| Objective:To explore the protective effects and mechanisms of Cilostazol on neuronal apoptosis in the hippocampus after global cerebral ischemia in rats.Methods:The model of transient global cerebral ischemia was produced by improved four-vessel occulusion as described by Pulsinelli in Sprague-Daewley rats. The electroencephalogram (EEG) was used to detect if the models were successful. 184 (the success rate of the model was 75%, and extra rats were supplemented)healthy adult male rats(about 300g) were randomly divided into 3 groups:sham-operated group(n=8 × 2), ischemia reperfusion group(n=8 × 9) and treatment group(n=8 ×9). The rats were sacrificed at 0,1,12, 24, 72h and 7d following the global cerebral ischemia for 10min and reperfusion in ischemia reperfusion group and treatment group. The rats in sham group were decapitated at Oh after the cerebral ischemia and reperfusion. Rats in treatment group received two oral administration of Cilostazol(30mg/kg)at 6h and 2h before the global cerebral ischemia, then they were given Cilostazol every 24h. Rats in other groups were given the same volume of dimethyl sulfoxide. After rat brain tissues were perfused and fixed, they were used to make paraffin sections for HE staining to observe the pathological changes of neurons in hippocampus CA1 region at 24, 72h and 7d after ischemia-reperfusion; the frozen sections were needed for the immunohistochemistry of caspase-3;immunohistochemistry(SP method)and Image-ProPlus 5.0 image analysis system were used to detect the mean optical density of p-Akt in hippocampus CA1 region at 0, 1, 12h after ischemia-reperfusion; immunohistochemistry (SP method)were used to calculate the mean caspase-3 positive neurons at 24, 72h and 7d after ischemia-reperfusion. The another 24 rats were randomly divided into 3 groups:sham-operated group(n=8), ischemia reperfusion group (n=8)and treatment group (n=8). They were used to study the learning and recalling ability of rats at 7d after global ischemia-reperfusion by Morris water maze.Results:1.HE staining: No neuron damage was found in sham-operated control group in hippocampal CA1 region. Compared with sham-operated group, there were respectively about 15%, 54%, 80% neuronal necrosis in the hippocampal CA1 region at 24,72h and 7d in ischemia reperfusion group. Whereas the neuronal damage in Cilostazol group was less than that in ischemia reperfusion group(24h subgroup:P< 0.05, the other two subgroups: P<0.01). 2. The behavior change: Within 5 days, the Escape Latency(EL) in ischemia reperfusion group and treatment group was markedly longer than that of in sham-operated group(P<0.01, P<0.05 respectively);within 4 days, the Escape Latency(EL) in treatment group was markedly shorter than that of in ischemia reperfusion group(P< 0.01);whereas on the fifth day, there was on significant difference in these two groups. The average number of rats passing the platform in ischemia reperfusion group and treatment group was markedly lower than that in sham-operated group(P<0.01, P<0.05 respectively);compared with ischemia reperfusion group, the average number of rats passing the platform in treatment group was markedly higher(P< 0.01)3. the Mean Optical Density of p-Akt:The difference in p-Akt level between ischemia reperfusion group and sham-operated group at 0,1,12h after ischemia-reperfusion was significant(P<0.01, P<0.05, P<0.01). The p-Akt level was obviously higher at 0,1h after ischemia-reperfusion in treatment group than that in ischemia control group(P< 0.01, P< 0.05). 4. caspase-3 mean positive cell numbers: the caspase-3 mean positive cell numbers in ischemia control group were obviously higher than those in sham-operated group(P< 0.01), and the positive cell numbers in treatment group were obviously lower than those in ischemia control group (P< 0.05).Conclusions:1. Cilostazol can reduce neuronal damage and the expression of caspase-3 in CA1 region after global cerebral ischemia and reperfusion in rats. 2. Cilostazol can improve the decreased learning and recalling ability after global cerebral ischemia in rats,which was connected with Cilostazol's lessening the apoptosis of neurons in hippocampal CA1 region. 3. Cilostazol can inhibit the rush decrease of p-Akt level in hippocampal CA1 region after ischemia-reperfusion in rats, which may be one of the mechanisms of the protective effects of Cilostazol on the neurons in hippocampal CA1 region.
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