Abstract
Purpose: :
Elevation of plasma HCY is a risk factor for neurodegenerative disease and has been implicated in certain forms of glaucoma. Exposure of primary RGCs to 50µM HCY induces death of 50-60% of cells within 18h (Dun et al, 2007) and endogenous elevation of HCY in an in vivo model results in loss of 20% of cells in the ganglion cell layer (Ganapathy et al, 2009). The mechanism of this toxicity has not been thoroughly investigated. It has been shown that L-homocysteic acid is capable of activating the NMDA receptor and inducing calcium influx in rat RGCs (Zhang et al, 1992). A hallmark of NMDA receptor-mediated excitotoxicity is rapid induction of superoxide (O2-) and nitric oxide (NO) due to increased intracellular calcium. This study examined the role of the NMDA receptor in HCY-induced RGC toxicity.
Methods: :
Primary RGCs were isolated from 2-4 day old mice. Control and HCY-exposed RGCs (50µM HCY for 18h) were pre-treated with 0, 25, 50, 100, 250, and 500 nM of MK801 (a potent NMDA receptor blocker) for 1h plus the duration of HCY exposure; cell death was analyzed by measuring cytosolic lactate dehydrogenase (LDH) levels. Control and HCY-exposed RGCs that were pre-treated with 250nM MK801 were also examined using TUNEL analysis. RGCs exposed to 50µM HCY for 0, 0.5, 1, and 3 h were analyzed for O2- and NO using electron paramagnetic resonance spectroscopy and an NO selective electrode, respectively.
Results: :
RGC exposure to HCY resulted in 59.7 ± 4.9 % cell death, similar to previous findings; pre-treatment of RGCs with 250nM MK801 significantly reduced cell death to 19.9 ± 3.0 % (p<0.001) as shown by cytosolic LDH measurement. TUNEL analysis showed that pre-treatment with 250nM MK801 reduced HCY-mediated RGC death from 48.9 ± 8.1 % to 21.9 ± 4.8 % (p<0.002). Cellular O2- and NO levels were not significantly altered within 3h of HCY exposure.
Conclusions: :
Our results implicate NMDA receptor involvement in the pathogenesis of HCY-induced RGC death. In contrast to the expected rapid induction of O2- and NO following HCY treatment, levels remained unaltered within 3h of insult. Studies are underway to assess O2- and NO levels following longer exposure to HCY and to further elucidate the temporal involvement of the NMDA receptor in HCY-induced RGC toxicity.
Keywords: ganglion cells • excitatory amino acid receptors • apoptosis/cell death