorum of arteries originates either from the parent artery or small neighboring vessels and is limited almost entirely to the adventitia (Arey, 1968, Bloom and Fawcett, 1968), however, in the femoral artery it is also seen in the outer third of the media. FEMORAL VEIN The femoral vein has been classified as both a medium sized (C0penhaver, 1964, Greep, 1966) and a large vein (Bloom and Fawcett, 10 1968, Arey, 1968). The three coats of the venous walls are very difficult to distinguish due to a lack of limiting membranes. Variations in the structure of veins do not appear to be related to the size of the vessel but to the local mechanical conditions. As a result the microscopic structure of veins of the same caliber, or even different sections of the same vein are quite different, if they exist under different stress conditions. Muscular tissue is usually scarce, but when it does occur, it may be in all three layers. In the inner and outer layers, it may occur longitudinally or circularly. Since hydrostatic pressure is greatest in the limbs, muscular tissue is more abundant in the femoral and saphenous veins, and for the same reason elastic tissue is also highly deve10ped (Pesonen, 1953). Franklin (1932, 1937) stated that the femoral vein possesses muscle and connective tissue in the wall, with longitudinal bundles in the adventitia, and Spirally arranged collagenous fibers. The veins of the extremities are usually well endowed with valves. Valves are formed from extensions of the intima, and in most cases are bicuspid, but occasionally a tricuspid valve is found (Powell and Lynn, 1951, Basmajian, 1952). The most common position for a valve is just distal to a tributary (Franklin, 1927, Arey, 1968). Valves at the entrance of a tributary are termed ostial valves, while those along the course of the vein are parietal valves (Rau, 1943). It has been shown that at the site of a valve the vein assumes 11 an elliptical shape with the major axis of this ellipse paralleling the surface of the skin. The cusps are parallel to the elastic force (Edwards, 1936). There seems to be an inverse proportion between the number of valves and the size of the vessel. They are very numerous in the deep veins and fewer in number in the femoral, p0pliteal and external iliac veins (Cockett, 1964). An examination of the external saphenous vein by‘Bleicher and Weber (1932) showed an average number of 9. 6 valves. This is quite a few more than the average of 3 found for the femoral (Basmajian, 1952). Basmajian found the following percentages: 0 valves - 1. 3%, lvalve - 7. 9%, 2 valves - 23.8%, 3 valves - 33%, 4 valves - 23. 8%, 5 valves - 3. 9%, 6 valves - 6.6%. In another study, Powell and Lynn (1951) found one valve in the common femoral vein of 72% of his subjects and 1-4 valves in the superficial femoral of 100% of the subjects. Using a comparative approach, Williams (1954) studied the valves in the limb veins of the dog, cat and rhesus macaque. He found the dog contained the most valves with an equal number in the fore and hind limbs. He felt that there should be more valves in the hind limbs of the monkey due to its erect posture but found an equal number here, also. This seems to support the views of Franklin (1937) and Kampmeier and Birch (1927) that the function of the valves is not to counter the affect of gravity. It seems likely that the lack of a compensatory mechanism in the lower limbs of erect animals would lead to an increase 12 in the disorders of their veins. There are two quite divergent views on the subject. It is felt by some that the absence or incompetence of valves in the lowe r limbs is an etiological factor of varicose veins (Edwards and Edwards, 1940, Egar and Wagner, 1949, Powell and Lynn, 1951). Basmajian (1952), on the other hand, felt that the presence or absence of valves proximal to the termination of the long saphenous vein is at most of theoretical importance. Van Cleave and Holman (1954) found a valve situated every 8. 81 centimeters in normal veins and every 16. 8 centimeters in varicose veins, thus indicating the number may affect the susceptibility of the vein to disease. In a later study by Mullarky (1964) two limbs with varicose veins were found to have normal valves above and below the sapheno-femoral junction as well as normal valves in the great saphenous vein. He found no evi- dence that the absence of valves in the femoral or iliac veins is a cause of varicose veins of the leg (Mullarky, 1964). The media of veins is better supplied with vasa vasorum than that of arteries and these vessels may extend as far as the intima. This fact has been attributed to the poor quality of blood flowing through the veins (Franklin, 1937, Arey, 1968). Venous vasa originate at the junction of the outer and middle thirds of the media, and terminal arteriovenous anastomoses also occur at this junction in the femoral vein (Clark, 1965). INNERVATION OF BLOOD VESSELS Dogiel (1898) says that His (1892), Kolliker (1863), Cajal and l3 Sola (1891), Retzius (1892) and Bietti (1895) observed nerve networks in blood vessel walls, especially the arteries. Muller and Glaser (1913) found a nerve plexus in the adventitia, and in 1914, Glaser found nerve fibers in the adventitia, media and intima. Woolard (1926) thought the intra-muscular plexus to be a true net and described a pericellular rather than an intracellular ending about the smooth muscle cells of the media. Lewis (1927) summed up the knowledge of the time of vasoconstrictor nerves, in saying that all the fibers branch upon reaching the vessel forming intricate plexuses in the adventitia with a few fine fibers running into the media. Ganglion cells were found in the adventitia of the aorta, renal artery, internal carotid and some arteries of organs, but they were not found in the vessels of the extremities (Muller and Glaser, 1913, Krogh, 1922, Truex, 1936). By using degeneration to isolate each of the nerve components, Hinsey (1929) observed afferent nerves in the adventitia of small arteries and veins, and he also observed branches of these fibers in the perivascular connective tissue and adipose tissue. Small skein- like structures and coarser nerve fibers, which formed brush like arrangements, were also found in the surrounding tissue (Truex, 1936). Truex also found that the end organs in the adventitia are coarser than those of the perivascular region. He observed no specialized nerve endings in the media but did find slender fibers ending freely in the connective tissue between the muscle cells. 14 Later, it was observed that non-medullated fibers extend into the adventitia, between the adventitia and media, and also a few finely beaded fibers in the media. The medullated fibers end in the fat of the perivascular tissue (Miller, 1948, Polley, 1955). Cheng and Kimura (1958) found thick undulated nerve fibers in the media of the dog's common carotid and abdominal aorta. Franklin (1937) found nerve fibers in close continuity with the sarc0plasm of muscle cells in the venous wall. He also observed Pacinian corpuscles in the vena cava, portal vein and certain of the cerebral veins. Terminal loops and encapsulated end organs were also observed in the adventitia of veins (Pereira, 1946). Thick undulating nerve fibers were found in the intima of the vena cava (Cheng and Kimura, 1958). MATERIALS AND METHODS The post-mortem study of the femoral artery and vein included 19 mongrel dogs, 10 males and 9 females, varying in weight from 6. 8 to 22 kilograms. All 19 were used for study of the artery, and 18 of these were used for study of the vein. The animals were obtained from the Department of Physiology and the Department of Surgery of the College of Veterinary Medicine. They were euthanatized by intra- venous or endocardial injection of either magnesium sulfate or sodium pentabarbitol. The age of the animals could not be determined in most cases, so only the weight and sex were recorded. Prior to the actual research, a short pilot study was conducted on several animals to determine the best surgical technique, the best fixative and the most beneficial stains. The results will be enumerated in the following account. Immediately after death, the femoral artery and vein on one side of the animal were removed. An incision was made on the medial surface of the hind limb, and the vessels were dissected free of the muscle and fascia. At this point, a U-shaped piece of aluminum (Figure l) was placed in the incision with the prongs extending between the artery and vein. The vessels were then clamped to the holder with hemostats, one being placed proximal to the inguinalligament, and 15 16 the other distal to the point where the p0p1iteal artery emerges. The use of the aluminum holder was necessary to eliminate longitudinal contraction of the vessels when they were cut free, since contraction markedly changed the thickness of the vessel wall. The choice of aluminum was made, because it is flexible enough to be fitted to the vessel length yet sturdy enough that its length will not be affected by the elasticity of the vessels. The vessels were then dissected free of the limb by cutting them on the outer edge of the aluminum holder. After removal from the body, the vessels, while still attached to the holder, were placed in Bouin's fixative where they remained from 8 to 24 hours. Bouin's fixative was chosen because it was a mild yet fast acting fixative and acted with a minimum of distortion to the tissues. After adequate fixation, a piece of both the artery and vein, 6-10 mm. in length, was removed from the upper third (near the deep femoral artery), the middle third, and the lower third (near the popliteal artery) of the vessels. These pieces were washed in several changes of 50% alcohol and stored overnight in 70% alcohol. After adequate washing, the tissue was cleared and dehydrated by the follow- ing method: two hours in tetrahydrafuran and water mixed in a 1:1 ratio, one and a half hours in each of two changes of pure tetrahydra- furan, and two hours in a 1:1 mixture of tetrahydrafuran and paraffin. The tissues were then infiltrated for 1-2 hours in a vacuum dessicator l7 and blocked in Paraplast’r‘. Sectioning of these blocks was done at 6 microns, and the sections were affixed to slides. One slide from each representative group (upper, middle and lower third) was stained with hematoxylin and eosin and one from each group was stained with Weigert's resorcin-fuchsin elastic stain. This proved to be a most useful stain in examining the elastic tissue and in differentiating the layers of the vessels. After the tissues were stained, the thickness of the tunica media and the tunica adventitia were measured by using a Bausch and Lomb microscope equipped with an occular micrometer. The criteria for the boundaries of the layers measured are as follows: The media was measured from the internal elastic membrane to the junction of the smooth muscle of the media and the elastic fibers of the adventitia. This point is quite clearly demonstrated with the resorcin-fuchsin stain. The measurement of the adventitia was from the muscle-elastic tissue junction to the point where the elastic fibers disappeared in the perivascular connective tissue. Since the adventitia blends so well with the surrounding fascia, this point was used as an arbitrary boundary. Four measurements of each of these layers was made, and an average figure attained. Since it is very difficult to accurately separate the coats of the vein wall (Pesonen, 1953), they were all in- cluded in one measurement, which was done in the same manner as those of the arteries, from the internal elastic membrane to the *Sherwood Medical Industries, St. Louis, Missouri. l8 disappearance of the elastic tissue in the fascia. When all the measurements had been taken, the figures were plotted on graphs, both linear and logarithmic, which compared the thicknesses to the weight of the animal. The method of the least squares was then used to determine the best straight line for the figures. PERFUSION According to Bunce (1965), blood vessels are altered more by post-mortem changes than any other organ in the body. They collapse, their walls thicken and the area of the lumen is reduced. After the study was completed on the structure of the post- mortem vessels of these 19 animals, 5 animals, 1 male and 4 females, were studied to determine any structural changes which might occur with an intraluminal pressure of 100 mm. mercury. To do this, a cannula was inserted in the common iliac artery for introduction of the fixative and another in the common iliac vein for drainage of blood and excess fixative. Physiological saline was then injected into the system to clean out the excess blood and 10% formalin was injected into the vessels at 100-105 mm. mercury. The vessels remained in the animal, uninterrupted, for four to five hours after injection and were then dissected free in the same manner as described in the pre- vious section. They were then placed in 10% formalin for at least 48 hours and processed in the same manner as the Bouin's fixed tissue with the alcohol washings being eliminated. These vessels were 19 measured in exactly the same manner as the non-perfused vessels, and they too were plotted on graphs and compared to the others. VALVES IN VEINS In order to determine the number of valves in the femoral vein, this vessel was dissected out of 15 limbs by fastening them to the aluminum holders and fixing them in 10% formalin for at least 48 hours. At the end of this time, the veins were placed under a dissect- ing microsc0pe and split lengthwise. This process adequately exposed the valves for enumeration. COLLATERAL STUDIES In conjunction with the aforementioned work, a number of closely related studies were also conducted. Several representative slides were stained with Bielchowsky's stain to demonstrate the reticular network of the vessel wall. For the demonstration of nerve fibers and nerve endings, the Davenport rapid silver method was employed (Conn and Darrow, 1946). Observations were also made on the number of layers of elastic tissue in the tunica media and the tunica adventitia and on the vasa vasorum. OPTICAL AND PHOTOGRAPHIC EQUIPMENT Observations for this study were made on a Bausch and Lomb binocular microsc0pe and on an American Optical binocular microscope. 20 The photomicrographs were made with a Carl Zeiss Photomicroscope>l< which was equipped with an automatic exposure setting device. The photographic film used was Adox KB14**. The film was enlarged in printing to the magnifications noted on the figures. ’1‘ Carl Zeiss, Oberkochen, Wuerttemberg, Germany. 95* Adox Fotowerke, Frankfurt, Germany. 21 FIGURE 1 Aluminum holder used for maintaining normal length of femoral artery (A) and vein (V). FIGURE 1 OBSERVATIONS AND CONCLUSIONS FEMORAL ARTERY INTIMA The intima of the femoral artery of the dog varies in appearance with changes in the intraluminal pressure. The intima of the vessel with no intraluminal pressure consists of an endothelium, two or three layers of connective tissue and is limited by a very prominent internal elastic membrane. The intima of the perfused vessel, on the other hand, consists of an endothelium and an internal elastic membrane, with only occasional connective tissue fibers between them. The nuclei in the endothelium of the relaxed vessel are quite prominent and rounded, while those of the perfused vessels are in close apposition to the internal elastic membrane and are elongated. The internal elastic membrane of the relaxed vessel is very convoluted, with the folds extending in both circular and longitudinal directions (Figure 2, 3). This configuration is caused by post-mortem contraction of smooth muscle in the vessel in both longitudinal and circumferential directions (Finerty and Cowdry, 1960, Galloway, 1936). These folds become less prominent in the perfused vessel, and in some cases completely dis- appear (Figure 4). At first glance, the internal elastic membrane appears to be one 23 24 thick elastic fiber, but upon closer examination, it can be seen that it is actually composed of several layers of finer fibers. This may be evidenced at certain points along the membrane where the layers are separating (Figure 5). Located in the internal elastic membrane are numerous fenestrations which are created when Openings in adjacent layers of elastic tissue coincide (Figure 6, 7). Only limited amounts of connective tissue appear between the endothelium and internal elastic membrane of the perfused vessel. An explanation for this might be that as the vessel relaxes, some of the tissue from the media, con- nective tissue and smooth muscle, migrates through the fenestrations into the intima, however, when the vessel is distended by an intra- luminal pressure, this material seems to be forced back into the media (Figure 8, 9). These observations indicate that the subendothelial layer described in many textbooks may be a product of tissue preparation rather than a naturally occurring morphological feature. MEDIA The media of the canine femoral artery is very well delineated with the internal boundary being the internal elastic membrane and the external boundary the junction between smooth muscle of the media and large elastic fibers of the adventitia (Figure 3). As in the intima, the structure of the media also varies slightly between relaxed and perfused vessels. The main constituent of the media is 18-40 layers of smooth muscle. In the relaxed vessel, the nuclei of the muscle 25 fibers are of the cork-screw shape, indicating contraction, while in the perfused artery, the nuclei are quite elongated (Figure 8, 9). Between the muscle fibers is a network of elastic, collagenous and reticular connective tissue. Viewed in cross section, the elastic tissue seems to be arranged in three to five distinct layers of fine fibers (Figure 4), but when the vessel is cut longitudinally, it can be seen that the elastic tissue is actually an interwoven net, running in all directions (Figure 10). Since the media contains this network of elastic fibers, the femoral artery of the dog appears to be of the transitional type rather than the muscular classification it is usually given. There is more collagenous connective tissue in the media than elastic, but like the elastic tissue, this does not occur in great amounts either. It is found coursing randomly between the muscle fibers. Also found in the media, is a network of reticular fibers around the muscle fibers. These fibers are not oriented in any one direction and appear to enclose the muscle fibers (Figure 11). ADVENTITIA The adventitia of the artery consists of collagenous connective tissue with a large number of thick elastic fibers coursing predominently in a longitudinal direction (Figure 3,10). These fibers are much thicker than those found in the media, and resemble the internal elastic membrane in appearance. Although they are very Sparse, reticular 26 fibers are also found in the adventitia. These fibers course randomly between the elastic and collagenous fibers (Figure 11). The collagenous fibers of the adventitia, which are quite extensive, lie between the large elastic fibers and extend into the connective tissue of the peri- vascular region. The collagenous fibers of the two layers blend very well and make the external boundary of the adventitia quite hard to distinguish (Figure 3). VASA VASORUM The vasa vasorum of the artery extends in a predominately longitudinal direction and is found almost exclusively in the adventitia (Figure 4, 12). In only one case a longitudinal nutrient vessel was observed in the media. Cases were observed, however, where nutrient arterioles originated from the parent vessel and ran obliquely through the media and ended in the outer adventitia (Figure 14, 15, 16, 17). The internal elastic membrane of the nutrient vessel is an extension of the external elastic membrane of the parent vessel and is incorporated in the nutrient arteriole over its entire length. Near the junction of the connective tissue of the adventitia and perivascular tissue, the vessel connects with a capillary network. It is necessary to inspect longitudinal sections to observe this type of arrangement, and it was seen in two of the Specimens examined. NERVES Several nerve fibers were observed coursing through both the adventitia and the media of the femoral artery. At intervals along 27 these fibers several small fibers branched from the main fiber. A few fibers appeared to extend as far as the intima but the majority ended in the media. The majority of these terminals were simply free end- ings but a few net-like endings were observed. MEASUREMENTS Measurement of the walls of the femoral arteries showed a decrease in thickness distal to the inguinal ligament. In the media there was a 13% decrease in the average thickness between the upper third and the middle third of the artery and a 19% drop between the upper third and the lower third. The adventitia decreased 14% in average thickness between the upper and middle thirds of the artery and 32% between the upper and lower thirds (Appendix 1). When plotted against body weight, the thickness of the layers at the three levels along the artery increased logarithmically as the weight increased (Figure 25-30). The perfused vessels showed a definite decrease in wall thick- ness. This decrease differed, however, between the media and the adventitia. The thickness of the media decreased 42% in the upper one third, 37% in the middle one third, and 49% in the lower third when compared to the thickness of comparably sized animals (Appendix 1, 3). When plotted against the body weight, the thickness of the media of the perfused vessels increased logarithmically as the weight increased and the calculated straight line ran approximately parallel to that of the 28 relaxed vessel but was diSplaced 600 to 1000 microns lower on the graph (Figure 25, 26, 27). A different arrangement was found in the adventitia of the artery. The adventitia of the animals observed showed a decrease in average thickness, but as the animal size increased, the decrease in thickness was much less pronounced. The average thickness for the group of animals observed showed a decrease of 37% in the upper third of the perfused vessel from that of the relaxed vessel, 36% in the middle third, and 34% in the adventitia of the lower third (Appendix 1, 3). The calculated straight line for the adventitia of the perfused animals is not parallel to that of the relaxed vessels, but intersects the latter at the 12-18 kg. point on the abscissa (Figure 28, 29, 30). FEMORAL VEIN INTIMA AND MEDIA The femoral vein of the dog closely resembles the generally accepted description of the medium sized vein (Figure 18, 19). It is very difficult to distinguish between the three layers of the wall, eSpecially between the intima and media. There is no internal elastic membrane, so the collagenous connective tissue of the intima and media blend together. IntersPersed between the collagenous fibers are 3-8 layers of smooth muscle and a few longitudinally directed elastic fibers (Figure 20, 21). Few reticular fibers were observed. 29 ADVENTITIA The adventitia, which constitutes the main portion of the vein wall, is composed of collagenous connective tissue with a network of large elastic fibers within it. These elastic fibers resemble quite closely those seen in the artery and are disposed in much the same manner (Figure 18, 20). The external boundary of the adventitia of the vein, like that of the artery is quite indistinct and for the same reason. The collagen fibers of the adventitia blend with those of the perivascular region preventing a clear line of demaracation (Figure 17). VASA VASORUM The vasa vasorum of the vein was located entirely in the adventitia and was more extensive than that seen in the artery (Figure 13, 20). It was also evident that there were more small vessels supplying the perivascular connective tissue of the vein than there were to the same region of the artery (Figure 19). Increasing the intraluminal pressure of the vein did not have nearly as much effect on its appearance as it did on the artery. One difference, however, was seen in the collagenous tissue of the adven- titia. In the relaxed vessel, this tissue was fairly compact in nature, but in the perfused vein it appeared to be much looser. In order to accommodate the increase in diameter, the fibers seemed to pull away from each other as the vessel was distended (Figure 13 vs 20). 3O NERVES The main network of nerves of the vein wall was observed in the adventitia but a few fibers extended into the media. These net- works appeared quite similar to those found in the artery but not as extensive. MEASUREMENTS It was felt that the thickness of the venous wall would decrease as the measurements proceeded distally from the inguinal ligament, for as one proceeds in this direction, the venous pressure increases, being greatest at the most distal point. This, however, was not the case. Measurement of the venous wall showed that, like the artery, it decreased in thickness as measurements proceeded distally from the inguinal ligament. The average thickness decreased 8% from the upper third to the middle third and 20% from the upper third to the lower third (Appendix 2). As in the arteries, the veins increased in thickness logarithmically as the body weight increased (Figure 31, 32 33). Perfusion caused a change in the thickness of the femoral vein wall comparable to the change noted for the adventitia of the femoral artery (Appendix 4). The average wall thickness was less for these animals than for the comparably sized group with relaxed veins, but the straight lines calculated for the two groups were not parallel but, as in the arterial adventitia, intersected. In this case the point of intersection was at the 9-11 kg. point on the abscissa. 31 VALVES The most common sites for valves in the femoral vein were the entrances of tributaries, the most prominent being the entrance of the deep femoral. The valves were all of the bicuspid type (Figure 22, 23) and in addition to the valves, an occasional small reservoir, or invagination, was found in the wall. Of the 15 femoral veins examined for valves, all had at lea st one, and the following distribution occurred: one (6. 7%) had one valve, four (26. 7%) had two valves, eight (53. 3%) _ had three valves, and two (13. 3%) had four valves (Appendix 5 and Figure 34). The average for this group was 2. 8, which is quite similar to the average of 3 found in the femoral vein of humans (Basmajian, 1952). SUMMARY A study was made of the microscopic structure of the femoral artery and vein of the dog in an attempt to elucidate the similarities and differences between their morphology and that of similar vessels in man. It was h0ped that by doing this study, vascular research on the dog may be more meaningful in its interpretation to human disease. The vessels were examined in both a normal post-mortem state and after perfusion with formalin at a pressure of 100 mm mercury. One morphological difference observed in the femoral artery of the dog was 4-6 layers of elastic tissue in the media. This feature indi- cates that the artery should be classified as a musculo-elastic type rather than the muscular class it is usually given. This type of classi- fication has been suggested by Dankmeijer and Haefsmit (1967) to distinguish the transitional arteries from those which are strictly muscular or elastic. Measurements were made on the thickness of the arterial and venous walls at various points. It was found that as the body weight of the animals increased, the thickness of the vessel walls increased logarithmically. After perfusion of the vessels, there were marked changes in the architecture of the vessels such as near elimination of the convo- lutions of the internal elastic membrane of the artery, a decrease in 32 33 sub-endothelial connective tissue, elongation of the nuclei of the endothelium and the muscle of both the artery and vein, .and an overall decrease in thickness of the vessel walls. This decrease varied in magnitude, however, between the arterial media, arterial adventitia and venous wall. The vasa vasorum of both the artery and vein were limited primarily to the adventitia with a few nutrient vessels extending into the media. Small nerve fibers and small nerve endings were observed in both the adventitia and media of the arteries and veins. After exami- nation of 15 veins, an average of 2. 8 valves were found with the most common position being distal to the entrance of a tributary. This number is in close agreement with that found for man. Other than the few layers of elastic tissue found in the media of the femoral artery, no distinct morphological differences were observed between these two vessels in the dog and the same two in man. LITERATURE CITED LITERATURE CITED Arey, L. B. 1968. Human Histology. 3rd ed. pp. 128-135. W. B. Saunders Co. , Philadelphia. Basmajian, J. V. 1952. The distribution of valves in the femoral, external iliac and common iliac veins, and their relation to varicose veins. Surg. Gyn and Obst. 95:537-542. Bleicher, M. and P. Weber. 1932. Number and distribution of valves in superficial veins of adults. Ann. d'anat. path. 9:1045-1047. Bloom, William and D. W. Fawcett. 1968. A Textbook of Histology. 9th ed. pp. 365-377. W. B. Saunders Co., Philadelphia. Blumenthal, H. T., A. I. Lansing, and S. H. Gray. 1950. The interrelation of elastic tissue and calcium in the genesis of arteriosclerosis. Am. J. Path. 26:989-1009. Bunce, D. F. M. 1965. Structural differences between distended and collapsed arteries. Angiology 16:53-56. Cheng, Y. M. and C. Kimura. 1958. A histological study on the afferent innervation of the large blood vessels. Acta Neurovegetativa 17:8-17. Clark, J. A. 1965. The vasa vasorum of normal human lower limb arteries. Acta Anat. 61:481-487. Clark, W. E. LeGros. 1958. The Tissues of the Body. 4th ed. pp. 187-189. Oxford University Press, London. Cockett, F. B. 1964. The significance of absence of valves in the deep veins. J. Cardiov. Surg. 5:722-727. Conn, H. J. and Mary A. Darrow. 1946. Staining Procedures used by the Biological Stain Commission. IC2-22. Biotech Publication, Geneva, N.Y. C0penhaver, W. M. 1964. Bailey's Textbook of Histology, 15th ed. pp. 258-269. The Williams and Wilkens Co. , Baltimore. 34 35 Dahme, E. 1962. Morphological changes in the vessel wall in Spontaneous animal arteriosclerosis. J. Atherosclerosis Res. 2:153-160. Dankmeijer, J. and E. C. M. Haefsmit. 1967. Some observations on the structure of the arteries. C. R. Ass. Anat. 138:381-386. Davies, R. F. and H. Reinert. 1965. Arteriosclerosis in the dog. J. Atherosclerosis Res. 5:181-188. Detweiler, D. K. 1963. Cardiovascular disease in the dog. A study in comparative cardiology. Proc. XVII World Vet. Congr. 1:47-55. Dock, W. 1950. The causes of arteriosclerosis. Bull. N.Y. Acad. Med. 26:182-188. Dogiel, A. S. 1898. Die sensiblen Nervenendigungen im Herzen und in den Blutgeféissen der Sfiugethiere. Arch. f. mikrosh. Anat. 52:44-70. Duff, G. L. 1954. Functional anatomy of the blood vessel wall, adaptive changes. Symposium on Atherosclerosis. pp. 33-41. National Academy of Sciences - National Research Council, Washington, D. C. Edwards, E. A. 1936. Orientation of valves in relation to body surfaces. Anat. Rec. 64:369-385. Edwards, J. E. and E. A. Edwards. 1940. The saphenous valves in varicose veins. Am. Heart J. 19:338-351. Egar, S. A. and J. B. Wagner, Jr. 1949. Etiology of varicose veins. Postgrad. Med. 6:234-238. Finerty, J. C. and E. V. Cowdry. 1960. A Textbook of Histology. 5th ed. p. 240. Lea and Febiger., Philadelphia. Franklin, K. J. 1927. Valves in veins, an historical survey. Proc. Roy. Soc. Med. 21:1-33. Franklin, K. J. 1932. Further notes on the arrangement of collagen fibers of veins together with certain other observations on veins and venous return. J. Anat. 66:602-609. Franklin, K. J. 1937. A Monograph on Veins. pp. 35-47. Charles C. Thomas, Springfield, Ill. 36 Galloway, R. J. M. 1936. The change in appearance of a muscular artery between diastolic and systolic blood pressures. Am. J. Path. 12:333-339. Glaser, W. 1914. Uber die Nervenverzweigungen innerhalb der Gefasswand. Deutsche Z. Nervenheilk. 50:305-310. Greep, Roy O. 1966. Histology. 2nd ed. pp. 273-292. McGraw- Hill Book Co. , New York. Gross, J. 1949. The structure of elastic tissue as studied with the electron microscope. J. Exp. Med. 89:699-708. Hall, D. A., R. Reed, and R. E. Tunbridge. 1955. Electron microscopic studies of elastic tissue. Exp. Cell. Res. 7:35-48. Hare, H. A. 1929. Blood vessels are deserving of more study. M. J. and Rec. 129:623-625. Harkness, M. L., R. D. Harkness and D. A. McDonald. 1955. The collagen and elastic content of the arterial wall. J. Physiol. 127:33P, 34P. Hass, G. M. 1939. Elastic tissue. Arch. Path. 27:334-365, 583-613. Hayes, J. R. 1967. Histological changes in constricted arteries and arterioles. J. Anat. 101:343. Hesse, J. 1926. Uber die pathologischen Veranderungen der Arterien der oberen Extremitat. Virchow's Arch. Path. Anat. 261: 225-252. Hinsey, J. C. 1929. Observations on the innervation of blood vessels in Skeletal muscle. J. Comp. Neur. 47:23-60. Huber, G. C. 1916. A note on the elastica interna of arteries. Anat. Rec. 11:169-175. Kampmeier, O. F. and C. L. F. Birch. 1927. The origin and development of venous valves with particular reference to the spahenous district. Am. J. Anat. 28:451. Klingelhtiffer, D. and W. W. Meyer. 1962. Comparative micrometric studies on the dilated and contracted muscular arteries of the upper and lower extremity. Virchow's Arch. Path. Anat. 335:529-543. 37 Krogh, A. 1922. The Anatomy and Physiology of Capillaries. p. 111. Hafner Publishing Co. , N. Y. Lansing, A. I. 1959. The Arterial Wall. p. VII. Williams and Wilkens Co. . Baltimore. Lansing, A. I. 1961. Atherosclerosis and the nature of the arterial wall. Circulation 24:1283-1285. Lewis, T. 1927. The Blood Vessels of the Human Skin and Their Responses. Shaw and Sons, Ltd. , London. MacWilliam, J. A. 1902. On the pr0perties of the venous and arterial walls. Proc. Roy. Soc. London 70:109-153. MacWilliam, J. A. and A. H. McKie. 1908. Arteries, normal and pathological. Brit. Med. J. 2:1477-1482. Maximow, Alexander A. and William Bloom. 1938. A Textbook of Histology, 3rd ed. p. 237. W. B. Saunders Co., Philadelphia. Miller, J. W. 1948. Observations on the innervation of blood vessels. J. Anat. 82:68-80. Mullarky, R. F. 1964. Valves of the iliac and femoral veins. Northwest Med. 63:230-231. Muller, L. R. and W. Glaser. 1913. Uber die innervation der Gefasse. Deutsche Z. Nervenhielk. 46:325-365. Pesonen, Nulo. 1953. The microscopic structure of veins functionally considered. Ann. Acad. Scient. Fennicae A. V. 38:3-10. Pereira, A.de Sousa. 1946. The innervation of veins. Surgery 5:731- 742. Polley, E. H. 1955. The innervation of blood vessels in striated muscle and skin. J. Comp. Neur. 103:253-267. Powell, T. and R. B. Lynn. 1951. The valves of the external iliac, femoral and upper third of the popliteal veins. Surg. Gyn. Obst. 92:453-455. Rau, R. K. 1943. Valves of the human body. Antiseptic 40:615—629. Roach, M. A. and A. C. Burton. 1959. The effect of age on the elasticity of humaniliac arteries. Canad. J. Biochem. and Physiol. 37:557-570. 38 Roy, C. S. 1880-1882. Elastic properties of the arterial wall. J. Physiol. 3:125-159. Strong, K. C. 1938. Structure of the media of the distributing arteries, by micro-dissection. Anat. Rec. 72:151-168. Tait, L. 1875-1876. Observations on the umbilical cord. Proc. Roy. Soc. London 24:417. Trautmann, A. and J. Fiebiger. 1957. Fundamentals of the Histology of Domestic Animals. Translated and revised by R. E. Habel and E. L. Biberstein. pp. 109-113. Comstock Publishing Associates, Ithaca, N.Y. Truex, R. C. 1936. Sensory nerve terminations associated with peripheral blood vessels. Proc. Soc. Exp. Biol. and Med. 34:699-700. Van Cleave, C. D. and R. L. Holman. 1954. Apreliminary study on the number and distribution of valves in normal and varicose veins. Am Surgeon 20:533-537. Wagner, B. M. 1962. Histologic observations of the arterial wall during vasoconstriction. Bull. N. Y. Acad. Med. 38:66-68. Wiedman, Mary P. 1962. Lengths and diameters of peripheral arterial vessels in the living animal. Circulation Res. 10:686-678. Williams, A. F. 1954. A comparative study of the venous valves in the limbs. Surg. Gyn. Obst. 99:676-678. Woolard, H. H. 1926. The innervation of blood vessels. Heart 13:319-336. Wright, I. 1963. The microsc0pica1 appearances of human peripheral arteries during growth and aging. J. Clin. Path. 16:499-522. 39 FIGURE 2 Longitudinal section of the femoral artery. Note the numerous circular folds of the internal elastic membrane (IEM). X 33 Weigert's resorcin-fuchsin. FIGURE 2 41 FIGURE 3 Cross section of femoral artery demonstrating the boundaries of the media with the adventitia (MB) and the adventitia with the perivascular connective tissue (AB). Note, also, the distribution of the dark staining elastic tissue and the highly convoluted internal elastic membrane (IEM). X 170 Weigert's resorcin-fuchsin. FIGURE 4 Cross section of femoral artery perfused at 100 mm. Hg demon- strating the network of elastic tissue (ET) in the media and adventitia and an internal elastic membrane (IEM) which is much less convoluted than the similar structure of the relaxed vessel (Figure 3). Note the nutrient vessel (NV) in the adventitia. X 210 Weigert's resorcin-fuchsin. FIGURE 3 o.g. 43 FIGURE 5 Separation of the internal elastic membrane (IEM) of the femoral artery. ET elastic tissue X 850 Weigert's resorcin-fuchsin. FIGURE 5 45 FIGURE 6 Internal elastic membrane of femoral artery. Note the fenestration. X 850 Weigert's resorcin-fuchsin. FIGURE 7 Same section as Figure 6. Due to adjustment of fine focus the fenestration has closed (F). X 850 Weigert's resorcin-fuchsin. FIGURE 6 FIGURE 7 47 FIGURE 8 Intima and media of relaxed femoral artery. Note the large amount of connective tissue and the round nuclei of the intima (I), and the corkscrew shaped nuclei (N) of the smooth muscle in the media. Note, also, the highly convoluted internal elastic membrane (IEM). X 530 Hematoxylin and Eosin. FIGURE 9 Intima and media of artery perfused at 100 mm. Hg. Note very thin intima (I), few convolutions of the internal elastic membrane (IEM) and the elongated nuclei of the smooth muscle in the media (N). X 530 Hematoxylin and Eosin. FIGURE 8 FIGURE 9 49 FIGURE 10 Longitudinal section of a perfused femoral artery. Note the pre- dominatly longitudinal direction of the dark elastic fibers (EF) and the flat internal elastic membrane (IEM). X 210 Weigert‘s resorcin-fuchsin. FIGURE 11 Longitudinal section of relaxed femoral artery. Note the abundance of the dark staining reticular fibers in the media (M) compared to the few fibers in the adventitia (A). X 210 Bielschowsky's reticular stain. FIGURE 10 ' ' c ‘,~ ‘ “: r. t 4 flat: (‘I’ . . ' {:45 13:3,. - “’0’ o ‘ . i , —.. ‘ . FIGURE 11 ' ‘~:-’\"".I‘/l’ )';; in], ' "' (‘1‘ ’Vl’Il " '1“":‘.(¥; 'I’ I“ (1‘ 51 FIGURE 12 Cross section of femoral artery. Note nutrient vessel (NV) in the adventitia and convoluted internal elastic membrane (IEM). X 210 Weigert's resorcin-fuchsin. FIGURE 13 Cross section of femoral vein. Note the nutrient vessels (NV) in the adventitia and the distribution of the dark elastic fibers (EF). X 330 Weigert's resorcin-fuchsin. FIGURE 12 FIGURE 13 53 FIGURE 14, 15 Longitudinal section of femoral artery. Note the origin of a nutrient arteriole from the intima of the parent vessel (NA). X 33 Weigert‘s resorcin-fuchsin. F IG URE 14 ..r% .".. s .... J... .. s a E . .I -r: IL. .9“. _._.oo._...9. IWGW .. ,., .5417. . FIGURE 15 55 F IGURE 16 , 17 Continuation of longitudinal series of Figure 14, 15 demonstrating a nutrient arteriole (NA) as it courses obliquely through the media (M) and approaches the adventitia (A). Figure 16 X 43 Weigert's resorcin-fuchsin. Figure 17 X 53 Weigert's resorcin-fuchsin. F IG URE 16 +1: v'h «.9 w .I ,. . 2.5,... 3.... v: ,' FIGURE 17 57 FIGURE 18 Cross section of femoral vein. Note the extent of media (M) and adventitia (A), and the distribution of the dark elastic fibers (EF). X 170 Weigert‘s resorcin-fuchsin. FIGURE 19 Cross section of the femoral vein. Note the abundance of peri- vascular vessels (PV). X 33 Hematoxylin and Eosin. 7 PM“ '.-.— FIGURE 18 FIGURE 19 59 FIGURE 20 Cross section of femoral vein. Note the extent of media (M) and adventitia (A), and the nutrient vessel (NV) in the adventitia. (EF) - elastic fibers. X 170 Weigert's resorcin-fuchsin. FIGURE 21 Cross section of perfused femoral vein. Note the smooth muscle of the media (SM) and elongated nuclei of the endothelium (N). (M) - media. (A) - adventitia. X 530 Hematoxylin and Eosin. FIGURE 20 FIGURE 21 61 FIGURE 22 Valve in femoral vein (V). See Figure 24 for level of section. X 33 Weigert's resorcin-fuchsin. F IGURE 2 3 Valve in femoral vein (V). Note origin from intima (O). X 170 Hematoxylin and Eosin. FIGURE 22 63 FIGURE 24 Level of section of Figure 22. FIGURE 24 MICRONS VFWW’” 65 FIGURE 25 THICKNESS OF TUNICA MEDIA OF FEMORAL ARTERY UPPER THIRD a “Tub? (fl ’ ’ I ’ F E: 15 KILOGRAMS 20 25 R E 1.3-. X E D o ..... PERFUSED MICRONS 66 . FIGURE 26 THICKNESS OF TUNICA MEDIA OF FEMORAL ARTERY MIDDLE THIRD L L t 7 C 5 4 1 2 ' - WM ”Li/I" ,. . . __ -_-_ __ _____ _ L 7 L 5' 3 1 up 5 10 15 20 25' KILOGRAMS . —.———RELAXED O-- --PERFUSED MICRONS 67 FIGURE 27 THICKNESS OF TUNICA' MEDIA OF FEMORAL ARTERY LOWER THIRD ._ 8 7 o ‘ ‘r 4 3 L - ‘M M O W ' o ’ ,. 1 ix- ’ 9 i I C 4’L 7 - - * ’ I ‘ o 5 . Lfi L L 109 5 10 15 20 2 5 KILOGRAMS . RELAXED o-----PERFUSED MIC RONS 68 FIGURE 28 THICKNESS OF ADVENTITIA OF FEMORAL ARTERY UPPER THIRD O L 7 I I’ 9 g L of” . // a / W O l . . O / / / O L : /1 - e 4 7 / L I 1‘ l O A 3 L up 5 10 15 20 25 KILOGRAMS - ——-—- RELAXED @- - - - PERFUSED MICRONS and. 69 FIGURE 2 9 THICKNESS OF ADVENTITIA OF FEMORAL ARTERY MIDDLE THIRD b HrVrr N \\ .10 15 KILOGRAMS 20 25 - —- RELAXED .- - -- PERFUSED MICRONS 70 FIGURE 30 THICKNESS OF ADVENTITIA OF FEMORAL ARTERY LOWER THIRD 5 10 15 20 25 KILOGRAMS - —- RELAXED o — --- PERFUSED ‘ MICRONS 71 FIGURE 31 THICKNESS OF THE FEMORAL VEIN UPPER THIRD 10 15 20 25 KILOGRAMS --——- RE LAXED o—- -— — PERFUSED MICRONS 72 FIGURE 32 THICKNESS OF THE FEMORAL VEIN MIDDLE THIRD 10 15 20 25' KILOGRAMS RELAXED @- - - - PERFUSED MICRONS 73 FIGURE 33 THICKNESS OF THE FEMORAL VEIN LOWER THIRD 10 15 20 25 KILOGRAMS RELAXED o ----- PERFUSED NUMBER or VESSELS 10 74 FIGURE 34 DISTRIBUTION OF VALVES IN THE FEMORAL VEIN 1 2 3 4 5 NUMBER OF VALVES APPENDICES 75 APPENDIX 1 MEASUREMENTS OF ARTERIAL WALLS Animal Weight (kg.) Stags Media 1E) Adventitia (i1) 2 *upper 13 Female 2665 2155 *middle 13 Female 2050 1640 *lower 13 Female 1847 820 2-1*upper 18 Female 2768 1435 >Fmiddle 18 Female 2050 1129 *lower 18 Female 2155 1026 3 upper 6. 8 Male 2461 1231 middle 6. 8 Male 2050 1078 lower 6. 8 Male 1796 820 4 upper 11 Female 1640 1435 middle 11 F emale 15 3 9 112 9 lower 11 Female 1435 923 5 upper 9 Female 1949 1385 middle 9 Female 1949 1231 lower 9 Female 1847 1332 6 upper 8. 5 Female 1847 1640 middle 8. 5 Female 1640 1332 lower 8. 5 Female 1847 1332 7 upper 22 Male 2665 2155 middle 22 Male 2258 2050 lower 22 Male 2155 1435 8 upper 10 Female 1847 1332 middle 10 Female 1745 1435 lower 10 Female 1640 1332 9 middle 11. 5 Male 1949 1332 lower 11. 5 Male 1590 820 10 upper 9. 5 Male 1640 1435 middle 9. 5 Male 1332 1180 lower 9. 5 Male 1435 924 11 *upper 11. 5 Male 2258 1692 *middle 11. 5 Male 2050 1500 *lower 11. 5 Male 1798 1435 1 lower 20 Male 2155 1231 1 middle 20 Male 2360 1540 12 upper 15 Female 2990 2165 middle 15 Female 2265 1545 lower 15 Female 1959 1236 13 upper 21 Male 2680 1750 middle 21 Male 2265 1545 lower 21 Male 1853 1133 76 Animal Weight (kg.) §2§ Media (1.1) Adventitia ()1) 14 upper 17 Male 2165 1750 middle 17 Male 216 5 1649 lower 17 Male 19 5 9 1441 15 upper 8 Male 1649 1340 middle 8 Male 1545 1030 lower 8 Male 1400 876 16 upper 8 Male 19 5 9 1441 middle 8 Male 1441 1236 lower 8 Male 1441 825 17 upper 7. 9 Female 2060 1340 middle 7. 9 Female 1649 1030 lower 7. 9 Female 1441 978 18 upper 8. 6 Female 2125 1855 middle 8. 6 Female 1910 1700 lower 8. 6 Female 1959 1133 *Fixed with formalin. AVERAGE WEIGHT 12. 44 kg. AVERAGE THICKNESS (11) Media Adventitia Upper 2198 1619. 88 Middle 1906 1385 Lower 1774 1108. 15 2 Animal *upper *middle *lower 2-1*upper 10 11 12 13 *middle *lower upper middle lower upper middle lower upper middle lower upper middle lower upper middle lower upper middle lower upper middle lower upper middle lower *upper *middle *lower upper middle lower upper middle lower 77 APPENDIX 2 MEASUREMENTS OF VEIN WALLS Weight (kg. ) 13 13 13 18 18 18 6. 6. 6. 11 ll 11 mU'lU'lmmmmU'lU'l 0000 U1 Sex Female Female Female Female Female Female Male Male Male Female Female Female Female Female Female Female Female Female Male Male Male Female Female Female Male Male Male Male Male Male Male Male Male Female Female Female Male Male Male Thickne s s (n) 2968. 5 2505 1641.5 1156 1621.5 1500 2660 2127 1487. 5 1959 1500 1212 1800 1700 1700 2364.5 2264.5 2055 3400 3390 3080 3300 2770 2258 2155 2461 1640 2155 1745 1640 3280 2538 2000 3090 2680 1855 2575 2895 2060 14 15 16 17 18 Animal upper middle lower upper middle lower upper middle lower upper middle lower upper middle lower 78 Weight (kg.) 17 17 17 CD O\O‘O\\O\O\O *Fixed with formalin. AVERAGE WEIGHT 12. 02 kg. AVERAGE THICKNESS (11) Upper 2506. 85 Middle 2403. 36 Lower 1920. 86 Sex Male Male Male Male Male Male Male Male Male Female F emale Female Female Female Female Thickne S s (u) 2100 2060 2060 2163 1750 1750 3295 2370 1959 2600 3000 1130 2800 2835 1599 79 APPENDIX 3 ANIMALS USED AND MEASUREMENTS OF THE WALLS OF PERFUSED ARTERIES Animal Weight (kg.) Eg Media 1E) Adventitia ()u) 19 upper 9 Female 1133 1100 middle 9 Female 979 875 lower 9 Female 824 875 20 upper 8. 2 Female 1236 1000 middle 8. 2 Female 1340 1100 lower 8. 2 Female 824 800 21 upper 10 Male 1030 824 middle 10 Male 8 7 5 92 6 lower 10 Male 762 669 22 upper 8. 2 Female 1441 1082 middle 8. 2 Female 1236 824 lower 8. 2 Female 1133 824 23 upper 5. 4 Female 875 567 middle 5 . 4 Female 927 412 lower 5. 4 Female 646 309 AVERAGE WEIGHT 8. 16 kg. AVERAGE THICKNESS ()1) Media Adventitia Upper 1143 914. 6 Middle 1071. 4 827. 4 Lower 837 695. 4 80 APP END IX 4 ANIMALS USED AND MEASUREMENTS OF THE WALLS OF PERFUSED VEINS Animal Weight (kg. ) _S_e_x_ Thickness (p) 19 upper 9 Female 3605 19 middle 9 Female 2470 19 lower 9 Female 2215 20 upper 8. 2 Female 2575 middle 8. 2 Female 2680 lower 8. 2 Female 1545 21 upper 10 Male 2060 middle 10 Male 1278 lower 10 Male 1390 22 upper 8. 2 Female 1649 middle 8. 2 Female 1546 lower 8. 2 Female 12 36 23 upper 5. 4 Female 1030 middle 5 . 4 Female 1030 lower 5. 4 Female 721 AVERAGE WEIGHT 8. 16 kg. AVERAGE THICKNESS (n) Upper 2183. 8 Middle 1800. 8 Lower 1421. 4 APPENDIX 5 81 ANIMALS USED AND NUMBER OF VALVES IN THE VEINS Anima 1 10 11 12 13 14 15 Weight (kg. ) ll. 5 9. 8. 15 22 22 22 22 18 18 15 15 5. 8. 8. 5 5 5 2 2 Sex Male Male Male Female Female Female Female Female Male Male Female Female Female Male Male Number of Valves MICHIGAN STATE UNIV RSITY LIB I E RAR ES I l 3 1293 030561J65