Enrique Alborch

Reduction of infarct size by the NO donors sodium nitroprusside and spermine/NO after transient focal cerebral ischemia in rats

Nitric oxide (NO) plays a dual role (neuroprotection and neurotoxicity) in cerebral ischemia. NO promoting strategies may be beneficial shortly after ischemia. Therefore, we have studied the hemodynamic and possible neuroprotective effects of two NO donors, the classical nitrovasodilator sodium nitroprusside (SNP) and the NONOate spermine/NO, after transient focal cerebral ischemia in rats. Parietal cortical perfusion was measured by laser-Doppler flowmetry. The effects of increasing intravenous doses (10-300 microgram) of sodium nitroprusside and spermine/NO on cortical perfusion and arterial blood pressure were assessed. Transient (2 h) focal cerebral ischemia was carried out by the intraluminal thread method. The effects of intraischemic intravenous infusion of SNP (0.11, 1.1 mg/kg) and spermine/NO (0.36, 3.6 mg/kg) on hemodynamic parameters and infarct size developed after 1 week reperfusion were assessed. In control conditions, SNP and, to a lesser extent, spermine/NO induced dose-dependent hypotension and concomitant reduction in cortical perfusion. In focal cerebral ischemia, infusion of SNP (0.11 mg/kg) and spermine/NO (0.36, 3.6 mg/kg) reduced the infarct size. In the case of spermine/NO, cortical perfusion was maintained above the control levels during the ischemic insult. No significant hypotension was elicited by NO donors at the dose-ratios infused. In conclusion, brain damage induced by transient focal ischemia is reduced by intravenous NO donors. Neuroprotective effects of spermine/NO are due at least in part to improvement of brain perfusion, while sodium nitroprusside must provide direct cytoprotection. These results give further support to the protective effect of NO in the early stages of cerebral ischemia and point to the therapeutic potential of NONOates in the management of brain ischemic damage.

Relaxant Effects of 17-β-Estradiol in Cerebral Arteries through Ca2+ Entry Inhibition

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-β-estradiol in rabbit isolated basilar artery. Cumulative doses of 17-β-estradiol (0.3 μmol/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-β-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 μmol/L), nor the protein synthesis inhibitor cycloheximide (1 μmol/L) affected 17-β-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-β-estradiol, which was not significantly modified by incubation with the K+ channel blockers charybdotoxin (1 nmol/L and 0.1 μmol/L) or glibenclamide (10 nmol/L and 1 μmol/L). Preincubation with 17-β-estradiol (3 to 100 μmol/L) produced concentration-dependent inhibition of CaCl2-induced contraction, with less potency than the Ca2+ entry blocker nicardipine (0.01 to 10 nmol/L). The authors conclude that 17-β-estradiol induces endothelium-independent relaxation of cerebral arteries with tissue and gender selectivity. The relaxant effect is because of inhibition of extracellular Ca2+ influx to vascular smooth muscle, but activation of estrogen receptors, protein synthesis, or K+ efflux are not involved. Relatively high pharmacologic concentrations of 17-β-estradiol causing relaxation preclude acute vascular effects of physiologic circulating levels on the cerebral circulation.

Dietary phytoestrogens improve stroke outcome after transient focal cerebral ischemia in rats

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 liquide 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.

Acute estradiol protects CA1 neurons from ischemia-induced apoptotic cell death via the PI3K/Akt pathway.

Global ischemia arising during cardiac arrest or cardiac surgery causes highly selective, delayed death of hippocampal CA1 neurons. Exogenous estradiol ameliorates global ischemia-induced neuronal death and cognitive impairment in male and female rodents. However, the molecular mechanisms by which a single acute injection of estradiol administered after the ischemic event intervenes in global ischemia-induced apoptotic cell death are unclear. Here we show that acute estradiol acts via the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling cascade to protect CA1 neurons in ovariectomized female rats. We demonstrate that global ischemia promotes early activation of glycogen synthase kinase-3beta (GSK3beta) and forkhead transcription factor of the O class (FOXO)3A, known Akt targets that are related to cell survival, and activation of caspase-3. Estradiol prevents ischemia-induced dephosphorylation and activation of GSK3beta and FOXO3A, and the caspase death cascade. These findings support a model whereby estradiol acts by activation of PI3K/Akt signaling to promote neuronal survival in the face of global ischemia.

Acute relaxant effects of 17-β-estradiol through non-genomic mechanisms in rabbit carotid artery

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.

Comparison of the anticonstrictor action of dihydropyridines (nimodipine and nicardipine) and Mg2+ in isolated human cerebral arteries

The isometric tension recorded from ring segments of branches of human middle cerebral artery was the parameter used to study the inhibition of spasmogen-induced contractions as model for cerebral vasospasm. Concentration-response curves to 5-hydroxytryptamine (10(-9)-3 x 10(-5) M) and prostaglandin F2 alpha (10(-7)-3 x 10(-5) M) were inhibited in Ca(2+)-free medium and in Ca(2+)-free medium to which EGTA (1 mM) had been added, respectively. Nimodipine (10(-7), 10(-5) M), nicardipine (10(-7), 10(-5) M) and Mg2+ (magnesium sulfate 10(-4), 10(-2) M) inhibited the 5-HT-elicited contractions, and this inhibition was similar for the highest concentrations tested. In contrast, nimodipine and nicardipine were more effective than Mg2+ to inhibit the prostaglandin F2 alpha-elicited contractions. Nimodipine (10(-9)-10(-5) M), nicardipine (10(-9)-10(-5) M) and Mg2+ (10(-5)-3 x 10(-2) M) relaxed the arteries precontracted with PGF2 alpha (10(-5) M), but nicardipine was the most potent relaxant drug. Because 5-hydroxytryptamine and prostaglandin F2 alpha may be involved in the pathogenesis of cerebral vasospasm, nimodipine, nicardipine, and Mg2+ could be used in the pharmacological treatment of this disorder. However, dihydropyridines (particularly nicardipine) are more potent anticonstrictors than Mg2+.

Endothelin receptors mediating contraction in goat cerebral arteries

1 The aim of the present study was to identify the subtype of receptor mediating contraction to endothelin‐1 and sarafotoxin S6b in goat isolated middle cerebral arteries. 2 Endothelin‐1, endothelin‐2 and endothelin‐3 contracted cerebral arteries in a concentration‐dependent manner. Although the three peptides were full agonists, the order of potency was endothelin‐1 = endothelin‐2 > endothelin‐3, with a relative potency of endothelin‐1 and endothelin‐2 versus endothelin‐3 of ∼280. Sarafotoxin S6b induced concentration‐dependent contractions with lower potency than endothelin‐1/endothelin‐2, higher potency than endothelin‐3 and a higher maximum response than the three endothelins. 3 The selective ETA‐receptor antagonist, BQ‐123, did not induce changes in either the resting tension or in the active tone developed by depolarization. In contrast, BQ‐123 produced concentration‐dependent relaxations of endothelin‐1‐precontracted cerebral arteries, and to a greater extent of sarafotoxin S6b‐precontracted arteries. 4 Concentration‐response curves to endothelin‐1 and sarafotoxin S6b were competitively antagonized by BQ‐123 (pA2 of 7.43 ± 0.12 and 8.41 ± 0.09, respectively). In contrast, BQ‐123 had no effect on 5‐hydroxytryptamine‐elicited contractions even at 10−6 M. 5 It is concluded that both the order of potency of endothelin isopeptides and the antagonism of BQ‐123 point to the existence of ETA receptors mediating vasoconstriction to endothelin‐1 and sarafotoxin S6b in the goat middle cerebral artery. The different antagonistic potency of BQ‐123 against endothelin‐1 and sarafotoxin S6b suggests the existence of subtypes of ETA receptors.

Impairment of the modulatory role of nitric oxide on the endothelin-1-elicited contraction of cerebral arteries: a pathogenetic factor in cerebral vasospasm after subarachnoid hemorrhage?

OBJECTIVE Nitric oxide (NO) and endothelin-1 (ET-1) are two endothelium-derived factors probably involved in the pathogenesis of cerebral vasospasm after subarachnoid hemorrhage (SAH). Our aim was twofold, i.e., to ascertain whether endothelial and nonendothelial NO modulates the contractile response of cerebral arteries to ET-1 and to analyze whether this relationship might be impaired after experimental SAH. METHODS Rings of middle cerebral artery from goats in the control group and from goats with SAH were set up for isometric tension recordings. SAH was induced 3 days before the experiments by infusion of 10 ml of autologous arterial blood through a catheter previously inserted into the subarachnoid space (basal cistern). In goats in the control group, the response to ET-1 was obtained as follows: 1) in control arteries (unrubbed and nonincubated arteries); 2) in rubbed arteries (arteries in which the endothelium was mechanically removed); 3) during incubation with NG-nitro-L-arginine (L-NOArg) alone or plus L- or D-arginine; and 4) in rubbed arteries plus incubation with L-NOArg. In goats with SAH, that response was obtained in control arteries, rubbed arteries, and during incubation with L-NOArg. Specimens of middle cerebral artery were processed for transmission electron microscopy study. RESULTS In goats in the control group, ET-1 elicited concentration-dependent contraction of the middle cerebral artery that was significantly potentiated after endothelium denudation or during incubation with L-NOArg. The latter effect was reversed by L-arginine but not by D-arginine. Combined endothelium denudation and incubation with L-NOArg produced a contractile response to ET-1 significantly higher than that induced by each treatment separately. Hyperreactivity to ET-1 was observed in goats with SAH. Endothelium denudation did not alter the enhanced response to ET-1, but it was further significantly increased after incubation with L-NOArg. CONCLUSION These results demonstrate that an ET-1-NO interaction exists in control cerebral arteries in such a way that endothelial and nonendothelial NO partially counteract the contractile response to ET-1 and that although SAH did not modify the effect of nonendothelial NO, the absence of endothelial NO after SAH may contribute to the hyperreactivity of cerebral arteries to ET-1 and, thereby, to the development of cerebral vasospasm.

Changes in the Cerebrovascular Effects of Endothelin-1 and Nicardipine after Experimental Subarachnoid Hemorrhage

The role of endothelium-related factors in the pathogenesis of cerebral vasospasm after subarachnoid hemorrhage (SAH) has gained interest since the discovery of endothelin-1 (ET-1). We have examined, before and after SAH, the responsiveness of the cerebrovascular bed of the goat to ET-1, the sources of Ca2+ in ET-1-induced responses, and the ability of the Ca2+ entry blocker nicardipine to counteract them. Before SAH, injection of ET-1 into the cerebral circulation increased cerebrovascular resistance, thereby producing dose-dependent reductions in cerebral blood flow (CBF), which were prevented by nicardipine. In isolated middle cerebral arteries, ET-1 induced concentration-dependent contractions, which were equally inhibited in Ca(2+)-free medium (without or with ethylene glycol tetraacetic acid) and by the Ca2+ entry blocker nicardipine. On the third day after SAH, CBF was reduced by 28% and cerebrovascular resistance increased by 39%. At the same time, both ET-1-induced reductions in CBF and the constricting effects of ET-1 in vitro were enhanced. The ability of nicardipine to increase CBF and to inhibit the effects of ET-1 was impaired as a result of reduced dependence of cerebral arteries on extracellular Ca2+. On the seventh day after SAH, CBF and cerebrovascular resistance returned to control values, and effects of ET-1 became normal. It is suggested that the hyperreactivity to ET-1 of the cerebrovascular bed induced by SAH could have a role in the development of vasospasm, which could reduce the vascular effects of Ca2+ entry blockers after SAH.

Delayed post-ischemic administration of CDP-choline increases EAAT2 association to lipid rafts and affords neuroprotection in experimental stroke

Glutamate transport is the only mechanism for maintaining extracellular glutamate concentrations below excitotoxic levels. Among glutamate transporters, EAAT2 is responsible for up to 90% of all glutamate transport and has been reported to be associated to lipid rafts. In this context, we have recently shown that CDP-choline induces EAAT2 translocation to the membrane. Since CDP-choline preserves membrane stability by recovering levels of sphingomyelin, a glycosphingolipid present in lipid rafts, we have decided to investigate whether CDP-choline increases association of EAAT2 transporter to lipid rafts. Flotillin-1 was used as a marker of lipid rafts due to its known association to these microdomains. After gradient centrifugation, we have found that flotillin-1 appears mainly in fractions 2 and 3 and that EAAT2 protein is predominantly found colocalised with flotillin-1 in fraction 2. We have also demonstrated that CDP-choline increased EAAT2 levels in fraction 2 at both times examined (3 and 6 h after 1 g/kg CDP-choline administration). In agreement with this, [(3)H] glutamate uptake was also increased in flotillin-associated vesicles obtained from brain homogenates of animals treated with CDP-choline. Exposure to middle cerebral artery occlusion also increased EAAT2 levels in lipid rafts, an effect which was further enhanced in those animals receiving 2 g/kg CDP-choline 4 h after the occlusion. Infarct volume measured at 48 h after ischemia showed a reduction in the group treated with CDP-choline 4 h after occlusion. In summary, we have demonstrated that CDP-choline redistributes EAAT2 to lipid raft microdomains and improves glutamate uptake. This effect is also found after experimental stroke, when CDP-choline is administered 4 h after the ischemic occlusion. Since we have also shown that this delayed post-ischemic administration of CDP-choline induces a potent neuroprotection, our data provides a novel target for neuroprotection in stroke.