Shinji Toshima

The Role of Inverted Internal Limiting Membrane Flap in Macular Hole Closure

Purpose To investigate the mechanism of macular hole (MH) closure following the inverted internal limiting membrane (ILM) technique. Methods We performed the inverted ILM flap surgical technique as an experimental MH model in monkeys, and investigated the process of MH closure immunohistochemically. We then investigated the effects of type IV collagen, fibronectin, and laminin, which are constituent proteins of the ILM, on the proliferation and migration of cultivated Müller cells (MIO-M1). We also investigated the expression of neurotrophic factors and basic fibroblast growth factor (bFGF) in human ILM and MIO-M1 cells, and the effect of MIO-M1 migration on the expression of these factors, via immunohistochemical staining and the real-time reverse transcription polymerase chain reaction. Results Ten days after inverted ILM flap surgery, the MH had closed and proliferating glial fibrillary acidic protein (GFAP)-positive cells surrounded the ILM. Type IV collagen, fibronectin, and laminin all enhanced the proliferation of MIO-M1 cells, and type IV collagen and fibronectin enhanced the migration of MIO-M1 cells. Neurotrophic factors and bFGF were present on the surface of the human ILM, and MIO-M1 cells produced these factors. Neurotrophic factors and bFGF were expressed to a significantly greater extent by migrating MIO-M1 cells than by these cells in their static state. Conclusions During MH closure, the ILM functioned as a scaffold for the proliferation and migration of Müller cells, and may promote Müller cell activation. Neurotrophic factors and bFGF produced by activated Müller cells and present on the surface of the ILM may contribute to MH closure.

Factors affecting foveal avascular zone in healthy eyes: An examination using swept-source optical coherence tomography angiography

Objective To examine factors affecting foveal avascular zone (FAZ) area in healthy eyes using swept-source optical coherence tomography angiography (OCTA). Methods This prospective, cross-sectional study included 144 eyes of 144 individuals (77 women, 67 men) with a best corrected visual acuity of at least 20/20 and no history of ocular disorders. The area of the superficial FAZ was assessed using OCTA. Age, gender, central retinal thickness (CRT), retinal vascular density, refractive error, and axial length were examined to determine associations with FAZ area. Results The mean age of the subjects was 42.1 ± 20.2 years (range: 10–79 years). The mean FAZ area was 0.32 ± 0.11 mm2, while the mean retinal vascular density was 35.53 ± 0.92%. Multivariate regression analysis was performed using FAZ area as the dependent variable and age, gender, CRT, retinal vascular density, refractive error, and axial length as independent variables. The results of this analysis demonstrate that CRT and retinal vascular density were significantly associated with FAZ area in our sample (P < 0.001, R2 = 0.425). Age, gender, refractive error, and axial length were not significantly correlated with FAZ area, while CRT and retinal vascular density were negatively correlated with FAZ area (CRT: P < 0.001, R2 = 0.356; retinal vascular density: P < 0.001, R2 = 0.189). Conclusions OCTA results suggest that CRT and retinal vascular density negatively affect FAZ area in healthy eyes.

Suppressive effect of AMP-activated protein kinase on the epithelial-mesenchymal transition in retinal pigment epithelial cells

The epithelial-mesenchymal transition (EMT) in retinal pigment epithelial (RPE) cells plays a central role in the development of proliferative vitreoretinopathy (PVR). The purpose of this study was to investigate the effect of AMP-activated protein kinase (AMPK), a key regulator of energy homeostasis, on the EMT in RPE cells. In this study, EMT-associated formation of cellular aggregates was induced by co-stimulation of cultured ARPE-19 cells with tumor necrosis factor (TNF)-α (10 ng/ml) and transforming growth factor (TGF)-β2 (5 ng/ml). 5-Aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), a potent activator of AMPK, significantly suppressed TNF-α and TGF-β2-induced cellular aggregate formation (p < 0.01). Dipyridamole almost completely reversed the suppressive effect of AICAR, whereas 5’-amino-5’-deoxyadenosine restored aggregate formation by approximately 50%. AICAR suppressed the downregulation of E-cadherin and the upregulation of fibronectin and α-smooth muscle actin by TNF-α and TGF-β2. The levels of matrix metalloproteinase (MMP)-2, MMP-9, interleukin-6, and vascular endothelial growth factor were significantly decreased by AICAR. Activation of the mitogen-activated protein kinase and mammalian target of rapamycin pathways, but not the Smad pathway, was inhibited by AICAR. These findings indicate that AICAR suppresses the EMT in RPE cells at least partially via activation of AMPK. AMPK is a potential target molecule for the prevention and treatment of PVR, so AICAR may be a promising candidate for PVR therapy.

Novel Technique for Subretinal Injection Using Local Removal of the Internal Limiting Membrane.

Novel Technique for Subretinal Injection Using Local Removal of the Internal Limiting Membrane Surgical procedures for subretinal injections have been used to treat a variety of retinal conditions, such as the displacement of subretinal hemorrhages, in gene therapy for retinal degeneration, in macular translocation in age-related macular degeneration, in the removal of hard foveal exudates, and in planned foveal detachments to resolve diffuse diabetic macular edema. Currently, these procedures use 38to 41gauge flexible cannulas to inject fluid into the subretinal space. However, the retina is resistant to puncture, making subretinal injections difficult to administer smoothly. Using excessive pressure to puncture the retina can damage it, as well as the retinal pigment epithelium (RPE) and the choroid, and damage to these tissues can lead to retinal and/or subretinal hemorrhage, tears in the RPE, or choroidal neovascularization. One approach to achieving a smooth injection technique is to create a sharp point on the cannula by cutting its tip obliquely so that the retina can be punctured more easily. However, it is still difficult to avoid trauma to the RPE and choroid, which are close to the injection site. We considered the possibility that the major resistance to subretinal injection is due to the internal limiting membrane (ILM) and that removing this at the site where the injection is to be performed might enable a smoother and safer insertion. In this report, we describe this novel technique for subretinal injection with local ILM removal.

Assessment of tilt and decentration of crystalline lens and intraocular lens relative to the corneal topographic axis using anterior segment optical coherence tomography

Purpose To investigate the tilt and decentration of the crystalline lens and the intraocular lens (IOL) relative to the corneal topographic axis using anterior segment ocular coherence tomography (AS-OCT). Methods A sample set of 100 eyes from 49 subjects (41 eyes with crystalline lenses and 59 eyes with IOLs) were imaged using second generation AS-OCT (CASIA2, TOMEY) in June and July 2016 at Okayama University. Both mydriatic and non-mydriatic images were obtained, and the tilt and decentration of the crystalline lens and the IOL were quantified. The effects of pupil dilation on measurements were also assessed. Results The crystalline lens showed an average tilt of 5.15° towards the inferotemporal direction relative to the corneal topographic axis under non-mydriatic conditions and 5.25° under mydriatic conditions. Additionally, an average decentration of 0.11 mm towards the temporal direction was observed under non-mydriatic conditions and 0.08 mm under mydriatic conditions. The average tilt for the IOL was 4.31° towards the inferotemporal direction relative to the corneal topographic axis under non-mydriatic conditions and 4.65° in the same direction under mydriatic conditions. The average decentration was 0.05 mm towards the temporal direction under non-mydriatic conditions and 0.08 mm in the same direction under mydriatic conditions. A strong correlation was found between the average tilt and decentration values of the crystalline lens and the IOL under both non-mydriatic and mydriatic conditions (all Spearman correlation coefficients, r ≥ 0.800; all P < 0.001). Conclusion When measured using second generation AS-OCT, both the crystalline lens and the IOL showed an average tilt of 4–6° toward the inferotemporal direction relative to the corneal topographic axis and an average decentration of less than 0.12 mm towards the temporal direction. These results were not influenced by pupil dilation and they showed good repeatability.

Assessment of Lamellar Macular Hole and Macular Pseudohole With a Combination of En Face and Radial B-scan Optical Coherence Tomography Imaging

PURPOSE To investigate lamellar macular hole (LMH) and macular pseudohole (MPH) using a combination of en face and radial B-scan OCT. DESIGN Retrospective observational case series. METHODS Setting: Institutional study. PATIENT POPULATION En face and radial B-scan OCT images of 63 eyes of 60 patients diagnosed with LMH or MPH based on an international classification were reviewed. OBSERVATION PROCEDURES Cases were classified using en face images based on the presence/absence of epiretinal membrane (ERM), retinal folds, parafoveal epicenter of contractile ERM (PEC-ERM), and retinal cleavage. We compared the en face imaging-based classification system with the international classification system using radial B-scan images. We quantitatively evaluated visual function and macular morphology. MAIN OUTCOME MEASURES Characterization of multimodal OCT-based subtypes of LMH and MPH. RESULTS All cases showed ERM and were classified into 4 groups. In the first group, which lacked retinal folds and showed significantly lower visual acuity than the other groups, 81% of eyes had degenerative LMH. In the second group, which lacked PEC-ERM and retinal cleavage and showed significantly lower retinal fold depth, all eyes had MPH. The third group, in which 95% of eyes had symmetric tractional LMH, included eyes with retinal cleavage but without PEC-ERM, and this group showed higher circularity of the foveal aperture and cleavage area than the group with both these features, in which all eyes had asymmetric tractional LMH. CONCLUSIONS Multimodal OCT enables classification of LMH and MPH based on pathologic conditions. Retinal traction in particular may be useful for determining treatment methods.

Planned Foveal Detachment Technique for the Resolution of Diabetic Macular Edema Resistant to Anti–Vascular Endothelial Growth Factor Therapy

Planned Foveal Detachment Technique for the Resolution of Diabetic Macular Edema Resistant to Anti–Vascular Endothelial Growth Factor Therapy Diabetic macular edema (DME) is the most common cause of vision loss in people with diabetes.1 In recent years, anti–vascular endothelial growth factor (VEGF) reagents have become the first-line treatment for DME based on numerous studies showing their beneficial effects.2–7 However, macular edema is reported to persist in some patients despite multiple intravitreal anti-VEGF injections.8 In addition, patients with diabetes with other systemic diseases, such as cardiovascular disease, cannot tolerate multiple antiVEGF injections over a long treatment period. In such cases, alternative treatments, including vitrectomy, are needed. Although vitrectomy does reduce retinal thickness, one of the major shortcomings of this procedure for the treatment of diffuse DME is that it does not consistently improve visual acuity.9 One possible reason for this is that vitrectomy only has a weak impact on the pathophysiology of DME. Improvement of the condition of the retina after vitrectomy takes time, and during that time, the photoreceptor cells may be damaged. Indeed, vitrectomy only reduces retinal thickness and improves edema gradually.9–14 It has been shown that chronic macular edema leads to permanent photoreceptor dysfunction and poor visual prognosis.15 Furthermore, recent optical coherence tomography (OCT) observations show that a shorter time from the onset of DME to its resolution is the major factor affecting the integrity of the ellipsoid zone and a good visual outcome,16–18 indicating the importance of a rapid resolution of DME after vitrectomy. Takagi et al19 published a procedure to remove foveal hard exudates in diabetic retinopathy by detaching the fovea and flushing out the hard exudates from the retina with a subretinally injected balanced salt solution (BSS). When we performed this procedure, we encountered one case in which we were unable to remove the foveal hard exudates, but the macular edema resolved the day after surgery. We thus hypothesized that subretinal BSS injection might resolve macular edema, and we conducted a pilot study to evaluate the therapeutic efficacy of subretinal BSS injections in conjunction with conventional vitrectomy and internal limiting membrane (ILM) removal for treating diffuse DME.20 We found that this surgical technique promoted a rapid resolution of diffuse DME and an improvement in visual acuity. However, at the time of that study, anti-VEGF therapy for DME was not approved in Japan, so the clinical effect of this surgical procedure on DME resistant to anti-VEGF therapy was unclear. In this study, we evaluated the therapeutic efficacy of subretinal BSS injections in conjunction with conventional vitrectomy for treating diffuse DME resistant to anti-VEGF therapy.

Embedding of lamellar hole-associated epiretinal proliferation combined with internal limiting membrane inversion for the treatment of lamellar macular hole: a case report

BackgroundWe recently reported that lamellar macular hole (LMH) with lamellar hole-associated epiretinal proliferation (LHEP) can be effectively treated by embedding the LHEP into the retinal cleavage to improve foveal contour and visual acuity. Here, we report a case of LMH with LHEP for which we performed embedding of the LHEP combined with internal limiting membrane (ILM) inversion. We then evaluated the effects of this surgery on macular morphology and visual functions.Case presentationA 62-year-old man presented with visual disturbance (20/29) and metamorphopsia in his right eye. B-scan optical coherence tomography (OCT) imaging revealed the presence of both partial-thickness defect of the macula with degenerative retinal cleavage and LHEP at the surface of the retina. En face OCT imaging showed the absence of retinal fold. We performed phacoemulsification with intraocular lens implantation, vitrectomy, embedding of LHEP into the retinal cleavage, and ILM inversion. Three months after the surgery, both foveal contour and visual acuity (20/20) were improved and metamorphopsia was reduced.ConclusionEmbedding of the LHEP combined with ILM inversion may be an effective treatment for LMH with LHEP.

A novel cell exclusion zone assay with a barrier made from room temperature vulcanizing silicone rubber

Objective To examine the usefulness of room temperature vulcanizing (RTV) silicone rubber as a barrier material for cell exclusion zone assays. Methods We created barriers using three types of RTV silicone rubber with differing viscosities. We then assessed the adherence of these barriers to culture dishes and their ease of removal from the dishes. We tested the effect of the newly created barriers on the extracellular matrix (ECM) protein fibronectin by attaching and then removing them from fibronectin-coated culture dishes. We also conducted cell exclusion zone assays with MIO-M1 cells using this new barrier in order to measure cell migration. We used real time reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemical staining to measure the effect of fibronectin on MIO-M1 cell migration and the effect of migration (with fibronectin coating) on basic fibroblast growth factor (bFGF) expression in MIO-M1 cells. Results Of the three types of RTV silicon rubber tested, KE-3495-T was the best in terms of adherence to the dish and ease of removal from the dish. When barrier attachment and removal tests were performed, this rubber type did not have an effect on the fibronectin that coated the dish. In the cell exclusion assay, removal of the barrier revealed that a cell-free area with a distinct margin had been created, which allowed us to conduct a quantitative assessment of migration. Fibronectin significantly promoted the migration of MIO-M1 cells (P = 0.02). In addition, both real time RT-PCR and immunohistological staining indicated that bFGF expression in migrating MIO-M1 cells was significantly higher than that in non-migrating cells (P = 0.03). Conclusions RTV silicone rubber can be used to create an effective barrier in cell exclusion zone assays and allows simple and low-cost multi-parametric analysis of cell migration.

Surgical outcome of idiopathic epiretinal membranes with intraretinal cystic spaces

Objective To investigate the occurrence ratio, localization, and surgical outcomes of intraretinal cystic spaces in idiopathic epiretinal membranes (ERMs). Methods We retrospectively reviewed the charts of 432 eyes of 398 consecutive patients with idiopathic ERM who underwent vitrectomy and ERM peeling from January 2012 to September 2015. We selected cases with intraretinal cystic space prior to surgery, detected by spectral-domain optical coherence tomography. We then evaluated the effects of ERM peeling on intraretinal cystic spaces, best corrected visual acuity, and central retinal thickness at 6 months after surgery. Results Twenty-four eyes (5.5%) showed intraretinal cystic spaces before surgery, present in the inner retinal layer (the inner group) in 9 eyes, in the outer retinal layer (the outer group) in 6 eyes, and in both the inner and the outer retinal layers (the combined group) in 9 eyes. Additionally, 30 eyes with ERM but without any presence of intraretinal cystic space were selected randomly and classified as the no cyst group. At 6 months after surgery, the disappearance rate of cystic spaces was significantly greater for the outer group than for the inner group (83.3% and 11.1%, respectively, P = 0.011). The mean best corrected visual acuity improved significantly after surgery in the inner group, the outer group, and the no cyst group (P < 0.05 for all three groups) but did not improve in the combined group (P = 0.58). The mean central retinal thickness decreased significantly after surgery in the inner group, the combined group, and the no cyst group (P < 0.05). Conclusions Intraretinal cystic spaces were observed in 5.5% of preoperative idiopathic ERM cases. Following surgery, the cystic spaces in the outer retinal layer disappeared at higher rates than those in the inner retinal layer, suggesting that the pathophysiologies of these cystic spaces are different.