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Y. Shinohara, M. Nakatani, M. Hirabayashi, C. Taki, H. Mori, S. Nishimura; Immortalized Rat Müller Cell Line (TR-MUL) Attenuates Neurotrophic Factor Deficient- and Glutamate-Mediated Death of Rat Retinal Ganglion Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):133.
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The purpose of this study was to determine the efficacy of an immortalized rat Müller cell line (TR-MUL) to support the survival of rat retinal ganglion cells (RGCs).
RGCs were isolated from 6-day-old rats by an immunopanning procedure and provided for (i) mixed-culture with TR-MUL or (ii) co-culture on a porous membrane insert, avoiding direct contact with TR-MUL. The cultures were incubated for 48 hours in neurobasal medium under conditions of neurotrophic factor deficiency. Experimental steps included: (1) exposure to 200 µM glutamate for 24 hours with or without methionine sulfoximine, a glutamine synthetase inhibitor; and (2) exposure to anti-ciliary neurotrophic factor (CNTF) neutralizing antibody. The effect on RGC survival was evaluated by counting DiI-labeled RGCs in the mixed culture or calcein-AM-stained RGCs in the co-culture. The mRNA expression levels of brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), and CNTF in TR-MUL were determined by real time quantitative polymerase chain reaction (PCR).
The confluent TR-MUL significantly enhanced RGC survival under conditions of neurotrophic factor deficiency with or without glutamate exposure. Regardless of the presence or absence of glutamate, methionine sulfoximine inhibited the protective effect of TR-MUL significantly in the mixed culture, but the inhibition was less in the co-culture. Compared with primary cultured Müller cells, mRNA expression in TR-MUL was 3.7-fold for CNTF and 0.5-fold for BDNF and GDNF. RGC survival promoted by the co-cultured TR-MUL with glutamate exposure was partially blocked by anti-CNTF neutralizing antibody.
Our results demonstrate that TR-MUL can protect RGCs from both neurotrophic factor deficient and excitoxic conditions. The neuroprotective activity of TR-MUL may be attributed to the release of factors such as CNTF, in addition to glutamate detoxification via glutamine synthesis.
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