Protective Effects Of Recombinant Human Erythropoietin On Injury Of Ratinal After Acute Ocular Hypertension In Rats

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 plan to make the SD rats model of acute intraocular hypertension (AIH) by increasing the intraocular pressure. Observe the expression of HIF-1αin retina. Study the effect of rhEPO or saline by intraperitoneal injection on expression of HIF-1αand count the positive cells of apoptosis in retina. To explore if rhEPO protect the optic nerve by decreasing the expression of HIF-1αin retina after acute ocular hypertension.Methods:1. Animals and modeling:Thirty-nine healthy SD rats were randomly divided into four groups:○1 Normal control group:3 rats(6 eyes)only puncture of anterior chamber without ocular hypertension.○2 experimental group:12 rats(24eyes)were created into acute intraocular hypertension (AIH) model by perfusing 0.9% normal saline into anterior chambers of rat's eyes . They were divided into 12h, 24h, 48h and 72h group according to the different time after AIH.○3 treatment group A: The method of dividing groups is similar to the above, each group has six eyes. We injected rhEPO 5000IU/kg (1ml) into their abdominal cavity.○4 treatment group B: The method of dividing groups is similar to the above. 12 rats(24 eyes)were createded into AIH model with same way, then normal saline(1ml) was injected into their abdominal cavity. There were 6 eyes in each group.2. Making the model of AIH:We elevate the intraocular pressure (IOP) by perfusing 0.9% normal saline into anterior chambers of rat's eyes. We can see the blood vessel of retinal was block with ophthalmoscope after pupils were dilated in successful models. We enucleated eyeballs of the rats of each group at the specified time after the formation of model and made paraffin slices.3. Pathohistology: We observed retinal pathological changes of every slice under optical microscope after performing HE staining.4. HIF-1αmeasurement with immunohistochemistry: That cytoplasm was stained brown or yellow was considered as positive expression of HIF-1αunder optical microscope. Degree of HIF-1αexpression was analyzed with technique of pathological image diagnosis (the 4th edition).5. Apoptosis measurement: That nucleus in the ganglion cell layer were stained brown was considered as positive expression of TUNEL under optical microscope. The ratio of positive RGCs number to ganglion cell layer length was considered as the exponent of apoptosis.6. Evaluation of visual function :We evaluate the optic nerve injury by flash visual evoked potential (fVEP).Results : 1. The change of retina histopathology: In experiment group, the thinningz of inner nuclear layer(INL) was seen at 12th hour after acute ocular hypertension; the number of RGCs decreased and the INL was in disorder at 24th hour; then the entoretina turn thin obviously at 72th hour. In treatment group A, the vacuolar degeneration and karyopycnosis were slighter than experiment group, and the number of RGCs was much more than experiment group. There was no obvious diference between treatment group B and experiment group.2. The expression of HIF-1α(foot print of HIF-1α): HIF-1αmainly appear in entoretina .The degree of brown staining in normal retina was very slight. In experiment group, the degree of staining was slight at the 12th hour in the entoretina; reached the peak at the 24th hour; reduced obviously at the 72th hour. The average gray density of experiment group was significantly lower compared with the normal group (P<0.01). In treatment group A, the trend of changes in HIF-1αexpression was similar to that of experiment group, but the average gray density was significantly higher (P<0.01); no distinguishable differences were found compared to normal group(P>0.05).In treatment group B, the trend of the expression of HIF-1αfollowed that of experiment group, when compared with normal group, the average gray density was significantly lower (P<0.05); no distinguishable differences were found between treatment group B and experiment group(P>0.05); it was lower when compared with treatment group A (P<0.01).3. 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 12th hour after acute ocular hypertension; reached the peak at the 24th hour; decreased at the 72th hour. In treatment group A, 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 B and experiment group(P>0.05); when compared with treatment group A, the exponent of apoptosis increased significantly in treatment group B(P<0.01).4. Detection of VEP: At the 24th hour after acute ocular hypertension, the latency time( P1) of experiment group was significantly longer compared with the normal group (P<0.01) . In treatment group A the latency time was siginificantly short compared with the experiment group (P<0.01). In treatment group B the latency time was significantly longer than treatment group A (P<0.05), and no distinguishable differences were found compared with experiment group (P>0.05).Conclusions:1. By perfusing 0.9% normal saline into anterior chambers of rat's eyes, we can established the model of AIH successfully.2. AIH induced the positive expression of HIF-1αand which maybe involved in the injury to retina.3. AIH induced the injury to retina and the apoptosis of retinal ganglion cells .4. rhEPO inhibited the expression of HIF-1α, decreased the positive expression of TUNEL and the losing of visual function by AIH. We can estimate that rhEPO is a new optic neuroprotective cytokine.