Abstract
Purpose: :
As BH3–only proteins have been shown to play a significant role in regulating cell death in the central nervous system, we wished to investigate the role of Bim, a prominent member of this protein family in the regulation of cell death in the retinal ganglion cell layer (GCL) using in vitro retinal explants
Methods: :
Retinal explant culture of wild–type and Bim knockout mice was carried out as previously described by this laboratory. Western blot and RT–PCR was used to analyse Bim expression following optic nerve axotomy in a purified GCL population. TUNEL staining was employed to identify dying cells at various time points and conditions in both wild–type and Bim knockout animals. The presence of cleaved caspases 3 & 9 was detected using immunohistochemistry of retinal explant sections.
Results: :
We show that in adult wild–type explants, cells in the ganglion cell layer are TUNEL positive at 72 and 96 hours, following axotomy. However, in similar experiments carried out on Bim deficient explants, TUNEL positive cells were absent at all time points investigated. Furthermore, while cleaved caspases 3 and 9 were detected in the GCL of wild–type retinae, staining for these proteases was not present in Bim deficient explants. Finally, the absence of Bim also delayed cell death in the GCL when treated with other death–inducing stimuli.
Conclusions: :
Mice deficient for the BH3 only protein Bim are protected from GCL degeneration induced by various death stimuli in retinal explant culture. These results indicate a key role for this protein in cell death. Apoptosis is most likely blocked upstream of caspase activation as axotomized Bim deficient retinae do not express cleaved caspases unlike their wild–type counterparts.
Keywords: apoptosis/cell death • ganglion cells • retinal degenerations: cell biology