Abstract
Purpose::
σRs are non-opioid, non-phencyclidine binding sites with robust neuro-protective properties. Previously, we used the RGC-5 cell line and very high doses (1 mM) of the excitatory amino acid glutamate (Glu) and demonstrated that high doses (10 µM) of the σR1 ligand PTZ could protect against cell death (Martin et al, 2004). The purpose of this study was to establish a physiologically relevant paradigm for testing the neuroprotective effects of PTZ.
Methods::
Primary ganglion cells (1ºGCs) were isolated by immunopanning from 1 day mouse retinas per our method (Dun et al, 2006). They were maintained in culture for 3 days and then exposed to 10, 20, 25 or 50 µM Glu for 6 or 18 h in the presence or absence of PTZ (0.5, 1.0, 3.0 µM). Cell viability was measured using (1) Live/Dead and (2) ApopTag Fluorescein In Situ Assays. Expression of σR1 was assessed by immunohistochemistry, RT-PCR and western blotting.
Results::
1°GCs showed robust σR1 expression. The cells are exquisitely sensitive to Glu toxicity (6 h treatment with 25 µM Glu induced 50% cell death; 50 µM Glu induced 75% cell death). 1ºGCs co-incubated 18 h with 25µM Glu plus 0.5 or 3 µM PTZ showed ~15% attenuation of cell death. 1ºGCs pre-treated 1 h with PTZ followed by 18 h co-treatment with 25 µM Glu + PTZ showed a marked decrease in % cell death: (25 µM Glu alone: 50%; 25 µM Glu + 0.5 µM PTZ: 38%; 25 µM Glu + 1.0 µM PTZ: 20%; 25 µM Glu + 3.0 µM PTZ: 18%). σR1 mRNA expression increased slightly after 4 h exposure to 25 µM Glu and protein levels increased after 18 h exposure to 25 µM Glu.
Conclusions::
1ºGCs express σR1. Their marked sensitivity to Glu toxicity mimics the sensitivity observed in vivo making them a highly relevant model for testing neuroprotection. Pre-treatment of cells with 1-3 µM PTZ affords significant protection against Glu-induced cell death. σR1 ligands may be very useful therapeutic tools in retinal diseases such as glaucoma and diabetic retinopathy in which ganglion cells die.
Keywords: neuroprotection • ganglion cells • excitatory neurotransmitters