| INTRODUCTIONCerebral ischemia-reperfusion-induced injury is a major contributor affecting therapy of cerebral ischemia patients. There are no specific treatments in the clinical practice at present. Apoptosis which is one of the main patterns of neuronal death plays an important role in cerebral ischemia-reperfusion induced injury. So anti-apoptosis treatment may become one of the important remedies for cerebral ischemia-reperfusion-induced injury. Sevoflurane is a new volatile anesthetic agent. Recently, it is found that sevoflurane-induced preconditioning protects against cerebral ischemic neuronal damage by the mechanism like ischemia preconditioning or KATP channel opener, but the mechanism of neuronal apoptosis was not clear up. Mitogen-activated protein kinase (MAPK) is an integration point of different signal transduction pathways. Many tyrosine kinases stimulate signaling cascades that lead to activation of MAPK pathways. In vertebrates, there are at least four different MAPKs that convey distinct biological responses. ERK/JNK play an essential role in regulating inflammatory responses, cytokine secretion and cell apoptosis. In the nervous system, activation of ERK/JNK is closely related to apoptosis and death in response to a variety of pathological conditions.In this study, We observed the difference of neurological deficits, examined survival neurons through light microscope and transmission electron microscope by HE staining and TTC(triphenyltetrazoliumchloride) staining, explored the expression of p-ERK/p-JNK by immunohistochemistry on the basis of the cerebral ischemic model to study the effects of sevoflurane against focal ischemia-reperfusion-induced injury and the possible mechanism of anti-apoptosis.METHODSPart one: Sixty Sprague-Dawlay health rats were randomly divided into ischemia-reperfusion group(IR) and control group (blank control, sham operation, PD98059 control, SP600125 control). First of all, We established right middle cerebral artery occlusion(MCAO) model based on Zea Longa method. Briefly, rats were anesthetized with intraperitoneal injection of pentobarbital sodium(45mg·kg-1, ip), the right common carotid artery(CCA), internal carotid artery(ICA) and external carotid artery(ECA) were exposed through amid line incision of the neck. A 4-0 nylon suture was used as an occluder and was inserted via the CCA. The ECA and the CCA were legated. The occluder advanced into the ICA about 17.5-18.5mm beyond the carotid bifurcation(The rats of sham operation group were received the same surgical procedure with only 9mm beyond the carotid bifurcation). At 60min after MCAO, reperfusion began with the suture withdrawn. The neurological scores were determined by the method described by Zea Longa at 1d,3d,5d after operation. Then rats were decapitated, the brains were stained using triphenyltetrazoliumchloride(TTC) to calculate infarct volume.Part two: Modified middle cerebral artery occlusion models of transient focal cerebral ischemia were established. Twenty SD rats were randomly allocated into three groups: control group (C), ischemia-reperfusion group(IR), sevoflurane preconditioning group(S). In C group, no suture was inserted, while, S group received 30min sevoflurane preconditioning (1MAC) before surgery. The neurological deficit levels were measured at 3 timepionts(1d,3d,5d after reperfusion), then rats were decapitated. Brains were removed, postfixed, embedded, sectioned and processed for HE staining. Hematoxylin and Eosin(HE) staining was used to count survival neutrons in hippocampus CA1. Transmission electron microscope was used to describe neurons apoptosis.Part three: Thirty-two SD rats were divided randomly as follows: control group (C),ischemia-reperfusion group(IR), sevoflurane preconditioning group(S), PD98059 group(PD), SP600125 group(SP). We established right middle cerebral artery occlusion(MCAO) model based on Zea Longa method. In C group, no suture was inserted, while, S group received 30min sevoflurane preconditioning (1MAC) before MCAO. In PD group, an ERK pathway inhibitor PD98059 was administered before sevoflurane preconditioning(1mg·kg-1, ip).In SP group, SP60015—JNK pathway inhibitor is used(6mg·kg-1, ip). The neurological behavior were measured by Longa Scores at 3 timepionts(1d,3d,5d after reperfusion), then rats were decapitated. Apoptosis neurons in hippocampus were observed by TEM and HE staining. ERK/JNK phosphorylation was detected by immunohistochemistry.All the data were expressed by x±s and analyzed statistically by One-Way ANOVA of variance among groups, t-test between two groups. The significant testing standard was P<0.05.RESULTS1. The study presents a modified model for the pathophysiological investigation of ischemia stroke, which is produced by nylon suture occlude the right middle cerebral artery. In all model groups, 47 of 60 rats(78%) showed mild to severe neurological deficits, and no neurological deficits were found in control group. Statistically, the Longa score(neurological deficit grading) is negative correlation with survival neurons.(r=-0.561,P<0.01)2. The neurological scores of sevoflurane preconditioning group were significantly lower than those of ischemia-reperfusion group. The survival neurons of IR group and S group was 46.0±1.4 and 58.0±1.4 perμm2, 1d(P<0.05); 29.0±2.8 and 47.0±2.8, perμm2, 3d(P<0.05); 17.0±2.8 and 32.5±2.1 perμm2, 5d(P<0.05). Sevoflurane preconditioning can improve neurological outcome induced by ischemia-reperfusion.3. It was observed clearly by HE staining and light microscope and transmission electron microscope that there were many apoptosis neurons in the hippocampus CA1 zone in ischemia-reperfusion group. Brain tissue section HE staining show that there was no infarction in control group, the shape and structure of the neurons were normal, there was no edema interstitial, and there was obvious infarction in ischemia-reperfusion group, the neurons were found deformed and shrunken, nucleus and cytoplasm were concentrated, edema was obvious. Apoptosis neurons in sevoflurane preconditioning group distribute sparsely.4. Compared the ischemia-reperfusion group with sevoflurane preconditioning group, the levels of p-ERK in hippocampus ischemia zone are 25.5±0.7 vs 19.5±0.7, 1d; 22.5±0.7 vs 16.0±1.4, 3d; 18.0±1.4 vs 9.0±1.4, 5d, the differences have significant(P<0.05); on the contrary, the levels of p-JNK in hippocampus ischemia zone are 13.5±0.7 vs 19.5±0.7, 1d; 10.5±0.7 vs 14.5±0.7, 3d; 8.5±0.7 vs 11.0±1.4, 5d, the differences have significant(P<0.05). Cerebral ischemia-reperfusion may be result in the activity of ERK up-regulation and JNK down-regulation, which maybe associated with the process of neurons apoptosis.5. Administered ERK pathway inhibitor—PD98059, the sevoflurane neuroprotective effect was blocked; furthermore, SP600125—JNK pathway inhibitor probably involved. The final results of the neuronal apoptosis were balanced between the two pathways.CONCLUSION1. The modified focal cerebral ischemia-reperfusion model (MCAO) in the rat was reliable and reproducible for cerebral vascular disease study.2. Neuronal apoptosis plays an important role in cerebral ischemia-reperfusion induced injury.3. Sevoflurane preconditioning can provides neuroprotection by inhibiting neuron apoptosis induced by ischemia-reperfusion.4. The sevoflurane neuroprotection against ischemia-reperfusion is possibly related to the ERK/JNK signal transduction pathways.
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