Phosphate binders prevent phosphate-induced cellular senescence of vascular smooth muscle cells and vascular calcification in a modified, adenine-based uremic rat model.

Clinical and experimental studies have reported that phosphate overload plays a central role in the pathogenesis of vascular calcification in chronic kidney disease. However, it remains undetermined whether phosphate induces cellular senescence during vascular calcification. We established a modified uremic rat model induced by a diet containing 0.3% adenine that showed more slowly progressive kidney failure, more robust vascular calcification, and longer survival than the conventional model (0.75% adenine). To determine the effect of phosphate on senescence of vascular smooth muscle cells (VSMCs) and the protective effect of phosphate binders, rats were divided into four groups: (1) normal control rats; (2) rats fed with the modified adenine-based diet (CKD); (3) CKD rats treated with 6% lanthanum carbonate (CKD-LaC); and (4) CKD rats treated with 6% calcium carbonate (CKD-CaC). After 8 weeks, CKD rats showed circumferential arterial medial calcification, which was inhibited in CKD-LaC and CKD-CaC rats. CKD rats showed increased protein expression of senescence-associated ß-galactosidase, bone-related proteins, p16 and p21, and increased oxidative stress levels in the calcified area, which were inhibited by both phosphate binders. However, serum levels of oxidative stress and inflammatory markers, serum fibroblast growth factor 23, and aortic calcium content in CKD-CaC rats were higher than those in CKD-LaC rats. In conclusion, phosphate induces cellular senescence of VSMCs in the modified uremic rat model, and phosphate binders can prevent both cellular senescence and calcification of VSMCs via phosphate unloading. Our modified adenine-based uremic rat model is useful for evaluating uremia-related complications, including vascular calcification.

Polymorphism in the sorbin and SH3-domain-containing-1 (SORBS1) gene and the risk of brain infarction in the Japanese population: the Fukuoka Stroke Registry and the Hisayama study.

BACKGROUND AND PURPOSE: Sorbin and SH3-domain-containing-1 (SORBS1) is an important adaptor protein in insulin-signalling pathway, and its genetic polymorphism may regulate the activity of insulin resistance. We investigated the association between the SORBS1 T228A polymorphism and ischaemic stroke. METHODS: Genotyping was achieved by a rapid-cycle PCR and melting curve analysis using fluorescent probes in 1049 incident cases of ischaemic stroke and 1049 age- and sex-matched control subjects recruited from the Hisayama study. RESULTS: The allele distributions of the SORBS1 T228A polymorphism were similar amongst cases and controls. The multivariate-adjusted odds ratio (OR) of the AA genotype for ischaemic stroke was 2.897 (95% CI, 0.907-8.018) compared with the TT genotype. In terms of stroke subtype, there was a trend toward a difference in the AA genotypes for lacunar infarction, compared with the TT genotype (OR = 8.740, P = 0.0510), and combined TT and TA genotypes (OR = 8.768, P = 0.0505). The other polymorphisms genotyped were not associated with any subtypes of ischaemic stroke. T228A polymorphism of SORBS1 was not associated with the prevalence of diabetes. CONCLUSIONS: The AA genotype of SORBS1 T228A polymorphism may play a role in lacunar infarction in the Japanese population.

NAD(P)H oxidase p22phox C242T polymorphism and ischemic stroke in Japan: the Fukuoka Stroke Registry and the Hisayama study.

The C242T polymorphism of p22phox, a component of NAD(P)H oxidase, may have an impact on cardiovascular diseases; however, the association between this polymorphism and brain infarction is not fully understood. Here, we investigate the relationship between the C242T polymorphism and brain infarction in Japan. We recruited 1055 patients with brain infarction and 1055 control subjects. A chi-squared test revealed that the T-allele frequency was lower in patients with cardioembolic infarction (5.6%) than in control subjects (11.0%, P < 0.001); however, allele frequencies in patients with lacunar and atherothrombotic infarction (11.2%) were not significantly different from those in control subjects (11.0%). A multivariate-adjusted conditional logistic regression analysis also revealed no association between CT + TT genotype, and lacunar and atherothrombotic infarction (odds ratio = 0.97, 95% confidence interval: 0.72-1.32). To investigate the functional effects of the C242T polymorphism, we examined superoxide production in COS-7 cells cotransfected with Nox4 and p22phox of each genotype. The superoxide-producing activity in those cells expressing p22phox with the T allele was not significantly different from that in cells expressing p22phox with the C allele. The present results suggest that the p22phox C242T polymorphism may have a protective effect against cardioembolic infarction, but is not related to lacunar and atherothrombotic infarction in Japan.

Selective induction of DeltaFosB in the brain after transient forebrain ischemia accompanied by an increased expression of galectin-1, and the implication of DeltaFosB and galectin-1 in neuroprotection and neurogenesis.

Transient forebrain ischemia causes selective induction of DeltaFosB, an AP-1 (activator protein-1) subunit, in cells within the ventricle wall or those in the dentate gyrus in the rat brain prior to neurogenesis, followed by induction of nestin, a marker for neuronal precursor cells, or galectin-1, a beta-galactoside sugar-binding lectin. The adenovirus-mediated expression of FosB or DeltaFosB induced expression of nestin, glial fibrillary acidic protein and galectin-1 in rat embryonic cortical cells. DeltaFosB-expressing cells exhibited a significantly higher survival and proliferation after the withdrawal of B27 supplement than the control or FosB-expressing cells. The decline in the DeltaFosB expression in the survivors enhanced the MAP2 expression. The expression of DeltaFosB in cells within the ventricle wall of the rat brain also resulted in an elevated expression of nestin. We therefore conclude that DeltaFosB can promote the proliferation of quiescent neuronal precursor cells, thus enhancing neurogenesis after transient forebrain ischemia.

Adenovirus-mediated gene transfer to ischemic brain: ischemic flow threshold for transgene expression.

BACKGROUND AND PURPOSE: Gene therapy may be a promising approach for treatment of brain ischemia, although protein synthesis is generally inhibited in ischemic conditions. Our goal in this study was to examine effects of brain ischemia on transgene expression of adenovirus-mediated gene transfer to ischemic brain. METHODS: Brain ischemia was produced by photochemical occlusion of the distal middle cerebral artery of spontaneously hypertensive rats (n=15). Ninety minutes after ischemia, adenoviral vectors encoding bacterial beta-galactosidase were injected into ipsilateral (nonischemic [I-n], peri-ischemic [I-p], and ischemic core [I-c] areas) and contralateral parietal (C) cortices. Cerebral blood flow before and during ischemia at each injected area was measured by laser-Doppler flowmetry. Expression of transgene was detected by histochemistry for semiquantitative scoring or by biochemical assay for quantitative analysis. RESULTS: Blood flow to the cortex decreased to 72+/-10% (mean+/-SEM) at I-n, 41+/-6% at I-p, and 23+/-3% at I-c after 10 minutes of ischemia. Expression of the reporter gene was consistently detected at C and I-n at each survival period. The semiquantitative score for transgene expression decreased according to severity of ischemia (C, 2.3; I-n, 2.6; I-p, 1.1; I-c, 0.3; mean values). beta-Galactosidase activity detected by chemiluminescent assay revealed that the values (mean+/-SEM) in the ischemic area (I-p, 15.9+/-9.2 mU/mg protein; I-c, 1.3+/-0.5) were significantly smaller than that of the nonischemic area (C, 45.4+/-6.9). Analysis of cerebral blood flow at I-p revealed that cerebral blood flow threshold for transgene expression was approximately 40% of the resting value. CONCLUSIONS: Adenovirus-mediated gene transfer into the ischemic brain provided effective expression of transgene at the nonischemic and peri-ischemic areas. Gene transfer to the ischemic brain may be a promising approach for treatment of ischemic penumbra.

Bradykinin mediates the acute effect of an angiotensin-converting enzyme inhibitor on cerebral autoregulation in rats.

BACKGROUND AND PURPOSE: In patients with stroke and long-standing hypertension, the autoregulation curve of cerebral blood flow (CBF) shifts toward higher blood pressure levels. Angiotensin-converting enzyme (ACE) inhibitors reduce blood pressure and shift the autoregulation curve back to normal in hypertensive patients. ACE inhibitors have 2 major pharmacological properties: they inhibit both the production of angiotensin II and the breakdown of kinins. Hence, we investigated whether the effect of an ACE inhibitor on the lower limit of CBF autoregulation is mediated by the potentiation of bradykinin-mediated vasodilatation. METHODS: In 28 male Sprague-Dawley rats, CBF was measured by laser-Doppler flowmetry during stepwise controlled hypotension. The lower limit of CBF autoregulation was defined as the mean arterial pressure at which CBF decreased by 20% of the baseline value. The rats were treated with an ACE inhibitor, captopril, in the captopril group; a bradykinin BK2-receptor antagonist, Hoe140, in the Hoe140 group; and both agents in the captopril+Hoe140 group. Other rats served as a control group. The lower limits of CBF autoregulation were compared among the 4 groups. RESULTS: In the captopril group, the lower limit of CBF autoregulation was 43+/-8 mm Hg (mean+/-SD), which was significantly lower than that in the control group (57+/-14 mm Hg). Inhibition of bradykinin abolished the effect of captopril on the lower limit of CBF autoregulation. Hoe140 alone had no significant effect on the lower limit of CBF autoregulation. CONCLUSIONS: These results suggest that the shift of the lower limit of CBF autoregulation by captopril is mediated, at least in part, by bradykinin.

Ischemia-induced release of amino acids in the hippocampus of aged hypertensive rats.

We have recently demonstrated the age-related vulnerability of hippocampal neurons to 20-min forebrain ischemia in spontaneously hypertensive rats (SHR). In the present study, we investigated the effect of aging on the release of amino acids in the hippocampus during transient cerebral ischemia for 20 min. Concentrations of extracellular amino acids and cerebral blood flow in the CA1 subfield were examined by an in vivo brain dialysis technique and a hydrogen clearance method, respectively, in adult (5-7 month) and aged (19-23 month) female SHR. During cerebral ischemia by bilateral carotid artery occlusion, cerebral blood flow to the hippocampus decreased to 20% of the resting values in both groups. After recirculation, both groups showed delayed hypoperfusion which was more prominent in the aged SHR. In the adult rats, concentrations of both aspartate and glutamate increased to approximately 8-fold of the resting values during ischemia. The elevation of these excitatory amino acids in the adult SHR was not significantly different from that in the aged rats. In contrast, the concentration of taurine increased 26-fold in the adult SHR but only 16-fold in the aged rats. Changes in other amino acids were not different between the two groups. These results indicate that an imbalance of excitatory and inhibitory amino acids, e.g., smaller release of taurine, during ischemia may, at least in part, contribute to the age-related vulnerability of hippocampal neurons to transient cerebral ischemia in SHR.

Adenovirus-mediated gene transfer to cerebral circulation.

Gene therapy may, be a promising approach for treatment of cerebrovascular disease. An adenoviral vector encoding beta-galactosidase was administered intracisternally or intraventricularly into the brain of rats. Efficient expression of the reporter gene was observed at the cerebral blood vessels and perivascular tissues. When the adenoviral vector was delivered into CSF of dogs suffering from subarachnoid hemorrhage, prominent expressions of transgene were observed. Introduction of the vector to the ischemic brain of rats provided efficient transgene expression in the peri-ischemic area. Therefore, gene transfer to the cerebral blood vessel and brain may be a promising approach for gene therapy of stroke. Atherosclerotic lesion plays an important role in stroke. We evaluated efficacy of adenovirus-mediated gene transfer to the atherosclerotic vessels from monkeys and rabbits using an ex vivo gene transfer system. Efficiency of transgene expression in the atherosclerotic endothelium was better than that of normal vessels in both animals. Thus, the endothelium of atherosclerotic vessels may be a good target for gene therapy. Next, we transfected atherosclerotic carotid arteries from rabbits with an adenoviral vector encoding endothelial nitric oxide synthase (eNOS). After overexpression of eNOS in the atherosclerotic arteries, the response to acetylcholine was augmented, showing similar relaxation with normal vessels. These results suggest that gene transfer to atherosclerotic vessels improves endothelial function, which may be a new therapeutic approach for cerebrovascular disease.

Increase in TUNEL positive cells in aorta from diabetic rats.

TUNEL staining, which allows detection of fragmented DNA in situ, is commonly used as an indication of apoptosis. Recent studies suggest that apoptosis is increased in several pathophysiological conditions. In this study we examined the hypothesis that chronic diabetes is associated with an increase in TUNEL staining of the aorta. Diabetic rats were studied 4-5 months after injection of streptozotocin (50 mg/kg). Aorta of diabetic and control rats were examined for TUNEL staining, morphology by electron microscopy, and DNA contamination in RNA preparation by polymerase chain reaction (PCR). TUNEL staining of aortic sections showed a 6 fold increase of positive cells in the media of diabetic aorta (22 +/- 6%) (mean +/- SE) compared with aorta from age-matched controls (3.6 +/- 0.9%, p < 0.05). Electron microscopy demonstrated typical apoptotic cells and bodies in the media of aorta from diabetic but not control rats. DNA contamination was found in RNA prepared from diabetic aorta, which was detected using PCR, which is consistent with increased DNA fragmentation. Increased TUNEL staining was not observed in rats with hyperglycemia 3 days after injection of streptozotocin. In conclusion, severe chronic diabetes is associated with an increase in TUNEL staining, and perhaps apoptosis, in the aorta. We speculate that increased apoptosis may compensate for increased proliferative activity in diabetic blood vessels.

Approaches to enhance expression after adenovirus-mediated gene transfer to the carotid artery.

The goal of this study was to enhance transgene expression after adenoviral-mediated gene transfer to the carotid artery. We used an adenoviral vector with a transgene that expresses beta-galactosidase, driven by the human cytomegalovirus (CMV) promoter/enhancer. The CMV promoter drives constitutive expression, and response elements within the enhancer allow inducible expression through binding of active transcription factors, such as cAMP response element binding protein (CREB) and nuclear factor kappa B (NFkappaB). Rings of rabbit carotid artery were incubated ex vivo with a replication-deficient adenovirus that expresses beta-galactosidase (AdCMV-betagal). Virus was removed from the medium, and forskolin or phorbol-12-myristate-13-acetate (PMA), which can induce activation of CREB or NFkappaB, respectively, were added to the medium. Pyrrolidine dithiocarbamate (PDTC) was used to inhibit activation of NFkappaB. Following incubation for 24 hours, beta-galactosidase activity was assessed by chemiluminescent reporter assay. Forskolin and PMA enhanced transgene expression in the carotid artery. Activity increased from 56+/-13 mU/mg protein (mean+/-SE) in rings of carotid treated with virus alone (10(9) pfu) to 159+/-23 mU/mg protein (P<0.05) in rings treated with forskolin, and to 189+/-40 mU/mg protein (P<0.05) in rings treated with PMA. Phorbol didecanoate, an inactive phorbol, did not affect expression of beta-galactosidase. After pre-incubation with PDTC prior to PMA, expression of beta-galactosidase was less than in rings incubated with PMA alone (29+/-11, P<0.05). Histochemical staining of carotid artery for beta-galactosidase demonstrated enhanced endothelial expression following administration of PMA. These findings suggest that expression after gene transfer to the carotid artery using an adenoviral vector with the CMV promoter/enhancer may be enhanced by PMA and forskolin, perhaps by activation of transcription factors.

Mortality and histological findings of the brain during and after cerebral ischemia in male and female spontaneously hypertensive rats.

Mortality and pathological changes of the brain during and after cerebral ischemia induced by bilateral carotid artery occlusion (BCO) were studied in male and female spontaneously hypertensive rats (SHR). Systolic arterial blood pressure at rest was significantly higher in male SHR (228 +/- 13 mm Hg, mean +/- S.E.M.) than female (192 +/- 12) (P less than 0.05). The average survival time during permanent occlusion was 11 +/- 6 h (mean +/- S.D.) in male SHR and 17 +/- 7 in female (P less than 0.005), though the cumulative mortality during 24-h ischemia was not different between male (88%) and female SHR (84%). Severe ischemic changes of nerve cells in the brain, especially in the cortex and hippocampus, were observed in 50% of male SHR at 3-h ischemia, while only 15% was observed in female SHR even after 7-h ischemia. After the temporary ischemia followed by reperfusion for 24 h, the mortality was varied between male and female SHR; 0, 31 and 100% after 1-, 3- and 5-h ischemia, respectively, in male SHR and 0% after 1- to 3-h ischemia and 33% after 5- to 7-h ischemia, respectively, in female. Ischemic changes of the brain tissue, such as acidophilic cytoplasm, nuclear degeneration and intercellular edema, were more frequent and severe in male SHR than female after recirculation following 3- or 5-h ischemia. It is concluded that the mortality and post-ischemic viability seem to be determined by the duration of ischemia and also by the degree of the neuronal damage, and female SHR is more tolerated for ischemic insult in comparison to male SHR.

Role of Na(+)/H(+) exchanger in dilator responses of rat basilar artery in vivo.

We tested the hypothesis that activation of Na(+)/H(+) exchanger is involved in dilator responses of the basilar artery to endothelium-dependent vasodilators in vivo. Using a cranial window in anesthetized rats, we examined responses of the basilar artery to acetylcholine and bradykinin. Topical application of acetylcholine and bradykinin increased diameter of the basilar artery in a concentration-related manner. Because N(G)-nitro-L-arginine, an inhibitor of nitric oxide synthase, almost abolished vasodilator responses to acetylcholine and bradykinin, vasodilatation produced by the agonists appears to be mediated primarily by nitric oxide. 5-N,N-Hexamethyleneamiloride, an inhibitor of Na(+)/H(+) exchanger, did not affect baseline diameter of the basilar artery, but inhibited vasodilatation in response to acetylcholine and bradykinin, without affecting vasodilatation produced by sodium nitroprusside. FR183998, another inhibitor of Na(+)/H(+) exchanger, also attenuated acetylcholine-induced dilatation of the basilar artery without affecting vasodilatation in response to sodium nitroprusside. Monomethylamine hydrochloride, which produces intracellular alkalinization, enhanced acetylcholine-induced dilatation of the basilar artery in the presence of 5-N,N-hexamethyleneamiloride. These results suggest that intracellular alkalinization produced by activation of Na(+)/H(+) exchanger may enhance nitric oxide production in the basilar arterial endothelium and thereby contribute to dilator responses of the artery in vivo.

Inhibition of Na+/H+ exchanger reduces infarct volume of focal cerebral ischemia in rats.

Activation of Na+/H+ exchanger (NHE) may have an important role in ischemic cell death by means of intracellular overload of Na(+) and Ca(2+). Recent evidence has suggested that inhibitors of NHE have protective effects on myocardial ischemia both in vivo and in vitro. In this study, we tested the hypothesis that FR183998, an inhibitor of NHE, reduces infarct volume produced by focal cerebral ischemia in rats. We used 20 male spontaneously hypertensive rats. Either FR183998 (1 mg/kg; n=10), or vehicle (n=10) was given intravenously to the rats and the distal middle cerebral artery of each animal was occluded using a photothrombotic technique. We measured regional cerebral blood flow using laser-Doppler flowmetry throughout the experiments. After 3 days, infarct volume was measured in each animal group. To estimate the brain edema, we also calculated the cortical volume in both hemispheres. The infarct volume in the FR183998-treated group (82+/-8 mm(3), mean+/-S.E.M.) was significantly smaller than that in the control group (115+/-12 mm(3)) (P=0.034). The cortical volume of the occluded side in the FR183998-treated group (359+/-7 mm(3)) tended to be smaller than that in the control group (378+/-9 mm(3)) (P=0.116). The regional cerebral blood flow and physiological variables during ischemia were not significantly different between the two groups throughout the experiments. These results suggest that inhibition of NHE by FR183998 may have beneficial effects in reducing infarct volume and brain edema during cerebral ischemia. Thus, NHE may play an important role in the development of neuronal damage during acute cerebral ischemia.

Protein kinase C modulates ischemia-induced amino acids release in the striatum of hypertensive rats.

The role of protein kinase C (PKC) in mediating the ischemia-induced release of amino acids in the striatum was studied using an in vivo brain dialysis technique in the striatum of spontaneously hypertensive rats (SHRs). Using HPLC combined with fluorescence detection methods, we investigated the concentrations of amino acids in the dialysates produced by 20 min of transient forebrain ischemia. We studied the effects of an inhibitor of PKC, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H7) and another isoquinoline analog (HA1004) with less inhibitory effect on the C kinase in ischemia-induced amino acids release. Bilateral carotid artery occlusion caused a marked reduction in the striatal blood flow by 91 +/- 6%. The extent of the cerebral blood flow (CBF) reduction were essentially the same among H7-, HA1004-, and the vehicle-treated groups. Forebrain ischemia produced a marked increase in glutamate (21-fold of the basal concentration), aspartate (19-fold) and taurine (16-fold). Pretreatment with H7 markedly attenuated the ischemia-in-duced release of these three amino acids to 3, 3 and 4-fold of the basal values, respectively. Increase of gamma-aminobutyric acid (GABA) was also attenuated by H7 (vehicle; 2.46 +/- 1.26 microM, H7; 0.62 +/- 0.75 mM). HA1004 did not affect the release of glutamate, aspartate or GABA during ischemia. The ischemia-induced release of taurine was significantly inhibited by HA1004 but the effect was much smaller than that of H7. These results thus indicate that PKC plays a major role in the ischemia-induced release of amino acids in the striatum of SHR.

Hypothermia inhibits ischemia-induced efflux of amino acids and neuronal damage in the hippocampus of aged rats.

Brain hypothermia has been reported to protect against ischemic damages in adult animals. Our goal in this study was to examine whether brain hypothermia attenuates ischemic neuronal damages in the hippocampus of aged animals. We also determined effects of hypothermia on ischemia-induced releases of amino acids in the hippocampus. Temperature in the hippocampus of aged rats (19-23 months) was maintained at 36 degrees C (normothermia), 33 degrees C (mild hypothermia) or 30 degrees C (moderately hypothermia) using a thermoregulator during 20 min of transient forebrain ischemia. Cerebral ischemia increased extracellular concentrations of glutamate and aspartate by 6- and 5-fold, respectively, in the normothermic group. Mild and moderate hypothermia, however, markedly inhibited the rise of these amino acids to less than 2-fold. Elevation of extracellular taurine, a putative inhibitory amino acid, was 16-fold in the normothermic rats. Mild hypothermia attenuated ischemia-induced increase in taurine (10-fold), and moderate hypothermia inhibited the increase. Ischemic damages, evaluated by histopathological grading of hippocampal CA1 area 7 days after ischemia, was significantly ameliorated in the mild (1.3+/-0.5, mean+/-S.E.M.) and moderate hypothermic rats (0.8+/-0.3) compared with the normothermic ones (3.4+/-0.4). These results suggest that brain hypothermia protects against ischemic neuronal damages even in the aged animals, and the protection is associated with inhibition of excessive effluxes of both excitatory and inhibitory amino acids.

Modulation of ischemia-evoked release of excitatory and inhibitory amino acids by adenosine A1 receptor agonist.

Adenosine has been reported to have beneficial effects against ischemic brain damage, although the mechanisms are not fully clarified. To examine the role of adenosine on the ischemia-evoked release of neurotransmitters, we applied a highly selective agonist for adenosine A1 receptor, 2-chloro-N6-cyclopentyladenosine (CCPA), into the ischemic brain using in vivo brain dialysis, which directly delivered the agonist to the local brain area. Concentrations of extracellular amino acids (glutamate, aspartate, gamma-aminobutyric acid (GABA) and taurine) and regional blood flow in the striatum of spontaneously hypertensive rats (SHRs) were monitored during cerebral ischemia elicited by bilateral carotid artery occlusion for 40 min and recirculation. Striatal blood flow and basal levels of amino acids were not affected by direct perfusion of CCPA (10 microM or 100 microM). During ischemia, concentrations of glutamate, aspartate, GABA and taurine increased up to 37-, 30-, 96- and 31-fold, respectively, when vehicle alone was administered. Administration of CCPA did not affect the changes in regional blood flow during ischemia and reperfusion. Perfusion of CCPA (100 microM), however, significantly attenuated the ischemia-evoked release of aspartate (by 70%) and glutamate (by 73%). The ischemia-induced increase of GABA tended to be decreased by CCPA, although it was not statistically significant. In contrast, both low and high concentrations of CCPA had little effect on the release of taurine during ischemia. These results suggest that stimulation of adenosine A1 receptors selectively attenuated the ischemia-evoked release of excitatory amino acids, but not of inhibitory amino acids without affecting blood flow. This modulation of the release of amino acids by adenosine A1 receptor agonists may play a protective role against ischemic neuronal damage.

Effect of thromboxane synthetase inhibitor on cerebral circulation and metabolism during experimental cerebral ischemia in spontaneously hypertensive rats.

The protective effect of thromboxane synthetase inhibitor, OKY-046, on brain ischemia was studied in spontaneously hypertensive rats. Cerebral ischemia was developed by bilateral carotid artery ligation (BCL) for 1 or 3 h and thereafter, circulation was restored for 15 min. OKY-046, 5 or 30 mg/kg, or saline as control was administered i.v. before BCL. Neither blood pressure nor blood gases were altered by OKY-046 or saline injection. During BCL, cerebral cortical blood flow was reduced to 25 and 15% of the resting value at 30 and 60 min, respectively, and these changes were not different among the groups. In rats with ischemia longer than 1 h, the blood flow was well preserved by OKY-046, 30 mg/kg, to 10-17% of the resting level, thus significantly higher than that (less than 5%) in non-treated rats. After 15 min recirculation, the supratentorial lactate level was lower and adenosine triphosphate (ATP) was higher in OKY-046-treated rats than in the saline-treated ischemic rats. Plasma thromboxane B2 was increased markedly in 1 h ischemic-reperfused rats without treatment and the increase was almost completely inhibited by OKY-046. In contrast, 6-keto-prostaglandin F1 alpha was increased 8.5-fold after ischemia and the increase was not affected by the treatment. OKY-046 seems to have an antiischemic effect on acutely induced cerebral ischemia. Selective inhibition of thromboxane A2 production and an inversely high level of prostaglandin I2 may be an important contribution to protection of the microcirculation during ischemia and preservation of ischemic cerebral metabolism.

Isradipine attenuates the ischemia-induced release of dopamine in the striatum of the rat.

We examined the effect of isradipine, a blocker of L-type voltage-sensitive Ca2+ channels (VSCCs), on the ischemia-induced release of dopamine in the rat striatum. Perfusion of 200 micrograms/ml isradipine in the striatum did not alter extracellular dopamine concentrations monitored by microdialysis. However, a marked increase (145-fold) in dopamine level during forebrain ischemia, developed by bilateral carotid artery occlusion, was attenuated significantly by 37% by isradipine whereas the intensity of ischemia, monitored by striatal blood flow, was unchanged. These results suggest that isradipine attenuates the ischemia-induced release of dopamine via blockade of L-type VSCCs on dopaminergic neurons.

Acute effects of antihypertensive agents on cerebral blood flow in hypertensive rats.

The acute effects of various antihypertensive agents on cerebral blood flow and mean arterial pressure (MAP) were studied in anesthetized (amobarbital 100 mg/kg) spontaneously hypertensive rats. Cerebral blood flow in the cortex and thalamus was measured by the hydrogen clearance method before and during a 60-min i.v. infusion of calcium antagonist (nifedipine), angiotensin converting enzyme inhibitor (captopril) or beta-blocker (propranolol). Nifedipine, 30 or 150 micrograms/kg per h, decreased dose dependently the MAP by 20 or 31%, and concomitantly increased cortical blood flow by 28 or 74%, and thalamic blood flow by 51 or 64%, respectively. Captopril, 10 or 100 mg/kg per h, decreased MAP by 7 or 14%, but changed cerebral blood flow minimally. In contrast, propranolol, 1.0 or 5.0 mg/kg per h, decreased MAP by 13 or 11%, with a concomitant reduction of cortical and thalamic blood flow by 20 or 15 and 33 or 37%, respectively. It is concluded that the changes in cerebral blood flow in response to hypotension are varied by antihypertensive drugs depending on the direct or indirect effect of the drugs (dilatation or constriction) on cerebral vessels. Nifedipine seems to dilate while propranolol constricts cerebral vessels.

Attenuation of ischemic and postischemic damage to brain metabolism and circulation by a novel Ca2+ channel antagonist, NC-1100, in spontaneously hypertensive rats.

We investigated the effect of a newly synthesized Ca2+ channel antagonist, NC-1100, on cerebral blood flow (CBF) and metabolism in spontaneously hypertensive rats. The rats received a bolus injection of 0.2 or 1.0 mg/kg NC-1100 i.v. and 1-h cerebral ischemia was then induced by bilateral carotid artery occlusion (group 1). The rats in group 2 were continuously infused with NC-1100 0.03 or 0.1 mg/kg per min, starting immediately after bilateral carotid artery occlusion, for the 1 h of ischemia and following 3-h recirculation. Group 1: during ischemia, CBF in all rats decreased to 6-8% of the resting values. At 1 h cerebral ischemia, brain tissue lactate increased 11.5-, 10.1- and 9.8-fold of the normal control given vehicle or NC-1100, 0.2 and 1.0 mg/kg, respectively. The ATP levels were better preserved by NC-1100 administration; 0.61 +/- 0.04 (mean +/- S.E.M.), 0.80 +/- 0.09 and 0.97 +/- 0.14 mmol/kg (P < 0.05 vs. vehicle), respectively. Group 2: during recirculation, CBF in NC-1100-treated rats returned to 83-90% of the resting values, but to only 65% in the vehicle group. Postischemic brain lactate at 3 h was less well preserved and ATP was dose dependently better preserved in NC-1100- than vehicle-treated rats. It is considered that pre- as well as postischemic administration of a Ca2+ channel antagonist, NC-1100, is beneficial to attenuate and also ameliorate the metabolic and circulatory derangement in the ischemic brain.