Aortic Distensibility and Structural Changes in Sinoaortic-Denervated Rats
Abstract The purpose of the present study was to determine the effects of chronic sinoaortic denervation on the mechanical properties and composition of the abdominal aorta in Wistar rats. We used a high-resolution echotracking system to determine in situ under physiological conditions of blood flow and arterial wall innervation the aortic diameter-, compliance-, and distensibility-pressure curves in 16-week-old anesthetized rats that had been denervated at 10 weeks of age for 6 weeks (n=8). Compared with sham-operated rats (n=8) we observed a marked reduction of baroreflex response and increase in overall mean blood pressure variability as measured by standard deviation and spectral analysis in sinoaortic-denervated rats. Mean blood pressure was not affected by sinoaortic denervation in both conscious and anesthetized rats. Sinoaortic denervation significantly shifted the distensibility-pressure curve toward lower levels of distensibility, indicating a decreased aortic distensibility for a given level of arterial pressure. Sinoaortic denervation produced a significant increase of aortic wall cross-sectional area and collagen content, one of the less-distensible components of the arterial wall. These results suggest that intact arterial baroreceptors are necessary for maintaining normal functional and structural properties of large arteries in rats. The reduction in arterial distensibility in chronic sinoaortic-denervated rats may have resulted from different factors, including the initial hypertensive phase, aortic wall hypertrophy, and increase in collagen content. The changes in aortic wall structure and related reduction in aortic distensibility, in addition to other mechanisms, might have been direct consequences of an increased blood pressure variability.
Increased APV is produced by SAD.1 2 3 Although the mechanisms involved in the generation and maintenance of APV remain largely unknown, increased APV in chronic SAD is usually attributed to increased variability in peripheral vascular resistance without increased MAP.3 4 Jacob et al5 demonstrated that 2 weeks after SAD in rats, increased APV was abolished by short-term ganglionic blockade combined with either captopril or a V1 vasopressin receptor antagonist and fully restored by vasoconstrictor agents such as phenylephrine, angiotensin II, epinephrine, and vasopressin. These results suggest that changes in vascular tone of neural and/or humoral origin are critical for the increased APV. To our knowledge, little attention has been focused on the structural and functional modifications of large arteries in chronic SAD, which may be altered by several factors including the neurohumoral changes observed in this model and the increased APV per se. The purpose of this study was to investigate the long-term effects of SAD on the mechanical properties of the rat abdominal aorta, via the in situ determination of diameter- and distensibility-pressure curves, and on the composition of the aorta.
All procedures were in accordance with institutional guidelines for animal experimentation. SAD was performed at 10 weeks of age in anesthetized male Wistar rats (Iffa-Credo) with the use of the method of Krieger with slight modifications.2 3 4 5 The effectiveness of SAD was studied 24 hours after the SAD procedure and 6 weeks later. For methodological reasons this was studied in two different rat groups. Twenty-four hours after SAD and intra-aortic catheter implantation, conscious rats (n=6) responded with bradycardia of less than 25 beats per minute to an increase in arterial pressure of at least 45 mm Hg induced by phenylephrine (5±1 μg). At 16 weeks of age the tachycardic and bradycardic responses to phenylephrine (6±1 μg) and sodium nitroprusside (5±1 μg) were studied in SAD (n=8) and sham (n=8) rats (Table 1⇓) 24 hours after a catheter was implanted in the lower abdominal aorta. Phenylephrine and sodium nitroprusside were administered at the same concentration in sham and SAD rats. At 16 weeks of age the weight of SAD rats (428±13 g) was significantly lower compared with that of sham rats (464±7 g, P<.05).
At week 16 BP was measured in the abdominal aorta in conscious rats and continuously recorded over a 1-hour period after at least a 30-minute equilibration period. Thirty minutes of the signal was then sampled at 1 kHz with a 12-bit analog-to-digital convertor and stored on an PC-486 microcomputer. An algorithm has been developed to identify the cardiac cycles and to calculate for each of them the values of pulsatile, diastolic, and systolic pressures; MAP; and the heart period (milliseconds). Bad data are removed through a statistical analysis; beat-to-beat records are split into 256-beat blocks. For each block, mean value and SD are computed for heart period; pulse, diastolic, and systolic pressures; and MAP. Beats with at least one of these parameters out of the interval of mean±3.3 SD (99.9% confidence interval for normal distribution) are removed. The SD of MAP, calculated beat-by-beat during the entire acquisition period, was used as an index of APV.
MAP spectral analysis was performed with a fast Fourier transform algorithm.6 7 8 The analysis consisted of the following steps: (1) the entire signal was interpolated with cubic splines; (2) the entire recording period was split into 100-second intervals without overlapping and without any more filtering; (3) for each interval the signal was resampled on 512 points (sample rate, 5.12 Hz close to the heart rate in rats); (4) the spectrum was computed on each interval and the average spectral modulus (mm Hg/Hz1/2) calculated over the 30-minute period; and (5) the power spectrum (mm Hg2/Hz) was also computed and the quantification obtained through the value of the integral of the power spectrum (mm Hg2) in the very-low-frequency band (0.015 to 0.25 Hz), the low-frequency band (0.25 to 0.75 Hz), and the high-frequency band (0.75 to 2 Hz).
Determination of the Diameter-Pressure Relationship
Twenty-four to 48 hours after the above measurements, the diameter-pressure relationship was established from the simultaneous recording of arterial diameter and BP in pentobarbital-anesthetized rats. The technique of arterial diameter measurement, with the use of an ultrasonic echotracking device (NIUS-01, Asulab SA), has been previously described in humans and rats.9 10 11 From the two simultaneous and continuous signals of pulsatile changes in arterial diameter and BP, the computerized acquisition system fits the diameter-pressure curve within the diastolic-systolic range of BP and then calculates the compliance- and distensibility-pressure curves. The relationship between the pressure, P, and the lumen cross-sectional area (LCSA) was fitted with the model of Langewouters et al9 12 with an arctangent function and three optimal-fit parameters (α, β, and γ):
Composition of the Abdominal Aorta
The rat abdominal aorta was fixed (saline solution with 4% formaldehyde) at each rat’s MAP to provide the tissue fixation closest to the physiological in situ state of the vessel. Three successive sagittal sections of 5-μm thickness were treated by specific staining to obtain a monochromatic color associated with the various structures studied in the aortic media. Sirius red was used for collagen staining, orcein for elastin, and hematoxylin after periodic acid oxidation for nucleus staining. As previously described, aortic thickness and composition were quantified with an automated image processor (NS 1500, Nachet-Vision) based on morphological principles.13
All values were averaged and are expressed as mean±SEM. An unpaired Student’s t test was performed to compare SAD rats with sham-operated rats. Differences were considered significant at values of P<.05. To compare diameter at the same BP level in SAD and sham-operated rats, we calculated the area under the curve of each diameter-pressure curve for the pulse pressure range common to both groups (108 to 132 mm Hg). We then compared the mean±SD of the area under the curve of SAD rats with that of sham-operated rats by an unpaired Student’s t test as though they were raw data. We did the same analyses for comparing compliance and distensibility between groups.
Effects of SAD on APV and Baroreflex Function
Table 1⇑ shows the effects of 6 weeks of SAD on MAP and heart rate. Compared with values in control rats, MAP and heart rate remained unchanged in SAD rats, and the bradycardic and tachycardic responses to phenylephrine and sodium nitroprusside, respectively, were markedly reduced. APV, assessed from the SD and spectral power, was increased in SAD rats. In the latter rats, the SD of MAP was significantly higher than in sham-operated rats. As indicated in Table 1⇑, the low-frequency peak in MAP spectra was markedly reduced, and there was a significant increase in the very-low-frequency peak in SAD rats.
Arterial Parameters in Anesthetized Rats
In anesthetized rats heart rate but not MAP was significantly reduced in SAD rats compared with sham-operated rats (Table 2⇓). No significant differences in mean aortic diameter, pulsatile change in diameter (arterial systolic diameter minus arterial diastolic diameter [Ds−Dd]), distensibility, and compliance were observed between the groups when they were compared at their respective MAPs. The diameter- and compliance-pressure curves of SAD and sham-operated rats were not significantly different. By contrast, the distensibility-pressure curve of SAD rats was significantly shifted downward (Table 2⇓ and Figure⇓) compared with sham-operated rats, indicating a lower distensibility for a given level of arterial BP.
Composition of the Abdominal Aorta
The media wall cross-sectional area was significantly increased in SAD rats compared with sham-operated rats (Table 3⇓). Collagen content and density were significantly increased in SAD rats, whereas elastin content and density and the size and number of nuclei of smooth muscle cells remained unchanged.
This study provides the first in situ determination of the elastic properties of a large artery, the abdominal aorta, after chronic SAD in Wistar rats. The main findings were that chronic SAD decreased aortic distensibility, compared with that measured at the same level of arterial pressure in sham-operated rats, and increased arterial wall collagen content.
Consideration of Methods
The method used to establish the in situ diameter-, compliance-, and distensibility-pressure curves in rats has been considered in detail previously in humans9 10 11 and rats.10 The simultaneous and continuous measurement of BP at the same site of the abdominal aorta with the use of a nonocclusive catheter allows the determination of the pressure-diameter curve, from which the compliance- and distensibility-pressure curves are derived. Distensibility and compliance are determined as dynamic cross-sectional parameters because they are calculated from changes in cross-sectional area within the systolic-diastolic range of BP. The application of this echotracking method presents three advantages and two potential limitations. First, blood flow and arterial wall innervation are maintained during the entire experimental procedure, thus giving optimal physiological conditions. Second, the diameter-pressure relationship is established over the systolic-diastolic range of BP rather than from the end points of the pressure-diameter curve generated during one cardiac cycle under varying levels of BP.12 The maneuvers used to vary BP can affect the pressure-diameter curve through changes in baroreflex activity and arterial vasomotor tone. Third, the in situ compliance and distensibility determinations were not significantly different from in vitro determinations performed in the same rats using dynamic recordings of pressure-diameter curves, with mean and pulsatile arterial pressure equal to those values observed under in situ conditions (unpublished observations, 1995). The potential limitations of the method are the use of pentobarbital anesthesia and the surgical exposure of the abdominal aorta during diameter measurements. This procedure is required to provide the best conditions of arterial measurements because our ultrasonic probe should be positioned strictly perpendicular to the arterial axis without direct contact with the arterial wall. Since dissection12 but not surgical exposure14 of the abdominal aorta was reported to alter wall stiffness, our experimental procedure consisted of surgical exposure devoid of any further dissection.
By performing SAD at 10 weeks of age and studying all rats at 16 weeks of age, we increased the likelihood of aortic wall structural changes being present in SAD rats. To our knowledge, the SAD duration is one of the longest reported in the literature and may explain some partial recovery of the phenylephrine-induced bradycardia (Table 1⇑).15
Consideration of Findings
After 6 weeks of denervation SAD rats showed an increased overall APV, without increased MAP, compared with sham-operated rats. Spectral analysis indicate that SAD induced a significant reduction in low-frequency power and a marked increase in very-low-frequency power. These results are consistent with previous studies.8 16 17
SAD significantly shifted the distensibility-pressure curve toward lower levels of distensibility, indicating a decreased aortic distensibility for a given level of arterial pressure. The mechanisms involved in the reduction of arterial distensibility are difficult to analyze because of the complexity of the pressure-diameter relationship and the number of its determinants, which include the passive connective tissue elements, vascular smooth muscle tone, and set point of distending pressure. The latter mechanism probably did not occur in the present study because MAP was not significantly different between SAD and sham-operated rats. An increase in aortic smooth muscle tone is unlikely because after an initial increase3 5 18 a normal vascular sympathetic tone has been reported in SAD rats.19 The absence of a reduction in aortic diameter favors this latter hypothesis. Another possible explanation for the decreased aortic distensibility is the structural modification of the arterial wall.
The main change in arterial wall composition was an increase of aortic wall cross-sectional area and collagen content, one of the less-distensible components of the arterial wall. A reduction in arterial distensibility has previously been related to an increase in collagen content in hypertensive rats.13 However, the relationship between the sympathetic nervous system and collagen is complex. Indeed, the sympathetic nervous system has been reported to exert an inhibitory influence on collagen synthesis, as an increase in arterial wall collagen content was observed in response to chemical sympathetic denervation.20 This finding suggests that the increase in collagen content that we observed in SAD rats was probably not related to the sustained increase in sympathetic tone that has been described in this model.3 5 18 We hypothesize that the initial hypertensive phase, which has been extensively reported during the first week after denervation, could have contributed to the increase in both arterial wall thickness and collagen content through the structural adaptation of the arterial wall to the increased wall stress. The very long turnover of collagen proteins may explain the sustained collagen abnormalities, despite the rapid normalization of BP in this model.
SAD rats, in which arterial distensibility was reduced, had an overall APV significantly higher than that of sham-operated rats, the distensibility of which was normal. This indicates that a lower distensibility and higher APV can be associated during long-term inhibition of arterial baroreflex function. These results are consistent with the hypothesis that an excessive variability of wall mechanical stress may enhance its fatiguing effect,21 22 23 thus favoring the alterations of wall material seen with aging and hypertension. In favor of this hypothesis, we recently reported that a decrease in arterial distensibility was associated with an increase in overall APV in long-term guanethidine-sympathectomized rats.24 To our knowledge, only a few clinical studies have suggested that APV per se could be a factor of target-organ damage.23
In summary, the present study indicates that chronic SAD reduced aortic distensibility and increased arterial wall thickness and collagen content. We suggest that an intact arterial baroreflex is necessary to maintain normal functional and structural properties of large arteries in rat. The decrease in aortic distensibility could have resulted from various factors, including initial BP elevation and the changes in aortic smooth muscle tone and/or wall composition.
Selected Abbreviations and Acronyms
|APV||=||arterial pressure variability|
|MAP||=||mean arterial pressure|
|SAD||=||sinoaortic denervation, sinoaortic-denervated|
Cowley AW, Liard JF, Guyton AC. Role of the baroreceptor reflex in daily control of arterial blood pressure and other variables in dogs. Circ Res. 1973;32:564-576.
Krieger EM. Neurogenic hypertension in the rat. In: de Jong, ed. Handbook of Hypertension: Experimental and Genetic Models of Hypertension. New York, NY: Elsevier; 1984;4:350-363.
Trapani AJ, Barron KW, Brody MJ. Analysis of hemodynamic variability after sinoaortic denervation in the conscious rat. Am J Physiol. 1986;251:R1163-R1169.
Jacob HJ, Alper RH, Grosskreutz CL, Lewis SJ, Brody MJ. Vascular tone influence arterial pressure lability after sinoaortic deafferentation. Am J Physiol. 1991;260:R359-R367.
Akselrod S, Eliash S, Oz O, Cohen S. Hemodynamic regulation in SHR: investigation by spectral analysis. Am J Physiol. 1987;253:H176-H183.
Cerutti C, Barres C, Paultre C. Baroreflex modulation of blood pressure and heart rate variabilities in rats: assessment by spectral analysis. Am J Physiol. 1994;266:H1993-H2000.
Hayoz D, Rutschmann B, Perret F, Niederberger M, Tardy Y, Mooser V, Nussberger J, Waeber B, Brunner H. Conduit artery compliance and distensibility are not necessarily reduced in hypertension. Hypertension. 1992;20:1-6.
Boutouyrie P, Lacolley P, Girerd X, Beck L, Safar M, Laurent S. Sympathetic activation decreases medium-size artery compliance in humans. Am J Physiol. 1994;267:H1368-H1376.
Megerman J, Hasson JE, Warnock DF, L’Italien GJ, Abbott W. Noninvasive measurements of nonlinear arterial elasticity. Am J Physiol. 1986;250:H181-H188.
Levy BI, Michel JB, Salzmann JL, Azizi M, Poitevin P, Safar M, Camilleri JP. Effects of chronic inhibition of converting enzyme on mechanical and structural properties of arteries in rat renovascular hypertension. Circ Res. 1988;63:227-239.
Barres C, Lewis SJ, Jacob HJ, Brody MJ. Arterial pressure lability and renal sympathetic nerve activity are dissociated in SAD rats. Am J Physiol. 1992;263:R639-R646.
Persson PB, Ehmke H, Köhler WW, Kirchheim HR. Identification of major slow blood pressure oscillations in conscious dogs. Am J Physiol. 1990;259:H1050-H1055.
Di Rienzo M, Parati G, Castiglioni P, Omboni S, Ferrari AU, Ramirez AJ, Pedotti A, Mancia G. Role of sinoaortic afferents in modulating BP and pulse-interval spectral characteristic in unanesthetized cats. Am J Physiol. 1991;261:H1811-1818.
Trapani AJ, Barron KW, Brody MJ. Increased neurogenic vasomotor tone persists chronically after sinoaortic baroreceptor deafferentation. Fed Proc. 1982;41:1094.
Laurent S. Mechanical stress of the arterial wall and hypertension. In: Safar M, O’Rourke MF, eds. The Arterial System in Hypertension. Dordrecht, Netherlands: Kluwer Academic; 1993:5-26.
Nichols WW, O’Rourke MF. Aging, high blood pressure and disease in human. In: Nichols WW, O’Rourke MF, eds. McDonald’s Blood Flow in Arteries. London, UK: Edward Arnold; 1990:398-420.
Lacolley P, Glaser E, Challande P, Boutouyrie P, Mignot JP, Duriez M, Levy B, Safar M, Laurent S. In situ aortic pressure-diameter relationship and structural changes in long-term chemical sympathectomized rats. Am J Physiol. In press.