Daidzein has neuroprotective effects through ligand-binding-independent PPARγ activation.

Phytoestrogens are a group of plant-derived compounds that include mainly isoflavones like daidzein. Phytoestrogens prevent neuronal damage and improve outcome in experimental stroke; however, the mechanisms of this neuroprotective action have not been fully elucidated. In this context, it has been postulated that phytoestrogens might activate the peroxisome proliferator-activated receptor-γ (PPARγ), which exerts neuroprotective effects in several settings. The aim of this study was to determine whether the phytoestrogen daidzein elicits beneficial actions in neuronal cells by mechanisms involving activation of PPARγ. Our results show that daidzein (0.05-5 µM) decreases cell death induced by exposure to oxygen-glucose deprivation (OGD) from rat cortical neurons and that improves synaptic function, in terms of increased synaptic vesicle recycling at nerve terminals, being both effects inhibited by the PPARγ antagonist T0070907 (1 µM). In addition, this phytoestrogen activated PPARγ in neuronal cultures, as shown by an increase in PPARγ transcriptional activity. Interestingly, these effects were not due to binding to the receptor ligand site, as shown by a TR-FRET PPARγ competitive binding assay. Conversely, daidzein increased PPARγ nuclear protein levels and decreased cytosolic ones, suggesting nuclear translocation. We have used the receptor antagonist (RE) fulvestrant to study the neuroprotective participation of daidzein via estrogen receptor and at least in our model, we have discarded this pathway. These results demonstrate that the phytoestrogen daidzein has cytoprotective properties in neurons, which are due to an increase in PPARγ activity not mediated by direct binding to the receptor ligand-binding domain but likely due to post-translational modifications affecting its subcellular location and not depending to the RE and it is not additive with the agonist rosiglitazone.

Soy-derived phytoestrogens as preventive and acute neuroprotectors in experimental ischemic stroke: influence of rat strain.

The ability of a soy-based high-phytoestrogen diet (nutritional intervention) or genistein (pharmacological intervention), to limit ischemic brain damage in Wistar, Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats, has been assessed. As to the nutritional intervention, two groups from each strain received either a phytoestrogen-free (PE-0) or a high-phytoestrogen (PE-600) diet from weaning to adulthood. As to the pharmacological intervention, all animals were fed the standard soy-free AIN-93G diet and subsequently separated into two groups from each strain to receive either pure genistein (aglycone form, 1mg/kg/day intraperitoneal) or vehicle at 30 min reperfusion. After an episode of 90 min ischemia (intraluminal thread procedure) followed by 3 days reperfusion, cerebral infarct volume was measured. Arterial blood pressure (ABP) was significantly higher at the basal stage (just before ischemia) in SHR (140 ± 7 mmHg, n=17, p<0.05) than in Wistar (113 ± 4mmHg, n=23) and WKY (111 ± 6mmHg, n=14) rats. No significant differences were shown among the three stages (basal, ischemia, reperfusion) within each rat strain for both PE-0 and PE-600 diets. Wistar, but not WKY or SHR, rats fed the PE-600 diet showed significantly lower infarct volumes than their counterparts fed the PE-0 diet (30 ± 3% vs. 17 ± 3%, p<0.01). Genistein-treated Wistar, but not WKY or SHR, rats showed significantly lower infarct volumes than their vehicle-treated controls (27 ± 2% vs. 15 ± 2%, p<0.01). Our results demonstrate that: (1) the neuroprotective action of either chronic or acute exposure to soy isoflavones is strain-dependent, since it was shown in Wistar but not WKY or SHR rats; and (2) the soy-based diet does not prevent development of hypertension in SHR rats.

Guía de recomendaciones en la aplicación de modelos animales para el estudio del ictus / Recommendations guide for experimental animal models in stroke research

Introducción: La búsqueda de una terapia neuroprotectora efectiva para el ictus sigue siendo un reto para investigadores y clínicos. Una de las causas principales por la que a nivel clínico han fracasado terapias eficaces en ensayos experimentales reside probablemente en el desarrollo y modo de evaluación en estudios preclínicos de los agentes neuroprotectores en los modelos animales de isquemia cerebral. Métodos: Para unificar la metodología en la aplicación de los modelos experimentales a nivel nacional y mejorar la investigación en este campo, se ha elaborado un documento entre varios grupos españoles expertos en investigación neurovascular que constituye una guía de recomendaciones para el uso de los mismos. Resultados: Sus aspectos fundamentales se basan en la selección del modelo más adecuado en función del objetivo del estudio, teniendo en cuenta el tipo de especie y la cepa animal, la edad, el sexo y los factores de riesgo. La realización del diseño experimental incluye un grupo sham control y el cálculo previo del tamaño muestral. Otros aspectos muy importantes a seguir son la aleatorización en la asignación de los animales en cada grupo, el análisis ciego de los parámetros estudiados, el registro de la temperatura y flujo sanguíneo cerebral, así como la notificación y causas de animales excluidos en el estudio y la tasa de mortalidad. Conclusiones: Es esencial adquirir compromisos metodológicos para la optimización del empleo de los modelos animales de isquemia cerebral que incremente el rendimiento de hallazgos positivos en la fase preclínica y puedan trasladarse a la práctica clínica (AU)

Introduction: The progress of effective therapies for stroke has become a challenging task for both researchers and clinicians. Some pitfalls in clinical trials might have their origins in the pre-clinical experimental ischaemic models for the evaluation of potential neuro-protective agents. Methods: We aim to standardise the methods for the development of stroke animal models throughout Spain, to produce document with appropriate recommendations and best practice in order to improve experimental methods in the field of stroke research. Results: Members of several experienced stroke research groups prepared a guide with recommendations in the application of focal cerebral ischaemic models. The main features of this guide are based on the selection of the most appropriate animal model, taking in account the objective of the study, the species, strain, age, sex of animals, as well as risk factors. The experimental design must include a sham control group and the sample size calculation. Animal randomisation and blind analysis, masked assessment of outcomes, monitoring of body temperature and cerebral blood flow, and the reporting of reasons for excluding animals from the study, as well as the mortality rate, are other main points to fulfil in the application of stroke models.Conclusions: Standardised methods are essential to increase the success of the pre-clinical findings in the stroke neuroprotection field to be able to translate to the clinical practice (AU)

Recommendations guide for experimental animal models in stroke research.

INTRODUCTION: The progress of effective therapies for stroke has become a challenging task for both researchers and clinicians. Some pitfalls in clinical trials might have their origins in the pre-clinical experimental ischaemic models for the evaluation of potential neuro-protective agents. METHODS: We aim to standardise the methods for the development of stroke animal models throughout Spain, to produce document with appropriate recommendations and best practice in order to improve experimental methods in the field of stroke research. RESULTS: Members of several experienced stroke research groups prepared a guide with recommendations in the application of focal cerebral ischaemic models. The main features of this guide are based on the selection of the most appropriate animal model, taking in account the objective of the study, the species, strain, age, sex of animals, as well as risk factors. The experimental design must include a sham control group and the sample size calculation. Animal randomisation and blind analysis, masked assessment of outcomes, monitoring of body temperature and cerebral blood flow, and the reporting of reasons for excluding animals from the study, as well as the mortality rate, are other main points to fulfil in the application of stroke models. CONCLUSIONS: Standardised methods are essential to increase the success of the pre-clinical findings in the stroke neuroprotection field to be able to translate to the clinical practice.

Administration of transforming growth factor-alpha reduces infarct volume after transient focal cerebral ischemia in the rat.

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.

Temporospatial expression of HSP72 and c-JUN, and DNA fragmentation in goat hippocampus after global cerebral ischemia.

The role of gene induction (expression of HSP72 and c-JUN proteins) and delayed ischemic cell death (in situ labeling of DNA fragmentation) have been investigated in the goat hippocampus after transient global cerebral ischemia. The animals were subjected to 20-min ischemia (bilateral occlusion of the external carotid arteries plus bilateral jugular vein compression) and allowed to reperfuse for 2 h, and then 1, 3, and 7 days. Histological signs of cell loss were not found in the hippocampus at 2 h, 1 day, or 3 days of reperfusion. However, such an ischemic insult produced extensive, selective, and delayed degeneration in the hippocampus, as 68% of the neurons in CA1 had died at 7 days, but cell loss was not detected in CA3 and dentate gyrus fields. Concomitantly, a high percentage of TUNEL-positive CA1 neurons (60+/-9%, mean +/- SEM) was seen at 7 days, but not at the earlier time points. Mild induction of HSP72 was detected in the goat hippocampus after ischemia. The maximum percentage of HSP72-positive neurons (10-15%) was shown at 3 days of reperfusion and was concentrated mainly in the CA3 field, subiculum, and hilus, rather than in the CA1 field, whereas HSP72 expression was hardly detected at 7 days. At this later time point, scattered induction of nuclear c-JUN was found in a few neurons. The results show that: 1) postischemic delayed neuronal death selectively affects the CA1 field in the goat hippocampus, a phenomenon which seems to take longer to develop than in previously reported rodent models; and 2) postischemic expression of c-JUN does not appear to be related to cell death or survival, while the inability of most CA1 neurons to express HSP72 could contribute to neuronal death.

Relaxant effects of 17-beta-estradiol in cerebral arteries through Ca(2+) 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-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.

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.

Cerebrovascular effects of dotarizine, a piperazine derivative, in the goat. An in vivo and in vitro complementary study.

The effects of dotarizine (1-(diphenylmethyl)-4-[3-(2-phenyl-1,3- dioxolan-2-yl)propyl]-piperazine, CAS 84625-59-2) on the cerebral circulation of goats were assessed in vivo by recording continuously global cerebral blood flow (gCBF) and cortical perfusion (CP), and in vitro by recording isometric tension in goat isolated middle cerebral artery (MCA). Administration of dotarizine (1 microgram-5 mg) directly into the cerebroarterial supply of goats produced transient increases in gCBF and CP, and decreases in cerebral vascular resistance (CVR) which were significant for the highest doses tested: 1, 3 and 5 mg. On the other hand, cumulative addition of increasing concentrations of dotarizine (10(-8)-3 x 10(-5) mol/l) to MCA segments subjected to the resting tone of 1 g did not induce sizeable changes in vascular tension; by contrast, dotarizine elicited concentration-dependent relaxations of MCA segments subjected to the active tone induced by either 5-hydroxytryptamine (10(-6) mol/l) or high-K+ medium (50 mmol/l). These results show that dotarizine exerts a direct relaxant action on cerebral arteries, which results in an improvement of cerebral perfusion.

Nitric oxide is involved in anoxic preconditioning neuroprotection in rat hippocampal slices.

Sublethal anoxia/ischemia protects against subsequent damaging insults in intact brain or hippocampal slices. To help further understand mechanisms underlying anoxic/ischemic preconditioning, we tested three hypotheses which were that: (a) anoxic preconditioning (APC) improves electrical recovery in rat hippocampal slices; (b) anoxic preconditioning requires nitric oxide (NO); and (c) anoxic preconditioning blocks mitochondrial dysfunction that occurs following re-oxygenation after anoxia. Control hippocampal slices underwent a single 'test' anoxic insult. Experimental slices were preconditioned by 3 short anoxic insults prior to the 'test' insult. Evoked potentials (EPs), and NADH redox status were recorded prior to, during and after preconditioning and/or 'test' anoxic insults. To examine the role of NO, studies sought to determine whether APC could be produced by the NO donor, DEA/NO, and whether APC could be inhibited by NO synthase (NOS) inhibitor (7-nitroindazole). EP amplitudes recovered significantly better after reoxygenation in preconditioned slices and after NO-emulated preconditioning (90.0+/-17.7% and 90.0+/-21.3%, respectively, n=9, ** p<0.01, vs. 17.0+/-7.9%, n=9, in control slices). Inhibition of NOS blocked APC protection (6.8+/-6.8%, n=9). The intensity of NADH hyperoxidation was not significantly different among groups following 'test' anoxia. These data confirm that preconditioning by anoxia improves electrical recovery after anoxia in hippocampal slices. Evidence supports that NO from constitutive hippocampal NOS may be involved in the neuroprotection afforded by preconditioning by a mechanism that does not change the apparent mitochondrial hyperoxidation after anoxia.

Relaxant effects of sodium nitroprusside and NONOates in goat middle cerebral artery: delayed impairment by global ischemia-reperfusion.

Global cerebral ischemia and subsequent reperfusion induce early impairment of the vasodilator responses to hypercapnia and vasoactive substances. Nitric oxide (NO) is involved in the regulation of cerebral blood flow (CBF) in both health and disease. The present study was designed to assess possible changes in the cerebrovascular reactivity to NO donors induced by cerebral ischemia-reperfusion in goats. Female goats (n = 9) were subjected to 20 min global cerebral ischemia under halothane/N2O anesthesia. Sixteen additional goats were sham-operated as a control group. One week later the effects of ischemia-reperfusion on relaxations to NO donors sodium nitroprusside (SNP), diethylamine/NO (DEA/NO), diethylenetriamine/NO (DETA/NO), and spermine/NO (SPER/NO) were studied in rings of middle cerebral artery (MCA) isolated in an organ bath for isometric tension recording. SNP, DEA/NO, DETA/NO, and SPER/NO induced concentration-dependent relaxations of MCA precontracted with KCl (DEA/NO > SPER/NO > SNP > DETA/NO) or with endothelin-1 (DEA/NO > SNP > SPER/NO > DETA/NO). Relaxations were always higher in endothelin-1-precontracted arteries. One week after cerebral ischemia concentration-response curves to SNP and DEA/NO were displaced to the right, indicating a reduction in relaxant potency of NO donors. The classical nitrovasodilator SNP and NONOates induce relaxation of isolated goat MCA which is partially inhibited by arterial depolarization. Global cerebral ischemia followed by reperfusion induces delayed impairment of the relaxant effects of NO on cerebrovascular smooth muscle, which results in reduced vasodilatory potency of NO donors in large cerebral arteries.

Comparative relaxant effects of the NO donors sodium nitroprusside, DEA/NO and SPER/NO in rabbit carotid arteries.

1. Sodium nitroprusside (SNP, 10(-9)-3x10(-4) M), diethylamine/NO complex (DEA/NO, 10(-9)-10(-4) M) and spermine/NO complex (SPER/NO, 10(-8)-3x10(-4) M) induced concentration-dependent relaxation of isolated rabbit carotid arteries precontracted with KCl (50 mM) or with histamine (3x10(-6) M). 2. In KCl-precontracted arteries the order of potency was SNP=DEA/NO>SPER/NO, and in histamine-precontracted arteries the order of potency was SNP>DEA/NO>SPER/NO. Relaxations to the three NO donors were significantly higher in histamine-precontracted arteries than in KCl-precontracted arteries. 3. The guanylyl cyclase inhibitor methylene blue (10(-5) M) significantly inhibited relaxations to the three NO donors in histamine-precontracted arteries and, to a lesser extent, in KCl-precontracted arteries. 4. In conclusion, the NO donors SNP, DEA/NO and SPER/NO induce quantitatively different relaxation of rabbit carotid artery. Both, lower relaxant effects in depolarized arteries and inhibition of relaxation by methylene blue indicate the mediation of cGMP formation in the relaxant effects of the three NO donors.

Relaxant effects of sodium nitroprusside and NONOates in rabbit basilar artery.

Abstract NONOates are a new class of NO donors that have proven useful for studying the effects of spontaneous and chemically predictable NO release in biologic systems. In order to assess their potential as vasodilatatory drugs in the cerebrovascular bed we have compared the relaxant effects of the classical nitrovasodilator sodium nitroprusside (SNP) and three NONOates, diethylamine/NO complex (DEA/NO), spermine/NO complex (SPER/NO), and diethylenetriamine/NO complex (DETA/NO) in isolated rabbit basilar arteries precontracted with UTP. The 4 NO donors induced full relaxation of the UTP-induced tone, with the following order of potency: SNP > DEA/NO > SPER/NO > DETA/NO. Relaxations induced by SNP and DETA/NO were not modified in rubbed (endothelium denuded) arteries in which acetylcholine-relaxations were almost abolished. On the other hand, relaxations to SNP and SPER/NO were more potent and effective in histamine-precontracted arteries than in KCl-precontracted arteries. Methylene blue significantly inhibited SPER/NO-induced relaxations in both KCl- and histamine-precontracted arteries while SNP-induced relaxations were only slightly inhibited by methylene blue in KCl-precontracted arteries. This study shows that the NO donors SNP, DEA/NO, SPER/NO and DETA/NO have quantitatively different relaxant effects in rabbit basilar arteries according to their rate of NO release. Relaxations are not mediated by endothelial factors, and are inhibited by arterial depolarization. Finally, cGMP formation is involved in relaxation induced by NONOates and much less in SNP-induced relaxation.

Characterization of the cortical laser-doppler flow and hippocampal degenerative patterns after global cerebral ischaemia in the goat.

Large-animal models offer several advantages in the study of cerebral ischaemia: easier control of physiological variables, easier neuropathological evaluation, etc. In the present study we have taken advantage of the unique cerebrovascular anatomy of the goat to reproduce a model of reversible, incomplete, global cerebral ischaemia in a large-sized animal species, in which the effects of successive manoeuvres to stop and re-start cerebral blood flow can be recorded continuously. Early cortical laser-Doppler flow response (up to 2 h) and delayed neuronal degeneration (7 days) in the hippocampal CA1 subfield have been analysed in goats undergoing 5, 10 or 20 min of transient, global cerebral ischaemia. Bilateral occlusion of the external carotid artery plus compression of jugular veins reduced cortical laser-Doppler flow to 11 +/- 8% of preischaemic values (P<0.01), flattened the electrocorticogram, and increased mean arterial blood pressure by 17 +/- 23% (P<0.01) and intracranial pressure by 161 +/- 136% (P<0.01). A rather heterogeneous response was obtained during reperfusion: 14 out of 31 goats showed the "classical" pattern consisting of hyperaemia followed by delayed hypoperfusion. The remaining goats showed neither hyperaemia (11 goats) nor delayed hypoperfusion (6 goats). The duration of the ischaemic insult did not correlate with the magnitude of hyperaemia or delayed hypoperfusion, but influenced neurodegeneration: while no loss of hippocampal CA1 neurons was observed at 7 days after 5 or 10 min ischaemia, a 68% cell loss was observed in the 20-min ischaemia group. Our goat model has thus proven to be very suitable for the induction of global cerebral ischaemia in a large-animal species without extensive surgery. It allows reproducible reductions of cerebral blood flow, long-term recovery, low mortality rate, and high incidence of neuronal damage. The results reported here support the view that delayed hypoperfusion is not an important determinant of neuronal injury.

Effects of magnesium sulphate on the noradrenaline-induced cerebral vasoconstrictor and pressor responses in the goat.

OBJECTIVE: To examine the ability of magnesium sulphate to counteract the noradrenaline-induced cerebral vasoconstrictor and pressor responses in goats by using both in vivo and in vitro techniques. DESIGN: Cerebral blood flow was measured in vivo by means of an electromagnetic flow probe around the internal maxillary artery. Isometric tension was recorded in vitro from rings of goat middle cerebral artery maintained in an organ bath. RESULTS: 1. In vivo. Continuous infusion of noradrenaline (10 micrograms/min) directly into the cerebral arterial supply elicited sustained decrease in cerebral blood flow (61% [SEM 3] of control values) and increase in cerebral vascular resistance (178% [SEM 9] of control values). Magnesium sulphate, injected directly into the cerebral arterial supply (10-300 mg) or infused intravenously (0.3 g and 3 g during 15 min) at the noradrenaline-induced steady state, increased cerebral blood flow by decreasing cerebral vascular resistance in a dose-dependent manner. A similar result was obtained when intravenous magnesium sulphate (3 g/15 min) was tested against the cerebral vasoconstrictor and pressor responses induced by intravenous infusion of noradrenaline (30 micrograms/min). 2. In vitro. When compared with the response obtained in a control medium (1 mmol/L Mg2+), 10 mmol/L Mg2+ significantly inhibited the maximum contraction elicited by noradrenaline (10(-8) to 3 x 10(-3) mol/L) from 45% [SEM 4] to 26% [SEM 4]. CONCLUSIONS: Magnesium sulphate reverses the noradrenaline-induced cerebral vasoconstrictor and pressor responses by a direct inhibitory action of Mg2+ on the actions of noradrenaline in the cerebral and peripheral vascular beds, which leads to a decrease in vascular resistance. These results could explain, at least in part, the beneficial effects of magnesium sulphate in the management of preeclampsia and eclampsia.

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.

Modulatory role of endothelial and nonendothelial nitric oxide in 5-hydroxytryptamine-induced contraction in cerebral arteries after subarachnoid hemorrhage.

OBJECTIVE: Endothelial dysfunction is claimed to play a role in the pathogenesis of delayed cerebral vasospasm after subarachnoid hemorrhage (SAH). We have examined the effect of experimental SAH on the modulatory action of endothelial and nonendothelial nitric oxide (NO) in the contractile response of goat middle cerebral artery to 5-hydroxytryptamine (5-HT). METHODS: We compared the 5-HT-induced contractile responses of cerebral arteries from control goats and from goats with SAH that had been experimentally induced 3 days earlier by delivery of autologous arterial blood into the subarachnoid space. Contractile responses were examined by recording the isometric tension in isolated cerebral arteries. To assess the influence of endothelium, this cell layer was mechanically removed in some of the arteria, segments (rubbed arteries) from both control goats and goats with SAH. RESULTS: In arteries from control goats, contractile responses to 5-HT were significantly higher in rubbed arteries than in arteries with intact endothelium; 5-HT-induced contractions were significantly enhanced by a competitive inhibitor of NO synthesis, NG-nitro-l-arginine, in arteries both with and without endothelium. In arteries from goats with SAH, 5-HT contracted cerebral arteries without showing significant differences between segments with endothelium and those that had been rubbed; in both cases, 5-HT-induced contractions were significantly higher than those obtained in arteries from control goats. NG-Nitro-l-arginine significantly enhanced the contractile response to 5-HT of cerebral arteries from goats with SAH. CONCLUSION: These results suggest that cerebral arteries after SAH exhibit hyperreactivity to 5-HT via a mechanism that involves the absence of the modulatory role of endothelial NO, that SAH does not modify the modulatory role of nonendothelial NO, and that impairment of the modulatory action of endothelial NO on vascular responses to 5-HT could contribute to the pathogenesis of cerebral vasospasm after SAH.

Effects of cocaine on human and goat isolated cerebral arteries.

Cocaine abuse has been increasingly associated with cerebrovascular disease. We have studied the vasoactive properties of cocaine in branches of human middle cerebral artery and in goat middle cerebral artery isolated in an organ bath for isometric tension recording. Cocaine (10(-5) - 3 x 10(-4) M) induced small contractions, while higher concentrations (10(-3) - 3 x 10(-3) M) induced relaxation of human arteries at resting tension. In human arteries precontracted with KCl (50 mM), prostaglandin F- (10(-5) M) or endothelin-1(10(-9) M), cocaine (10(-6) - 3 x 10(-3) M) induced concentration-dependent relaxations which differed in terms of EC50 or maximum effect (Emax). With regard to goat arteries, cocaine (10(-6) - 3 x 10(-3) M) induced almost negligible changes in resting tension, and induced concentration-dependent relaxations of the arterial tone induced with KCl (50 mM). By contrast, goat arteries precontracted with prostaglandin F2 alpha (10(-5) M) or endothelin-1 (10(-9) M) showed biphasic concentration-response curves with concentration-dependent contractions to cocaine (10(-5) - 10(-3) M) and relaxation to the highest concentration (3 x 10(-3) M). Preincubation with cocaine (10(-4) - 10(-3) M) inhibited the contractile responses to CaCl2 (10(-6) - 10(-2) M) in depolarizing, Ca(2+)-free medium, and this inhibition was reversed by preincubation with the Ca2+ entry activator Bay K8644 (10(-10) - 10(-8) M). Therefore, cocaine induces tension changes in cerebral arteries which depend on the species, the arterial tone and the contractile agent inducing it. The relaxant effects could be attributed to the interference of cocaine with the role of Ca2+ in the maintenance of arterial tone, at least in part by blocking Ca2+ entry through membrane channels.

Characterization of 5-hydroxytryptamine receptors in goat cerebral arteries.

1. In isolated goat middle cerebral artery segments, 5-hydroxytryptamine (5-HT, 10(-8)-3 x 10(-5) M) elicited concentration-dependent contractions with EC50 = 2.1 (1.9-2.5) x 10(-7) M and Emax = 64 +/- 2% of 50 mM KCl-induced contraction. 2. Several 5-HT receptor agonists were used: (a) the agonist of 5-HT2 receptors alpha-methyl-5-hydroxy-tryptamine (10(-7)-3 x 10(-4) M) induced strong contraction (51 +/- 6%); (b) the selective agonists of 5-HT1 receptors sumatriptan (10(-8)-10(-5) M) and 5-carboxamidotryptamine (10(-9)-10(-4) M) and the agonist of 5-HT1A receptors 8-hydroxy-2-(di-n-propylamino)tetralin (10(-7)-3 x 10(-5) M) induced weak contractions (8, 18 and 14%, respectively); and (c) the agonist of 5HT3 receptors 2-methyl-5-hydroxytryptamine (3 x 10(-6)-10(-4) M) induced almost negligible contraction. 3. Pretreatment with the antagonist of 5-HT1A and 5-HT1B receptors cyanopindolol (10(-8), 10(-6) M), the antagonist of 5-HT1/5-HT2 receptors methysergide (10(-11), 10(-9) M) and the antagonist of 5-HT2 receptors ketanserin (10(-11), 10(-9) M) induced non-competitive inhibition of the concentration-response curve to 5-HT. The antagonist of 5-HT3 receptors 3-trophanyl-3,5-dichlorobenzoate (10(-7), 10(-5) M) did not inhibit the contractile curve to 5-HT. 4. These results suggest that 5-HT contracts the goat middle cerebral artery by acting mainly on 5-HT2 receptors.

Calcium channels in cerebral arteries.

Electrophysiological evidence shows the existence of voltage-operated Ca2+ channels of the L- and, in some cases, T- and B-, type in the smooth muscle cells of major cerebral arteries and arterioles. Current intensity through L-type Ca2+ channels is higher in cerebral than in peripheral arteries, which points to a greater dependence on extracellular Ca2+ of contractile responses in cerebral arteries. The increase in cytosolic Ca2+ concentration is the key event leading both to maintenance of basal cerebrovascular tone and to contraction of cerebral arteries in response to depolarization and agonist-receptor interaction. Such an increase results from increased transmembrane influx of Ca2+ through L-type Ca2+ channels, as well as from the release of Ca2+ from intracellular Ca2+ stores. Ca2+ entry modulators (dihydropyridines, phenylalkylamines, benzothiazepines, and diphenylpiperazines) bind to allosterically coupled sites in the Ca2+ channel, thus inhibiting (Ca2+ entry blockers) or stimulating (Ca2+ entry activators) Ca2+ influx and, therefore, contractile responses of the cerebral arteries. In vivo, Ca2+ entry blockers increase pial vascular caliber and cerebral blood flow by their direct action on the cerebroarterial wall. However, such an action also takes place on several peripheral vascular beds, which leads to hypotension. Therefore, the brain cannot be considered a "privileged" organ when the vasodilatatory action of Ca2+ entry blockers is considered. Since increased cytosolic Ca2+ concentration (and, therefore, activation of Ca2+ channels) plays a crucial role in the pathogenesis of ischemic brain damage (e.g., acute stroke and subarachnoid hemorrhage), Ca2+ entry blockers could be useful cytoprotective drugs. However, with the exception of nimodipine in the management of subarachnoid hemorrhage, clinical trials have yielded results that are not so promising as one could expect from those obtained in experimental research.