The In Vivo Experiment Studies On The Neuroprotective Effect Of Estrogen Via GPER1 After Global Cerebral Ischemia

Ischemic cerebrovascular disease(ICVD) accounts for about 8% of all cerebrovascular diseaseand is a common and frequent neurology disease, threating the expectancy and quality of life, especially in the elderly. In China, with the improvement of living standards, hypertension and diabetes increases and the incidence of ischemic stroke also increased year by year with an annual rate of 9 percent.The mortality of ICVD is as high as 60%-80%, which is much higher than that of United States, Canada, France, Japan, Switzerland and other countries. Patients surviving from cerebral ischemic stroke will inevitablybecome a heavy burden on families and society. Therefore, exploring the drug for the treatment of cerebral ischemia will have great practical significance.The neuroprotective agents are of great importance for reducing the mortality and morbidityof stroke. Currently, according to the epidemiological study and animal research, estrogen has been proved havingneuroprotection effect for the neurons after ischemia. Based on the epidemiological study, the incidence and prognostic of stroke and cardiovascular diseases have significant difference between genders. Compared to the male counterparts of same age, pre-menopausal female has lower incidence for diseases like hypertension, stroke and myocardial ischemia.While post-menopausal women have higher risk for stroke and poorer outcomecomparing to men of the same age. Evidences from animal study have confirmed that whether it is a long-term prophylactic physiological doses of estrogen or immediate administration of estrogen after ischemia, the area of ischemic infarction or hippocampal cell damage induced by global hemisphere ischemia could be greatly reduced by both ways. The underlying mechanisms of neuroprotection of estrogen are complex. Act as a sex hormone, estrogen’s effects are involved in several physiological and pathological processes in vivo. Hence applying estrogen as a neuroprotective agent might bring series of side effects. For example, estrogen administration has positive relationship with the risk of breast cancer and reproductive system tumors for postmenopausal women. Therefore, estrogen used as a neuroprotective agents in clinical is still in great controversy. To better understanding the mechanism of neuroprotective effect of estrogen is the key for solving this controversy.This research is divided into four parts. In the first part of Experiment I, The normal female rats were ovariectomized bilaterally and the blood were collected by cutting tails at 1, 3, 5, 7 day after the ovariectomized surgery. The changes of E2 concertration in serum of ovariectomized rats were detected by electrochemical methods. Then we observed the concentration curves of 1, 3, 5, 7 day after the ovariectomized surgery. Compared with the control group, E2 concentration in serum of most ovariectomized animals decreased rapidly.E2 concertration in serum fell nearly 75% at 5days after operation and it is below 5pg/ml at 7days after operation. Consequently, according to the results of serum detection we can determine whether the ovariectomized surgery was successful. E2 concertration in serum can be used as a standard about whether the ovariectomized surgery was useful. In order to guarantee the reliability of the experimental result,the success of ovariectomized surgery is the basic of establishing an animal model with controllable, constant E2 level. In the second part of the Experiment I, we observed the localisation of GPER1 in the ovariectomised adult female rat hippocampus by immunochemical tissue staining. We found that GPER1 has a wide tissue distribution in the hippocampus. Cells positive for GPER1 based on immunoreactivity were found in the CA1-3 and dentate gyrus in the hippocampus. Immunostaining was expressed on the cell membrane.Experiments II studied the global cerebral ischemia model induced by four-vessel occlusion. We applied microsurgical technique and EEG monitoring support technologies to judge the success of the global ischemia model.Our results showed that the hippocampal CA1 neurons were severely damaged and most of which died.The pyramidal cells were significantly reduced. the normal structure was lost and the cell organization was in disorder. Nissl staining fades or disappears, only a very small amount of relatively morphologyintact neurons remain. All the above results proved the success of the global cerebral ischemia model and ensured the smooth progress of the subsequent experiments.In experiment III, we researched that the activation of GPER1 by G-1 protected morphology of hippocampus and behavior of rats after global cerebral ischemia. The model of ovariectomized and global cerebral ischemia was established. By injecting different doses of G-1 at different time points before ischemia or after reperfusion, we observed the Nissl staining result and counted and compared the viable cells in hippocampal CA1 area from different doses and time groups at 72 h after reperfusion. Morris water maze of each group was tested after 7 days. The results provided morphological and behavioral evidence of the neuroprotective effect on hippocampal neurons of GPER1 activation. The present study showed that G1 protected CA1 neurons against ischemic injury in a dose- and time-dependent manner and ameliorated the cognitive dysfunction caused by GCI.In Section 4, we managed to discover the mechanism of GPER1 activation for protecting the rat hippocampal neurons after global cerebral ischemia. By using western blotting technique, we examined the p-Akt、p-ASK1 and p-JNK protein expression level at 1h、4h、12h after reperfusion in following groups: sham, ischemia + vehicle, ischemia + G1(50μg,-1h) and ischemia + G1(50μg,-1h). we also examined the p-Akt、p-ASK1 and p-JNK protein expression level at 1h after reperfusion in sham、 ischemia + vehicle、ischemia + G1(50μg,-1h) and ischemia + G1(50μg,-1h) + LY294002 groups. Our results showed that GPER1 activation could significantly increase p-Akt expression and decrease p-ASK1 and p-JNK expression in CA1 area of rat hippocampus at 1 h after reperfusion. These effects were further enhanced by G1 at 1 h after reperfusion. LY294002 significantly reduced the phosphorylation of Akt and inhibited the neuroprotective effect and cognitive protection of G1 against ischemia. LY294002 also significantly increased the phosphorylation of ASK1 and JNK.These findings demonstrated that G1 is effective in reducing hippocampal CA1 neuron damage and ameliorates the cognitive dysfunction caused by GCI in SD rats by inhibiting the ASK1/JNK pathway via activation of PI3K/AKT, suggesting that GPER1 activation has a neuroprotective effect after GCI and may represent a potential therapeutic target.