/. 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 coeliaco- mesenteric 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 per- formed 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 circumfer- ential 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.