Cellular and circuit remodeling of the primate foveal midget pathway after acute photoreceptor loss.

The retinal fovea in human and nonhuman primates is essential for high acuity and color vision. Within the fovea lies specialized circuitry in which signals from a single cone photoreceptor are largely conveyed to one ON and one OFF type midget bipolar cell (MBC), which in turn connect to a single ON or OFF midget ganglion cell (MGC), respectively. Restoring foveal vision requires not only photoreceptor replacement but also appropriate reconnection with surviving ON and OFF MBCs and MGCs. However, our current understanding of the effects of cone loss on the remaining foveal midget pathway is limited. We thus used serial block-face electron microscopy to determine the degree of plasticity and potential remodeling of this pathway in adult Macaca fascicularis several months after acute photoreceptor loss upon photocoagulation. We reconstructed MBC structure and connectivity within and adjacent to the region of cone loss. We found that MBC dendrites within the scotoma retracted and failed to reach surviving cones to form new connections. However, both surviving cones and ON and OFF MBC dendrites at the scotoma border exhibited remodeling, suggesting that these neurons can demonstrate plasticity and rewiring at maturity. At six months postlesion, disconnected OFF MBCs clearly lost output ribbon synapses with their postsynaptic partners, whereas the majority of ON MBCs maintained their axonal ribbon numbers, suggesting differential timing or extent in ON and OFF midget circuit remodeling after cone loss. Our findings raise rewiring considerations for cell replacement approaches in the restoration of foveal vision.

DISCREPANCY BETWEEN FUNDUS AUTOFLUORESCENCE ABNORMALITY AND VISUAL FIELD LOSS IN BIETTI CRYSTALLINE DYSTROPHY.

PURPOSE: The aim of this study was to explore the potential benefits of retinal pigment epithelium replacement therapy in patients with Bietti crystalline dystrophy (BCD) by assessing the disease pathology with the distinctive relationship between fundus autofluorescence (FAF) abnormality and visual field defect. METHODS: Sixteen eyes from 16 patients with BCD and 16 eyes from 16 patients with RHO-associated retinitis pigmentosa were included. Fundus autofluorescence, optical coherence tomography, and Goldmann perimetry results were retrospectively reviewed and assessed using image analyses. RESULTS: In patients with BCD, the FAF abnormality area was not correlated with the overall visual field defect area and median overall visual field defect area (57.5%) was smaller than FAF abnormality area (98.5%). By contrast, the ellipsoid zone width was significantly correlated with the central visual field area (r = 0.806, P < 0.001). In patients with RHO-associated retinitis pigmentosa, the FAF abnormality area and ellipsoid zone width were significantly correlated with the overall visual field defect area (r = 0.833, P < 0.001) and central visual field area (r = 0.887, P < 0.001), respectively. CONCLUSION: The FAF abnormality shown in patients with BCD involves retinal pigment epithelium degeneration without complete loss of photoreceptors or visual function. These results suggest that patients with BCD are good candidates for retinal pigment epithelium replacement therapy for preservation of residual visual function.

Gene and Induced Pluripotent Stem Cell Therapy for Retinal Diseases.

Given the importance of visual information to many daily activities, retinal degenerative diseases-which include both inherited conditions (such as retinitis pigmentosa) and acquired conditions (such as age-related macular degeneration)-can have a dramatic impact on human lives. The therapeutic options for these diseases remain limited. Since the discovery of the first causal gene for retinitis pigmentosa almost three decades ago, more than 250 genes have been identified, and gene therapies have been rapidly developed. Simultaneously, stem cell technologies such as induced pluripotent stem cell-based transplantation have advanced and have been applied to the treatment of retinal degenerative diseases. Here, we review recent progress in these expanding fields and discuss the potential for precision medicine in ophthalmic care.

Stem-cell-based therapies for retinal degenerative diseases: Current challenges in the establishment of new treatment strategies.

Various advances have been made in the treatment of retinal diseases, including new treatment strategies and innovations in surgical devices. However, the treatment of degenerative retinal diseases, such as retinitis pigmentosa (RP) and age-related macular degeneration (AMD), continues to pose a significant challenge. In this review, we focus on the use of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to treat retinal diseases by harnessing the ability of stem cells to differentiate into different body tissues. The retina is a tissue specialized for light sensing, and its degradation leads to vision loss. As part of the central nervous system, the retina has very low regenerative capability, and therefore, treatment options are limited once it degenerates. Nevertheless, innovations in methods to induce the generation of retinal cells and tissues from ESCs/iPSCs enable the development of novel approaches for these irreversible diseases. Here we review some historical background and current clinical trials involving the use of stem-cell-derived retinal pigment epithelial cells for AMD treatment and stem cell-derived retinal cells/tissues for RP therapy. Finally, we discuss our future vision of regenerative treatment for retinal diseases with a partial focus on our studies and introduce other interesting approaches for restoring vision.

A ROCK Inhibitor Promotes Graft Survival during Transplantation of iPS-Cell-Derived Retinal Cells.

Currently, retinal pigment epithelium (RPE) transplantation includes sheet and single-cell transplantation, the latter of which includes cell death and may be highly immunogenic, and there are some issues to be improved in single-cell transplantation. Y-27632 is an inhibitor of Rho-associated protein kinase (ROCK), the downstream kinase of Rho. We herein investigated the effect of Y-27632 in vitro on retinal pigment epithelium derived from induced pluripotent stem cells (iPS-RPE cells), and also its effects in vivo on the transplantation of iPS-RPE cell suspensions. As a result, the addition of Y-27632 in vitro showed suppression of apoptosis, promotion of cell adhesion, and higher proliferation and pigmentation of iPS-RPE cells. Y-27632 also increased the viability of the transplant without showing obvious retinal toxicity in human iPS-RPE transplantation into monkey subretinal space in vivo. Therefore, it is possible that ROCK inhibitors can improve the engraftment of iPS-RPE cell suspensions after transplantation.

Autologous Induced Stem-Cell-Derived Retinal Cells for Macular Degeneration.

We assessed the feasibility of transplanting a sheet of retinal pigment epithelial (RPE) cells differentiated from induced pluripotent stem cells (iPSCs) in a patient with neovascular age-related macular degeneration. The iPSCs were generated from skin fibroblasts obtained from two patients with advanced neovascular age-related macular degeneration and were differentiated into RPE cells. The RPE cells and the iPSCs from which they were derived were subject to extensive testing. A surgery that included the removal of the neovascular membrane and transplantation of the autologous iPSC-derived RPE cell sheet under the retina was performed in one of the patients. At 1 year after surgery, the transplanted sheet remained intact, best corrected visual acuity had not improved or worsened, and cystoid macular edema was present. (Funded by Highway Program for Realization of Regenerative Medicine and others; University Hospital Medical Information Network Clinical Trials Registry [UMIN-CTR] number, UMIN000011929 .).

Assessment of the deformation of the outer nuclear layer in the Epiretinal membrane using spectral-domain optical coherence tomography.

BACKGROUND: We aimed to investigate the deformation of the outer nuclear layer using optical coherence tomography in patients with epiretinal membrane (ERM) and its relationship with metamorphopsia. METHODS: Thirty-nine eyes from 39 patients with ERM were included in the study. Patients with the subtypes of pseudo macula hole and lamellar hole were excluded. Twenty-one fellow eyes without macular disease were included as normal controls. Forty-nine B-scan images were obtained in the range of 20 degrees around the macula using SD-OCT. The outer nuclear layer (ONL) was evaluated as a three-dimensional image (3D-ONL) reconstructed using the distance between the ONL and retinal pigment epithelium (RPE) line. The deformation of the ONL was figured at the reference plane and evaluation plane (ONL-B). The characteristic parameters of the ONL-B were defined as circularity, area ratio, and axis ratio. The correlations between these parameters and visual acuity and MCHART score ratio (MH/MV) were then evaluated. RESULTS: ONL height was significantly higher in ERM patients than in normal controls (54.1 ± 5.3 µm and 84.1 ± 12.9 µm, respectively; P < 0.001). In ERM patients, the MV score was 0.53 ± 0.50, the MH score was 0.71 ± 0.61, and the distance from the RPE line to the ONL-B was 153.5 ± 13.5 µm. The axis of the ONL-B in normal controls and ERM patients was - 6.25 ± 21.8 and - 1.28 ± 29.1, respectively, which indicates that the ONL is horizontally long in both normal individuals and ERM patients. The circularity and area ratio were significantly smaller in ERM patients than in normal controls. In all ERM patients, MH/MV had a significant correlation with axis (r = - 0.29, p = 0.034), circularity (r = - 0.28, p = 0.044), and area ratio (r = - 0.47, p = 0.001). Moreover, we found that the correlation was more significant if the subjects had an axis of the ONL within ±10 degrees (n = 16); the correlations of MH/MV with axis (r = - 0.29, p = 0.034), circularity (r = - 0.53, p = 0.021), and area ratio were more significant (r = - 0.78, P < 0.0001). CONCLUSION: The ONL is horizontally long in normal individuals and ERM patients. The direction of metamorphopsia is correlated with the direction of ONL deformation.

Transplantation of human embryonic stem cell-derived retinal tissue in two primate models of retinal degeneration.

Retinal transplantation therapy for retinitis pigmentosa is increasingly of interest due to accumulating evidence of transplantation efficacy from animal studies and development of techniques for the differentiation of human embryonic stem cells (hESCs) and induced pluripotent stem cells into retinal tissues or cells. In this study, we aimed to assess the potential clinical utility of hESC-derived retinal tissues (hESC-retina) using newly developed primate models of retinal degeneration to obtain preparatory information regarding the potential clinical utility of these hESC-retinas in transplantation therapy. hESC-retinas were first transplanted subretinally into nude rats with or without retinal degeneration to confirm their competency as a graft to mature to form highly specified outer segment structure and to integrate after transplantation. Two focal selective photoreceptor degeneration models were then developed in monkeys by subretinal injection of cobalt chloride or 577-nm optically pumped semiconductor laser photocoagulation. The utility of the developed models and a practicality of visual acuity test developed for monkeys were evaluated. Finally, feasibility of hESC-retina transplantation was assessed in the developed monkey models under practical surgical procedure and postoperational examinations. Grafted hESC-retina was observed differentiating into a range of retinal cell types, including rod and cone photoreceptors that developed structured outer nuclear layers after transplantation. Further, immunohistochemical analyses suggested the formation of host-graft synaptic connections. The findings of this study demonstrate the clinical feasibility of hESC-retina transplantation and provide the practical tools for the optimization of transplantation strategies for future clinical applications.

Protective Effects of Human iPS-Derived Retinal Pigmented Epithelial Cells in Comparison with Human Mesenchymal Stromal Cells and Human Neural Stem Cells on the Degenerating Retina in rd1 mice.

Retinitis pigmentosa (RP) is a group of visual impairments characterized by progressive rod photoreceptor cell loss due to a genetic background. Pigment epithelium-derived factor (PEDF) predominantly secreted by the retinal pigmented epithelium (RPE) has been reported to protect photoreceptors in retinal degeneration models, including rd1. In addition, clinical trials are currently underway outside Japan using human mesenchymal stromal cells and human neural stem cells to protect photoreceptors in RP and dry age-related macular degeneration, respectively. Thus, this study aimed to investigate the rescue effects of induced pluripotent stem (iPS)-RPE cells in comparison with those types of cells used in clinical trials on photoreceptor degeneration in rd1 mice. Cells were injected into the subretinal space of immune-suppressed 2-week-old rd1 mice. The results demonstrated that human iPS-RPE cells significantly attenuated photoreceptor degeneration on postoperative days (PODs) 14 and 21 and survived longer up to at least 12 weeks after operation than the other two types of graft cells with less immune responses and apoptosis. The mean PEDF concentration in the intraocular fluid in RPE-transplanted eyes was more than 1 µg/ml at PODs 14 and 21, and this may have contributed to the protective effect of RPE transplantation. Our findings suggest that iPS-RPE cells serve as a competent source to delay photoreceptor degeneration through stable survival in degenerating ocular environment and by releasing neuroprotective factors such as PEDF.

Developing rods transplanted into the degenerating retina of Crx-knockout mice exhibit neural activity similar to native photoreceptors.

Replacement of dysfunctional or dying photoreceptors offers a promising approach for retinal neurodegenerative diseases, including age-related macular degeneration and retinitis pigmentosa. Several studies have demonstrated the integration and differentiation of developing rod photoreceptors when transplanted in wild-type or degenerating retina; however, the physiology and function of the donor cells are not adequately defined. Here, we describe the physiological properties of developing rod photoreceptors that are tagged with green fluorescent protein (GFP) driven by the promoter of rod differentiation factor, Nrl. GFP-tagged developing rods show Ca(2 +) responses and rectifier outward currents that are smaller than those observed in fully developed photoreceptors, suggesting their immature developmental state. These immature rods also exhibit hyperpolarization-activated current (Ih ) induced by the activation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. When transplanted into the subretinal space of wild-type or retinal degeneration mice, GFP-tagged developing rods can integrate into the photoreceptor outer nuclear layer in wild-type mouse retina and exhibit Ca(2 +) responses and membrane current comparable to native rod photoreceptors. A proportion of grafted rods develop rhodopsin-positive outer segment-like structures within 2 weeks after transplantation into the retina of Crx-knockout mice and produce rectifier outward current and Ih upon membrane depolarization and hyperpolarization. GFP-positive rods derived from induced pluripotent stem (iPS) cells also display similar membrane current Ih as native developing rod photoreceptors, express rod-specific phototransduction genes, and HCN-1 channels. We conclude that Nrl-promoter-driven GFP-tagged donor photoreceptors exhibit physiological characteristics of rods and that iPS cell-derived rods in vitro may provide a renewable source for cell-replacement therapy.

Adaptive Optics Optical Coherence Tomography Analysis of Induced Pluripotent Stem Cell-Derived Retinal Organoid Transplantation in Retinitis Pigmentosa.

This study evaluates the transplantation of induced pluripotent stem cell (iPSC)-derived retinal organoids into patients with advanced retinitis pigmentosa using adaptive optics optical coherence tomography (AO-OCT) to monitor retinal changes over two years post transplantation. Our results confirmed successful engraftment and increased retinal thickness, with AO-OCT providing detailed visualization of cellular structures such as an outer plexiform layer-like line and highly reflective particles within rosette-like formations, indicative of photoreceptor development. Immunohistological analysis in a parallel monkey model confirmed these structures as mature, functional photoreceptor rosettes. The integration of high-resolution AO-OCT with immunohistology provides critical insights into the structural and functional outcomes of transplantation and represents a promising advancement in the treatment of retinal degenerative diseases.

Label-free enrichment of human pluripotent stem cell-derived early retinal progenitor cells for cell-based regenerative therapies.

Pluripotent stem cell-based therapy for retinal degenerative diseases is a promising approach to restoring visual function. A clinical study using retinal organoid (RO) sheets was recently conducted in patients with retinitis pigmentosa. However, the graft preparation currently requires advanced skills to identify and excise suitable segments from the transplantable area of the limited number of suitable ROs. This remains a challenge for consistent clinical implementations. Herein, we enabled the enrichment of wild-type (non-reporter) retinal progenitor cells (RPCs) from dissociated ROs using a label-free ghost cytometry (LF-GC)-based sorting system, where a machine-based classifier was trained in advance with another RPC reporter line. The sorted cells reproducibly formed retinal spheroids large enough for transplantation and developed mature photoreceptors in the retinal degeneration rats. This method of enriching early RPCs with no specific surface antigens and without any reporters or chemical labeling is promising for robust preparation of graft tissues during cell-based therapy.

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.

Relationship between residual visual field and full-field stimulus testing in patients with late-stage retinal degenerative diseases.

This study aimed to investigate how the extent and central/peripheral location of the residual visual field (VF) in patients with late-stage inherited retinal diseases (IRDs) are related to retinal sensitivity detected using full-field stimulus testing (FST). We reviewed the results of Goldmann perimetry and FST from the medical records of patients with IRDs whose VF represents central (within 10°) and/or peripheral islands, or undetectable. In total, 19 patients (19 eyes) were analyzed in this study. The median value of residual VF area was 1.38%. The median values of rod and cone sensitivities were - 14.9 dB and 7.4 dB, respectively. Patients with only the peripheral island (- 33.9 dB) had better median rod sensitivity than other groups (only central, - 18.9 dB; both, - 3.6 dB). VF area significantly correlated with rod sensitivity (r = - 0.943, p = 0.005) in patients with only peripheral island, but not with cone sensitivity. Peripheral VF islands were significant contributors to FST results, especially rod sensitivity. With reduced or loss of central vision, the extent of residual peripheral VF significantly affected rod sensitivity, suggesting that FST can be useful in quantitatively estimating the overall remaining vision in patients with late-stage IRD.

Host-Graft Synapses Form Functional Microstructures and Shape the Host Light Responses After Stem Cell-Derived Retinal Sheet Transplantation.

Purpose: Retinitis pigmentosa represents a leading cause of blindness in developed countries, yet effective treatments for the disease remain unestablished. Previous studies have demonstrated the potential of stem cell-derived retinal organoid (SC-RO) sheet transplantation to form host-graft synapses and to improve light responsiveness in animal models of retinal degeneration. However, the detailed microstructures of these de novo synapses and their functional contribution have not been well elucidated. This study aims to (1) elucidate the microstructures of the host-graft synapse, and (2) investigate the overall distribution and contribution of these synapses to host retinal light responses. Methods: We identified host-graft synapses using a reporter system in mouse SC-RO and rd1 mice, a well-established model of end-stage retinal degeneration. Correlative array tomography was used to reveal the microstructure of host-graft synapses. Furthermore, we developed a semi-automated algorithm that robustly detects the host-graft photoreceptor synapses in the overall grafted area using the same reporter system in flat-mount retinas. We then integrated the spatial distribution of the host-graft synapses with light responses detected by multi-electrode array recording. Results: Correlative array tomography revealed that host-graft synapses recapitulate the developmental process of photoreceptor synapse formation involving horizontal cells first and then rod bipolar cells. By integrating the spatial distribution of host-graft synapse and multi-electrode array recording, we showed that the number of light-responsive host retinal ganglion cells is positively correlated with the local density of host-graft synapses. Conclusions: De novo host-graft synapses recapitulate the developmental microstructure of the photoreceptor synapse, and their formation contributes to the light responsiveness after SC-RO transplantation.

Transplantation of human pluripotent stem cell-derived retinal sheet in a primate model of macular hole.

Macular hole (MH) is a retinal break involving the fovea that causes impaired vision. Although advances in vitreoretinal surgical techniques achieve >90% MH closure rate, refractory cases still exist. For such cases, autologous retinal transplantation is an optional therapy showing good anatomic success, but visual improvement is limited and peripheral visual field defects are inevitable after graft harvesting. Here, using a non-human primate model, we evaluated whether human embryonic stem cell-derived retinal organoid (RO) sheet transplantation can be an effective option for treating MH. After transplantation, MH was successfully closed by continuous filling of the MH space with the RO sheet, resulting in improved visual function, although no host-graft synaptic connections were confirmed. Mild xeno-transplantation rejection was controlled by additional focal steroid injections and rod/cone photoreceptors developed in the graft. Overall, our findings suggest pluripotent stem cell-derived RO sheet transplantation as a practical option for refractory MH treatment.

Graft cell expansion from hiPSC-RPE strip after transplantation in primate eyes with or without RPE damage.

Clinical studies using suspensions or sheets of human pluripotent cell-derived retinal pigment epithelial cells (hiPSC-RPE) have been conducted globally for diseases such as age-related macular degeneration. Despite being minimally invasive, cell suspension transplantation faces challenges in targeted cell delivery and frequent cell leakage. Conversely, although the RPE sheet ensures targeted delivery with correct cell polarity, it requires invasive surgery, and graft preparation is time-consuming. We previously reported hiPSC-RPE strips as a form of quick cell aggregate that allows for reliable cell delivery to the target area with minimal invasiveness. In this study, we used a microsecond pulse laser to create a local RPE ablation model in cynomolgus monkey eyes. The hiPSC-RPE strips were transplanted into the RPE-ablated and intact sites. The hiPSC-RPE strip stably survived in all transplanted monkey eyes. The expansion area of the RPE from the engrafted strip was larger at the RPE injury site than at the intact site with no tumorigenic growth. Histological observation showed a monolayer expansion of the transplanted RPE cells with the expression of MERTK apically and collagen type 4 basally. The hiPSC-RPE strip is considered a beneficial transplantation option for RPE cell therapy.

Recovery of photoreceptor outer segments after anti-VEGF therapy for age-related macular degeneration.

PURPOSE: To evaluate whether the status of the external limiting membrane (ELM) or inner segment/outer segment junction (IS/OS) improves after intravitreal injection of ranibizumab for age-related macular degeneration (AMD). We also evaluated whether the pre-operative values of these parameters are associated with the visual prognosis. METHODS: This was a hospital-based, cross-sectional study. Seventy-six eyes of 76 treatment-naive AMD patients who received three monthly intravitreal injections of ranibizumab followed for more than 6 months with additional as-needed injections were investigated. Spectral domain OCT was used to evaluate the length of ELM, IS/OS, and foveal thickness pre- and post-operatively. Changes of ELM and IS/OS length were evaluated postoperatively. Correlation coefficients between pre-operative parameters and post-operative visual acuity were also analyzed. RESULTS: Significant changes were noted in mean logMAR (0.66 to 0.53), foveal thickness (231.1 to 151.1 µm), and IS/OS length (514.9 to 832.3 µm) after the treatment. ELM length did not improve significantly (1,312.4 to 1,376.7 µm). Restoration of IS/OS occurred where ELM is retained. Although pre-operative ELM length, IS/OS length, and foveal thickness showed correlation with post-operative logMAR (R = -0.51, -0.39, and 0.46, respectively), the most powerful predictive factor for visual prognosis was pre-operative logMAR (R = 0.77, p < 0.001). CONCLUSIONS: IS/OS status improves in response to anti-VEGF therapy but ELM seems to have less plasticity. The status of IS/OS and ELM can be used as prognostic factors but the predictive power is inferior to that of baseline visual acuity.

Early changes in foveal thickness in eyes with central serous chorioretinopathy.

PURPOSE: To investigate when and how fast the foveal thinning occurs in central serous chorioretinopathy. METHODS: Outer nuclear layer (ONL) thickness was measured in 60 eyes of 60 patients with active central serous chorioretinopathy. Patients were divided into 3 groups based on the duration of symptoms; within 1 month (Group A, 25 eyes), 1 to 6 months (Group B, 17 eyes), and >6 months (Group C, 18 eyes). Outer nuclear layer thickness and visual acuity were compared between each group. Some of the patients underwent several examinations and serial changes were analyzed. RESULTS: The ONL thickness was correlated with the duration of symptoms (R = -0.61, P < 0.001). Visual acuity was worse in Group C compared with Group A (P = 0.003). The mean ONL thickness of each group and healthy contralateral eyes was 82.4, 70.2, 53.5, and 89.3 µm, respectively. Comparisons between each group showed statistical significances. Seventeen eyes in Group A were re-examined between 3 and 6 months after the first visit. The mean ONL thickness significantly decreased from 81.9 µm to 75.0 µm (P = 0.020). CONCLUSION: In central serous chorioretinopathy, ONL thinning starts in the early stage and may continue as long as the subretinal fluid persists.