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Abstract
PURPOSE: Retinal ganglion cells die by apoptosis after axotomy, and this process may reflect altered expression of cell-death genes. Several of these genes, including bcl-2, bcl-x, and bax, share homology at the amino acid level in the BH1 and BH2 domains, through which they also interact. To understand their role in the neuronal response to axotomy, the authors studied their expression in the adult rat retina and after optic nerve crush. METHODS: An initial survey was conducted with reverse transcription-polymerase chain reaction (RT-PCR), using oligonucleotides against identified members of this family and against the conserved BH1 and BH2 domains. Retinal bcl-xl expression at the messenger RNA (mRNA) and protein level was studied by RT-PCR, Northern blotting, RNase protection analysis, in situ hybridization, Western blotting, and immunofluorescence staining. The effect of retinal ganglion cell axotomy on the steady-state level of bcl-x mRNA was investigated. RESULTS: RT-PCR results indicated that rat retinal cells predominantly express the long form of bcl-x. Both clonal analysis and quantitative measurements using RNase protection assays demonstrated that bcl-xL message was at least 16 times more abundant than that of bcl-2. In situ hybridization and indirect immunofluorescence demonstrated that nearly all neuronal cells of the retina express bcl-x. Northern and RNase protection analyses showed a moderate decrease in bcl-xL message shortly after optic nerve crush. CONCLUSIONS: These findings suggest that the antideath gene bcl-xL is the predominant member of the bcl-2 family in the adult retina, and that its level decreases after optic nerve crush. Changes in bcl-xL expression may correlate with increased retinal ganglion cell apoptosis after axotomy.