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.

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.

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.

Heterogeneity of P2-purinoceptors in brain circulation.

The existence of P2-purinoceptors in the cerebrovascular bed was examined by testing the effects of ATP and its stable analog, alpha, beta-methylene-ATP, on CBF in the unanesthetized goat as well as on isometric tension in isolated goat middle cerebral artery. When injected directly into the cerebral circulation, ATP increased and alpha, beta-methylene-ATP decreased CBF. Indomethacin did not modify either of these effects. The vasoconstrictor action of alpha, beta-methylene-ATP was reduced by nicardipine. "In vitro," both ATP and alpha, beta-methylene-ATP contracted the cerebral arteries at resting tone, but the analog was more potent than ATP. Repeated application of alpha, beta-methylene-ATP as well as indomethacin significantly reduced the ATP-induced contractions. Nicardipine inhibited both the alpha, beta-methylene-ATP- and the ATP-induced contractile response. In preconstricted arteries, ATP produced relaxation and alpha, beta-methylene-ATP induced further contraction. The relaxant response to ATP was not modified by indomethacin. These results show the existence of two subtypes of P2-purinoceptors in brain circulation: P2x, more sensitive to alpha, beta-methylene-ATP than to ATP, which elicits cerebral vasoconstriction; and P2y, sensitive to ATP, which elicits cerebral vasodilation.

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.

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 approximately 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-I and sarafotoxin S6b suggests the existence of subtypes of ETA receptors.

Comparison of the contractile effects of endothelin-1 and sarafotoxin S6b in goat isolated cerebral arteries.

1. The effects of endothelium-derived endothelin-1 and snake venom-derived sarafotoxin S6b, peptides with striking structural and functional similarities, were examined and compared in isolated middle cerebral arteries of goats. 2. Endothelin-1 and sarafotoxin S6b contracted cerebral arteries in a concentration-dependent manner. The potency of endothelin-1 (EC50 = 4.9 (3.9-6.2) x 10(-10) M) was about ten times higher than that of sarafotoxin S6b (EC50 = 5.5 (4.4-6.9) x 10(-9) M). The tension returned to basal values after repeated washings and contraction with endothelin-1 could be reproduced. Endothelin-1 and sarafotoxin S6b induced further contraction in arteries precontracted with prostaglandin F2 alpha (10(-5) M). 3. Mechanical removal of the endothelium or incubation with indomethacin (10(-5) M) displaced the concentration-response curves to endothelin-1 and, more pronouncedly, to sarafotoxin S6b to the left. The maximum response to sarafotoxin S6b was also increased by either of these two treatments. 4. Incubation in 'nominally' Ca(2+)-free medium attenuated the vasoconstrictor response to endothelin-1 but not to sarafotoxin S6b, which was inhibited after incubation in Ca(2+)-free medium to which EGTA (10(-4) M) had been added. Pretreatment with caffeine (2 x 10(-2) M) in Ca(2+)-free medium abolished responses to endothelin-1 and sarafotoxin S6b. 5. Bay K 8644 (10(-10) M, 10(-8) M) enhanced and nicardipine (10(-10) M, 10(-8) M) inhibited in a concentration-dependent manner vasoconstrictor response to endothelin-1. Response to sarafotoxin S6b was only affected by 10(-8) M Bay K 8644 or nicardipine.6. It is concluded that endothelin-1 and sarafotoxin S6b are potent vasoconstrictors of goat cerebral arteries, having direct effects on smooth muscle which are counteracted by the endothelium through the release of a vasodilatator substance, probably prostacyclin. Both endothelin-l and sarafotoxin S6b depend on extracellular Ca2+ and on intracellular, caffeine-sensitive Ca2+ stores to develop vasoconstriction.However, endothelin-l depends to a larger extent than sarafotoxin S6b on free extracellular Ca2+.

Inhibitory effect of GABA on cerebrovascular sympathetic neurotransmission.

The possibility that gamma-aminobutyric acid (GABA) could modulate sympathetic neurotransmission in the cerebrovascular bed of the goat has been investigated by means of 3 experimental approaches: measurement of cerebral blood flow in the anesthetized animal, recording of isometric tension in isolated cerebral arteries, and measurement of tritium efflux from cerebral arteries preloaded with [3H]noradrenaline. Electrical stimulation of cervical sympathetic nerve produced reductions in cerebral blood flow which were significantly diminished during continuous infusion of GABA (20-40 micrograms/min) into the internal maxillary artery. Picrotoxin (3 mg) did not change the inhibitory effect of GABA. Exogenously administered noradrenaline (1-9 micrograms) and tyramine (50-500 micrograms) reduced cerebral blood flow as well, but this effect was unchanged by GABA infusion. Transmural electrical stimulation elicited frequency-dependent contractile responses in isolated cerebral arteries which were significantly blocked when GABA was present, at a dose (10(-4) M) which did not modify the contractile response to exogenous noradrenaline (10(-8)-10(-4) M). Moreover, GABA (10(-5)-10(-4) M) inhibited transmural electrical stimulation-evoked tritium efflux from arteries preloaded with [3H]noradrenaline. These results show that GABA inhibits adrenergic neurotransmission in cerebral arteries by a mechanism involving inhibition of transmitter release. Probably, specific presynaptic GABA-B receptors mediate this inhibitory effect.

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.

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.

P1-purinoceptors in the cerebrovascular bed of the goat in vivo.

The possible existence and function of specific P1-purinoceptors in the cerebrovascular bed of the unanesthetized goat have been investigated. Blood flow to one cerebral hemisphere (cerebral blood flow) was measured by means of an electromagnetic flow probe previously implanted around the ipsilateral internal maxillary artery. The injection of adenosine, AMP, ADP and ATP (3-30 micrograms) directly into the internal maxillary artery increased cerebral blood flow and decreased cerebrovascular resistance in a dose-dependent manner. Continuous infusion of 8-phenyltheophylline (8-PT), 100 micrograms/min, into the internal maxillary artery did not alter the resting cerebral blood flow or the cerebrovascular resistance, but significantly inhibited the cerebral vasodilation induced by adenosine, AMP, ADP and, to a lesser degree, ATP. The acetylcholine- and histamine-induced cerebral vasodilation was unaffected by 8-PT. These results indicate that adenosine, AMP, ADP and, at least in part, ATP increase cerebral blood flow by acting on specific P1-purinoceptors located in the cerebrovascular wall. These P1-purinoceptors do not appear to be tonically activated under physiological conditions.

Effects of Ca2+ entry blockers on CaCl2-, KCl- and noradrenaline-induced contractions of goat cerebral arteries.

The effects of three Ca2+ entry blockers, nicardipine, nimodipine and verapamil, on CaCl2-, KCl- and noradrenaline-induced contractions were examined in isolated goat middle cerebral artery. The relationship between the effects of Ca2+ entry blockers and the extracellular Ca2+ dependence of the contractions was also examined. In 'nominally' Ca2(+)-free medium, addition of CaCl2 induced concentration-dependent contractions of previously depolarized arteries. Withdrawal of Ca2+ from the extracellular medium caused strong inhibition of the KCl- and noradrenaline-induced arterial contractions. Addition of EGTA to the Ca2(+)-free medium almost abolished the noradrenaline-response but did not increase the inhibition of the KCl-induced contractions. The Ca2+ entry blockers induced concentration-dependent relaxation of the precontracted arteries (100 mM KC1) with the following order of potency: nimodipine greater than nicardipine greater than verapamil. The CaCl2-, KCl- and noradrenaline-induced contractions were depressed in a concentration-related manner by nicardipine, nimodipine and verapamil. Dihydropyridines showed a greater inhibitory effect than verapamil. These results show that Ca2+ entry blockers are able to inhibit the contractile responses of goat cerebral arteries to KCl and noradrenaline, an effect which may be explained by the strong dependence of both responses on extracellular Ca2+.

Effects of endothelin-1 on the cerebrovascular bed of the goat.

The aim of the present study was to analyze the effects of endothelin-1 (ET-1) in the cerebrovascular bed of the goat, the importance of endothelial integrity and the contribution of extracellular Ca2+ to these responses. We measured isometric tension and 45Ca2+ uptake in isolated middle cerebral arteries, and cerebral blood flow (CBF) in unanesthetized animals. ET-1 elicited concentration-dependent contractions which were potentiated in arteries without endothelium. Ca2(+)-free medium and nicardipine inhibited, and Bay K 8644 potentiated the ET-1-induced contractions. ET-1 enhanced 45Ca2+ uptake in isolated arteries. Injections of ET-1 directly into the cerebral circulation decreased CBF and increased cerebrovascular resistance in a dose-dependent manner. Infusion of nicardipine inhibited the ET-1-induced reductions in CBF. These results suggest that ET-1 reduces CBF of goats because of contraction of cerebral arteries by a direct action on smooth muscle, which is modulated by the endothelium and depends partially on the activation of Ca2+ influx through dihydropyridine-sensitive channels.

Ca2+ entry blockers inhibit prostaglandin F2 alpha-induced cerebrovascular contractile responses in goats.

We examined the effects of extracellular Ca2+ withdrawal and of Ca2+ entry blockers on goat cerebrovascular responses to prostaglandin F2 alpha (PGF2 alpha). We measured isometric tension in isolated middle cerebral arteries, and cerebral blood flow (CBF) in unanesthetized animals. PGF2 alpha produced concentration-dependent contractions of isolated arteries. The contractions were partially inhibited by incubation in Ca(2+)-free medium (by 63.1 +/- 1.8% without ethyleneglycol-bis-(beta-amino-ethylether)-N,N,N',N'-tetra-a cetate (EGTA), and by 82.4 +/- 3.7% with EGTA). The Ca2+ entry blockers inhibited PGF2 alpha-elicited contraction and relaxed PGF2 alpha-precontracted arteries (nicardipine, 91.4 +/- 9.8%; nimodipine, 73.1 +/- 2.0%; and verapamil, 50.5 +/- 4.5% relaxation of the active tone). Injection of PGF2 alpha into the cerebral circulation produced dose-dependent reductions in CBF (34.4 +/- 2.1% after 30 micrograms) which were inhibited during infusion of Ca2+ entry blockers (nicardipine 10 micrograms/min, 14.7 +/- 1.5%; nimodipine 10 micrograms/min, 13.6 +/- 1.3%; and verapamil 100 micrograms/min, 13.7 +/- 2.3% of flow reduction). We conclude that PGF2 alpha-elicited contraction of goat cerebral arteries is mainly mediated by extracellular Ca2+ influx through Ca2+ channels sensitive to Ca2+ entry blockers. The anticonstrictor effects of Ca2+ entry blockers observed in vitro are consistent with their inhibitory effect on the PGF2 alpha-induced CBF reductions.

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+.

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.

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.

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 adrenergic mechanisms of cerebral arteries after subarachnoid hemorrhage in goats.

We have examined the effects of experimental subarachnoid hemorrhage (SAH), induced by delivering autologous blood into the subarachnoid space, on the adrenergic mechanisms of the goat cerebrovascular bed. To achieve this, the response to noradrenaline was recorded both in vivo, by measuring cerebral blood flow in unanesthetized animals, and in vitro, by recording isometric tension in isolated cerebral arteries. In addition, we checked the function of adrenergic innervation by measuring the tritium efflux evoked by electrical stimulation in cerebral arteries preloaded with [3H]-noradrenaline, and we examined this innervation by using both fluorescent and electron transmission microscopy. All studies were performed before and 3, 7, and 14 days after SAH. Injections of noradrenaline (0.1-10 micrograms) directly into the cerebro-arterial supply produced reductions in cerebral blood flow, with no concomitant changes in mean arterial blood pressure and heart rate, which were significantly enhanced (P < 0.01) 3 and 7 days after SAH and returned to control values 14 days after hemorrhage induction. In isolated cerebral arteries, noradrenaline (10(-8)-10(-4) mol/L) produced concentration-dependent contractions, which were also significantly enhanced (P < 0.05) 3 and 7 days after SAH and returned to control values in cerebral arteries obtained 14 days after SAH. On the other hand, increases in the release of tritium induced by electrical stimulation in cerebral arteries preloaded with [3H]-noradrenaline were significantly lower (P < 0.01) after SAH. Moreover, microscopical studies showed a reduction in catecholamine fluorescence and signs of sympathetic degeneration in some perivascular axons after SAH.(ABSTRACT TRUNCATED AT 250 WORDS)

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.