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Atherosclerosis, 32 (1979) 397-402 @ Elsevier/North-Holland Scientific Publishers, Ltd.

SEASONAL VARIATIONS IN THE BIOPHYSICAL PROPERTIES OF RABBIT AORTA AND ITS SUSCEPTIBILITY TO ARTERIOSCLEROSIS

H. OXLUND, P. HELIN and I. LORENZEN Anatomy Department C, University of Aarhus, and Division of Immunology and Rheumatology, Depcrtment of Medicine, Hvidovre Hospital, University of Copenhagen (Denmark) (Received 17 July, 1978) (Revised, received 18 October, 1978) (Accepted 18 October, 1978)

The physiological variations in the mechanical properties of rabbit aortae in relation to the periods of hair shedding were studied. The load-strain curves of the eight proximal thoracic segment in 15 shedding and 15 non-shedding male albino rabbits were analysed. The slope of the curves (tangent of the angle between the linear region of the load-strain curve and the strain axis) was decreased in the shedding animals compared to that of the non-shedding animals and the toe of the load-strain curves was significantly lower towards the x-axis in the shedding animals. The observations indicate a lower stiffness, that is increased elasticity, of the aortae of rabbits during hair shedding. The increased elasticity during hair shedding may explain the previously reported resistance to experimental arteriosclerosis caused by the hemodynamic strain elicited by exposure to systemic hypoxia. A decrease in the aortic content of collagen and of sulfated glycosaminoglycans, and an increase in the content of hyaluronic acid, may be of importance in the alterations of the mechanical properties of rabbit aorta during hair shedding. Key words:

Arteriosclerosis - Collagen - Giycosaminoglycans Susceptibility to atherosclerosis

Reprint requests to Hvidovre

Hospital,

- Mechanical properties

P. Helin, Division of Immunology and Rheumatology, Kette&ds Alle 30,265O Hvidovre. Denmark.

Department

--

of Medicine,

398

Introduction In a previous study Helin et al. [l] demonstrated a decreased susceptibility of the aortic wall to injury in rabbits during the period of hair shedding. The injury was elicited by intermittent exposure of the rabbits to systemic hypoxia which caused a short-lasting arterial hypertension followed by dilatation and stretching of the aorta [2]. In a subsequent investigation [3] it was observed that the content of hyaluronic acid in the aortic wall of intact rabbits was increased while the content of sulfated glycosaminoglycans and hydroxyproline was decreased during hair shedding compared to aortae of non-shedding animals. On the basis of these observations it was suggested that the biochemical alterations could lead to changes in the mechanical properties of the aortic wall. Such changes during the period of hair shedding may imply an improved resistance to the increased hemodynamic strain induced by the arterial hypertension elicited by the exposure to systemic hypoxia. The present study was therefore undertaken to analyse the mechanical properties of the thoracic aorta in intact rabbits during and outside the period of hair shedding. Materials and Methods The study included two groups, each of 15 male albino rabbits of the Danish country strain. One group was studied between 19 August and 24 August, outside the period of hair shedding. The other group was studied between 28 September and 12 October, when the animals had been shedding for one or two weeks. The animals were 5 months old at the time of death. They were fed standard laboratory pellets containing 12% protein, 54% carbohydrate and 4% fat with only traces of cholesterol. The temperature in the animal quarters was 20°C. The daily light period for the shedding animals was 6 h in contrast to the 13 h for the non-shedding animals, The animals were killed by i.v. injection of 300 mg pentobarbital. The thoracic aorta was dissected free and transferred immediately to a Krebs-Ringer bicarbonate solution at 37°C (pH 7.4) saturated with 95% oxygen and 5% carbon dioxide. The thoracic aorta was placed on a filter paper, the adventitial surface downwards. Eight strips, 6 mm wide, were cut from each aorta between the intercostal arteries and at a right-angle to the axis of the aorta. The filter paper with the specimen was fixed between two clamps. The distance between the clamps was 6.5 mm. The filter paper was then cut and the aortic specimen gradually stretched by increasing the load on the movable clamp. The loading was achieved by pumping water into a bag attached to the clamp at a constant rate, 38 g/min. The resulting deformation of the specimen, that is increase in strip length, was continuously recorded by means of a linear potentiometer, the signal being fed to a strip-chart recorder. The deformation curves were read on-line into a computer by a digitizer and load-strain curves were computed for the specimens from each animal as well as for each group [ 51. Strain values were calculated by normalizing the deformation values to units of the original length of the specimen. The original length was calculated as the sum of the distance between the clamps, 6.5 mm,

and the deformation that occurred before a load value of 0.04 newton was reached. Load values were calculated on the basis of a constant loading rate, 38 g/min. The load-strain curve has a toe-part, followed by a part which is fairly linear before it bends off towards the strain axis and reaches the point of maximum load and failure [4,6]. From the load-strain curves the following parameters were calculated: (1) the strain and load value, respectively, for the beginning of the linear region (eLB, FL& (2) the strain and load value for the end of the linear region (eLE, FLE); (3) the strain and load value for the point of maximum load (eFmax, F ,,,); and (4) the elastic stiffness -tangent of the angle between the linear region of the load-strain curve and the strain axis (tan ff). For statistical analysis the Student’s t-test was used. Differences were regarded as statistically significant at the 5% level of probability (two-tailed test). Results The body weights of the animals in the two groups did not differ. The shedding animals weighed 3.13 + 0.06 kg, the non-shedding 3.11 + 0.06 kg (mean + SEM). The mean load-strain curves from the strips are shown in Fig. 1. In Table 1 the data from the curves are given. No significant differences were found between strain values at the beginning or at the end of the linear part of the curve in the two groups, nor at maximum load. Neither were any differences found between load values for the beginning and the end of the linear part of the curve. The maximum load value of the shedding group tended (2P < 0.10) to be reduced compared with the non-shedding group. The toe-part for the aortae in the shedding group (Fig. 2) was significantly lowered towards

,_+ non-shedding

Oi

0:6

0:6

1.b strain (elongation per original length)

Fig. 1. Load-strain curves for thoracic aorta calculated from the mean values of eight cranial intercostal segments of 15 non-shedding and 15 shedding male rabbits. The beginning and the end of the linear region and the maximum values are indicated by mean 2 SEM.

400 TABLE 1 LOAD-STRAIN VALUES FOR THORACIC VALUES FOR 8 CRANIAL INTERCOSTAL AND 15 SHEDDING RABBITS

RABBIT AORTA CALCULATED SEGMENTS OF AORTAE FROM

Thoracic aorta

Non-shedding

Shedding

eLB eLE eF ’ max FLB FLE Fmax tano.

0.14 0.88 1.03 1.30 1.91 2.21 4.14

0.15 0.94 1.03 1.18 1.85 2.02 3.62

* 0.03 ?r 0.03 * 0.02 + 0.06 k 0.08 ? 0.07 + 0.25

k * f r * f +

FROM THE MEAN 15 NON-SHEDDING

0.02 0.03 0.02 0.05 0.01 0.08 * 0.11 **

The values given are mean ? standard error of mean * 2P < 0.1. ** 2P < 0.01.

Load (Newton) 1 non-shedding shedding

I 0.1

0.2

0.3

0.4

0.5 Strain (elongation per original length)

Fig. 2. Toe-part of the load-strain curves for thoracic aortae. SEM for the load values for each strain increment of 0.02.

the strain axis compared with that for the non-shedding group. Tan a (that is the inclination of the linear part of the curve) was also significantly decreased for the shedding group compared with the non-shedding group. Discussion The aorta act8 as an elastic reservoir which is filled during cardiac systole and is in part emptied during diastole according to the windkessel effect. The magnitude of the distension of the aorta during each systole is determined by the mechanical properties of the aortic wall. The aorta becomes stiffer with age, and the storage function of the arterial tree is decreased. The result is a higher pulse pressure. In the present study the lowered toe-part and the reduced inclination of the linear part of the load-strain curve of the aorta of rabbit8 during the hair-shedding period compared to that outside this period demonstrates a decreased stiffness of the aortic wall during shedding. When the stiff-

401

ness of the aorta is reduced the increase in pulse pressure in the aorta during the hypertension induced by systemic hypoxia [2] will be smaller and the hemodynamic strain of the aortic wall consequently lower. The findings of the present study may therefore support our hypothesis of an alteration in the mechanical properties of the aorta as an explanation of the resistance to experimental arteriosclerosis observed in a previous study [l] during shedding. At normal blood pressure the region of interest of the load-strain curve is the initial, toe, part [7]. The static mechanical properties of the aorta are mainly attributed to the elastin and collagen components. The initial slope of the loadstrain curve corresponds mainly to the elastic fibers and the final slope, when the wall is stretched, to the collagenous fibers. As the load is increased collagenous fibers bear more and more of the load giving the aortic wall a much higher modulus of elasticity [8]. This concept of the function of different components of the aortic wall is supported by Hoffmann et al. [9], who studied the contribution of the individual components to the mechanical properties of the whole tissue by removing each component selectively by means of enzymes. They found that collagen contributed to stress development throughout the entire strain region, but especially in the high-strain region, while elastin was found to be the major stress-bearing component in the lowstrain re’gion. Obrink [lo] studied the interactions between collagen and glycosaminoglycans and found that dermatan sulphate preparations completely precipitated collagen molecules from solution, accelerated fiber formation and influenced the polymerization pattern of collagen molecules in vitro. If a corresponding effect of dermatan sulphate could occur in vivo, its reduced content might influence aggregation and polymerization of newly synthesized collagen. At present it is difficult to explain the mechanical findings in our study on the basis of the biochemical alteration in the rabbit aorta found during the shedding period. Information on proportional amounts of collagen and elastin and also on the chemical characteristics and structural arrangement of these proteins is needed. However, the reduced stiffness in the high-strain region corresponds well with the reduced hydroxyproline content. Due to the viscoelastic properties of hyaluronic acid the increased content of this glycosaminoglycan during shedding may also contribute to a lowered stiffness of the aortic wall. However, changes in viscoelastic properties caused by changes in glycosaminoglycans are probably more easily detectable at a higher velocity than that used in stress-strain studies due to the rheological properties of the glycosaminoglycans [ll]. Under in vivo conditions the primary mechanical stress is supported by the collagen and elastic fibers. Secondarily, however, part of the energy is transferred to the glycosaminoglycans for storage and dissipation through frictional coupling between the two biological networks, the collagen and elastic fibers on the one hand and glycosaminoglycans on the other. The present study has demonstrated a physiological variation in the mechanical properties of the aortic wall in rabbits. The stiffness of the aorta is reduced during the hair-shedding period compared to that outside this period. The information available does not allow any conclusive linkage of the biomechanical and biochemical changes but the alterations in the content of collagen and the glycosaminoglycans may be of importance. Changes in the bio-

402

mechanical properties in the collagenous as well as pharmacological means, such costeroid administration [6,13], suggest the molecular stability of the components

framework induced by physiological as during pregnancy [12] and cortithat not only the proportions but also is of importance.

References 1 HeIin, P., Lorenzen, I., Garbsrsch, C. and Matthiesscn, M.E., Relative immunity to arteriosclerosis in rabbit during the hair-shedding period, J. Atheroscler. Res.. 10 (1969) 369-369. 2 HeIin, P. and Loranzen, I.. Arteriosclerosis in rabbit aorta induced by systemic hypoxia, Angiology. 20 (1969) l-12. 3 Hehn. P. and Lorenzen. I., Seasonal variations in the susceptibility of the aortic waB to arterioscleroais, Atherosclerosis, 24 (1976) 269-266. 4 Viidik. A., Functional properties of collagenotis tissues. In: D.A. HaB and D.S. Jackson (Eds.). hit. Rev. Conn. Tim. Res.. Vol. 6. Academic Press. New York, NY, pp. 127-215. 5 Andreassen. T.T., Fogdestam. I. and Rundgrcn, A., A biomechanical study of healing of skin incisions in rats during pregnancy, Surg. Gynecol. Obstet.. 146 (1977) 175-178. 6 Oxlund. H.. The influence of corticotropin on temporal changes in the biomechanical properties of skin and aorta. In: U.J. Schmidt (Ed.), 5th European Symposium on Basic Research in Gerontology, Perimed. ErIangen. 1977. pp. 342-350. 7 BjBrkerud. S.. Viidik, A. and Bondjers, G.. Mechanical properties of structurally defined regions of aortic atherosclerotic lesions compared with normal aorta in the rabbit, In manuscript. 8 Burton, A.C., Relation of structure to function of the tissues of the wall of blood vessels, Physiol. Rev., 34 (1954) 619-642. 9 Hoffmann, A.S.. Grande. L.A.. Gibson, P.. Park, J.B.. Daly. C.H.. Bornstein, P. and Ross, R., Sequential ensymolysis of human aorta and resultant stress-strain behavior. In: R.M. Kenedi (Ed.), Perspectives in Biomedical Engineering, MacmiBan. New York, NY, 1973. PP. 173-176. 10 Gbrink, B.. Studies on the interactions between collagen and glycosaminoglycans, Acta Univ. Ups&. 108 (1971). 11 Balass, E.A. and Gibbs, D.A., The rheological properties and biological function of hyaiuronic acid. In: E.A. BaIazs (Ed.), Chemistry and Molecular Biology of the InterceIiuIar Matrix. Vol. 3. Academic Press, New York, NY, 1970, PP. 1241-1253. 12 Rundgren, A., Physical properties of connective tissue as influenced by single and repeated pregnancies in the rat, Acta Physiol. &and., Suppl. 417 (1974). 13 Vogel, H.G., Correlation between tensile strength and collagen content in rat skin - Effect of age and cortisol treatment, Corm. Tiss. Res.. 2 (1974) 177-182.

Seasonal variations in the biophysical properties of rabbit aorta and its susceptibility to arteriosclerosis.

397 Atherosclerosis, 32 (1979) 397-402 @ Elsevier/North-Holland Scientific Publishers, Ltd. SEASONAL VARIATIONS IN THE BIOPHYSICAL PROPERTIES OF RAB...
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