Alterations in the corneal nerve and stem/progenitor cells in diabetes: preventive effects of insulin-like growth factor-1 treatment.

This study aimed to investigate whether corneal nerve and corneal stem/progenitor cells are altered in insulin-like growth factor-I (IGF-I-) treated individuals with diabetes. A group consisting of db/db mice with type 2 diabetes mellitus (DM) and a wild-type group were assessed by neural and corneal stem/progenitor cell markers immunostaining and real-time PCR. Moreover, the expression of corneal nerve and stem/progenitor cell markers was examined in IGF-1-treated diabetic mice. Compared with a normal cornea, swelling and stratification of the corneal epithelium were noted in db/db mice. Beta-III tubulin immunostaining revealed that the corneal subbasal plexuses in diabetic mice were thinner with fewer branches. mRNA expression levels of Hes1, Keratin15, and p75 (corneal stem/progenitor cell markers) and the intensity and number of positive cells of Hes1 and Keratin19 immunostaining diminished in the diabetic corneas. Compared with the subbasal nerve density in the normal group, a decrease in the diabetic group was observed, whereas the corneal subbasal nerve density increased in IGF-1-treated diabetic group. The decreased expression of Hes1 and Keratin19 was prevented in IGF-1-treated diabetic group. Our data suggest that corneal nerve and stem/progenitor cells are altered in type 2 DM, and IGF-I treatment is capable of protecting against corneal damage in diabetes.

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.

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.

Axonal protection via modulation of the amyloidogenic pathway in tumor necrosis factor-induced optic neuropathy.

PURPOSE: To examine the changes in and localization of phosphorylated presenilin1 (p-PS1) and amyloid precursor protein (APP) in the optic nerve after intravitreal injection of TNF and to investigate the role of γ-secretase in the cleavage of APP in optic nerve degeneration. METHODS: Groups of rats were euthanatized at 1 or 2 weeks after intravitreal injection of TNF. Levels of p-PS1 protein in the optic nerve were determined by immunoblotting and immunohistochemistry. The localization of APP was determined by immunohistochemistry, and its downstream cleavage was determined by immunoprecipitation using 6E10 antibody followed by immunoblotting with an APP intracellular domain (AICD) antibody. The effect of a γ-secretase inhibitor on TNF-induced optic nerve degeneration was determined by counting the number of axons. RESULTS: p-PS1 was increased in the optic nerve after TNF injection and was found to colocalize with vimentin and glial fibrillary acidic protein, markers of astrocytes. Immunoprecipitation using 6E10 antibody followed by immunoblotting with AICD antibody revealed an increase in γ-secretase activation in the optic nerve after TNF injection, which was inhibited by treatment with the γ-secretase inhibitor. Moreover, γ-secretase inhibition significantly prevented the loss of axons in the optic nerve after TNF injection. CONCLUSIONS: The increase in p-PS1 and activation of γ-secretase in the optic nerve may be associated with TNF-induced axonal degeneration. Modulation of γ-secretase activity may be useful for the treatment of TNF-related optic neuropathy.

[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.

Effects of a long-acting ophthalmic formulation of carteolol containing alginic acid on the corneal epithelial barrier function and water retentive effect.

PURPOSE: Effects of a long-acting ophthalmic formulation of carteolol containing alginic acid on the corneal epithelial barrier function and its water retentive effect were investigated. METHODS: Using 10 healthy adult subjects, 2% Mikelan Ophthalmic Solution(®) (MK) was instilled in the eye once daily for 7 days (MK group) and then after a washout period of at least 28 days, 2% Mikelan LA Ophthalmic Solution(®) (MKLA) was instilled in the eye once daily for 7 days (LA group). As an index of the corneal epithelial barrier function, the fluorescein uptake was measured using Kowa FL-500. A Schirmer test was conducted to evaluate the tear dynamics. In another 10 subjects, 0.5% Timoptol(®) (TM) was instilled in the eye unilaterally twice daily for 7 days (TM group), and the tests were conducted in the same manner. RESULTS: Concerning the fluorescein uptake before and after initiation of instillation, the levels before and at 7 days after initiation of instillation were 20.7 and 26.5 ng/mL, respectively, in the LA group and 20.6 and 26.4 ng/mL, respectively, in the MK group, showing no significant difference between levels before and after initiation of instillation in either group. In the TM group, the levels were 21.4 and 65.5 ng/mL, respectively, showing a significant increase after initiation of instillation. In the Schirmer test, the values before and after initiation of instillation were 16.8 and 20.7 mm, respectively, in the LA group and 13.7 and 12.7 mm, respectively, in the MK group, showing a trend toward increase in the LA group. CONCLUSIONS: The findings suggest that the long-acting ophthalmic formulation of carteolol containing alginic acid does not affect the corneal epithelial barrier function and that it may possess a water retentive action.

17ß-estradiol prevents reduction of retinal phosphorylated 14-3-3 zeta protein levels following a neurotoxic insult.

Previous studies demonstrated the substantial protective role of 17ß-estradiol (E2) in several types of neuron, although its mechanism of action remains to be elucidated. In this study, we found that the levels of 14-3-3 zeta mRNA and phosphorylated and total 14-3-3 zeta proteins were significantly decreased in the rat retina after intravitreal injection of N-methyl-d-aspartate (NMDA). 17ß-E2 implantation significantly inhibited NMDA-induced decreases in phosphorylated but not in total 14-3-3 zeta protein levels in the retina. There was a decrease in both phosphorylated and total 14-3-3 protein levels in RGC-5 cells, a retinal ganglion cell line, after glutamate and buthionine sulfoximine (BSO) exposure, and 17ß-E2 treatment significantly inhibited only the decrease in phosphorylated but not in total 14-3-3 zeta protein levels. The cell viability assay showed substantial cell death after glutamate and BSO exposure and that 17ß-E2 treatment significantly protects against this cell death. 17ß-E2 treatment also significantly increased the level of phosphorylated 14-3-3 protein in RGC-5 cells without other treatments. These results suggest that a decrease in 14-3-3 zeta expression may be associated with retinal neurotoxicity induced by NMDA or the combination of glutamate and BSO. The regulation of 14-3-3 zeta phosphorylation is one possible mechanism of the protective effect of 17ß-E2 in the retina.

Imaging of a severe case of acute hydrops in a patient with keratoconus using anterior segment optical coherence tomography.

AIM: To investigate the clinical and diagnostic findings of a patient with acute hydrops using anterior segment optical coherence tomography (AS-OCT). METHODS: The AS-OCT findings of a 43-year-old patient with acute hydrops associated with keratoconus were examined. At the initial examination and during follow-up, evaluation of the anterior segment was performed. RESULTS: The patient presented with decreased visual acuity, pain, and redness in the right eye. The symptoms, clinical presentation, and topographical findings of the right eye confirmed the diagnosis of acute corneal hydrops. Changes in the stroma and Descemet's membrane during the healing process of acute hydrops could be demonstrated by high-resolution AS-OCT. The use of contact lenses was improved at the last follow-up visit after 8 months and increased visual acuity to 20/20 with correction. CONCLUSIONS: AS-OCT is a useful tool for studying the morphologic features of acute hydrops.

Axonal protection by 17ß-estradiol through thioredoxin-1 in tumor necrosis factor-induced optic neuropathy.

Axonal degeneration often leads to the death of neuronal cell bodies. Previous studies demonstrated the substantial protective role of 17ß-estradiol (E2) in several types of neuron. However, most studies examined cell body protection, and the role of 17ß-E2 in axonal degeneration of retinal ganglion cells (RGC) remains unclear. In this study, we showed the presence of thioredoxin-1 (Trx1) in the optic nerve axons and found that the levels of Trx1 protein were significantly decreased in isolated RGC and the optic nerve after intravitreal injection of TNF, which was shown previously to induce optic nerve degeneration and subsequent loss of RGC. These changes were concomitant with disorganization of the microtubules with neurofilament accumulation, which were blocked by 17ß-E2 implantation. 17ß-E2 treatment also totally abolished TNF-induced decreases in Trx1 protein levels in isolated RGC and the optic nerve. The induction of Trx1 by 17ß-E2 in the optic nerve was significantly inhibited by simultaneous injection of Trx1 small interfering RNA (siRNA) with TNF. Up-regulation of Trx1 by 17ß-E2 in RGC-5 cells was prevented by Trx1 siRNA treatment. 17ß-E2 significantly prevented TNF-induced axonal loss, and this axonal-protective effect was inhibited by intravitreal injection of Trx1 siRNA. This finding was also supported by the quantification of microtubules and neurofilaments. These results suggest that a Trx1 decrease in RGC bodies and their axons may be associated with TNF-induced optic nerve axonal degeneration. Axonal protection by 17ß-E2 may be related to its regulatory effect on Trx1 induction.

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.

Kinesin-1 and degenerative changes in optic nerve axons in NMDA-induced neurotoxicity.

We examined the histologic findings of optic nerve axons and changes in kinesin-1, which is involved in axonal flow, in N-methyl-d-aspartate (NMDA)-induced neurotoxicity in rats. Substantial degenerative changes visualized as black profiles and pale large axons were observed 72h after NMDA injection, but those degenerative changes were not apparent in axons 12 and 24h after injection. Morphometric analysis showed a significant, approximately 40% reduction in the number of axons 72h after NMDA injection. Immunohistochemical study showed that there was a recognizable loss of neurofilament-immunopositive dots, but myelin basic protein immunostaining was unchanged 72h after NMDA injection. Western blot analysis showed early elevation of kinesin-1 (KIF5B) protein levels in the retina 24 and 72h after NMDA injection. Conversely, significant decreases in KIF5B protein levels in the optic nerve were seen during the same time course. Immunohistochemical study also showed that there was a reduction in KIF5B immunoreactivity in axons, but neurofilament immunostaining was unchanged 24h after NMDA injection. These findings suggest that the intravitreal injection of NMDA causes neurofilament loss without myelin alteration in the early stage. The depletion of kinesin-1 precedes axonal degeneration of the optic nerve in NMDA-induced neurotoxicity.

Induction of corneal epithelium-like cells from cynomolgus monkey embryonic stem cells and their experimental transplantation to damaged cornea.

PURPOSE: We previously reported the successful transplantation of corneal epithelium-like cells derived from mouse embryonic stem (ES) cells onto injured mouse cornea. Here, we tested whether nonhuman primate ES cells have ability to differentiate into corneal epithelial cells and whether monkey ES cell-derived corneal epithelium-like cells were applicable for the experimental transplantation to damaged cornea. METHODS: Monkey ES cells were cultivated on type IV collagen-coated dishes for various days to induce differentiation into corneal epithelium-like cells. The differentiation was evaluated by reverse transcription-polymerase chain reaction and immunostaining. The corneal epithelium-like cells were transplanted to the injured mouse cornea. Reconstitution of the corneal epithelium was evaluated by immunostaining. RESULTS: The cells cultured on type IV collagen showed cobblestone-like appearance resembling epithelial cells. They expressed messenger RNA of pax6, p63, E-cadherin, CD44, proliferating cell nuclear antigen, keratin 3, and keratin 12. Protein expressions of pax6, keratin 3/12, p63, proliferating cell nuclear antigen, E-cadherin, and CD44 were confirmed by immunostaining. When the corneal epithelium-like cells were transplanted, they adhered to the corneal stroma, leading to formation of multiple cell layers. The grafted cells were stained with anti-human nuclear protein antibody, which cross-reacted with nuclei of monkey cells but not with those of mouse cells. They retained the expressions of keratin 3/12, E-cadherin, and CD44. CONCLUSIONS: We induced corneal epithelium-like cells from monkey ES cells with moderate efficiency. The cells were successfully transplanted onto the injured mouse cornea. This is the first demonstration that nonhuman primate ES cells were induced to differentiate into corneal epithelium-like cells, which were applicable for transplantation to an animal model of corneal injury.

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.

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.

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.

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.

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.

Asymmetric dimethylarginine (ADMA) in the aqueous humor of diabetic patients.

Asymmetric dimethylarginine (ADMA) is an endogenous NO synthase (NOS) inhibitor whose production is enhanced by oxidative stress. Recent studies have shown that ADMA may also directly stimulate the production of reactive oxygen species (ROS) by up-regulation of the renin-angiotensin system independently of NOS inhibition. In this study, to investigate the clinical association of ADMA with diabetic retinopathy, we evaluated the levels of ADMA and NO oxides (NO2- and NO3-) in serum and aqueous humor obtained during cataract surgery from non-diabetic subjects (n = 21) and diabetic patients (n = 17). We found that the ADMA existed in aqueous humor and its level was similar to that in serum. The ADMA levels in both serum and aqueous humor were higher in diabetic patients, especially those with severe retinopathy, than in the non-diabetic group (serum ADMA: 0.67 +/- 0.26 vs. 0.53 +/- 0.08 micromol/l, p<0.05; aqueous humor ADMA: 0.55 +/- 0.20 vs. 0.32 +/- 0.16 micromol/l, p<0.05). Also, the aqueous humor level of ADMA, but not the serum level, was correlated with HbA1c on analysis of all the patients (R = 0.33, p<0.05 by simple regression analysis). However, a correlation between the ADMA levels in serum and aqueous humor was not observed in either the non-diabetic group or the diabetic group. Furthermore, serum and aqueous humor levels of NOx did not differ between the two groups, and no correlation with ADMA levels was observed in either group. These results suggest that ROS production may be enhanced in the eyes of diabetics. Since ADMA may act to potentiate ROS production independently of its inhibition of NOS, further investigation is required to clarify the possible contribution of ADMA to the development or progression of retinopathy.