CNTF inhibition of the ERG a– and b–waves is reversible and correlates with down–regulation of rhodopsin and photo–transduction protein expression.

Abstract

Abstract: : Purpose: Several studies have found that over–expression of CNTF (via viral vectors) in the retina protects photoreceptors from degeneration. However this also decreases the ERG a– and b–waves. ERG suppression has raised concerns as to whether CNTF might be detrimental to photoreceptor function. This study investigates the ERG after a single bolus intravitreal injection of recombinant CNTF protein. Methods: CNTF protein derivation: mature human CNTF cDNA was PCR–cloned, expressed in E. coli, and recombinant protein was purified on Ni⁺ columns under native conditions. Right eyes of Long Evans rats were injected intravitreally with CNTF (10 µg in 5 µl) or PBS (5 µl). Left eyes served as untreated controls. ERGs were recorded from both eyes simultaneously at 6 days or 3 weeks after injection. For biochemical and morphological studies, CNTF was injected into the right eyes and PBS into the left, and retinas were harvested 3, 6 days or 3 weeks after injection. Levels of rod specific proteins were evaluated by Western blot analyses, and photoreceptor morphology was examined on semi–thin plastic sections. Results: The CNTF treated group showed a significant ERG decrease of both scotopic a– and b– wave amplitudes 6 days after injection compared with the contralateral untreated eyes. No significant difference was found between PBS–treated and untreated eyes. Significant decreases were found in rhodopsin and transducin α and ß subunits at 6 days, and an increase in arrestin protein both at 3 and 6 days. ROS (rod outer segments) were 30–50% shorter than controls at 6 days. However, at 3 weeks after CNTF injection, the ERG a– and b–wave amplitudes, rhodopsin, transducin α and ß subunits, and arrestin, as well as ROS length had returned to control levels. Conclusions: These results indicate that the initial ERG suppression, shortened ROS and down–regulation of photo–transduction proteins by a single intravitreal dose of CNTF protein is reversible over time. While the dose used was considerably larger than previous therapeutic doses the results support the hypothesis that CNTF protects photoreceptors, at least in part, by regulating the photo–transduction machinery.

Keywords: neuroprotection • electroretinography: non–clinical • growth factors/growth factor receptors