Immunochemical changes of calbindin, calretinin and SMI32 in ischemic retinas induced by increase of intraocular pressure and by middle cerebral artery occlusion

The reaction of neuroactive substances to ischemic conditions in the rat retina evoked by different methods was immunochemically evaluated in adult Sprague-Dawley rats. Ocular ischemic conditions were unilaterally produced by elevating intraocular pressure (EIOP) or by middle cerebral artery occlusion (MCAO). Two EF-hand calcium binding proteins, calbindin D28K (CB) and calretinin (CR), in the normal retina showed similar immunolocalization, such as the amacrine and displaced amacrine cells, the ganglion cells, and their processes, particularly CB in horizontal cells. CB immunoreactive neurons in the ganglion cell layer in both types of ischemic retinas were more reduced in number than CR neurons compared to those in a normal retina. The CB protein level in both ischemic retinas was reduced to 60-80% of normal. The CR protein level in MCAO retinas was reduced to about 80% of normal but increased gradually to the normal value, whereas that in the EIOP showed a gradual reduction and a slight recovery. SMI32 immunoreactivity, which detects a dephosphorylated epitope of neurofilaments-M and -H, appeared in the axon bundles of ganglion cells in the innermost nerve fiber layer of normal retinas. The reactivity in the nerve fiber bundles appeared to only increase slightly in EIOP retinas, whereas a moderate increase occurred in MCAO retinas. The SMI32 protein level in MCAO retinas showed a gradual increasing tendency, whereas that in the EIOP showed a slight fluctuation. Interestingly, the MCAO retinas showed additional SMI32 immunoreactivity in the cell soma of presumed ganglion cells, whereas that of EIOP appeared in the M$\ddot{u}$ller proximal radial fibers. Glial fibrillary acidic protein (GFAP) immunoreactivity appeared in the astrocytes located in the nerve fiber layer of normal retinas. Additional GFAP immunoreactivity appeared in the Muller glial fibers deep in EIOP retinas and at the proximal end in MCAO retinas. These findings suggest that the neurons in the ganglion cell layer undergo degenerative changes in response to ischemia, although EIOP retinas represented a remarkable M$\ddot{u}$ller glial reaction, whereas MCAO retinas had only a small-scaled axonal transport disturbance.