Murilo F. Roggia

Impact of Robotic Assistance on Precision of Vitreoretinal Surgical Procedures

Purpose To elucidate the merits of robotic application for vitreoretinal maneuver in comparison to conventional manual performance using an in-vitro eye model constructed for the present study. Methods Capability to accurately approach the target on the fundus, to stabilize the manipulator tip just above the fundus, and to perceive the contact of the manipulator tip with the fundus were tested. The accuracies were compared between the robotic and manual control, as well as between ophthalmologists and engineering students. Results In case of manual control, ophthalmologists were superior to engineering students in all the 3 test procedures. Robotic assistance significantly improved accuracy of all the test procedures performed by engineering students. For the ophthalmologists including a specialist of vitreoretinal surgery, robotic assistance enhanced the accuracy in the stabilization of manipulator tip (from 90.9 µm to 14.9 µm, P = 0.0006) and the perception of contact with the fundus (from 20.0 mN to 7.84 mN, P = 0.046), while robotic assistance did not improve pointing accuracy. Conclusions It was confirmed that telerobotic assistance has a potential to significantly improve precision in vitreoretinal procedures in both experienced and inexperienced hands.

Protective Role of Glutathione Peroxidase 4 in Laser-Induced Choroidal Neovascularization in Mice

Purpose To evaluate the influence of glutathione peroxidase 4 (GPx4) expression in retinal pigment epithelium (RPE)/choroid tissue using a mouse model of laser-induced choroidal neovascularization (CNV). Methods In this study, GPx4+/−, GPx4+/+, and GPx4-overexpressing transgenic mice were created for comparison. The mRNA and protein expression of vascular endothelial growth factor (VEGF)-A in RPE/choroid tissue were evaluated before and after CNV induction by laser. Moreover, we investigated the changes in the VEGF-A mRNA level in RPE/choroid tissue in the CNV model that have not been clearly shown previously. Lipid peroxidation in RPE/choroid tissue was evaluated by immunohistochemistry using antibody against 4-hydroxy-2-nonenal. To investigate the protective role of GPx4, the size of laser-induced CNV was compared on day 7 among the mice expressing different levels of GPx4. Results In the laser-induced CNV mouse model, laser treatment reduced the VEGF-A mRNA level in RPE/choroid tissue, while it increased the VEGF-A protein level. Evaluation of VEGF-A expression in RPE/choroid tissue of the GPx4+/−, GPx4+/+, and GPx4 transgenic mice revealed that GPx4 increased the VEGF-A protein level under physiological conditions (i.e., without laser treatment), while GPx4 suppressed the increase in the VEGF-A protein level under pathological conditions (i.e., after CNV induction by laser). In addition, GPx4 reduced the CNV size in a dose-dependent manner in vivo. Conclusions GPx4 suppresses the increase in the VEGF-A protein level, which occurs during the development of pathological CNV, thus partly explaining the protective effect of GPx4 against CNV.

αvβ5 Integrin/FAK/PGC-1α Pathway Confers Protective Effects on Retinal Pigment Epithelium

Purpose To elucidate the mechanism of the induction of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) by photoreceptor outer segments (POS) and its effects on retinal pigment epithelium (RPE). Methods PGC-1α upregulation by POS was confirmed in ARPE-19 cells and in RPE ex vivo. To elucidate the mechanism, siRNAs against β5 integrin, CD36, Mer tyrosine kinase (MerTK), and Atg5, blocking antibodies against CD36 and MerTK, and a specific inhibitor for focal adhesion kinase (FAK) were used. We examined the effect of POS-induced PGC-1α upregulation on the levels of reactive oxygen species (ROS), mitochondrial biogenesis, senescence-associated β-galactosidase (SA-β-gal) after H2O2 treatment, and lysosomal activity. Lysosomal activity was evaluated through transcriptional factor EB and its target genes, and the activity of cathepsin D. Lipid metabolism after POS treatment was assessed using Oil Red O and BODIPY C11. RPE phenotypes of PGC-1α-deficient mice were examined. Results POS-induced PGC-1α upregulation was suppressed by siRNA against β5 integrin and a FAK inhibitor. siRNAs and blocking antibodies against CD36 and MerTK enhanced the effect of POS on PGC-1α. The upregulation of PGC-1α increased the levels of mRNA for antioxidant enzymes and stimulated mitochondrial biogenesis, decreased ROS levels, and reduced SA-β-gal staining in H2O2-treated ARPE-19 cells. PGC-1α was critical for lysosomal activity and lipid metabolism after POS treatment. PGC-1α-deficient mice demonstrated an accumulation of type 2 lysosomes in RPE, thickening of Bruch’s membrane, and poor choriocapillaris vasculature. Conclusions The binding, but not the internalization of POS confers protective effects on RPE cells through the αvβ5 integrin/FAK/PGC-1α pathway.

EFFECT OF INTERNAL LIMITING MEMBRANE PEELING DURING VITRECTOMY FOR DIABETIC MACULAR EDEMA: Systematic Review and Meta-analysis.

Purpose: To evaluate the effect of internal limiting membrane (ILM) peeling during vitrectomy for diabetic macular edema. Methods: MEDLINE, EMBASE, and CENTRAL were systematically reviewed. Eligible studies included randomized or nonrandomized studies that compared surgical outcomes of vitrectomy with or without ILM peeling for diabetic macular edema. The primary and secondary outcome measures were postoperative best-corrected visual acuity and central macular thickness. Meta-analysis on mean differences between vitrectomy with and without ILM peeling was performed using inverse variance method in random effects. Results: Five studies (7 articles) with 741 patients were eligible for analysis. Superiority (95% confidence interval) in postoperative best-corrected visual acuity in ILM peeling group compared with nonpeeling group was 0.04 (−0.05 to 0.13) logMAR (equivalent to 2.0 ETDRS letters, P = 0.37), and superiority in best-corrected visual acuity change in ILM peeling group was 0.04 (−0.02 to 0.09) logMAR (equivalent to 2.0 ETDRS letters, P = 0.16). There was no significant difference in postoperative central macular thickness and central macular thickness reduction between the two groups. Conclusion: The visual acuity outcomes using pars plana vitrectomy with ILM peeling versus no ILM peeling were not significantly different. A larger randomized prospective study would be necessary to adequately address the effectiveness of ILM peeling on visual acuity outcomes.

Role of Glutathione Peroxidase 4 in Glutamate-Induced Oxytosis in the Retina

Purpose The purpose of the present study was to investigate the role of glutathione peroxidase 4 (GPx4) in glutamate-induced oxytosis in the retina. Methods For in vitro studies, an immortalized rat retinal precursor cell line R28 was used. Cells were transfected with siRNA specifically silencing GPx4 or with scrambled control siRNA. Lipid peroxidation was evaluated by 4-hydroxy-2-nonenal (4-HNE) immunostaining. Cytotoxicity and cell death were evaluated using an LDH activity assay and annexin V staining, respectively. Cells transfected with GPx4 siRNA or control siRNA were treated with glutamate (1 or 2 mM), and the cytotoxicity was evaluated using the LDH activity assay. For in vivo studies, retinal ganglion cell damage was induced by intravitreal injection of 25-mM N-methyl-D-aspartate (NMDA, 2 μL/eye) in GPx4+/+ and GPx4+/− mice. The evaluation of lipid peroxidation (4-HNE immunostaining), apoptosis (TUNEL staining), and cell density in the ganglion cell layer (GCL) were performed at 12 h, 1 day, and 7 days after the NMDA injection. Results GPx4 knockdown significantly increased LDH activity by 13.9-fold (P < 0.01) and increased peroxidized lipid levels by 3.2-fold in R28 cells (P < 0.01). In cells transfected with scrambled control siRNA, treatment with glutamate at 1 or 2 mM did not increase LDH activity; whereas, in cells transfected with GPx4 siRNA, glutamate treatment significantly increased LDH activity (1.52-fold, P < 0.01). GPx4+/− mice exhibited higher levels of lipid peroxidation in retinas treated with NMDA than GPx4+/+ mice (1.26-fold, P < 0.05). GPx4+/− mice had more TUNEL-positive cells induced by NMDA in GCL (1.45-fold, P < 0.05). In addition, the cell density in GCL of GPx4+/− mice was 19% lower than that in GPx4+/+ mice after treatment with NMDA (P < 0.05). Conclusion These results suggest that defective GPx4 expression is associated with enhanced cytotoxicity by glutamate-induced oxytosis in the retina.

SUPPLEMENTAL SCLERAL BUCKLE IN VITRECTOMY FOR THE REPAIR OF RHEGMATOGENOUS RETINAL DETACHMENT: A Systematic Review of Literature and Meta-Analysis.

Purpose: To evaluate the effect of supplemental scleral buckle (SB) in pars plana vitrectomy (PPV) for rhegmatogenous retinal detachment. Methods: MEDLINE, EMBASE, and CENTRAL were searched to identify studies comparing PPV with supplemental SB (PPV + SB) to PPV alone for the repair of rhegmatogenous retinal detachment. The outcome measures were primary and final reattachment rates, and postoperative complications. Odds ratio with 95% confidence interval in random effects for the comparison of outcomes between PPV + SB and PPV alone was calculated. Results: Ten studies consisting of 1,704 patients were included. Meta-analysis showed that the overall primary reattachment rate was significantly higher in PPV + SB than PPV alone (odds ratio, 1.70; 95% confidence interval, 1.21–2.39; P = 0.002). The final reattachment rate was equally high in both groups. Postoperative development of epiretinal membrane was more frequent in PPV + SB than in PPV alone (odds ratio, 1.89; 95% confidence interval, 1.30–2.76; P = 0.001), whereas no significant difference in postoperative development of macular edema, proliferative vitreoretinopathy, or elevation of intraocular pressure was found. Conclusion: Supplemental SB increases the primary reattachment rate in PPV for rhegmatogenous retinal detachment, although final reattachment rate was equally high with or without SB.

Oxidative stress induces ferroptotic cell death in retinal pigment epithelial cells

ABSTRACT The dysfunction and cell death of retinal pigment epithelial (RPE) cells are hallmarks of late‐stage dry (atrophic) age‐related macular degeneration (AMD), for which no effective therapy has yet been developed. Previous studies have indicated that iron accumulation is a source of excess free radical production in RPE, and age‐dependent iron accumulation in RPE is accelerated in patients with dry AMD. Although the pathogenic role of oxidative stress in RPE in the development of dry AMD is widely accepted, the mechanisms of oxidative stress‐induced RPE cell death remain elusive. Here, we show that ferroptotic cell death, a mode of regulated necrosis mediated by iron and lipid peroxidation, is implicated in oxidative stress‐induced RPE cell death in vitro. In ARPE‐19cells we observed that the ferroptosis inhibitors ferrostatin‐1 and deferoxamine (DFO) rescued tert‐butyl hydroperoxide (tBH)‐induced RPE cell death more effectively than inhibitors of apoptosis or necroptosis. tBH‐induced RPE cell death was accompanied by the three characteristics of ferroptotic cell death: lipid peroxidation, glutathione depletion, and ferrous iron accumulation, which were all significantly attenuated by ferrostatin‐1 and DFO. Exogenous iron overload enhanced tBH‐induced RPE cell death, but this effect was also attenuated by ferrostatin‐1 and DFO. Furthermore, mRNA levels of numerous genes known to regulate iron metabolism were observed to be influenced by oxidative stress. Taken together, our observations suggest that multiple modes of cell death are involved in oxidative stress‐induced RPE cell death, with ferroptosis playing a particularly important role. HIGHLIGHTSOxidative stress‐induced RPE cell death is rescued by ferroptosis inhibitors.Lipid peroxidation, glutathione depletion, and Fe2+ accumulation are observed.Iron overload enhances oxidative stress‐induced RPE cells death.Gene expressions of iron metabolism regulators are affected by oxidative stress in RPE cells.

The extent of stretched lamellar cleavage and visual acuity in macular pseudoholes

Background/aims To evaluate the extent of lamellar cleavage and its association with preoperative and postoperative visual acuity (VA) in macular pseudoholes. Methods One eye each of 50 patients with macular pseudohole who underwent vitrectomy was retrospectively investigated. Preoperative macular pseudoholes were evaluated using spectral-domain optical coherence tomography (SD-OCT) images taken radially around the central fovea at 30° intervals. The macular pseudoholes were categorised into stage 1 (no cleavage), stage 2 (localised cleavage with (2b) and without (2a) crossing central fovea) and stage 3 (diffuse cleavage). Results Among the 50 macular pseudoholes, 14, 13, 9 and 14 were categorised into stages 1, 2a, 2b and 3, respectively. The extent of stretched cleavages was associated with worse baseline VA (p=0.0049 by multiple regression model). After surgery, the stretched lamellar cleavage disappeared in 32 patients out of 36 who were postoperatively examined by SD-OCT. In addition, the extensive cleavage (stage 2b/3) independently predicted larger postoperative VA recovery at 3 months by 0.105 logMAR compared with no/mild cleavage (stage 1/2a, p=0.030 by multiple regression model). Conclusions Although advanced cleavage in macular pseudohole is associated with worse VA before surgery, even an advanced pseudohole could show favourable visual recovery after surgery.