| Objective To study the change expression of tissue-type transglutaminase and caspase-3 and the relationship between them and Cystamine after cerebral ischemia-reperfusion in rats. Thus to explore the possible mechanism of its neuroprotection of rats.Methods 104 adult male Sprague-Dawley rats(the model achievement ratio was 75% and dead rats were supplemented) were randomly divided into sham-operated group (n=8), global cerebral ischemia group(n=48) and global cerebral ischemia treatment group (n=48). The two latter groups were randomly divided into 6,12h,1,3,5,7d six subsets according to the time point with 8 rats. All rats were about 280-320g. The experimental global cerebral ischemia-reperfusion injury model was established by the method of 4-vessel occlusion.The electroencephalogram (EEG) was used to detect if the models were successful. Cystamine was injected intraperitoneally (150mg/kg/day) 10 minutes after cerebral ischemia for global cerebral ischemia treatment group. Cystamine was injected only once for 6,12,24h time course group. Other groups were given only once by Cystamine on the same time a day. Saline was injected intraperitoneally for the global cerebral ischemia group. Saline 1ml was injected intraperitoneally for the sham-operated group and the rats were sacrificed on the third day. The global cerebral ischemia group and global cerebral ischemia treatment group were sacrificed by decapitation after the global cerebral ischemia-reperfusion injury on 6,12h,1,3,5 and 7d time points. Neurons apoptosis was detected by TUNEL technology.The expression of tTG and caspase-3 were detected by Immunohistochemistry(SP). The other 78 rats were randomly divided into 3 groups:sham-operated group (n=6), global cerebral ischemia group(n=6) and global cerebral ischemia treatment group (n=6).The rats were sacrificed by decapitation which were takien the hippocampus to refrigerator in 80 degrees below zero. The protein expression of tTG and caspase-3 were detected by Western blotting.Results 1) TUNEL staining:About 24h after ischemia-reperfusion injury,the positive cells of TUNEL staining were significantly increased. Compared to the sham-operated group, the positive cells of TUNEL at the subset 1,3,5,7d after global cerebral ischemia group were significantly increased (P＜0.05).The number of apoptotic cells in subset 1,3,5,7d of global cerebral ischemia treatment group were significantly less than that of global cerebral ischemia group (P<0.05).2) Immunohistochemistry(SP) The expression of tTG immunopositive cells were small amount in the sham-operated group. The number of positive cells in subset 1,3,5,7d of global cerebral ischemia group were significantly more than that of the sham-operated group (P<0.05). In global cerebral ischemia treatment group,tTG immunopositive cells began to decrease in the CA1 region of hippocampus in subset 12h time point,and significantly decreased than that in 3,5,7d subset of global cerebral ischemia group (P<0.05). The positive cells expression of caspase-3 in CA1 area of hippocampus increased only a little in the sham-operated group. In global cerebral ischemia group,caspase-3 immunopositive cells began to increase in the CA1 region of hippocampus in subset 6 h time point, peaked at 3d time point,and then decreased to a high leveer at 7d time piont,and significantly decreased in 3,5,7d subset of global cerebral ischemia group (P<0.05). Compared to the corresponding subsets between global cerebral ischemia group and treatment group after global cerebral ischemia, the expression level of caspase-3 in 1,3,5,7d were significantly decreased(P<0.01).3) Western blotting:The protein expression of tTG from global cerebral ischemia group began to increase at 1d time point and significantly more increase in 3,5,7d subset of the sham-operated group (P<0.05).In global cerebral ischemia treatment group,tTG protein level began to increase 1 days after ischemia-reperfusion,and significantly decreased in 3,5,7d subset of global cerebral ischemia group (P<0.05).The protein expression of caspase-3 in hippocampus began to significantly increase at 6h time point and increased nearby to a high lever at 7d time point (P<0.05).In global cerebral ischemia treatment group,caspase-3 protein level significantly decreased in 1,3,5,7d subset of global cerebral ischemia group (P<0.05)Conclusion The number of apoptotic cells were significantly increased after cerebral ischemia-reperfusion.At the same time,the expression of tTG and caspase-3 were also increased obviously, which might be one possible mechanism of neuronal apoptosis after cerebral ischemia-reperfusion. The use of Cystamine at the acute of cerebral ischemia-reperfusion can reduce the number of apoptotic cells and have a certain degree of neuroprotection.One of the probable mechanisms was that Cystamine treatment restrained the expression of tTG and caspase-3,which decreased the apoptosis of neurons.
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