Experimental Study On Neuroprotective Effect Of Memantine On The RGC Injured By Acute High Intraocular Pressure In The Rats

Objective:Glaucoma is one of the main reasons of blindness, in which intraocular hypertension result in injure of papilla optica and defect of visual field,and is characterized by optic nerve injury and loss of retinal ganglion cell(RGC). The final purpose of therapy is to prevent RGC from injury and protect visual function. Many factors play important roles in the occurrence of loss of retinal ganglion cell(RGC), lots of investigations suggested that loss of RGC is involed in excitoxicity of glutamate.It is reported that memantine is the noncompetitive NMDA receptor antagonist.The aim of the study is to provide a theory basis for clinical treatment of glaucoma through observing the pathomorphologic characteristics of the eye, the expression of caspase-3 and apoptosis of RGC and the effect of Memantine on the RGC of the cats after acute high intraocular pressure.Methods: 48 female Wistar adlult wistar rats which weighed from 200 to 300g were enrolled in our study and allocated to three groups:healthy healthy control group(A group)8 rats,acute ocular hypertension group(B group)20 rats and memantine treatment group(C group)20 rats.Briefly,the rats were anesthetized with intraperitoneal injections of 30mg/kg 3% sodium pentobarbital.After total application,the anterior chamber was punctured with a 5.5-gauge needle connected to a bottle containing normal saline(NS). Intraocular pressure was raised to 110 mmHg by elevating the saline container 146cm which was from the eye of the animal. The infusion needle was removed from the anterior chamber 60 minutes later. Reperfusion of the retinal vasculature was confirmed by fundus examination.Memantine was intraperitoneal injected in C group,NS was injected in B group,and intraocular pressure was not raised in A group. The rats were then euthanatized at 6 hours,24 hours,and 72 hours after ocular hypertension,and their eyes were enucleated to observe the pathomorphology characteristics of the eye and detect the expression of caspase-3 and the apoptosis of RGC by immunohistochemical method.Results:1,Ocular conditionIn B group and C group,1 hour after experimental eyes were affused cornea edema and turbidness, anterior chamber not bleeding;after 48 hours cornea's edema alleviation.In A group,eyes'cornea was transparence and anterior chamber was not bleeding.2,Conventional histopathology examinationIn B group, the edema of nerve fiber layer and inner plexiform layer was severity at the 6th hour after acute ocular hypertension,but their structures were still integral. The edema of retina was ingravescent and some cells of nerve fiber layer were denaturalized at the 24th hour. At the 72th hour,the edema of retina alleviated much more than it at the 24th hour,most nerve fiber layer showed vacuolar degeneration and the edema basically disappeared,nucleus was dissolved and dead, the structures was turbulent.At the 6,24,72th hour in C group, the edema of retina were slighter than those of the experiment group;at the 72th hour; the trauma of RGC layer decreased.In A group, the structures of retina were natural.3,The expression of caspase-3 in retinaNo specific staining was detected by using the caspase-3 antibody in the retina of A group. In B group and C group,there were positive staining cells in the RGC,positive staining materials were scattered in the cytoplasm with brown or light brown colors.A few of caspase-3 positive cells began to express after 6 hours in B group,and increased in 24 hours later,and then decreased in 72 hours.C group had the same trend with B group in each index,but the expressive intensity of each period was weaken inevidence. Compare both of them,there was statistics significance in difference between both of the groups in 6,24,72 hours after acute ocular hypertension. 4,TUNEL stainingThe positive cells TUNEL staining were not detected in A group.The positive cells of TUNEL staining were principle RGC. It started to a occurrence in 6 hours after reperfusion in B group increase strikingly in 24 hours,and then decreased in 72 hours. It also started to a occurrenc in 6 hours after reperfusion in C group, increased strikingly in 24 hours,and then decreased in 72 hours;while the positive expression in C group was lower than in B group. Compare both of them,there was statistics significance in difference between both of the groups in 6,24,72 hours after acute ocular hypertension.Conclusions:1,The high positive expression of Caspase-3 is detected in the acute high intraocular pressure injured RGC. It shows that the activation of the apoptic gene Caspase-3 was in relation to the apoptosis of RGC.2,The positive TUNEL staining RGC are detected early after injured by acute high intraocular pressure. It shows that acute ocular hypertension was an important agent in the apoptosis of RGC.3,The positive expression of Caspase-3 and the positive TUNEL staining RGC are much more slightly in C group than in B group. It shows that memantine restrain high expression of Caspase-3,and can play a key role in protecting the RGC after acute ocular hypertension injury.