Protective Effect Of Ciliary Neurotrophic Factor On Experimental Optic Nerve Injury

Objective (1) To study the expression and cellular localization of ciliary neurotrophic factor (CNTF) and its a receptor subunit (CNTFR a ) in the rat retina after optic nerve injury.(2) To explore the biodistribution of I rhCNTF in eyes and organs of SD rats by intravitreous injection, retrobulbar injection, hyodemic injection, for finding out the best and acceptable administration method. (3) To observe the protective effect of exogenous CNTF on the experimental crushing injury of optic nerve. (4) To assess the effect of CNTF on the mitosis of human retinal pigmental epithelial cells (RPE), Retinal glial cells (RGC), and Lens epithelial cells (LEG). (5) To investigate the toxiciry of topical administrated CNTF to rabbit eyes, livers and kidneies.Methods (1) Expression of CNTFmRNA and CNTFRamRNA in rat retina after optic nerve injury was detected by reverse transcription-polymerase chain reaction (RT-PCR), and their protein products were detected by a Western Blot test. Immunofluorescence was used to localize cells those expressed CNTF. (2) Labeled rhCNTF with 125I by lodogen method. The biodistribution of 125IrhCNTF in tissue was investigated respectively at 30min, 1 hour, 2 hours, 3 hours and 24 hours after injection. (3) SD rats were used for intraorbital optic nerve crush models. The rats were divided into CNTF treating groups and control groups. Morphological change of retina was observed by electron microscope and light microscope. Optic nerve function was evaluated by F-VEP test. (4) Fourth passage cultured human RPE cells and RGC were inoculated into 96-well-plate. Different concentration levels of CNTF or bFGF were added into culture media and the proliferation of cells was detected by an MTT test after 48 hours. Only CNTF was added into LEG culture media, and morphological change was observed by light microscope. (5) Rabbits were divided into 3 groups according to different concentration levels of CNTF intraocular injected. Direct ophthalmolscopic examination was taken every day after injection. Light microscope and transmission electron microscope examination was taken for the microstructure of retina. Retinal function was tested by ERG examination. The microstructure of livers and kidneies were observed with light microscope .(6) SPSS10.0 software package served as statistical analyze tool.Results (1) The signal of CNTFmRNA increased at 1 day and 7 days, then decreased at 14 days and 28 days after injury (KO. 001), but some signal of CNTFR a mRNA can be found at 1,7, 14, 28 days after injury. Signal of CNTF appeared in a layer-specific and time-dependent manner. (2) Biodistribution in rats indicated rapid uptake and rapid clearance of 125IrhCNTF. It displayed a higher uptake ratio in liver and kidney. The value of uptake in eyes was very little in hyodemic injection. While there were higher uptake in eyes in intravitreous injection group and retrobulbar injection group. (3) CNTF treating groups showed a significantly lower lesion of retina and optic nerve than control groups. In control groups the thickness of retina decreased in all layers, and the inner retina was most sensitive; Latency of P1 in control groups significant increased compared to that of CNTF groups, and with significantly decreased P1 amplitude. (4) CNTF (1-100 ng/ml) increased mitosis of RGC, but had no effect on RPE cells, LEG. (5) There was no apparent retinal and corneal change under the direct ophthalmolscope, electron microscope and light microscope in all groups. The amplitude of ERG was not changed in all groups (P >0. 05).Conclusion (1) As a result of crushing injury, the expression level of CNTF down-regulated soon after a transitorily up-regulation, but CNTFR a is constitutively expressed in retina after injury. These facts suggest that exogenous CNTF might provide a supportive environment for axonal regeneration and might play a protective role in the retinal recovery event after crushing injury. (2) Retrobulbar injection of rhCNTF with a higher dose than used in intraocular injection will easily be ab...