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PurposeIn a previous cross-sectional study, we reported that the sexes can be distinguished using known factors obtained from color fundus photography (CFP). However, it is not clear how sex differences in fundus parameters appear across the human lifespan. Therefore, we conducted a cohort study to investigate sex determination based on fundus parameters in elementary school students.MethodsThis prospective observational longitudinal study investigated 109 right eyes of elementary school students over 4 years (age, 8.5 to 11.5 years). From each CFP, the tessellation fundus index was calculated as red/red + green + blue (R/[R+G+B]) using the mean value of red-green-blue intensity in eight locations around the optic disc and macular region. Optic disc area, ovality ratio, papillomacular angle, and retinal vessel angles and distances were quantified according to the data in our previous report. Using 54 fundus parameters, sex was predicted by L2 regularized binomial logistic regression for each grade.ResultsThe right eyes of 53 boys and 56 girls were analyzed. The discrimination accuracy rate significantly increased with age: 56.3% at 8.5 years, 46.1% at 9.5 years, 65.5% at 10.5 years and 73.1% at 11.5 years.ConclusionsThe accuracy of sex discrimination by fundus photography improved during a 3-year cohort study of elementary school students.

Vascular endothelial growth factor A (VEGF-A) plays pivotal roles in regulating tumor angiogenesis as well as physiological vascular function. The major VEGF-A isoforms, VEGF-A121 and VEGF-A165, in serum, plasma, and platelets have not been exactly evaluated due to the lack of the appropriate assay system. Antibodies against human VEGF-A121 and VEGF-A165 (hVEGF-A121 and hVEGF-A165) were successfully produced and Enzyme-Linked ImmunoSorbent Assay (ELISA) for hVEGF-A121 and hVEGF-A165 were separately created by these monoclonal antibodies. The measurement of recombinant hVEGF-A121 and hVEGF-A165 by the created ELISA showed no cross-reaction between hVEGF-A121 and hVEGF-A165 in conditioned media from HEK293 cells transfected with either hVEGF-A121 or hVEGF-A165 expression vector. The levels of VEGF-A121 and VEGF-A165 in serum, plasma, and platelets from 59 healthy volunteers proved that VEGF-A121 level was higher than VEGF-A165 in both plasma and serum in all the cases. VEGF-A121 or VEGF-A165 in serum represented higher level than that in plasma. In contrast, the level of VEGF-A165 was higher than VEGF-A121 in platelets. The newly developed ELISAs for hVEGF-A121 and hVEGF-A165 revealed different ratios of VEGF isoforms in serum, plasma, and platelets. Measuring these isoforms in combination provides useful information as biomarkers for diseases involving VEGF-A121 and VEGF-A165.

Aim/backgroundTo aim of this study is to develop an artificial intelligence (AI) that aids in the thought process by providing retinal clinicians with clinically meaningful or abnormal findings rather than just a final diagnosis, i.e., a "wayfinding AI."MethodsSpectral domain optical coherence tomography B-scan images were classified into 189 normal and 111 diseased eyes. These were automatically segmented using a deep-learning based boundary-layer detection model. During segmentation, the AI model calculates the probability of the boundary surface of the layer for each A-scan. If this probability distribution is not biased toward a single point, layer detection is defined as ambiguous. This ambiguity was calculated using entropy, and a value referred to as the ambiguity index was calculated for each OCT image. The ability of the ambiguity index to classify normal and diseased images and the presence or absence of abnormalities in each layer of the retina were evaluated based on the area under the curve (AUC). A heatmap, i.e., an ambiguity-map, of each layer, that changes the color according to the ambiguity index value, was also created.ResultsThe ambiguity index of the overall retina of the normal and disease-affected images (mean ± SD) were 1.76 ± 0.10 and 2.06 ± 0.22, respectively, with a significant difference (p < 0.05). The AUC used to distinguish normal and disease-affected images using the ambiguity index was 0.93, and was 0.588 for the internal limiting membrane boundary, 0.902 for the nerve fiber layer/ganglion cell layer boundary, 0.920 for the inner plexiform layer/inner nuclear layer boundary, 0.882 for the outer plexiform layer/outer nuclear layer boundary, 0.926 for the ellipsoid zone line, and 0.866 for the retinal pigment epithelium/Bruch's membrane boundary. Three representative cases reveal the usefulness of an ambiguity map.ConclusionsThe present AI algorithm can pinpoint abnormal retinal lesions in OCT images, and its localization is known at a glance when using an ambiguity map. This will help diagnose the processes of clinicians as a wayfinding tool.

PurposeTo explore the effect of photodynamic therapy (PDT) on the choroid of medial area from optic disc and factors correlated with treatment outcomes, we evaluated choroidal changes using ultra-widefield optical coherence tomography (UWF-OCT) after PDT for central serous chorioretinopathy (CSC).MethodsIn this retrospective case-series, we included CSC patients who received a standard-dose of full-fluence PDT. UWF-OCT were examined at baseline and 3 months after treatment. We measured choroidal thickness (CT), classified into central, middle, and peripheral sectors. We examined CT changes after PDT by sectors and treatment outcome.ResultsTwenty-two eyes of 21 patients (20 males; mean age 58.7 ± 12.3 years) were included. CT reduction after PDT was significant in all sectors, including peripheral areas: supratemporal, 330.5 ± 90.6 μm vs. 237.0 ± 53.2 μm; infratemporal, 240.0 ± 89.4 μm vs. 209.9 ± 55.1 μm; supranasal, 237.7 ± 59.8 vs 209.3 ± 69.3 μm; infranasal, 172.6 ± 47.2 μm vs. 155.1 ± 38.2 μm (P < 0.001, for all). In patients with retinal fluid resolution, despite no apparent difference in baseline CT, there was more significant reduction after PDT in supratemporal and supranasal peripheral sectors, compared with patients without resolution: supratemporal, 41.9 ± 30.3 μm vs. -1.6 ± 22.7 μm; supranasal, 24.7 ± 15.3 μm vs. 8.5 ± 3.6 μm (P < 0.019, for both).ConclusionsWhole CT decreased after PDT, including in medial areas from optic disc. This may be associated with the treatment response of PDT for CSC.

PurposeTo evaluate the clinical characteristics, treatment trends, and visual prognosis of submacular hemorrhage (SMH) secondary to neovascular age-related macular degeneration (nAMD) and retinal arterial macroaneurysm (RAM).MethodsThis retrospective study enrolled 187 Japanese patients with SMH at 10 institutions from 2015 to 2018. Medical records including SMH etiology, best-corrected visual acuity (BCVA), fundus photographs, optical coherence tomography images, and selected treatments were analyzed.ResultsMajor causes of SMH were typical nAMD (tnAMD) (18%), polypoidal choroidal vasculopathy (PCV) (50%) and RAM (29%). Age, male/female ratio, baseline BCVA, central retinal thickness, and involved retinal layers were significantly different between etiologies (all PConclusionsThe characteristics of, and treatment strategy for, SMH were different between the underlying diseases. Anti-vascular endothelial growth factor treatment with or without expansile gas was mainly chosen for SMH in tnAMD and PCV, whereas vitrectomy with gas was the most common treatment for RAM, and the higher rate for vitrectomy might result in the greater BCVA improvement in the RAM group than in the other groups. Final BCVA was better in PCV, RAM, and tnAMD, in that order, because patients with PCV were younger and had better baseline BCVA.

PurposeMyopia is a known risk factor of pigmentary glaucoma (PG), and the increased prevalence of myopia in Asian countries indicates that more cases of PG will likely develop soon. However, there are no diagnostic criteria for PG for Asians. Therefore, the purpose of this study is to determine the characteristics of PG in Japanese individuals and establish three diagnostic signs for PG.MethodsThis was a single-center, retrospective, case series study of glaucoma patients who visited the Kagoshima University Hospital between January 2015 and January 2020. The inclusion criteria were age ResultsTen eyes of 5 Japanese men and 10 eyes of 5 Japanese women were studied. Their age ranged from 13 to 46 years at the time of diagnosis. One eye had posterior corneal pigmentation and 6 eyes had pigmentation of the trabecular meshwork. None had mid peripheral iris transillumination defect. The Sampaolesi line, iris concavity, and midperipheral iris depigmentation were found in all patients except one patient who lacked the mid peripheral depigmentation. Two eyes had the pigment reversal sign, none had lens pigmentation, and 2 eyes had peripheral retinal degeneration.ConclusionThe presence of the Sampaolesi line, iris concavity, and midperipheral iris depigmentation may be appropriate signs for the diagnosis of PG in Asians.

PurposeTo investigate retinal vessel shift (RVS) and its association with axial length (AL) elongation in junior high school students.MethodsTotal 161 eyes of 161 healthy junior high school students were prospectively studied. Optical AL and anterior chamber depth (ACD) measurements, and fundus photography were performed in the first and third grades. Eyes of subjects in the first and third grade that had perfect matching among all the retinal vessels were allocated to the RVS(-) group, otherwise allocated to the RVS(+) group. In the RVS(+) group, the peripapillary retinal arteries angle (PRAA) was measured for quantitative analysis of RVS; the angle between the major retinal arteries. The variables related to PRAA were identified using model selection with the corrected Akaike information criterion.ResultsForty-two eyes (26.1%) were allocated to the RVS(+) group. There were seven patterns in the RVS of those in the RVS(+) group, including clockwise shift in the supra temporal area (5 eyes), infra temporal area (7 eyes), and nasal area (9 eyes); anticlockwise shift in the supra temporal area (7 eyes), infra temporal area (5 eyes), and nasal area (2 eyes); and distal shift in the temporal area (7 eyes). The optimal model for the PRAA narrowing included larger AL and body weight in the first grade, and greater AL elongation.ConclusionVarious (seven) RVS patterns were observed in about 25% of the junior high school students within two years. RVS was associated with AL elongation, and useful to reveal the mechanism of myopic retinal stretch.

PurposeAbnormalities of the running pattern of choroidal vessel have been reported in eyes with pachychoroid diseases. However, it is difficult for clinicians to judge the running pattern with high reproducibility. Thus, the purpose of this study was to compare the degree of concordance of the running pattern of the choroidal vessels between that determined by artificial intelligence (AI) to that determined by experienced clinicians.MethodsThe running pattern of the choroidal vessels in en face images of Haller's layer of 413 normal and pachychoroid diseased eyes was classified as symmetrical or asymmetrical by human raters and by three supervised machine learning models; the support vector machine (SVM), Xception, and random forest models. The data from the human raters were used as the supervised data. The accuracy rates of the human raters and the certainty of AI's answers were compared using confidence scores (CSs).ResultsThe choroidal vascular running pattern could be determined by each AI model with an area under the curve better than 0.94. The random forest method was able to discriminate with the highest accuracy among the three AIs. In the CS analyses, the percentage of certainty was highest (66.4%) and that of uncertainty was lowest (6.1%) in the agreement group. On the other hand, the rate of uncertainty was highest (27.3%) in the disagreement group.ConclusionAI algorithm can automatically classify with ambiguous criteria the presence or absence of a symmetrical blood vessel running pattern of the choroid. The classification was as good as that of supervised humans in accuracy and reproducibility.

PurposeIntravitreal anti-VEGF injection (IVI) is administered before vitrectomy to assist management of proliferative diabetic retinopathy (PDR)-related complications. In the clinical setting, retinal surgeons determine the use of preoperative IVI based on individual criteria. In this study, we investigated factors related to the potential bias of retinal surgeons in using IVI prior to vitrectomy for PDR-related complications, and evaluated the real-world outcomes of surgeon-determined preoperative IVI.MethodsMedical records of 409 eyes of 409 patients who underwent 25-gauge vitrectomy for PDR complications at seven Japanese centers (22 surgeons) were retrospectively reviewed. Ocular factors, demographic and general clinical factors, surgical procedures, and postoperative complications were compared between IVI group (patients who received preoperative IVI; 87 eyes, 21.3%) and non-IVI group (patients who did not receive preoperative IVI; 322 eyes, 78.7%). In addition, baseline HbA1c in IVI group and non-IVI group was compared between eyes with and without postoperative complications.ResultsAt baseline, IVI group was younger (PConclusionsIn this multicenter real-world clinical study, younger age and presence of NVG and TRD were identified as potential biases in using IVI before vitrectomy for PDR complications. Eyes that received preoperative IVI had more intraoperative retinal breaks requiring tamponade than eyes not receiving IVI, but postoperative outcome was not different between the two groups.

The purpose of this study is to determine the factors related to anxiety and depression in patients with retinitis pigmentosa (RP). The status of anxiety and depression was determined in RP patients with the Hospital Anxiety and Depression Scale (HADS) questionnaire which consisted of subscales for HADS-anxiety (HADS-A) and HADS-depression (HADS-D). The vision-specific quality of life (VSQOL) was assessed with the National Eye Institute Visual Function Questionnaire 25 (NEI-VFQ25). The correlations between the HADS-A or HADS-D scores and vision-related clinical parameters such as the best-corrected visual acuity (BCVA), Functional Acuity Score, Functional Field Score, Functional Vision Score, the NEI- VFQ25 subscale score were determined. The socioeconomic status, such as the work status and membership in the RP society, was investigated to determine the factors related to the HADS-A and HADS-D scores. One hundred and twelve RP patients (46 men and 66 women) with mean age of 60.7±15.4 (standard deviation) years were studied. The HADS-A score was not significantly correlated with any visual functions but was significantly correlated with the general health condition (r = -0.34, P

Optical coherence tomography (OCT) has made it possible for clinicians to measure the peripapillary choroidal thickness (ppCT) noninvasively in various ocular diseases. However, the ocular factors associated with the ppCT have not been conclusively determined. The purpose of this study was to determine the relationship between the local ppCT and the axial length, optic disc tilt, and the angle of the papillo-macular position (PMP) in healthy eyes. This was a prospective, observational cross-sectional study of 119 right eyes of 119 healthy Japanese volunteers. The ppCT was manually measured at eight sectors around the optic disc using the B-scan images of the Topcon 3D OCT RNFL 3.4 mm circle scan. The trajectory of the retinal pigment epithelium in the B-scan image was fitted to a sine curve using ImageJ, and the amplitude of the sine curve was used to determine the degree of the optic disc tilt. The PMP angle was determined in the color fundus photographs. The relationships between the ppCT and the axial length, the optic disc tilt, and PMP angle were determined by Spearman and multiple correlation analyses. The mean age was 25.8 ± 3.9 years and the mean axial length was 25.5 ± 1.4 mm. The ppCT was significantly and negatively associated with the axial length (R = -0.43 to -0.24, P

The location of the peaks of the circumpapillary retinal nerve fiber layer (cpRNFL) thickness is affected by several ocular parameters. In this study, we have generated equations that can determine the peaks of the cpRNFL. This study was a prospective, observational, cross sectional study of 118 healthy right eyes. The axial length, optic disc tilt, superiortemporal (ST)- and inferiortemporal (IT)-peaks of the cpRNFL thickness, and angles of the ST and IT retinal arteries (RA) and veins (RV) were determined. The correlations between the location of the ST- and IT-peaks and ocular structural parameters and the sex, body height and weight were calculated. The best fit equations to generate the location of the ST/IT-peaks were determined using corrected-Akaike Information Criteria. The location of the ST-peak was 0.72+(0.40 x ST-RA)+(0.27 x ST-RV)+(0.14 x height)-(0.47 x papillo-macular-position)-(0.11 x disc tilt) with a coefficient of correlation of 0.61 (P

Tessellated fundus is found as common and early-phase characteristic of myopic eyes and their locations are varied among patients. However, the relationship between their locations and morphological parameters of the eyes is still unknown. The purpose is this study is to determine the locations of the tessellations in the ocular fundus of young healthy eyes, and to determine relationships between their locations and morphological parameters of the eyes. This is a prospective observational cross sectional study of 126 eyes of 126 healthy volunteers (mean age 26.0±4.1 years). The eyes were classified into eight groups based on the location of the tessellations; no tessellation, temporal, infra-temporal, inferior, nasal, peripapillary, whole retina, and unclassified tessellations. The degree of optic disc tilt was quantified using a sine curve fitting program on the optical coherence tomographic circle scan images. The correlations between each tessellation location and the axial length, area of the optic disc plus conus (AOC), and optic disc tilt were determined. Forty-four eyes were place in the no tessellation group, 12 eyes in the temporal, 21 eyes in the infra-temporal, 9 eyes in the inferior, 8 eyes in the nasal, 15 eyes in the peripapillary, 11 eyes in the whole, and 6 eyes in the unclassified groups. The differences in the axial lengths between the no tessellation group and the infra-temporal groups were significant. A significant difference was found in the AOC between the no tessellation and the inferior, infra-temporal, and peripapilalry groups. A significant difference was found in the optic disc tilt between the no tessellation and infra-temporal groups (P

Humanin (HN) is a small mitochondrial-encoded peptide with neuroprotective properties. We have recently shown protection of retinal pigmented epithelium (RPE) cells by HN in oxidative stress; however, the effect of HN on endoplasmic reticulum (ER) stress has not been evaluated in any cell type. Our aim here was to study the effect of HN on ER stress-induced apoptosis in RPE cells with a specific focus on ER-mitochondrial cross-talk. Dose dependent effects of ER stressors (tunicamycin (TM), brefeldin A, and thapsigargin) were studied after 12 hr of treatment in confluent primary human RPE cells with or without 12 hr of HN pretreatment (1-20 μg/mL). All three ER stressors induced RPE cell apoptosis in a dose dependent manner. HN pretreatment significantly decreased the number of apoptotic cells with all three ER stressors in a dose dependent manner. HN pretreatment similarly protected U-251 glioma cells from TM-induced apoptosis in a dose dependent manner. HN pretreatment significantly attenuated activation of caspase 3 and ER stress-specific caspase 4 induced by TM. TM treatment increased mitochondrial superoxide production, and HN co-treatment resulted in a decrease in mitochondrial superoxide compared to TM treatment alone. We further showed that depleted mitochondrial glutathione (GSH) levels induced by TM were restored with HN co-treatment. No significant changes were found for the expression of several antioxidant enzymes between TM and TM plus HN groups except for the expression of glutamylcysteine ligase catalytic subunit (GCLC), the rate limiting enzyme required for GSH biosynthesis, which is upregulated with TM and TM+HN treatment. These results demonstrate that ER stress promotes mitochondrial alterations in RPE that lead to apoptosis. We further show that HN has a protective effect against ER stress-induced apoptosis by restoring mitochondrial GSH. Thus, HN should be further evaluated for its therapeutic potential in disorders linked to ER stress.

The purpose of this study was to develop a method of quantifying the degree of optic disc tilt in normal eyes. This was a prospective, observational cross sectional study of 126 right eyes of 126 healthy volunteers. The optic disc tilt was determined from the circular peripapillary optical coherence tomographic (OCT) scan images. The course of the retinal pigment epithelium (RPE) layer in the peripapillary cross sectional scan images was fit to a sine wave curve, and the amplitude of the sine curve was used to reflect the degree of the optic disc tilt in the optical axis. The repeatability of the amplitude determinations was calculated. The correlation between the amplitude and the ovality ratio of the optic disc was determined. The correlation between the amplitude and the body height was also calculated. The mean amplitude was 36.6 ± 17.5 pixels, which was significantly and inversely correlated with the ovality ratio of the optic disc (R = -0.59, P < 0.001). The intra-rater and inter-rater correlation coefficients of the amplitude were significant high (P < 0.001, both). The amplitude was significantly and inversely correlated with the body height (R = -0.38, P < 0.001), but not with the axial length. In conclusion, a sine wave function can be used to describe the course of the RPE in the circumpapillary OCT images. The results indicate that the amplitude of the sine wave can be used to represent the degree of optic disc tilt. Thus, the sine wave analyses can be used as a quantifiable and repeatable method to determine the optic disc tilt.

PURPOSE: To determine by objective methods the minimum number of spectral-domain optical coherence tomographic (SD-OCT) images to average to obtain the clearest retinal image. METHODS: SD-OCT Images were obtained from 9 healthy eyes and also from a phantom eye model. The SD-OCT images were obtained by averaging 1, 5, 20, 60, and 100 B-scan images. The reflectivity (mean gray value) of the different retinal layers was evaluated in these images. The image quality was evaluated by the size of the standard deviations (SDs) and the contrast-to-noise ratios (CNRs). A phantom eye model made by TiO2 silicone plates was also examined. RESULTS: The SDs decreased significantly when the number of images averaged increased from 1 to 5 and also from 5 to 20 (P

Asymmetrical secretion of vascular endothelial growth factor (VEGF) by retinal pigment epithelial (RPE) cells in situ is critical for maintaining the homeostasis of the retina and choroid. VEGF is also involved in the development and progression of age-related macular degeneration (AMD). We studied the effect of tumor necrosis factor-α (TNF-α) on the secretion of VEGF in polarized and non-polarized RPE cells (P-RPE cells and N-RPE cells, respectively) in culture and in situ in rats. A subretinal injection of TNF-α caused a decrease in VEGF expression and choroidal atrophy. Porcine RPE cells were seeded on Transwell™ filters, and their maturation and polarization were confirmed by the asymmetrical VEGF secretion and trans electrical resistance. Exposure to TNF-α decreased the VEGF secretion in P-RPE cells but increased it in N-RPE cells in culture. TNF-α inactivated JNK in P-RPE cells but activated it in N-RPE cells, and TNF-α activated NF-κB in P-RPE cells but not in N-RPE cells. Inhibition of NF-κB activated JNK in both types of RPE cells indicating crosstalk between JNK and NF-κB. TNF-α induced the inhibitory effects of NF-κB on JNK in P-RPE cells because NF-κB is continuously inactivated. In N-RPE cells, however, it was not evident because NF-κB was already activated. The basic activation pattern of JNK and NF-κB and their crosstalk led to opposing responses of RPE cells to TNF-α. These results suggest that VEGF secretion under inflammatory conditions depends on cellular polarization, and the TNF-α-induced VEGF down-regulation may result in choroidal atrophy in polarized physiological RPE cells. TNF-α-induced VEGF up-regulation may cause neovascularization by non-polarized or non-physiological RPE cells.

Photoreceptor degeneration is the most critical cause of visual impairment in age-related macular degeneration (AMD). In neovascular form of AMD, severe photoreceptor loss develops with subretinal hemorrhage due to choroidal neovascularization (CNV), growth of abnormal blood vessels from choroidal circulation. However, the detailed mechanisms of this process remain elusive. Here we demonstrate that neovascular AMD with subretinal hemorrhage accompanies a significant increase in extracellular ATP, and that extracellular ATP initiates neurodegenerative processes through specific ligation of Purinergic receptor P2X, ligand-gated ion channel, 7 (P2RX7; P2X7 receptor). Increased extracellular ATP levels were found in the vitreous samples of AMD patients with subretinal hemorrhage compared to control vitreous samples. Extravascular blood induced a massive release of ATP and photoreceptor cell apoptosis in co-culture with primary retinal cells. Photoreceptor cell apoptosis accompanied mitochondrial apoptotic pathways, namely activation of caspase-9 and translocation of apoptosis-inducing factor (AIF) from mitochondria to nuclei, as well as TUNEL-detectable DNA fragmentation. These hallmarks of photoreceptor cell apoptosis were prevented by brilliant blue G (BBG), a selective P2RX7 antagonist, which is an approved adjuvant in ocular surgery. Finally, in a mouse model of subretinal hemorrhage, photoreceptor cells degenerated through BBG-inhibitable apoptosis, suggesting that ligation of P2RX7 by extracellular ATP may accelerate photoreceptor cell apoptosis in AMD with subretinal hemorrhage. Our results indicate a novel mechanism that could involve neuronal cell death not only in AMD but also in hemorrhagic disorders in the CNS and encourage the potential application of BBG as a neuroprotective therapy.