CNTF Increases Gene Expression of Neurofilament-200 in Retinal Ganglion Cells After ET-1 Treatment

Abstract

Purpose:: A single intravitreal injection of 5ul (500uM) ET-1 into rat eyes results in the death of 40% of retinal ganglion cells (RGCs) within 4 wks. We have previously shown that injections of CNTF (5ul; 20uM) 2 days after ET-1 injection resulted in a 30% increase in RGC survival. Few surviving RGCs had inreased phosphorylated neurofilament 200 (NF-200) immunoreactivity (-IR) in their somas and dendrites, a characteristic observed in developing and regenerating neurons. However, because ET-1 has been shown to inhibit axonal transport, the localization of NF200-IR in RGC somas and dendrites may be due to a lack of transport of NF200 into the axon. The purpose of this experiment was to determine if CNTF caused an increase in NF-200 gene expression, which would be attributed to its affects on promoting RGC regeneration.

Methods:: Rat RGCs were retrogradely labeled with Fluorogold (FG) from the superior colliculus (SC) 2 days prior to intraocular injection with ET-1. Two days later the vitreous was injected with 5ul saline, CNTF (20uM), or CNTF+cAMP (20uM;60nM). Rats were killed after 4 wks and the eyes were enucleated in RNA Later. RNA integrity and concentration was assessed using the AGilent 2100 Bioanalyzer. NF200 gene expression from recovered cDNA was analyzed with RT-PCR using a 96 well TaqMan optical reaction plate format on an ABI Prism 7700 sequence detection system. RNA was normalized to GAPDH for each reaction.

Results:: In control retinas, 2430 cells/mm² were retrogradely labeled 2 days after FG injection. Only 1520 RGC/mm² were FG labeled when eyes were treated with ET-1 one day before FG injection, indicating that ET-1 slows axonal transport. There was no difference in the number of PGP9.5-IR RGC somas, indicating that the lack of FG labeling must have been due to decreased FG transport rather than RGC death. ET-1 treatment decreased NF200 gene expression by 60% after 4 weeks when compared to control animals. Administration of CNTF and CNTF/cAMP increased NF200 gene expression by 20% and 55%, respectively, when compared to ET-1 treated retinas. CNTF/cAMP treatment induced NF200 gene expression to 95% if control levels.

Conclusions:: These studies show that ET-1 slows RGC axonal transport, which may be a cause or consequence of subsequent RGC loss. NF200 gene expression was reduced 4 wks after ET-1 mediated RGC death. CNTF and CNTF/cAMP increased gene expression of NF200, and was directly proportional to the increased NF200-IR obserevd in RGC somas and dendrites. These studies demonstrate that the increase in NF200-IR observed in RGC somas after ET-1, ET-1/CNTF, or ET-1/CNTF/cAMP treatment is due to intra-retinal regeneration by surviving RGCs.

Keywords: gene/expression • neuroprotection • regeneration