Circulatory Mechanics in the Toad Bufo Marinus I

/. exp Biol. 158, 275-289 (1991) 275 Printed in Great Britain © The Company of Biologists Limited 1991

CIRCULATORY MECHANICS IN THE TOAD BUFO MARINUS I. STRUCTURE AND MECHANICAL DESIGN OF THE AORTA

BY CAROL A. GIBBONS1 AND ROBERT E. SHADWICK2* 1 Department of Biology, University of Calgary, Calgary, Alberta, Canada, T2N1N4 and 2Marine Biology Research Division A-004, Scripps Institution of Oceanography, La Jolla, CA 92093, USA

Accepted 28 March 1991

Summary This study describes several important mechanical design features of the aorta of a typical poikilothermic vertebrate. A strong functional similarity to the aorta of mammals is apparent, but some structural and mechanical differences are seen that reflect the lower pressure and simpler haemodynamics of the poikilothermic circulation. 1. The aorta is highly distensible, resilient and non-linearly elastic, giving it the requisite properties to act as an effective storage element in the arterial circulation. 2. An abrupt transition from high compliance (low elastic modulus) to relatively low compliance (high elastic modulus) takes place at pressures above the resting physiological range of 2-4 kPa. This behaviour reflects the composite nature of the artery wall in which rubbery elastin fibres and relatively rigid collagen fibres are the predominant elements. 3. The longitudinal tethering of the aorta when inflated is due primarily to anisotropy in elastic properties, rather than to links to the axial skeleton by branch vessels or connective tissue. 4. No significant changes in elastic properties or connective tissue content occur along the length of the toad arterial tree, in contrast to the situation in mammals.

Introduction The aorta is an important elastic element in the arterial circulation. The vessel wall expands during systole and recoils during diastole, thereby storing and releasing elastic strain energy and smoothing the pulsatile output of the heart. An important benefit is the reduction of the arterial pressure pulse and, consequently, protection of the small exchange vessels from the high shear forces associated with pulsatile flow, and a reduction of the total energy required to circulate the blood (Taylor, 1964). To perform this function effectively, the aorta must have non- *To whom reprint requests should be addressed, rftey words: aorta, elasticity, circulatory system, mechanical design, Bufo marinus. 276 C. A. GIBBONS AND R. E. SHADWICK linear elastic properties in order to be a compliant reservoir at low pressures but be stiff enough to resist rupture at high pressures. Most of our information on structure and mechanical properties of arteries comes from studies on mammalian tissues (Bergel, 1961; Milnor, 1982). The artery wall is a composite structure whose non-linear elastic properties result from the combination of rubber-like elastic and relatively inextensible collagen fibres. The transition from a highly compliant to a relatively stiff vessel takes place at about 10-12 kPa, the mean resting blood pressure. In mammals, the distensibility of the aorta decreases as the ratio of collagen to elastin increases along the arterial tree. The elastic properties of the artery wall are important in determining the haemodynamic behaviour of the arterial system. Poikilothermic vertebrates generally have much lower blood pressures and often lower heart rates than do mammals (Shelton and Jones, 1968; Jones et al. 191 A; Shelton and Burggren, 1976; Langille and Jones, 1977). Nevertheless, the aortas of reptiles, amphibians and fish appear to have non-linear elastic properties (Goto and Kimoto, 1966; Satchell, 1971; Gibbons and Shadwick, 1989) that are comparable to those observed in mammals, but presumably appropriate for function in a lower-pressure system. However, very little is known about the relationship between structure, connective tissue composition and the elastic properties of the aorta in any lower vertebrate species. The purpose of this investigation is to make a detailed study of the aortic mechanical properties and their structural basis in the toad Bufo marinus, and to compare the design features of this arterial system to that of mammals. In a subsequent study (Gibbons and Shadwick, 1991), the effects of arterial elasticity on haemodynamic properties in the toad will be examined.

Materials and methods Animals The experiments were performed on the toad, Bufo marinus L. Animals weighing 200-500 g were maintained in tanks at room temperature with access to water. They were fed mealworms weekly. The animals were killed by injection of MS-222 into the peritoneal cavity (Sandoz, 1:1000, 0.022 ml g~x body mass). The heart, aortic arches and dorsal aorta were exposed by a ventral midline incision. Four regions were arbitrarily designated, as shown in Fig. 1, and their in situ lengths determined before excision of the whole vessel. Experiments were performed on these aortic segments at room temperature within a few hours of death.

Mechanical testing Artery segments (about 5 cm long) were placed in a chamber containing amphibian saline, and cannulated at one end with a blunt 18-gauge syringe needle and connectors leading to a pressure reservoir and a variable-speed pump. Once the air bubbles had been cleared, the distal end and the branch segments wei^ Mechanics of the toad aorta 277

Subclavian artery

Site II

Coeliacomesenteric artery Site III

Site IV

Sciatic artery

Fig. 1. Diagram of the aorta and associated branches in the toad, Bufo marinus, approximately to scale. Four testing sites were used and are labelled as shown. Site I is the proximal aortic arch, from the branching of the truncus arteriosus to the subclavian artery. Site II is the distal aortic arch, between the subclavian artery and the coeliacomesenteric artery. Site in is the proximal dorsal aorta, from the coeliacomesenteric artery to the urogenital arteries. Site IV is the distal dorsal aorta, between the urogenital arteries and the sciatic arteries. The distance from the beginning of the systemic arch to the sciatic artery in a 350g animal is approximately 11 cm. ligated, and the vessel was extended to its in vivo length. The preparations were generally leak-free. Slow inflation-deflation cycles (lasting 1-2 min) were performed using a variable-speed pump. Conditioning cycles (usually 2-3) were run until the pressure-diameter curves were stable. The pressure during one cycle was raised to approximately lOkPa and lowered back to zero. Pressure was measured continuously using a P23Db Gould pressure transducer, while diameter was determined simultaneously with a video dimension analyzer system or VDA (Instrumentation for Physiology and Medicine, model 303), as described by Fung (1981). Some inflation tests were also performed on untethered vessel segments to measure the effect of increasing pressure on longitudinal extension. In this case, lengths were measured for step-wise pressure increments by using a microscope digital micrometer (Wild-Leitz MMS235). Pressure and radius data were collected on-line by a PDP11/23 laboratory computer (Digital Equipment Corporation) for the inflation-deflation cycles at four positions along the vessel from the arches to the sciatic arteries (Fig. 1). Values for radius at zero pressure were taken when the artery was stretched and it was unstretched. Frozen-cut sections were made of each vessel segment 278 C. A. GIBBONS AND R. E. SHADWICK after the inflation tests. From these, wall thickness and internal radius for the unstretched, unpressurized condition were measured using the digital micrometer. Assuming that the vessel wall is a constant-volume material, the internal radius and wall thickness could then be calculated at each pressure from the measured external radius and length. With these values, the luminal volume and circumferential stress, strain and elastic modulus were calculated at 0.5 kPa intervals for each cycle and position, using the laboratory computer. Circumferential wall stress was defined as: a = Pr/h, (1) where r is the inside radius, h is the wall thickness and P is the pressure. The circumferential strain was calculated at mid-wall radius as: e = AR/Ro , (2) where R=(R+r)/2, R is the outer wall radius and Ro is the unstressed mid-wall radius. The elastic modulus describes the relationship between stress and strain and is a measure of material stiffness. For non-linear materials, such as the artery wall, the modulus varies with the level of strain. We used an incremental formula to calculate the elastic modulus (E) from biaxial stress-strain data obtained at constant vessel length (Bergel, 1961; Dobrin, 1983): £ = (l-^)(l + e)(Aa/Ae), (3) where fj. is the Poisson ratio, assumed to be 0.5 (see Dobrin, 1983). This formula uses an incremental strain that is based on the average radius at each pressure increment, and is therefore equivalent to e/(l + e) (Shadwick and Gosline, 1985). Pressure-strain, stress-strain and modulus-pressure relationships for each aortic position were calculated as mean curves from data pooled from several animals. Standard errors were calculated, and the mean curves were compared at each 0.5 kPa interval using multiple comparison tests to determine whether they differed significantly (Zar, 1984). Uniaxial force-extension tests were made on a tensile testing machine (Mon- santo Tensometer T10) in both the circumferential and longitudinal directions. For the circumferential testing, arterial rings were cut with 2 mm widths. They were placed around two metal L-shaped hooks. One hook was anchored at the base, while the other was connected to the force transducer attached to the moveable head. Longitudinal testing was done on vessel segments (2-3 cm long) held in vice- type clamps in the tensometer. Stress was then calculated as o=F/A, where F is the tensile force applied and A is the tissue cross-sectional area perpendicular to the force. Length changes (AL) were measured between surface markers by the video dimension analyzer. The markers were placed in the central region of the specimen to avoid any clamp effects. Strain was calculated as e=AL/L0, where Lo was the initial length. Connective tissue content Flistological sections were made from vessel segments fixed in Bouin's solutio^ Mechanics of the toad aorta 279

(Humason, 1979) at constant pressures ranging from 0 to 4kPa. Sections were stained with aldehyde-fuchsin or Verhoeff s stain to identify elastin fibres. Picro- ponceau or Mallory's Trichrome stain were used to identify collagen and muscle (Humason, 1979). The content and arrangement of the collagen and elastin were then examined in both the transverse and longitudinal directions. Sections were stained from arterial segments at the four arterial sites where inflation tests were made. For comparison, a fresh rat aorta was fixed at OkPa and stained using the same methods. To determine the smooth muscle disposition of the aortic wall, sections were stained with haematoxylin and counterstained with eosin. The elastin content was determined by Lansing's method. Freeze-dried vessel segments were rehydrated and then treated with 0.1 moll"1 sodium hydroxide for 45min at 100 °C, followed by subsequent washings in distilled water. The residue was then dried and weighed (Lansing et al. 1952). The elastin content was this fraction's dry mass expressed as a percentage of the total dry mass. Collagen content was estimated from the hydroxyproline content, which was determined with an automated amino acid analyser (LKB Alpha Plus, model 1215) or by a manual colorimetric assay (Berg, 1982). Values for collagen content were corrected to account for the hydroxyproline present in the elastin according to published information on the composition of elastin in Amphibia (Sage and Gray, 1979). The collagen content was calculated as a percentage of the total dry mass. A collagen to elastin ratio was also calculated.

Results Mechanical properties Upon removal from the animal, the aorta shortened by approximately 30% in each of the designated regions, causing a corresponding increase in diameter. Interestingly, this is comparable to values reported by Bergel (1961) for the dog thoracic and abdominal aorta, which showed shortening of 32-34%. When left untethered during an inflation, the length increased with distending pressure until the upper physiological pressure was reached (Fig. 2). At this point, the aorta had regained its in vivo length. Increasing the pressure beyond the physiological range resulted in virtually no further lengthening. The vessels were stretched to the in vivo length for subsequent inflation tests. A typical inflation cycle (Fig. 3) illustrates the non-linear and viscoelastic behaviour of the aorta. Compliance is relatively high up to the physiological pressure range (2-4 kPa), but thereafter the slope of the pressure-volume curve increases sharply as the artery wall becomes very stiff. The difference between the inflation and deflation portions of this curve indicates that some viscous energy losses occur during each cycle. The area under the ascending curve represents the total work done on inflation, while the area under the descending curve represents the strain energy recovered by elastic recoil. Hysteresis, the proportion of energy lost through viscous processes during each cycle, is the ratio of the area within the Dp to the total area beneath the inflation limb. For the toad aorta, the hysteresis 280 C. A. GIBBONS AND R. E. SHADWICK was low at each of the arterial sites tested (17% in this example). Resilience, the proportion of strain energy recovered, was correspondingly high, at 80-85 %. Fig. 4 shows the mean values of pressure versus circumferential strain from inflation tests of the four arterial sites, as shown in Fig. 1, in 10 animals. These curves are not statistically different, suggesting that the toad aorta has uniform elastic properties along its length. The circumferential strain of about 0.75 at the

1.0 1.2 1.4 1.6 Longitudinal extension (L/Lo)

Fig. 2. A typical longitudinal extension (L/Lo) of an untethered aortic segment upon inflation in vitro. The curve shown is from data for a 4cm segment from site II (see Fig. 1). The resting physiological blood pressure range is indicated by arrows on the pressure axis.

17%

12 3 4 Relative volume (V/K»)

Fig. 3. A typical pressure-volume curve for an inflation-deflation cycle on a vessel segment in vitro. The physiological pressure range is shown by arrows on the pressure scale, while arrows on the curves indicate the direction of loading. The value for mechanical hysteresis is shown beside the loop. Luminal volume was calculated from measured radius values and is expressed relative to the starting volume, Vo. Mechanics of the toad aorta 281

10- I T11

III | IV 8-

i* 6- a - 3

~~o- III III 1 I 1 1 0.2 0.4 0 6 0.8 1.0 Circumferential strain~~

Fig. 4. Pressure-circumferential strain plots from the four sites along the aorta indicated in Fig. 1. Mean values for strain at each pressure increment are plotted from data for 10 animals. The standard errors for each of these plots are also shown. There are no significant differences between the curves at the four testing sites, as indicated by the Mest. mean physiological pressure of 3 kPa is comparable to the level of strain in the rat thoracic aorta at its mean blood pressure of llkPa (Cox, 1978). Stress-strain relationships, calculated from the data in Fig. 4, were all J-shaped and also showed no significant differences among the four arterial sites. The level of circumferential wall stress attained (approximately lxlf^Pa) at mean blood pressure was nearly equal to that in the aorta of the rat at its much higher mean blood pressure (Gibbons and Shadwick, 1989). Fig. 5 shows the incremental elastic modulus as a function of distending pressure. These plots demonstrate that the arterial wall stiffness is highly dependent on pressure and increases considerably over the physiological range, from about 0.2 to 0.7 MPa. At the mean resting blood pressure of 3 kPa, the elastic modulus is approximately 0.4 MPa. This is similar to values reported for the aorta of other poikilotherms (Gibbons and Shadwick, 1989) as well as for the aorta and upper thoracic aorta of mammals (See Fig. 10) at their respective blood pressures. The similarity of the four plots in Fig. 5 indicates that the toad aorta, unlike that of mammals, has uniform elastic properties along its entire length. Potential vascular muscle activators, such as acetylcholine and epinephrine (West and Burrgren, 1984), were applied to the arterial wall and perfused through the vessel in an attempt to stimulate muscle activity. These tests were made within a few minutes of death. Although many different concentrations of these drugs were tested, the dorsal aorta showed no measurable mechanical response to these chemicals. The stress-strain curves resulting from uniaxial tests (Fig. 6) show that the aorta 282 C. A. GIBBONS AND R. E. SHADWICK is much stiffer longitudinally than drcumferentially. This is in agreement with biaxial tests in which changes in pressure produced larger strain increments circumferentially than longitudinally (Figs 2, 4).

~~Structure and histology Table 1 shows average dimensions of the outside radius (R) and wall thickness~~

~~2 4 6 Pressure (kPa)~~

Fig. 5. The incremental elastic modulus as a function of inflation pressure for the four arterial sites along the aorta shown in Fig. 1. No significant differences were found between the means at any pressure level. Arrows on the curves indicate the resting physiological pressure range. Standard errors are shown; JV=10.

~~IV I 0.4-! IV III,~~

~~03-~~

~~0.2-~~

~~0.1-~~

~~0.4 0.6 Strain~~

Fig. 6. Uniaxial longitudinal and circumferential stress-strain curves for each of the four arterial sites indicated in Fig. 1. Typical plots are shown from the data obtained by tensile tests on longitudinal segments or arterial rings. Mechanics of the toad aorta 283

Table 1. Average values for radius (R), wall thickness (h) and the R/h ratio at a mean pressure of 3 kPa from six different animals at the four arterial sites R h Elastin Collagen Site (mm) (mm) R/h (%) (%) I 1.34 0.057 23.5 15 34 n 1.23 0.054 22.8 15 35 in 1.29 0.058 22.2 14 35 IV 1.20 0.051 23.5 14 35

Elastin and collagen contents are shown as a percentage of total dry mass along the aorta. Each value is the mean of four determinations, each containing arterial segments pooled from at least 10 animals.

(h) of the toad aorta at a mean pressure of 3 kPa. Both R and h are important in determining the mechanical properties of the arterial wall. This aorta is surprisingly uniform in these dimensions and in the R/h ratio along its length, i.e. there is virtually no 'geometric taper' as there is in the mammalian aorta (see Fig. 9). The average radius of 1.27 mm and wall thickness of 0.055 mm give an R/h ratio of 23 at mean blood pressure. In contrast, the R/h ratio for a mammalian thoracic aorta at mean blood pressure is only about 10 (McDonald, 1974). The relative thinness of the toad aorta is illustrated in Fig. 7; while the diameter of the aorta from the toad and a similar-sized rat are about the same, the wall of the former is only half as thick as that of the latter. In both the transverse and longitudinal sections of the toad aorta, the elastin (stained black) appears as 4-5 layers within the wall (Fig. 7). These sections together show that the elastin must be arranged as concentric cylindrical sheets. In uninflated vessels, the luminal elastica is thicker and wavier than the other elastin layers found throughout the wall. Smooth muscle cells, indicated by the stained nuclei in Fig. 7D, are not very abundant. In both transverse and longitudinal planes, the elastin layers were relatively thin and sometimes appeared incomplete or fused to adjacent layers. In a rat aorta of similar diameter, the elastin lamellae were more numerous (9-11), more uniform and well-defined, and thicker than in the toad aorta (Fig. 7). In the latter there is no distinct outer layer of collagen; rather, it occurs in large amounts among the elastin lamellae. There is relatively less elastin and more collagen in the arterial wall of the toad than in that of the rat. Fig. 8 shows sections of toad aorta fixed at pressures of 1.0, 1.7, 2.5 and 4.0 kPa. With increasing pressure, the elastin fibres become straighter and more taut as they are loaded within the physiological pressure range. Concomitantly, the interlamellar space and lamellar thickness decrease. The elastin content of the toad aorta was approximately 15 % of the total dry mass, while the collagen content was about 35 % of the dry mass. These values were consistent at all four sites (Table 1), indicating that the aorta has a uniform imposition along its length. The collagen to elastin ratio of 2.3 in the toad aorta is 284 C. A. GIBBONS AND R. E. SHAD WICK

~~/ ir»~~

Fig. 7. Micrographs of a toad aorta fixed at OkPa and stained for elastin, showing a transverse section (A) and a longitudinal section (B). For comparison, a transverse section of a rat aortic wall is shown in C. (D) A transverse section of a toad aorta fixed at lOkPa and stained to show cell nuclei. / indicates the luminal side of the wall. Scale bar, 50^m. more than five times that found at the level of the aortic arch in mammals (see Fig. 10).

Discussion This study describes several important mechanical design features of the aorta of a typical poikilothermic vertebrate. The high distensibility, resilience and non- linear elasticity are the requisite properties for the vessel to act as an effective storage element in the arterial circulation. In this respect, a strong functional similarity to the aorta of mammals is apparent, although specific structural and mechanical differences are seen that reflect the much lower level of pressure and simpler haemodynamics for which the toad vessel is suited. Mechanics of the toad aorta 285

Fig. 8. Micrographs of transverse sections of the aorta from four different toads, fixed at distending pressures of 1.0kPa (A), 1.7kPa (B), 2.5 kPa (C) and 4.0kPa (D), and stained to show the elastin lamellae. Scale bar, 50/an. /, lumen.

Our initial observation that a longitudinal recoil of about 30 % occurred when the aorta was excised is surprisingly similar to the behaviour of the aorta of mammals. With inflation to the normal physiological pressure of about 3kPa, an unrestrained toad aorta regains its in vivo length (Fig. 2), i.e. the longitudinal stress due to pressure alone causes the lengthening, and tethering to the axial skeleton is unimportant. In fact, there are no branch vessels between the subclavian and coeliacomesenteric arteries, and very few along the rest of the aorta. During a normal pressure pulse, the aorta will lengthen only slightly, owing to the relatively limited longitudinal extensibility at pressures above 2kPa. Similarly, in the mammalian thoracic aorta, longitudinal strains of only 1 % or less occur with each heart beat (Patel and Fry, 1964), although this behaviour has generally been attributed to a high degree of tethering by the intercostal arteries and connective tissues (Milnor, 1982). However, Van Loon et al. (1977) demon- ^rated that there is a maximal length to which mammalian arteries elongate when 286 C. A. GIBBONS AND R. E. SHADWICK

pressurized without tethering, and that this is very close to the in vivo length. Thus, to a large extent, the relatively constant aortic length in vivo results from the mechanical anisotropy of the wall material, i.e. the stiffness is greater longitudinally than it is circumferentially (Fenn, 1957; Patel et al. 1969; Vaishnav et al. 1972; Dobrin, 1983). This type of anisotropy in the toad aorta is evident from the uniaxial tests shown in Fig. 6. Interestingly, similar mechanical behaviour has been observed for the aorta of octopods and squid (Gosline and Shadwick, 1982; Shadwick and Gosline, 1985; Shadwick and Nilsson, 1990), soft-bodied animals in which no skeletal tethering of the aorta is possible. The non-linear and viscoelastic properties of the toad aorta reflect its composite structure; it consists mainly of highly extensible elastin and relatively inextensible collagen. With inflation, the elastic lamellae are loaded and become straightened as the pressure approaches the physiological range (Fig. 8). Above 3kPa there is an abrupt decrease in the vessel distensibility and an increase in the circumferential elastic modulus (Figs 3, 5), above what could be attributed to elastin alone (Aaron and Gosline, 1981). In the mammahan aorta, an abrupt transition from high compliance to high stiffness also occurs, but at much higher mean blood pressures of 11-12 kPa (Bergel, 1961; Gibbons and Shadwick, 1989). In both toad and mammal, inflation of the thoracic aortas to their respective physiological mean pressures results in circumferential strains of 0.6-0.8, an elastic modulus of about 0.4 MPa and mechanical hysteresis of 15-20% (Bergel, 1961; Cox, 1978; Gibbons and Shadwick, 1989). Thus, the aorta of the toad appears to be designed to have essentially the same functional properties as that of mammals, but at much lower pressures. A significant finding in this study is that, in contrast to mammals, there is no geometric or elastic 'tapering' in the arterial tree of the toad. The dimensions and elastic properties are relatively uniform along the length of the aorta, whereas the mammahan aorta exhibits a progressive decrease in radius and increase in elastic modulus peripherally (Figs 9, 10). The distribution of mechanical properties in these aortas is correlated with their connective tissue composition, i.e. the toad aorta has a relatively constant quantity of elastin and collagen along its length, while in the mammahan aorta an increase in the ratio of collagen to elastin occurs distal to the heart (McDonald, 1974; Fig. 10). In the mammahan system, this elastic and geometric tapering together cause a continuous increase in pressure- wave velocity and aortic impedance distal to the heart, giving the aorta the properties of a non-uniform transmission line (McDonald, 1974; Milnor, 1982). The lack of any significant elastic or geometric taper in the toad aorta, by contrast, suggests that this vessel is more suited to function as a simple Windkessel than as a complex transmission line. These haemodynamic features are considered in more detail in the following paper. The question of how the non-linear elastic properties occur at much lower pressures in the toad aorta than in that of the mammal can be addressed by considering structural and connective tissue compositional differences. The aortic R/h ratio is nearly 2.5 times higher in the toad, resulting in higher wall stresj| Mechanics of the toad aorta 287

(equation 1) and, consequently, higher modulus for any given pressure than in the mammalian vessel. The higher fraction of collagen in the former may also contribute to the transition from high to low compliance at the relatively low physiological pressure in the toad compared to that in the mammalian system. In a comparative study, Wolinsky and Glagov (1967) established that the number of

~~1.25-1~~

~~1.0- Toad~~

~~2 0.75-~~

~~1 0.50-~~

~~0.25-~~

~~Arch Thoracic Abdominal Relative position~~

Fig. 9. A comparison of the change in aortic radius with increasing distance from the heart, for the toad and the dog. In both cases the external radius at mean blood pressure was normalized to the maximal value, which occurs at the top of the arch, and was plotted as a function of the relative position along the aortic tree. Values for the dog aorta were taken from McDonald (1974).

~~1.5-i~~

~~1.2- 2.5~~

~~a. 20 ? S 0.9- C 15 | 0.6- c QJ oo LL) n3 0.3- -0.5 ^~~

~~Arch Thoracic Abdominal -0 Relative position~~

Fig. 10. A comparison of the elastic modulus at mean blood pressure (solid lines), and the connective tissue content (broken lines) as a function of the relative position along the arterial tree, for the toad and the dog. Values for the dog aorta were taken from Milnor (1982). 288 C. A. GIBBONS AND R. E. SHADWICK elastin layers, N, in the mammalian aorta was a function of body size, or specifically of aortic radius at mean blood pressure. Consequently, the circumferential wall tension per layer (=Pr/N) remains nearly constant at about 2 Pa m in all species. In contrast, the quantity and mechanical behaviour of the elastin layers in the toad aorta do not fit this general pattern, instead becoming straightened at a relatively low pressure (Fig. 8) that generates a tension per layer of only about 0.5Pam. In conclusion, specific differences in structure, connective tissue architecture and mechanical properties are demonstrated in the toad aorta compared to that of mammals. Overall, however, the mechanical behaviour of the aorta is functionally comparable in the two taxa, although their range of physiological pressures is very different. Thus, the aorta has the same capability to act as an effective elastic energy storage element in the circulation of this poikilothermic vertebrate as in that of mammals. The contribution of these elastic properties to arterial haemodynamics is explored in the following study.

This work was supported by a Graduate Research Grant from the University of Calgary to C.A.G. and by an operating grant and Research Fellowship from the Natural Science and Engineering Research Council of Canada to R.E.S.

References AARON, B. B. AND GOSLINE, J. M. (1981). Elastin as a random network elastomer. Biopolymers 20, 1247-1260. BERG, A. R. (1982). Determination of 3- and 4-hydroxyproline. Meth. Enzymol. 82, 372-398. BERGEL, D. H. (1961). The static elastic properties of the arterial wall. /. Physiol., Lond. 156, 458-469. Cox, R. H. (1978). Comparison of carotid artery mechanics in the rat, rabbit, and dog. Am. J. Physiol. 234, H280-H288. DOBRIN, J. B. (1983). Vascular mechanics. In Handbook of Physiology, section 2, The Cardiovascular System, vol. II (eds. D. F. Bohr, A. P. Somylo and H. V. Sparks, Jr), pp. 65-102. Bethseda, MD: American Physiological Society. FENN, W. O. (1957). Changes in length of blood vessel on inflation. In Tissue Elasticity (ed. J. Remington), pp. 154-167. Washington: American Physiological Society. FUNG, Y. C. (1981). Biomechanics. Mechanical Properties of Living Tissues. New York: Springer-Verlag. GIBBONS, C. A. AND SHADWICK, R. E. (1989). Mechanical design in the arteries of lower vertebrates. Experientia 45, 1083-1089. GIBBONS, C. A. AND SHADWICK, R. E. (1991). Circulatory mechanics in the toad Bufo marinus. II. Haemodynamics of the arterial Windkessel. /. exp. Biol. 158, 291-306. GOSLINE, J. M. AND SHADWICK, R. E. (1982). The biomechanics of the arteries of Nautilus, Notodarus, and Sepia. Pacific Sci. 36, 283-2%. GOTO, M. AND KIMOTO, Y. (1966). Hysteresis and stress-relaxation of the blood vessels studied by a universal tensile testing instrument. Jap. J. Physiol. 76, 169-184. HUMASON, G. L. (1979). Animal Tissue Techniques. San Francisco: W. H. Freeman and Company. JONES, D. R., LANGILLE, B. L, RANDALL, D. J. AND SHELTON, G. (1974). Blood flow in dorsal and ventral aortas of the cod, Gadus morhua. Am. J. Physiol., Lond. 226, 90-95. LANGILLE, B. L. AND JONES, D. R. (1977). Dynamics of blood flow through the hearts and arterial systems of anuran amphibia. J. exp. Biol. 68, 1-17. LANSING, A. I., ROSENTHAL, T. B., ALEX, M. AND DEMPSEY, E. W. (1952). The structure and| Mechanics of the toad aorta 289 chemical characterization of elastic fibres as revealed by elastase and electron microscopy. Anat. Rec. 114, 555-575. MCDONALD, D. A. (1974). Blood Flow in Arteries. London: Edward Arnold. MILNOR, W. R. (1982). Hemodynamics. Baltimore: Williams and Wilkens. PATEL, D. J. AND FRY, D. L. (1964). In situ pressure-radius-length measurements in the ascending aorta of anaesthetized dogs. /. appl. Physiol. 19, 413-416. PATEL, D. J., JANICKI, J. S. AND CAREW, T. E. (1969). Static anisotropic elastic properties of the aorta in living dogs. Circulation Res. 25, 765-769. SAGE, E. H. AND GRAY, W. R. (1979). Studies on the evolution of elastin. I. Phylogenetic distribution. Comp. Biochem. Physiol. 66B, 313-327. SATCHELL, G. H. (1971). Circulation in Fishes. Cambridge: Cambridge University Press. SHADWICK, R. E. AND GOSLLNE, J. M. (1985). Mechanical properties of the octopus aorta. /. exp. Biol. 114, 259-284. SHADWICK, R. E. AND NILSSON, E. K. (1990). The importance of vascular elasticity in the circulatory system of the cephalopod Octopus vulgaris. J. exp. Biol. 152, 471-484. SHELTON, G. AND BURGGREN, W. W. (1976). Cardiovascular dynamics of the chelonia during apnoea and lung ventilation. J. exp. Biol. 64, 323-343. SHELTON, G. AND JONES, D. R. (1968). A comparative study of central blood pressures in five amphibians. /. exp. Biol. 49, 631-643. TAYLOR, M. G. (1964). Wave travel in arteries and the design of the cardiovascular system. In Pulsatile Blood Flow (ed. E. O. Attinger), pp. 343-372. New York: McGraw-Hill. VAISHNAV, R. N., YOUNG, J. T., JANICKI, J. S. AND PATEL, D. J. (1972). Non-linear anisotropic elastic properties of the canine aorta. Biophys. J. Yl, 1008-1027. VAN LOON, P., KLIP, W. AND BRADLEY, E. L. (1977). Length-force and volume-pressure relationships of arteries. Biorheology 14, 181-201. WEST, N. H. AND BURGGREN, W. W. (1984). Factors influencing pulmonary and cutaneous arterial blood flow in the toad, Bufo marinus. Am. J. Physiol., Lond. 247, R884-R894. WOLINSKY, H. AND GLAGOV, S. (1964). Structural basis for the static mechanical properties of the aortic media. Circulation Res. 14, 400-413. WOLINSKY, H. AND GLAGOV, S. (1967). A lamellar unit of aortic medial structure and function in mammals. Circulation Res. 20, 99-111. ZAR, J. H. (1984). Biostatistical Analysis. New Jersey: Prentice-Hall.