Intraocular Gene Transfer Of Ciliary Neurotrophic Factor Repairs Optic Nerve Injury In Rats

Background:The trunk of the optic nerve consists of millions of axons that arise from the ganglion cells of the retina. Since the optic nerve is a part of central nervous system, there is not any effective therapy for optic nerve injury. It was traditional viewpoint that central nervous system can not regenerate after the axons injured. Studies in this field recently reported that injured retinal ganglion cells (RGCs) have somewhat survival and regenerative ability. It has been proved that supplying exterior neurotrophic factors can promote the survival and regeneration of RGCs in adult mammalian. Recently Cui et al reported that ciliary neurotrophic factor (CNTF) was an unique of all the neurotrophic factors which could take effects on promoting regeneration of adult hamster RGCs, and supplying additional CNTF may be a new strategy to curing optic nerve trauma. Stockli et al reported that high levels of CNTF messenger RNA (mRNA) were found in the optic nerve, and no CNTF mRNA could be detected in the retina in 1991. Chun et al reported that CNTF immunoreactivity was restricted to profiles in the ganglion cell layer in the normal retina. Walsh et al studied the distributions of CNTF protein in the normal rat retina in 2001. Retinas of rats were immunolabelled with antibodies to CNTF and analysed by confocal microscopy. CNTF was generally in macroglial cells (astrocytes, Muller cells). CNTF was distributed in the somas of these cells and along the processes of macroglial cells, in granular form. CNTF was not detected in neurons. Zou Qian et al detected the expression of CNTF mRNA in the retina by reverse transcriptase-polymerase chain reaction (RT-PCR), and Western blotting was employed in measure of CNTF protein in the retina, and found that the signals of CNTF mRNA and protein were present in normal rat retina. From above all, different studies about the distribution of CNTF mRNA in normal retina still have different results. Injuried RGCs can be rescued by a single intravitreal injection of CNTF at the time of optic nerve transection. However, the effect is transient. Repeated administration of CNTF can prolong the protection of axotomized RGCs, but posterior subcapsular cataract and retinal folds will occure in the eyes. With the development of gene therapy, efficient transgene methods are new choices to repair optic nerve injury. Replication defective adenoviral vectors (AdV) have been demonstrated as an effective method for transfecting genes into the retina. Previous studies were successed in prolonging neuroprotective effects of CNTF by intravitreal adenoviral gene transfer in the rat retina following optic nerve transection. But crush injury models of optic nerves are more like the patients following optic nerve trauma. No study about intravitreal Ad-CNTF gene transfer into the retina following optic nerve crush is found. Objective: The aim of this experiment is to study the distribution of CNTF mRNA in the normal rat retina, and the neuroprotective effects of intravitreal Ad-CNTF after optic nerve crush. Methods: 1. The distribution of CNTF mRNA in the normal rat retina and optic nerve was studied by in situ hybridization histochechemistry. 2. After recombinant adenovirus vectors were reproduced and purified, the expressions of recombinant adenovirus vectors with target gene were detected by histochemistry. 3. After optic nerves of adult rats crushed, Ad-CNTF eyes injections were made. Retrogradely labeled RGCs by fluorogold (FG) were counted in retina. P1 wave amplitude and latency of the flash visual evoked potential (F-VEP) was measured after optic nerve injured. Results: 1. In situ hybridization histochemistry displayed: CNTF mRNA generally distributed in normal rat optic nerve. In retina, CNTF mRNA was found in inner nuclear layer where the somas of Muller cells were distributed. 2. Adenovirus vectors with β-galactosidase gene were trasfected in cultured Hela cells. Gene expression was detected by enzymatic color reaction using X-gal stain. That resulted in efficient transfection of Ad-LacZ in vitro. Ad-LacZ can be as control Ad of the experiment in vivo. Immunohistochemistry displayed: Following intravitreal injection ofAd-CNTF, CNTF immunostaining increased in retina significantly, and distributed in diffuse forms. Expression of CNTF in the retina following intravitreal injection of Ad-CNTF was still higher in 28 day post-injection than other controls. 3. After adult rat optic nerve crushed, intravitreal injection was made. Retrogradely labeled RGCs by fluorogold were counted in retina. The mean RGCs density in the retina following intravitreal injection of Ad-CNTF was higher in 28 day post-injury than other controls. P1 wave amplitude of F-VEP in the retina following intravitreal injection of Ad-CNTF was higher than other controls. Conclusion: Our results showed: In normal retina, CNTF mRNA was found in inner nuclear layer where the somas of Muller cells were distributed. Following intravitreal injection of Ad-CNTF, Expression of CNTF in the retina increased significantly, distributed in diffuse forms, and expression of CNTF was still higher in 28 day post-injection than other controls. Following adult rat optic nerve crushed, a single intravitreal injection of Ad-CNTF had obviously neuroprotective effects to injured RGCs.The protective effects were better than a single intravitreal injection of CNTF.