Morphological changes in cerebral vascular smooth muscle cells in stroke-prone spontaneously hypertensive rats (SHRSP). A scanning and transmission electron microscopic study

The ultrastructure of the vascular smooth muscle cells of the middle cerebral artery in 6-month-old male stroke-prone spontaneously hypertensive rats (SHRSP) was studied by scanning (SEM) and transmission electron microscopy (TEM) and compared with that of age-matched normotensive Wistar Kyoto rats (WKY). Although the smooth muscle cells of WKY rats by SEM had a typical spindle shape and smooth surface texture, those of SHRSP were structurally modified by numerous surface invaginations and projections, bearing some structural resemblance to the myotendinous junction of skeletal muscle. Structural modifications affected more than half the surface of medial smooth muscle cells in SHRSP, but less than 0.6% of the surface of these cells in WKY rats. About 10% of medial smooth muscle cells were necrotic in SHRSP, but no necrotic cells were identified in WKY rats. By TEM, smooth muscle cells in SHRSP were shown to be irregular in profile with deep indentations of the plasma membrane and were surrounded by many layers of basal lamina-like material. The present study suggests that most smooth muscle cells in the middle cerebral artery of SHRSP may be modified to adapt to chronic hypertension by increasing the junctional area between muscle cells and connective tissue and that some cells may undergo necrosis.     

A high-potassium diet reduces infarct size and improves vascular structure in hypertensive rats

High-potassium diets can improve vascular function, yet the effects of potassium supplementation on ischemic stroke have not been studied. We hypothesized that dietary potassium supplementation would reduce ischemic cerebral infarct size by reversing cerebral artery hypertrophy. Six-week-old male stroke-prone spontaneously hypertensive rats (SHRSP) were fed diets containing 0.79% potassium (LK) or 2.11% potassium (HK) for 6 wk; Wistar-Kyoto (WKY) rats were fed the LK diet. The HK diet did not reduce blood pressure, as measured by telemetry, in the SHRSP. Cerebral ischemia was induced by middle cerebral artery (MCA) occlusion. The resultant infarct was smaller in the HK-SHRSP than in the LK-SHRSP: 55.1 +/- 6.3 vs. 71.4 +/- 2.4% of the hemisphere infarcted (P < 0.05). Infarcts were smaller in WKY rats (33.5 +/- 4.8%) than in LK-SHRSP or HK-SHRSP. The vessel wall of MCAs from LK-SHRSP was hypertrophied compared with WKY rats; this was reversed in HK-SHRSP. RT-PCR analysis of the cerebral vessels showed that expression of platelet-derived growth factor receptors-alpha and -beta, epidermal growth factor receptor, and collagen I and III was increased in the vessels from LK-SHRSP compared with WKY rats and reduced in HK-SHRSP. These results suggest that potassium supplementation provides neuroprotection in a model of ischemic stroke independent of blood pressure and possibly through changes in vascular structure.     

Elevated production of 20-HETE in the cerebral vasculature contributes to severity of ischemic stroke and oxidative stress in spontaneously hypertensive rats

Hypertension is a major risk factor for stroke, but the factors that contribute to the increased incidence and severity of ischemic stroke in hypertension remain to be determined. 20-hydroxyeicosatetraenoic acid (20-HETE) has been reported to be a potent constrictor of cerebral arteries, and inhibitors of 20-HETE formation reduce infarct size following cerebral ischemia. The present study examined whether elevated production of 20-HETE in the cerebral vasculature could contribute to the larger infarct size previously reported after transient middle cerebral artery occlusion (MCAO) in hypertensive strains of rat [spontaneously hypertensive rat (SHR) and spontaneously hypertensive stroke-prone rat (SHRSP)]. The synthesis of 20-HETE in the cerebral vasculature of SHRSP measured by liquid chromatography-tandem mass spectrometry was about twice that seen in Wistar-Kyoto (WKY) rats. This was associated with the elevated expression of cytochrome P-450 (CYP)4A protein and CYP4A1 and CYP4A8 mRNA. Infarct volume after transient MCAO was greater in SHRSP (36+/-4% of hemisphere volume) than in SHR (19+/-5%) or WKY rats (5+/-2%). This was associated with a significantly greater reduction in regional cerebral blood flow (rCBF) in SHR and SHRSP than in WKY rats during the ischemic period (78% vs. 62%). In WKY rats, rCBF returned to 75% of control following reperfusion. In contrast, SHR and SHRSP exhibited a large (166+/-18% of baseline) and sustained (1 h) postischemic hyperperfusion. Acute blockade of the synthesis of 20-HETE with N-hydroxy-N'-(4-butyl-2-methylphenyl)-formamidine (HET0016; 1 mg/kg) reduced infarct size by 59% in SHR and 87% in SHRSP. HET0016 had no effect on the fall in rCBF during MCAO but eliminated the hyperemic response. HET0016 also attenuated vascular O2*- formation and restored endothelium-dependent dilation in cerebral arteries of SHRSP. These results indicate the production of 20-HETE is elevated in the cerebral vasculature of SHRSP and contributes to oxidative stress, endothelial dysfunction, and the enhanced sensitivity to ischemic stroke in this hypertensive model.     

Enhanced contractile effect of phorbol dibutyrate in portal veins from hypertensive rats

The effects of phorbol 12,13-dibutyrate (PDBu) on portal veins from hypertensive (SHRSP0 and normotensive (WKY) rats were examined. PDBu contracted the strips from SHRSP and WKY in a concentration-dependent manner. However, both twitch contraction and tonic contraction of strips in response to PDBu were enhanced in SHRSP. Treatment with staurosporine reduced contractile response to PDBu in strips from SHRSP. It appears that the activity of protein kinase C in vascular smooth muscle is increased in SHRSP.     

Spironolactone reduces cerebral infarct size and EGF-receptor mRNA in stroke-prone rats

Remodeling of the cerebral vasculature contributes to the pathogenesis of cerebral ischemia. Remodeling is caused by increased smooth muscle proliferation and may be due to an increase in the responsiveness of vascular cells to epidermal growth factor (EGF). Aldosterone is a risk factor for stroke, and the literature suggests it may play a role in increasing the expression of the receptor for EGF (EGFR). We hypothesized that mRNA for the EGF-stimulated pathway would be elevated in the vasculature of stroke-prone spontaneously hypertensive rats (SHRSP) and that this and experimental ischemic cerebral infract size would be reduced by aldosterone inhibition with spironolactone. We found that spironolactone treatment reduced the size of cerebral infarcts after middle cerebral artery occlusion in SHRSP (51.69 +/- 3.60 vs. 22.00 +/- 6.69% of hemisphere-infarcted SHRSP vs. SHRSP + spironolactone P < 0.05). Expression of EGF and EGFR mRNA was higher in cerebral vessels and aorta from adult SHRSP compared with Wistar-Kyoto rats. Only the expression of EGFR mRNA was elevated in the young SHRSP. Spironolactone reduced the EGFR mRNA expression in the aorta (1.09 +/- 0.25 vs. 0.56 +/- 0.11 phosphorimage units SHRSP vs. SHRSP + spironolactone P < 0.05) but had no effect on EGF mRNA. In vitro incubation of aorta with aldosterone +/- spironolactone produced similar results, suggesting a direct effect of aldosterone. Thus spironolactone may reduce the size of cerebral infarcts via a reduction in the expression of the EGFR mRNA, leading to reduced remodeling.     

Increased medial smooth muscle cell length is responsible for vascular hypertrophy in young hypertensive rats

Large mesenteric arteries from 3- to 4-wk-old spontaneously hypertensive rats (SHR) showed medial hypertrophy and an increased contractile response to various agonists before significant blood pressure increase. Here we determined the cellular nature of this vascular hypertrophy. Large mesenteric arteries from SHR and Wistar-Kyoto (WKY) rats were fixed at maximal relaxation either with an in situ perfusion fixation or an in vitro fixation method. With the use of morphometric protocols and confocal microscopy, the volume of the medial wall and lumen, numerical density of smooth muscle cell nuclei in the medial layer, and smooth muscle cell and nuclear length were measured. Both methods of fixation yielded similar results, showing significant medial volume expansion in SHR than WKY without lumen change. Numerical density of medial smooth muscle cells was significantly less in SHR than WKY, and their total number per 100 microm length were similar between the strains. Average smooth muscle nuclear and cell length from SHR was significantly longer than that of WKY. Regression analysis showed that the increase in smooth muscle cell length explained 80% of the medial volume increase. We concluded that increased smooth muscle cell length in prehypertensive SHR is responsible for increased medial volume in the mesenteric arteries.     

Sex-specific differences in cerebral arterial myogenic tone in hypertensive and normotensive rats

Cerebral blood flow (CBF) is maintained constant despite changes in systemic blood pressure (BP) through multiple mechanisms of autoregulation such as vascular myogenic reactivity. Our aim was to determine myogenic characteristics of cannulated middle cerebral arteries (MCA) in male and female stroke-prone spontaneously hypertensive rats (SHRSP) and Wistar-Kyoto rats (WKY) at 12 wk of age under pressurised no-flow conditions. MCA pressure-diameter relationships (20-200 mmHg) were constructed in active (with calcium) and passive (without calcium) conditions, and myogenic and mechanical properties were determined. Myogenic reactivity in WKY (P < 0.05) and SHRSP (P < 0.05) males was impaired compared with their female counterparts. Comparison of SHRSP with WKY in males revealed similar myogenic reactivity, but in females SHRSP exhibited augmented myogenic reactivity (P < 0.05). In both sexes, myogenic tone yielded at lower pressure in SHRSP compared with WKY vessels (120-140 vs. 140-180 mmHg). Stress-strain relationships and elastic moduli in WKY rats showed that vessels were stiffer in females than in males. Conversely, in SHRSP, male vessels were stiffer than female vessels. Comparison of strains in males indicated that stiffness was increased in SHRSP compared with WKY vessels, whereas the converse was observed in females. These findings demonstrate that MCA myogenic and distensibility characteristics exhibit significant sex- and strain-dependent differences. Inappropriate myogenic adaptation and augmented vascular stiffness, particularly in male SHRSP, are potential limiting factors in blood flow autoregulation and may increase the predisposition for stroke-related cerebrovascular events.     

Pathological alterations of astrocytes in stroke-prone spontaneously hypertensive rats under ischemic conditions

Stroke-prone spontaneously hypertensive rats (SHRSP/Izm) develop severe hypertension, and more than 95% of them die of cerebral stroke. We showed the vulnerability of neuronal cells of SHRSP/Izm rats. Furthermore, we analyzed the characteristics of SHRSP/Izm astrocytes during a stroke. It is known that the proliferating ability of SHRSP/Izm astrocytes is significantly enhanced compared with those in the normotensive Wistar Kyoto rats (WKY/Izm) strain. Conversely, the ability of SHRSP/Izm astrocytes to form tight junctions (TJ) was attenuated compared with astrocytes from WKY/Izm rats. During the stress of hypoxia and reoxygenation (H/R), lactate production, an energy source for neuronal cells, decreased in SHRSP/Izm astrocytes in comparison with the WKY/Izm strain. Moreover, during H/R, SHRSP/Izm astrocytes decreased their production of glial cell line-derived neurotrophic factor (GDNF) in comparison with WKY/Izm astrocytes. Furthermore, SHRSP/Izm rats decreased production of l-serine, compared with WKY/Izm rats following nitric oxide (NO) stimulation. Additionally, in H/R, astrocytes of SHRSP/Izm rats expressed adhesion molecules such as VCAM-1 at higher levels.It is possible that all of these differences between SHRSP/Izm and WKY/Izm astrocytes are not associated with the neurological disorders in SHRSP/Izm. However, attenuated production of lactate and reduced GDNF production in astrocytes may reduce required energy levels and weaken the nutritional status of SHRSP/Ism neuronal cells. We suggest that the attenuation of astrocytes’ functions accelerates neuronal cell death during stroke, and may contribute to the development of strokes in SHRSP/Izm. In this review, we summarize the altered properties of SHRSP/Izm astrocytes during a stroke.     

Accumulation of 8-oxo-deoxyguanosine in cardiovascular tissues with the development of hypertension

Accumulation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) in DNA is associated with mutagenesis and cell death. Little attention has been given to the biological significance of 8-oxo-dG accumulation in cardiovascular tissues during the different stage of hypertension and its prevention. We thus investigated the levels and localization of both 8-oxo-dG accumulation and expression of MTH1, which hydrolyzes 8-oxo-dGTP to prevent its incorporation into DNA, in the thoracic aorta prepared from stroke-prone spontaneously hypertensive rats (SHRSP) and age-matched Wister-Kyoto rats (WKY), aged 5-32 weeks. HPLC-MS/MS analysis revealed that the levels of nuclear 8-oxo-dG in the aorta increased significantly in SHRSP, but not WKY, with aging. Immunohistochemical study revealed that both TUNEL reactivity and 8-oxo-dG immunoreactivity were increased in smooth muscle cells (SMC) and endothelial cells (EC) of the aorta with aging, and they exhibited similar distributions in serial sections. The number of 8-oxo-dG and TUNEL positive cells in EC, but not in SMC, was significantly higher in SHRSP than WKY at 32 weeks of age. In contrast, the expression levels of Mth1mRNA and MTH1 protein in the aorta were similarly decreased both in SHRSP and WKY with aging. However, the number of MTH1 expressing EC was remarkably increased in the older SHRSP compared to the younger ones or age-matched WKY. Hypertension significantly increased not only 8-oxo-dG accumulation but also the expression of MTH1 in EC of the aorta during aging. While accumulation of 8-oxo-dG in SMC of the aorta was slightly increased, the expression of MTH1 protein in SMC was rather decreased by hypertension. We thus suggest that MTH1 may protect EC in the aorta from the oxidative damage increased by hypertension.     

Increased density of fluorescent adrenergic fibers around the middle cerebral arteries of stroke-prone spontaneously hypertensive rats.

The distribution of fluorescent adrenergic nerve fibers in the proximal portion (horizontal segment, Hs) and the three distal portions (major branches) of the middle cerebral arteries (MCA) was examined in stroke-prone spontaneously hypertensive rats (SHRSP) aged 10, 30, 60, 90, and 180 days, by the glyoxylic acid method. The results were compared with those in age-matched normotensive Wistar Kyoto (WKY) rats. While the distribution pattern of fluorescent nerve fibers in the proximal portion of WKY rats changed from a straight linear arrangement at 10 and 30 days of age to a network-like arrangement after 60 days, those from SHRSP showed a constant meshwork pattern throughout the entire examination period. In the distal portions of the MCA of both SHRSP and WKY rats at all ages examined, fluorescent nerve fibers formed a coarse network. The distribution densities of adrenergic nerve fibers in the proximal and distal portions of the MCA of SHRSP were significantly higher (P less than 0.01 and 0.05) than those of WKY rats at all ages examined, except in the proximal portion at 90 and 180 days of age. The difference in nerve fiber density between SHRSP and WKY rats reached a peak at 30 days of age in both proximal and distal portions, and then gradually decreased with age. The present study suggests that sympathetic hyperinnervation is an important factor in the development of hypertension, and is involved in its maintenance in SHRSP.     

Increased density of fluorescent adrenergic fibers around the middle cerebral arteries of stroke-prone spontaneously hypertensive rats

The distribution of fluorescent adrenergic nerve fibers in the proximal portion (horizontal segment, Hs) and the three distal portions (major branches) of the middle cerebral arteries (MCA) was examined in stroke-prone spontaneously hypertensive rats (SHRSP) aged 10, 30, 60, 90, and 180 days, by the glyoxylic acid method. The results were compared with those in agematched normotensive Wistar Kyoto (WKY) rats. While the distribution pattern of fluorescent nerve fibers in the proximal portion of WKY rats changed from a straight linear arrangement at 10 and 30 days of age to a network-like arrangement after 60 days, those from SHRSP showed a constant meshwork pattern throughout the entire examination period. In the distal portions of the MCA of both SHRSP and WKY rats at all ages examined, fluorescent nerve fibers formed a coarse network. The distribution densities of adrenergic nerve fibers in the proximal and distal portions of the MCA of SHRSP were significantly higher (P<0.01 and 0.05) than those of WKY rats at all ages examined, except in the proximal portion at 90 and 180 days of age. The difference in nerve fiber density between SHRSP and WKY rats reached a peak at 30 days of age in both proximal and distal portions, and then gradually decreased with age. The present study suggests that sympathetic hyperinnervation is an important factor in the development of hypertension, and is involved in its maintenance in SHRSP.     

Estrogen status affects sensitivity to focal cerebral ischemia in stroke-prone spontaneously hypertensive rats

Estrogen treatment has been shown to reduce ischemic brain damage. Because endogenous estrogen levels fluctuate markedly during the estrous cycle, we investigated the effect of stage of estrous cycle on ischemic brain damage. Halothane anesthetized 3- to 5-mo-old female Wistar-Kyoto rats (WKY) and stroke-prone spontaneously hypertensive rats (SHRSP) in proestrus (high estradiol levels) or metestrus (low estradiol levels) underwent permanent middle cerebral artery occlusion. In SHRSP, infarct volume at 24 h postocclusion was 24% smaller in proestrus compared with metestrus [208.6 +/- 9.5 mm(3) (n = 7) vs. 272.7 +/- 23.8 mm(3) (n = 7), respectively, means +/- SE; P = 0.0278, unpaired t-test]. In WKY, infarct volumes were similar in proestrus and metestrus [157.0 +/- 5.4 mm(3) (n = 5) and 131.5 +/- 16.5 mm(3) (n = 8), respectively; P = not significant (NS)]. Brain swelling (ipsilateral minus contralateral hemispheric volumes) was similar in proestrus and metestrus for SHRSP [138 +/- 9 mm(3) (n = 6) and 136 +/- 10 mm(3) (n = 7), respectively] and for WKY [103 +/- 15 mm(3) (n = 5) and 90 +/- 11 mm(3) (n = 8), respectively]. Thus the reduction in infarct size in SHRSP is caused by a true attenuation of the infarct volume and not simply by a reduction in brain edema.     

Increased sympathetic innervation in the cerebral and mesenteric arteries of hypertensive rats

The density of catecholamine-containing nerve fibers was studied in the cerebral and mesenteric arteries from normotensive Wistar-Kyoto rats (WKY), spontaneously hypertensive rats (SHR), and stroke-prone SHR (SHRSP) in the growing (SHR, WKY) and adult (SHR, SHRSP, WKY) animals. Cerebral arteries from SHR showed an increased adrenergic innervation from day 1. The nerve plexuses reached an adult pattern earlier in SHR than in WKY. The arteries from adult SHR and SHRSP (22 weeks old) showed a markedly higher nerve density than WKY. There was a positive linear correlation between blood pressure and nerve density for four cerebral arteries. The mesenteric arteries were not innervated at birth. However, hyperinnervation of these arteries in the SHR was already present at 10 days of age as compared with WKY. Sympathectomy with anti-nerve growth factor and guanethidine caused a complete disappearance of fluorescent fibers in the mesenteric arteries from SHR and WKY, and in the cerebral arteries of WKY. The same procedure caused only partial denervation of the cerebral arteries from hypertensive animals. We postulate that the increase in nerve density in the cerebral arteries from the hypertensive rats may contribute to the development of arterial hypertrophy in chronic hypertension through the trophic effect of the sympathetic innervation on vascular structure.     

Resveratrol inhibits AGEs-induced proliferation and collagen synthesis activity in vascular smooth muscle cells from stroke-prone spontaneously hypertensive rats

Advanced glycation end-products (AGEs) of plasma proteins and/or matrix proteins are candidate mediators for various vascular complications such as atherosclerosis. We previously reported a significantly larger accumulation of AGEs of the aorta in stroke-prone spontaneously hypertensive rats (SHRSP) than in age-matched Wistar-Kyoto rats (WKY). In this study, we examined the effects of AGEs on vascular smooth muscle cells (VSMC) from SHRSP and WKY rats. We also studied the in vitro effects of resveratrol (3, 4',5-trihydroxystilbene), a natural phytestrogen, on VSMC proliferation, DNA synthesis, and collagen synthesis activity in SHRSP-VSMC. AGEs accelerated the proliferation of SHRSP- or WKY-VSMC in a time- and dose-dependent manner. VSMC from SHRSP were more sensitive to AGEs than VSMC from normotensive WKY. AGEs also significantly increased DNA synthesis and prolyl hydroxylase activity, a marker for collagen synthesis, in SHRSP-VSMC. AGEs-induced increases in TGF-beta1 mRNA in SHRSP-VSMC were significantly greater than in WKY-VSMC. Resveratrol inhibited AGEs-stimulated proliferation, DNA synthesis, and prolyl hydroxylase activity in SHRSP-VSMC in a dose-dependent manner. ICI 182780, a specific estrogen receptor antagonist, partly blocked the inhibitory effects of resveratrol on AGEs-stimulated proliferation, DNA synthesis, and prolyl hydroxylase activity. Resveratrol significantly inhibited AGEs-induced TGF-beta1 mRNA increases in a dose-dependent manner. Thus, resveratrol may confer protective effects on the cardiovascular system by attenuating vascular remodeling and may be clinically useful as a safer substitute for feminizing estrogens in preventing cardiovascular disease.     

Decreased content in left atrium and increased plasma concentration of atrial natriuretic polypeptide in spontaneously hypertensive rats (SHR) and SHR stroke-prone

The atrial contents and concentrations, and the plasma concentrations of atrial natriuretic polypeptide (ANP) in spontaneously hypertensive rats (SHR) and SHR stroke-prone (SHRSP) were measured and compared with those of age-matched Wistar Kyoto rats (WKY) using a specific radioimmunoassay (RIA) for alpha-rat ANP (alpha-rANP). The contents of alpha-rANP-LI in the atria of SHR (19.0 +/- 0.9 micrograms, mean +/- SEM) and SHRSP (19.3 +/- 0.6 micrograms) were significantly lower than that of WKY (22.8 +/- 1.4 micrograms) (p less than 0.05). The atrial concentration of alpha-rANP-LI was also significantly lower in SHR (248.2 +/- 11.3 ng/mg, p less than 0.05) and tended to be lower in SHRSP (272.2 +/- 12.4 ng/mg) than that of WKY (300.0 +/- 14.2 ng/mg). Furthermore, the concentrations in the left auricles of SHR and SHRSP were significantly lower than that of WKY (p less than 0.01 and p less than 0.05, respectively). In contrast, no significant difference was observed in the alpha-rANP-LI concentrations in the right auricles of WKY, SHR and SHRSP. Gel filtration studies coupled with RIA showed that gel filtration profiles of the extracts from the right and left auricles of WKY, SHR and SHRSP were essentially identical. The plasma alpha-rANP-LI levels in SHR (260 +/- 34 pg/ml) and SHRSP (319 +/- 19 pg/ml) were significantly higher than that in WKY (170 +/- 17 pg/ml) (p less than 0.05 and p less than 0.01, respectively). These results suggest that the secretion of ANP from the heart is increased in SHR and SHRSP compared with WKY.     

Involvement of iNOS in postischemic heart dysfunction of stroke-prone spontaneously hypertensive rats

We investigated the possible contribution of inducible nitric oxide synthase (iNOS) to postischemic heart dysfunction and injuries in stroke-prone spontaneously hypertensive rats (SHRSP). SHRSP, 13-14 wk of age, had significantly higher systolic blood pressure and greater heart weight than age-matched Wistar-Kyoto rats (WKY). Permanent occlusion of the left anterior descending coronary artery (LAD) caused significant and long-lasting increases in the activity and mRNA expression of myocardial iNOS in SHRSP compared with WKY. However, there was no significant difference in the LAD occlusion-induced expression of interleukin-1beta mRNA between SHRSP and WKY. Hemodynamic deterioration and myocardial fibrosis were also observed in SHRSP at 4 wk after LAD occlusion. Continuous administration of 2-amino-5,6-dihydro-6-methyl-4H-1,2-thiazin (AMT) completely blocked the LAD occlusion-induced increase in the myocardial iNOS activity of SHRSP. Moreover, postischemic heart dysfunction and injuries were also significantly ameliorated by 2-amino-5,6-dihydro-6-methyl-4H-1,2-thiazin (AMT). These results suggest that the increased activity of myocardial iNOS plays a pivotal role in the development of postischemic cardiac dysfunction and injuries in SHRSP with the hypertensive and hypertrophic heart.     

Incidence of multinucleated and polyploid aortic smooth muscle cells cultured from different age groups of spontaneously hypertensive rats.

Cell size and incidence of multinucleated, polyploid cells in cultured aortic smooth muscle cells from different age groups of spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY) were compared. Smooth muscle cells from SHR were generally larger than those from WKY, and the percentage of multinucleated smooth muscle cells was always higher in SHR than WKY in the three age groups of rats studied (3-4, 10-12, and 28-30 weeks). In smooth muscle cells from the 3- to 4-week group, there was a positive correlation between cell diameter and the percentage of multinucleated smooth muscle cells. Microdensitometric measurements also showed that the incidence of polyploid smooth muscle cells was always higher in SHR than WKY in the three age groups. There was a positive correlation between DNA density and nuclear area measurements in all the age groups of SHR and WKY. We conclude that cultured aortic smooth muscle cells from different age groups of SHR and WKY contained heterogeneous populations of cells and that, under our culture conditions, the polyploidy of the smooth muscle cells found in vivo was maintained in the SHR and WKY.     

Blood-brain barrier disruption in the hypothalamus of young adult spontaneously hypertensive rats

Vascular permeability and endothelial glycocalyx were examined in young adult spontaneously hypertensive rats (SHR), stroke-prone SHR (SHRSP), and Wistar Kyoto rats (WKY) as a control, in order to determine earlier changes in the blood-brain barrier (BBB) in the hypothalamus in chronic hypertension. These rats were injected with horseradish peroxidase (HRP) as an indicator of vascular permeability. Brain slices were developed with a chromogen and further examined with cationized ferritin, a marker for evaluating glycocalyx. Staining for HRP was seen around vessels in the hypothalamus of SHR and SHRSP, but was scarce in WKY. The reaction product of HRP appeared in the abluminal pits of endothelial cells and within the basal lamina of arterioles, showing increased vascular permeability in the hypothalamus of SHR and SHRSP, whereas there were no leaky vessels in the frontal cortex of SHR and SHRSP, or in both areas of WKY. The number of cationized ferritin particles binding to the capillary endothelial cells was decreased in the hypothalamus of SHR and SHRSP, while the number decreased in the frontal cortex of SHRSP, compared with those in WKY. Cationized ferritin binding was preserved in some leaky arterioles, while it was scarce or disappeared in other leaky vessels. These findings suggest that BBB disruption occurs in the hypothalamus of 3-month-old SHR and SHRSP, and that endothelial glycocalyx is markedly damaged there without a close relationship to the early changes in the BBB.     

Inhibition of NOS enhances pulmonary vascular changes in stroke-prone spontaneously hypertensive rats

To determine the effects of chronic nitric oxide (NO) blockade on the pulmonary vasculature, 58-day-old spontaneously hypertensive rats of the stroke-prone substrain (SHRSP) and Wistar-Kyoto rats (WKY) received N(omega)-nitro-L-arginine (L-NNA; 15 mg. kg(-1). day(-1) orally for 8 days). Relaxation to acetylcholine (ACh) in hilar pulmonary arteries (PAs), the ratio of right ventricular (RV) to body weight (RV/BW) to assess RV hypertrophy (RVH), and the percent medial wall thickness (WT) of resistance PAs were examined. L-NNA did not alter the PA relaxation, RV/BW, or WT in WKY. Although the PA relaxation and RV/BW in control SHRSP were comparable to those in WKY, the WT was increased (31 +/- 2 vs. 19 +/- 1%). L-NNA-treated SHRSP showed two patterns: in one group, the relaxation, RV/BW, and WT were comparable to those in the control SHRSP; in the other, impaired relaxation (36 +/- 7 vs. 88 +/- 4% for WKY) was associated with an increase in WT (37 +/- 1%) and RV/BW (0. 76 +/- 0.05). Thus the abnormal pulmonary vasculature in SHRSP at <10 wk of age is not accompanied by impaired relaxation in PAs or RVH; however, impaired relaxation is associated with increased WT and RVH.