The Research Of Signal Mechanism Of Nerve Injury Induced By Reactive Oxygen Species In Status Epilepticus

Objective:To establish rat SE model by useing lithium chloride-pilocarpine and to observe the alteration of rat behavior, the change of reactive oxygen species and endogenous antioxidant in the pathological process of status epilepticus.Methods:24 adult female SD rats were randomly divided into the control group and SE model group. The model group were divided into 3 groups:30min,60min,90min group after SE. Each group is 6. Observe the alteration of rat behavior during SE and measure concentration of ROS and endogenous antioxidant in rat cerebral cortex by chemical colorimetric method, compare with the control group.Results:1. Behavior results:control group without epilepsy behavior, model group reach grade IV and above, continue for more than 30min.Seizure latency is 38.78±9.63min.2. Measure results of ROS and endogenous antioxidant activation:Concentration of MDA in the SE30min group is no statistical difference as compared to control group (P＞0.05). In the SE60min and SE90min group, concentration of MDA is significantly higher than the SE30min and the control group (P＜0.01). Concentration of MDA reaches peak in SE90min group. Concentration of H2O2 rapidly increases and reached peak in SE30min group. Concentration of H2O2 gradually decreases in SE60min and SE90min group. But the three groups were higher than the control group (P＜0.05).SOD,CAT and GR activation rapidly decrease in SE30min, SE60min and SE90min group, which are lower than the control group (P＜0.05). GSH activation decreases significantly in SE30min, SE60min and SE90min groups, which are lower than the control group (P＜0.05). GSH activation in SE90min is higher than SE30min and SE60min group, but still lower than the control group level (P＜0.05).Conclusions:1. Model of Lithium chloride-pilocarpine induced SE is simple, high success rate and low mortality. It has similar behavior change with status epilepticus of human. It is ideal model.2. In the model of Lithium chloride-pilocarpine induced status epilepticus, concentration of ROS increased and levels of endogenous antioxidants decrease in cerebral cortex. ROS participate in the pathological process of status epilepticus. The longer times of SE take placed, the stronger the injury of oxidative stress induced. The body has some ability to compensate, but the compensation is limited. Objective:To investigate the effect of resveratrol on alteration of behavior and to observe concentration of ROS and endogenous antioxidant in rat SE model. Investigate the signal mechanism of ROS induce neuronal damage. Explore the possible neuroprotection mechanism of resveratrol.Methods:54 adult female SD rats were randomly divided into model group, resveratrol pretreatment group and control group. Each group is 18. Establish the SE rat model by peritoneal injection of lithium chloride-pilocarpine. Meantime, rats are pretreated by resveratrol. Observe alteration of behavior during SE and measure concentration of ROS and endogenous antioxidant of rats brain cortex by chemical colorimetric method after SE. Observe expression of apoptosis-related protein bcl-2, bax, caspase-3 by immunohistochemistry and Western blot methods.Results:1. Behavior results:the control group without SE behavior. After injection of pilocarpine., seizure latency of the SE group is 40.72±9.75 min. The rats of resveratrol pretreatment group appear the behavior similar to the model group. Seizure latency is 64.61±11.54min.2. Measure results of ROS and endogenous antioxidants:In rat cerebral cortex the concentration of SOD, GSH-PX, GSH and GR in model group decrease.. Compared with the control group, there were significant differences (P＜0.01). The concentration of MDA increases significantly in model group. Compared with the control group, there were differences (P＜0.05). The concentrations of SOD, GSH-PX, GSH, GR in resveratrol pretreatment group is significantly higher than the model group (P＜0.05). The concentration of MDA is lower than the model group and reaches the level of the control group. Compared with the control group, there are no significant differences (P＞0.05).3. Immunohistochemical results:①Caspase-3 positive cell count result shows that in the model group the number of caspase-3 positive cells is higher than the control group. In resveratrol pretreatment group the number of caspase-3 positive cells significantly decreases, compared with the model group (P＜0.05).②Bcl-2 positive cell count result shows in the control group that in resveratrol pretreatment group the number of bcl-2 positive cells compared with model group increases (P＜0.05).③Bax positive cell count result shows that in the model group the number of bax positive cells increases, compared with the control group. In resveratrol pretreatment group the number of bax positive cells compared with the model group decreases (P＜0.05).④The ratio of bax/bcl-2 in the model group is higher than in the control group. The ratio of bax/bcl-2 in resveratrol pretreatment group is higher than in the model group (P＜0.05).4. Western blot analysis:①In rat brain cortex, the model group caspase-3 protein content are significantly higher than the control group (P＜0.05), whereas the resveratrol pretreatment group caspase-3 protein content are significantly lower than model group (P＜0.05).②The resveratrol pretreatment group bcl-2 protein content increased, compared with the model group(P＜0.05).③The model group bax protein was significantly higher than the normal control group (P＜0.05), whereas the resveratrol pretreatment group bax protein was significantly lower than model group (P＜0.05).Conclusions:1. The rats of model group epileptic seizure fromⅠgrade toⅤgrade, eventually into status epilepticus. Although resveratrol can not prevent the occurrence of status epilepticus, but it can significantly prolong the latency of status epilepticus. Resveratrol has evident function of delay SE.2. The rats significantly reduced antioxidant capacity after SE. It can not remove excess oxygen free radicals, leading to accumulation of free radicals. It is confirmed that oxidative stress participate in mechanisms of brain cell damage in pilocarpine-induced SE. Resveratrol not only significantly reduce ROS levels, but also significantly increased the activity of endogenous antioxidants. Resveratrol can raise content of endogenous antioxidant. The function enhances the body's ability to eliminate reactive oxygen species and inhibit oxidative stress. It play the role of neuroprotective. This may be important way to inhibit apoptosis of resveratrol after SE. 3. All of apoptosis-related protein bcl-2, bax, caspase-3 change after status epilepticus. Resveratrol can the expression of anti-apoptotic protein bcl-2 and inhibit the expression of pro-apoptotic protein bax, decrease the ratio of bax/bcl-2, inhibit the expression of caspase-3 activity. It suggests that resveratrol inhibiting neuronal apoptosis may be through the regulation of apoptosis-related factors. Resveratrol play neuroprotective role.