Abstract
Abstract: :
Purpose: Members of the IL–6 family of cytokines, including CNTF, CT–1, and OsM, protect photoreceptors in several animal models of retinal degeneration. Little is known as to how this protection is achieved. The present work examines phototransduction–related proteins and photoreceptor morphology after intravitreal injection of recombinant CNTF. Methods: The cDNA encoding mature human CNTF was PCR cloned and expressed in E. coli. Recombinant protein was purified on Ni+ columns under native conditions. Long Evans rats were injected intravitreally with CNTF protein (10 µg in 5 µl) into the left eyes and PBS (5 µl) into right eyes. Retinas were harvested 3, 6 days and 3 weeks after injection. Levels of phototransduction–related proteins were evaluated on western blots and expression of mRNAs was assessed using real–time PCR. CNTF induced Phosphorylation of STAT3 was localized by immunohistochemistry. Photoreceptor morphology was examined on semi–thin plastic sections. ERGs were recorded at 6 days and 3 weeks after CNTF or PBS was injected into the right eye. The left eyes were untreated. Results: Significant decreases in rhodopsin, transducin α and ß subunits were found 6 days after CNTF injection. An increase in arrestin protein was detected at both 3 and 6 days. Real–time PCR showed a significant decrease in rhodopsin mRNA expression 3 days after injection. Rod outer segments were 30–50% shorter than control at 6 days. In addition, both scotopic a– and b– wave were significantly reduced in CNTF treated eyes 6 days after injection as, compared to contralateral untreated eyes. All changes were fully restored to control levels 3 weeks after injection. CNTF induced increase in STAT3 phosphorylation was localized to cells in the inner nuclear layer, but was absent in photoreceptors. Conclusions: CNTF treatment decreases the sensitivity of rod photoreceptors to light by negatively regulating phototransduction machinery. This reduces the metabolic burden on rods by decreasing the amount of circulating dark current and the amount of materials synthesized for outer segment renewal, which may help to protect photoreceptors. The effects of CNTF on photoreceptors are likely mediated through cells in the inner nuclear layer, including Muller cells.
Keywords: growth factors/growth factor receptors • photoreceptors • neuroprotection