Abstract
Abstract: :
Purpose: Endothelin-1 (ET-1) is a small peptide which has vasoconstrictive properties, has been localized in the retina, and is elevated in glaucomatous eyes. Injection of ET-1 has been shown to slow retinal ganglion cell (RGC) axonal transport and cause their death. The purpose of this study was to determine the time-course of RGC death and study glial fibrillary acidic protein (GFAP) immunoreactivity after a single injection of ET-1. Methods: The RGCs of rats were retrogradely labeled with Fluorogold . Two days later the eyes were injected with 5µl ET-1 (0.05µg) or saline. The effect of ET-1 on RGC survival was determined over a 3 week time course by fixing injected eyes at 1, 4, 7, 10, 14, and 21 days postinjection. Surviving RGCs were quantified from confocal micrographs of the RGC layer after anti-Fluorogold immunohistochemistry. Changes in the thickness of the retinal layers was determined by incubating retinas in YoPro-1 (0.1µM) overnight to label retinal cell nuclei. The thickness of retinal layers was measured in transverse sections cut orthogonal to the retinal layers reconstructed from serial confocal sections of flat mounted retinas. Astrocytes in flatmounted retinas were examined by confocal microscopy after GFAP immunohistochemistry. Results: The density of retrogradely labeled RGCs remained unchanged for 7 days after ET-1 injection and was not significantly different from saline injected control retinas (2400 cells/mm2). At 10 days the density of RGCs began to decline (56%) and was decreased to 30% of the original population by 14 days. There was no significant additional RGC loss between 14 and 21 days. The thickness of the ONL (photoreceptors) and INL (amacrine, bipolar, horizontal cells) was not significantly different from saline injected eyes at 7 and 14 days post-injection. However, there was a 19% and 25% decrease in the thickness of the IPL (amacrine, bipolar, RGC processes) at 7 and 14 days, respectively, which was consistent with RGC loss at these time points. The density of GFAP immunoreactivity in the astrocyte layer increased by 24% and 98% at 7 and 14 days, respectively. There was also an increase in the GFAP labeling of Muller cells, which usually express GFAP in response to stress. Conclusions: These studies demonstrate that a single injection of ET-1 causes the death of RGCs in a timecourse that is similar to optic nerve transection. There was no cell loss apparent in the ONL or INL, suggesting that the effect of ET-1 was specifically on RGCs. Increases in GFAP immunoreactivity in astrocytes and Muller cells suggest that glial reactions accompany RGC loss.
Keywords: ganglion cells • retinal glia • cell death/apoptosis