Study On Effect Of Calcium Transduction Pathway In The Mechanism Of Cerebral Ischemic Preconditioning

There is the highest incidence of ischemic cerebrovascular disease in china. Stroke has been the first cause of death. The death rate of stroke is higher 4 or 5 multiples than that in the united states. The number of disability to work amount to threes of forth in all strokers in our country,which may be hemiplegia, aphasia, intelligence is low, the life cannot provide for oneself, etc .The object of treatment of stroke in the acute period limit the seriousness of ischemia and reduce the duration of the ischemia. But the effect of the intervention in the acute period is dissatisfied. In order to less the injury of brain tissue, we should intervene the adaptation mechanism of ischemic stress damage. Cerebral ischemic preconditioning （CIPC） is a phenomenon in which the brain protects itself against lethal injury by adapting to sub-lethal insults. CIPC can decrease neuron injury and promote neuron survival.We established rat models with the technique of the occlusion of middle cerebral artery , The concentration of Ca²⁺ in the hippocampal neurons of the each group were detected by relative fluorescent value（using the Fluo-3/AM as the fluorescent indicator） under the confocal laser scanning microscope. The apoptotic neurons was detected by TUNEL apoptotic detection assay. We discussed expression changes in CaN, cbl-b and p-AKT after CIPC or pretreatment with a Ca²⁺-regulator,such as nimodipine, MK801 or cyclosporine A before CIPC. We also discussed a possible transduction pathway of Ca²⁺.Objective:1.We established rat models with the technique of the occlusion of middle cerebral artery, and we improved the operation method, so as to raise the success rateof rat models in our study.2.The neurons apoptosis was detected by TUNEL apoptosis assay, so as to understand the effect of CIPC and Ca²⁺-regulator on neurons apoptosis.3.The concentration of Ca²⁺ in the hippocampal neurons of the each group were detected by relative fluorescent value（using the Fluo-3/AM as the fluorescent indicator） under the confocal laser scanning microscope, so as to understand the effect of CIPC and Ca²⁺-regulator on the concentration of Ca²⁺ in the hippocampal neurons.4.The neurological deficit scores and volume of the cerebral infarction were assessed, so as to understand the effect of CIPC and Ca²⁺-regulator .5.The expression of CaN, cbl-b and p-AKT by Western blotting assay were detected, so we can know the change of apoptotic protein in CIPC and pretreatment of Ca²⁺-regulator.6.The transcription of cbl-b in hippocampus neurons by RT-PCR was detected ,we can understand weather cbl-b regulate apoptosis from the transcription level of cbl-b.Methods:Part one:The improvement of MCAO rat model According to the technique of Koizumi and jiao zuo min, we established the rat models and changed the following :1) the partial right by the neck of incision;2)the diameter of the fish line is 0.26mm,according to the weight of rat;3)we used the purchased fish lines. The rat model of CIPC,sham and ischemic groups were established. We judged the successful model with Horner syndrome, bederson neurologic deficits score and TTC staining method.Part two: The detection of apoptosis neurons and the relative fluorescent value of intracellular free calcium in hippocampus neurons in each group.1.The sham,cerebral ischemia,CIPC,nimodipine-pretreated,MK801- pretreated models were established . The apoptosis of the hippocampus neurons was detected in each group with TUNEL apoptosis detection kit.2.The brain tissue in all groups were removed at the scheduled time, then the hippocampus neurons were separated, the fluorescence was marked, and the intensity of fluorescence was detected by relative fluorescent value under the confocal laser scanning microscope.Part three:Effect of CIPC and intervention of Ca²⁺-regulated factors on CaN, cbl-b and p-AKT expression in neurons.We established rat models including sham, ischemia, CIPC, nimodipine, MK801 and cyclosporine A. The neurological deficit scores were processed. The right hippocampus was removed and stained with TTC, and the volume of cerebral infarction was calculated. The expressions of CaN, cbl-b and p-AKT at the protein level were examined by Western blotting, and the transcription of cbl-b by RT-PCR, respectively. Results:Part one:The judgement of the success of rats model1.The neurological deficit scores in three groups: The scores in the sham group were 0. There were the highest scores in the ischemia group, the scores in CIPC group were lower than those in the ischemia group（P<0.05）.2.The volume of the cerebral infarction in three groups: The volume of the cerebral infarction in the sham group were 0. There were the largest volume in the ischemia group, the volumes of the cerebral infarction in CIPC group were less than those in the ischemia group（P<0.05）.Part two:1.Apoptotic neurons: Apoptotic neurons were most in the ischemia and MK801 groups, and there was no difference between these two groups（P>0.05）;Apoptotic neurons in the CIPC groups were less than those in the ischemia group（P<0.05）;and were much less in the nimodipine group than those in the CIPC group（P<0.01 ）.2.The relative fluorescent values of free calcium in hippocampus neurons: The relative fluorescent values of free calcium in hippocampus neurons were highest in the ischemia and MK801 groups, and there was no difference between these two groups（P>0.05）;The relative fluorescent values in the CIPC groups were lower than those in the ischemia group（P<0.05）;and were much lower in the nimodipine group than those in the CIPC group（P<0.05 ）.Part three:1.The neurological deficit scores: the neurological deficit scores were the highest in the ischemia and MK801 groups, there were no difference between the two groups（P>0.05）; this factors in CIPC group were lower than those in the ischemia group（P<0.05）; and much lower in the nimodipine and cyclosporine A group than those in the CIPC group （P<0.05）, but no significant difference existed between the nimodipine and cyclosporine A groups（P>0.05）.2.The volume of the cerebral infarction: The volumes of the cerebral infarction were the largest in the ischemia and MK801 groups, there were no difference between the two groups（P>0.05）; this factor in CIPC group was smaller than that in the ischemia group（P<0.05）; and much smaller in the nimodipine and cyclosporine A group than that in the CIPC group （P<0.01）, but no significant difference existed between the nimodipine and cyclosporine A groups（P>0.05）.3.The protein expression of CaN, cbl-b and p-AKT: The protein expression of CaN, cbl-b were high in the ischemia and MK801 groups, there were no difference between the two groups（P>0.05）; these factors in CIPC group were lower than those in the ischemia group（P<0.05）; and much lower in the nimodipine and cyclosporine A group than those in the CIPC group （the expression of CaN in nimodipine group P<0.01, others were P<0.05）, but no significant difference existed between the nimodipine and cyclosporine A groups（P>0.05）. The expression of AKT in all groups was alike, the expression of p-AKT was the lowest in the ischemia and MK801 groups, and there was no difference between the two groups （P>0.05）, This factor was higher in CIPC group than that in the ischemia group （P<0.05）; it was the highest in the nimodipine and cyclosporine A groups among these groups （the nimodipine group P<0.01, the cyclosporine A group P<0.05）, no significant difference existed between the nimodipine and cyclosporine A groups （P>0.05）.4.The transcription of cbl-b:The transcription of cbl-b were the highest in the ischemia and MK801 groups, there were no difference between the two groups（P>0.05）; this factor in CIPC group were lower than that in the ischemia group（P<0.05）; and much lower in the nimodipine and cyclosporine A group than that in the CIPC group （P<0.05）, but no significant difference existed between the nimodipine and cyclosporine A groups（P>0.05）.Conclusions1.The improvement of the operation in rat models brings about the following advantages:shorten operation time, increasing embolism rate, decreasing rats damage ratio,and improving the success rate of MCAO models .2.CIPC can moderately increase the free calcium in the neurons, decreasing the apoptosis of the hippocampus neurons, and protecting the neurons from ischemic damage, inducing the cerebral ischemic tolerance;MK801 can increase free calcium in the neurons, raising the apoptotic neurons in the hippocampus, blocking neuroprotection. Nimodipine can reduce the concentration of the free calcium in the hippocampus neurons, decreasing the apoptosis of the neurons, increasing neuroprotection.3.During the continuous ischemic period, calcium overload increases the expression of CaN by the activation of calmodulin, then promoting the transcription and the protein expression of cbl-b in the neuron nucleus. Cbl-b reduces the content of PI3K, decreasing the phosphorylated expression of AKT through ubiquitinating the subunit p85 of PI3K, ultimately activating apoptosis. CIPC inhibits above process and reduces the expression of CaN and cbl-b, and increases the expression of p-AKT, thereby inhibiting apoptosis in neurons. Nimodipine reduce the concentration of free calcium in the neurons, and cyclosporine A inhibit the activity of CaN, which can reduce the expression of CaN and cbl-b, and increase the expression of p-AKT,leading to neuroprotection. MK801 counteracts the effect of CIPC by getting to calcium overload during the ischemic period, and increasing the expression of CaN and cbl-b as the ischemic process, then inhibiting the the phosphorylated expression of AKT ,promoting the apoptosis of neurons.