| Objectives:1.To establish the model of optic nerve clamping in rats at different time points;2.To observe the expression of inflammatory factors and glial cells after optic nerve injury;3.To explore the role of retinal inflammatory factors and glial cells after optic nerve injury;4.To provide new treatment ideas and methods for optic nerve injury.Methods:99 rats of adult SPF grade SD rats were randomly divided into normal group(n=3)?surgery group(n = 48)and sham group(n = 48).In surgery group,optic nerve was separated and clamped to make optic nerve contusion model,but the optic nerve of sham group was only separated not clamped.The rats in surgery group and sham group were sampled at the time point after injury,1 d group,3 d group,5 d group,7 d group(12 rats in each subgroup).The protein levels of IL-1? and TNF-a in the retina was determined by Western blot(n = 4).Immunofluorescence staining was used to observe the optic nerve and retinal glia cell at different time points after optic nerve injury(n = 4).Immunofluorescent localization were used to observe the co-localization of TNF-a,IL-1? and glia cells in the retina(n = 4).Results:The data of Western blot showed that the expression of IL-1? and TNF-a was changed after TON,and IL-1? reached the peak at 3 d.The difference of IL-1? in 3 day was statistically significant compared with the sham group(p<0.05),but the difference at other time points was not significant compared with the sham operation group(p>0.05).The expression of TNF-a increased significantly at one day after injury,and reached a peak at 3 days.5-7 days slightly decreased but the difference compared with the sham operation group was also statistically significant(p<0.05).The results of immunofluorescence staining of glial cells on the optic nerve showed that the structure of the cells on the optic nerve of the optic nerve was irregular,the distribution was not uniform,and the cells of the clamping position were missing.the fluorescence signal expression in microglial was enhanced significantly than the sham group after optic nerve injury,and the distribution of the number of microglia was increased;the astrocytes compared with the sham group,the expression of GFAP fluorescence signal was also enhanced.The axons were getting longer.The results of immunofluorescence showed that the morphology?number and distribution of glia cells changed after TON.Microglia was mainly distributed in GCL layer and IPL layer.The microglia began to show activation at one day after injury,microglia's synapses became shorter and shorter,the secondary branch decreased or disappeared simultaneously.Microglia at 5-7 days began to appear ameboid,also the number of activated microglia was become more and more,and had significantly difference compared with sham operation group(p<0.05).Astrocytes mainly distributed out of the GCL layer.The astrocytes became hypertrophy,the axons were thickened and the GFAP fluorescence was enhanced.Immunofluorescence co-localization exhibited that TNF-a and IL-1? in the retina was significantly increased after TON,mainly secreted by the microglia of the retina.The expression of IL-1? secreted by astrocytes was evident at 3 day,but no TNF-a was detected in retinal astrocytes.Conclusions:1)The expression of IL-1? and TNF-a in the retina was increased after optic nerve injury.2)Retinal microglia and astrocytes also on optic nerve were activated after optic nerve injury.3)Both microglia and astrocytes can espress IL-1? after being activated,TNF-?in the retina is mainly derived from microglia. |
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