Adenovirally Delivered BDNF To Murine Ocular Tissues Hepls Improve The Repairment Of Crushed Optic Nerve

PURPOSEOptic nerve injury occurs commonly among clinical cases, which results in severe visual loss and could not be treated effectively.The progressed death of retinal ganglion cell is considered the main reason.Brain-derived neorotrophic factor can prevent injured neurons from PCD in vivo and ex vivo.So we apply adenovirus to diliver exogenous BDNF into the injured rat's eye and transfect retinal cells such as RGCs and muller cells.From the optic nerve crushed-model rats we investigate the expression of BDNF and its effects on RGCs or optic nerve after injury to study a new therapy way for clinic.METHODS(1)A dilution series of adenovirus with beta-galactosidase gene ranging from 109 to 10" p.f.u./ml was injected into the vitreous, anterior chamber, peribulbar space or applied topically to eyes of adult rats. Gene expression was detected by enzymatic color reaction using (3 -gal stain as a substrate in tissue sections of enucleated eyes. Cytopathic effect was detected simultaneously.(2) Adenovirus with BDNF gene was injected into the vitreous. Gene expression was detected by immunofluorescence staining,and quantitative analysis was performed after injury or(and) transfection by ELISA.(3) The F-VEP of normal rats was monitored with visual electrophysiology testing instrument. Adenovirus with BDNF gene was injected into vitreous of the injured eye of optic nerve crushed-model rats. The F-VEP was reexamined in BDNF group and control groups 2 hours later and at the lst,2nd,3rd and 4th week after crush. The PI wave amplitude and latency were observed.RESULTS(l)The efficiency of transfection increased as a function of concentration of virus. Injection did not result in any detectable cytopathic effect. Cell positive for beta-galactosidase expression showed bluestaining of enzymatic color reaction in tissue sections. Adenovirus ?mediated gene transfer was detected in murine cornea, iris, ciliary body and retinal ganglion cells, photoreceptors, and retinal pigment epithelium after injected into the vitreous. The gene was transferred into murine corneal, iris, ciliary body through injecting into the anterior chamber. However, only the comeal epithelial layer was transfected by topical application. Oculomotor muscle cells was transfected by peribulbar injection. Gene expression in murine ocular tissues was observed after 3d following injection or topical application. The expression lasted at least 4 weeks. Blue staining indicating an enzymatic color reaction was not observed in all of control eyes.(2)The results of expression of BDNF were similar to 3 -gal by immunofluorescence staining; Quantitative analysis showed that the two BDNF groups were higher than control group at every time point and the injured group without transfection was higher than normal group at 3d and Iwk.(3) The normal SD rats PI wave amplitude and latency were 15.51 + 3.67uV and 86.20?.30ms(n=20) based on our experimental system. Optic nerve postinjuryed at 2hrs, the PI wave amplitude decreased and latency increased. The PI wave amplitude of BDNF group were higher than the control group's and the latency of BDNF group were shorter meantime. There were significent differences at the 1st. 2nd and 3rd weeks.CONCLUSIONEfficient and stable transfer of functional genes could be achieved by adenovirus delivery into the cornea, iris, ciliary body and retina of rats. Adenovirally delivered BDNF to murine ocular tissues can increase the expression of BDNF in retina and help improve electrophysiology of vision during earlier period.