| Objective:Neonatal hypoxic-ischemic encephalopathy (HIE) is a common neonatal disease, caused by perinatal asphyxia, it can cause neonatal death and permanent nervous system injury after neonatal period, and there are no effective therapeutic methods at present. Our study established hypoxic-ischemic brain damage (HIBD) model and gave mild hypothermia treatment to observe the expression of Caspase-3,bcl-2 and bax in brain tissue at different time points, thereby to reveal the possible mechanism of neuroprotection of mild hypothemia and develop new method for the therapy of HIE.Methods:A total of 120 female or male Wistar rats aged 7d, with the birth weight of 12～18 gram were randomly divided into three groups:sham operation group, HIBD model group, mild hypothermia group (n=40 in each group), HIBD model of neonatal rats were established in HIBD model group and mild hypothermia group. Then each group was randomly divided into five subgroups (n=8 in each subgroup) based on different time points at 6h,12h,24h,48h,72h after HIBD. HIBD model rats were prepared by keeping rats into hypoxic environment of 8% O2 concentration for 2 hours after clamping left common carotid artery. Rats in mild hypothermia group were given mild hypothermia treatment instantly after HIBD. Rats in three groups were sacrificed at different time points and brain tissue were collected. Then morphologic changes of brain tissue in general obtained through macroscopic observation, the pathological changes of left brain tissue were observed under light microscope, and the expressions of Caspase-3,bcl-2 and bax in left brain tissue were detected with immunohistochemistry.Results:(1) Neonatal rats had various abnormal behaviors after HIBD. (2) Various morphologic abnormality of brain tissue of rats in HIBD model group at different time points were observed, but there were no obvious changes of morphology of brain tissue of rats in mild hypothemia group. (3) HE staining:Cellular layer was distinct, cellular morphous was normal and there were no obvious nerve cell loss in brain tissue in sham operation group. Different extent cellular swelling and degeneration were found at 6h,12h after HIBD in HIBD model group, cellular necrosis became apparent at 24h,48h:cellular nuclear fragmentation, solution, cellular necrosis was more significant at 72h. In mild hypothermia group, the pathological changes of brain tissue lessened significantly compared with HIBD model group, and cell arranged regularly. (4) Expressions of Caspase-3,Bax and Bcl-2:Caspase-3,Bax and Bcl-2 positive staining showed buffy fine grain deposition, positive cells were found in cerebral cortex and hippocampus, and positive staining was mainly in cytoplasm and axon process of nerve cell.①Expressions of Caspase-3:Caspase-3 protein in sham-operated group showed a low level of expression. There were no statistical significant changes at different time points(P>0.05).The positive expression of Caspase-3 in HIBD model group were increased with time,reached peak at 48h,and reduced after that point. After 72 hours, the expression of Caspase-3 in brain tissue was slightly higher than that of sham-operated group. Expressions of Caspase-3 in brain tissue at different time points in mild hypothermia groups decreased significantly compared with HIBD group.There was statistical significant difference between them(P<0.01).②Expression of Bcl-2:Bcl-2 protein in sham-operated group showed a low level of expression. There were no statistical significant changes at different time points(P>0.05). The expression of Bcl-2 at different time points in HIBD group rats was increased compared with that of sham-operated group. There was statistical significant difference between them (P<0.01). The expression of Bcl-2 at different time points in mild hypothermia therapy group was increased compared with that of HIBD group, there was statistical significant difference between them (P<0.01). The peak value was at the 24h in HIBD group and at the 48h in mild hypothermia therapy group,which suggests that mild hypothermia therapy can prolong the peak value time course of antiapoptosis protein:Bcl-2.③Expression of Bax protein:Bax in sham-operated group rats expressed weakly. The expression of Bax at each time point in HIBD group was increased compared with that of sham-operated group, there was statistical significant difference between them (P<0.01). The expression of Bax at each time point in mild hypothermia therapy group was decreased compared with that of HIBD group, there was statistical significant difference between them (P<0.01).Conclusions:(1) Mild hypothermia after HIBD in neonatal rats can reduce pathological changes of brain tissue, which suggests that mild hypothermia has neuroprotective effect. (2) Mild hypothermia could up-regulate the expression of Bcl-2 and down-regulate the expression, of Bax, decrease the expression of Caspase-3,alleviate pathological injuries in the course of HIBD. It may inhibit neuronal apoptosis and provide a protective effect for hypoxia-ischemia-induced neuronal injury of neonatal rats. |
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