| Lots of evidences have shown that Copolymer-1(Cop-1) has the ability of triggering the autoimmunity reaction, resulting in the neuroprotective efficacy to the CNS and optic nerve. Studies have indicated that Cop-1could induce T-cell-mediated protective autoimmunity and played neuroprotective role in glaucoma neural injury. But the autoimmune response evoked by Cop-1is complex, serial and ordered, and possibly activation of T-cells is just an initial response. In the protective autoimmunity induce by Cop-1in the CNS, microglia were activated by Cop-1specific T cells (Tcop-1) and played the neuroprotective role. Thus we hypothesize that the microglia in retina were also activated by Cop-1specific T cells in model of glaucoma, which can play a critical role in protective autoimmune response.In our research, we attempt to observe the expression and activation of microglia and macrophages in the retina in autoimmune response evoked by Cop-1in a rat model of glaucoma. We also try to examine the possible interaction between Tcop-1and microglia and the correlation between microglia and RGCs survival, to probe into the possible mechanisms through which microglia play its role.In our study, we found that microglia and macrophages were accumulated and activated in the retina of the rat model of glaucoma immunized by Cop-1, playing a neuroprotective role to RGCs survival. Tcop-1, rather than the direct impact of Cop-1, played a critical role in microglia activation. The activated microglia could secret a series of cytokines (such as TNF-a, BDNF and IL-10), which might be a probable mechanism that microglia played its neuroprotectino function. Part I The changes of microglia/macrophages and RGCs survival in the retina of a normal rat immuzined by Cop-1Aims:To investigate the morphological and quantitative changes of microglia and macrophage in the retina of a normal rat immunized by Cop-1and study the RGCs survival in retina.Methods:48female Wistar rats were divided into two groups randomly:24rats were immunized by Cop-1(Cop-1group) and the other24rats were immunized by PBS (PBS group). Another4Wistar rats without any treatment were used as blank control. The activation and distribution of microglia and macrophages were observed by immunohistocytochemistry after immunization. RGCs in retina were labeled by2%FluoroGold injection into the superior colliculus. Seven days later the retina stretched preparation were made. RGCs were observed under microscope and the RGCs number was counted. All data collected were compared among groups using T test or unequal T test in randomized block design. A P value of≤0.05was taken as statistical significance.Results:There were activation of microglia and macrophages in both groups. But the numbers of microglia and macrophages in Cop-1group on the3rd, the7th, the10th, the17th and the24th day after immunization were43.14±9.27/mm2,34.35±4.35/mm2,38.25±9.53/mm2,23.11±5.16/mm2and24.94±3.82/mm2, greater than those in PBS group (each P<0.05). The mean RGCs counts at all observation time were more than those of PBS group, and on the24th day after immunization, the RGCs survival count was2583.61±339.70/mm2, statically higher than that of PBS group (P=0.0198).Conclusion:Microglia and macrophages were accumulated and activated in the retina of the normal rat after immunized by Cop-1, and reducing RGCs apoptosis was also observed at the same time, suggesting the neuroprotective effect of Cop-1. Part Ⅱ The changes of microglia and macrophages in the retina of a rat glaucoma model immuzined by Cop-1Aims:To investigate the morphological and quantitative changes of microglia and macrophage in the retina of a rat model of glaucoma immunized by Cop-1and speculate the relation between RGCs survival and activated microglia and macrophage in retina.Methods:64female Wistar rats were used to establish glaucoma model. They were divided into2groups randomly,32rats were immunized by Cop-1(Cop-1group) and32rats were immunized by PBS (PBS group). Another4Wistar rats without any treatment were used as blank control. The activation and distribution of microglia and macrophages were observed by immunohistocytochemistry and Western-blot was used to measure the MHC-Ⅱ expression on microglia and macrophages in the retina on the observation days. Spearman correlation study was made to describe the relevance between the microglia count and RGCs count. A P value of≤0.05was taken as statistical significance.Results:The numbers of microglia and macrophages in the retina increased in the experimental eyes and they reached the summit on the7th day both in Cop-1and PBS groups (105.28±7.87/mm2and82.14±3.47/mm2respectively). The numbers of microglia and macrophages in Cop-1group were greater than those in PBS group on the7th and10th day (both P<0.05). With the prolongation of the observation time, the numbers of microglia and macrophages in the experimental eyes decreased gradually and didn’t show statistical significance within groups. The results of Western-blot also showed the activation of microglia in Cop-1group. Spearman correlation study showed that in Cop-1group the microglia count and RGCs count were positively correlated (coefficient=1.0, P<0.001).Conclusion:Microglia and macrophages were accumulated and activated in the retina of the rat model of glaucoma immunized by Cop-1, playing a neuroprotective role to RGCs survival. Part III The activation of microglia derived from retina by Cop-1specific T cells (Tcop-1)Aims:To investigate the morphological and functional changes of microglia derived from retina after co-culture with Cop-1specific T cells (Tcop-1) and speculate the relation between Tcop-1and activated microglia and the probable mechanisms of activated microglia play its neuroprotective function.Methods:The T-cell line (Tcop-1) was generated from draining lymph node cells obtained from Wistar rats immunized with Cop-1emulsified in CFA. Microglia was produced from retina of neonatal Wistar rats (2-3days old). Then co-culture of Tcop-1and microglia (Tcop-1+MG) was made in vitro. The co-culture of normal T cells and microglia (Tcell+MG group), the culture of Cop-1stimulated microglia (Cop-l+MG group) and the culture of Tcop-1(Tcop-1group) were made as controlled groups. The morphological and amount changes of microglia were observed. After co-culture of12h, the secretions of TNF-a, the BDNF and the IL-10in the supernatant were measured by ELISA method. One-way ANOVA and Boffironni method were used in the statistical analysis. A P value of≤0.05was taken as statistical significance.Results:Co-culturing of Tcop-1and microglia in vitro proved that microglia were activated with increased number and more ameboid shaped cells, while Cop-1+MG group and Tcell+MG group didn’t show significant activation of microglia. After co-culture for12h, the secretion of TNF-a, BDNF and IL-10were the highest in Tcop-1+MG group (50.16±9.60pg/ml,437.42±5.01pg/ml and67.12±10.06pg/ml respectively) and were statically higher than the other controlled groups (P<0.001, P<0.05and P<0.05respectively).Conclusion:In vitro study further proved that the microglia derived from retina were activated after Cop-1stimulation, that Tcop-1, rather than the direct impact of Cop-1, played a critical role in microglia activation. The activated microglia could secret a series of cytokines (such as TNF-α, BDNF and IL-10), which might be a probable mechanism that microglia played its neuroprotectino function. |
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