Neuro-protection Of Retinal Stem Cells Transplantation Combined With Copolymer-1Immunization On Retinal Ganglion Cell Of Glaucoma Model In Rat

Chapterl Establishment of glaucoma animal model and analysis of retinal stem cells in ciliary margin of the adult rat eye in vitroAIM:To develop a glaucoma animal model and culture the retinal stem cells(RSCs) of the adult rat eye in vitro.METHODS:A diode laser with wavelength of532-nm was used to burn directed at three of the four episcleral veins and at270°of the limbal plexus, the rat model with chronic glaucoma was established. Explants of ciliary margin of the adult SD rat eye were cultured in serum-free medium containing bFGF(Basic fibroblast growth factor), EGF(Epidermal growth factor) and B27. The phenotypes of cultured cells were identified by immunohistochemical analyses.RESULTS:21rat models with intraocular hypertension were obtained successfully. The peak value of IOP was at d14and the mean IOP of experimental eyes was31.6±3.2mmHg, the control group was16.8±1.7mmHg(P<0.001). The mean IOP of experimental eyes was no difference in each group at d21. The majority of cells in the sphere were BrdU immunoreactive and expressed the mark of RSCs(Nestin). Subcultured cells differentiated and expressed neuronal marker(NSE), astrocytic marker(GFAP),respectively.CONCLUSION:The rat model caused by a laser photocoagulation to the episcleral veins represents a useful and efficient experiment glaucoma model. The RSCs can generate sucessfully from the ciliary margin of the adult rat eye in vitro. Chapter2IFN-y expression after RSCs transplantation combined with COP-1immunization treatment in glaucoma model ratAIM:To explore the effect of immunization with copolymer-1(COP-1) and retinal stem cells (RSCs) transplantation on interferon-gamma (IFN-y) levels in the rat experimental glaucoma model. To understand the mechanism of combined treatment in glaucoma.METHODS:An experimental glaucoma was induced by532-nm laser photocoagulation on the right eye of SD rats and the left eye as control. Six groups of SD rats were treated as follows:(1) rats were vaccinated with COP-1emulsified in CFA after laser-induced ocular hypertension and received RSCs marked with Lentivirus-GFP via posterior vitreous cavity injection seven days later (COP-1/RSCs);(2) rats were vaccinated with COP-1and were injected with PBS (emulsified in CFA) seven days later (COP-1/PBS);(3) rats were injected with PBS in CFA and transplanted with RSCs marked with Lentivirus-GFP seven days later (PBS/RSCs);(4) rats were injected with PBS at both times (PBS/PBS);(5) glaucoma model group:laser-induced ocular hypertension only; and (6) a normal control group-not any treatment. The concentration of IFN-y in aqueous humor (AH) and serum were measured by enzyme-linked immunosorbent assay (ELISA) in each of the six groups. Retinal ganglion cells (RGCs) survival was assessed by quantifying apoptosis using Hoechst staining. Immunohistochemical analyses of frozen sections and whole-mount retina were performed on RSCs Transplanted Retinas.RESULTS:The concentrations of IFN-γ in AH and serum of the RSCs/COP-1treated group were determined to be2371.9ng/L and710.9ng/L respectively, which were significantly lower than those in the other treated groups (P<0.05). The RSCs/COP-1group had lower number of apoptotic RGCs than the other groups.(P<0.05). The transplanted GFP+-RSCs were scattered throughout the whole-mount glaucomatous retina and integrated into the nerve fiber layer (NFL) and retinal ganglion cell layer (GCL).CONCLUSION:Combined treatment of RSCs transplantation and COP-1may reduce levels of IFN-y in AH and serum of the SD glaucoma rat, and prevente apoptosis of RGCs. It may be related to synergistic effects between RSCs transplantation and COP-1immune modulation. The transplanted GFP+-RSCs were integrated into the rat retinas.Chapter3Synergistic neuro-protection of RSCs transplantation and COP-1immunization in an experimental model rat of glaucomaAIM:To explore the mechanism of the neuroprotection of rentinal stem cells (RSCs) transplantation and copolymer-1(COP-1) in an experimental model rat of glaucoma.METHODS:An experimental glaucoma was induced by argon laser photocoagulation on the right eye of SD rats. Four groups of SD rats were treated as follows:(1) rats were vaccinated with COP-1emulsified in CFA after laser-induced ocular hypertension and received RSCs via posterior vitreous cavity injection seven days later (COP-1/RSCs);(2) rats were vaccinated with COP-1and were injected with PBS (emulsified in CFA) seven days later (COP-1/PBS);(3) rats were injected with PBS in CFA and transplanted with RSCs seven days later (PBS/RSCs);(4) rats were injected with PBS at both times (PBS/PBS). The expression of brain-derived neurotrophic factor (BDNF) and insulin-like growth factor I(IGF-I) were detected by immunohistochemical staining, Real-time RT-PCR, and Western blot. RGCs survival was assessed by TUNEL staining. The number of RGCs was counted by immunohistochemical staining on freezen section of retinas.RESULTS:The expression of BDNF and IGF-I in the RSCs/COP-1group were significantly higher than that in other groups (P<0.05). In addition, the number of the apoptotic RGCs in the RSCs/COP-1group were notably lower than that in other groups (P<0.05), and the number of RGCs in the RSCs/COP-1group were higher than that in other groups (P<0.05).CONCLUSION:Combined treatment of RSCs transplantation and COP-1may increase levels of BDNF and IGF-I of the SD glaucoma rat, prevente apoptosis of RGCs and provide Neuro-protection on RGCs.