Characterization of a transformed rat retinal ganglion cell line

The purpose of the present study was to establish a rat retinal ganglion ce ll line by transformation of rat retinal cells. For this investigation, ret inal cells were isolated from postnatal day 1 (PN1) rats and transformed wi th the psi2 E1A virus. In order to isolate retinal ganglion cells (RGC), si ngle cell clones were chosen at random from the transformed cells. Expressi on of Thy-1 (a marker for RGC), glial fibrillary acidic protein (GFAP, a po sitive marker for Muller cells), HPC-1/syntaxin (a marker for amacrine cell s), 8A1 (a marker for horizontal and ganglion cells) and neurotrophins was studied using reverse transcriptase-polymerase chain reaction (RT-PCR), imm unoblotting and immunocytochemistry. One of the retinal cell clones, design ated RGC-5, was positive for Thy-1, Brn-3C, Neuritin, NMDA receptor, GABA-B receptor, and synaptophysin expression and negative for GFAP, HPC-1, and 8 A1, suggesting that it represented a putative RGC clone. The results of RT- PCR analysis were confirmed by immunocytochemistry for Thy-1 and GFAP. Upon further characterization by immunoblotting, the RGC-5 clone was positive f or Thy-1, negative for GFAP, 8A1 and syntaxin. RGC 5 cells were also positi ve for the expression of neurotrophins and their cognate receptors. To esta blish the physiological relevance of RGC-5, the effects of serum/trophic fa ctor deprivation and glutamate toxicity were analyzed to determine if these cells would undergo apoptosis. The protective effects of neurotrophins on RGC-5 after serum deprivation was also investigated. Apoptosis was studied by terminal deoxynucleotidyl transferase-mediated fluoresceinated dUTP nick end labeling (TUNEL). Serum deprivation resulted in apoptosis and suppleme ntation with both BDNF and NT-4 in the growth media, protected the RGC-5 ce lls from undergoing apoptosis. On differentiation with succinyl concanavaIi n A (sConA), RGC-5 cells became sensitive to glutamate toxicity, which coul d be reversed by inclusion of ciplizone (MK801). In conclusion, a transform ed rat retinal cell line, RGC-5, has certain characteristics of retinal gan glion cells based on Thy-1 and Brn-3C expression and its sensitivity to glu tamate excitotoxicity and neurotrophin withdrawal. These cells may be valua ble in understanding of retinal ganglion cell biology and physiology includ ing in vitro manipulations in experimental models of glaucoma. (C) 2001 Els evier Science B.V. All rights reserved.