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Nell2 is a neuron-specific protein containing six epidermal growth factor-like domains. We have identified Nell2 as a retinal ganglion cell (RGC)-expressed gene by comparing mRNA profiles of control and RGC-deficient rat retinas. The aim of this study was to analyze Nell2 expression in wild-type and optic nerve axotomized retinas and evaluate its potential role in RGCs. Nell2-positive in situ and immunohistochemical signals were localized to irregularly shaped cells in the ganglion cell layer (GCL) and colocalized with retrogradely-labeled RGCs. No Nell2-positive cells were detected in 2 weeks optic nerve transected (ONT) retinas characterized with approximately 90% RGC loss. RT-PCR analysis showed a dramatic decrease in the Nell2 mRNA level after ONT compared to the controls. Immunoblot analysis of the Nell2 expression in the retina revealed the presence of two proteins with approximate MW of 140 and 90 kDa representing glycosylated and non-glycosylated Nell2, respectively. Both products were almost undetectable in retinal protein extracts two weeks after ONT. Proteome analysis of Nell2-interacting proteins carried out with MALDI-TOF MS (MS) identified microtubule-actin crosslinking factor 1 (Macf1), known to be critical in CNS development. Strong Macf1 expression was observed in the inner plexiform layer and GCL where it was colocalizied with Thy-1 staining. Since Nell2 has been reported to increase neuronal survival of the hippocampus and cerebral cortex, we evaluated the effect of Nell2 overexpression on RGC survival. RGCs in the nasal retina were consistently more efficiently transfected than in other areas (49% vs. 13%; n = 5, p

Purpose: To evaluate the effectiveness of selective laser trabeculoplasty (SLT) on steroid-induced glaucoma., Methods: The study included 46 eyes of 41 subjects who were followed up for at least 12 months after SLT. The included 10 eyes with steroid-induced glaucoma, 16 eyes with primary open angle glaucoma (POAG), 10 eyes with pseudoexfoliation glaucoma (PEX.G) and 10 eyes with mixed glaucoma (Mixed. G). The range of the SLT laser was 360 degrees. Intraocular pressure (IOP) before and after SLT, and cumulative survival rate after SLT were determined., Results: Significant decreases in IOP were observed after SLT in the steroid-induced glaucoma group, the POAG group and the PEX.G group. At 12 months after SLT, preoperation IOP decreased by 35.9% (29.9 +/- 7.5 mmHg to 17.9 +/- 2.2 mmHg) in the steroid-induced glaucoma group, 13.2% (20.0 +/- 3.0 mmHg to 17.3 +/- 3.1 mmHg) in the POAG group, 10.7% (21.1 +/- 4.0 mmHg to 18.1 +/- 4.1 mmHg) in the PEX.G group and 6.9% (21.3 +/- 1.9 mmHg to 19.9 +/- 3.4 mmHg) in the Mixed.G group. Cumulative survival rates were 80%, 56.3%, 50.0%, 40.0% in the steroid-induced glaucoma, POAG, PEX.G, and Mixed. G groups, respectively, at 12 months after SLT (Logrank test, p = 0.467)., Conclusion: These data suggest that SLT increased IOP reduction rates for steroid-induced glaucoma more than for any other group.

Purpose: Oxidative damage is implicated in retinal ganglion cell (RGC) death after optic nerve transection (ONT) and in glaucoma. We analyzed the expression and protective effects of thioredoxins (TRXs), regulators of the cellular reduction-oxidative (redox) state, in RGCs damaged by pharmacologically induced oxidative stress, ONT, and elevated intraocular pressure (IOP)., Methods: ONT and glaucoma models in the rat were used. The glaucoma model was generated in albino rats by intracameral injection of india ink followed by ab externo laser applications to the pigmented trabecular band. Retrograde labeling of RGCs was performed with dextran tetramethylrhodamine. RGC isolation from rat retinas was performed with magnetic beads coated with Thy-1 monoclonal antibody. Immunoblot analysis, RGC-5 culture and transfection, and cell viability assays were used. Gene delivery was performed with in vivo electroporation., Results: Endogenous levels of thioredoxin-2 (TRX2) in RGCs after axotomy and in RGC-5 cells after glutamate/buthionine sulfoximine (BSO) treatment were up-regulated. An increased level of TRX-interacting protein (TXNIP) in the retina was observed 2 and 5 weeks after IOP elevation. TRX1 levels were decreased at 2 weeks and more prominently at 5 weeks after IOP elevation. No change in TRX2 levels in response to IOP elevation was detected. Overexpression of TRX1 and TRX2 in RGC-5 treated with glutamate/BSO increased the cell survival by twofold and threefold 24 and 48 hours after treatment, respectively. Overexpression of these proteins in the retina in vivo increased the survival of RGCs by 35% and 135% at 7 and 14 days after ONT, respectively. In hypertensive eyes, RGC loss was approximately 27% after 5 weeks of IOP elevation compared to controls. TRX1 and TRX2 overexpression preserved approximately 45% and 37% of RGCs, respectively, in the glaucoma model compared to controls., Conclusion: Thioredoxin overexpression protects RGCs from death after optic nerve axotomy, in pharmacologically induced oxidative stress in vitro and in an animal model of glaucoma.

Purpose: Stress-induced crystallin expression is commonly viewed as activation of the cell survival mechanism. The authors analyzed the expression of alphaA- and alphaB-crystallins in a rat optic nerve transection (ONT) model characterized by specific retinal ganglion cell (RGC) degeneration and determined their role in RGC survival., Methods: ONT was performed on adult Wistar rats. Quantitative and spatial expression were examined with Western blot analysis and immunohistochemistry, respectively. Electroporation was used to deliver alphaA and alphaB expression constructs to RGCs. Cell-protective effects of alphaA and alphaB overexpression after ONT were determined by RGC density analysis., Results: Expression of alphaA and alphaB in the retina was observed predominantly in the ganglion cell layer, where most crystallin-positive cells were colocalized with RGCs. Levels of alphaA and alphaB proteins after ONT were decreased 1.6-fold. The effect of alphaA and alphaB overexpression on RGC survival was evaluated 7 and 14 days after axotomy. At day 7 after ONT, 1426 +/- 70 and 1418 +/- 81 RGCs/mm(2) were present in retinas electroporated with alphaA and alphaB expression constructs, respectively, compared with 1010 +/- 121 RGCs/mm(2) in sham-transfected or 1016 +/- 88 RGCs/mm(2) in nontransfected retinas. Numbers of surviving RGCs at 14 days were 389 +/- 57 and 353.57 +/- 60 cells/mm(2) after alphaA and alphaB transfection, respectively, compared with 198 +/- 29 cells/mm(2) after transfection with the vector alone or 206 +/- 60 cells/mm(2) in nontransfected retinas., Conclusions: Increases of approximately 95% and 75% in RGC survival mediated by alphaA and alphaB overexpression, respectively, were observed 14 days after ONT. At day 7, the RGC protective effect of alphaA and alphaB overexpression was approximately 40%.

We investigated whether the neuroprotective effect of estrogen is mediated by the estrogen receptor (ER) and whether extracellular signal-regulated kinase (ERK) is involved in the protective effect of estrogen against N-methyl-D-aspartate (NMDA)-induced retinal neurotoxicity. Retrograde labeling of retinal ganglion cells (RGCs) showed that pretreatment with 17beta-estradiol (E2) using a silastic implant significantly attenuated the loss of RGCs induced by intravitreal injection of NMDA. Simultaneous administration of U0126, an ERK inhibitor, with NMDA completely abolished the protective effect of E2. Moreover, ICI182,780, an ER antagonist, also significantly diminished the protective effect of E2. Pretreatment with E2 significantly reduced the number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells in the retinal ganglion cell layer (RGCL) and the inner nuclear layer (INL) 12 hr after NMDA injection. Moreover, ICI182,780 inhibited the ameliorative effect of E2 on TUNEL-positive cells in a dose-dependent manner. Immunostaining of anti-ERalpha monoclonal antibody was observed mainly in the RGCL and the INL. Western blot analysis showed a significant increase in the level of phosphorylated ERK (p-ERK) 6 hr after NMDA injection. However, NMDA did not increase the level of p-ERK protein 7 days after injection. Pretreatment of E2 induced further increases of p-ERK expression 6 hr and 7 days after NMDA injection, and U0126 and ICI182,780 significantly inhibited E2-induced p-ERK expression after 6 hr. These results suggest that E2 has an ER-mediated neuroprotective effect against NMDA-induced retinal neurotoxicity and that this effect may be associated with induction of p-ERK in the retina.

Transcription factors of the nuclear factor-kappa B (NF-kappaB)/Rel family may be involved in neuronal cell death or survival. We examined the role of NF-kappaB p65 in N-methyl-D-aspartate (NMDA)-induced neurotoxicity in the rat retina. Western blot analysis showed that elevated levels of retinal NF-kappaB p65 protein at days 1 and 5 after intravitreal NMDA injection. Immunohistochemistry localized increased NF-kappaB p65 immunoreactivity in the ganglion cell layer (GCL) and the inner nuclear layer (INL) after NMDA injection especially in retinal ganglion cells (RGCs), displaced amacrine cells, and amacrine cells. Concomitant with the early increase in NF-kappaB p65 protein levels, there was an increase in NF-kappaB DNA binding activity after NMDA injection as shown by electrophoretic mobility shift assay (EMSA). These increases in NF-kappaB p65 protein levels and NF-kappaB DNA binding activity were totally abolished by simultaneous injection of NF-kappaB p65 antisense oligodeoxynucleotide (AS ODN). A partial but significant protective effect on the inner retina was noted when the AS ODN was given together with NMDA as shown by morphological analysis, morphometry of cells in the GCL and morphometry of inner plexiform layer thickness as well as quantitative real-time PCR of Thy-1 mRNA levels. These results suggest that activated NF-kappaB p65 may participate in NMDA-induced retinal neuronal cell death and that inhibition of NF-kappaB activation such as the use of AS ODN may be a viable neuroprotective strategy for protective RGCs and other inner retinal neurons.