Abstract
Abstract: :
Purpose: A corollary of the hypothesis that excessive glutamate levels in the retina contribute to glaucomatous retinal ganglion cell (RGC) death is that the glutamate clearance mechanisms in the retina are compromised. Under such conditions, RGCs should be more sensitive to exogenous glutamate. Therefore, the purpose of this work was to determine if RGCs in a rat model of pressure–induced optic neuropathy are more sensitive to exogenously applied glutamate. Methods: Intraocular pressure (IOP) was elevated in one eye of Brown Norway rats (n=4) by injecting hypertonic saline into an episcleral vein. IOP was monitored twice–weekly with a Tonopen and animals sacrificed after 7–16 weeks of elevated IOP. The retinas were carefully removed, cut into 4 pieces and then mounted on Millipore filters. Dextran–conjugated calcium indicator dye (Fura–2) was injected into the flat–mounted retinas, and following incubation for 7–15 hrs, the dye was retrogradely transported to RGC somata. Changes in the Fura–2 fluorescence ratio, indicative of changes of free intracellular calcium concentration, were monitored with a cooled CCD camera during exposure to glutamate and the agonist NMDA. Results:Relative to the fellow control eye, the peak and integral IOP elevation in the experimental eyes ranged from 20.2–21.6 mm Hg and 767–1102 mm Hg days, respectively. The ratio of Fura–2 labelled RGCs in the experimental to control eyes ranged from 0.1–0.8, consistent with the nerve damage in the experimental eyes. In total, 787 RGCs were imaged: 238 from the experimental eyes and 549 RGCs from the fellow control eyes. Application of 200 µM NMDA (not recognized by endogenous glutamate transporters) induced an elevation of calcium in nearly all (>95%) RGCs from both experimental and control eyes. The application of 100 or 500 µM glutamate did not affect calcium levels in any RGC from either experimental or control eyes, indicating that glutamate clearance mechanisms were functional and able to clear sufficiently glutamate at these concentrations. Conclusions: These results suggest that in a model of chronic pressure–related optic nerve damage there is no evidence of a global and irreversible defect in the glutamate clearance mechanisms, even in eyes with very severe RGC loss. This work represents a novel attempt to directly assess the vulnerability of surviving RGCs to glutamate excitotoxicity in an animal model of glaucoma.
Keywords: excitatory neurotransmitters • ganglion cells • calcium