Evaluation of photoreceptor features in retinitis pigmentosa with cystoid macular edema by using an adaptive optics fundus camera.

OBJECTIVE: Cystoid macular edema (CME) in retinitis pigmentosa (RP) is an important complication causing visual dysfunction. We investigated the effect of CME on photoreceptors in RP patients with previous or current CME, using an adaptive optics (AO) fundus camera. METHODS: We retrospectively observed the CME and ellipsoid zone (EZ) length (average of horizontal and vertical sections) by optical coherence tomography. The density and regularity of the arrangement of photoreceptor cells (Voronoi analysis) were examined at four points around 1.5° from superior to inferior and temporal to nasal. We also performed a multivariate analysis using CME duration, central macular thickness and transversal length of CME. RESULTS: We evaluated 18 patients with previous or current CME (18 eyes; age, 48.7 ± 15.6 years) and 24 patients without previous or current CME (24 eyes; age, 46.0 ± 14.5 years). There were no significant differences in age, logMAR visual acuity, or EZ length. In groups with and without CME, cell density was 11967 ± 3148 and 16239 ± 2935 cells/mm2, and sequence regularity was 85.5 ± 3.4% and 88.5 ± 2.8%, respectively; both parameters were significantly different. The correlation between photoreceptor density and age was more negative in group with CME. The CME group tended toward greater reductions in duration of CME. CONCLUSION: Complications of CME in RP patients may lead to a decrease in photoreceptor density and regularity. Additionally, a longer duration of CME may result in a greater reduction in photoreceptor density.

Investigation of the effectiveness of gelatin hydrolysate in human iPS-RPE cell suspension transplantation.

Introduction: The retinal pigment epithelium (RPE) plays essential roles in maintaining retinal functions as well as choroidal capillaries and can lead to visual disorders if dysfunctional. Transplantation of human-induced pluripotent stem cell-derived RPE (hiPSC-RPE) is a promising therapy for such RPE impaired conditions including age-related macular degeneration. The challenge with cell suspension transplantation is targeted delivery of graft cells and undesired cell reflux. Gelatin hydrolysate, a soluble variant with specific molecular weight distribution, is examined in this study for its potential use in hiPSC-RPE suspension transplantation, particularly in reducing cell reflux and enhancing RPE engraftment. Methods: A retinal bleb model was created using polydimethylsiloxane (PDMS) soft lithography to quantify cellular reflux. We examined the effects of gelatin hydrolysate on the hiPSC-RPE of various aspects of cell behavior and performance such as cell viability, hypoxia reaction, morphology, induction of inflammation and immune responses. Results: Gelatin hydrolysate at 5 % concentration effectively mitigated cell reflux in vitro mimic, improved cell viability, reduced cell aggregation, and had an inhibitory effect on hypoxic reactions due to cell deposition with hiPSC-RPE. Additionally, gelatin hydrolysate did not affect cell adhesion and morphology, and decreased the expression of major histocompatibility complex class II molecules, which suggests reduced immunogenicity of hiPSC-RPE. Conclusion: Gelatin hydrolysate is considered a valuable and useful candidate for future regenerative therapies in hiPSC-RPE suspension transplantation.

Retinal haemorrhages on ultra-widefield red channel images and perfusion status in central retinal vein occlusion.

PURPOSE: To evaluate the relationship between retinal haemorrhages detected on Ultra-widefield (UWF) red channel images and perfusion status in eyes with acute central retinal vein occlusion (CRVO). METHODS: UWF fundus images were split into green and red channels using ImageJ software. The retinal haemorrhages were calculated quantitatively with both the green and red channel images, resulting in green channel haemorrhages (GCH) and red channel haemorrhages (RCH). The nonperfusion area (NPA) was also calculated from fluorescein angiography in each eye. The relationships between both the GCH and RCH with the NPA were investigated. RESULTS: Thirty-two eyes of 32 patients with acute CRVO (18 men, 14 women) were included. The mean GCH and RCH values were 10.4% ± 8.2% and 1.7% ± 1.7%, respectively. The mean NPA was 39.2% ± 28.8%. Significant correlations were seen between the GCH and NPA (r = 0.38; P = 0.022) and RCH and NPA (r = 0.44; P = 0.010, linear regression analysis). Multivariate analysis suggested that only the RCHs were correlated significantly with the NPA. CONCLUSIONS: Retinal haemorrhages detected by UWF red channel imaging were less compared to green channel imaging and associated closely with retinal NPAs in eyes with acute CRVO. UWF red channel imaging allowed us to identify ischaemia-related haemorrhage.

Retinal Morphologic Features in Patients with Large Macular Holes Treated by Autologous Neurosensory Retinal Transplantation.

PURPOSE: To investigate the time courses of morphologic changes in the transplanted grafts, including the retinal layer, the ellipsoid zone (EZ) and the visual acuity (VA) after autologous retinal transplantation (ART) in patients with primary large macular holes (MHs). DESIGN: Single-center, retrospective cohort study. SUBJECTS: The study included 17 eyes of 17 patients who had undergone ART. All patients fulfilled the following criteria: (1) MH was the only disease-causing anatomic abnormality of the macula; (2) they could be followed up for at least 12 months after ART surgery; (3) they had no other systemic disorders; and (4) the MH was > 400 µm in diameter. METHODS: Data of all patients who underwent assessment of the VA and spectral-domain (SD)-OCT at 1, 3, 6, and 12 months after surgery were analyzed. The morphologic features of the graft and the EZ were measured by SD-OCT. MAIN OUTCOME MEASURES: Changes in the morphologic features of the graft (graft thickness, overall graft area, graft length, area of outer retina, and EZ) and VA over 1 year after surgery. RESULTS: Closure of the MH was achieved in all patients. The VA was 0.59 ± 0.27 (logarithm of the minimal angle of resolution units) at 1-year postsurgery compared with 0.84 ± 0.22 before surgery (P < 0.05). The retinal layers gradually became smoothly connected. Although a decrease in graft thickness and overall graft area was observed, the length and area of the outer retina were maintained throughout the follow-up period. The presence of the EZ was observed in 9 patients (52.9%) and our analyses revealed significantly greater improvement of VA in these patients than in those without the EZ (P < 0.05). Moreover, the incidence of macular edema (ME) was higher in the group without the EZ (P = 0.04; chi-square test). CONCLUSION: Patients showed significant improvements of the VA. Morphologically, the layers were gradually connected to each other, and the outer retina was particularly maintained. Especially, patients in whom an EZ was restored in the graft showed a low prevalence of ME and greater improvement of the VA. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Angiogenesis and Anastomosis on Graft Retina after Autologous Retinal Transplantation.

Anastomosis on graft retina was studied after autologous retinal transplantation (ART) on 26 eyes with primary and refractory macular holes. Angiogenesis and anastomosis were seen in 35% of the eyes with successful ART, likely contributing to the survival of the transplanted retina.

Using ultra-widefield red channel images to improve the detection of ischemic central retinal vein occlusion.

PURPOSE: To examine the usefulness of red channel fundus imaging to detect the ischemic status in eyes with central retinal vein occlusion (CRVO). METHODS: Ultra-widefield (UWF) fundus images were obtained from 42 eyes with CRVO. Twenty-one eyes were ischemic, and 21 eyes were non-ischemic. Rubeosis was found in 11 ischemic eyes. UWF images were split into red and green channels using ImageJ software. Both the color and red channel images were used to predict the presence or absence of ischemia when examined by masked graders. The sensitivity and specificity of UWF imagings for the detection of ischemia were calculated in Group A (total 42 eyes), Group B (32 eyes excluding non-rubeotic ischemic CRVO) and Group C (31 eyes excluding rubeotic ischemic CRVO), respectively. Moreover, a linear mixed model was conducted to investigate the relationship between the type of images and the accuracy of prediction in each group. RESULTS: No significant difference in the sensitivity of color fundus imaging was seen between Group A and Group B. By contrast, a significant difference in the sensitivity of red channel imaging was seen between Group A and Group B (p = 0.031). The accuracies of the predictions were not associated with the type of image in Group A and Group B, but were significantly associated in Group C (p = 0.026). CONCLUSIONS: UWF red channel imaging enabled more accurate detection of the ischemic status, compared with color fundus images, especially in non-rubeotic CRVO eyes.

Transient rho-associated coiled-coil containing kinase (ROCK) inhibition on human retinal pigment epithelium results in persistent Rho/ROCK downregulation.

Retinal pigment epithelium (RPE) cells is the outermost layer of the retina and RPE dysfunction is a key factor in the disease pathogenesis of age-related macular degeneration (AMD). Transplantation therapy using induced pluripotent stem cell (iPSC)-derived RPEs has recently received much attention as a treatment for AMD. Preserving these cells under the best possible conditions is important, and preservation methods using Y-27632 have been reported. Rho-associated coiled-coil containing kinase (ROCK) inhibitors are known to inhibit cell death, emerging as important drug candidates for stem cell differentiation and regenerative medicine. However, it has recently been shown that ROCK inhibitors may have a vasodilatory effect on human retinal arterioles, a side effect that should ideally be avoided in RPE transplantation. Although ROCK inhibitors hold great potential, optimizing efficacy while minimizing adverse reactions is critical for translation into a clinical treatment. We examined the effect of transient exposure of RPE cells to ROCK inhibitor Y-27632 to determine whether the extracellular presence of the drug is necessary for ongoing Rho/ROCK downregulation. Human RPE cells were subcultured as a suspension for 4 h in drug-free medium following exposure to Y-27632 for 2 h. A Y-27632 concentration of >10 µM improved cell survival beyond 4 h and cell proliferation in recovery culture medium. ROCK2 expression levels were specifically downregulated by Y-27632 in the Rho/ROCK signaling pathway. In conclusion, we demonstrated that the effect of Y-27632 is not dependent on its extracellular availability and can last beyond the 2 h of exposure. The lasting Rho/ROCK signaling pathway downregulation by Y-27632 suggests that RPE cell transplantation with ROCK inhibitor-free media is possible, which can minimize side effects to host tissue and have wider implications for transplantation methods requiring ROCK inhibition.

Long-term follow-up of a case of amyloidosis-associated chorioretinopathy.

PURPOSE: To describe the findings of long-term follow-up of a case of amyloidosis-associated chorioretinopathy by multimodal imagings, including optical coherence tomography (OCT). OBSERVATIONS: A 47-year-old woman who had been diagnosed as having systemic amyloidosis was found to have a best corrected visual acuity of 20/13 in both eyes at the age of 41, which subsequently decreased to 20/100 in the left eye and 20/20 in the right eye at age 47. Visual field examination demonstrated worsening of the central scotoma during the follow-up period. Funduscopic examination revealed bilateral white deposits along the choroidal vessels, which became more pronounced over time, along with diffuse pigmentary changes. The fluorescein angiography and indocyanine green angiography findings were consistent not only with atrophic lesions, but also with amyloid deposition (i.e., staining of the vessels).At the baseline, macula OCT revealed a thick hyporeflective band at the choriocapillaris, however, at the last follow-up, it revealed an absent ellipsoid zone, and bilateral progressive retinal pigment epithelium irregularities in both eyes. CONCLUSIONS: Patients diagnosed as having amyloidosis-related chorioretinopathy may have maintained visual function at the first detection of retinal amyloid deposition, and a number of years may elapse before the patient manifests visual deterioration.

Optical Coherence Tomography-Based Deep-Learning Models for Classifying Normal and Age-Related Macular Degeneration and Exudative and Non-Exudative Age-Related Macular Degeneration Changes.

INTRODUCTION: The use of optical coherence tomography (OCT) images is increasing in the medical treatment of age-related macular degeneration (AMD), and thus, the amount of data requiring analysis is increasing. Advances in machine-learning techniques may facilitate processing of large amounts of medical image data. Among deep-learning methods, convolution neural networks (CNNs) show superior image recognition ability. This study aimed to build deep-learning models that could distinguish AMD from healthy OCT scans and to distinguish AMD with and without exudative changes without using a segmentation algorithm. METHODS: This was a cross-sectional observational clinical study. A total of 1621 spectral domain (SD)-OCT images of patients with AMD and a healthy control group were studied. The first CNN model was trained and validated using 1382 AMD images and 239 normal images. The second transfer-learning model was trained and validated with 721 AMD images with exudative changes and 661 AMD images without any exudate. The attention area of the CNN was described as a heat map by class activation mapping (CAM). In the second model, which classified images into AMD with or without exudative changes, we compared the learning stabilization of models using or not using transfer learning. RESULTS: Using the first CNN model, we could classify AMD and normal OCT images with 100% sensitivity, 91.8% specificity, and 99.0% accuracy. In the second, transfer-learning model, we could classify AMD as having or not having exudative changes, with 98.4% sensitivity, 88.3% specificity, and 93.9% accuracy. CAM successfully described the heat-map area on the OCT images. Including the transfer-learning model in the second model resulted in faster stabilization than when the transfer-learning model was not included. CONCLUSION: Two computational deep-learning models were developed and evaluated here; both models showed good performance. Automation of the interpretation process by using deep-learning models can save time and improve efficiency. TRIAL REGISTRATION: No15073.

Critical Functionality Effects from Storage Temperature on Human Induced Pluripotent Stem Cell-Derived Retinal Pigment Epithelium Cell Suspensions.

Human induced pluripotent stem cell (hiPSC)-derived retinal pigment epithelium (hiPSC-RPE) cells suspension have the potential for regenerative treatment. However, practical regenerative applications with hiPSC-RPE cells require the development of simple and cost-effective non-freezing preservation methods. We investigated the effect of non-freezing temperatures on suspended hiPSC-RPE cells in various conditions and analysed mechanisms of cell death, anoikis, Rho GTPases, hypoxia, microtubule destruction, and cell metabolism. Cells stored at 37 °C had the lowest viability due to hypoxia from high cell metabolism and cell deposits, and cells preserved at 4 °C were damaged via microtubule fragility. Cell suspensions at 16 °C were optimal with drastically reduced apoptosis and negligible necrosis. Moreover, surviving cells proliferated and secreted key proteins normally, compared to cells without preservation. hiPSC-RPE cell suspensions were optimally preserved at 16 °C. Temperatures above or below the optimal temperature decreased cell viability significantly yet differentially by mechanisms of cell death, cellular metabolism, microtubule destruction, and oxygen tension, all relevant to cell conditions. Surviving cells are expected to function as grafts where high cell death is often reported. This study provides new insight into various non-freezing temperature effects on hiPSC-RPE cells that are highly relevant to clinical applications and may improve cooperation between laboratories and hospitals.

Deep Learning Classification Models Built with Two-step Transfer Learning for Age Related Macular Degeneration Diagnosis.

The objective of this study was to build deep learning models with optical coherence tomography (OCT) images to classify normal and age related macular degeneration (AMD), AMD with fluid, and AMD without any fluid. In this study, 185 normal OCT images from 49 normal subjects, 535 OCT images of AMD with fluid, and 514 OCT mages of AMD without fluid from 120 AMD eyes as training data, while 49 normal images from 25 normal eyes, 188 AMD OCT images with fluid and 154 AMD images without any fluid from 77 AMD eyes as test data, were enrolled. Data augmentation was applied to increase the number of images to build deep learning models. Totally, two classification models were built in two steps. In the first step, a VGG16 model pre-trained on ImageNet dataset was transfer learned to classify normal and AMD, including AMD with fluid and/or without any fluid. Then, in the second step, the fine-tuned model in the first step was transfer learned again to distinguish the images of AMD with fluid from the ones without any fluid. With the first model, normal and AMD OCT images were classified with 0.999 area under receiver operating characteristic curve (AUC), and 99.2% accuracy. With the second model, AMD with the presence of any fluid, and AMD without fluid were classified with 0.992 AUC, and 95.1% accuracy. Compared with a transfer learned VGG16 model pre-trained on ImageNet dataset, to classify the three categories directly, higher classification performance was achieved with our notable approach. Conclusively, two classification models for AMD clinical practice were built with high classification performance, and these models should help improve the early diagnosis and treatment for AMD.

Efficacy of additional topical betamethasone in persistent cystoid macular oedema after carbonic anhydrase inhibitor treatments in retinitis pigmentosa.

OBJECTIVE: We investigated the efficacy of additional topical betamethasone in persistent cystoid macular oedema (CMO) after carbonic anhydrase inhibitors (CAIs) therapy. METHODS AND ANALYSIS: This retrospective cohort study included 16 eyes of 10 patients with retinitis pigmentosa (RP). All patients were previously administered CAI for at least 3 months to treat CMO secondary to RP and lacking an effective reduction (≥11%) of central foveal thickness (CFT). We administered topical 0.1% betamethasone daily in each affected eye following a preceding course of the CAI medication as a first treatment. CMO was diagnosed using spectral-domain optical coherence tomography. CFT was regarded as the average of vertical and horizontal foveal thickness. Best-corrected visual acuity (BCVA) and intraocular pressure (IOP) were obtained from patient medical records. We compared the CFT and BCVA between baseline and the average of 1-3, 5-7, 10-14 and 16-20 months period. RESULTS: In treatments with brinzolamide in 14 eyes, dorzolamide in 2 eyes and bromfenac in 2 eyes, CFT effectively decreased in 12 of 16 eyes (81%). CFT decreased significantly in 1-3 months (326±102 µm; n=16; P=0.029) and 5-7 months (297±102 µm; n=12; P=0.022) compared with baseline but not within 10-14 months (271±96 µm; n=9; P=0.485) or 16-20 months (281±134 µm; n=9; P=0.289). There were no significant intergroup differences in BCVA throughout the study. Betamethasone treatment was stopped in three patients because of IOP elevation. CONCLUSION: Our data suggested that additional betamethasone might improve treatments for persistent CMO. Topical steroids could be an alternative option for managing persistent CMO in RP.