Photoreceptor outer segment length: a prognostic factor for idiopathic epiretinal membrane surgery.

PURPOSE: To investigate prognostic factors for visual improvement in patients undergoing vitrectomy for epiretinal membrane (ERM) using spectral domain (SD) optical coherence tomography (OCT). DESIGN: Prospective cohort study. PARTICIPANTS: A total of 41 eyes of 38 patients. METHODS: A total of 41 eyes of 38 patients with idiopathic ERM underwent ERM resection. Ophthalmic evaluations included best-corrected visual acuity (BCVA) and OCT parameters before and 1, 3, and 6 months after surgery. Correlations between OCT parameters and BCVA were assessed at each time point. Correlations between postoperative BCVA and preoperative factors were evaluated, including age, preoperative BCVA, photoreceptor outer segment (PROS) length, central foveal thickness (CFT), outer foveal thickness (OFT), and outer nuclear layer thickness (ONLT). The factors influencing postoperative BCVA were evaluated using multiple regression analysis. MAIN OUTCOME MEASURES: The BCVA at 6 months postoperatively. RESULTS: The PROS length had the most significant correlation with BCVA at each time point (baseline: P = 0.0098, r = -0.409; 1 month: P = 0.0002, r = -0.586; 3 months: P < 0.0001, r = -0.642; 6 months: P = 0.0002, r = -0.577). The PROS length 1 month postoperatively was significantly decreased compared with that preoperatively (P = 0.0325), and the PROS length at 3 months recovered to the baseline length. Preoperative BCVA and PROS length were significantly correlated with postoperative BCVA at 6 months (P = 0.0055, r = 0.439 and P = 0.0089, r = -0.414, respectively). Other parameters, including age, CFT, OFT, and ONLT, were not significantly correlated with postoperative BCVA. Multiple regression analysis showed that preoperative PROS length yielded the highest regression coefficient with postoperative BCVA (P = 0.0363, standard regression coefficient = -0.335, overall R(2) = 0.289). CONCLUSIONS: Imaging of PROS length with SD-OCT was found to be a good indicator of BCVA at each time point after surgery and a predictor of postoperative BCVA in patients with idiopathic ERM. The PROS length changes after surgery may indicate surgical injury and restoration of the macular outer layer.

Effects of indocyanine green staining on the recovery of visual acuity and macular morphology after macular hole surgery.

PURPOSE: To evaluate whether indocyanine green (ICG)-assisted internal limiting membrane peeling affects visual outcome and macular morphologic changes in spectral-domain optical coherence tomography images after macular hole (MH) surgery. METHODS: A retrospective analysis was performed of 34 eyes in 34 patients who had undergone surgical treatment for MH. Best-corrected visual acuity (BCVA) and optical coherence tomography parameters including central foveal thickness, length of the external limiting membrane (ELM) defect, and length of the inner segment and outer segment (IS/OS) defect were analyzed pre- and postoperatively. RESULTS: The eyes were divided into 2 groups based on ICG use (ICG+/-). The changes in BCVA did not differ significantly between the 2 groups at 6 months. However, the ICG+ group had poorer changes compared with the ICG- group at 1 and 3 months (p = 0.038, p = 0.012, respectively). Central foveal thickness and ELM defect did not differ between the 2 groups at each period. The IS/OS defect in the ICG+ group was significantly greater at 1 and 3 months than that in the ICG- group (p = 0.026, p = 0.048, respectively). CONCLUSIONS: ICG staining may affect the recovery process of macular morphology and visual acuity in the first several months after MH surgery.

[Effects of selective laser trabeculoplasty treatment in steroid-induced glaucoma].

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.

Modulation of mitochondria in the axon and soma of retinal ganglion cells in a rat glaucoma model.

Mitochondrial abnormality has been implicated in various models of retinal ganglion cell (RGC) degeneration. We investigated modulation of mitochondrial membrane permeability and apoptosis-inducing factor (AIF) translocation in a rat experimental glaucoma model. A decrease in MitoTracker-labeled mitochondria around the lamina area of the optic nerve was observed in the glaucomatous eye. Immunoblot analysis for axonal motor proteins showed that a significant decrease in kinesin 1 and myosin Va levels in the glaucomatous optic nerve. A significant decrease in mitochondrial thioredoxin 2 (Trx2) level was observed in the optic nerve after intraocular pressure (IOP) elevation. Translocation of AIF from the mitochondria to the axoplasm and nucleus was observed in the axon and cell body, respectively. Trx2 over-expression in the mitochondrial membrane of RGC-5 cells inhibited AIF translocation, resulting in cytoprotective effect against neurotoxicity induced by TNF-α/buthionine sulfoximine treatment. In vivo transfection was performed with EGFP-Trx2 plasmid and electroporation. Over-expression of Trx2 in the retina and optic nerve indicated the protective effect against high IOP induced axonal degeneration. Thus, the decreased mitochondrial membrane potential and subsequent AIF translocation were involved in the glaucomatous neurodegeneration. Furthermore, modulation of mitochondria through the inhibition of AIF translocation may become a new treatment strategy for neurodegenerative disease, such as glaucoma.

Transfection with pax6 gene of mouse embryonic stem cells and subsequent cell cloning induced retinal neuron progenitors, including retinal ganglion cell-like cells, in vitro.

OBJECTIVE: It is theoretically possible to induce various cell types, including retinal neurons, from embryonic stem cells (ESCs). pax6 regulates early events in eye development, including the generation of retinal ganglion cells (RGCs). We previously reported the successful induction of corneal epithelial cells from ESCs transfected with the pax6 gene. Here, we attempted to establish cloned RGC-like cells from ESCs transfected with the pax6 gene. METHODS: Undifferentiated mouse ESCs were transfected with pax6 cDNA by electroporation, followed by selection with G418. We conducted limiting-dilution culture of pax6-transfected cells. We expanded the cloned pax6-transfected cells, which expressed nestin and musashi-1, for further characterization in culture media containing fibronectin. The cells were characterized using RT-PCR, immunostaining, electron microscopy, renal subcapsular transplantation assay and Ca imaging. RESULTS: We obtained clonally expanding pax6-transfected cells, all of which were positive for six3, sonic hedgehog (shh), math5, brn3, thy1 and melanopsin, by using several ESCs. When transplanted into a mouse renal capsule, they differentiated into neurons with elongated axons, expressing betaIII tubulin and neurofilament middle chain, and were free from teratoma development. Electron-microscopic examination showed neurotubules and neurofilaments in the axon-like processes of the cloned pax6-transfected cells. High KCl stimulation increased free Ca influx on Ca2+ imaging. CONCLUSIONS: ESCs were applicable for the induction of retinal progenitor cells, including RGC-like cells, by transfection with the pax6 gene and subsequent limiting-dilution culture. Cloned cell lines may be useful to analyze the requirements for retinal progenitor cell differentiation, and our study suggests the clinical application of this cell type.

Axonal and cell body protection by nicotinamide adenine dinucleotide in tumor necrosis factor-induced optic neuropathy.

Axonal degeneration often leads to the death of neuronal cell bodies. Previous studies have demonstrated the crucial role of nicotinamide adenine dinucleotide (NAD) biosynthesis in axonal protection of motor neurons, but the role of nicotinamide mononucleotide adenylyltransferase 1 and NAD in optic nerve degeneration is unclear. Intravitreal injection of tumor necrosis factor (TNF) induces optic nerve degeneration and subsequent loss of retinal ganglion cells. We found that the levels of nicotinamide mononucleotide adenylyltransferase 1 mRNA and protein and of NAD were significantly decreased in the optic nerve after intravitreal injection of TNF in rats. The concomitant disorganization of microtubules with vacuoles and neurofilament accumulations in the axons were blocked by exogenous NAD treatment. Nicotinamide adenine dinucleotide also prevented TNF-induced axonal loss and delayed retinal ganglion cell loss 2 months after TNF injection. Microglia identified by immunohistochemistry were increased in the optic nerves after TNF injection; this increase was inhibited by NAD treatment. These results suggest that axonal nicotinamide mononucleotide adenylyltransferase 1 and NAD declines are associated with TNF-induced optic nerve axonal degeneration and that axonal protection of NAD may be related to its inhibitory effect on microglial activation.

Axonal protection by brain-derived neurotrophic factor associated with CREB phosphorylation in tumor necrosis factor-alpha-induced optic nerve degeneration.

Brain-derived neurotrophic factor (BDNF) is a potent survival and developmental factor that is regulated by cyclic AMP-response element binding protein (CREB) and has a protective effect against retinal ganglion cell (RGC) death. However, the effect of BDNF on the optic nerve axonal degeneration remains to be examined. In this study, we show that intravitreal injection of tumor necrosis factor (TNF)-alpha induces transient increases in phosphorylated-CREB (p-CREB) and BDNF expression in the optic nerve. Administration of exogenous BDNF further increased the p-CREB and endogenous BDNF level and exerted a neuroprotective effect against TNF-alpha-induced axonal loss. The increases in BDNF mRNA and protein induced by TNF-alpha were inhibited significantly by a CRE decoy oligonucleotide. The protective effect of exogenous BDNF on axons was also inhibited by the CRE decoy oligonucleotide. These results suggest that the protective effect of exogenous BDNF may be associated with increases in CREB phosphorylation and endogenous BDNF in the optic nerve.

Effects of unoprostone on phosphorylated extracellular signal-regulated kinase expression in endothelin-1-induced retinal and optic nerve damage.

Endothelin-1 (ET-1), a potent vasoconstrictor peptide, has been implicated in the development of normal- and high-tension glaucoma. We investigated the effects of unoprostone on extracellular signal-regulated kinase (ERK) in ET-1-induced retinal ganglion cell (RGC) death and optic nerve injury. Our morphometric study showed that intravitreal injection of ET-1 led to cell loss in the RGC layer (RGCL) in 28 days. Western blot analysis showed decreased neurofilament (NF) protein in the optic nerve 28 days after ET-1 injection. In this in vivo model, increased phosphorylated ERK (p-ERK) was observed in the retina on 1 day and subsequently in the optic nerve from 7 days after ET-1 injection. Simultaneous injection of M1, as a metabolite of unoprostone, showed further increased p-ERK levels compared with ET-1 injection alone. Our morphometric study of flat-mount preparations stained with cresyl violet or retrograde labeling with a neuro-tracer and Western blot analysis of NF showed that inhibition of ERK phosphorylation led to acceleration of ET-1-induced RGC death and optic nerve damage. In addition, M1 significantly attenuated both RGC loss and the decrease in NF protein induced by ET-1. The protective effects of M1 were significantly inhibited by U0126, an ERK inhibitor. These results suggest that unoprostone has neuroprotective effects against ET-1-induced neuronal injury through ERK phosphorylation.

The additive effects on intraocular pressure of combining nipradilol 0.25% and latanoprost 0.005% ophthalmic solutions: a prospective, randomized, multicenter study.

PURPOSE: To investigate the effectiveness of combining nipradilol 0.25% and latanoprost 0.005% ophthalmic solutions in improving the intraocular pressures (IOPs) in glaucoma patients. METHODS: We divided the 53 patients into two groups, those who had been treated with latanoprost and those who had been treated with nipradilol. We administered to the first group one dose of latanoprost daily for 12 weeks and to the second group one dose of nipradilol daily for 12 weeks. Each group then received both solutions for another 12 weeks; the latanoprost group received nipradilol and the nipradilol group received latanoprost. IOPs were measured at each 4-week visit. RESULTS: In the patients previously treated with latanoprost, the mean IOP was 19.6+/-2.5 mmHg at baseline, and 14.9+/-2.4 mmHg (23.7% reduction) after 12 weeks of latanoprost monotherapy. The addition of nipradilol decreased the IOP to 13.8+/-1.9 mmHg (29.0% reduction). In the group previously treated with nipradilol, the mean IOP was 20.2+/-3.1 mmHg at baseline, and 16.7+/-3.5 mmHg (17.1% reduction) after 12 weeks of nipradilol monotherapy. Addition of latanoprost decreased the IOP to 14.2+/-3.2 mmHg (29.5% reduction). CONCLUSION: Latanoprost and nipradilol are more effective as a combination therapy than each one by itself.

NMDA-induced interleukin-1beta expression is mediated by nuclear factor-kappa B p65 in the retina.

Transcription factors of the nuclear factor-kappa B (NF-kappaB) p65/RelA may be involved in neuronal cell death. We examined the involvement of NF-kappaB p65 in N-methyl-D-aspartate (NMDA)-induced upregulation of interleukin (IL)-1beta, a proinflammatory cytokine, and subsequent neurotoxicity in the rat retina. Immunohistochemistry showed that IL-1beta is localized not only in glial cells, but also in neurons, especially retinal ganglion cells (RGCs) after intravitreal injection of NMDA. Semi-quantitative real-time PCR showed that NMDA induces an increase in IL-1beta mRNA levels. Preinjection of NF-kappaB p65 antisense oligodeoxynucleotide (AS ODN) ameliorated the NMDA-induced increase in IL-1beta mRNA expression. Western blot analysis showed elevated levels of retinal IL-1beta protein 12 h after intravitreal NMDA injection and this elevation was significantly inhibited by NF-kappaB p65 AS ODN. Neurotracer labeling showed that the inhibition of NF-kappaB p65 by AS ODN or siRNA exerted a protective effect against NMDA-induced RGC loss. IL-1beta siRNA also had a protective effect on RGC number in NMDA-treated eyes. Penetration of AS ODN and siRNA to cells in the RGC layer and inner nuclear layer was confirmed after labeling with rhodamine or Cy3. These results suggest that NF-kappaB p65 may participate in the induction of IL-1beta expression in NMDA-induced retinal neuronal cell death and that the inhibition of NF-kappaB p65 and IL-1beta with the use of AS ODN or siRNA may be a viable neuroprotective strategy for RGC survival.

Calcium/calmodulin-dependent protein kinase II regulates the phosphorylation of CREB in NMDA-induced retinal neurotoxicity.

We examined the role of the phosphorylation of calcium/calmodulin-dependent protein kinase II (CaMKII) and cyclic AMP-response element binding protein (CREB) in N-methyl-d-aspartate (NMDA)-induced neurotoxicity in the rat retina. Western blot analysis showed early elevation of phosphorylated CaMKII (p-CaMKII) protein levels and subsequential elevation of phosphorylated CREB (p-CREB) protein after NMDA injection. Immunohistochemistry showed that p-CaMKII was colocalized with Thy-1-positive retinal ganglion cells (RGCs) after NMDA injection. The increase in the p-CaMKII protein level was significantly inhibited by the preinjection of CaMKII small interfering RNA (siRNA), whereas negative control siRNA did not affect. Moreover, the increase in the p-CREB protein level after NMDA injection was also prevented by preinjection of CaMKII siRNA. In addition, our morphometric study of neurotracer retrograde labeling and Thy-1-positive cells showed that CaMKII siRNA significantly accelerated NMDA-induced RGC loss. Furthermore, the prevention of CREB binding by CRE decoy oligonucleotide also exacerbated RGC loss. These results suggest that the activation of CaMKII may regulate CREB phosphorylation and that the transient phosphorylation of CaMKII and CREB may be a neuroprotective response against NMDA-induced neurotoxicity.

Direct, real-time, simultaneous monitoring of intravitreal nitric oxide and oxygen in endotoxin-induced uveitis in rabbits.

Nitric oxide (NO) plays a critical role in the pathogenesis of endotoxin-induced uveitis (EIU). Since NO is a labile free radical, it is difficult to examine the dynamics of NO directly in vivo. In this study, we established a system for direct monitoring of the dynamics of NO and partial pressure of oxygen (pO(2)) in EIU in rabbits. The currents (calculated concentrations) of NO and pO(2) in the vitreous were monitored after the intravitreal injection of lipopolysaccharide (LPS). In addition, the protein concentrations and nitrite levels in the aqueous humor were analyzed. The eyes were enucleated, and a histologic study was performed on their posterior segments. The tissue slices were also immunostained with anti-nitrotyrosine as a marker of peroxinitrite and/or nitrogen-related oxidants. The NO level decreased temporarily after LPS injection and then increased from 1 to 7 h. pO(2) increased temporarily for about 30 min after LPS injection. The change in NO current was inversely proportional to pO(2) after LPS injection and vice versa. The protein concentration and nitrite level after LPS injection increased significantly. These changes were suppressed by pretreatment with N(G)-nitro-l-arginine-methyl-ester. Immunohistochemical study showed enhanced immunoreactivity of nitrotyrosine in the inflamed retina. Since nitrotyrosine was detected, it appears that NO readily reacts with oxygen to produce cytotoxic species, peroxynitrite, and/or nitrogen-related oxidants. This process may be related to the retinal injury in EIU. This monitoring system can provide useful information on dynamic changes in intravitreal NO and pO(2) for understanding EIU.

Experimental transplantation of corneal epithelium-like cells induced by Pax6 gene transfection of mouse embryonic stem cells.

PURPOSE: Corneal epithelial stem cells are deficient in cases of limbal disorders, leading to conjunctival epithelial ingrowth, vascularization, and eventually visual disturbance. We introduced the eye development-associated transcription factor pax6 to embryonic stem (ES) cells and tested whether pax6-transfected cells resembling purified corneal epithelial cells were applicable as a cell source for corneal transplantation. METHODS: pax6 cDNA with green fluorescence protein was electrotransfected to ES cells and the cells were cultured with G418 for 14 days. They were characterized by reverse transcription-polymerase chain reaction and immunohistochemistry. The cells were transplanted onto experimentally damaged mouse corneas. Histologic reconstitution of the corneal epithelium was assessed. RESULTS: pax6-transfected cells formed a monolayer of epithelium-like cells in vitro. They expressed cytokeratin12, a specific keratin of corneal epithelial cells, E-cadherin, and CD44, which are important adhesion molecules of corneal epithelial cells on the cell membrane. They accumulated to make a colony that gave a staining pattern of reticular configuration for cytokeratin 12, E-cadherin, and CD44. When the cells were transplanted onto damaged cornea, they have been kept alive on the cornea. CONCLUSIONS: The purified corneal epithelium-like cells derived from ES cells transfected with pax6 gene adapted to the injured cornea and were kept alive on it. These results suggested application of ES cell-derived corneal epithelial cells for treating corneal injuries.

Involvement of TNF-alpha in glutamate-induced apoptosis in a differentiated neuronal cell line.

We examined the involvement of tumor necrosis factor (TNF)-alpha on glutamate-induced cytotoxicity in a differentiated neuronal cell line. In this study, we used nerve growth factor (NGF)-differentiated PC12h cells. Glutamate cytotoxicity was assessed using the MTS and TUNEL assays. To detect TNF-alpha levels in culture supernatants after glutamate exposure, we used ELISA methods. The involvement of caspase-8, which is downstream from TNF receptor 1 (TNF-R1) in glutamate-induced cytotoxicity, was determined by Western blot analysis. The MTS assay showed that the addition of glutamate resulted in dose-dependent cell death, while the TUNEL assay showed that glutamate induced apoptosis in differentiated PC12h cells in a dose-dependent manner. TNF-alpha levels in the supernatant of glutamate-exposed cells were significantly increased compared with those in unexposed cells. In addition, glutamate caused increases in the levels of caspase-8 protein. The increases in caspase-8 levels were ameliorated by pretreatment with soluble TNF-R1. Moreover, soluble TNF-R1 significantly ameliorated the cell death induced by glutamate. These results suggest that TNF-alpha released from neuronal cells may be associated with glutamate-induced neuronal cell death.

Neuroprotective effect of atrial natriuretic peptide against NMDA-induced neurotoxicity in the rat retina.

Atrial natriuretic peptide (ANP) can regulate aqueous humor production in the eye and has recently been suggested to play some functional roles in the retina. It has also been reported that ANP increases tyrosine hydroxylase (TH) mRNA levels and intracellular dopamine levels in PC12 cells. The effect of ANP on TH levels and the role of ANP in retinal excitotoxicity remain unknown. In this study, we investigated the effects of ANP on TH expression and dopamine levels in rat retina after intravitreal injection of NMDA. Immunohistochemistry localized natriuretic peptide receptor-A (NPRA) in the ganglion cell layer (GCL), the inner nuclear layer (INL) and the outer nuclear layer (ONL) in the rat retina. Quantitative real-time PCR and Western blot analysis showed a dramatic reduction in retinal TH levels 5 days after NMDA injection, while ANP, at a concentration of 10(-4) M, ameliorated this reduction in TH mRNA and TH protein levels. High-performance liquid chromatography (HPLC) analysis showed that NMDA reduced dopamine levels in the retina, and that ANP attenuated this reduction. Moreover, morphological analysis showed that ANP ameliorated NMDA-induced neurotoxicity through NPRA. The ameliorative effect of ANP was inhibited by a dopamine D(1) receptor antagonist. These results suggest that ANP may have a neuroprotective effect through possible involvement of dopamine induction.

Contribution of mitogen-activated protein kinases to NMDA-induced neurotoxicity in the rat retina.

We examined the contributions of the mitogen-activated protein kinases (MAPKs) family [extracellular signal-regulated kinase (ERK), p38 kinase (p38), and c-Jun N-terminal kinase (JNK)] to N-methyl-D-aspartate (NMDA)-induced neurotoxicity in the rat retina. Detection of apoptotic cell death in the retinal ganglion cell layer (RGCL) and the inner nuclear layer (INL) by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) staining began 6 h after intravitreal NMDA (100 nmol) injection and continued to increase thereafter. Western blot analysis showed that phosphorylated MAPKs (p-MAPKs) were expressed in the retina following a temporal manner: maximal expression of phosphorylated ERK (p-ERK) at 1 h, maximal expression of phosphorylated p38 (p-p38) at 6 h, and beginning of phosphorylated JNK (p-JNK) significant increase at 6 h after injection. An immunohistochemical/TUNEL co-localization study showed that p-JNK- and p-p38-positive cells in the RGCL were frequently TUNEL-positive, whereas few p-ERK-positive cells were TUNEL-positive. Moreover, co-injection of inhibitors for JNK (0.2 nmol SP600125) and/or p38 (2.0 nmol SB203580) with NMDA was effective in ameliorating NMDA-induced apoptotic cell loss in the RGCL 12 h after injection, as shown by TUNEL-positive cell counts. These inhibitors also protected the inner retina as shown by morphometric studies such as cell counts in the RGCL and measurement of the IPL thickness 7 days after injection. On the other hand, an ERK inhibitor (2.0 nmol U0126) did not suppress NMDA-induced cell death in the RGCL nor thinning of the IPL. These findings suggest that JNK and p38 are proapoptotic in NMDA-induced cell death in the RGCL, but not ERK.

Induction of epithelial progenitors in vitro from mouse embryonic stem cells and application for reconstruction of damaged cornea in mice.

PURPOSE: Severe ocular surface diseases and injuries cause loss of the corneal limbal epithelium, leading to re-epithelialization by bulbar conjunctival cells, resulting in vascularization of the cornea, conjunctival scarring, and loss of visual acuity. In this study, the optimal culture condition for induction of differentiation of epithelial progenitor cells from embryonic stem (ES) cells was determined for use in transplantation to damaged cornea in mice. METHODS: Mouse ES cells were cultured on Petri dishes coated with several extracellular matrix proteins, and the markers for epithelial cells were analyzed with RT-PCR and Western blot analysis. The optimal condition for induction of epithelial progenitor cells was determined, and the progenitors were transplanted onto mouse eyes with corneal epithelia that had been damaged by exposure to n-heptanol. RESULTS: Epithelial progenitors were successfully induced by culturing mouse ES cells on type IV collagen for 8 days. These progenitors expressed keratin (K)12, which is specific to corneal epithelial cells, and cell surface CD44 and E-cadherin, both of which are essential in corneal epithelial wound healing. Complete re-epithelialization of the corneal surface occurred within 24 hours after transplantation. The resultant corneal epithelial cells expressed markers of the grafted cells, and no teratomata were observed during the follow-up period. CONCLUSIONS: Epithelial progenitors were successfully induced in vitro from ES cells and were applicable as grafts for treating corneal epithelial injury. ES cells may become an unlimited donor source of corneal epithelial cells for corneal transplantation and may restore useful vision in patients with a deficiency of limbal epithelial cells. This is an important first trial toward assessing the use of ES cells to reconstruct corneal epithelial cells.

Nuclear factor-kappa B p65 in NMDA-induced retinal neurotoxicity.

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.

Neuroprotective effect of nitric oxide against NMDA-induced neurotoxicity in the rat retina is associated with tyrosine hydroxylase expression.

N-methyl-D-aspartate (NMDA) may affect dopaminergic cells, which contain tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis. To clarify the involvement of TH in the neuroprotective effects of nitric oxide (NO), we investigated whether NMDA alters TH mRNA and TH protein levels and whether NO inhibits NMDA-induced changes in the rat retina. Dopamine levels in the retina were measured by high-performance liquid chromatography (HPLC). Reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR showed that intravitreal injection of NMDA caused a significant reduction in TH mRNA levels in the retina. Similarly, Western blot analysis showed that NMDA decreased the production of TH protein. These reductions in TH mRNA and TH protein levels were attenuated by concomitant injection of NOC 18, an NO donor. HPLC analysis showed that NMDA reduced dopamine levels in the retina and that NO attenuated this reduction. Furthermore, morphological analysis showed that NO prevents NMDA-induced neurotoxicity through dopamine D(1) receptors. These results suggest that the neuroprotective effect of NO may be associated with the induction of TH expression and increased levels of dopamine.

Involvement of RhoA and possible neuroprotective effect of fasudil, a Rho kinase inhibitor, in NMDA-induced neurotoxicity in the rat retina.

RhoA, a key protein involved in cytoskeleton regulation modulating neurogenesis and neural plasticity, has been implicated in a variety of cellular functions including the modulation of N-methyl-D-aspartate (NMDA) receptor activity. We examined its possible involvement in NMDA-induced excitotoxicity in the retina, and evaluated the neuroprotective effect of fasudil, a Rho kinase inhibitor, in this model of neurotoxicity. RhoA protein levels in NMDA-treated retinas were assessed by Western blot analysis and localized by immunohistochemistry. Fasudil (10(-6)-10(-4) M together with 4 x 10(-2) M NMDA) was given intravitreally and its effect was evaluated by counting the number of cells in the ganglion cell layer (GCL), measuring the thickness of the inner plexiform layer (IPL), and measuring retinal Thy-1 mRNA levels at 5 days after injection. Western blot analysis showed a transient increase in the level of retinal RhoA and ROCKII proteins at 1 day after NMDA injection, and that this increment was significantly prevented by simultaneous injection of fasudil. Immunohistochemistry showed that NMDA induced a substantial increase in RhoA immunoreactivity in the GCL and the IPL. Fasudil injection reduced cell loss in the GCL and the reduction in IPL thickness after NMDA injection. The reduction in Thy-1 mRNA levels by NMDA was also significantly attenuated by concomitant injection of fasudil. These results suggest that RhoA and ROCKII are upregulated and may be involved in NMDA-induced retinal neurotoxicity, and that fasudil is neuroprotective against glutamate-related excitotoxicity.