Temporary middle cerebral artery occlusion in the rat: consistent protocol for a model of stroke and reperfusion.

The intraluminal suture method of middle cerebral artery occlusion (MCAO) in the rat (the suture model) is a model of stroke which readily lends itself to studying the pathophysiology of post-ischaemic reperfusion. Unfortunately, variability of outcome has compromised the potential of the model, but systematic studies might characterize a consistent protocol. Therefore, the clinical and neuropathological outcome of temporary MCAO and reperfusion in the suture model were systematically investigated. Two hours or 4 h of MCAO were employed, measuring the extent of infarction at 24 h with triphenyltetrazolium chloride or at 72 h with histopathological techniques. Outcome was compared in three rat strains. Following 2 h of MCAO, motor function improved during reperfusion in Sprague-Dawley, but not in Wistar or Fischer-344 rats. All Sprague-Dawley and Wistar rats survived the protocol to 72 h, but 33% of Fischer-344 rats died. The extents of infarction and oedema were greater and less variable in Wistar and Fischer-344 than Sprague-Dawley rats, and in all three strains, the extent of infarction increased with reperfusion time. Following 4 h of MCAO, there was no improvement in motor function during reperfusion in Sprague-Dawley rats, and mortality was high at 24 h in Wistar (33%) and Fischer-344 rats (83%). Outcome was only pursued in Sprague-Dawley rats to 72 h, where the extent of infarction was quite variable. It was concluded that the extent and variability of outcome following temporary MCAO in the suture model is strain-dependent, and a consistent protocol with zero mortality was found in Wistar rats using 2 h of MCAO and 70 h of reperfusion.

Middle cerebral artery occlusion in the rat: consistent protocol for a model of stroke.

Variability in the effects of the intraluminal suture method of middle cerebral artery occlusion (MCAO) in the rat has been a common and disadvantageous finding. Therefore, we systematically investigated the effects of suture type and rat strain on outcome. First, the clinical and neuropathological effects of permanent MCAO with either an uncoated or a silicone-coated nylon suture were studied over 7 days in Sprague-Dawley rats (n = 36 for each type of suture). Outcome was less severe with the uncoated compared with the silicone-coated suture (e.g. total cerebral infarct volume at 24 h before any fatalities was 119.9 +/- 79.8 mm3, cf. 183.0 +/- 36.5 mm3, n = 12 for each, P < 0.05; and overall mortality rate was 12.5% cf. 33%, respectively), but much more variable (coefficient of variation was 66.6% cf. 19.9%, respectively). Second, being more consistent in its effects, the silicone-coated suture was further studied in Wistar and Fischer-344 rats (n = 12 for each). Seventy-five per cent of the Wistar's died prematurely from gross hemispheric oedema. Motor deficit and extent of infarction in the Fischer-344 rats were both significantly greater compared with Sprague-Dawley rats (e.g. total cerebral infarct volume at 24 h in the former was 253.6 +/- 25.4 mm3, n = 11, P < 0.05), and more consistent (coefficient of variation was only 10.0%). It was concluded that the silicone-coated suture and the Fischer-344 rats strain produced the most consistent results and their novel combination provides a reliable acute stroke model.

Effects of combined glutamate and platelet-activating factor inhibition on the outcome of focal cerebral ischaemia - an initial screening study.

Since both glutamate excitotoxicity and inflammatory responses have been implicated in ischaemic neuronal death, we questioned whether joint inhibition of both processes would be more neuroprotective than either on its own. Therefore we assessed the effects of combined inhibition of both glutamate release (with a use-dependant sodium channel blocker, 619C89) and inflammatory processes (with a platelet-activating factor (PAF) receptor antagonist, BB-823) on the degree of motor deficit and the extent of cerebral (cortical and sub-cortical grey matter) infarction produced by middle cerebral artery occlusion (MCAO) in the rat, and compared results to appropriate single agent, vehicle and positive controls. The combination of both agents produced the greatest reduction in motor deficit, but the effect was only significant (p<0.05) acutely (4 to 6 hours post-MCAO). The extent of cortical infarction at 24 hours post-MCAO was significantly reduced in all experimental groups compared to vehicle-controls (p<0.05) and the greatest reduction occurred in the combination group (55%), though it was not significantly better than either of the single agent groups. Similarly the greatest reduction in sub-cortical infarction was in the combination group, but this was also not significantly better than the single agents. The results of this novel combination of pharmacological interventions suggest that inhibition of both glutamate excitotoxicity and inflammatory responses afforded an overall enhanced, if modest, neuroprotective effect, compared to inhibition of either process alone. The possible mechanisms involved are discussed, but warrant further clarification before therapeutic strategies are developed.

Focal cerebral ischaemia increases the levels of several classes of heat shock proteins and their corresponding mRNAs.

The induction of focal cerebral ischaemia in rats by middle cerebral artery occlusion has previously been shown to increase, over time, the mRNA levels of the heat shock proteins (HSPs) 27 and 70. However, the levels of HSP90 mRNA remain constant. In contrast, during global ischaemia, HSP70 and HSP90 mRNA levels are both raised, particularly in the CA1 neurons in the hippocampus, an area that is resistant to the insult in comparison to the surrounding regions. HSP27 mRNA is raised in the neuroglia in the subregions of the hippocampus. However, the protein levels of HSP27, 70 and 90 have not been characterised in focal ischaemia. With this data in mind, we have carried out a comparative study of HSP27, 56, 60, 70 and 90 mRNA and protein levels during focal cerebral ischaemia in rats, up to 24 h post-occlusion. We have shown that HSP70 and HSP27 mRNA levels are increased and also that HSP60 mRNA levels (which had also not previously been characterised in this model of focal ischaemia) are significantly raised. HSP90 and HSP56 mRNAs were not significantly elevated. On Western blot analysis, the inducible HSP72 protein was first detected at 8 h post-occlusion, HSP27 protein was detected only at 24 h post-occlusion and HSP60 protein, although constitutive, appeared to increase at 24 h post-occlusion. HSP56 protein levels appeared to rise on the occluded side, but HSP90 protein levels remained constant.

Focal ischemia causes an extensive induction of immediate early genes that are sensitive to MK-801.

BACKGROUND AND PURPOSE: There is strong evidence to implicate glutamate in the cerebral damage caused by ischemia. In this study we investigated the role of glutamate receptors in mediating effects of middle cerebral artery occlusion (MCAO) on immediate early gene expression in the rat by quantitation of mRNA levels. METHODS: The effect of MCAO on the induction of immediate early genes was studied in five regions, both ipsilateral and contralateral to the occlusion: the "core" ischemic area of the cortex in the central region of the middle cerebral artery territory, the surrounding area, frontal cortex, occipital cortex, and hippocampus. Levels of c-fos, c-jun, zif-268, and krox-20 mRNA were measured by Northern and slot blot analysis. RESULTS: A large induction of c-fos mRNA was obtained in all four cortical regions ipsilateral to the occlusion, with the greatest effect detected in the core area. Little effect was detected in the ipsilateral hippocampus and in all contralateral regions. Pretreatment with MK-801 (3 mg/kg) largely inhibited the induction of c-fos mRNA, indicating that the induction was mediated through an N-methyl-D-aspartate subtype of glutamate receptor. MCAO also produced a significant induction of c-jun and zif-268 mRNA in ipsilateral cortical regions. CONCLUSIONS: These results indicate that MCAO causes a profound modulation of the expression of multiple genes in an extensive area of cerebral cortex extending beyond the immediate area supplied by the middle cerebral artery. The marked effect of MK-801 indicates the potential importance of glutamate antagonists in restricting the widespread deleterious effects of glutamate.

Effect of bezafibrate on the sequelae of acute experimental focal cerebral ischaemia.

The possibility that the ability of bezafibrate to lower the oxygen affinity of haemoglobin might lead to an increased oxygen delivery to ischaemic cerebral tissue was explored in preliminary studies in two animal models. The combined morbidity and mortality of unilateral carotid ligation in the gerbil appeared to be reduced at two hours in bezafibrate treated animals. By four hours and thereafter the outcome was unaffected. Two hours after MCA occlusion bezafibrate-treated rats showed a significantly reduced rise in tissue lactate concentration (p less than 0.01) suggesting less anaerobic metabolism had occurred in the ischaemic tissue.

Do leukocytes have a role in the cerebral no-reflow phenomenon?

The possible role of leukocytes in the cerebral microcirculation following ischaemia was assessed in the gerbil. The no-reflow phenomenon seen after 30 minutes of severe bilateral hemispheric ischaemia during hypotensive reperfusion was compared in control animals and in a group made leukopenic by pretreatment with cyclophosphamide. Neither the incidence nor the severity of the no-reflow phenomenon differed between the two groups. The evidence from this study casts doubt on the hypothesis that leukocyte plugging plays a major role in the cerebral microcirculation's response to ischaemia.

Sex difference in antithrombotic effect of aspirin.

A number of clinical trials suggest that the antithrombotic effect of aspirin is limited to men. To test the possibility that this is due to a sex difference in the inhibitory effect of aspirin on platelet behavior, we studied whole-blood platelet aggregation in men and women and in male patients with carcinoma of the prostate receiving hormone therapy. The in vitro inhibitory effect of aspirin on so-called spontaneous platelet aggregation induced by stirring whole blood and monitored by the decrease in the number of singleton platelets was greater in men (mean +/- SD inhibitory ratio 1.54 +/- 0.30 in men, 1.23 +/- 0.22 in women; p less than 0.001). The inhibitory effect of aspirin was reduced in orchiectomized male patients and was restored by the addition of testosterone to blood samples. Estradiol had no detectable influence on the inhibitory effect of aspirin. Testosterone thus seems to influence platelet aggregation and its inhibition by aspirin as assessed by whole-blood in vitro aggregometry. Possible mechanisms for this effect of testosterone and its relevance to the choice of antithrombotic therapy are discussed.

The combined effects of hypertension, hemodilution, and osmotherapy on the metabolic sequelae of acute experimental cerebral ischemia.

The effects of induced hypertension, hemodilution, and osmotherapy (mannitol) have been assessed singly and in combination on the metabolic sequelae 2 hr after middle cerebral artery (MCA) occlusion in the anesthetized rat. All regimes that included hypertension and monotherapy with mannitol significantly reduced the rise in hemispheric lactate produced by vessel occlusion. No treatment caused increase cerebral edema, and mannitol produced a slight reduction in the hemisphere water content. The significance of the results is discussed.

The effect of increased blood pressure on hemispheric lactate and water content during acute cerebral ischaemia in the rat and gerbil.

Infusions of metaraminol and angiotensin were used to test the effect of increased perfusion pressure on tissue metabolism and oedema after induction of regional cerebral ischaemia in the rat and the gerbil. An increase of mean arterial blood pressure of 30-40 mm Hg in the rat over the first 2 hours after diathermy of the middle cerebral artery prevented the 100% rise in hemisphere lactate seen in normotensive control animals. Angiotensin infusion also prevented early hemispheric oedema in this model. In the gerbil, 4 hours after placing a clip on one carotid artery, metaraminol-induced increases in blood pressure had no such protective effect on the metabolic changes or on oedema. When the clip was removed after 3 hours to permit 1 hour of reperfusion, lactate levels returned to normal but the degree of oedema was unchanged. Hypertension in this reperfusion model caused a slight but not statistically significant increase in oedema. The evidence suggests that moderate increases in blood pressure may be protective against the early metabolic sequelae of focal cerebral ischaemia, but there are potential problems with oedema formation. It is argued that a clinical trial should study the potentially beneficial effects of a brief early increase in blood pressure in the acute aftermath of ischaemic stroke.

An assessment of the possible protective effect of allopurinol in acute stroke.

Allopurinol has been shown to have a protective effect on ischaemic tissue by the indirect prevention of excessive purine loss. This property was tested in the gerbil model of acute stroke. A total of 69 animals were pretreated with an intraperitoneal injection of either allopurinol (50 mg/kg) or sterile water and then subjected to unilateral ligation of the left common carotid artery under general anaesthesia. The clinical effect of the ligation was grouped into three categories of normal, mild to moderate defect (splayed leg, turning behaviour) and severe defect (death, nonresponsiveness and seizures). More normal animals and fewer severely affected animals were present in the allopurinol treated group compared to controls, but only at 2 to 4 hours after carotid ligation (p less than 0.05). Histological examination of brain tissue from the normal category failed to reveal any difference in subclinical ischaemic damage between the two groups. It was concluded that allopurinol may have a protective effect in acute stroke and that this property warrants further elucidation.