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(Investigative Ophthalmology and Visual Science. 2004;45:1531-1543.)
© 2004 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.03-0406
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Acetylcholine Protection of Adult Pig Retinal Ganglion Cells from Glutamate-Induced Excitotoxicity

Erica Wehrwein, Sean A. Thompson, Sylvie F. Coulibaly, David M. Linn, and Cindy L. Linn

From the Department of Biological Sciences, Western Michigan University, Kalamazoo, Michigan.

PURPOSE. To determine which glutamate receptor (GluR) subtypes are responsible for glutamate-induced excitotoxicity in cultured adult pig retinal ganglion cells (RGCs) and to characterize the neuroprotective effect of acetylcholine (ACh) on pig RGCs.

METHODS. Adult pig RGCs were isolated from other retinal tissue by a modified panning technique using Thy 1.1 antibody. Isolated RGCs were cultured in control media and media containing: glutamate, NMDA, or KA; glutamate and CNQX, MK-801, or AP-7; ACh, nicotine or muscarine; ACh and {alpha}-bungarotoxin (Bgt) or methyllycaconitine (MLA); and glutamate and choline or glutamate, choline, and MLA. To determine cell viability, cells were loaded with calcein and counted.

RESULTS. Ninety-eight percent of isolated cells were immunolabeled with Thy 1.1 antibody. Chronic exposure to 500 µM glutamate decreased the number of surviving large and small RGCs, compared to control conditions. This glutamate-induced excitotoxicity was mediated through both NMDA and non-NMDA GluRs. In neuroprotective studies, ACh, nicotine, and choline significantly reduced glutamate-induced excitotoxicity in adult pig RGCs through {alpha}-Bgt-sensitive nicotinic ACh receptors (nAChRs).

DISCUSSION. This was the first report of a modified panning technique to isolate adult pig RGCs. Cell viability was relatively high using this method, and both large and small RGCs grew extensive neurites in culture. The finding that both NMDA and non-NMDA GluRs were involved in glutamate-induced excitotoxicity suggests that isolated pig RGCs provide a good model for glaucoma. In addition, activation of AChRs may be useful in protecting RGC from excitotoxic insults occurring in neurodegenerative diseases such as glaucoma.




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