THE ANATOMICAL RECORD 229:103-108 (1991I
Sympathetic Innervation of the Hindlimb Arterial System in the Giraffe (Giraffa carnelopardalis) JAMES KIRUMBI KIMANI, REGINA N. MBUVA, Department
of
AND
RICHARD M KINYAMU
Human Anatomy, University of’Nairobi,NaLrobi, Kenya
ABSTRACT
We report the distribution of sympathetic nerves in the hindlimb arterial system of the giraffe based on the histochemical demonstration of monoamines by the sucrose-potassium phosphate-glyoxylic acid method. It is noted t h a t the hindlimb arterial system shows regional variations in its sympathetic innervation with regard to the density and the penetration of the nerves into the tunica media not hitherto described. The femoral and popliteal arteries showed a paucity of sympathetic innervation. Distally the dorsal pedal and great metatarsal arteries showed sparse sympathetic innervation characterized by a tendency toward exclusion of the nerves toward the outer layers of the tunica media. In contrast, the anterior (cranial) tibia1 artery in the leg revealed a relatively rich pattern of sympathetic innervation and a greater penetration of the nerves into the tunica media. The latter part of the arterial system showed a marked thickening of the tunica media and luminal narrowing, thus suggesting a “sphincteric” function. It is conceivable that this sphincter subserves a dual function, namely, to modulate blood flow to the distal parts of the limbs, and secondly to channel blood to the thigh and crural musculature. Pertinent to this is the fact that the presumptive sphincter occurs immediately after the crural muscular branches are given off.
The giraffe stands a t a height of 5-6 m and has a n average systemic blood pressure of 2601160 mm Hg (Goetz and Keen, 1957; Goetz et al., 1960; Van Citters et al., 1966,1968,1969;Warren, 1974).This high blood pressure, added to the effects of gravitational pull on a blood column of 2-3 m, would lead to a hydrostatic pressure of 500 mm Hg in the region of the hooves, which may cause rupture of the capillaries andlor filtration oedema (Goetz and Budtz-Olsen, 1955). The late August Krogh (in Warren, 1974),while delivering the Silliman Lecture a t Yale University in 1929, is reported to have suggested that the giraffe blood would be expected to have a high viscosity, but subsequent studies revealed that the chemistry of its blood is not grossly different from t h a t of human blood (Goetz and Budtz-Olsen, 1955; Warren, 1974; Hargens et al., 1987). Warren (1974) and Badeer (1986) postulated that the high intravascular pressure in this animal is counterbalanced by a correspondingly high extravascular pressure, which is maintained by its thick, tight skin termed the antigravity suit (Hargens et al., 1987).Goetz and Keen (1957)considered the thickened wall and pinpoint lumen of the metatarsal arteries a s the necessary adaptation of the limb arteries. Not much is known a t present about the sympathetic innervation of the blood vessels of the giraffe in general and in particular those of the limbs. In this study the innervation of the femoral arterial system is described and its functional significance discussed in the light of the special haemodynamic characteristics of this animal.
c
1991 WII,EY-l,ISS. IN(’
MATERIALS AND METHODS
Materials used in this study were obtained from four adult giraffes. The animals were obtained during routine culls conducted by the Department of Wildlife Management and Conservation of the Ministry of Tourism and Wildlife of the Government of Kenya. The specimens were transferred from the field to the laboratories wrapped in aluminum foil and stored in dry ice. Embedding of the tissues was done using OCT compound (Tissue-Tek 11) in a cryostat chamber a t -30°C. Sections 16 pm thick were cut and picked off the knife by use of clean but nontreated room-temperature glass slides. Cut sections were prepared for the demonstration of tissue monoamines by the sucrose-potassium phosphate-glyoxylic acid (SPG)method a s described by de la Torre and Surgeon (1976).The sections were then given three dips ( 1 dip1sec) in the SPG solution. Excess solution was drained off and the slides dried a t a temperature of about 40°C with a hair drier. The slides were then placed in an oven maintained a t 100°C under liquid paraffin for 5 minutes. They were then removed and coverslipped using fresh liquid paraffin. Examination of the slides was done under a Leitz Ortholux flu-
Received October 4. 1989; accepted J u n e 6. 1990. Address reprint requests to Prof. J a m e s K Kimani, Department of H u m a n Anatomy. University of Nairobi. P.O. Box 30197. Nairobi. Kenva.
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Fig. I . Photograph of a giraffe and micrographs of selected zones of the hind limb arterial axis (insets). Note the enormous medial hypertrophy and the slit-like lumen of the anterior tibial artery (B) and the gradual widening of the lumen towards the metatarsal and digital arteries (C-E). The femoral and popliteal arteries also have a wide lumen (A).Specimens fixed by perfusion except for femoral artery (A). Inserts, x 7 .
orescent microscope, using a 25014 ultra-high pressure mercury lamp with a Leitz BP 546120 filter block. Photographs were taken using Kodak Tri-X400 black and white film. RESULTS
The arterial system of the giraffe hindlimb is characterized by a segment of marked hypertrophy of the tunica media and a slit-like lumen in the region of the leg that involves the anterior tibial artery which resembles a sphincter (Fig. 1).Proximal and distal to this the vessels in the thigh and metartarsal regions show
a wide lumen relative to the wall thickness (Fig. 1). The arteries in the thigh, namely, the terminal part of the external iliac and femoral artery, were characterized by a paucity of sympathetic nerves (Fig. 2). Sympathetic nerve fibres represented by green-yellow fluorophores were sparsely distributed largely a t the media-adventitia junction (Fig. 2). The femoral artery terminates at the level of the knee joint by dividing into about two muscular branches and then continues through the leg a s the anterior tibial artery (Fig. 1).The latter showed variations in the density of sympathetic innervation and in the depth of penetration of the nerves into the tunica media. The proximal 3-4 cm showed a wide lumen and paucity of sympathetic nerves a s demonstrated in the femoral artery (Fig. 2). Distal to this region of the anterior tibial artery is the muscular cuff, or “sphincteric” portion, characterised by a marked decrease in the luminal diameter and a thick muscular tunica media (Fig. 1B). This segment of the limb arterial system showed a rich sympathetic innervation in which the adrenergic nerve profiles penetrated the tunica media to about three fourths of its thickness (Fig. 3). However, no adrenergic fluorophores were evident in the juxtaintimal medial layer and/or in the tunica intima (Fig. 3a). As the artery was followed distally toward the ankle joint, one observed a general reduction in medial sympathetic innervation a s well as in the depth of penetration of the nerves into the tunica media (Fig. 4). A large number of the adrenergic nerve terminals entered the tunica media through the connective tissue septa (Fig. 4). The anterior tibial artery continues into the metatarsal region to form the great metatarsal artery. Near the hooves the artery divides into two main digital arteries. Neither the metatarsal or the digital arteries were densely innervated nor did the sympathetic nerves penetrate the tunica media to as much a n extent as they did in the “sphincteric” portion of the anterior tibial artery (Fig. 5 ) . In the metatarsal artery, adrenergic fluorophores could be seen entering the media to about 1/4 of its thickness (Fig. 5a). Again, as demonstrated in other areas, sympathetic nerves enter the tunica media along fibroelastic septa (Fig. 5b). Innervation of the digital arteries was relatively sparse compared with that of the metatarsal artery, with the nerves being restricted largely to the media-adventitia junction (Fig. 5c). DISCUSSION
The findings of this study have revealed that the hindlimb arteries of the adult giraffe show a region of luminal constriction and a n increased thickness of the tunica media between the knee and ankle joints. Proximal and distal to this region the artery showed a wide lumen and a concomitant reduction in thickness of the tunica media. Fluorescent histochemical findings revealed that, whereas the main arterial trunk in the thigh is poorly innervated, the parts of the anterior tibial artery, which has a relatively constricted lumen and a thick wall, had a strikingly rich density of sympathetic nerves and a greater extent of penetration of the nerves into the tunica media. Distal to this, the density of innervation decreased, whereas the sympathetic neuronal profiles became restricted more to the
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Fig. 2. Photomicrographs showing the distal part of the femoral artery. a: Note the characteristic absence of adrenergic nerve fluorophores and the autofluorescence of elastic fibres in the tunica adventitia (A) and a few elastic lamellae (E) in the tunica media (M). L, lumen; arrowheads, media-adventitia junction. X 125. b The media-
adven-titia zone in which a few sympathetic varicosities (arrows) are seen in the outer layers of the tunica media (MI. Note also the prominent autofluorescence of elastic tissue in the tunica adventitia (A). Arrowheads indicate media-adventitia junction. x 250.
outer layers of the tunica media and the media-adventitia border. The view generally held is that, a s arteries get smaller, the density of their innervation increases, with a peak in the small arteries (Burnstock and Iwayama, 1971; Burnstock, 1975; Griffith e t al., 1982). Rather, in the limb arteries of the giraffe, the pattern of innervation is such that the arterial segment in the leg is well innervated, beyond this, the density of innervation diminishes considerably. It is interesting, however, that the well-innervated segments of the arterial wall are coextensive with areas showing the greatest hypertrophy of the tunica media and constriction of the lumen. The findings of this study are a t variance with the statement in a recent study that the sympathetic innervation of the gravitation-dependent arteries in the limbs of the giraffe is restricted to the media-adventitia border, in contrast to the medial innervation of the carotid arteries (Nilsson et al., 1988). It is conceivable, however, that Nilsson et al. (1988) obtained their material from the distal segment of the tibial artery, metatarsal, andlor digital arteries in which the sympathetic nerves are located in the outer layers of the tunica media and media-adventitia border. In a previous study, these vessels were believed to have the largest content of smooth muscle (Goetz and Keen, 1957). However, as reported in this study, the arterial segment in the limbs that present the so-called gravita-
tion-dependent features, as described by Goetz and Keen (1957), is in the region of the leg and involves mainly the anterior tibial artery. Even then, the findings reported in this study have revealed that, although the metatarsal and digital arteries are generally sparsely innervated, sympathetic nerve fibres are not as a rule restricted to the media-adventitia border, as the same may be found in the peripheral fourth of the tunica media. The findings of this study are also a t variance with the suggestion that there is a n increasing density of innervation of large arteries from foot to head that is inversely proportional to the blood vessel thickness (Nilsson et al., 1988). In our view, the studies of Pettersson et al. (1986) and Nilsson e t al. (1988)may have been based on limited material, in which case the structural variability of the nature reported in our study based on glyoxylic acid-induced histofluorescence of catecholamines could have been missed. It is possible also that the inability to demonstrate sympathetic nerves in the tunica media of the thick-walled arteries by Nilsson et al. (1988) was largely due to inadequate fixation of their material, which is particularly crucial in immunofluorescence techniques. The specimens used in their study were fixed by immersion in 4% formaldehyde in phosphate buffer for 4-6 hours, which according to our experience is insufficient for proper fixation of the thick-walled arteries of this animal, particularly those of the limbs. This implies,
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Fig.3. Photomicrograph of sections of the anterior tibial artery a t levels corresponding to the area shown in the Figure 1(B)to illustrate its pattern of sympathetic innervation. a: The luminal zone of the tunica media showing the deepest extent of sympathetic nerves (arrows) and absence of nerves in the adluminal zone. Note the presence
of autofluorescing elastic fibres (e). E, internal elastic lamina. x 250. b The outer zone of the tunica media (MI to demonstrate the rich sympathetic innervation (arrows).Note the numerous varicosities associated with the sympathetic nerves. TA, tunica adventitia; arrowheads indicate media-adventitia border. x 250.
therefore, t h a t the efficacy of tissue fixation-and, conceivably, therefore preservation of the labile enzymes in the sympathetic nerves-would be inversely proportional to the wall thickness and luminal diameter. It is not unusual, therefore, that Nilsson et al. (1988) reported a n increasing density of innervation of the large arteries from foot to head. It is generally believed that in the small laboratory animals sympathetic nerves are confined to the mediaadventitia border, with few, if any intramural fibres (Burnstock and Iwayama, 1971; Burnstock, 1975; Burnstock and Griffith, 1983). In this scheme, neurogenic vasoconstriction is believed to be mediated by the spread of excitation from cell to cell (electrotonic coupling) in the tunica media (Burnstock and Iwayama, 1971; Burnstock, 1975). However, numerous investigations have demonstrated sympathetic nerve fibres in the tunica media, for example, in the carotid arteries of the giraffe (Nilsson et al., 1988), in the saphenous artery of the fetal and newborn guinea pigs (Kimani, 1984), in the saphenous artery of the rabbit (Bevan and Purdy, 1973), sheep carotid artery (Keatinge, 1966), dog aorta (Dolezel, 1972) and mesenteric artery (Mohri et al., 1969), in the aorta of fish and amphibians (Kirby and Burnstock, 1969), and in the proximal arteries of diving animals such a s the seal (White et al., 1973).
The deep penetration of sympathetic nerves into the tunica media of the supply arteries of the viscera and limbs in the seal is associated with constriction of these arteries in diving so that blood can be directed to the vital organs, namely, the brain and the heart as part of
Fig.4. Photomicrograph showing the distal segment of the anterior tibial artery close to the ankle joint as shown in Figure l(C). Note the decrease of sympathetic nerves (arrows)in the tunica media compared with Figure 3b and their lesser penetration into the tunica media (M). fs, fibrous septa; arrowhead indicates internal elastic lamina. x 125. Flg. 5. Photomicrograph showing the metatarsal artery (a,b) at a level corresponding to Figure 1(D)and the digital artery (c)as shown in Figure 1(E).a: Note the paucity of sympathetic nerves (arrows)and their exclusion toward the outer layers of the tunica media (MI compared with Figure 4. TA, tunica adventitia; L, lumen; arrowheads indicate internal elastic lamina. x 125. b: A fibrous septum (fs) within the outer layers of the tunica media running between bundles of smooth muscle (sm).Note the presence of sympathetic nerves in the fibrous septum (arrow),whose bright fluorescence is discernable from the dull fluorescence of elastic fibres within the septum. x 250. c: The outer zone of the tunica media (MI showing the paucity of sympathetic nerves (arrow)in the digital artery and their presence a t the mediaadventitia junction. Note the presence of numerous elastic fibres ( e )in the tunica media. TA, tunica adventitia; arrowheads indicate mediaadventitia border. x 250.
HINDLIMB ARTERIAL SYSTEM OF T H E G I R A F F E
Figs. 4 and 5
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the diving reflex (White et al., 1973). A similar mechanism may operate in the giraffe limb vasculature, whereby the enormous hypertrophy of the tunica media in the anterior tibia1 artery and the deep penetration of sympathetic nerves into the tunica media form part of the host of mechanisms that underly the ability of this animal to cope with the relatively high systemic blood pressure. Recent physiological findings are, i t seems, in concordance with the existence of a “gatevalve” control mechanism (Hargens et al., 1987). According to Hargens et al. (1987) the interstitial fluid and blood pressure in the extremities of the giraffe decline when the animal is walking and rises in a stationary animal. It is possible, therefore, that when the animal is active, sympathetic stimulation closes the arterial “sphincter” and thereby shunts blood to the muscles in the thigh and leg portions of the limbs. Concomitantly, this breaks the blood column, thus reducing the gravitational pressure in the extremities and hence the excessive formation of interstitial fluid. The contracting limb muscles may also act a s a peripheral pump, squeezing the veins so that blood in them flows back to the heart, reflux being prevented by the venous valves. This effect may ensure not only a n efficient venous return, but counteracts gravitational venous pooling and results in reduced venous pressures (Hargens et al., 1987). These mechanisms, together with the structural adaptations of the arterioles and the capillary basement membrane (Williamson et al., 1971), may abolish the tendency toward formation of filtration oedema. The observations made in this study clearly indicate that, in addition to the “antigravity” role of the tight skin in the lower extremities, other mechanisms intrinsic to blood vessels should be considered in explaining the haemodynamic adaptations of this animal. ACKNOWLEDGMENTS
The authors are grateful to the Department of Wildlife Conservation and Management in the Ministry of Tourism and Wildlife of the Government of Kenya for granting permission to cull the animals used in this study; to the technical staff in the Department of Human Anatomy, University of Nairobi, for their technical assistance; and to Miss Hydah Were and Mrs. Mary Kiarie for typing the manuscript. This work was supported by a grant from the University of Nairobi, Deans Committee research grant. LITERATURE CITED Badeer, H.S. 1986 Does gravitational pressure of blood hinder flow to the brain of the giraffe? Comp. Biochem. Physiol., 83A:207-211. Bevan, J.A., and R.E. Purdy 1973 Variation in the adrenergic innervation and contractile responses of the rabbit saphenous artery. Circ. Res., 32r746-751.
Burnstock, G. 1975 Innervation of vascular smooth muscle: histochemistry and electron microscopy. Clin. Exp. Pharmacol. Physiol. [Suppl.] 2:7-20. Burnstock, G., and S.G. Griffith 1983 Neurohumoral control of the vasculature. In: Biology and Pathology of the Vessel Wall. A Modern Appraisal. N. Woolf, ed., Praeger Pub., Eastbourne, pp. 1540. Burnstock, G., and T. Iwayama 1971 Fine-structural identification of autonomic nerves and their relation to smooth muscle. In: Progress In Brain Research. Histochemistry of Nervous Transmission. 0. Eranko, ed. vol. 34. Elsevier, Amsterdam, pp. 389404. de la Torre, J.C., and J.W. Surgeon 1976 A methodological approach to rapid and sensitive monoamine histofluorescence using a modified glyoxylic acid technique: The SPG method. Histochemistry, 49%-93. Dolezel, S. 1972 Monoaminergic innervation of the aorta. Folia Morphol., 20t14-20. Goetz, R.H., and 0. Budtz-Olsen 1955 Scientific safari. The circulation of the Giraffe. S.Afr. Med. J., 29r773-776. Goetz, R.H., and E.N. Keen 1957 Some aspects of the cardiovascular system in the giraffe. Angiology, 8r542-564. Goetz R.H., J.V. Warren, O.H. Gauer, J.L. Petterson Jr., J.T. Doyle, E.N. Keen, and M. McGregor 1960 Circulation in the giraffe. Circ. Res., 8t1049-1058. Griffith, S.G., R. Crowe, J. Lincoln, A.J. Haven, and G. Burnstock 1982 Regional differences in the density of perivascular nerves and varicosities, noradrenaline content and responses to nerve stimulation in the rabbit ear artery. Blood Vessels, 19r41-52. Hargens, A.R., R.W. Millard, K. Petterson, and K. Johansen 1987 Gravitational haemodynamics and oedema prevention in the giraffe. Nature, 239t59-60. Keatinge, W.R. 1966 Electrical and mechanical responses of arteries to stimulation of sympathetic nerves. J. Physiol., (Lond.), 185; 701-7 15. Kimani, J.K. 1984 Adrenergic innervation of the tunica media in the saphenous artery of the fetal and newborn guinea pig. Cell Tissue Res., 237r383-385. Kirby, S., and G. Burnstock 1969 Comparative pharmacological studies of isolated strips of large arteries from lower vertebrates. Comp. Biochem. Physiol., 28r307-319. Mohri, K., N. Ohgushi, M. Ikeda, K. Yamamoto, and K. Tsunekawa 1969 Histochemical demonstration of adrenergic fibres in the smooth muscle layer of media of arteries supplying abdominal organs. Arch. Jpn. Chir., 38:236-248. Nilsson, O., S. Booj, A. Dahlstrom, A.R. Hargens, R.W. Millard, and K.S. Pettersson 1988 Sympathetic innervation of the cardiovascular system in the giraffe. Blood Vessels, 25:299-307. Petterson, K., A.R. Hargens, R.W. Millard, K. Johansen, D.H. Gershuni, R. Burroughs, D.G.A. Meltzer, and W. van Hoven 1986 Dependent hypertension and arterial wall hypertrophy without interstitial oedema in the giraffe. Proc. Int. Union Physiol. Sci., 16r411. Van Citters, R.L., D.L. Franklin, D.F. Vatner, T. Patrick, and J.V. Warren 1969 Cerebral haemodynamics in the giraffe. Trans. AsSOC. Am. Physicians, 82:293-303. Van Citters, R.L., W.S. Kemper, and D.L. Franklin 1966 Blood pressure responses of wild giraffes studied by radio telemetry: Science, 152r384-386. Van Citters., R.L..,~W.S. Kemuer. and D.L. Franklin 1968 Blood flow ~~. and pressure in the giraffe carotid artery. Comp. Biochem. Physiol., 24:1035-1042. Warren, J.V. 1974 The physiology of the Giraffe. Sci. Am., 231r961_--. n!i White, F.N., M. Ikeda, and R.W. Eisner 1973 Adrenergic innervation of large arteries in the seal. ComD. Gen. Pharmacol.. 4271-276. Williams&, J.R., N.J. Vogler, andC. Kilo 1971 Regional variations in the width of the basement membrane of muscle capillaries in man and giraffe. Am. J. Pathol.. 63r359-370. L
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Sympathetic Innervation of the Hindlimb Arterial System in the Giraffe (Giraffa carnelopardalis) JAMES KIRUMBI KIMANI, REGINA N. MBUVA, Department
of
AND
RICHARD M KINYAMU
Human Anatomy, University of’Nairobi,NaLrobi, Kenya
ABSTRACT
We report the distribution of sympathetic nerves in the hindlimb arterial system of the giraffe based on the histochemical demonstration of monoamines by the sucrose-potassium phosphate-glyoxylic acid method. It is noted t h a t the hindlimb arterial system shows regional variations in its sympathetic innervation with regard to the density and the penetration of the nerves into the tunica media not hitherto described. The femoral and popliteal arteries showed a paucity of sympathetic innervation. Distally the dorsal pedal and great metatarsal arteries showed sparse sympathetic innervation characterized by a tendency toward exclusion of the nerves toward the outer layers of the tunica media. In contrast, the anterior (cranial) tibia1 artery in the leg revealed a relatively rich pattern of sympathetic innervation and a greater penetration of the nerves into the tunica media. The latter part of the arterial system showed a marked thickening of the tunica media and luminal narrowing, thus suggesting a “sphincteric” function. It is conceivable that this sphincter subserves a dual function, namely, to modulate blood flow to the distal parts of the limbs, and secondly to channel blood to the thigh and crural musculature. Pertinent to this is the fact that the presumptive sphincter occurs immediately after the crural muscular branches are given off.
The giraffe stands a t a height of 5-6 m and has a n average systemic blood pressure of 2601160 mm Hg (Goetz and Keen, 1957; Goetz et al., 1960; Van Citters et al., 1966,1968,1969;Warren, 1974).This high blood pressure, added to the effects of gravitational pull on a blood column of 2-3 m, would lead to a hydrostatic pressure of 500 mm Hg in the region of the hooves, which may cause rupture of the capillaries andlor filtration oedema (Goetz and Budtz-Olsen, 1955). The late August Krogh (in Warren, 1974),while delivering the Silliman Lecture a t Yale University in 1929, is reported to have suggested that the giraffe blood would be expected to have a high viscosity, but subsequent studies revealed that the chemistry of its blood is not grossly different from t h a t of human blood (Goetz and Budtz-Olsen, 1955; Warren, 1974; Hargens et al., 1987). Warren (1974) and Badeer (1986) postulated that the high intravascular pressure in this animal is counterbalanced by a correspondingly high extravascular pressure, which is maintained by its thick, tight skin termed the antigravity suit (Hargens et al., 1987).Goetz and Keen (1957)considered the thickened wall and pinpoint lumen of the metatarsal arteries a s the necessary adaptation of the limb arteries. Not much is known a t present about the sympathetic innervation of the blood vessels of the giraffe in general and in particular those of the limbs. In this study the innervation of the femoral arterial system is described and its functional significance discussed in the light of the special haemodynamic characteristics of this animal.
c
1991 WII,EY-l,ISS. IN(’
MATERIALS AND METHODS
Materials used in this study were obtained from four adult giraffes. The animals were obtained during routine culls conducted by the Department of Wildlife Management and Conservation of the Ministry of Tourism and Wildlife of the Government of Kenya. The specimens were transferred from the field to the laboratories wrapped in aluminum foil and stored in dry ice. Embedding of the tissues was done using OCT compound (Tissue-Tek 11) in a cryostat chamber a t -30°C. Sections 16 pm thick were cut and picked off the knife by use of clean but nontreated room-temperature glass slides. Cut sections were prepared for the demonstration of tissue monoamines by the sucrose-potassium phosphate-glyoxylic acid (SPG)method a s described by de la Torre and Surgeon (1976).The sections were then given three dips ( 1 dip1sec) in the SPG solution. Excess solution was drained off and the slides dried a t a temperature of about 40°C with a hair drier. The slides were then placed in an oven maintained a t 100°C under liquid paraffin for 5 minutes. They were then removed and coverslipped using fresh liquid paraffin. Examination of the slides was done under a Leitz Ortholux flu-
Received October 4. 1989; accepted J u n e 6. 1990. Address reprint requests to Prof. J a m e s K Kimani, Department of H u m a n Anatomy. University of Nairobi. P.O. Box 30197. Nairobi. Kenva.
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Fig. I . Photograph of a giraffe and micrographs of selected zones of the hind limb arterial axis (insets). Note the enormous medial hypertrophy and the slit-like lumen of the anterior tibial artery (B) and the gradual widening of the lumen towards the metatarsal and digital arteries (C-E). The femoral and popliteal arteries also have a wide lumen (A).Specimens fixed by perfusion except for femoral artery (A). Inserts, x 7 .
orescent microscope, using a 25014 ultra-high pressure mercury lamp with a Leitz BP 546120 filter block. Photographs were taken using Kodak Tri-X400 black and white film. RESULTS
The arterial system of the giraffe hindlimb is characterized by a segment of marked hypertrophy of the tunica media and a slit-like lumen in the region of the leg that involves the anterior tibial artery which resembles a sphincter (Fig. 1).Proximal and distal to this the vessels in the thigh and metartarsal regions show
a wide lumen relative to the wall thickness (Fig. 1). The arteries in the thigh, namely, the terminal part of the external iliac and femoral artery, were characterized by a paucity of sympathetic nerves (Fig. 2). Sympathetic nerve fibres represented by green-yellow fluorophores were sparsely distributed largely a t the media-adventitia junction (Fig. 2). The femoral artery terminates at the level of the knee joint by dividing into about two muscular branches and then continues through the leg a s the anterior tibial artery (Fig. 1).The latter showed variations in the density of sympathetic innervation and in the depth of penetration of the nerves into the tunica media. The proximal 3-4 cm showed a wide lumen and paucity of sympathetic nerves a s demonstrated in the femoral artery (Fig. 2). Distal to this region of the anterior tibial artery is the muscular cuff, or “sphincteric” portion, characterised by a marked decrease in the luminal diameter and a thick muscular tunica media (Fig. 1B). This segment of the limb arterial system showed a rich sympathetic innervation in which the adrenergic nerve profiles penetrated the tunica media to about three fourths of its thickness (Fig. 3). However, no adrenergic fluorophores were evident in the juxtaintimal medial layer and/or in the tunica intima (Fig. 3a). As the artery was followed distally toward the ankle joint, one observed a general reduction in medial sympathetic innervation a s well as in the depth of penetration of the nerves into the tunica media (Fig. 4). A large number of the adrenergic nerve terminals entered the tunica media through the connective tissue septa (Fig. 4). The anterior tibial artery continues into the metatarsal region to form the great metatarsal artery. Near the hooves the artery divides into two main digital arteries. Neither the metatarsal or the digital arteries were densely innervated nor did the sympathetic nerves penetrate the tunica media to as much a n extent as they did in the “sphincteric” portion of the anterior tibial artery (Fig. 5 ) . In the metatarsal artery, adrenergic fluorophores could be seen entering the media to about 1/4 of its thickness (Fig. 5a). Again, as demonstrated in other areas, sympathetic nerves enter the tunica media along fibroelastic septa (Fig. 5b). Innervation of the digital arteries was relatively sparse compared with that of the metatarsal artery, with the nerves being restricted largely to the media-adventitia junction (Fig. 5c). DISCUSSION
The findings of this study have revealed that the hindlimb arteries of the adult giraffe show a region of luminal constriction and a n increased thickness of the tunica media between the knee and ankle joints. Proximal and distal to this region the artery showed a wide lumen and a concomitant reduction in thickness of the tunica media. Fluorescent histochemical findings revealed that, whereas the main arterial trunk in the thigh is poorly innervated, the parts of the anterior tibial artery, which has a relatively constricted lumen and a thick wall, had a strikingly rich density of sympathetic nerves and a greater extent of penetration of the nerves into the tunica media. Distal to this, the density of innervation decreased, whereas the sympathetic neuronal profiles became restricted more to the
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Fig. 2. Photomicrographs showing the distal part of the femoral artery. a: Note the characteristic absence of adrenergic nerve fluorophores and the autofluorescence of elastic fibres in the tunica adventitia (A) and a few elastic lamellae (E) in the tunica media (M). L, lumen; arrowheads, media-adventitia junction. X 125. b The media-
adven-titia zone in which a few sympathetic varicosities (arrows) are seen in the outer layers of the tunica media (MI. Note also the prominent autofluorescence of elastic tissue in the tunica adventitia (A). Arrowheads indicate media-adventitia junction. x 250.
outer layers of the tunica media and the media-adventitia border. The view generally held is that, a s arteries get smaller, the density of their innervation increases, with a peak in the small arteries (Burnstock and Iwayama, 1971; Burnstock, 1975; Griffith e t al., 1982). Rather, in the limb arteries of the giraffe, the pattern of innervation is such that the arterial segment in the leg is well innervated, beyond this, the density of innervation diminishes considerably. It is interesting, however, that the well-innervated segments of the arterial wall are coextensive with areas showing the greatest hypertrophy of the tunica media and constriction of the lumen. The findings of this study are a t variance with the statement in a recent study that the sympathetic innervation of the gravitation-dependent arteries in the limbs of the giraffe is restricted to the media-adventitia border, in contrast to the medial innervation of the carotid arteries (Nilsson et al., 1988). It is conceivable, however, that Nilsson et al. (1988) obtained their material from the distal segment of the tibial artery, metatarsal, andlor digital arteries in which the sympathetic nerves are located in the outer layers of the tunica media and media-adventitia border. In a previous study, these vessels were believed to have the largest content of smooth muscle (Goetz and Keen, 1957). However, as reported in this study, the arterial segment in the limbs that present the so-called gravita-
tion-dependent features, as described by Goetz and Keen (1957), is in the region of the leg and involves mainly the anterior tibial artery. Even then, the findings reported in this study have revealed that, although the metatarsal and digital arteries are generally sparsely innervated, sympathetic nerve fibres are not as a rule restricted to the media-adventitia border, as the same may be found in the peripheral fourth of the tunica media. The findings of this study are also a t variance with the suggestion that there is a n increasing density of innervation of large arteries from foot to head that is inversely proportional to the blood vessel thickness (Nilsson et al., 1988). In our view, the studies of Pettersson et al. (1986) and Nilsson e t al. (1988)may have been based on limited material, in which case the structural variability of the nature reported in our study based on glyoxylic acid-induced histofluorescence of catecholamines could have been missed. It is possible also that the inability to demonstrate sympathetic nerves in the tunica media of the thick-walled arteries by Nilsson et al. (1988) was largely due to inadequate fixation of their material, which is particularly crucial in immunofluorescence techniques. The specimens used in their study were fixed by immersion in 4% formaldehyde in phosphate buffer for 4-6 hours, which according to our experience is insufficient for proper fixation of the thick-walled arteries of this animal, particularly those of the limbs. This implies,
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Fig.3. Photomicrograph of sections of the anterior tibial artery a t levels corresponding to the area shown in the Figure 1(B)to illustrate its pattern of sympathetic innervation. a: The luminal zone of the tunica media showing the deepest extent of sympathetic nerves (arrows) and absence of nerves in the adluminal zone. Note the presence
of autofluorescing elastic fibres (e). E, internal elastic lamina. x 250. b The outer zone of the tunica media (MI to demonstrate the rich sympathetic innervation (arrows).Note the numerous varicosities associated with the sympathetic nerves. TA, tunica adventitia; arrowheads indicate media-adventitia border. x 250.
therefore, t h a t the efficacy of tissue fixation-and, conceivably, therefore preservation of the labile enzymes in the sympathetic nerves-would be inversely proportional to the wall thickness and luminal diameter. It is not unusual, therefore, that Nilsson et al. (1988) reported a n increasing density of innervation of the large arteries from foot to head. It is generally believed that in the small laboratory animals sympathetic nerves are confined to the mediaadventitia border, with few, if any intramural fibres (Burnstock and Iwayama, 1971; Burnstock, 1975; Burnstock and Griffith, 1983). In this scheme, neurogenic vasoconstriction is believed to be mediated by the spread of excitation from cell to cell (electrotonic coupling) in the tunica media (Burnstock and Iwayama, 1971; Burnstock, 1975). However, numerous investigations have demonstrated sympathetic nerve fibres in the tunica media, for example, in the carotid arteries of the giraffe (Nilsson et al., 1988), in the saphenous artery of the fetal and newborn guinea pigs (Kimani, 1984), in the saphenous artery of the rabbit (Bevan and Purdy, 1973), sheep carotid artery (Keatinge, 1966), dog aorta (Dolezel, 1972) and mesenteric artery (Mohri et al., 1969), in the aorta of fish and amphibians (Kirby and Burnstock, 1969), and in the proximal arteries of diving animals such a s the seal (White et al., 1973).
The deep penetration of sympathetic nerves into the tunica media of the supply arteries of the viscera and limbs in the seal is associated with constriction of these arteries in diving so that blood can be directed to the vital organs, namely, the brain and the heart as part of
Fig.4. Photomicrograph showing the distal segment of the anterior tibial artery close to the ankle joint as shown in Figure l(C). Note the decrease of sympathetic nerves (arrows)in the tunica media compared with Figure 3b and their lesser penetration into the tunica media (M). fs, fibrous septa; arrowhead indicates internal elastic lamina. x 125. Flg. 5. Photomicrograph showing the metatarsal artery (a,b) at a level corresponding to Figure 1(D)and the digital artery (c)as shown in Figure 1(E).a: Note the paucity of sympathetic nerves (arrows)and their exclusion toward the outer layers of the tunica media (MI compared with Figure 4. TA, tunica adventitia; L, lumen; arrowheads indicate internal elastic lamina. x 125. b: A fibrous septum (fs) within the outer layers of the tunica media running between bundles of smooth muscle (sm).Note the presence of sympathetic nerves in the fibrous septum (arrow),whose bright fluorescence is discernable from the dull fluorescence of elastic fibres within the septum. x 250. c: The outer zone of the tunica media (MI showing the paucity of sympathetic nerves (arrow)in the digital artery and their presence a t the mediaadventitia junction. Note the presence of numerous elastic fibres ( e )in the tunica media. TA, tunica adventitia; arrowheads indicate mediaadventitia border. x 250.
HINDLIMB ARTERIAL SYSTEM OF T H E G I R A F F E
Figs. 4 and 5
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the diving reflex (White et al., 1973). A similar mechanism may operate in the giraffe limb vasculature, whereby the enormous hypertrophy of the tunica media in the anterior tibia1 artery and the deep penetration of sympathetic nerves into the tunica media form part of the host of mechanisms that underly the ability of this animal to cope with the relatively high systemic blood pressure. Recent physiological findings are, i t seems, in concordance with the existence of a “gatevalve” control mechanism (Hargens et al., 1987). According to Hargens et al. (1987) the interstitial fluid and blood pressure in the extremities of the giraffe decline when the animal is walking and rises in a stationary animal. It is possible, therefore, that when the animal is active, sympathetic stimulation closes the arterial “sphincter” and thereby shunts blood to the muscles in the thigh and leg portions of the limbs. Concomitantly, this breaks the blood column, thus reducing the gravitational pressure in the extremities and hence the excessive formation of interstitial fluid. The contracting limb muscles may also act a s a peripheral pump, squeezing the veins so that blood in them flows back to the heart, reflux being prevented by the venous valves. This effect may ensure not only a n efficient venous return, but counteracts gravitational venous pooling and results in reduced venous pressures (Hargens et al., 1987). These mechanisms, together with the structural adaptations of the arterioles and the capillary basement membrane (Williamson et al., 1971), may abolish the tendency toward formation of filtration oedema. The observations made in this study clearly indicate that, in addition to the “antigravity” role of the tight skin in the lower extremities, other mechanisms intrinsic to blood vessels should be considered in explaining the haemodynamic adaptations of this animal. ACKNOWLEDGMENTS
The authors are grateful to the Department of Wildlife Conservation and Management in the Ministry of Tourism and Wildlife of the Government of Kenya for granting permission to cull the animals used in this study; to the technical staff in the Department of Human Anatomy, University of Nairobi, for their technical assistance; and to Miss Hydah Were and Mrs. Mary Kiarie for typing the manuscript. This work was supported by a grant from the University of Nairobi, Deans Committee research grant. LITERATURE CITED Badeer, H.S. 1986 Does gravitational pressure of blood hinder flow to the brain of the giraffe? Comp. Biochem. Physiol., 83A:207-211. Bevan, J.A., and R.E. Purdy 1973 Variation in the adrenergic innervation and contractile responses of the rabbit saphenous artery. Circ. Res., 32r746-751.
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