Protective Effect Of Combined Ganglioside GM1 And Shenfu Injection In Newborn Rats With Hypoxia-ischemic Brain Damage

Objective: Neonatal Hypoxic-ischemic brain damage (HIBD) means infant suffocation occurs in the perinatal period which causes hypoxic brain damage, it is also the important causes of early neonatal death in children. Pathogenesis of HIBD was very complicated. Apoptosis is the main cause of a late nerve cell loss and dysfunction. Therefore, according to the pathogenesis of the disease to take appropriate treatment measures on the prognosis of HIE has a very important significance. This study mainly focuses on the Bcl-2, Bax expression trend after the treatment of ganglioside and Shenfu in brain tissue for neonatal rats have hypoxic ischemic brain injury. From a theoretical point of view, whether it has a more significant improvement on functional recovery after the combination of the two drugs on the nervous system of HIBD rats.Methods: (1) Animal group: seven-day-old Wistar rats were randomly divided into five groups: sham-operated group; HIBD group; GM1 treatment group; Shenfu treatment group; combination treatment group, 18 rats each. HIBD models were prepared of all groups except the sham-operated group. And rats in each group were divided into three subgroups further (n=6 each) based on different time points: 6h, 24h and 48h group. (2) Model preparing: Sham-operated group rats received cervical median incision and left common carotid arteries were isolated without hypoxia-ischemia. Model group rats were ligated the left common carotid artery and placed in hypoxia box for 2h. Rats of GM1 and Shenfu groups were given immediately intraperitoneal injection of GM1 and Shenfu after hypoxia-ischemia damage. Combination-treated group rats were intraperitoneal injected the two drugs after the hypoxia ischemia damage. (3) To observe changes in brain tissues: All the rats were killed at corresponding time points and brain tissue were collected. The macroscopical morphous changes of brain tissue of all rats were observed. HE staining was performed. And the pathological changes of left brain tissue were observed under light microscope. Bcl-2, Bax expression of the left brain were detected by immunohistochemistry semi-quantitative method.Results: (1) Behavior changes: Neonatal rats had various abnormal behaviors after HIBD. (2) Visual observation: Sham group rats have no significant changes in brain tissue in general. Brain tissues of HIBD group rats have different degrees of abnormalities. Changes in brain tissues of treatment group rats were less than that of the HIBD group significantly. (3) HE staining showed: Hippocampus of the sham operation group was normal, cells closely aligned in normal morphous. HIBD group rats were with loose organizational structure in hippocampus, hippocampus cells were in disorder and with vacuoles, degeneration and necrosis. The numbers of normal neurons decreased. The level of injury of intervention group rats was significantly less than that of HIBD group, especially in the combination group rats. (4) Immunohistochemical staining showed:â‘ Bcl-2 expression: Bcl-2 positive cells were widely expressed in the cerebral cortex and hippocampus, mainly the cytoplasm colored, showed with brownish yellow or brown particles. Bcl-2 expression of the HIBD group and every treatment group was peaked at 24h after the HIBD. However, only weak expressions showed in the sham group, and there were no significant differences among each time points. Bcl-2 expression at each time point in the HIBD group increased, the average gray value of positive cells was lower than that of the sham operation group rats. There were statistical significant differences among them (P<0.01). In GM1 group, the Shenfu group and combination group, the positive expression of Bcl-2 at all time points were stronger than that of HIBD group. And the average gray value of positive cells compared with HIBD group reduced. There were statistical significant differences among them (P <0.01). The positive expressions of Bcl-2 in the combination group at all time points were more obvious increased than the GM1 group and Shenfu group. There were statistical significant differences among them (P <0.01).â‘¡Bax expression: Bax-positive cells expressed in the cytoplasm, showed with brown particles. Bax expression in HIBD group and all treatment groups was peaked at 24h after HIBD. Sham-operated group had a very small amount of Bax protein expression. Bax protein expressions at each time point in HIBD group rats were apparently increased, which had the deepest dye. And the average gray value of the positive expression of HIBD group rats was significantly lower than that of the sham group. There were statistical significant differences among them (P <0.01). The positive expression of Bax in GM1 group, the Shenfu group and combination group at all time points were less than that of HIBD group. The average gray value of the positive expression was higher than that of the HIBD group. There were statistical significant differences among them (P <0.01). Compared with GM1 and Shenfu group, the positive expression of Bax at all time points in the combination group were more apparently decreased. There were statistical significant differences among them (P <0.01). Conclusion: (1) After the hypoxic ischemic brain injury model established successfully, a single intraperitoneal injection of ganglioside or injection of Shenfu can reduce brain injury in neonatal rats. The combination application of the two drugs intraperitoneal injection can improve their brain injury obviously. (2) Both gangliosides and Shenfu can up-regulate Bcl-2 expression and down-regulate Bax expression. Changing Bcl-2 / Bax ratio in brain may reduce the pathological damage of HIBD and inhibit the neurons apoptosis.