The Neuroprotective Effect And Mechanism Of Estrogen On Injury Of Ratinal After Induction Of Chronic Ocular Hypertension In Rabbits

Objective: Glaucoma is a common cause of blindness,whose injury pathogenesis of optic nerve is the loss of retinal ganglion cells(RGCs)and nerve fiber.Many factors play important roles in the occurrence of glaucoma.Elevated intraocular pressure not only can lead to retinal ischemia and hypoxia,but also can make RGCs degenerate and cause apoptosis. We designed a study to explore the feasibility of using ovarectomy and sham-ovarectomy for studying the effect of estrogen on ocular diseases ,This study was designed to investigate the protective effect of estrogen on injury of retinal by establishing chronic ocular hypertension model ,which was induced by injecting 3 g/ L carbomer into anterior chamber of the eye.Observe the expression of Bcl-2 in retina.This study also investigated whether estrogen inhibited the cell of apoptosis by increaing the expression of Bcl-2 in retina, which provides theory basis for the study in clinic in the future.Methods:1 Animal and groups: Twenty-eight big healthy white female rabbits from New Zealand were randomly divided into three groups:①Normal control group(six eyes);②Experimental group(ovarectomy group);③Control group(sham-ovarectomy group) ; Rabbits were ovariectomized in experment group and only opened the abdomens in the control group .Both experimental group and control group were established chronic ocular hypertension model,It was divided into 4wk, 6wk, 8wk, groups according to the different time after the chronic ocular hypertension respectively, each group had six eyes.2 Standard and method to induce chronic ocular hypertension:The standard of chronic ocular hypertension model is that the IOP is higher than 2.93kPa(22mmg)and long for 1 week.The chronic ocular hypertension model was induced by injecting 3 g/ L carbomer into anterior chamber of the eye.We enucleated eyeballs of the rabbits of each group at the specified time after the formation of model and made paraffiln slices.3 Measurement of estrogen level of each rabbit in blood: The estrogen level of each rabbit in blood was measured by Beckman Acess Immunoassy System by the vein of ear edge in 9 AM at 1day before operation,2wk,4wk,6wk,8wk after operation.4 Histopathology: We observed the pathological changes of every slice under optical microscope after performing HE staining.5 Measurement of Bcl-2 by immunohistochenistry: It was regarded as positive expression of Bcl-2 that cytoplasn was stained brown when it was observed under optical microscope.6 Measurement of Bax by immunohistochenistry: It was regarded as positive expression of Bax that cytoplasn was stained brown when it was observed under optical microscope.7 Measurement of apoptosis by TUNEL: TUNEL (terminal deoxynucleotidy transferase mediated x-dUTP nick end labeling) was used to measure the apoptosis of retinal cells. It was regarded as positive expression of apoptosis that nuclear was stained brown when it was observed under optical microscope.8 Measurement of F-VEP: We evaluate the optic nerve injury by flash visual evoked potential(F-VEP) .Results:1 IOP : The method of IOP to express is means±standard deviation( x±s)。The average IOP was 27.89±1.56mmHg in the whole experiment ,the peak value was35~48 mm Hg .There was no distinguishable differences in this study about the IOP of two eyes,the average IOP was 14.50±1.35mmg in the whole rabbits before experiment.2 Histopathology of retina: The structure of normal rabbit was similar on the whole ,each layer tissue was clear,well-arranged,There was no cellular necrosis and degeneration. Each layer of retina of ovarectomy and sham-ovarectomy group were thinner than that of normal group, when compared with the ovarectomy group ,the ganglion cell layer and optic nerve fibre layer increased significantly in the sham-ovarectomy group.3 Measurement of estrogen level of each rabbit in blood: There was no significant difference in the average level of estrogen of the sham-ovarectomy group before and after operation;the estrogen level of ovarectomy group after operation was remarkably lower than that before operation.4 Expression of Bcl-2 in the retina: There was significant difference in the expression of Bcl-2 at the corresponding time between each group.when compared with the ovarectomy group ,the expression of Bcl-2 in the sham-ovarectomy group increased significantly in the sham-ovarectomy group.5 Expression of Bax in the retina: There was significant difference in the expression of Bax at the corresponding time between each group.when compared with the ovarectomy group ,the expression of Bax in the sham-ovarectomy group decreased significantly in the sham-ovarectomy group.6 The expression of positive TUNEL cells in the retina: There was significant difference in the expression of positive TUNEL cells at the corresponding time between each group. when compared with the ovarectomy group ,the expression of TUNEL in the sham-ovarectomy group decreased significantly in the sham-ovarectomy group.7 The latency time of F-VEP: The latency time of F-VEP in the ovarectomy group and sham-ovarectomy group was significantly prolonged, which was longer than that of the normal group at the specified time after the formation of model,the latency time of the sham-ovarectomy was siginigicantly shorter compared with the ovarectomy.Conclusion:1 Ovarectomy and sham-ovarectomy are reliable methods in studying the effect of estrogen on eye diseases.2 The ocular hypertension model induced by injection of 3 g/ L carbomer into anterior chamber of rabbit is a stable ocular hypertension model.3 Chronic ocular hypertension induced the injury to retina and the apoptosis of retinal ganglion cells.4 Estrogen has protective effect on the retinal tissue during the course of chronic ocular hypertension, which relieves the trauma of retinal tissue and visual function in this course.5 The neuroprotective mechanism of Estrogen may be related to up- regulating the expression of Bcl- 2 protein, down- regulating the expression of Bax protein and inhibition of the expression of positive TUNEL cells.