Experimental Study Of The Expression Of Matrix Metalloproteinase-2 In Oxygen-Induced Retinal Neovascularization Of Mouse And Inhibitory Effect Of Captopril On Retinal Neovascularization In The Mouse

ObjectiveTo explore the expression of matrix metalloproteinase-2 (MMP-2) in oxygen-induced retinal neovascularization of mouse and the correlation between the expression of MMP-2 and pigment epithelium derived factor (PEDF);and to evaluate the disturbing effect of Captopril, an inhibitor of angiotensin converting enzyme, in the growth of retinal neovascularization.MethodsPart I:Sixty 7-day-old C57BL/6J mice were randomly divided into normal control groupand hyperoxic group with thirty mice in each group.The norml control group were fed in the normal environment without any treatment and the hyperoxic group were exposed to 75% oxygen for 5 days and then to room air.The mice were sacrificed on the 17th day after birth and the eyes were enucleated to make sample. Adenosine diphosphate-ase(ADPase) stained retina flat-mounts was performed to assess the retinal vascular profiles, HE staining method was applied to count the number of new vascular cell nuclei and the expression of MMP-2 and PEDF was detected by immunohistochemical method.Part II:Thirty 7-day-old C57BL/6J mice were randomly divided into treated group and untreated group with fifteen mice in each group.These mice were exposed to 75% oxygen for 5 days and then to room air.The treated group had been injected captopril (5.4ml/kg), while untreated group had been injected the equal physiological saline by peritoneum for 5 days.The mice were sacrificed on the 17th day after birth and the eyes were enucleated to make sample.HE staining method was applied to count the number of new vascular cell nuclei and immunohistochemical for MMP-2 and PEDF was performed on retinas.ResultsPart I:ADPase-stained retina flat-mounts:The retina vessels of the normal control grouphave a fine radial branching pattern that extend from the optic nerve to the periphery.The retinal vascular pattern in the mice exposed to hyperoxia is characterized by retinal vessel obvious tortuous,bigger non-perfusion area and increased neovascularization.HE staining:There are less or no new vessels vascular endothelial cell nuclei breaking through the internal limiting membrane into vitreous (the mean value of nuclei of new vessels vascular endothelial cell is about 1.27±0.20 pieces in the each slice) of the normal control group than of the hyperoxic group (the mean value of nuclei of new vessels vascular endothelial cell is about 33.51±2.55 pieces in the each slice. t=10. 345, P<0.05).Immunohistochemical:There is weak protein expression of MMP-2 in the ganglion cell layer , the inner plexiform layer, some cells of the inner nuclear layer and the outer nucler layer in the normal control group. PEDF protein is mainly expressed in the nerve fiber layer, the ganglion cell layer layer, the inner nuclear cell layer,the photoreceptor cells layer and the RPE layer in the normal control group. MMP-2 protein is mainly expressed in the ganglion cell layer, the inner plexiform layer, the inner nuclear layer and the neovascularization breaking through the internal limiting membrane in the hyperoxic group. PEDF protein is faintly expressed in the nerve fiber layer,the ganglion cell layer layer , the inner plexiform layer,the photoreceptor cells layer and the RPE layer in the hyperoxic group.And there is an obvious negative correlation between the expression of MMP-2 and PEDF(r=-0. 825, P <0. 05).Part II:HE staining:The nuclei of endothelial cells were found to break through the inner retinal.Compared with the untreated group,the number of the nucllei of endothelial cell in treated group reduced significantly (32.43±0.45 vs 7.39±1.52 , t=9. 238, P <0. 05).Immunohistochemical:In the untreated group,the expression of MMP-2 was mainly showed in the ganglion cell layer, the inner plexiform layer, the inner nuclear layer and the neovascularization breaking through the internal limiting membrane;There is weak protein expression of PEDF in the nerve fiber layer,the ganglion cell layer, the inner plexiform layer ,the photoreceptor cells layer and the RPE layer in the untreated group.In the treated group,there are no expression of MMP-2 in the inner nuclear layer and the outer nucler layer and little expression only in intracytoplasm of some ganglion cell layer and the inner plexiform layer. PEDF protein is mainly expressed in the nerve fiber layer,the ganglion cell layer layer, the inner nuclear layer,the photoreceptor cells layer and the RPE layer in the treated group.Compared with the untreated group, the protein expression of MMP-2 and PEDF of the treated group was different (P<0. 05).Conclusion1.This animal model of oxygen-induced mouse retinal neovascularization is similar to retinopathy of prematurity(ROP).Because of its characteristic of repeatability and fix quantity,it is useful for the study of pathogenesis of retinal neovascularization and therapeutic intervention for ROP.2.There exist higher expression of MMP-2 and lower expression of PEDF in the oxygen-induced retinal neovascularization of mouse,both showing significant negative correlation,and both maintaining retinal neovascularization.3.Intraperitoneal injection of captopril may block the retinal neovascularization in the oxygen-induced mouse model and may provide an effective method for preventing ROP.