Alpinumisoflavone ameliorates choroidal neovascularisation and fibrosis in age-related macular degeneration in in vitro and in vivo models.

Age-related macular degeneration (AMD) is a major cause of vision loss in the elderly population. Anti-vascular endothelial growth factor (VEGF) antibody therapy is applicable to neovascularisation of AMD; however, the prevention of fibrosis after anti-VEGF monotherapy is an unmet medical need. Subretinal fibrosis causes vision loss in neovascular age-related macular degeneration (nAMD) even with anti-VEGF therapy. We report the anti-fibrotic and anti-neovascularisation effects of alpinumisoflavone (AIF), an isoflavonoid derived from unripe Maclura tricuspidata fruit, in in vitro and in vivo models. For in vitro study, we treated H2O2 or THP-1 conditioned media (TCM) following activation with phorbol 12-myristate 13-acetate (PMA) and lipopolysaccharide (LPS) in a human retinal pigment epithelial cell line (ARPE-19). Choroidal neovascularisation (CNV) was induced by laser photocoagulation in mice, immediately followed by intravitreal administration of 25 µg AIF. CNV area and fibrosis were measured 7 days after laser photocoagulation. AIF showed anti-fibrosis and anti-neovascularisation effects in both the models. The laser induced CNV area was reduced upon AIF administration in nAMD mouse model. Additionally, AIF decreased the levels of the cleaved form of crystallin alpha B (CRYAB), a chaperone associated with VEGF stabilisation and fibrosis. Our results demonstrate a novel therapeutic application of AIF against neovascularisation and fibrosis in nAMD.

USING RETeval SYSTEM FLICKER ELECTRORETINOGRAPHY FOR EVALUATION OF DENSE VITREOUS HEMORRHAGE.

PURPOSE: To determine whether the RETeval electroretinography (ERG) system can be used to evaluate eyes with dense vitreous hemorrhage (VH). METHODS: This retrospective case series study included 69 eyes of 69 patients with acute dense VH. Flicker ERGs were recorded by the RETeval system, an ERG device with adhesive skin electrodes. We evaluated the flicker ERG amplitudes in eyes with VH and the ratio of the VH eye amplitudes compared with the fellow eye amplitudes for each VH cause. RESULTS: In patients with rhegmatogenous retinal detachment, the amplitude ratios were extremely low (0.08 ± 0.03). To detect rhegmatogenous retinal detachment, the area under the receiver operating characteristic curve was 0.977 (95% confidence interval, 0.943-1.000) (best rhegmatogenous retinal detachment cutoff value, 0.14; sensitivity, 100.0%; and specificity, 95.4%). The flicker ERG amplitude was not significantly correlated with the initial visual acuity (ρ = -0.189, P = 0.120) but was positively correlated with the postoperative visual acuity in eyes with VH (ρ = -0.328, P = 0.006). CONCLUSION: The RETeval ERG system was found to be a useful diagnostic option in situations where dense VH precluded fundus examination or posterior vitreous detachment was indistinguishable from rhegmatogenous retinal detachment.

Development of a Fundus Image-Based Deep Learning Diagnostic Tool for Various Retinal Diseases.

Artificial intelligence (AI)-based diagnostic tools have been accepted in ophthalmology. The use of retinal images, such as fundus photographs, is a promising approach for the development of AI-based diagnostic platforms. Retinal pathologies usually occur in a broad spectrum of eye diseases, including neovascular or dry age-related macular degeneration, epiretinal membrane, rhegmatogenous retinal detachment, retinitis pigmentosa, macular hole, retinal vein occlusions, and diabetic retinopathy. Here, we report a fundus image-based AI model for differential diagnosis of retinal diseases. We classified retinal images with three convolutional neural network models: ResNet50, VGG19, and Inception v3. Furthermore, the performance of several dense (fully connected) layers was compared. The prediction accuracy for diagnosis of nine classes of eight retinal diseases and normal control was 87.42% in the ResNet50 model, which added a dense layer with 128 nodes. Furthermore, our AI tool augments ophthalmologist's performance in the diagnosis of retinal disease. These results suggested that the fundus image-based AI tool is applicable for the medical diagnosis process of retinal diseases.

Changes in the Foveal Avascular Zone Area and Retinal Vessel Density after Anti-VEGF Therapy for Neovascular Age-Related Macular Degeneration.

PURPOSE: To investigate changes in the foveal avascular zone (FAZ) area and retinal vessel density (VD) in the macula of patients receiving multiple anti-vascular endothelial growth factor (VEGF) therapy for neovascular age-related macular degeneration (N-AMD). METHODS: This study included 54 eyes of 54 treatment-naïve N-AMD patients. Thirty-three eyes were treated with intravitreal aflibercept injections, and 21 eyes were treated with intravitreal ranibizumab injections. Unaffected fellow eyes (54 eyes) were used as controls. All image scans were acquired after the macular architecture had recovered with drying up of the subretinal fluid/hemorrhage after treatment. RESULTS: Both the superficial and deep FAZ areas were significantly larger in the aflibercept group than in the control group. The VD was also significantly reduced in the aflibercept group. CONCLUSIONS: Prolonged and repeated anti-VEGF therapy may cause an increase in the FAZ area and a decrease in the VD in patients with N-AMD, indicating ischemic damage.

Comparison of Retinal Layer Thickness and Capillary Vessel Density in the Patients with Spontaneously Resolved Acute Central Serous Chorioretinopathy.

We investigate retinal layer thickness and capillary vessel density (VD) in the patients with central serous chorioretinopathy (CSC) who recovered spontaneously and evaluate the correlation between the changes in these values and visual outcomes using swept-source optical coherence tomography (SS-OCT) and OCT angiography (OCTA). This retrospective case-control study included 34 eyes of 34 patients with spontaneously resolved acute CSC. The changes in retinal layer thickness and capillary VD were examined using SS-OCT and OCTA after complete resolution of subretinal fluid (SRF). The fellow eyes and 34 healthy eyes were used as controls. In the eyes with CSC, the outer retinal layer was significantly thinner than in the eyes of fellow and healthy controls. The foveal avascular zone area and VDs in the superficial and deep capillary plexus in the eyes with CSC were not significantly different from those in the eyes of fellow and healthy controls. The VD of the choriocapillaris in the eyes with CSC was significantly lower than that in the eyes of fellow and healthy controls. Correlation analyses revealed that the outer retinal layer thickness and initial visual acuity were positively correlated with the final visual acuity. Furthermore, the initial SRF area and height were negatively correlated with the outer retinal layer thickness after SRF resolution. Attenuation of outer retinal layer thickness and decreased VD of the choriocapillaris were observed in the eyes with spontaneously resolved acute CSC. The outer retinal layer thickness could be an important visual predictor of CSC.

Development of a Deep-Learning-Based Artificial Intelligence Tool for Differential Diagnosis between Dry and Neovascular Age-Related Macular Degeneration.

The use of deep-learning-based artificial intelligence (AI) is emerging in ophthalmology, with AI-mediated differential diagnosis of neovascular age-related macular degeneration (AMD) and dry AMD a promising methodology for precise treatment strategies and prognosis. Here, we developed deep learning algorithms and predicted diseases using 399 images of fundus. Based on feature extraction and classification with fully connected layers, we applied the Visual Geometry Group with 16 layers (VGG16) model of convolutional neural networks to classify new images. Image-data augmentation in our model was performed using Keras ImageDataGenerator, and the leave-one-out procedure was used for model cross-validation. The prediction and validation results obtained using the AI AMD diagnosis model showed relevant performance and suitability as well as better diagnostic accuracy than manual review by first-year residents. These results suggest the efficacy of this tool for early differential diagnosis of AMD in situations involving shortages of ophthalmology specialists and other medical devices.

Elevated vitreous αB-crystallin in patients with rhegmatogenous retinal detachment and association with proliferative vitreoretinopathy and retinal detachment area.

PURPOSE: To investigate the relationship between αB-crystallin levels in the vitreous of patients with rhegmatogenous retinal detachment (RRD) and correlate these levels with the presence of proliferative vitreoretinopathy (PVR) and to the extent of RRD. METHODS: Prospective, cross-sectional study. Vitreous samples were collected from twenty-six patients with RRD at the surgical repair. PVR was evaluated using the Updated Proliferative Vitreoretinopathy Grade Classification system. RRD area was measured using ultra-widefield retinal photographs. The concentration of αB-crystallin in the vitreous was measured using an enzyme-linked immunosorbent assay kit. RESULTS: The average concentration of αB-crystallin was significantly higher in the PVR Grade B and C group (97.83 ± 69.47) than in the PVR (-) and Grade A group (39.43 ± 13.53 ng/mL, P = 0.005). The level of αB-crystallin was also significantly higher in patients with large RD area (RD area > 1/4, 85.52 ± 66.07 ng/mL) than in patients with small RD area (RD area ≤ 1/4, 39.20 ± 13.02 ng/mL, P = 0.016). CONCLUSIONS: The level of αB-crystallin was significantly increased in the vitreous of RRD patients with high-grade PVR and large RD area. This finding suggests that αB-crystallin may function as an endogenous mediator in fibrotic processes in RRD patients.

Comparison of retinal layer thickness measurements obtained using two different swept-source optical coherence tomography imaging modes.

PURPOSE: To compare the retinal layer thickness values obtained using two swept-source optical coherence tomography (OCT) wide modes. METHODS: This study included fifty-four healthy eyes. Three-dimensional (3D) wide and 12 radial wide OCT scans were performed in each eye on the same day. Full retinal, retinal nerve fiber layer (RNFL), and ganglion cell-inner plexiform layer (GC-IPL) thicknesses with fully automated segmentation obtained in 3D wide mode, and with semi-automated correction and fully automated segmentation obtained in 12 radial wide scan. RESULTS: The intraclass correlation coefficients for full retinal thickness measurements obtained by the two protocols with fully automated segmentation were from 0.958 to 0.996, 0.754-0.918 for GC-IPL thickness measurements, and 0.013-0.727 for RNFL thickness measurements, in the nine ETDRS subfields. CONCLUSIONS: The full retinal thickness measurements determined using both fully automated segmentation algorithms were reliable and clinically acceptable. However, segmentation errors are shown when using the 12 radial wide scanning protocol with fully automated segmentation for measurement of RNFL and GC-IPL thickness.

Retinal and Choroidal Changes after Anti Vascular Endothelial Growth Factor Therapy for Neovascular Age-related Macular Degeneration.

OBJECTIVE: To investigate changes in retinal nerve fiber layer, ganglion cell-inner plexiform layer, and choroidal thickness in the macular area in patients with neovascular age-related macular degeneration who received repeated intravitreal ranibizumab and aflibercept treatments. METHODS: This retrospective study included 90 eyes of 90 treatment-naive patients. Fifty eyes were treated with intravitreal injections of aflibercept, and 40 were treated with intravitreal injections of ranibizumab. Unaffected fellow eyes (71 eyes) were used as controls. The dosage was one injection per month for 3 consecutive months as an initial treatment. The patients were examined monthly for 6 months following the initial injection. Additional intravitreal injections were given reactively in an optical coherence tomography-guided "pro re nata" protocol. Measurements of the retinal nerve fiber layer, ganglion cell-inner plexiform layer, full retina, and choroidal thickness were simultaneously obtained via swept-source optical coherence tomography in the nine Early Treatment Diabetic Retinopathy Study subfields. RESULTS: The retinal nerve fiber layer thickness in the nine Early Treatment Diabetic Retinopathy Study subfields did not differ significantly among the three study groups (aflibercept vs. ranibizumab vs. control). The ganglion cell-inner plexiform layer thickness was significantly reduced in the aflibercept group, while the choroidal thickness was reduced in both the aflibercept and ranibizumab groups. CONCLUSION: Excessive long-term vascular endothelial growth factor inhibition by an anti-vascular endothelial growth factor agent that is trapped by neuronal and retinal pigment epithelium cells may adversely affect the function of physiological vascular endothelial growth factor and harm retinal cells and vessels.

Superficial foveal avascular zone area changes before and after idiopathic epiretinal membrane surgery.

To investigate the foveal morphological changes and foveal avascular zone (FAZ) area before and after epiretinal membrane (ERM) surgery. Twenty-two eyes with treatment-naive ERM were included in this retrospective study. The central foveal thickness (CFT) and FAZ area were measured via swept-source optical coherence tomography (SS-OCT) and OCT angiography pre- and postoperatively. The unaffected fellow eyes were used as controls. The preoperative superficial FAZ area was significantly smaller in patients (0.08±0.04 mm2) than in controls (0.33±0.09 mm2; P<0.001). The postoperative superficial FAZ (0.12±0.06 mm2) area was significantly greater than the preoperative area (P<0.001). The preoperative superficial FAZ area was strongly negatively correlated with CFT (P<0.001, rho=-0.763). ERM induced significant foveal morphological changes and reduction of the superficial FAZ area. Foveal thickness was restored and FAZ area increased postoperatively. However, the process is rather slow and the recovery is incomplete.

Foveal Avascular Zone Area Changes Analyzed Using OCT Angiography after Successful Rhegmatogenous Retinal Detachment Repair.

PURPOSE: To investigate foveal avascular zone (FAZ) area after surgery in patients with rhegmatogenous retinal detachment (RRD) that involved or uninvolved the macula, and to evaluate the correlation between FAZ area and visual outcomes using swept-source optical coherence tomography angiography (OCTA). MATERIALS AND METHODS: This retrospective case-control study included 34 eyes (34 patients) with recent onset RRD that were successfully repaired with a single, uncomplicated surgical procedure (pars plana vitrectomy with gas tamponade). The changes of FAZ area were examined by OCTA after surgery. The unaffected fellow eye was used as a control for additional comparison. RESULTS: Both superficial and deep FAZ area were significantly larger in the macula-off group (superficial: 0.374 ± 0.112, deep: 0.702 ± 0.193 mm2) than in the macula-on group (superficial: 0.282 ± 0.105, deep: 0.543 ± 0.114 mm2) following surgery. The deep FAZ area was also markedly larger in the macula-off group than in the control group (0.532 ± 0.124 mm2). Correlation analyses revealed that both superficial (ρ = 0.555, P = 0.015) and deep FAZ (ρ = 0.616, P = 0.005) areas were negatively correlated with postoperative best-corrected visual acuity in the macula-off RRD group. CONCLUSIONS: The FAZ area enlargement after successful surgical repair in macula-off RRD eyes may indicate that there is an ischemic damage to retinal capillary plexus in fovea.

Effects of Diabetic Macular Edema on Repeatability of Retinal Nerve Fiber Layer Thickness Measurements at the Macular and Peripapillary Area Using Swept-Source Optical Coherence Tomography.

PURPOSE: To investigate the repeatability of macular and peripapillary retinal nerve fiber layer (RNFL) thickness measurements made using swept-source optical coherence tomography (SS-OCT) and automated segmentation. Measurements were made in non-diabetic controls and in patients with diabetic retinopathy (DR) with or without diabetic macular edema (DME). MATERIALS AND METHODS: A total of 131 eyes of 131 participants were included. Fifty-one eyes with DR had no DME (DME[-]), 45 eyes with DR had DME (DME[+]), and 35 eyes were healthy. Measurements of RNFL and full retinal thickness were simultaneously obtained with SS-OCT in the peripapillary area and in the nine Early Treatment Diabetic Retinopathy Study (ETDRS) subfields using the wide three-dimensional mode. All measurements were made twice on the same day by a single examiner to test intra-observer repeatability. Intraclass correlation coefficients (ICCs) and coefficients of repeatability were examined to evaluate repeatability. RESULTS: Average macular and temporal peripapillary RNFL thickness values were greater in the DME[+] group (36.4 ± 13.2 and 83.8 ± 19.4 µm, respectively) than in the control (27.4 ± 3.5 and 73.5 ± 11.4 µm, respectively) and DME[-] (27.9 ± 3.4 µm and 70.3 ± 11.3 µm, respectively) groups (both P < 0.001). The ICCs of average macular (control: 0.982, DME[-]: 0.913, and DME[+]: 0.970) and peripapillary (control: 0.972, DME[-]: 0.973, and DME[+]: 0.958) RNFL thickness measurements indicated good repeatability in all three study groups. CONCLUSIONS: Although the ICCs of average RNFL thickness measurements were relatively lower in eyes with DR than in healthy controls, the intra-observer repeatability of SS-OCT RNFL and full retinal thickness measurements is sufficiently reliable for them to be clinically useful.

Elevated Plasma Pentraxin3 Levels and Its Association with Neovascular Age-related Macular Degeneration.

PURPOSE: To evaluate pentraxin3 (PTX3) levels in patients with neovascular age-related macular degeneration (N-ARMD) and to investigate its role as a predictive biomarker. METHODS: Thirty individuals with N-ARMD and 30 controls without N-ARMD were studied. Plasma concentrations of C-reactive protein (CRP) and PTX3 were measured in frozen samples using an enzyme-linked immunosorbent assay kit. RESULTS: PTX3 concentration was 1341 ± 625 pg/mL (mean ± standard deviation) in N-ARMD patients, which was significantly higher than in control subjects (887 ± 478, p = 0.003). The mean CRP level was also significantly higher in N-ARMD (2121 ± 2300) than in control (748 ± 618, p = 0.004). Pearson's correlation analysis showed a significant positive correlation between PTX3 and CRP (r = 0.407, p = 0.002). CONCLUSIONS: Our data support the role of chronic inflammation in the development of ARMD. They also show PTX3 may contribute to efforts to understand pathogenesis of N-ARMD.

A new technique for Nd:YAG laser posterior capsulotomy.

AIM: To investigate the effects of a new opening pattern in neodymium:yttrium-aluminum-garnet (Nd:YAG) laser posterior capsulotomy on visual function. METHODS: This technique was conducted along a circular pattern. The energy ranged between 0.8 and 1.2 mJ/pulse was consumed and mean total energy levels were 74±21 mJ (mean±standard deviation: SD, from 40 to 167) and laser shots aimed at 150 µm away behind a datum point and went along an imaginary line which extends 0.5 mm inside from optic margin and into the circular en bloc pattern. Vitreous stands were attached with fragment and then they were cut off by the laser after circular application. The circular fragment was completely separated from vitreous, and then this fragment was quickly sunk in intravitreal space. RESULTS: The follow-up period ranges from at least a week to 40mo, making 15.8mo on average. The procedural outcome showed 96% (74 eyes out of the 77 eyes) enhancement in patients' visual acuity. Cystoid macular edema or retinal detachment was not observed in any of the patients during follow-up periods. CONCLUSION: This new technique is expected to improve the weaknesses that the conventional procedures have by adding the process to cut off vitreous stands attached with the fragment by the laser to the circular application.

Acute vision loss associated with retinal circulatory disturbances after intravitreal injection of bevacizumab.

PURPOSE: Bevacizumab, a monoclonal antibody targeting vascular endothelial growth factor (VEGF), specifically binds to VEGF protein, and inhibits angiogenesis. Intravitreal/intracameral injection of bevacizumab has been reported as another treatment option for patients with various ocular ischemic conditions. However, we report 4 cases of acute vision loss after bevacizumab intravitreal injection. METHODS: Intravitreal bevacizumab injections were administrated to 2 ocular ischemic syndrome (OIS) patients and 2 central retinal vein occlusion (CRVO) patients. Best-corrected visual acuity (BCVA), intraocular pressure (IOP), funduscopic findings, and fluorescein angiography were evaluated before and after the treatments. RESULTS: All 4 cases presented with acute vision loss within 1 week after bevacizumab injection, before its clearance from the eye, and showed that microcirculatory disturbances occurred in the retina. CONCLUSIONS: We believe that intravitreal injection of bevacizumab should be undertaken with extreme caution in patients with a history of cerebral infarction, especially with OIS or nonischemic CRVO, and with diabetic retinopathy and vitrectomized eye with pseudophakia.

Surgical outcome of mitomycin C-soaked collagen matrix implant in trabeculectomy.

PURPOSE: To assess the surgical outcomes and wound healing reaction of a filtering bleb after trabeculectomy using mitomycin C-soaked biodegradable collagen matrix (ologen). METHODS: Thirty eyes, in which the intraocular pressure (IOP) could not be controlled within the proper range by medication or laser treatment, were targeted in this study. After trabeculectomy with mitomycin C (0.2 mg/mL, 0.1 mL)-soaked ologen, surgical outcomes were assessed by evaluating bleb morphology by the Moorfield bleb grading system, as well as internal reflectivity and wound healing response of the bleb using a slit lamp and ultrasound biomicroscopy. The follow-up period continued for 12 months after surgery. RESULTS: Twelve months after surgery, 12/30 (40.0%) eyes were considered complete successes and 24/30 (80%) eyes as qualified successes. This rate is similar to the rate of our conventional trabeculectomy. The encapsulated bleb was the most frequent complication (9 eyes) and generally occurred at approximately 2 weeks after surgery. No avascular blebs were observed at final follow-up. CONCLUSIONS: Trabeculectomy with mitomycin C-soaked ologen do not seem to exert any synergistic effect with antimetabolites in terms of a reduction in IOP. However, the mitomycin C-soaked collagen matrix implant used in trabeculectomy resulted in comparatively stable IOP and did not aggravate wound healing or scar formation. Encapsulated blebs were generated at a more rapid pace in larger amounts compared with conventional trabeculectomy. The management of encapsulated blebs may be crucial to improving the success rate of surgery.

Sphingosine 1-phosphate in amniotic fluid modulates cyclooxygenase-2 expression in human amnion-derived WISH cells.

The metabolism of arachidonic acid, in particular the generation of prostaglandins (PGs), has been proposed to play a key role in the regulation of labor. Moreover, several extracellular proteins have been reported to modulate PG synthesis in amnion cells. In this study, we found that lipid components dissolved in the amniotic fluid modulate PG synthesis in WISH human amnion cells and identified one of these components as a sphingosine 1-phosphate (S1P). WISH cells express several S1P receptors including S1P1, S1P2, and S1P3. When WISH cells were stimulated with S1P, PGE2 synthesis increased in a concentration-dependent manner, showing maximal activity at around 100 nM. S1P treatment also caused the up-regulation of cyclooxygenase-2 (COX-2) mRNA and protein, which was apparent within 3-12 h of stimulation. In terms of the intracellular signaling pathway of S1P-induced WISH cell activation, we found that S1P stimulated two kinds of MAPK, ERK, and p38 kinase. We examined the roles of these two MAPKs in S1P-induced COX-2 expression. S1P-induced COX-2 expression was blocked completely by PD-98059 but not by SB-203580, suggesting that ERK has a critical role in the process. Transfection of S1P1 or S1P3 but not of S1P2 antisense oligonucleotide inhibited S1P-induced COX-2 expression and PGE2 production in WISH cells, indicating the involvements of S1P1 and S1P3 in the processes. This study demonstrates the physiological role of S1P in amniotic fluid and its effect on the modulation of COX-2 expression and PGs synthesis in WISH cells.

Comparative study of naphthalene-2,3-dicarboxaldehyde and o-phthalaldehyde fluorogenic reagents for chromatographic detection of sphingoid bases.

Naphthalene-2,3-dialdehyde (NDA) was developed as a precolumn labeling reagent for the fluorescent determination in a HPLC system of bioactive sphingoid bases, including sphingosine, sphinganine, and C20-sphinganine. Cellular sphingoid bases generally exist in the range of 10 to approximately 100 pmol/10(6) cells in a wide variety of cell types and tissues. This study aimed to obtain stable fluorescent derivatives of sphingoid bases and to increase their detectability at low concentrations. Sphingoid bases were reacted with NDA in the presence of cyanide ion to readily make an intensely fluorescent structure, 1-cyano-2-alkyl-benz[f]isoindole (CBI) and were then eluted separately on a reversed-phase C18 column with a simple mobile phase of 90% acetonitrile. For evaluating the NDA method, we compared the fluorescent intensity, elution profile, stability, and detectability of NDA derivatives with those of corresponding o-phthalaldehyde (OPA) derivatives. By monitoring the fluorescent intensity at the excitation wavelength of 252 nm and emission wavelength of 483 nm, NDA derivatives were sensitively determined at concentrations below 1.0 pmol of sphingoid bases in 1 x 10(5) U937 cells and were more stable than OPA derivatives. Linear calibration plots were obtained in the range studied (0.5 to approximately 500 nM). The limit of detection for NDA derivatives of sphingoid bases was approximately 0.1 pmol (S/N=3). The method successfully measured the accumulation of sphingosine in U937 cells following N,N-dimethylsphingosine treatment, and of sphinganine following fumonisin B1 treatment.