This study used organ cultures to examine whether retinal ganglion cells (RGCs) retain their ability to regenerate axons in
buphthalmos. A rat mutant with hereditary
buphthalmos was used to (1) determine whether the extent of RGC loss corresponds to the severity and duration of elevated intraocular pressure (IOP), (2) examine whether RGCs exposed to an elevated IOP are able to regenerate their axons in a retina culture model, and (3) analyze the
proteome of the regenerating retina in order to identify putative regeneration-associated
proteins. Retrograde labeling of RGCs revealed a decrease in their numbers in the retinas of buphthalmic eyes that increased with age. Quantification of axons growing out of
retinal explants taken at different stages of the disease demonstrated that buphthalmic RGCs possess a remarkable potential to regrow axons. As expected, immunohistochemistry and immunoblotting revealed that elevated IOP was associated with upregulation of certain known
proteins, such as
growth-associated protein 43,
glial fibrillary acidic protein, and
endothelin-1. In addition, two-dimensional
polyacrylamide gel electrophoresis and mass spectrometry revealed several spots corresponding to
proteins that were specifically regulated when buphthalmic RGCs were permitted to regrow their axons. Out of the
proteins identified,
heat-shock protein (HSP)-60 was constantly expressed during axonal growth at all stages of the disease.
Antibodies against HSP-60 reduced axonal growth, indicating the involvement of this
protein in regenerative axonal growth. These data are the first to show that diseased retinal neurons can grow their axons, and that HSP-60 supports neuritogenesis. This model may help to elucidate the fundamental mechanisms of
optic neuropathy at stages preceding death caused by chronic injury, and aid in the development of neuroprotective strategies.