Yasuya Inomata

Vitreous fluid levels of β-amyloid(1–42) and tau in patients with retinal diseases

PurposeA decrease in β-amyloid1–42 (Aβ42) and an increase in tau in the cerebrospinal fluid are reported to be characteristic phenomena in Alzheimer’s disease patients. To test the idea that Aβ42 and tau contribute to the development of retinal diseases, we measured Aβ42 and tau concentrations in the vitreous fluid from patients with macular hole (n = 13), diabetic retinopathy (n = 15), or glaucoma concurrent with other ocular diseases (n = 8).MethodsVitreous samples were collected from patients who underwent vitrectomy, and sensitive and specific enzyme-linked immunosorbent assays were used to determine the concentrations of Aβ42 and tau.ResultsBy comparison with the levels in the control macular-hole patients (33.9 ± 7.1 pg/ml for Aβ42; 3.3 ± 3.2 pg/ml for tau), there was a significant decrease in the Aβ42 level and a significant increase in the tau level in patients with diabetic retinopathy (1.8 ± 1.9 pg/ml for Aβ42, P = 0.002; 153.7 ± 71.6 pg/ml for tau, P = 0.041) or glaucoma concurrent with other ocular diseases (2.8 ± 1.8 pg/ml for Aβ42, P = 0.006; 113.6 ± 43.1 pg/ml for tau, P = 0.023).ConclusionsOur findings indicate the possibility of a role for Aβ42 and tau in the pathogenesis of some retinal diseases.

Elevated erythropoietin in vitreous with ischemic retinal diseases

The aim of the current study was to measure the concentrations of erythropoietin in the vitreous fluid and analyze its association with vascular endothelial growth factor (VEGF) in ischemic vitreoretinal diseases. Vitreous fluid samples were collected from patients with proliferative diabetic retinopathy, branch retinal vein occlusion and idiopathic macular hole. Concentrations of erythropoietin and VEGF in vitreous fluid were significantly elevated in patients with proliferative diabetic retinopathy and branch retinal vein occlusion as compared to patients with macular hole. There were no differences in serum concentrations of erythropoietin and VEGF among patient groups. There was significant correlation between erythropoietin and VEGF concentrations in vitreous fluid. Erythropoietin was up-regulated in ischemic disorders and may act as an endogenous neuroprotective factor against ischemic retinal disorders.

Persistent subretinal indocyanine green induces retinal pigment epithelium atrophy

PURPOSE To describe a case of a patient with macular hole with subretinal indocyanine green (ICG) during vitrectomy. DESIGN Interventional case report. METHODS A 66-year-old woman with macular hole underwent a vitrectomy with ICG. RESULTS After application of ICG into the vitreous, ICG was introduced in the subretinal space. Indocyanine green was found to be present for more than 6 months. Retinal pigment epithelium atrophy appeared at the site of the lesion. CONCLUSIONS Although ICG may be a useful tool for distinguishing the internal limiting membrane and other tissues careful application is required to prevent side effects.

Neuroprotective effects of interleukin-6 on NMDA-induced rat retinal damage.

This study shows that interleukin-6 (IL-6) combined with soluble interleukin-6 receptors (sIL-6R) modulates N-methyl-D-aspartate (NMDA)-induced retinal damage. Eyes pretreated with a combined injection of IL-6 and sIL-6R had NMDA administered into the vitreous cavity. Morphometric analysis and retrograde labeling analysis found that pretreatment with either IL-6 or sIL-6R alone did not bring about any neuroprotective effect. However, pretreatment with a combined administration of IL-6 and sIL-6R induced a significant neuroprotective effect against NMDA-induced retinal damage. Apoptotic changes in the retina were assessed by the TUNEL method. The results indicated that pretreatment with IL-6 combined with sIL-6R prevents NMDA-induced apoptosis. Western blotting studies demonstrated upregulation of gp130 expression in the NMDA-injected retina. Present studies suggest that IL-6 combined with sIL-6R provides a neuroprotective effect on NMDA-induced retinal damage.

NMDA-induced retinal injury is mediated by an endoplasmic reticulum stress-related protein, CHOP/GADD153

We investigated the role of an endoplasmic reticulum stress‐associated protein, CHOP/GADD153, after NMDA‐induced mouse retinal damage. After injection of NMDA into the vitreous, TUNEL‐positive cells were detected in the retinal ganglion cell layer (GCL) and inner nuclear layer (INL) at 6 h after NMDA injection, and these gradually increased in number up to 24 h. Analysis by real‐time RT‐PCR revealed that CHOP mRNA was induced by about 3‐fold, at 2 h after NMDA injection. Immunoreactivity for the CHOP protein was intense in cells of the GCL following NMDA treatment. Immunoblot analysis showed that NMDA injection increased the expression of CHOP protein in the retina. Compared with wild‐type mice, CHOP–/– mice were more resistant to NMDA‐induced retinal cell death as determined by TUNEL assay. At 7 days after NMDA treatment, the thickness of the inner plexiform layer and INL were larger in CHOP–/– mice than in wild‐type mice. The number of residual cells in the GCL following NMDA treatment was significantly higher in CHOP–/– mice than in wild‐type mice. In conclusion, CHOP is induced in mouse retina by NMDA treatment, and CHOP–/– mice are more resistant to NMDA‐induced retinal damage, suggesting that CHOP plays an important role in NMDA‐induced retinal cell death.

Thioredoxin inhibits NMDA-induced neurotoxicity in the rat retina.

Thioredoxin (TRX) plays a variety of redox‐related roles in organisms. To investigate its function as an endogenous redox regulator in NMDA‐induced retinal neurotoxicity, we injected NMDA with TRX, mutant TRX or saline into the vitreous cavity of rat eyes. Retinal ganglion cells were rescued by TRX, compared with saline, when evaluated by retrograde labeling analysis at 7 days after NMDA injection. TRX, but not its mutant form, prevented NMDA‐induced apoptosis in the retina, as measured by terminal deoxynucleotidyl transferase‐mediated UTP nick‐end labeling. The induction of caspase 3 and 9, but not caspase 8, by NMDA was significantly lower in TRX‐treated eyes than in saline‐treated eyes. NMDA‐induced activation of the MAPKs, p38 kinase and c‐Jun N‐terminal kinase after 6 h and of the MAPK kinases (MKKs) MKK3/6 and MKK4 after 3 h was markedly suppressed in retinal ganglion cells by TRX but not by the mutant form. NMDA‐induced increases in protein carbonylation, nitrosylation and lipid peroxidation were also suppressed in TRX‐treated eyes. We concluded that the intravitreous injection of TRX effectively attenuated NMDA‐induced retinal cell damage and that suppression of oxidative stress and inhibition of apoptotic signaling pathways were involved in this neuroprotection.

Plasmin-assisted vitrectomy for management of proliferative membrane in proliferative diabetic retinopathy: a pilot study.

Purpose: To demonstrate the feasibility of autologous plasmin for treatment of proliferative diabetic retinopathy. Methods: The study examined prospectively six patients with bilateral proliferative diabetic retinopathy. Comparisons of the surgical time and the incidence of retinal tears were made between the eyes treated with autologous plasmin and their respective opposite eyes, which were treated without plasmin. Results: All eyes treated with autologous plasmin required significantly less surgical time (68 versus 89 minutes, P = 0.04, paired t-test). In the plasmin group, no additional surgical procedures for removing the proliferative membrane were needed, including membrane delamination or segmentation. Moreover, with plasmin pretreatment, there were no retinal tears, which was in contrast to the control group, where three eyes with retinal tears were observed. There was no significant difference found between the two groups for final visual outcomes. Conclusion: Autologous plasmin may be beneficial in the surgical management of proliferative diabetic retinopathy.

Suppression of choroidal neovascularization by thioredoxin-1 via interaction with complement factor H.

PURPOSE To examine the role of thioredoxin-1 (TRX-1), an endogenous protein with a variety of redox-related roles, in the formation of choroidal neovascularization (CNV). METHODS CNV was induced by laser photocoagulation of the ocular fundus in wild-type and transgenic mice overexpressing human TRX-1 (TRX-1 Tg). Mice were injected intraperitoneally with TRX-1, mutant TRX, or vehicle. The incidence of CNV was evaluated by lectin staining. Leukocyte recruitment and C3b deposition after laser injury were determined by immunohistochemistry and Western blotting. Moreover, TRX-1-associated proteins from human plasma were isolated by two-dimensional gel electrophoresis with the use of a column coupled with a mutant TRX-1 and were identified by mass spectrometry and proteomics analysis. Complement activation was determined by a fluid-phase METHOD RESULTS The incidence of laser-induced CNV was reduced in TRX-1 Tg mice (56.1%) and in C57B/6 mice treated with TRX-1 (46.7%) but not in mutant TRX-1 (79.2%) compared with wild-type mice (85.7%). Furthermore, leukocyte recruitment was prevented in TRX-1-treated mice; C3b deposition was decreased in these and TRX-1 Tg mice. In human plasma, five proteins associated with TRX-1 were identified as apolipoprotein A-I, the CD5 antigen-like member of the scavenger receptor, cysteine-rich superfamily fibrinogen, albumin, and complement factor H (CFH). TRX-1 inhibited the alternative pathway C3 convertase, and its effect was additive with CFH. CONCLUSIONS These findings show that TRX-1 interacts with CFH, regulates complement activity, and inhibits CNV, suggesting novel preventive and interventional therapeutic strategies for AMD.

Suppression of Choroidal Neovascularization by N-Acetyl-Cysteine in Mice

Purpose: N-acetyl-cysteine (NAC) is a potent antioxidant known to be a precursor of glutathione. The purpose of this study was to investigate the role of NAC in the development of choroidal neovascularization (CNV). Methods: CNV was induced in C57BL/6 mice by laser photocoagulation of the ocular fundus. Mice were injected intraperitoneally with NAC or vehicle alone. The levels of 4-hydoroxy-2-nonenal (4-HNE)-modified protein and nucleus factor (NF)-κB were determined by wester blotting. The recruitment of macrophages and neutrophils after laser injury was analyzed immunohistochemically and in myeloperoxidase (MPO) assays. Enzyme-linked immunosorbent assays (ELISA) were used to measure monocyte chemotactic protein (MCP)-1, CXCL1, vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR)-1, and VEGFR-2. The extent of CNV was evaluated 7 d after laser injury by lectin staining. Results: In NAC-treated mice with laser-induced injuries, the induction of 4-HNE-modified protein after 3 hr and the activation of NF-κB in nuclear extracts after 6 hr were markedly suppressed compared to vehicle-treated mice. Macrophage and neutrophil recruitment were inhibited and the levels of MCP-1, CXCL1, VEGF, and VEGFR-1 were also lower in NAC-treated mice compared to vehicle-treated mice. Furthermore, the extent of CNV induced was significantly lower in NAC-treated compared to vehicle-treated mice (p = 0.027). Conclusions: Our results clearly showed that NAC inhibited indicators of oxidative stress and the activation of NF-κB induced by laser injury, and, consequently, suppressed macrophage and neutrophil infiltration and the development of CNV. This suggests novel preventative and interventional therapeutic strategies for age-related macular degeneration.

Lens epithelium-derived growth factor: neuroprotection on rat retinal damage induced by N-methyl-d-aspartate

The purpose of this study is to investigate possible neuroprotective effects of lens epithelium-derived growth factor (LEDGF) against cell death induced by N-methyl-D-aspartate (NMDA) in the rat retina. LEDGF and/or NMDA were intravitreally injected into rat eyes. NMDA-induced retinal death and protective effects of LEDGF were evaluated by morphometric analysis, cell numbers in the ganglion cell layer (GCL) and the thickness of the inner plexiform layer (IPL). Retrograde labeling with a fluorescent tracer (Fluoro-Gold) was applied for counting retinal ganglion cells (RGCs) that survived after NMDA injection. Terminal deoxyribonucleotidyl transferase (TdT)-mediated fluroscein-16-dUTP nick end-labeling (TUNEL) staining was used to evaluate of retinal cell death. Morphometric analysis and retrograde labeling analysis showed that retinal damage induced by NMDA was protected significantly by LEDGF. TUNEL assay revealed that pretreatment with LEDGF prevents NMDA-induced apoptosis. Retinal damage (ganglion and amacrine cells) induced by NMDA was protected by an intravitreal injection of LEDGF.