Abstract
Abstract: :
Purpose: Injection of 5ul (500uM) ET–1 into the vitreous of rat eyes results in the death of 40% of retinal ganglion cells (RGCs) and loss of axons in the optic nerve (ON) within 4 wks. We have previously shown that injections of Ciliary–derived Neurotrophic Factor (CNTF) (5ul; 20uM) 2 days after ET–1 injetion resulted in a 30% increase in RGC survival but did not preserve axons in the ON. In the present study we examined the effects of CNTF on intra–retinal sprouting after ET–1 mediated injury. 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 fixed in phospate buffered 4% formaldehyde and processed for immunihistochemistry. Regenerating RGCs were identified by immunohistochemical localization of highly phosphorylated neurofilament protein (pNF–H) in somas and dendrites (RT–97; Chemicon 1:500). The regional distribution of regenerating RGCs was determined by dividing the flatmounted retina into 4 concentric zones around the ON. The number of regenerating RGCs in each zone and the diameter of their somas was measured in confocal micrographs taken from the entire retina. Results: In control retinas, only the axons of RGCs were pNF–H immunoreactive (–IR). In ET–1/saline injected eyes, there was a 40% decrease in RGCs and a few spontaneously regenerating pNF–H–IR RGCs (12 RGCs/retina). These RGCs were large (avg. 25um diameter) and mostly located in the periphery (zones 1–4; 2%, 14%, 32%, 52%). There were over 6 times the number of regenerating RGCs in CNTF treated retinas (75 RGCs/retina). The average cell diameter was smaller (avg. 19um) and more evenly distributed across the retina (zones 1–4; 10%, 31%, 32%, 27%). cAMP augmented the effect of CNTF by causing a 21 times increase in regenerating RGCs (250 RGCs/retina) in each zone. Conclusions: Only a few, large RGCs, mostly located in the retinal periphery are capable of spontaneous regeneration after ET–1 treatment. CNTF caused an increase in the number of regenerating RGCs throughout the retina and induced regeneration in small RGCs. cAMP dramatically increased the efficiency of CNTF. These studies suggest that cAMP/CNTF treatment enhances both the survival and regeneration of ET–1 injured RGCs.
Keywords: regeneration • ganglion cells • second messengers