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1Department of Medicine, Cardiovascular Research Group, Manchester Royal Infirmary, Manchester M13 9WL; and 2Division of Cardiovascular and Medical Sciences, BHF Glasgow Cardiovascular Research Centre, University of Glasgow, Glasgow, G11 6NT United Kingdom
Submitted 14 April 2003 ; accepted in final form 9 June 2003
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ABSTRACT |
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cerebral circulation; arteriograph; tone
Hypertension is associated with an increase in the wall-to-lumen ratio of the cerebral arteries due to the processes of remodeling and growth (1). Remodeling leads to a reduced lumen diameter as well as an increased wall-to-lumen ratio but does not involve necessarily an increase in cross-sectional area of the vessel wall; this can be due to "true" remodeling or a consequence of reduced arterial wall distensibility (2). On the other hand, growth is defined by the presence of an increased wall cross-sectional area (4). Previous studies (3, 15) have suggested that the increase in the wall-to-lumen ratio in the proximal cerebral resistance arteries attenuates the increased pressure in the cerebral microcirculation in hypertension; this may be because the increased wall-to-lumen ratio offsets increased wall stress and so enables the arteries to regulate diameter at higher pressure. In support of this idea, sympathetic denervation, which causes a reduction in the wall-to-lumen ratio of the cerebral arteries, is associated with an accelerated incidence of stroke in the denervated cerebral hemisphere of the SHRSP (9). Therefore, the predisposition to stroke in the SHRSP may be, at least in part, a consequence of an inadequate increase in the wall-to-lumen ratio of the cerebral arteries in response to raised arterial pressure.
The myogenic and structural properties of small arteries can be assessed by constructing pressure-diameter relationships in the presence and absence of myogenic tone, respectively; thus the aims of this study were to compare the myogenic and structural properties of MCAs from prestroke SHRSP with SHR in vitro, after the rats had received a Japanese diet for 1 mo.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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10 mm distal to the circle of Willis and proximal to the first major branching point was removed, placed in a dissection dish, and cleared of adherent meninges. Pressure arteriography. Leak-free segments of the MCA were tied onto two glass micropipettes in an arteriograph as described previously (5, 8) and pressurized to 60 mmHg. Lumen diameter and wall thickness were continually monitored using a Video Dimension Analyzer (Living Systems Instrumentation; Burlington, VT). All signals were digitized and stored on a computer loaded with WinDaq data-acquisition software (Dataq Instruments; Akron, OH). The arteriograph chamber was superfused with PSS from a reservoir, which was gassed with 5% CO2 in air (pH 7.4–7.45), and the temperature was increased to 37°C. After an equilibration period of 1–1.5 h, the vessels had developed a stable level of spontaneous myogenic tone. To determine the myogenic properties of the arteries, intraluminal pressure was reduced to 20 mmHg and then increased to 300 mmHg in 40-mmHg increments. Each pressure step lasted for 3–4 min, which allowed the lumen diameter to stabilize. To determine the structural properties of the arteries, the arteries were superfused with Ca2+-free PSS containing 2 mM EGTA to eliminate myogenic tone. Intraluminal pressure was reduced to 3 mmHg to determine the unstressed diameter, and, subsequently, the previous pressure steps were repeated.
Histopathology. Brains were fixed in formalin, and eight coronal sections were prepared and assessed for the presence of cerebral lesions.
Collagen staining. In a separate series of experiments, MCAs were removed, fixed in 4% paraformaldehyde in PBS for 1 h, and embedded in OCT compound (R. A. Lamb; Eastbourne, UK). Cryosections (5 µm thick) were allowed to adhere to silanted microscope slides. After the removal of OCT compound in water, collagen was stained for 30 min with gentle agitation using picrosirius red [0.1% (wt/vol) Sirius red 3FB (Aldrich; Dorset, UK) in saturated aqueous picric acid]. Color images were captured with a x40 objective using a digital camera (1,920 x 1,600 resolution). To quantitate red staining, images were converted from RGB to CMYK color mode, and the magenta channel was extracted as grayscale and inverted (Adobe Photoshop software; San Jose, CA). Pixel intensity values along four radial line scans, each beginning outside the artery, traveling across the artery wall, and terminating in the vessel lumen, were obtained for each artery section imaged using KS300 software (Zeiss; Welwyn Garden City, UK). These intensity profiles were spatially aligned using an arbitrary intensity threshold (50 units) corresponding to the outer edge of the adventitia. Aligned intensity profiles were then averaged and represent the distribution of collagen (red intensity) across the vessel wall. For each SHRSP and SHR group (both n = 6 animals), a total of 20 sections taken from 10 arteries was quantitated as described. Care was taken when staining sections and when obtaining and quantitating images to ensure identical procedures applied to both groups.
Calculations. Wall cross-sectional area (CSA) was calculated as
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Values are expressed as means ± SE. Student's unpaired t-test and two-way ANOVA were used where appropriate. The slopes of the pressure-diameter relationship were compared using analysis of covariance. In all cases, P < 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Figure 1 shows the lumen diameter of MCAs in the presence of myogenic tone and under passive conditions. In the presence of myogenic tone, there was no significant difference in the pressure-diameter relationship between strains across the entire pressure range [not significant (NS), n = 13 and 14, SHRSP and SHR, respectively]. However, between 60 and 180 mmHg, the steady-state diameter of SHR vessels was essentially constant (slope –0.007 ± 0.008), i.e., it displayed a myogenic range (8). In contrast, there was no myogenic range in MCAs from SHRSP; between 60 and 180 mmHg, the slope was 0.18 ± 0.06, which was significantly different from SHR (P < 0.05). The passive pressure-lumen diameter relationship was significantly different between the two groups (P < 0.001); arteries from SHRSP had a reduced diameter at higher pressures.
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Figure 2 shows significant increases in wall thickness, the wall-to-lumen ratio, and wall cross-sectional area of MCAs from SHRSP compared with SHR. The increase in the wall cross-sectional area indicates significant arterial growth. The "remodeling index," which is the percent difference in lumen diameter between SHRSP and SHR, not involving an increase wall cross-sectional area, was 57% at 100 mmHg. Lumen diameters were not significantly different between strains at 3 mmHg; in fact, numerically, diameter was increased in SHRSP compared with SHR. Therefore, "true" remodeling did not contribute to the reduced lumen diameter of MCAs from SHRSP at higher pressure. To compare arterial distensibility between SHRSP and SHR, stress-strain relationships were constructed. The leftward shift in the stress-strain relationship indicates a decrease in distensibility of MCAs from SHRSP compared with SHR (Fig. 3). Thus both growth and a reduced distensibility, in nearly equal proportions, account for the structural differences in MCAs from SHRSP compared with SHR.
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Collagen staining was located primarily in the outer region of the vessel wall in arteries from SHRSP (Fig. 4A), whereas there was a more uniform distribution of collagen staining throughout the vessel wall in MCAs from SHR (Fig. 4B). Quantification of collagen staining revealed significant differences in the distribution of collagen in MCAs from SHRSP compared with SHR (Fig. 4C).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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However, significant differences were observed in the slope of the pressure-diameter relationship between 60 and 180 mmHg. Across this pressure range, lumen diameter of MCAs from SHRSP increased as a linear function of pressure (as noted in all parts of the pressure-diameter relationship). In contrast, a myogenic range was observed between 60 and 180 mmHg in MCAs from SHR, which is the likely physiological pressure range for the MCA from the SHR (Fig. 1). The myogenic range is the range of pressures at which steady-state lumen diameter at higher distending pressure is equal to or less than lumen diameter at lower pressure, and thus it reflects the autoregulatory pressure range (8). Although this and a previous study (8) demonstrate weak autoregulatory behaviour at the level of the MCA from SHR, i.e., a nearly flat pressure-diameter relationship, autoregulatory responses are completely absent in MCAs from SHRSP. The latter observation is also in agreement with a previous study (14), although in that study the myogenic properties of MCAs from SHRSP were compared with SHRSP treated with perindopril, not with MCAs from SHR. Interestingly, in that study, perindopril treatment, which prevented stroke but had little effect on blood pressure, led to the development a myogenic range in MCAs studied in vitro. Our data, in conjunction with data from previous studies (11, 12, 14), suggest impaired cerebral autoregulation, through a defect in the myogenic mechanism, as a key step in stroke development in the SHRSP. Thus, although high blood pressure is an essential element in the development of stroke in the SHRSP, it is likely that the increased blood pressure of SHRSP compared with SHR will exacerbate stroke development, but is not in itself a likely explanation for the impaired autoregulatory responses in the cerebral vasculature
The increased wall-to-lumen ratio of MCAs from SHRSP compared with SHR may act to normalize wall stress in the face of the higher blood pressure and suggests that an inadequate increase the wall-to-lumen ratio, in response to raised arterial pressure, is not a likely explanation for stroke development in SHRSP. Although one study (6) has shown that the wall-to-lumen ratio is reduced in some cerebral arteries from SHRSP compared with SHR (6), the rats were not placed on a Japanese diet, and so the findings cannot be related to rapid stroke development.
Passive lumen diameter was significantly reduced in MCAs from SHRSP compared with SHR at physiological pressures. At 100 mmHg, the remodeling index was 57%, which implies that just over half of the structural differences in the MCAs between the two groups does not involve growth; this is a consequence of a reduced distensibility of the MCA from SHRSP compared with SHR rather than true remodeling, as the lumen diameter of MCAs from the two groups did not differ at 3 mmHg. Consequently, almost half the reduction in lumen diameter of MCAs from SHRSP compared with SHR can be ascribed to growth; this conclusion is confirmed by the significant increase in wall cross-sectional area. The contribution of growth to the structural differences observed is somewhat larger than noted in cerebral vessels from SHRSP compared with normotensive Wistar-Kyoto rats (WKY) (1, 2). With the use of similar in vitro methodology as the present study (2), no difference in wall cross-sectional area was noted in segments of large or small posterior cerebral arteries from SHRSP and WKY; the caliber of large posterior cerebral arteries from SHRSP was similar to the MCA in the present study. Although this may suggest that different regions of the cerebral vasculature exhibit differing structural changes in hypertension, it is important to note that in the aforementioned studies, SHRSP were not fed a Japanese diet, and blood pressure is lower in these studies than the present study and other studies using a high-salt diet (11–13). Also, if our reasoning is correct, it is unlikely that the myogenic properties of cerebral arteries from SHRSP are impaired in the absence of a Japanese diet, which is required for rapid stroke development; in vivo studies support this contention (15). Both increased pressure and impaired myogenic constriction will increase wall stress, and wall stress may be the stimulus for growth (7); this may explain our findings of greater growth in SHRSP compared with the studies discussed above.
Distensibility of cerebral arteries, but not the arterioles, is reduced in SHRSP compared with WKY (2); hence, it is likely that the reduced distensibility of MCAs from SHRSP compared with SHR was a consequence of the higher pressure of SHRSP. The stress-strain relationship is independent of vessel geometry; thus the leftward shift observed in MCAs from SHRSP compared with SHR reflects an increase in less distensible components in the vessel wall. As collagen is a major nondistensible component in the artery wall, collagen staining may be expected to be increased in MCAs from SHRSP. However, collagen staining with picrosirius red revealed the opposite to be true: there was less collagen in MCAs from SHRSP compared with SHR (Fig. 4C). Also, collagen was specifically located in the outer half of the vessel wall in SHRSP (Fig. 4A), as opposed to the more uniform distribution in SHR (Fig. 4B). We speculate that the higher blood pressure of the SHRSP compared with the SHR results in a different distribution of tensile stress within the artery, because it has been shown that at high pressures, the advential layer carries over 50% of the total wall stress (10), and it is known that changes in tensile forces influence collagen biosynthesis in blood vessels (16).
A potential confounding factor in this study is that brain weight was reduced in SHRSP compared with SHR, so the size of the cerebral arteries may be expected to be reduced. However, as discussed above, lumen diameter did not differ between strains at 3 mmHg, and wall thickness and wall cross-sectional area were increased in SHRSP compared with SHR; such differences would not be expected if arteries from SHRSP were merely of a smaller caliber.
In summary, in the presence of myogenic tone, no difference was observed in the pressure-diameter relationship of MCAs from SHRSP versus SHR between 20 and 300 mmHg. However, diameter of the arteries from SHRSP increased with increasing pressure, whereas arteries from SHR maintained a constant diameter between 60 and 180 mmHg, i.e., they displayed a myogenic range. Structural differences observed in MCAs from SHRSP compared with SHR were a consequence of growth and reduced distensibility. However, collagen staining with picrosirius red revealed a reduction in collagen in MCAs from SHRSP compared with SHR. We speculate that the impaired myogenic properties play a causative role in stroke development, whereas structural differences are due to differences in wall stress.
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DISCLOSURES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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significant difference the in slope of the pressure-diameter relationship from 60 to 180 mmHg between strains, P < 0.05.
D is the change in lumen diameter, and D0 is the lumen diameter at 3 mmHg.