The present study explores the contribution of alterations in resting tone to cerebral artery narrowing in SHRs (spontaneously hypertensive rats) and the role of hypertension development. Young pre-hypertensive and adult fully hypertensive SHRs and age-matched Wistar–Kyoto rat controls were used. The contribution of basal vasoactive factors to resting tone was studied in middle cerebral arteries with pressure myography. Basal NO and O2− (superoxide anion) availability were determined with fluorescent indicators using confocal microscopy and lucigenin-enhanced chemiluminescence. Basal O2− was also assessed in mesenteric resistance arteries. Middle cerebral arteries from adult rats, but not young pre-hypertensive rats, had augmented myogenic responses and resting tone and decreased relaxation to sodium nitroprusside compared with their normotensive counterparts. Cerebral arteries from adult SHRs also had an increase in tonic NO associated with a decrease in basal O2− availability. Basal O2− was instead increased in mesenteric arteries from SHRs. The present results indicate that large cerebral arteries from SHRs have an increase in their resting tone as a consequence of sustained hypertension and that this is related to a decrease in NO responsiveness. We suggest that this increase in resting tone and myogenic responses could act as a protective mechanism against the development of stroke in SHRs. The present study also demonstrates some unusual findings regarding the current understanding of the NO/O2− balance in hypertension with important differences between vascular beds and draws attention to the complexity of this balance in cardiovascular health and disease.
It is well known that large cerebral arteries are important contributors to cerebrovascular resistance and CBF (cerebral blood flow) regulation . Brain vessels exhibit autoregulatory properties through several mechanisms. In addition to traditional considerations, the endothelium also participates in cerebral vascular autoregulation . The endothelium, through the tonic release of vasoactive factors, mainly NO, contributes to the resting tone of cerebral arteries and, thus, participates in the control of cerebrovascular resistance and CBF [3,4].
Increased peripheral vascular resistance to blood flow due to a decreased resistance artery lumen is a common finding in hypertension [5,6]. This alteration is also present in brain vessels, and cerebral artery narrowing has been demonstrated in several animal models of hypertension [7–12]. In addition to structural and mechanical abnormalities, functional changes might also participate in arterial narrowing ; however, despite the importance of resting tone for cerebrovascular resistance, its contribution to cerebral artery narrowing in hypertension and the role of tonic endothelial factor availability has not been thoroughly studied.
There is evidence that the structural and mechanical alterations mentioned above might be present in some vascular beds before the development of hypertension. Our own findings  demonstrate that, in MRAs (mesenteric resistance arteries) from SHRs (spontaneously hypertensive rats), vessel stiffness is already present in 1-month-old rats before a significant increase in blood pressure. Moreover, there is recent evidence that MCA (middle cerebral artery) narrowing due to a structural defect is also present in SHRs at this age .
On the basis of this background, the first aim of the present study was to assess, in pressurized MCAs from SHRs, the contribution of resting tone to ID (internal diameter) narrowing in young and adult rats. The second aim was to assess the mechanism implicated in resting tone and to evaluate the contribution of tonic vasoactive factor availability, with particular attention to NO/O2− (superoxide anion) balance, which is known to be altered in hypertension [15–19].
MATERIALS AND METHODS
Male 1-month-old and 6-month-old SHRs and WKY (Wistar–Kyoto) rats were obtained from colonies inbred at the Animal House of our Institution. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH publication No. 85-23, revised in 1996) and with guidelines set by Spanish legislation (RD 1201/2005).
MCA segments with endothelium were mounted on a pressure myograph (Model P100; J.P. Trading), as described previously . Briefly, the artery was set to 10 mmHg, allowed to equilibrate for 60 min at 37 °C in gassed PSS (physiological salt solution) in the absence of calcium [115 mmol/l NaCl, 4.6 mmol/l KCl, 25 mmol/l NaHCO3, 1.2 mmol/l KH2PO4, 1.2 mmol/l MgSO4, 10 mmol/l EGTA and 11 mmol/l glucose (pH 7.4)]. Thereafter ID and ED (external diameter) were measured at 20, 40, 60, 80, 100 and 120 mmHg. The segment was then set and incubated with PSS containing calcium (2.5 mmol/l CaCl2 omitting EGTA), and the ID and ED were again measured at each pressure. From these results, resting tone was calculated as follows: resting tone (%)=100×(ID0Ca−ID)/ID0Ca, where ID0Ca and ID are internal diameters in PSS in the absence or presence of calcium respectively.
Vascular function was studied in segments pressurized at 70 mmHg after a 60 min equilibration period in PSS. All experiments were performed in the presence of 0.1 μmol/l dexamethasone to avoid the contribution of iNOS [inducible NOS (NO synthase)]. The involvement of tonic release of COX (cyclo-oxygenase) metabolites or NO to resting ID was determined comparing the ID before and after the addition of the specific inhibitors indomethacin (5 μmol/l), L-NAME (NG-nitro-L-arginine methyl ester; 0.1 mmol/l) or SMTC (S-methyl-L-thiocitrulline; 10 μmol/l) to block COX, NOS and nNOS (neuronal NOS) respectively. Arterial contractility was determined by the response to 75 mmol/l KCl as follows: KCl response (%)=100×(ID0Ca−IDKCl)/ID0Ca, where ID0Ca and IDKCl are internal diameters in PPS in the absence of calcium and after the addition of 75 mmol/l KCl respectively. Thereafter the arteries were washed with PSS and cGMP-dependent vasodilation was studied by the increase in the ID induced by the cumulative addition of SNP (sodium nitroprusside; 0.01–10 μmol/l) in the presence of 0.1 mmol/l L-NAME (to exclude the contribution of endogenous NO). SNP response=IDSNP−IDL-NAME, where IDSNP and IDL-NAME are internal diameters in the presence of L-NAME with or without SNP respectively. All drugs were purchased from Sigma.
Determination of basal NO and O2− availability in adult rats
Basal NO availability was determined in MCAs by the fluorescent NO indicator DAF-2 DA (4,5-diaminofluorescein diacetate) as described previously . Briefly, MCA segments were stabilized in PSS (30 min at 37 °C) and stained with 10 μmol/l DAF-2 DA (15, 30 or 45 min in the dark at 37 °C in a shaking water bath). Negative controls for DAF-2 DA were incubated in 0.1 mmol/l L-NAME throughout the experimental period. Thereafter the segments were washed three times for 1 min each in PSS and fixed in 4% (w/v) paraformaldehyde. The MCA segments were mounted intact on slides and were visualized with a Leica TCS SP2 confocal microscope using the 488 nm/515 nm line. Serial images of the medial layer were captured at identical conditions of brightness, contrast and laser power for all of the experimental groups. Fluorescence intensity was quantified from confocal projections of the serial images with Metamorph image analysis software (Universal Imaging).
DHE (dihidroethidium; 3 μmol/l) was used to determine basal O2− availability in MCA and third-order MRAs using the 488 nm/590–620 nm line of the microscope . MCA segments were studied at three incubation times, as described previously for the DAF-2 DA protocol . MRAs were only studied at 30 min of incubation. Negative controls were incubated throughout the experimental period with 15 units/ml SOD (superoxide dismutase).
In MCA segments, O2− availability was also tested using the lucigenin-enhanced chemiluminiscence assay, as described previously . Isolated MCA segments did not yield enough signal and, therefore, all of the arteries from the circle of Willis were used. MCAs were dissected, cleaned from surrounding tissue and incubated in PSS (30 min at 37 °C). Background RLU (relative luminiscence units) were measured with a luminometer (OPTOCOMP I, Bacterial Systems; GEM Biomedical) in tubes containing 5 μmol/l lucigenin before addition of the tissue. Thereafter the arteries were transferred to the tubes and RLU emitted were integrated over 30 s intervals for 20 min. Finally, RLU were measured for another 20 min after the addition of the O2− scavenger tiron (10 mmol/l). Basal levels of O2− were calculated as: (value of tissue+lucigenin-containing PSS−background)/tissue weight.
Results are expressed as means±S.E.M., and n denotes the number of animals used in each experiment. Statistical analysis was performed with GraphPad prism 4 software using paired or unpaired Student's t test, or two-way ANOVA with repeated measures on pressure factor, where appropriate, followed by a Bonferroni post-hoc test. A value of P<0.05 was considered significant.
Changes in intrinsic tone and myogenic responses with age
In the presence of calcium, MCAs from adult SHRs, but not from young SHRs, had a significantly smaller ID when compared with age-matched WKY rats (Figure 1A). MCAs from adult SHRs maintained a constant diameter between 80–120 mmHg, with a slope of the ID pressure curve near zero values (SHRslope=0.02±0.1). However, there was no steady-state diameter in age-matched WKY rats (WKYslope=0.51±0.1; P<0.01 when compared with SHRs). In young rats there was no difference between strains in the slope of the ID pressure curve (SHRslope=0.24±0.1 and WKYslope=0.16±0.1; P=0.87).
ID and resting tone in young and adult SHRs and WKY rats
MCAs from adult SHRs had significantly larger resting tone compared with age-matched WKY rats. This difference was not observed in young rats (Figure 1B).
Factors involved in resting ID
Indomethacin did not modify the ID in adult SHRs or WKY rats (Figure 2A). However, the non-specific NOS inhibitor L-NAME significantly decreased the ID in both strains (Figure 2B). This decrease was smaller in SHRs (21.0±3%) when compared with their WKY rat counterparts (36.6±4%; P<0.01). As a result L-NAME abolished the differences in ID between strains. The specific nNOS blocker SMTC produced a negligible decrease in the ID, which was similar in both strains (SHR, 3.0±2.0 and 1.0±0.5% in SHRs and WKY rats respectively; P=0.18).
Effect of COX and NOS inhibition on the resting ID of MCAs in adult or young SHRs and WKY rats
In young rats, L-NAME decreased the ID to a similar extent in SHRs (38.8±6%) and WKY rats (30.5±4%; P=0.25) (Figure 2C).
Responses to vasoactive factors
There was no difference between strains in the contraction elicited by 75 mmol/l KCl in young or adult rats (Figure 3A).
Response to KCl and SNP in young and adult SHRs and WKY rats
Responses to the exogenous NO donor SNP were studied in the presence of 0.1 mmol/l L-NAME to avoid interference with endogenous NO production. In young rats, SNP concentration–response curves were similar between strains; however, in adult rats responses to SNP were significantly decreased in SHRs when compared with age-matched WKY rats (Figure 3B). Adult WKY rats had significantly greater SNP responses than their young WKY rat counterparts. There was no difference in the SNP response between old and young SHRs (Figure 3B).
Basal NO availability in MCAs from adult rats
The fluorescence emitted by DAF-2 DA was located in the cytoplasm of SMCs (smooth muscle cells). Fluorescence intensity was markedly decreased by pre-incubation with 0.1 mmol/l L-NAME (Figure 4A), and it was directly proportional to the incubation time (Figure 4B). DAF-2 DA fluorescence intensity was significantly larger in MCAs from SHRs compared with age-matched WKY rats (Figure 4C).
NO bioavailability in MCAs from adult SHRs and WKY rats
Basal O2− availability in MCAs and MRAs from adult rats
DHE fluorescence was located in the cell nuclei (Figures 5A and 6A), it was decreased by pre-incubation with the O2− scavenger SOD (Figure 5A) and fluorescence intensity was directly proportional to the incubation time in MCAs from both WKY rats and SHRs (Figure 5B). In MCAs, DHE fluorescence intensity was significantly smaller in arteries from SHRs when compared with age-matched WKY rats (Figure 5C). The lucigenin assay also showed significantly smaller RLU in cerebral arteries from SHRs compared with age-matched WKY rats (Figure 5D). This luminiscence was due to O2− production, as shown by its decrease with the O2− scavenger tiron (results not shown).
O2− availability in MCAs from adult rats
O2− availability in MRAs from adult rats
In MRAs, the opposite effect was found, with increased DHE-derived fluorescence in arteries from SHRs compared with those from their age-matched WKY rat counterparts (Figure 6).
The present study demonstrates the contribution of increased resting tone to MCA narrowing in adult SHRs. The alteration in resting tone is not present in young prehypertensive SHRs and is, therefore, possibly a consequence of hypertension. These changes might contribute to the suggested protection against haemorrhagic stroke in this strain. The present study also demonstrates a decrease in responsiveness of SMCs to NO, but a larger tonic NO availability related to the unexpected finding of a decrease in the presence of O2−, which appears to be specific to the cerebral vasculature of SHRs. To the best of our knowledge, the present study is the first report, assessing under non-stimulated conditions, the role of vasoactive factor availability to resting cerebral artery tone in hypertension.
The decrease in the ID due to structural alterations has been reported in brain vessels from several rat models of hypertension, including SHRs [7–12]. In the present study, we have investigated the possible contribution of resting tone to cerebral artery narrowing with pressure myography: the method which best resembles vascular physiological conditions in vitro. Our present results show an increase in resting tone in adult SHRs, similar to previous findings in cerebral vessels from Brattleboro hypertensive rats . In addition to an increase in resting tone, MCAs from adult SHRs also had elevated myogenic responses, as described previously . We aimed to determine whether these alterations were a consequence of high blood pressure or if they were present before the development of hypertension. We therefore studied 1-month-old SHRs, which, as shown by several authors [12,24,25], are in a pre-hypertensive phase. In the rat colonies inbred at our Institution, we have demonstrated previously that, at this age, SHRs have similar blood pressure levels compared with age-matched WKY rat counterparts . Resting tone and myogenic responses were similar in MCAs from young SHRs and WKY rats, suggesting that the alterations observed in adult SHRs are a consequence of high blood pressure.
We analysed the implication of tonic vasoactive factor release in the enhanced level of resting tone in adult SHRs. The COX inhibitor indomethacin did not modify the ID in SHRs or WKY rats, suggesting no major contribution of COX-derived metabolites on MCA resting tone.
We also assessed the role of tonic NO availability on resting tone. In mammals, there are three NOS genes: NOS1 (encoding nNOS), NOS2 (encoding iNOS) and NOS3 [encoding eNOS (endothelial NOS)], and non-specific and selective inhibitors have been developed to study the contribution of each of these enzymes to vascular tone regulation and vascular pathology. Regarding pharmacological agents, L-NAME and L-NMMA (NG-monomethyl-L-arginine) have been commonly used as non-specific NOS inhibitors [26–28]. With respect to iNOS, both the specific blocker aminoguanidine and dexamethasone, through inhibition of iNOS expression, have been used [26,28]. On the other hand, SMTC is a selective nNOS inhibitor, which has been shown to be approx. 17-fold more selective for nNOS compared with eNOS . We have demonstrated previously  that iNOS can be induced in arteries during prolonged experiments. Therefore to study the contribution of NO to vascular tone in MCAs we used dexamethasone throughout the experimental protocol to avoid interference with iNOS. The MCA ID was decreased effectively by L-NAME in both strains at the two age points studied, whereas the specific nNOS inhibitor SMTC had a negligible effect. The dose of SMTC used in the present study (10 μmol/l) has been shown previously  to elicit a pronounced and selective blockade of nNOS. As eNOS is the major NOS isoform expressed in the cardiovascular system and we avoided iNOS, we can conclude that L-NAME is inhibiting endothelial NO, which appears to be the main factor regulating resting tone in MCAs. This is in accordance with previous studies showing that NO is key in the regulation of CBF and basal cerebrovascular tone [3,4,31].
In adult rats, L-NAME had a smaller effect on arteries from SHRs compared with their WKY rat counterparts, and it abolished the differences in ID between the strains. Thus increased resting tone in SHRs appears to be related to an alteration in the NO pathway. The decrease in ID by L-NAME is the consequence of: (i) the responsiveness of SMCs to NO, and (ii) the availability of NO to SMCs, which, in turn, depends on how much is produced and how much is destroyed by O2−. We therefore analysed whether the smaller effect of L-NAME on MCAs from SHRs was due an alteration in the responsiveness of MCAs to NO and/or to a defect in NO availability.
To study the responsiveness of SMCs to NO we analysed the relaxation elicited by an exogenous NO donor (SNP). In these experiments, we blocked NOS with L-NAME to avoid interference with endogenous NO. Under these experimental conditions, MCAs from adult SHRs had a smaller relaxation to SNP when compared with their WKY rat counterparts. However, responses to the non-specific contractile agent KCl were not different between strains. These results suggest that the cerebral vasculature of SHRs has a decreased responsiveness to NO compared with normotensive adult WKY rats, which is not related to a general impairment of SMC reactivity. This is consistent with previous studies that have demonstrated that the local CBF increase to infusion of the NO donor SIN-1 was decreased in SHRs compared with WKY rats . The decreased response to NO observed in MCAs has also been reported in other vascular beds from SHRs and stroke-prone SHRs and it has been linked to a decreased expression of soluble guanylate cyclase [33–35]. The analysis of the responses to SNP in relation to age demonstrated that, in MCAs from 1-month-old rats, there are no differences between the two strains. Thereafter MCAs from WKY rats have increased sensitivity to the NO donor with age, whereas MCAs from SHRs do not. Similar results were found in aorta from SHRs, where the YC-1-induced content of cGMP in aortic rings was similar in young SHRs and WKY rats and increased with age in WKY rats, but not in SHRs .
We also analysed the second possibility, i.e. that a decrease in NO availability could also contribute to the reduction in responses to L-NAME. To determine the NO available to SMCs, we used the fluorescent indicator DAF-2 DA. This compound is captured by vascular cells and, in the presence of NO and oxygen, produces a highly fluorescent compound in the cytoplasm, which is proportional to the amount of NO [36,37]. We have used confocal microscopy and image analysis software previously to quantify NO availability from several vascular beds [21,38]. This method was sufficiently sensitive to enable the quantification of NO tonic availability in MCA segments, as demonstrated by the cumulative increase in fluorescence, which was blocked by L-NAME. Fluorescence was significantly greater in MCAs from SHRs compared with those from their WKY rat counterparts, indicating that, under basal conditions, cerebral arteries from SHRs have a greater NO availability. The amount of NO available depends on the balance between NO production and inactivation, mainly due to O2− [17,39]. In order to determine whether the differences observed in tonic NO between the strains were related to an alteration in O2−, we used DHE, a sensitive method that enables the detection of O2− in single arterial segments . Our results showed a decrease in basal O2− in MCAs from SHRs. This was confirmed by lucigenin-enhanced chemiluminescence. This method is less sensitive than DHE and a larger amount of tissue (all the arteries from the circle of Willis) was required to detect luminescence under basal conditions. This decrease in O2− explains the higher NO availability in MCAs from SHRs and might be surprising given the fact that increased oxidative stress has been thoroughly reported in hypertension [15–19]. The decrease in the presence of O2− in SHRs appears to be specific to the cerebral vasculature as, in third-order MRAs from age-matched rats, basal O2− was increased in SHRs instead. These findings are in accordance with several other studies performed in both conduit [40,41] and resistance  arteries from this strain.
The decrease in basal O2− in cerebral vessels in SHRs could be explained by a higher SOD activity in the hypertensive strain, able to quickly scavenge tonic O2− production. In this regard, Paravicini et al.  have demonstrated that it was not possible to detect basal production of O2− with chemiluminescence in single basilar artery segments from SHRs and WKY rats. They also demonstrated that, when SOD activity was blocked, basal O2− production could be detected, but only in SHRs. It has also been demonstrated that the application of NADPH to basilar arteries caused larger vasodilation in SHRs than WKY rats . It has to be taken into account that O2− dismutated by SOD produces H2O2, which is a powerful dilator in cerebral vessels [43,45–47]. As NADPH oxidase activity is higher in cerebral vessels of SHRs , the increased vasodilation to NADPH in SHRs  is probably associated with a parallel increase in SOD activity in the hypertensive strain.
Regarding the consequences of the present findings for cerebrovascular regulation, we suggest that the functional changes observed in adult SHRs could act as a protective mechanism against the development of haemorrhagic stroke. In accordance with this hypothesis is the demonstration that the auto-regulatory properties of the MCA are impaired in stroke-prone SHRs compared with the stroke-resistant SHRs . This has also been observed in uraemic hypertension , and it has been proposed that an alteration in auto-regulatory properties might be a key step in stroke development [11,49]. We therefore suggest that the observed changes in resting tone and myogenic responses are a functional adaptation that takes place along with the increase in high blood pressure in SHRs. Regarding the influence of the oxidative stress/NO balance in the regulation of cerebrovascular tone, the conclusion is less evident. In the cerebral circulation, O2− exerts a dual role on vasodilator balance, contributing to NO destruction on one hand, and as source of H2O2 generation, a powerful vasodilator for brain vessels, on the other . In this context, the term ‘oxidative stress’, and its association with cardiovascular pathology, may be an over-simplification that hides the complexity and diversity of reactive oxygen species. Therefore further studies are needed to understand the role of ‘oxidative stress’ and its implications for cardiovascular health in general and, in particular, for cerebrovascular disease.
cerebral blood flow
- DAF-2 DA
NG-nitro-L-arginine methyl ester
middle cerebral artery
mesenteric resistance artery
physiological salt solution
relative luminiscence units
spontaneously hypertensive rat
smooth muscle cell
This work was funded by Ministerio de Educación y Ciencia, Spain (BFI 2001-0638 and BFU2004-04148). We are grateful to Carmen Fernández Criado, who is in charge of the Animal House, and Diego Megías for his technical assistance with the confocal microscopy.