(Hypertension. 1995;26:337-340.)
© 1995 American Heart Association, Inc.
Articles |
Aortic Distensibility and Structural Changes in Sinoaortic-Denervated Rats
From the Institut National de la Santé et de la Recherche Médicale (INSERM) U337 (P.L., Y.B., X.G., A.B., N.G., B.L., R.A.); the Department of Pharmacology (P.B., S.L.), Paris; and URA CNRS 879, Saint-Cyr l'Ecole (P.C.), France.
Correspondence to Stéphane Laurent, MD, PhD, Service de Pharmacologie, Hôpital Broussais, 96, rue Didot, 75674 Paris Cedex 14, France.
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Abstract |
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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.
Key Words: denervation • compliance • aorta • sympathetic nervous system • hypertrophy • collagen
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Introduction |
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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.
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Methods |
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SAD
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).
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BP Measurement
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 (
,
ß, and 
):
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where D is the internal diameter assuming a cylindrical vessel. The calculation of the compliance- and distensibility-pressure curves was done by deriving Equation 1:
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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
Statistical Analysis
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.
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Results |
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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.
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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.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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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.
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Selected Abbreviations and Acronyms |
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