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.

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.

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.

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.

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)

Different effects of Mg2+ on endothelin-1- and 5-hydroxytryptamine-elicited responses in goat cerebrovascular bed.

Mg2+ influences the response of cerebral arteries to several agonists, but until now its effects on endothelin-1 (ET-1) had not been studied. We recorded and compared the responses of goat cerebrovascular bed to ET-1 and 5-hydroxytryptamine (5-HT) during various Mg2+ treatments. We performed experiments in vitro by recording isometric tension in isolated goat middle cerebral arteries and in vivo by recording cerebral blood flow (CBF) and other physiologic parameters in conscious goats. Cumulative addition of ET-1 (10(-11)-3 x 10(-8) M) and 5-HT (10(-9) -10(-5) M) contracted cerebral arteries concentration dependently in bath media containing 0 (Mg(2+)-free medium), 1 (control), and 10 mM Mg2+, but the influence of Mg2+ was different: Mg2+ deprivation increased sensitivity (EC50) and Mg2+ overload reduced contractility (Emax) of cerebral arteries to 5-HT, whereas the ET-1 response did not change in these conditions. Cumulative addition of Mg2+ (10(-4)-3 x 10(-2) M) at the active tone induced by ET-1 (10(-9) M) and 5-HT (10(-5) M) elicited concentration-dependent relaxations of cerebral arteries, but the relaxant response was lower at the ET-1 precontraction. Infusions of ET-1 (0.1 nmol/min) and 5-HT (10 micrograms/min) directly into the cerebroarterial supply of the unanesthetized goats elicited a sustained decrease in CBF and an increase in cerebral vascular resistance. Magnesium sulfate, administered as increasing doses (10-300 mg) in the same way increased CBF and decreased cerebral vascular resistance, although this effect was less on ET-1-induced than on 5-HT-induced cerebral vasoconstriction.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Endothelial modulation of 5-hydroxytryptamine-induced contraction in goat cerebral arteries.

1. In isolated goat middle cerebral artery segments, 5-hydroxytryptamine (5-HT, 10(-8) -3 x 10(-5)M) caused concentration-dependent contractions, with EC50 = 2.1 (1.9-2.5) x 10(-7) M and Emax = 60 +/- 2% of 50 mM KCl-induced contraction. 2. Mechanical removal of endothelium significantly increased the Emax (91 +/- 8%) and did not change the EC50 value of 5-HT-elicited contractions. 3. Incubation of unrubbed arteries with the irreversible inhibitor of EDRF, gossypol (10(-5) M), significantly increased the contractile response to 5-HT (Emax = 77 +/- 4%). 4. Incubation of unrubbed arteries with the competitive inhibitor of the NO synthesis, NG-nitro-L-arginine (L-NOARG) (10(-5) M), significantly enhanced the arterial response to 5-HT (Emax = 71 +/- 5%). The effects of L-NOARG were reversed by L-arginine (10(-4) M) but not by D-arginine (10(-4) M). 5. Pretreatment with the inhibitor of cyclooxygenase, indomethacin (10(-5) M), significantly increased the response of unrubbed arteries to 5-HT, with an Emax of 69 +/- 3%. 6. These results suggest that endothelium modulates the constrictor effect of 5-HT in goat cerebral arteries by producing both EDRF, probably NO, and prostacyclin.