Study Of Estradiol Benzoate Protection In Chronic Cerebral Ischemia Rats

Background and ObjectiveChronic ischemic brain impairment is a common pathological state,always complicated in many diseases such as vascular dementia ,Binswanger disease and Alzheimer disease and so on.In the early stages,its cardinal manifestation is cognitive disorder.At last,it can cause eternal or progressive impairment of cognitive and neurological function.Overwhelming numbers of studies have proved that comprehensive loss of neurons and white matter lesions resulting from chronic cerebral ischemia can cause cognitive disorder.Hippocampus is important parts of nervous system involved in learning and memory that loss of them can lead to impairment of learning and memory.The central cholinergic nervous system is closely related to study and memory.Studies showed that the decreasing activity of CHAT,the marker enzyme of cholinergic system,is an important cause leading to dysfunction in study and memory.The nervous anatomical base of study and memory is the plasticity of synapse structure,and the NO and synaptophysin have a close relationship with the plasticity of nervous synapse.The Epidemiological studies have shown that pre-menopausal women are at relatively decreased risk of cerebral stroke when compared with men in the same age,and the serious degree of ischemic injury is much lower than the matched men and post-menopausal women in the same age.But the stroke incidence becomes higher when women go into the post-menopausal period.It has been found in many studies that the relative risk and mortality of stroke decreased in post-menopausal women who had received estrogen replacement therapy. So people presume that estrogen has acerebral protective function.Recent study showed estrogen can ameliorate ischemiadamage and improve cognitive function.However,it has not been reported thatwhether estrogen has protective effect to patients with chronic cerebral hypoperfusion.The aim of this experiment by permanent occlusion of bilateral common carotidarteries was adopted to establish mad chronic cerebral ischemia rats model.The ratswere intra-abdomina infused with estradiol benzoate. Learning and memory of eachgroup of rats were determined by MG-2Ymaze;and immunohistochemical techniquesmethod was used to observe the change of CHAT,nNOS and SYN protein expressionin the hippocampal CA1 region to explore the mechanism of effect on the cognitivedysfunction of chronic cerebral ischemia of estrogen treatment.It will Provide someexperimential and theoretical supports for prevention and treatment of some diseasewhich are related to chronic cerebral ischemia.Materials and Methods1. Thirty healthy female SD rats weighing from 300-350 grams were randomly divided into 3 groups.GroupA:sham-operated group GroupB:chronic forebrain ischemia group;GroupC: chronic forebrain ischemia treatment group.There are 10 rats in each group.ischemia treatment group were intra-abdomina infused with estradiol benzoate 1mg/kg(0.3mI) after ovariotomy operation;sham operated group and pure ischemia group were injected with boiled oil (that is dissolvent which can dissolve estradiol)after operation,and every rat was intra-abdomina injected with boiled oil about 0.3ml,Every group were injected twice a week in 60 days period;2. Rat models of chronic cerebral ischemia were established by occluding and snipping of bilateral common carotid arteries via using 0# thread permanently in only ischemia group and ischemia treatment group.Bilateral common carotid arteries were isolated but not ligated or snipped in sham group. About one week after operation ,the ovarian of every rat of ischemia group and ischemia treatment group was removed bilateral.The rats of sham operation group were operated anaologly,but the ovarian of this rats were not removed.All rats were raised in common condition after the operation;3. Cognitive function of all rats was observed at 60d after operation by Y-maze test.And then,they were anesthetized and perfused with 200ml normal saline through heart.subsequently,brains of them were removed and fixed in 4% polyformaldehyde. Funally,brains were embeded,sectioned and processed for HE staining and immunohistochemical staining;4. (1) Maze of MG-2Y was used to detect the cognitive function of every rat.The rats were trained run to the exit passageway according to the signal lamp.Each test went on at 8:00-10:00 a.m.(2) HE staining was performed to observe the change of hippocampal histopathology under light microscope.(3) By using immunohistochemical method,we deteced the number of nNOS positive cell.The value of positive cells of CHAT,SYN were measured by pathological image analysis system.5. All the data were expressed by(x|-±s) and analyzed statistically with SPSS10.0 statistic software.The difference of every two groups was compared with one-way analysis of variance.The significant standard isα=0.05.Results(1) After completely blocking of the bilateral common carotid artery of the model rats,the rats firstly showed transient convusion and the righting reflex disappeared with the decrease of temperature and slowering respiration .5-7 days after operation,the creeping of every rat in each group basically returned to be normal with no obvious dyskinesia;(2) The results of cognitive ability.The times of electric attack in sham group was 34.50±7.29;in only ischemia group was 81.67±11.02 and in ischemia treated group was 60.83±15.90.There was significance between ischemia group and sham group in cognitive ability(P<0.01).The difference between only ischemia group and ischemia treated group in cognitive ability was also significant(P<0.01);(3) The results of pathological section with HE staining.only a few degenerated and dead neurons were found,while most neurons were normal in morphology in sham group.In ischemia group,the pathological impairment of the hippocampal neuron showed ingravescence,the stratification of cone cell in CA1 region reduced and the neuron of this area were more sparse and the cell-loss were more obvious.The karyon volume shrank with deep staining and pyknosed into triangle or polygon.The arrangement of nerve fiber was in disturbance.Comparing with ischemia group,the brain tissue injury in ischemia treated group got a remarked alleviation; (4) The expression of CHAT in every group:the analytical system of pictures was used to analyze the average gray scale value of the positive district of CHAT.A significantly time-dependent decrease of CHAT protein could be measured in ischemia group and ischemia treated group compared with sham group (P<0.01).CHAT expression was lower in ischemia group than in ischemia treated group(P<0.01);(5) The expression of synaptophysin in every group:the analytical system of pictures was used to analyze the average gray scale value of the positive district of synaptophysin. A significantly time-dependent decrease of synaptophysin protein could be measured in ischemia group and ischemia treated group compared with sham group(P<0.01). The synaptophysin gray scale value of treatment groups increased gradually;There are significant difference between ischemia group and treatment group(P<0.01);(6) The marks of the nNOS concerned in ischemia group was lower than the other groups(P<0.01).The marks increased remarkably in the rats of ischemia treatment group(P<0.01).Conclusions(1) The estradiol benzoate can improve the cognitive dysfunction of chronic cerebral ischemia rats;(2) The estradiol benzoate can alleviating the impairment of neuron ischemia in the hippocampal CA1 region of chronic cerebral ischemia rats;(3) The estradiol benzoate can obviously increase the expression of CHAT protein in the hippocampal CA1 region and it can comparatively increase the expression of nNOS and SYN protein.This suggest that estradiol benzoate can improve cognitive dysfunction of chronic cerebral ischemia rats by increasing the expression of synaptophysin,ACH,NO.