| The loss of vision induced by glaucoma influence human’s livelihood and productivity seriously. Reducing the intraocular pressure is the primary treatment for glaucoma, while some kinds of methods to protect the optic nerve could be used as subordinate cures. Although the intraocular pressure was under controlled, the losing of visual field of some patients were going on.The past few years have seen evidence which supports the view that the auto-immune induced by the optic neural damage has the both opposite impacts to the neural cells. The auto-immune would be activated once while the optic nerve was injured, the immunity which was too excessive or too weak may hurt the nerve or weaken the neuroprotection. The moderate autoimmune is obligatory for the neural protection.The protective autoimmune which is the internal opposing to injury has the compositive effects to the nerve. The effects are the ideal ways which are close to physiology condition and have less side-effect. Myelin basic protein(MBP) was confirmed could induce the autoimmune which was neuroprotective in the brain、spain、and optic nerve crush models, but MBP could induce the autoimmunity diseases commonly.Copolymer-1(Cop-1) is an artificially synthesized analogue for myelin basic protein(MBP).It was shown to be neuroprotective in rodent models of acute or chronic neurodegenerative conditions when administered together with an adjuvant, while it could not induce EAE.In this study Cop-1 was used to induce autoimmune in the glaucoma models and the morphological and expressional changes of Th1 cells and Th2 cells in the retina were observed to explore the possible neuroprotection role played by Th1/Th2; the interaction between Cop-1 special T cells and microglia for RGCs was testified, and some kinds of cytokines were detected for exploring the probable mechanisms for copolymer-1-induced neuroprotection. Part I The expressional changes of Thl and Th2 cells in the retina of a rat glaucoma model vaccinated by Cop-1Objective:To investigate the morphological and expressional changes of Thl cells and Th2 cells in the retina of a rat model of glaucoma vaccinated by Cop-1 and explore the possible neuroprotection role played by Thl/Th2.Method:Randomized controlled trials. The aqueous outflow from the rats’right eyes was blocked by ligation of three of the four episcleral veins.There are 48 rats with elevated IOP immunized by Cop-1 (Cop-1 group),48 rats with elevated IOP immunized by PBS (PBS group), and 10 rats without any treatment (normal group). Experimental rats were immunized with Cop-1/PBS emulsified in a total volume of complete freund’s adjuvant 0.4ml. The immunization was administered subcutaneously at the base of the trail. The immunohistochemistry methods were used to test the distribution and activation of Thl and Th2 cells in the retina on the 3rd,7th,10th,17th,24th and 31th day after the inmmunization respectively of each group. Western-blot was selectively performed according to the results of the immunohistochemistry in order to verify if there was a similar variation of the expression of IL-4 protein in the retina.Result:The results of immunohistochemical tests showed the numbers of Thl cells in the retina of the two experimental groups (Cop-1 group=137±15/mm2,PBS group=139±15/mm2)were similar to those of normal group (137±15/mm2) on the 3rd day after immunization. On the 7th day, the Thl cells numbers reached the summit in both Cop-1 (216±21/mm2)and PBS group (194±27/mm2), while without statistical significance between the two groups (P>0.05). The numbers of Th2 cells in the experimental groups (Cop-1 group=116±19/mm2,PBS group=114±15/mm2)were greater than those of normal group (28+17/mm2) on the 3rd day after immunization and reached the summit on the 7th day with statistical significance (Cop-1 group=300±28/mm2,PBS group=129±27/mm2) (P<0.01). With the prolongation of experimental period, the number of Th2 cells in the retina of Cop-1 group decreased gradually but still greater than that of PBS group at the following time point (p<0.05). The Western-blot result showed that the expression of IL-4 in Cop-1 group (1.91±0.05) was significantly higher than that of PBS group (0.51±0.04) from the 3rd day of observation and reached the highest on the 7th day (Cop-1 group=2.11±0.06,PBS group=0.57±0.05). Then IL-4 expression in COP-1 group decreased gradually but still represented statistical significance until the 31th day after immunization compared with PBS group (p<0.001).Conclusion:The activation and accumulation of Th2 cells were found in the retina of a rat model of chronic glaucoma immunized by Cop-1, thus Th2 cells may play a critical role in neuroprotective autoimmunity response induced by Cop-1.PartⅡCop-1 special T cell-microglia interaction to protect the RGCsObjective The apoptosis rate of rat retinal ganglion cells (RGCs) was detected to explore the effect of Cop-1 specific T cell-microglia to the RGCs.Methods Ten days after 0.2mg Copolymer-1 (Cop-1) or phosphate buffered solution(PBS) was injected into the rat s’hind foot pads, their draining lymph nodes were surgically removed and Tcop-1,TPBS were respectively cultured. The retina of the Wistar rats, which were born in the first 2-3days,was taked for culturing the microglia. After 72 hours in cultured RGCs and the supernatants of Tcop-1-microglia、T cell-microglia and microglia groups, TUNEL method was used to observed the changing of apoptosis rates in RGCs. RT-PCR was used to detect the mRNA variation of Caspase-3, Caspase-8. All data collected were compared among groups using multiple comparisons test and Boffironni (one-way ANOVA, Bonferroni test). A P value of≤0.05 was taken as statistical significance.Results TUNEL-positive cells of RGCs were lower in the culture Tcop-1-microglia supernatant (25.36%) than Tcop-1 group (31.03%) and microglia group (61.54%).The levels of caspase-3 and caspase-8’s mRNA of RGCs decreased accordingly in the Tcop-1-microglia group (6.22×10-3、1.26×10-5), were lower than microglia group (6.86×10-3、1.53×10-5) (P=0.136, P=0.001) and T cell-microglia group (9.04×10-3.12×10-5) (P=0.04, P<0.001)Conclusion The supernatant of Tcop-1-microglia could postpone the apoptosis of RGCs. Part III The cytokine production in Cop-1 special T cell-microglia interactionObjective:Some kinds of cytokines of the supernatants were detected for exploring the probable mechanisms of Cop-1 specific T cell-microglia protective interaction for RGCs.Methods Ten days after 0.2mg Copolymer-1 (Cop-1) or phosphate buffered solution(PBS) was injected into the rat s’hind foot pads, their draining lymph nodes were surgically removed and Tcop-1,TPBS were respectively cultured. The retina of the Wistar rats, which were born in the first 2-3days,was taked for culturing the microglia. The supernatant of T cell、Tcop-1 -microglia、T cell-microglia、microglia and Tcop-1 groups was collected after 12 hours,24 hours,48 hours, and examined by enzyme-linked immune sorbent assays(ELISAs) for IGF-1,BDNF and IL-10, TNF-αlevels. All data collected were compared among groups using multiple comparisons test (One-way ANOVA, Bonferroni test). A P value of≤0.05 was taken as statistical significance.Results After culturing for 12 hours, the secretory volume of IGF-1, BDNF TNF-αand IL-10 secreted by the Tcop-1-microglia supernatant (281.66 pg/ml、437.52 pg/ml、50.16 pg/ml、67.12 pg/ml) were higher than the TPBS group (171.50 pg/ml、198.11 pg/ml、18.25 pg/ml、18.09 pg/ml) and microglia group (219.58 pg/ml、31.14 pg/ml、15.03 pg/ml、10.87 pg/ml) evidently; After 24 hours, the secretory volume of BDNF was highest in the Tcop-1-microglia supernatant (495.52 pg/ml), and there were statistical differences with the control groups(P<0.05); After culturing for 48 hours, the secretory volume of IGF-1、IL-10 was highest in the Tcop-1-microglia group (1005.574 pg/ml、184.88 pg/ml),which were higher than the Tcop-1 group (343.54pg/ml、57.04 pg/ml), microglia group (233.45 pg/ml、16.11 pg/ml), TPBS-microglia group(61.90 pg/ml、51.99±3.96 pg/ml), there were statistical differences with these control groups.Conclusion:The interaction between Cop-1 specific T cells and microglia could make the secretory product of some cytokines changed. This appearance indicates that the interaction between Cop-1 specific T cells and microglia could be one of the mechanisms of Cop-1’s protective action for RGCs. This effect is evidently accompanied by a increasing of some neurotrophic factors and the regulating of some anti- and pro- inflammatory cytokine. But the detailed mechanisms are still unknown, which need to be studied intensively. |
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