Elevation of Primate IOP Is Associated With a Fall in EAAT1 but Not EAAT2 Expression

Abstract

Abstract: : Purpose: To determine whether expression of the glutamate transporters EAAT1 or EAAT2 is altered in the retinas of primates with elevated intraocular pressure (IOP). Methods: IOP was elevated unilaterally in cynomolgus monkeys by laser photocoagulation of the trabecular meshwork. IOP was monitored weekly. Oral memantine (8 mg/kg per diem) was given to 5 of 14 monkeys. For immunoblot experiments, eyes were fast frozen, retinal proteins purified using standard techniques and run on a SDS–PAGE. Gels were blotted with antibodies to EAAT1 or EAAT2 and appropriate secondary antibodies and visualized with ECL. Expression was quantified from each blot using Image Pro Plus software and 4 replicates were run for each sample. For immunohistochemical studies, tissue was perfused with 2% paraformaldehyde and sectioned at 12 microns. Sections were processed for immunohistochemistry using EAAT1 and 2 antibodies and staining was quantified as above. Staining from three regions in up to 9 corresponding sections were analyzed from each eye. Results: Expression of EAAT1 was decreased in eyes exposed to elevated IOP. Immunoblots gave the mean ratio of protein in lasered vs non–lasered (L/NL) eye as 0.80 ± 0.06 (n=7). To determine where this decrease was occurring, immunohistochemical analysis was undertaken. In control eyes, highest expression of EAAT1 was in the nerve fiber layer. When expression throughout the retina was quantified, the L/NL was 0.67 ± 0.05 (n=7). EAAT2 levels were not significantly altered by elevated pressure. Immunoblotting gave a mean of 0.98 ± 0.08 (n=7). Histochemical analysis showed EAAT2 was not as concentrated in the nerve fiber layer, with a cross–retinal L/NL of 1.43±0.53 (n=7). Preliminary analysis suggests that the effect of memantine was minimal. Conclusions: These preliminary findings suggest elevated IOP does not affect expression of EAAT2 in the primate retina but is associated with a decreased expression of EAAT1. As the predominant expression of EAAT1 was in the nerve fiber layer and this structure is preferentially lost in glaucoma, it is not clear whether the decrease in transporter levels is a cause or an effect of cell death.

Keywords: excitatory neurotransmitters • intraocular pressure • retinal glia