Ciliary Neurotrophic Factor And Adenovirally Delivered Brain-derived Neurotrophic Factor Protect The Crushed Optic Nerve

Both the optic nerve damnification caused by trauma and death of retina ganglion cells caused by glaucoma can induce unresumable loss of visual acuity. There aren't clinical efficient treatment to deal with it. How to initiate and improve the repair and regeneration of damaged RGC and promote the function of the optic nerve is our ultimate goal. The experiment before have showed that survival, repair, regeneration and functional recovery of RGC is definitely associated with its local environment and neurotrophic factors in it. In our study: (1) We observed the variable expression of GAP-43 and its gene after optic nerve crush. And we observed the effect of introvitreal injection of CNTF and Ad-BDNF to repair and regeneration of crushed optic nerve. (2) In order to count the number of RGC under the fluorescence microscope, we injected the fluoro gold into the superior colliculi of the animals. So we could draw the conclusion that if intravitreal injection of CNTF and Ad-BDNF could affect survival of RGC after optic nerve injury.METHODS:Part 1: Effects of CNTF and Ad-BDNF on the expression of GAP-43 in retina after optic nerve crush1. Establishment of Optic nerve crush model in rats. The- 14 -optic nerve of adult Spraque-Dawley rats was exposed. The optic nerve was crushed at 2 mm behind the eyeball using special forceps for 5 seconds.2. Intravitreal injection. After the optic nerve crushed, CNTF 50 ng (10uL), Ad-BDNF10uL or saline 10uLwere injected into the vitreous respectively through the sclera 1.5 mm away from the limbus.3. Get the retina. The animal were sacrificed 1, 2, 3 and 4 weeks after crush by disjoint of neck vertebra. The whole retina were fetched and preserved in -80癈 refrigeratory.4. Protein electrophoresis and western-blotting. Quantitative wet retina (15g) were homogenized and prepared for vertical SDS-PAGE. The protein were transported to the NC membrane. The protein in NC membrane were analyzed by Western Blot method. And the results were proceeded with imaging service.Part 2: Effects of CNTF and Ad-BDNF to expression of GAP-43 gene in retina after optic nerve crush1. Establishment of Optic nerve crush model in rats and intravitreal injection of neurotrophic factors. Same as that in the first part.2. Tissue prepared. The animal were sacrificed 1, 2, 3 and 4 weeks after crush by disjoint of neck vertebra. The eyeball were preserved in -80癈 refrigeratory. The whole eyeball were cut into sections 12 mm thick on a cryostat, after which sections were fixed in buffered 4% paraformaldehyde for 15 min at room temperature, rapidly air-dried, and stored desiccated at -4癈 refrigeratory.3. In situ hybridization. The slices were hybridized with GAP-43 cRNA. The results were observed under microscope.Part 3: Effects of CNTF and Ad-BDNF to survival of RGC after optic nerve crush1. Establishment of Optic nerve crush model in rats and intravitreal injection of neurotrophic factors. Same as that in the first part.2. Retrograde the RGC with fluoro-gold. 7 day before sacrificed, the animals were anaesthetized and fluoro-gold were injected into the the superior colliculi, 0.15uL in each superior colliculi.3. Retina spreading. The animals were sacrificed by given overdose of 846. The whole retina were spreaded after label. The tissue were sealed with Vectashield and stored avoid of light in -4癈 refrigeratory.4. RGC counting. The retrograde labeled RGC were counted under fluorescent microscope in 400?RESULTS:1. There is low expression of GAP-43 in normal SD rat's retina. Molecular weight is about 47KD. There was a little GAP-43mRNA in ganglion cell layer of normal rat. The number of RGC counted by retrograde labeling of fluoro-gold is about 2155?65 ?/mm2(n=12) 7 days after label.2. After the crush, expression of GAP-43 increased. It reached its peak about 1 week after the crush and come back to normal level 4 week after crush. The...