Protection Of Retinal Ganglion Cells In Diabetes Rats Induced By AAV Mediated BDNF Expression

Objectives:To construct recombinant adeno-associated virus of BDNF and GFP, determine viral titer,and conduct intravitreal injection of rats in order to monitor its expression after retinal infection. It also tried to investigate the feasibility for the treatment of diabetic retinopathy via the introduction of BDNF gene to the adeno-associated virus.Methods:1.TO clone target gene segment of CBA promoter,construct the recombinant plasm id of pAAV-EGFP and pAAV-BDNF.2.180 SD Rats were included in the experiment,diabetes was induced by intraperitioneal injection of streptzotocin(STZ),followed by intravitreal injection of AAV2-BDNF/His-tag and AAV-enhanced green fluorescent protein(EGFP).Survival of RGCs was estimated by counting FG-positive RGCs.Transgene expression was assessed by immunohisto-chemistry of BDNF or His-tag and direct visualization of GFP.Visual evoked potential(VEP) was performed to evaluate visual function in various groups.3. Diabetic model was induced by streptozotocin in 120 adult male Sprague-Dawley (SD) rats.Rat RGCs were labeled retrogradely by Fluorogold(FG) injection in superior colliculi region. After intravitreal injection of AAV2-BDNF/His-tag, survival of RGCs was estimated by counting FG-positive RGCs in retina. ERG and VEP in the normal and diabetic rats were recorded and analysed.Results:1.The recombinant plasmid of pAAV-EGFP and pAAV-BDNF was constructed; After the adeno-associated viruses (rAAV-BDNF and AAV-GFP) were constructed and purified and condensed, the titer of rAAV-EGFP was 3.0×1010/ml. The frozen sections showed the fluorescent expression under the fluorescent microscope and western blot analysis revealed high BDNF protein expression in the retinal tissues. 2. Among FG-positive RGCs,the number of both AAV-transfected cells and non AAV-transfected cells were all significant higher in AAV-BDNF/His-tag group than in AAV-GFP group(P<0.05),suggesting that several non-AAV-transfected cells could be rescued by secreted BDNF from AAV-transfected cells, which were identified by the following TrkB experiment. After gene therapy, both the latency and the amplitude of P wave in VEP and amplitude of OPS wave were recording in the control group were significantly superior to those in either AAV-BDNF/His-tag group or AAV-GFP group(P<0.05). The similar results were acquired in surving-RGC cell-counting.Conclusions:1. The constructed rAAV-EGFP and rAAV-BDNF can infect retina and successfully express the BDNF protein and green fluorescentprotein on the retina, which paves the way for the treatment ofopticneuropathy and retinopathy.2.There were significant increase numbers of RGCs in a rat diabetes model treated by AAV-BDNF. AAV-mediated BDNF expression could protect RGC in the diabetic retinopathy, and also recovery of visual function to some extent.