Characterization of a transformed rat retinal ganglion cell line

Raghu Krishnamoorthy, P. Agarwal, G. Prasanna, K. Vopat, W. Lambert, H. J. Sheedlo, Iok-Hou Pang, D. Shade, R. J. Wordinger, Thomas Yorio, Abbot Clark, N. Agarwal

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Abstract

The purpose of the present study was to establish a rat retinal ganglion cell line by transformation of rat retinal cells. For this investigation, retinal cells were isolated from postnatal day 1 (PN1) rats and transformed with the ψ2 E1A virus. In order to isolate retinal ganglion cells (RGC), single cell clones were chosen at random from the transformed cells. Expression of Thy-1 (a marker for RGC), glial fibrillary acidic protein (GFAP, a positive marker for Muller cells), HPC-1/syntaxin (a marker for amacrine cells), 8A1 (a marker for horizontal and ganglion cells) and neurotrophins was studied using reverse transcriptase-polymerase chain reaction (RT-PCR), immunoblotting and immunocytochemistry. One of the retinal cell clones, designated 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 8A1, 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 for Thy-1, negative for GFAP, 8A1 and syntaxin. RGC 5 cells were also positive for the expression of neurotrophins and their cognate receptors. To establish the physiological relevance of RGC-5, the effects of serum/trophic factor 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 supplementation with both BDNF and NT-4 in the growth media, protected the RGC-5 cells from undergoing apoptosis. On differentiation with succinyl concanavalin A (sConA), RGC-5 cells became sensitive to glutamate toxicity, which could be reversed by inclusion of ciplizone (MK801). In conclusion, a transformed rat retinal cell line, RGC-5, has certain characteristics of retinal ganglion cells based on Thy-1 and Brn-3C expression and its sensitivity to glutamate excitotoxicity and neurotrophin withdrawal. These cells may be valuable in understanding of retinal ganglion cell biology and physiology including in vitro manipulations in experimental models of glaucoma.

Original languageEnglish
Pages (from-to)1-12
Number of pages12
JournalMolecular Brain Research
Volume86
Issue number1-2
DOIs
StatePublished - 31 Jan 2001

Fingerprint

Retinal Ganglion Cells
Cell Line
Nerve Growth Factors
Clone Cells
Apoptosis
Glutamic Acid
Reverse Transcriptase Polymerase Chain Reaction
Immunoblotting
Serum
Immunohistochemistry
Syntaxin 1
Qa-SNARE Proteins
GABA-B Receptors
Ependymoglial Cells
Amacrine Cells
Cell Physiological Phenomena
Synaptophysin
DNA Nucleotidylexotransferase
Glial Fibrillary Acidic Protein
Brain-Derived Neurotrophic Factor

Keywords

  • Apoptosis
  • Excitotoxicity
  • Glaucoma
  • Glial fibrillary acidic protein
  • Neurotrophin
  • Serum deprivation
  • Thy-1
  • ψ2E1A virus

Cite this

Krishnamoorthy, R., Agarwal, P., Prasanna, G., Vopat, K., Lambert, W., Sheedlo, H. J., ... Agarwal, N. (2001). Characterization of a transformed rat retinal ganglion cell line. Molecular Brain Research, 86(1-2), 1-12. https://doi.org/10.1016/S0169-328X(00)00224-2
Krishnamoorthy, Raghu ; Agarwal, P. ; Prasanna, G. ; Vopat, K. ; Lambert, W. ; Sheedlo, H. J. ; Pang, Iok-Hou ; Shade, D. ; Wordinger, R. J. ; Yorio, Thomas ; Clark, Abbot ; Agarwal, N. / Characterization of a transformed rat retinal ganglion cell line. In: Molecular Brain Research. 2001 ; Vol. 86, No. 1-2. pp. 1-12.
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abstract = "The purpose of the present study was to establish a rat retinal ganglion cell line by transformation of rat retinal cells. For this investigation, retinal cells were isolated from postnatal day 1 (PN1) rats and transformed with the ψ2 E1A virus. In order to isolate retinal ganglion cells (RGC), single cell clones were chosen at random from the transformed cells. Expression of Thy-1 (a marker for RGC), glial fibrillary acidic protein (GFAP, a positive marker for Muller cells), HPC-1/syntaxin (a marker for amacrine cells), 8A1 (a marker for horizontal and ganglion cells) and neurotrophins was studied using reverse transcriptase-polymerase chain reaction (RT-PCR), immunoblotting and immunocytochemistry. One of the retinal cell clones, designated 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 8A1, 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 for Thy-1, negative for GFAP, 8A1 and syntaxin. RGC 5 cells were also positive for the expression of neurotrophins and their cognate receptors. To establish the physiological relevance of RGC-5, the effects of serum/trophic factor 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 supplementation with both BDNF and NT-4 in the growth media, protected the RGC-5 cells from undergoing apoptosis. On differentiation with succinyl concanavalin A (sConA), RGC-5 cells became sensitive to glutamate toxicity, which could be reversed by inclusion of ciplizone (MK801). In conclusion, a transformed rat retinal cell line, RGC-5, has certain characteristics of retinal ganglion cells based on Thy-1 and Brn-3C expression and its sensitivity to glutamate excitotoxicity and neurotrophin withdrawal. These cells may be valuable in understanding of retinal ganglion cell biology and physiology including in vitro manipulations in experimental models of glaucoma.",
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Krishnamoorthy, R, Agarwal, P, Prasanna, G, Vopat, K, Lambert, W, Sheedlo, HJ, Pang, I-H, Shade, D, Wordinger, RJ, Yorio, T, Clark, A & Agarwal, N 2001, 'Characterization of a transformed rat retinal ganglion cell line', Molecular Brain Research, vol. 86, no. 1-2, pp. 1-12. https://doi.org/10.1016/S0169-328X(00)00224-2

Characterization of a transformed rat retinal ganglion cell line. / Krishnamoorthy, Raghu; Agarwal, P.; Prasanna, G.; Vopat, K.; Lambert, W.; Sheedlo, H. J.; Pang, Iok-Hou; Shade, D.; Wordinger, R. J.; Yorio, Thomas; Clark, Abbot; Agarwal, N.

In: Molecular Brain Research, Vol. 86, No. 1-2, 31.01.2001, p. 1-12.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Characterization of a transformed rat retinal ganglion cell line

AU - Krishnamoorthy, Raghu

AU - Agarwal, P.

AU - Prasanna, G.

AU - Vopat, K.

AU - Lambert, W.

AU - Sheedlo, H. J.

AU - Pang, Iok-Hou

AU - Shade, D.

AU - Wordinger, R. J.

AU - Yorio, Thomas

AU - Clark, Abbot

AU - Agarwal, N.

PY - 2001/1/31

Y1 - 2001/1/31

N2 - The purpose of the present study was to establish a rat retinal ganglion cell line by transformation of rat retinal cells. For this investigation, retinal cells were isolated from postnatal day 1 (PN1) rats and transformed with the ψ2 E1A virus. In order to isolate retinal ganglion cells (RGC), single cell clones were chosen at random from the transformed cells. Expression of Thy-1 (a marker for RGC), glial fibrillary acidic protein (GFAP, a positive marker for Muller cells), HPC-1/syntaxin (a marker for amacrine cells), 8A1 (a marker for horizontal and ganglion cells) and neurotrophins was studied using reverse transcriptase-polymerase chain reaction (RT-PCR), immunoblotting and immunocytochemistry. One of the retinal cell clones, designated 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 8A1, 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 for Thy-1, negative for GFAP, 8A1 and syntaxin. RGC 5 cells were also positive for the expression of neurotrophins and their cognate receptors. To establish the physiological relevance of RGC-5, the effects of serum/trophic factor 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 supplementation with both BDNF and NT-4 in the growth media, protected the RGC-5 cells from undergoing apoptosis. On differentiation with succinyl concanavalin A (sConA), RGC-5 cells became sensitive to glutamate toxicity, which could be reversed by inclusion of ciplizone (MK801). In conclusion, a transformed rat retinal cell line, RGC-5, has certain characteristics of retinal ganglion cells based on Thy-1 and Brn-3C expression and its sensitivity to glutamate excitotoxicity and neurotrophin withdrawal. These cells may be valuable in understanding of retinal ganglion cell biology and physiology including in vitro manipulations in experimental models of glaucoma.

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KW - Glial fibrillary acidic protein

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