DBA/2J (D2) mice develop a form of progressive pigmentary glaucoma with increasing age. We have compared retinal cell populations of D2 mice with those in control C57BL/6J mice to provide information on retinal histopathology in the D2 mouse. The D2 mouse retina is characterized by a reduction in retinal thickness caused mainly by a thinning of the inner retinal layers. Immunocytochemical staining for specific inner retinal neuronal markers, viz., calbindin for horizontal cells; protein kinase C (PKC) and recoverin for bipolar cells, glycine, gamma-aminobutyric acid (GABA), choline acetyltransferase (ChAT), and nitric oxide synthase (NOS) for amacrine cells, and osteopontin (OPN) for ganglion cells, was performed to detect preferentially affected neurons in the D2 mouse retina. Calbindin, PKC, and recoverin immunoreactivities were not significantly altered. Amacrine cells immunoreactive for GABA, ChAT, and OPN were markedly decreased in number, whereas NOS-immunoreactive amacrine cells increased in number. However, no changes were observed in the population of glycine-immunoreactive amacrine cells. These findings indicate a significant loss of retinal ganglion and some amacrine cells, whereas glycinergic amacrine cells, horizontal, and bipolar cells are almost unaffected in the D2 mouse. The reduction in amacrine cells appears to be attributable to a loss of GABAergic and particularly cholinergic amacrine cells. The increase in nitrergic neurons with the consequent increase in NOS and NO may be important in the changes in the retinal organization that lead to glaucomain D2 mice. Thus, the D2 mouse retina represents a useful model for studying the pathogenesis of glaucoma and mechanisms of retinal neuronal death and for evaluating neuroprotection strategies.

Sodium nitroprusside (SNP), an NO donor, was studied for its effects on apoptosis in rat retinal neurons. TUNEL-positive cells were observed in the outer nuclear layer (ONL), but not in the inner retina after SNP treatment. Inner retinal neurons died by necrosis. No photoreceptor cells were found in the ONL after seven days. Immunoblotting confirmed that neurnal NO synthase expression increased up to 5 days (approximately 170% of control levels), and then declined by 7 days, suggesting that NO induces apoptosis in the ONL, and that inner retinal neurons die by necrosis due to glutamate from damaged photoreceptors.

We have investigated the expression and cellular localization of clusterin in the rat retina following ischemia induced by transiently increasing the intraocular pressure. In the normal retina, weak clusterin immunoreactivity was visible in Müller cell profiles located in the inner nuclear layer. Following ischemia and reperfusion, strong immunoreactivity appeared in Müller cell somata and processes up to 3 days postlesion. Quantitative evaluation by immunoblotting confirmed that clusterin expression continuously increased and showed a peak value at 3 days after ischemic injury (to 1300% of control levels), and then decreased again to 400% of controls at 4 weeks postlesion. Immunocytochemistry using antisera against clusterin or glutamine synthase combined with the TUNEL method or immunocytochemistry using antisera activated caspase 3 and electron microscopy revealed that some clusterin-labeled Müller cells underwent apoptotic cell death. Our findings demonstrate that some Müller cells die by apoptosis, and suggest that clusterin produced and released by Müller cell may play an important role in the pathogenesis of ischemic injury in the rat retina.

We investigated the expression and cellular localization of growth-associated protein (GAP)-43 in the rat retina following ischemia induced by transiently increased intraocular pressure. In the normal retina, GAP-43 immunoreactivity was restricted to profiles in the inner plexiform layer. Following ischemia and reperfusion, immunoreactivity appeared in ganglion cells. The cell density of labeled ganglion cells peaked three days post-lesion and then decreased at seven days. Quantitative evaluation by immunoblotting confirmed that GAP-43 expression increased at three days (to 190% of control levels) and then slightly decreased at seven days. Our findings suggest that some ganglion cells have the potential to regenerate through the up-regulation of GAP-43 in the ischemic rat retina.

Using immunoblot analysis and immunocytochemistry, we investigated expression and cellular localization of endothelial nitric oxide synthase (eNOS) and proliferating cell nuclear antigen (PCNA) in the l-arginine treated ischemic rat retina. In parallel, we tested whether the blood-retinal barrier was intact by immunocytochemistry using an antiserum against IgG. In the l-arginine-treated ischemic retina, the magnitude of the increased eNOS was higher, and PCNA was expressed in endothelial cells as well as in neurons in the inner retina during the whole experimental period. Finally, IgG leakage was not detectable in the l-arginine-treated ischemic retina. Our results clearly suggest that the increased NO production by eNOS may be essential for the survival of endothelial cells in the rat retina following transient ischemia.