Protrusion of KCNJ13 Gene Knockout Retinal Pigment Epithelium Due to Oxidative Stress-Induced Cell Death.

Purpose: This study was performed to elucidate the mechanisms of morphological abnormalities in a Leber congenital amaurosis 16 (LCA16) cell model using KCNJ13 knockout (KO) retinal pigment epithelial cells derived from human iPS cells (hiPSC-RPE). Methods: In KCNJ13 KO and wild-type hiPSC-RPE cells, ZO-1 immunofluorescence was performed, and confocal images were captured. The area and perimeter of each cell were measured. To detect cell death, ethidium homodimer III (EthD-III) staining and LDH assay were used. Scanning electron microscopy (SEM) was used to observe the cell surface. The expression levels of oxidative stress-related genes were examined by quantitative PCR. To explore the effects of oxidative stress, tert-butyl hydroperoxide (t-BHP) was administered to the hiPSC-RPE cells. Cell viability was tested by MTS assay, whereas oxidative damage was monitored by oxidized (GSSG) and reduced glutathione levels. Results: The area and perimeter of KCNJ13-KO hiPSC-RPE cells were enlarged. EthD-III-positive cells were increased with more dead cells in the protruded region. The KO RPE had significantly higher LDH levels in the medium. SEM observations revealed aggregated cells having broken cell surfaces on the KO RPE sheet. The KCNJ13-deficient RPE showed increased expression levels of oxidative stress-related genes and total glutathione levels. Furthermore, t-BHP induced a significant increase in cell death and GSSG levels in the KO RPE. Conclusions: We suggest that in the absence of the Kir.7.1 potassium channel, human RPE cells are vulnerable to oxidative stress and ultimately die. The dying/dead cells form aggregates and protrude from the surviving KCNJ13-deficient RPE sheet.

Graft Size and Double Scroll Formation Rate in Descemet Membrane Endothelial Keratoplasty.

PURPOSE: This study aimed to evaluate the usefulness of intentional double scroll formation of donor Descemet membrane (DM) inside a glass tube inserter (the Fogla technique) in DM endothelial keratoplasty (DMEK) for controlled insertion and unfolding of grafts. METHODS: Eleven consecutive patients who underwent DMEK were included in this study. We sought to specify graft characteristics in which double scroll configuration was successfully formed using the Fogla technique. We compared donor age, graft size, surgical time, unfolding time, and visual outcomes between patients with and without double scroll configuration. The ability to form double scroll formation of DM grafts of various diameters and unfolding time of DM grafts was evaluated using total seven eye-bank eyes in ex vivo experiments. RESULTS: A double scroll configuration inside a glass tube was successfully obtained in six DMEK grafts (54.5%). When comparing clinical features between those with and without double scroll configuration, only graft size was significantly larger in those with double scroll configuration (7.9 ± 0.2 mm) than in those without (7.4 ± 0.4, P = 0.03). There were no significant differences in other features and clinical outcomes, although unfolding-time was shorter in eyes with double scroll configuration (4.6 ± 2.0 min) compared to those without (8.6 ± 8.1, P = 0.21). Ex vivo experiments showed that unfolding time was significantly shorter in double scroll configuration (2.71 ± 0.49 min) than in single scroll (5.02 ± 0.79, P = 0.01). CONCLUSIONS: A double scroll configuration within a glass tube can be obtained more frequently in larger DMEK grafts (8.0 mm in diameter), which may result in easier and faster DMEK procedures.

KCNJ13 Gene Deletion Impairs Cell Alignment and Phagocytosis in Retinal Pigment Epithelium Derived from Human-Induced Pluripotent Stem Cells.

Purpose: The purpose of this study was to establish and analyze a cell model of Leber congenital amaurosis type 16 (LCA16), which is caused by mutations in the KCNJ13 gene encoding Kir7.1, an inward-rectifying potassium ion channel. Methods: The two guide RNAs specific to the target sites in the KCNJ13 gene were designed and KCNJ13 knock-out (KO) human-induced pluripotent stem cells (hiPSCs) were generated using the CRISPR/Cas9 system. The KCNJ13-KO hiPSCs were differentiated into retinal pigment epithelial cells (hiPSC-RPEs). The KCNJ13-KO in hiPSC-RPEs was confirmed by immunostaining. Phagocytic activity of hiPSC-RPEs was assessed using the uptake of fluorescently labeled porcine photoreceptor outer segments (POSs). Phagocytosis-related genes in RPE cells were assessed by quantitative polymerase chain reaction. Results: Most of the translated region of the KCNJ13 gene was deleted in the KCNJ13-KO hiPSCs by the CRISPR/Cas9 system, and this confirmed that the Kir7.1 protein was not present in RPE cells induced from the hiPSCs. Expression of RPE marker genes such as BEST1 and CRALBP was retained in the wild-type (WT) and in the KCNJ13-KO hiPSC-RPE cells. However, phagocytic activity and expression of phagocytosis-related genes in the KCNJ13-null hiPSC-RPE cells were significantly reduced compared to those of WT. Conclusions: We succeeded in generating an RPE model of LCA16 using hiPSCs. We suggest that Kir7.1 is required for phagocytosis of POSs by RPE cells and that impaired phagocytosis in the absence of Kir7.1 would be involved in the retinal degeneration found in LCA16.

Inverted Internal Limiting Membrane Flap versus Internal Limiting Membrane Peeling for Macular Hole Retinal Detachment in High Myopia.

PURPOSE: To compare surgical outcomes between the inverted internal limiting membrane (ILM) flap technique and ILM peeling for macular hole retinal detachment (MHRD) in eyes with high myopia. DESIGN: Multicenter cohort study. PARTICIPANTS: We retrospectively reviewed medical records of consecutive patients treated between June 2008 and September 2018 at 7 hospitals and included 100 eyes with MHRD associated with high myopia in our study. All eyes underwent vitrectomy with the inverted ILM flap technique (57 eyes) or ILM peeling (43 eyes) and were followed up for more than 6 months. METHODS: We estimated odds ratios and their 95% confidence intervals (CIs) for macular hole (MH) closure using multivariate logistic regression analysis. We also examined factors associated with the postoperative best-corrected visual acuity (BCVA) at the final visit using multiple linear regression analysis. MAIN OUTCOME MEASURES: Macular hole closure and postoperative BCVA at the final visit. RESULTS: The MH closure rate was significantly higher in the inverted ILM flap group (80.7%) than in the ILM peeling group (37.2%; P < 0.001). Moreover, postoperative BCVA at the final visit was significantly better in the former group (0.88 ± 0.48 vs. 0.99 ± 0.48; P = 0.03). The retinal attachment rate (ILM flap, 91.2%; ILM peeling, 79.5%; P = 0.229) and recovery rates for the external limiting membrane and ellipsoid zone line (ILM flap, 10.9%; ILM peeling, 0%; P = 0.12) showed no significant intergroup differences. After adjustment for age, axis, tamponade substance, and dye for ILM staining, the inverted ILM flap technique was associated strongly and positively with MH closure (odds ratio, 7.14; 95% CI, 2.72-18.7; P = 0.001). Moreover, the inverted ILM flap technique and preoperative BCVA were associated significantly and positively with the postoperative BCVA at the final visit. CONCLUSIONS: Our findings suggest that the MH closure rate and postoperative visual outcome for eyes with high myopia-associated MHRD are better with the inverted ILM flap technique than with ILM peeling. Thus, vitrectomy with the inverted ILM flap technique should be considered as the initial surgery for MHRD associated with high myopia.

The influence of subretinal injection pressure on the microstructure of the monkey retina.

PURPOSE: To investigate the influence of subretinal injection pressure on the microstructure of the retina in a monkey model. METHODS: After vitrectomy, balanced salt solution was injected subretinally into one eye each of four cynomolgus monkeys while controlling the injection pressure. Initially, a pressure of 2 psi was used, and this was gradually increased to determine the minimum required pressure. Subsequent injections were performed at two pressures: minimum (n = 13) and high (n = 6). To compare the influence of these injection pressures on retinal structure, optical coherence tomography (OCT) was performed before surgery and every week afterwards. The monkeys were euthanized and their eyes were enucleated at 1 or 6 weeks after the injections. The eyes were processed for light microscopy and transmission electron microscopy (TEM) as well as for TdT-mediated dUTP nick end labeling. RESULTS: The minimum pressure required to perform subretinal injection was 6 psi. After injection at this pressure, both OCT and microscopy showed that the retinal structure was well-preserved throughout the experimental period at all injection sites. Conversely, after injection at high pressure (20 psi) OCT images at all injection sites showed disruption of the ellipsoid zone (EZ) after 1 week. Microscopy indicated damage to the photoreceptor outer segment (OS) and stratification of the retinal pigment epithelium (RPE). After 6 weeks, OCT demonstrated that the EZ had become continuous and TEM confirmed that the OS and RPE had recovered. Photoreceptor apoptosis was absent after subretinal injection at both pressures. CONCLUSIONS: The retinal damage caused by subretinal injection increases depending on pressure, indicating that clinicians should perform subretinal injection at pressures as low as possible to ensure safety.