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Oxidative and nitrosative stress are targets for intervention after ischemia/reperfusion. The aim of this study was to explore the effect of CR-6, a vitamin-E analogue that is antioxidant and scavenger of nitrogen-reactive species. Sprague-Dawley rats had the middle cerebral artery (MCA) occluded either for 90 mins or permanently. Cortical perfusion was continuously monitored by laser-Doppler flowmetry. CR-6 (100 mg/kg) was administered orally either at 2 and 8 h after MCA occlusion, or at 2 h only. Infarct volume, neurological deficit, and signs of reperfusion injury were evaluated. CR-6 was detected in plasma and brain by HPLC. CR-6 reduced glutathione consumption in the ischemic brain and superoxide generation in the isolated MCA. CR-6 decreased infarct volume and attenuated the neurological deficit at 1 and 7 days after ischemia/reperfusion, but not after permanent ischemia. Immediately after reperfusion, cortical blood flow values returned to their baseline (+/-20%) in several animals, whereas others showed hyper-perfusion (>20% of baseline). Reactive hyperemia was associated with adverse events such as increased cortical BBB leakage, edema, protein nitrotyrosination, COX-2 expression, and neutrophil accumulation; and with a poorer outcome, and CR-6 attenuated these effects. In conclusion, oral CR-6 administration after transient ischemia protects the brain from reperfusion injury.

Cerebral ischemia followed by reperfusion alters vessel properties of brain arteries in rats, inducing an inflammatory response and excessive generation of reactive oxygen species. This study investigated the participation of oxidative stress on vessel properties after ischemia/reperfusion and the beneficial effects of 3,4-dihydro-6-hydroxy-7-methoxy-2,2-dimethyl-1(2H)-benzopyran (CR-6). The right middle cerebral artery was occluded (90 min) and reperfused (24 h). Sham-operated animals were used as controls. Ischemic rats were treated either with CR-6 (100 mg/kg in 1 ml olive oil) or vehicle (1 ml olive oil) administered orally at 2 and 8 h after the onset of ischemia. The structural, mechanical, and myogenic properties of the middle cerebral artery (MCA) were assessed by pressure myography. Superoxide anion ( ) production was evaluated by ethidium fluorescence, and protein tyrosine nitrosylation was determined by immunofluorescence. Infarct volume was smaller in rats treated with CR-6. In MCA, ischemia/reperfusion increased wall thickness, cross-sectional area, wall/lumen, and decreased wall stress. CR-6 treatment prevented all of these changes induced by ischemia/reperfusion. However, impaired myogenic response and larger lumen diameter in active conditions observed after ischemia/reperfusion were not modified by CR-6. Treatment with CR-6 prevented the increase in production and partially prevented the enhanced protein tyrosine nitrosylation that occurred in response to ischemia/reperfusion. Our findings suggest that oxidative stress is involved in the alterations of MCA properties observed after ischemia/reperfusion and that CR-6 induces protection.

Stroke triggers a local and systemic inflammatory response leading to the production of cytokines that can influence blood vessel reactivity. In this study, we aimed to assess whether cerebral ischemia/reperfusion could affect vasoconstriction and vasodilatation on mesenteric resistance arteries (MRA) from Wistar Kyoto rats. The right middle cerebral artery was occluded (90 min) and reperfused (24 h). Sham-operated animals were used as controls. Plasma levels of interleukin (IL)-6 and IL-1beta were measured at 24 h. Vasoconstrictor and vasodilator responses were recorded in a wire myograph. Protein expression was determined by Western blot and immunofluorescence, and superoxide anion (O(2)(.)) production was evaluated by ethidium fluorescence. In MRA, ischemia/reperfusion increased plasma levels of IL-6, O2. production, protein expression of cyclooxygenase-2, and protein tyrosine nitrosylation, but it impaired acetylcholine (ACh) vasodilatation without modifying the vasodilatations to sodium nitroprusside or the contractions to phenylephrine and KCl. Superoxide dismutase (SOD) and indomethacin reversed the impairment of ACh relaxation induced by ischemia/reperfusion. However, N(omega)-nitro-l-arginine methyl ester affected similarly ACh-induced vasodilatations in MRA of ischemic and sham-operated rats. Protein expression of endothelial and inducible nitric-oxide synthase, copper/zinc SOD, manganese SOD, and extracellular SOD was similar in both groups of rats. Our results show MRA endothelial dysfunction 24 h after brain ischemia/reperfusion. Excessive production of O2. in MRA mediates endothelial dysfunction, and the increase in plasma cytokine levels after brain ischemia/reperfusion might be involved in this effect.

Phytoestrogens and mycoestrogens are naturally occurring plant and fungus secondary metabolites with estrogen-like structure and/or actions. We aimed to check the hypothesis that phytoestrogens and mycoestrogens, due to their ability to elicit cerebral vasodilation, can induce acute increases in brain blood perfusion. For this purpose, we continuously recorded cerebrocortical perfusion by laser-Doppler flowmetry in anesthetized rats receiving intracarotid infusions (1 mg/kg) of one of the following estrogenic compounds: biochanin A, daidzein, genistein or zearalanone. We have shown the ability of two isoflavone class phytoestrogens (daidzein and biochanin A) and the mycoestrogen zearalanone to induce acute increases in brain blood flow when locally infused into the cerebral circulation of anesthetized rats. The isoflavone genistein failed to induce a significant increase in brain perfusion. No concomitant changes in blood pressure were recorded during the cerebral effects of the estrogenic compounds. Therefore, these microcirculatory effects were due to direct actions of the estrogenic compounds on the cerebrovascular bed.

Phospholipase A2s (PLA2s) seem to be involved in the pathophysiology of ischemic brain injury, but their specific role is far from being completely understood. The present study was carried out to ascertain how and to what extent secretory PLA2s (sPLA2s) activity influences outcome after cerebral ischemia-reperfusion, and to correlate this with the inflammatory response. To do this we used the potent and selective sPLA2 inhibitor, 12-epi-scalaradial. Male Wistar rats were separated into three groups: a control group receiving intracerebroventricular vehicle, and two groups receiving intracerebroventricular 0.005 or 0.5 microg/h 12-epi-scalaradial. Every animal was subjected to middle cerebral artery (MCA) occlusion (90 min, intraluminal thread technique) under continuous moni-torization of cerebrocortical perfusion (CP, laser-Doppler flowmetry), followed by reperfusion (3 days). Neurological status, infarct volume, and myeloperoxidase (MPO) activity were the main end points. Three days after the 90-min ischemia period, neurological examination did not reveal significant differences between the three groups of rats. Control rats showed a mean infarct volume of 145.9 +/- 24.7 mm3 (21 +/- 4.1% of the ipsilateral hemisphere volume), while mean infarct volume in rats treated with 0.005 or 0.5 microg/h 12-epi-scalaradial increased to 164.8 +/- 86.8 mm3 (22.0 +/- 10.9%) and 211.5 +/- 12.2 mm3 (28 +/- 3%, P < 0.05), respectively. Treatment with the highest dose of 12-epi-scalaradial (0.5 microg/h) increased MPO activity in the ipsilateral hemisphere by about 140% (from 0.59 +/- 0.59 to 1.42 +/- 1.03 units of activity/g of tissue in comparison with the control ischemic hemisphere, P < 0.05). Overall, our results point to a positive rather than a negative influence of sPLA2 activity during ischemia. This, along with its inability to decrease the inflammatory response, does not allow to propose the use of 12-epi-scalardial as a potential drug for stroke therapy.

As phytoestrogens are postulated as being neuroprotectants, we assessed the hypothesis that dietary isoflavone-type phytoestrogens are neuroprotective against ischemic stroke. Transient focal cerebral ischemia (90 min) was induced by middle cerebral artery occlusion (MCAO) following the intraluminal thread technique, both in rats fed with soy-based diet and in rats fed with isoflavone-free diet. Cerebro-cortical laser-Doppler flow (cortical perfusion, CP), arterial blood pressure, core temperature, PaO2, PaCO2, pH and glycemia were measured before, during and after MCAO. Neurological examination and infarct volume measurements were carried out 3 days after the ischemic insult. Dietary isoflavones (both glycosides and aglycones) were measured by high-performance liquid chromatography. Neither pre-ischemic, intra-ischemic nor post-ischemic CP values were significantly different between the soy-based diet and the isoflavone-free diet groups. Animals fed with the soy-based diet showed an infarct volume of 122 +/- 20.2 mm3 (19 +/- 3.3% of the whole ipsilateral hemisphere volume). In animals fed with the isoflavone-free diet the mean infarct volume was significantly higher, 191 +/- 26.7 mm3 (28 +/- 4.1%, P < 0.05). Neurological examination revealed significantly higher impairment in the isoflavone-free diet group compared with the soy-based diet group (3.3 +/- 0.5 vs. 1.9 +/- 0.5, P < 0.05). These results demonstrate that dietary isoflavones improve stroke outcome after transient focal cerebral ischemia in such a way that a higher dietary isoflavone content results in a lower infarct volume and a better neurological status.

We hypothesized that sildenafil, inhibitor of phosphodiesterase-5 (PDE-5), interacts with the nitric oxide (NO)-cGMP pathway in the cerebral arteries and shows vasoactive effects. To prove it in the isolated rabbit basilar artery, we compared the effects of sildenafil with other PDE-5 inhibitors, assessed the endothelial dependence of the vasoactive responses, and used modulators of the cGMP and cAMP signaling processes. Sildenafil (10 nM-0.1 mM) induced concentration-dependent relaxations of endothelin-1 (10 nM)-precontracted basilar artery, which were partially inhibited both in endothelium-denuded arteries and in arteries precontracted by depolarization with KCl (50 mM). Endothelin-1 (1 pM-30 nM) induced concentration-dependent contractions that were inhibited by sildenafil (0.1-100 microM). Zaprinast (10 nM-0.1 mM) and MBCQ (1 nM-0.1 mM), PDE-5 inhibitors, induced concentration-dependent relaxations with lower and higher potency than sildenafil, respectively. Sildenafil-induced relaxation was inhibited in arteries preincubated with the NO synthase inhibitor L-NAME (0.1 mM) or the soluble guanylyl cyclase inhibitor ODQ (10 microM). Preincubation with sildenafil (0.1 microM) enhanced the relaxations induced by acetylcholine (0.1 nM-0.1 mM) and the NO donor sodium nitroprusside (0.1 nM-0.1 mM), but not those induced by the cell-permeable cGMP analogue 8-Br-cGMP (1 nM-0.1 mM) and the adenylyl cyclase activator forskolin (0.1 nM-10 microM). These results show that sildenafil has vasoactive effects in isolated cerebral arteries. By enhancing the NO-cGMP signaling pathway in the cerebrovascular wall, sildenafil induces vasodilation, prevents vasoconstriction, and potentiates the effect of other NO-dependent vasodilators.

Background and Purpose: It has been shown that the reversed operation of glutamate transporters when ATP levels fall accounts for most glutamate release induced by severe cerebral ischemia. Nitric oxide (NO) is formed after ischemia and causes ATP depletion. Our purpose is to test if NO release from inducible NO synthase (iNOS) after stroke may cause a delayed glutamate release due to ATP depletion that might underlie progression of the ischemic infarct. We have studied the effect of the highly selective inhibitor of iNOS activity 1400W on brain ATP levels, extracellular glutamate, and stroke outcome after transient focal cerebral ischemia in rats., Methods: To induce focal ischemia, the middle cerebral artery (MCA) was occluded by using the intraluminal thread technique (tMCAO). 1400W was administered, after tMCAO, by using an Alzet osmotic pump to yield a drug delivery rate of 2.5 mg/kg/h. Results. Postischemic treatment with 1400W induced a reduction in the neurofunctional impairment and in the total volume of brain infarct. Western blot analysis showed ischemia-induced expression of iNOS. Treatment with 1400W partially prevented delayed ATP reduction and produced inhibition of the subsequent delayed increase in glutamate levels caused by the ischemic insult., Conclusions: Our data indicate that 1400W improves stroke outcome, an effect concomitant to the inhibition of both ischemia-induced decrease in brain ATP levels and increase in glutamate release. These results provide evidence indicating that the expression of iNOS induced by ischemia may contribute to the progression of the ischemic infarct and have important therapeutic implications for the management of stroke.

Oxygen free radicals have been involved in the pathophysiology of cerebral ischemia, especially after spontaneous or thrombolytic reperfusion. In this study with rats, we have combined a severe focal ischemic insult (2 h) and a prolonged reperfusion time (7 days) to assess the possible sustained neuroprotective effect of ebselen (10 or 100 mg/kg), a small, lipophilic organoselenium compound which mimics glutathione peroxidase. Parietal cortical perfusion was measured by laser-Doppler flowmetry, and focal cerebral ischemia was carried out by the intraluminal thread method. We have measured plasma selenium levels, brain reduced glutathione levels, as a marker of oxidative stress, and infarct volume associated with cerebral ischemia. Focal ischemia did not alter reduced glutathione levels, while 60 min reperfusion following ischemia induced a significant (P < 0.05) decrease in reduced glutathione levels of the ipsilateral hemisphere. Pretreatment with ebselen, which induced significant (P < 0.05) increase in plasma selenium levels, did not significantly alter the decrease in reduced glutathione levels. The ischemic insult induced 30% mortality on average, with deaths always occurring within 12-48 h. Surviving rats suffered up to 25% body weight loss 1 week after the ischemic insult. Infarct volumes were 26.8 +/- 4.7% of the hemisphere in placebo-treated rats, 26.6 +/- 3.6% in 10 mg/kg ebselen-treated rats, and 25.6 +/- 6.4% in 100 mg/kg ebselen-treated rats (not significantly different). Single-dose administration of ebselen does not reduce the size of brain infarct resulting from severe focal cerebral ischemia in rats. In contrast to previous studies with relatively earlier endpoints, we have delayed the measurement of infarct volume to 1 week after the ischemic insult., (Copyright 2004 Elsiever B.V.)

As a previous step to consider their use in the pharmacology for stroke, we investigated the effects of four phytoestrogens (i.e. genistein, daidzein, zearalanone and biochanin A) on cerebral vessels. Cerebral vascular responses were analyzed by conventional recording of isometric tension in rabbit basilar artery segments kept in organ bath under standard conditions. The four phytoestrogens elicited concentration-dependent relaxant responses of different potency in basilar artery segments previously contracted with either 5x10(-2) M KCl or 10(-4) M UTP. Neither endothelium removal, 10(-4) M N(omega)-nitro-L-arginine methyl ester (L-NAME, nitric oxide (NO) synthase inhibitor), 10(-5) M1 H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, selective inhibitor of NO-sensitive guanylyl cyclase), 10(-5) M 4H-8-bromo-1,2,4-oxadiazolo(3,4-d)benz(b)(1,4)oxazin-1-one (NS2028, specific soluble guanylyl cyclase inhibitor), nor 10(-5) M indomethacin (prostaglandin biosynthesis inhibitor) modified the phytoestrogen-elicited vasorelaxant responses. On the other hand, Ca(2+)-elicited contractile responses were effectively inhibited in the presence of phytoestrogens. Phytoestrogens act as cerebrovascular relaxants by a mechanism which involves Ca(2+) entry blockade in the vascular smooth muscle rather than stimulation of vasorelaxant endothelium-related mechanisms such as NO/cGMP or prostaglandins.

Estrogens could play a cardiovascular protective role not only by means of systemic effects but also by means of direct effects on vascular structure and function. We have studied the acute effects and mechanisms of action of 17-beta-estradiol on vascular tone of rabbit isolated carotid artery. 17-Beta-estradiol (10, 30, and 100 microM) elicited concentration-dependent relaxation of 50 mM KCl-induced active tone in male and female rabbit carotid artery. The stereoisomer 17-alpha-estradiol showed lesser relaxant effects in male rabbits. Endothelium removal did not modify relaxation induced by 17-beta-estradiol. The NO synthase inhibitor L-NAME (100 microM) only reduced significantly relaxation produced by 30 microM 17-beta-estradiol. Relaxation was not modified by the estrogen receptor antagonist ICI 182,780 (1 microM), the protein synthesis inhibitor cycloheximide (1 microM), and the selective K(+) channel blockers charybdotoxin (0.1 microM) and glibenclamide (1 microM). CaCl(2) (30 microM -10 mM) induced concentration-dependent contraction in rabbit carotid artery depolarized by 50 mM KCl in Ca(2+) free medium. Preincubation with 17-beta-estradiol (3, 10, 30, or 100 microM) or the L-type Ca(2+) channel blocker nicardipine (0.01, 0.1, 1, or 10 nM) produced concentration-dependent inhibition of CaCl(2)-induced contraction. In conclusion, 17-beta-estradiol induces endothelium-independent relaxation of rabbit carotid artery, which is not mediated by classic estrogen receptor and protein synthesis activation. The relaxant effect is due to inhibition of extracellular Ca(2+) influx to vascular smooth muscle, but activation of K(+) efflux is not involved. Relatively high pharmacological concentrations of estrogen causing relaxation preclude acute vasoactive effects of plasma levels in the carotid circulation.

Growth factors promote cell growth and survival and protect the brain from developing injury after ischemia. In this article, the authors examined whether transforming growth factor-alpha (TGF-alpha) was protective in transient focal ischemia and whether alteration of cerebral circulation was involved. Rats received intraventricular TGF-alpha (50 ng, either split into 2 doses given 30 minutes before and 30 minutes after middle cerebral artery occlusion (MCAO), or 1 dose given 30 minutes after MCAO) or vehicle. Rats were subjected to 1-hour intraluminal MCAO and cerebral blood flow was recorded continuously by laser-Doppler flowmetry. Infarct volume was measured 1 and 4 days later. The effects of TGF-alpha on arterial tone were assessed in isolated rabbit basilar and common carotid arteries. Transforming growth factor-alpha before and after ischemia reduced infarct volume by 70% at 1 day and 50% at 4 days. Transforming growth factor-alpha given only after ischemia also did reduce infarct volume by 70% at 1 day and 80% at 4 days. The protective effect was more marked in cortex than in striatum. Transforming growth factor-alpha did not change cortical microvascular perfusion and did not modify arterial passive tone nor agonist-induced active tone. It can be concluded that TGF-alpha reduces infarct volume, even when the factor is exclusively administered at reperfusion, and that this effect is not mediated by changes in microvascular perfusion or cerebral arteries. It is therefore suggested that TGF-alpha has a protective effect against neuronal cell death after transient focal ischemia.

Estrogens account for gender differences in the incidence and outcome of stroke, but it remains unclear to what extent neuroprotective effects of estrogens are because of parenchymal or vascular actions. Because reproductive steroids have vasoactive properties, the authors assessed the effects and mechanisms of action of 17-beta-estradiol in rabbit isolated basilar artery. Cumulative doses of 17-beta-estradiol (0.3 micromol/L to 0.1 mmol/L) induced concentration-dependent relaxation that was larger in basilar than carotid artery, in male than female basilar artery, and in KCl-precontracted than UTP-precontracted male basilar artery. Endothelium removal did not modify relaxation induced by 17-beta-estradiol in basilar artery, whereas relaxation induced by acetylcholine (1 nmol/L to 0.1 mmol/L) was almost abolished. Neither the estrogen receptor antagonist ICI 182,780 (1 micromol/L), nor the protein synthesis inhibitor cycloheximide (1 micromol/L) affected 17-beta-estradiol-induced relaxations. Relaxations induced by the K(+) channel openers NS1619 and pinacidil in the same concentration range were greater and lower, respectively, when compared with relaxation to 17-beta-estradiol, which was not significantly modified by incubation with the K(+) channel blockers charybdotoxin (1 nmol/L and 0.1 micromol/L) or glibenclamide (10 nmol/L and 1 micromol/L). Preincubation with 17-beta-estradiol (3 to 100 micromol/L) produced concentration-dependent inhibition of CaCl(2)-induced contraction, with less potency than the Ca(2+) entry blocker nicardipine (0.01 to 10 nmol/L). The authors conclude that 17-beta-estradiol induces endothelium-independent relaxation of cerebral arteries with tissue and gender selectivity. The relaxant effect is because of inhibition of extracellular Ca(2+) influx to vascular smooth muscle, but activation of estrogen receptors, protein synthesis, or K(+) efflux are not involved. Relatively high pharmacologic concentrations of 17-beta-estradiol causing relaxation preclude acute vascular effects of physiologic circulating levels on the cerebral circulation.