Glutamate Aspartate Transporter (GLAST) Content in Glaucomatous Monkey Retinas

Abstract

Abstract: : Purpose: Previous studies showed an accumulation of glutamine in Müller cells of monkeys with experimental glaucoma (Carter-Dawson et al., 1998). This finding indicates that glutamate transport and metabolism was increased in those retinas. The purpose of the current study is to determine whether the amount of GLAST is increased in glaucomatous monkey retinas. Methods: Experimental glaucoma was induced unilaterally in two monkeys and bilaterally in three monkeys by argon laser treatments to the trabecular meshwork. The intraocular pressures were measured weekly by hand-held applanation tonometry and visual field defects were assessed by behavioral perimetry in all five monkeys using a standard clinical field analyzer (Humphrey Field Analyzer). After the monkeys had developed visual field defects, their eyes were removed and a retinal sample 5mm in diameter was collected from the inferior retina beginning at 20 degrees from the center of the fovea from each eye. The retina was detached from the pigment epithelium, placed in small conical tubes, quickly frozen in liquid nitrogen and stored at -80C°. To determine GLAST content, samples were prepared for SDS-PAGE and proteins transferred to nitrocellulose membrane. The blots were probed with GLAST and ß-actin antibodies followed by HRP conjugated secondary antibodies. Immunolabeled proteins were visualized by chemiluminescence. Results: Two of the monkeys showed mild visual field defects and the other three exhibited moderate to severe visual field defects. The immunoblots consistently showed bands for GLAST at approximately 70KDa. In the three monkeys with moderate to severe glaucoma, GLAST content was dramatically increased compared to controls, but there were no significant differences in GLAST content between the eyes with mild visual field defects and the control eyes. Probing blots with anti-ß-actin showed equal loading of samples. Conclusions: In addition to an accumulation of glutamine in Müller cells, GLAST content also increases in glaucomatous eyes with moderate to severe visual field defects. However, the mechanism underlying the increase is not yet determined.

Keywords: Muller cells • pathology: experimental • visual fields