Objective: To investigate the effect of recombinant adeno-associated virus conducted NgRDN on the axonal regeneration of optic nerve after trauma.
Methods: Two kinds of adeno-associated virus (AAV), AAV-NgRDN-EGFP containing dominant negative form of Nogo receptor and enhanced green fluorescent protein (EGFP) and rAAV-NgR-EGFP containing Nogo-66 receptor (NgR) and EGFP, were constructed. 45 adult Wistar male rats were randomly divided into three equal groups, all with both eyes as experimental eyes: Groups A, B, and C to undergo injection of rAAV-EGFP, rAAV-NgR-EGFP, and rAAV-NgRDN-EGFP respectively into the vitreous; and each group was subdivided into 3 equal subgroups: subgroups 1 underwent injection of rAAV only, subgroups 2 underwent injection of rAAV and lens trauma, and subgroups 3 underwent injection of rAAV and zymosan. The rats in the Subgroups A2, B2, and C2 underwent. Crush of the optic nerve 2 mm behind the eyeball with optic nerve forceps 3 weeks after the injection. Four days after the crush the right eyes were taken out and the retinal explants were cultured in 2 kinds of culture fluid: with or without myelin. The growth of axons at the edge of retinal explants was observed by immunofluorescent staining with betaIII tubulin. Two weeks after the crush the other eyes were taken out to isolate the optic nerves. Immunofluorescence assay was used to detect the expression of growth associated protein-43 (GAP-43) of optic nerve. The axonal regeneration of optic nerve was observed.
Results: betaIII tubulin staining showed that on the condition of culture fluid without myelin both rAAV-NgR-EGFP and rAAV-NgRDN-EGFP showed no effects on the axonal regeneration of retinal ganglion cells (RGCs). However, on the condition of culture fluid with myelin the count of axonal regeneration and the length of regenerated axons of Group B were (13+/-4) and (36 microm+/-4 microm), both significantly lower than those of Group A [(21+/-4) and (83 microm+/-11 microm) respectively, both P<0.01]. There were not significant differences in count of axonal regeneration and length of regenerated axons between Subgroups C1 and A1. The count of axonal regeneration and length of regenerated axons of Subgroups C2 were (317+/-45) and (508 microm+/-44 microm), both significantly higher than those of Subgroup C3 [(238+/-30) and (365 microm+/-48 microm) respectively, both P<0.01], and the values of both Subgroups C2 and C3 were significantly higher than those of Subgroups A2 and A3. The GAP43-positive area in the optic nerve of Group C was significantly larger than that of Group A (P<0.01), and that of Group B was significantly smaller than that of Group A (P<0.01). The GAP43-positive area in the optic nerve of Subgroup A2 was (18.71+/-1.72)x100 microm2, significantly larger than that of Subgroup A3 [(12.75+/-1.02)x100 microm2, P<0.01], and that of Subgroup A3 was significantly larger than that of Subgroup A1 (P<0.01). There were not significant differences in the GAP43-positive area among the subgroups in Group B.
Conclusion: Transfection of rAAV-NgRDN-EGFP into RGC in an activated status enhances axonal regeneration of optic nerve. NgRDN AAV can inhibit effectively the role of NgR.