GDNF gene therapy for retinal injuries

• Main Authors: WEI-CHI WU, 吳為吉
• Other Authors: YEOU-PING TSAO
• Format: Others
• Language: zh-TW
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/46801113242450168071

博士 === 長庚大學 === 臨床醫學研究所 === 91 === ntroduction Neuroprotection describes the process whereby an agent interacts with specific cellular components to attenuate the process of death. As retina is a part of the central nervous system and its cells are terminally differentiated, it cannot be regenerated when injured. Therefore, current treatments for retinal diseases largely aim at stopping the disease progressions. Tremendous progress has been made in the past decade in delineating the molecular bases of ocular diseases, which allows for the development of rational strategies to resolve them. One of the most important break-through is the advent of gene therapy, as it offers many benefits unparalleled by traditional therapies. Although there has not, as yet, a demonstration of cure using gene therapy, proof-of-principle has been established in a number of animal models for ocular diseases. As attempts to “cure” retinal injuries with retinal stem cell transplantation or retinal prosthesis are largely inconclusive and their clinical value being uncertain, our current study intends to achieve neuroprotection against retinal injuries via gene therapy. We transfected retinal cells with recombinant adeno-associated virus (RAAV) carrying a gene for glial cell-line derived neurotrophic factor (GDNF), in an attempt to achieve neuroprotection. We evaluated this gene therapy’s protective potential against retinal detachment (RD)-induced and retinal ischemia-reperfusion (I/R)-induced injuries. We also examined the long-term effects of this therapy and its possible underlying mechanism. Experiment 1: Long-term safety of GDNF Gene Therapy We examined the long-term effects of GDNF gene therapy by comparing 4 criteria between the treated eyes and the control eyes. Sprague-Dawley (SD) rats’ retinal cells were transfected with RAAV carrying GDNF gene via intravitreal injection. The right eyes served as the experimental group while the left served as the control. First, retinal morphology was analyzed via microscopy 1 year after transfection. Second, retinal inflammation was assessed using antibodies against phagocytes. Third, Retinal ganglion cell (RGC) were labeled with neuro-tracer dye to allow for its quantification. Finally, retinal function was evaluated with electroretinogram (ERG). In this experiment, we found that neither retinal morphology nor RGC counts were significantly affected by GDNF gene therapy. In addition, immunohistochemical staining detects no additional inflammatory cells in the experimental group. Most importantly, ERG studies demonstrated that b-wave was not significantly decreased in the transfected eyes. Hence, we concluded that long-term expression of GDNF poses insignificant toxic effects to retina. Experiment 2. GDNF Gene Therapy’s Protective Potential against Retinal Detachment (RD)-induced photoreceptor injury In this study, we examined GDNF gene therapy’s protective potential against injuries induced by RD. RAAV carrying GDNF gene and RAAV carrying E. coli LacZ gene were injected into subretinal space in the right and left eyes of Lewis rats respectively to establish the experimental group and the control. Three weeks following gene delivery, we induced RD through subretinal injection of high-density vitreous substitute. The synthesis and accumulation of GDNF within retina was examined 3 weeks after transfection via immunohistochemical staining and enzyme-linked immunosorbent assay (ELISA) respectively. Subsequently, the lengths of photoreceptors outer segments (OS) and the thickness of retina’s outer nuclear layers (ONL) were used as evaluation criteria for photoreceptor integrity. Photoreceptor apoptosis was studied using TdT-dUTP terminal nick-end labeling (TUNEL) 2 days after RD. Finally, Müller cell activation was observed immunohistochemically with antibodies against glial fibrillary acidic protein (GFAP) 28 days after RD induction. Immunohistochemical analysis and ELISA both demonstrated successful gene delivery. Although photoreceptor OS degeneration and ONL thinning were noted in both experimental and control groups, it was observed that the experimental group sustained less severe damages both 7 days and 28 days after RD. More over, GDNF gene therapy-treated eyes showed statistically less apoptosis than control eyes in the photoreceptor layer (p=0.043). Lastly, Müller cell activation was less prominent in the experimental group, indicating less scar formation. These results have led us to conclude GDNF gene therapy as a good method to protect photoreceptors from RD-induced degeneration. In addition to preserving OS and ONL integrities, GDNF gene therapy may also exert its protective effect by attenuating photoreceptor apoptosis. Experiment 3. GDNF Gene Therapy’s Protective Effect against Retinal Ischemia-Reperfusion (I/R)-induced injury We assessed GDNF gene therapy’s protective potential against retinal I/R-induced injuries. RAAV carrying GDNF gene and RAAV carrying E. coli LacZ gene were injected intravitreally into the right and left eyes of SD rats respectively to establish the experimental and control groups. Subsequently, ischemic injury was induced 3 weeks following gene delivery. The synthesis and accumulation of GDNF within retina were studied 3 weeks after gene delivery via immunohistochemical staining and ELISA. The neuroprotective effects were evaluated1 week after reperfusion, using inner retina thickness and RGC counts as evaluation criteria. Retinal function study was performed with ERG. Finally, TUNEL method was used to assess RGC apoptosis 6 hours after reperfusion. Immunohistochemical analysis and ELISA both confirmed successful GDNF transfection. We also found that inner retina thickness and RGC counts were both better preserved in rAAV-GDNF-treated eyes than in rAAV-LacZ-treated eyes 7 days after reperfusion (P=0.038 and P=0.003, respectively). In addition, rAAV-GDNF-treated eyes demonstrated larger b-wave amplitudes than rAAV-LacZ-treated eyes 7 days after reperfusion (P=0.003). Finally, rAAV-GDNF-treated eyes demonstrated statistically less RGC apoptosis (P=0.021). We concluded that GDNF gene therapy can effectively protect retina against I/R injury and may exert its protective role through the attenuation of retinal cell apoptosis. Experiment 4. Mitogen-Activated Protein Kinase (MAPK) Pathway’s Involvement in GDNF’s Neuroprotective Mechanism In this section, we compared retinas at various time periods after their detachments in eyes that underwent GDNF gene therapy and control eyes. Immunohistochemical study was used to identify phospho-MAPK positive (active form of MAPK) cells in retina at various time periods. Our results indicated that there were significantly more phospho-MAPK positive cells in retinas transfected with RAAV carrying GDNF as compared to the control group (p<0.05). However, as the duration of RD increased, the number of phospho-MAPK-positive cells decreased in both groups. It may be possible that GDNF’s neuroprotective mechanism against RD is related to the activation of MAPK pathway. Conclusion Recombinant adeno-associated virus can effectively transfect retinal cells with GDNF gene for over one year without significant toxic effects. GDNF gene therapy can protect retina from injuries induced by retinal detachment and retinal ischemia-reperfusion. The neuroprotective effect of GDNF may be related to MAPK activation. Further studies are needed to elucidate the underlying mechanisms of GDNF gene therapy.