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Y Song, L Zhao, M Jiang, AM Laties, W Tao, R Wen; Recombinant Human CT-1 Protects Photoreceptors in Transgenic Rats with Rhodopsin Mutation S334ter . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3950.
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Purpose: CNTF and CLC, two members of the IL6 family of cytokines, protect photoreceptors in several animal models of retinal degeneration. In the present study, we examined CT-1 (cardiotrophin-1), another cytokine of this family, for its potential to protect photoreceptors in a line of transgenic rats (S334ter-3 rats) carrying a murine rhodopsin mutation S334ter. Methods: CT-1 cDNA was cloned by PCR from a human heart cDNA pool and expressed in E. coli. Recombinant protein was purified under native conditions. Heterozygous S334ter-3 rats were used in all experiments. The left eye of an animal was injected with 2 µg of CT-1 protein (in 2 µl PBS) intravitreally every three days starting at PD9. The right eye was injected with 2 µl of PBS in the same fashion. Eyes were collected at PD21 or 30, and retinas were examined histologically to evaluate photoreceptor survival. For immunoblot analysis, retinas were collected 0.5, 1, 2, 6, 12, 24, and 48 hours after a single intravitreal injection of 2 µg of CT-1 protein (in 2 µl PBS) at PD9. Total protein from each retina was subjected to SDS-PAGE. Phosphorylated Stat3 was detected using phospho-Stat3 specific antibodies. Results: In untreated animals most photoreceptors die from PD8 through PD20. The peak of photoreceptor death occurs on PD11 and 12. In PBS injected control eyes at PD21, there is only one row of nuclei in the ONL in the superior retina, and 1-2 rows in the inferior retina, similar to the untreated eyes. In CT-1 treated eyes at PD21, there are 5-6 rows of nuclei in the ONL in both the superior and the inferior retina. At PD30, the PBS injected eyes have 0-1 row of nuclei in the ONL in the entire retina. The CT-1 treated eyes, however, still have 5-6 rows. Immunoblot analysis shows a remarkable increase in Stat3 phosphorylation a single injection of CT-1 at PD9. The increase is observed as early as 30 min after injection, and lasts for 24 hr. A slight increase in Stat3 phosphorylation is also observed in PBS injected retinas 30 min to 12 hr after injection. Conclusion: Intravitreal injection of recombinant human CT-1 protein protects photoreceptors and this protection is extended by repeated dosing. Since CT-1 is a member of the IL6 family of cytokines, along with CNTF and CLC, our results likely indicate a common mechanism through which these cytokines protect photoreceptors from degeneration.
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