Upregulation of Retinal Glutamate in Chronic Experimental Glaucoma

Abstract

Purpose : The role, if any, of glutamate excitotoxicity in glaucoma is controversial. Previous attempts at measuring vitreous glutamate levels in both human and chronic experimental glaucoma (ExGl) in macaques have yielded contradictory results. Here, we investigate the local modulation of glutamate levels within the retina.

Methods : Ten macaques underwent laser trabecular destruction in the right eye. The intraocular pressures were elevated to above 25 mmHg for from 1 to 8 years. Six of the animals underwent bilateral enucleation at the time of euthanasia. The anterior segments were promptly removed and two 6 mm punches were removed from each retina—one from the superior and one from the inferior arcuate region. The removed tissue was then flash frozen in liquid nitrogen. Levels of free glutamate and taurine were then determined by mass spectrometry (MS). The remaining 4 macaques underwent perfusion fixation with glutaraldehyde. Segments of retina in the perimacular region were removed, embedded in epon and underwent immunohistochemical (IHC) staining for glutamate.

Results : MS showed no statistically significant difference in glutamate levels between the control and ExGl eyes. However, IHC revealed marked upregulation in glutamate staining in all 4 of the perfusion fixed ExGl eyes at the level of the rod spherules and cone pedicles. There was also increased glutamate staining in the inner plexiform layer. The retinal ganglion cell (RGC) and nerve fiber (NF) layers were nearly absent in the ExGl eyes.

Conclusions : The apparently contradictory MS and IHC results may be because the ExGl retinas are much thinner than the controls—missing their RGC and NF layers, which normally contain glutamate. Glutamate upregulation on IHC was present mostly in the rod spherules and cone pedicles (perhaps due to photoreceptor ischemia). These findings could explain the results of previous studies that failed to consistently show elevated glutamate levels in the vitreous. Furthermore, upregulation of glutamate at the critical photoreceptor and possibly bipolar synapses leaves open the possibility that excitotoxicity contributes to RGC injury in glaucoma.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

 

IHC for glutamate in the control retina. Arrows show rod spherules and cone pedicles.

 

IHC for glutamate in the ExGl fellow eye. There is markedly increased staining in the rod spherules and cones pedicles (arrows).