Abstract
Purpose::
To clarify whether endoplasmic reticulum (ER) stress involved in retinal cell death using cultured retinal ganglion cells (RGC-5, a rat ganglion cell line transformed with E1A virus) in vitro and mice in vivo.
Methods::
RGC-5 damage was induced by tunicamycin at 1, 2 and 4 µg/ml, and cell viability was measured by Hoechst 33342 and YO-PRO-1 or propidium iodide double nuclear stainings. The expressions of glucose-regulated protein 78(GRP78)/BiP, the phosphorylated form of eukaryotic initiation factor 2α (p-eIF2α), and C/EBP-homologous (CHOP) protein after tunicamycin at 2 µg/ml (in vitro and in vivo) were measured using immunoblot. To induce ER stress in vivo, tunicamycin at 0.1 and 1 µg was intravitreously administered in mice. Seven days after the injection, cell number in ganglion cell layer (GCL) and thickness of inner plexiform layer (IPL) were evaluated.
Results::
Treatment with tunicamycin induced apoptotic cell death in RGC-5. GRP78/BiP, a biomarker of ER stress, increased time-dependently throughout the 24 h tunicamycin treatment period. Treatment with tunicamycin time-dependently induced eIF2α phosphorylation, while total eIF2α levels were not changed during the 24-h observation period. CHOP was first detected at 6 h after addition of tunicamycin and persisted thereafter. In vivo, intravitreal injection of tunicamycin at 0.1 µg/eye (a low dose) induced a significant loss of cells in the GCL, but no thinning of the IPL. At a high dose of 1µg/eye, tunicamycin significantly decreased both the cell count in GCL and IPL thickness.
Conclusions::
These data indicate that ER stress may play a pivotal role in retinal cell death.
Keywords: retinal culture • retina • apoptosis/cell death