The Expression Of Peroxynitrite And The Effect Of Puerain In Retina Of Rabbits With Acute Ocular Hypertension

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. The final purpose of therapy is to prevent RGCs from injury and protect visual function. Many factors play important roles in the occurrence of glaucoma. Elevated intraocular pressure not only can interrupt the axonal transport of RGCs, but also can make RGCs degenerate and cause apoptosis. With the profound study on nitric oxide (NO), peroxynitrite (ONOO-) was found to play an important role in the injury process of NO. ONOO-, a strong oxide, is the production of NO and superoxide (O2-). Neufeld found excessive nitrotyrosine (foot print of ONOO-) in patients'retina with glaucoma, but the underlying mechanism that ONOO-cause injury to RGCs remains unclear. This study was designed to investigate the expression and injury mechanism of ONOO-in retina with acute ocular hypertension and evaluate the effect of puerarin, in order to provide valuable theories for studying the mechanism of injury and protection. Methods: 1 Animals and model: Thirty-three New Zealand white rabbits were randomly divided into four groups: ⑴Normal control group(six eyes); ⑵Experiment group: It was divided into 6h, 12h, 24h and 72h groups according to the different time after the acute ocular hypertension, each group has six eyes; ⑶Treatment groupⅠ:The method of dividing groups is similar to the above. We injected puerarin(50mg/kg)through the vein on rabbits'ears 12 hours and 10 minutes before making model, and giving puerarin of equal quantity every 5 hour after making model. ⑷Treatment groupⅡ: The method of dividing groups is similar to the above. We injected vitamin C (VitC)(40mg/kg) through the vein on ears at the same time as above. The experimental model of acute ocular hypertension was induced by perfusing normal saline into anterior chamber. We enucleated eyes of each group at the specified time after the formation of model and made paraffin slices. 2 Histopathology: We observed retina pathological chan-ges of every slice under optical microscope after performing HE staining. 3 Apoptosis measurement with TUNEL: Positive cells in the ganglion cell layer were observed under optical micro-scope (cell was positive whose nuclear was stained brown). Four visual fields were randomly elected from every slice under microscope. The ratio of positive RGCs number to ganglion cell layer length was considered as the exponent of apoptosis. 4 NT (foot print of ONOO-) measurement with immun-ohistochemistry: That cytoplasm was stained brown wasconsidered as positive expression of NT. Degree of NT expression was analyzed with technique of pathological image diagnosis (the 4th edition). Results: 1 The change of retina histopathology: In experiment group, the edema of nerve fiber layer and inner plexiform layer was severity at the 6th hour after acute ocular hypertension; RGCs showed vacuolar degeneration and nucleus pycnosis at the 12th hour; the edema disappeared and nerve fiber layer turned thin and atrophied at the 24th hour; the number of RGCs decreased. In treatment group Ⅰand Ⅱ, the edema, the vacuolar degeneration and nucleus pycnosis of retina were slighter than those of the experiment group, and the number of RGCs was much more than that of the above. 2 The expression of positive TUNEL cells: No positive TUNEL cell was present in normal retina. In experiment group, apoptosis began to appear in ganglion cell layer and inner plexiform at the 6th hour after acute ocular hypertension; increased at the 12th hour; reached the peak at the 24th hour; decreased at the 72th hour. In treatment groupⅠ, the expression of apoptosis followed the trend of the experiment group, but the exponent of apoptosis was less than that of the above significantly(P<0.01). There was no significant difference in the exponent of apoptosis at the corresponding time between treatment group Ⅱand experiment group(P > 0.05); when compared with treatment groupⅠ, the exponent of apoptosisincreased significantly in treatment groupⅡ(P<0.01). 3 The expression of NT(foot print of ONOO-): NT mainly appear in internal limiting, nerve fiber layer and ganglion cell layer. The degree of brown staining in normal retina was very slight. In experiment group, the degree of staining was slight at the 6th hour in the layers as above; increased with time; reached the peak at the 24th hour; reduced at the 72th hour. The average gray density (average optical density) of experiment group was significantly lower (higher) compared with the normal group(6h:P<0.05,others:P<0.01). In treatment groupⅠ, the trend of changes in NT expression was similar to that of experiment group, but the average gray density (average optical density) was significantly higher (lower)(P < 0.01); no distinguishable differences were found compared to normal group(P > 0.05). In treatment group Ⅱ, the trend of the expression of NT followed that of experiment group, when compared with normal group, the average gray density (average optical density) was significantly lower (higher)(P<0.05); no distinguishable differences were found between treatment group Ⅱand experiment group(P>0.05); it was lower (higher) when compared with treatment groupⅠ(6h:P<0.05,others: P<0.01). Conclusions: 1 The method of perfusing normal saline into anterior chamber can elevate intraocular pressure of rabbit's eye sharply, and which established the model of acute ocular hypertension...