The Protective Mechanisms Of Estrogen On Central Nerve System In Ovariectomized Rats

Alzheimer's disease (AD) is an age-related neuro-degenerative disease,characterized by progressive neuropathology with a corresponding loss oflearning and memory and other cognitive processes. The causes andmechanisms of AD are very complicated. At present, it's considered to berelated to the cooperation of heredity, metabolism and environmental factors.Central cholinergic nerve system is the important biologic basis of learningand memory. In seventies, loss of cholinergic neuron in the brain of ADpatients was noticed, and cholinergic hypothesis was suggested. Recently,oxidative stress and free radical toxicity have been becoming focus ofresearches. Mitochondrial lesion in AD leads to oxidative stress, then oxidativestress aggravates mitochondrial lesion. AD maybe the result of theirinteractions. The epidemiological data make it clear that the prevalence rate offemale is 3 times to that of male, which has been confirmed to be resulted fromlow estrogen level in postmenopausal women. Studies show that estrogensubstitution therapy (ERT) can help to reduce the risk and severity ofAD-related dementia in some postmenopausal women. But there are also manycontroversies. Further researches and more systemic reseaches are needed onthis background. In this research, we studyed the protective mechanisms ofestrongen by observing the protective effect of estrogen on central cholinergicnerve system and mitochondria oxidation system .We hope it can provideprecise theory basis for the treatment of AD in postmenopausal women. 1.The establishment of the ovariectomized rat model and the effect ofestrogen on praxiologic and histologic changes (1) The establishment of the ovariectomized rat model After the Morris Water Maze pre-test, we selected 75 female rats anddivided them into three groups(sham group, OVX group and OVX+EB group)in random, each 25 rats. In sham group, rats'fat around ovary was resected,while in the other two groups, ovaries were resected. After two weeks, rats inOVX+EB group were injected with benovocylin 0.20mg/ kg, once every threedays, for six weeks. The rats in sham group and OVX group were injected withthe same dosage of olive oil. (2) The changes of learning -memory capacity Morris Water Maze was used to detect the changes of spatial learning-memory capacity of the rats. The result showed: the escape latency of OVXgroup was significantly longer than that before operation and that of shamgroup, the escape latency of OVX+EB group was significantly shorter than thatof OVX group, but was not apparently different from that before operation andthat of sham group. (3) The morphologic changes ①HE staining The pyramidal cells in hippocampus CA1 of the rats in sham group andOVX+EB group distributed evenly and their appearance were nearly normal;the hippocampal pyramidal cell layers in OVX group were disturbed andinterrupted. ②Ultrastructure of hippocampus CA1regionThe ultra-structure of hippocampus neurons in sham group and OVX+EBgroup were nearly normal; In OVX group nuclei pyknosis, chondriosomesswelling, broken crista and becomed emptey bubble, the chromatins condenseand tended to the side. The nucleus and cytoplasmas turned densing, andlipofuscin deposited in cytoplasm. The constitution of synapse was not clear. The results suggested that estradiol can protective the pyramidal cells inhippocampus CA1 and enhance the learning-memory ability of femaleovariectomized rats. 2.The changes of central cholinergic nerve system in ovariectomizedrats and the effect of estrogen (1) The changes of cholinergic neurons in Meynert basal nuclei ofovariectomized rats and the effect of estrogen The cholinergic neurons of Meynert basal nuclei were observed by usingimmunohistofluorescent staining and confocal laser microscopy. The resultshowed: the number, the fluorescent intensity of the cholinergic neurons inMeynert basal nuclei of OVX group was evidently lower than those in thesham group, and there was no evident difference between the OVX+EB groupand sham group. The result suggests that estrogen can decrease the loss anddegeneration of cholinergic neurons. (2) The changes of ChAT activity of cholinergic neurons in hippocampusCA1 area of ovariectomized rats and the effect of estrogen The central cholinergic neurotransmitter is the important biological basis.Acetylcholine(ACh), the important central cholinergic neurotransmitter, issynthesized by choline acetyltransferse(ChAT). ChAT is the marker enzyme ofcholinergic nervous system and often taken as the indirect marker of Achcontent. The expression of ChAT was measured with immunohistochemistryand image-analysis. The ChAT immunoreaction showed buffy products in thecytoplasm. The result showed: the ChAT level in the OVX group was evidentlylower than those in the sham group, and there was no evident differencebetween the OVX+EB group and sham group. The results suggested thatestrogen replacement may enable the neurons maintain elevated level of AChrelease in OVX rats and help to improve cognitive deficits by enhancing ChATactivity of cholinergic projections to the hippocampus. (3) The expression of NGFmRNA and TrkAmRNA of the cholinergicneuron in basal forebrain of ovariectomized rats and the effect of estrogen NGF is a classical neurotrophin, it's produced in the hippocampus andcortex and transported to the cholinergic neuron in basal forebrain. TrkA ishigh-affinity binding site of NGF. Studies have shown that binding to TrkA,activation of the receptor tyrosine kinase, and autophosphorylation of thereceptor are essential for mediating biological effects of NGF. NGF has beenshown to promote both survival and fuction of basal forebrain cholinergicneuron by binding to TrkA. In order to study the mechanisms ofestrogen-mediated effects on central cholinergic nerve system, the expressionof NGFmRNA and TrkAmRNA of the cholinergic neuron in basal forebrain ofovariectomized rats was measured by the RT–PCR technique. The resultswere processed with a computerized image-analysis system. The results wereas followed: the NGF mRNA and TrkA mRNA expression level of OVX groupwas significantly lower than those of the sham group and OVX+EB group,there was no evident difference between the sham group and OVX+EB group.The result suggested that the estrogen replacement can significantly increasethe ChAT activity by enhancing the expression of NGFmRNA andTrkAmRNA. Estrogen can indirectly execute protective effects on central cholinergicnerve system by enhancing the expression of NGFmRNA and TrkAmRNA. 3 . Effect of estrogen on brain antioxidation capacity inovariectomized female rats. Superoxide dismutase(SOD) is a free radical cleaner which can cleansuperoxide anion and protect cell against reactive oxygen species (ROS)overload and subsequent damage. It can maintain the balance between theoxidation and antioxidation, and it is one of the markers to reflect the cleaningability of free radical. Malonialdehyde(MDA) is the metabolite of unsaturatedfatty acid in oxidation response in biomembrane resulted from free radical. Soit can indirectly reflect the severity degree of the injury of cell. Mitochondrialmembrane mainly consist of phospholipid molecule. It is very important formitochondrion membrane to execute normal function that keeping its fluidityand permeability. Increased production of free radicals can destructmitochondrion membrane 's fluidity and permeability, thus metabolites ofmitochondrion are released into intercellular substance, which can lead toapoptosis and necrosis of cells. Cytochrome C oxidase(COX) is the endenzyme of mitochondrion electron transport chain, and also is the rate-limitingenzyme of mitochondrion electron transport chain which have important effecton energy metabolism of cell. Thus activity of COX is closely correlated withthe function of neuron and is the marker of neuron metabolism. We measurethe activity of SOD, MDA and COX by UV-spectrophotometer and measurethe fluidity of mitochondrial membrane by fluorescence spectrophotometer.