Changes in Thy1 gene expression associated with damaged retinal ganglion cells

Purpose: The temporal series of molecular events that occur in dying retina l ganglion cells is poorly understood. We have examined the change in expre ssion of a normally-expressed ganglion cell marker gene, Thy1, relative to the kinetics of cell loss caused by acute and chronic damaging stimuli. Methods: For acute experiments, mice were subjected to optic nerve crush or intravitreal injections of N-methyl-D-aspartate (NMDA) to induce ganglion cell death. RNase protection analysis was used to quantify Thy1 mRNA levels from total retina RNA and in situ hybridization was used to monitor the pa ttern of Thy1 positive cells. Changes in Thy1 expression were compared to t he time course of cell loss induced by each treatment. To induce elevated i ntraocular pressure (IOP), the episcleral veins of rats were injected with hypertonic saline, which scleroses Schlemm's Canal and the trabecular meshw ork. Elevated IOP was monitored every day for 35 days after which the anima ls were sacrificed and the retinas harvested for quantitative RT-PCR or fix ed for in situ hybridization studies. Evaluation of glaucomatous damage cau sed by elevated IOP was determined from histological sections of the optic nerves of all rat eyes. Results: After optic nerve crush, Thy1 mRNA levels decreased within 24 h, a lthough the number of expressing cells did not decline until 7 days. Both m easures showed a loss of Thy1 well in advance of cell loss, which was detec ted by 2 weeks after surgery. This change in expression was not dependent o n execution of the cell death program since a similar decrease was detected in Bax-/- ganglion cells, which are resistant to cell death induced by opt ic nerve crush. Thy1 mRNA levels and the number of expressing cells also de creased within 6 h after NMDA injection, in advance of cell loss, which was detected by 24 h. Similarly, elevated intraocular pressure was associated with a decrease in mRNA and expressing cells in a pressure-dependent manner . In moderately hypertensive rat eyes, the number of cells expressing Thy1 decreased before significant cell loss in the retina. Virtually no Thy1-exp ressing cells were detected in eyes with severe disease. Conclusions: Thy1 mRNA abundance and expressing cells, decreased in advance of detectable ganglion cell loss caused by three different modalities of d amage. This change is independent of the committed step of cell death.