Retinal Pigment Epithelium Layer Research Articles

Association Between Arch-Shaped Hypo-Autofluorescent Lesions Detected Using Fundus Autofluorescence and Postoperative Hypotony.

Background: Chorioretinal folds are observed after vitrectomy due to ocular collapse caused by low intraocular pressure. The purpose of this study is to investigate the relationship between the postoperative hypotony, chorioretinal folds, and the fundus autofluorescence (FAF) findings. Methods: Two-hundred-and-seventy consecutive eyes that had undergone 25- or 27-gauge vitrectomy were examined. The associations between the arch-shaped hypo-autofluorescent lesions in the FAF images and the postoperative hypotony with intraocular pressure (IOP) ≤ 4 mmHg were determined on the day after the surgery. Results: Arch-shaped hypo-autofluorescent lesions were seen in 4 of the 270 eyes (1.5%), and hypo-autofluorescence was observed in 3 of 14 hypotonic eyes (18.5%). This was significantly more frequent than in the non-hypotony group (0.4%, p = 0.0004). Optical coherence tomography showed a loss of the ellipsoid zone and retinal pigment epithelial layer in the region of the arch-shaped lesions. None of the arch-shaped hypo-autofluorescent lesions involved the fovea, and the vision recovered in all cases. The hypo-autofluorescent lesions did not disappear during the 4 to 16 month observation period. Conclusions: The postoperative arch-shaped hypo-autofluorescent lesions were associated with postoperative hypotony and RPE damage due to chorioretinal folds. These findings remained even when the IOP was normalized and chorioretinal folds disappeared.

Lycium barbarum L. and Salvia miltiorrhiza Bunge extract ameliorates retinitis pigmentosa in rd10 mice by affecting endoplasmic reticulum stress

Ethnopharmacological relevanceLycium barbarum L. and Salvia miltiorrhiza Bunge (Gouqi and Danshen, LS) have led to their inclusion in the pharmacopoeia and healthcare systems of numerous countries globally. Traditional herbs known as LS are used in China to treat retinitis pigmentosa (RP). However, the mechanism is not clear. Aim of the studyThis study is to investigate the mechanism by which LS improves RP using rd10 mice as a model. Materials and methodsLS extract was used to treat the rd10 mice for four weeks. Fundus photographs, optical coherence tomography, electroretinography, histopathological examination, TUNEL apoptosis assay, digital PCR analysis, western blotting, and immunofluorescence double staining were performed. ResultsThe outer nuclear layer (ONL) thickness of the retina was significantly increased by the LS extract, improving atrophy, and both the ONL and the retinal pigment epithelium (RPE) layer were visible. Following treatment with LS extract, there was a notable increase in the magnitudes of ERG a- and b-waves in the retina, along with a significant reduction in the quantity of TUNEL-positive cells. Additionally, LS extract significantly reduced the levels of ER stress-related factors in rd10 mice. The results of immunofluorescence double staining further confirm that LS extract inhibits the GRP78/PERK/ATF4/CHOP pathway. ConclusionIn this study, the protective effects of LS extract on the retina were uncovered, suggesting that its mechanism could involve decreasing retinal cell apoptosis through the inhibition of the ER stress pathway.

Regualtion of LAMTOR1 by Oxidative Stress in Retinal Pigment Epithelium: Implications for Age-Related Macular Degeneration Pathogenesis

Oxidative stress is a critical pathogenic factor for age-related macular degeneration (AMD). Autophagy serves as a mechanism to counteract oxidative stress. LAMTOR1 regulates mTORC1 activity by recruiting or disassembling it on the lysosome under the addition or deprivation of amino acids. This regulation inhibits or enhances autophagy. Our study investigates whether oxidative stress impacts LAMTOR1, thereby adapting to oxidative conditions. We employed oxidative stressors, menadione (VK3) and 4-hydroxynonenal (4-HNE), and observed a reduction of LAMTOR1 in both human and mouse retinal pigment epithelium (RPE) following short-term (1h) and prolonged exposures (24h). Nrf2 overexpression increased both lamtor1 mRNA and LAMTOR1 protein in the RPE. To determine if Nrf2 regulates lamtor1 transcription, we cloned the deletion mutants of the lamtor1 promoter into a luciferase reporter. Although the promoter contained antioxidant response elements, transcriptional activity depended on the interaction between Nrf2 and the constructs containing the transcriptional start site. Moreover, Nrf2-driven transcription was significantly reduced by an inhibitor of histone acetyltransferase, p300. Correspondingly, Nrf2 overexpression increased levels of acetylated histone3 and p300. The reduction in LAMTOR1 by 4-HNE was reversed by pepstatin A and NH4Cl which block lysosomal degradation. 4-HNE increased TFEB nuclear translocation which was reversed by LAMTOR1 overexpression. In vivo, LAMTOR1 levels decreased in the photoreceptor and RPE layers of NaIO3-injected mice, compared to PBS-injected controls. In conclusion, oxidative injury reduces LAMTOR1, predominantly through lysosomal degradation although Nrf2-mediated histone acetylation enhances lamtor1 transcription. This study reveals a previously unrecognized regulatory mechanism of lamtor1 by oxidative stress, suggesting a novel role for LAMTOR1 in the pathogenesis of AMD.

Importance of OCT-derived biomarkers for the recurrence of central serous chorioretinopathy using statistics and predictive modelling

Central serous chorioretinopathy (CSCR) is a retinal disease characterised by the accumulation of subretinal fluid, which often resolves spontaneously in acute cases. However, approximately one-third of patients experience recurrences that may cause severe and irreversible vision. This study aimed to identify parameters derived from optical coherence tomography (OCT) that are associated with CSCR recurrence. Our dataset included 5211 OCT scans from 344 eyes of 255 patients diagnosed with CSCR. 178 eyes were identified as recurrent, 109 as non-recurrent, and 57 were excluded. We extracted parameters using artificial intelligence algorithms based on U-Nets, convolutional kernels, and morphological operators. We applied inferential statistics to evaluate differences between the recurrent and non-recurrent groups, and we used a logistic regression predictive model, reporting the coefficients as a measure of biomarker importance. We identified nine predictive biomarkers for CSCR recurrence: age, intraretinal fluid, subretinal fluid, pigment epithelial detachments, choroidal vascularity index, integrity of photoreceptors and retinal pigment epithelium layer, choriocapillaris and choroidal stroma thickness, and thinning of the outer nuclear layer, and of the inner nuclear layer combined with the outer plexiform layer. These results could enable future developments in the automatic detection of CSCR recurrence, paving the way for translational medical applications.

Quantitative comparison of automated OCT and conventional FAF-based geographic atrophy measurements in the phase 3 OAKS/DERBY trials

With the approval of the first two substances for the treatment of geographic atrophy (GA) secondary to age-related macular degeneration (AMD), a standardized monitoring of patients treated with complement inhibitors in clinical practice is needed. Optical coherence tomography (OCT) provides high-resolution access to the retinal pigment epithelium (RPE) and neurosensory layers, such as the ellipsoid zone (EZ), which further enhances the understanding of disease progression and therapeutic effects in GA compared to conventional fundus autofluorescence (FAF). In addition, artificial intelligence-based methodology allows the identification and quantification of GA-related pathology on OCT in an objective and standardized manner. The purpose of this study was to comprehensively evaluate automated OCT monitoring for GA compared to reading center-based manual FAF measurements in the largest successful phase 3 clinical trial data of complement inhibitor therapy to date. Automated OCT analysis of RPE loss showed a high and consistent correlation to manual GA measurements on conventional FAF. EZ loss on OCT was generally larger than areas of RPE loss, supporting the hypothesis that EZ loss exceeds underlying RPE loss as a fundamental pathophysiology in GA progression. Automated OCT analysis is well suited to monitor disease progression in GA patients treated in clinical practice and clinical trials.

VEGF and ELAVL1/HuR protein levels are increased in dry and wet AMD patients. A new tile in the pathophysiologic mechanisms underlying RPE degeneration?

Age-related macular degeneration (AMD) is a common retinal pathology characterized by degeneration of macula’s retinal pigment epithelium (RPE) and photoreceptors, visual impairment, or loss. Compared to wet AMD, dry AMD is more common, but lacks cures; therefore, identification of new potential therapeutic targets and treatments is urgent. Increased oxidative stress and declining antioxidant, detoxifying systems contribute to the pathophysiologic mechanisms underlying AMD. The present work shows that the Embryonic Lethal Abnormal Vision-Like 1/Human antigen R (ELAVL1/HuR) and the Vascular Endothelial Growth Factor (VEGF) protein levels are higher in the RPE of both dry and wet AMD patients compared to healthy subjects. Moreover, increased HuR protein levels are detected in the retina, and especially in the RPE layer, of a dry AMD model, the nuclear factor erythroid 2-related factor 2 (Nrf2) / peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) double knock-out mouse. The crosstalk among Nrf2, HuR and VEGF has been also studied in ARPE-19 cells in basal and stressful conditions related to the AMD context (i.e., oxidative stress, autophagy impairment, Nrf2 deficit), offering new evidence of the mutual influence between Nrf2 and HuR, of the dependence of VEGF expression and secretion by these two factors, and of the increased susceptibility of cells to stressful conditions in Nrf2- or HuR-impaired contexts. Overall, this study shows evidence of the interplay among Nrf2, HuR and VEGF, essential factors for RPE homeostasis, and represents an additional piece in the understanding of the complex pathophysiologic mechanisms underlying AMD.

Selective nanosecond laser removal of retinal pigment epithelium for cell therapy

Retinal pigment epithelial (RPE) cells play a crucial role in the health of the retina, and their dysfunction is associated with various ocular diseases. The transplantation of RPE cells has been proposed as a potential treatment for numerous degenerative diseases, including geographic atrophy from macular degeneration. However, current models to induce RPE damage in animal models prior to transplantation involve mechanical scraping, chemical administration, or laser photocoagulation techniques, which can damage the overlying neurosensory retina. This study aims to investigate the feasibility and efficacy of nanosecond duration laser treatment to safely remove large areas of RPE cells without causing damage to the adjacent tissue or affecting the retinal architecture. Twelve pigmented rabbits were treated with a nanosecond laser on each eye at a laser energy ranging from 200 to 800 nJ with a treated area of 5 × 5 mm2. Human induced pluripotent stem cells-differentiated to RPE (hiPSC-RPE) cells labeled with indocyanine green (ICG), an FDA approved dye, were transplanted subretinally into the damaged RPE areas at day 14 post-laser treatment. The RPE atrophy and hiPSC-RPE cell survival was evaluated and monitored over a period of 14 days using color photography, fluorescein angiography (FA), photoacoustic microscopy (PAM), and optical coherence tomography (OCT) imaging. All treated eyes demonstrated focal RPE loss with a success rate of 100%. The injured RPE layers and the transplanted hiPSC-RPE cells were visualized in three dimensions using PAM and OCT. By performing PAM at an optical wavelength of 700 nm, the location of hiPSC-RPE cells were identified and distinguished from the surrounding RPE cells, and the induced PA signal increased up to 18 times. Immunohistochemistry results confirmed the grafted hiPSC-RPE replaced regions of RPE damage. This novel technique has the potential to serve as an animal model of RPE degeneration, to improve models of RPE transplantation, and may help accelerate translation of this therapeutic strategy for clinical use.

Inducible RPE-specific GPX4 knockout causes oxidative stress and retinal degeneration with features of age-related macular degeneration

Age-related macular degeneration (AMD) is one of the leading causes of vision loss in the elderly. This disease involves oxidative stress burden in the retina leading to death of retinal pigment epithelial (RPE) cells and photoreceptors. The retina is susceptible to oxidative stress, in part due to high metabolic activity and high concentration of polyunsaturated fatty acids that undergo lipid peroxidation chain reactions. Antioxidant enzymes exist in the retina to combat this stress, including glutathione peroxidase 4 (GPX4). GPX4 specifically reduces oxidized lipids, protecting against lipid peroxidation-induced oxidative stress, which is noted in dry AMD. We hypothesize that Gpx4 knockout within the RPE will result in an environment of chronic oxidative stress yielding degeneration akin to AMD. C57BL/6J mice with a floxed Gpx4 gene were mated with Rpe65Cre/ER mice. Offspring containing Rpe65Cre ± alleles and either Gpx4 WT or Gpx4 fl/fl alleles were administered tamoxifen to induce Gpx4 knockout in Gpx4 fl/fl mice. At sequential timepoints, retinal phenotypes were assessed via in vivo imaging utilizing confocal scanning laser ophthalmoscopy and optical coherence tomography (OCT), and visual function was probed by electroretinography. Retinas were studied post-mortem by immunohistochemical analyses, electron microscopy, plastic sectioning, and quantitative polymerase chain reaction and Western analyses. The RPE-specific Gpx4 knockout model was validated via Western analysis indicating diminished GPX4 protein only within the RPE and not the neural retina. Following Gpx4 knockout, RPE cells became dysfunctional and died, with significant cell loss occurring 2 weeks post-knockout. Progressive thinning of the photoreceptor layer followed RPE degeneration and was accompanied by loss of visual function. OCT and light microscopy showed hyperreflective foci and enlarged, pigmented cells in and above the RPE layer. Electron microscopy revealed decreased mitochondrial cristae and loss of basal and apical RPE ultrastructure. Finally, there was increased carboxyethylpyrrole staining, indicating oxidation of docosahexaenoic acid, and increased levels of mRNAs encoding oxidative stress-associated genes in the RPE and photoreceptors. Overall, we show that RPE-localized GPX4 is necessary for the health of the RPE and outer retina, and that knockout recapitulates phenotypes of dry AMD.

Disease Activity and Therapeutic Response to Pegcetacoplan for Geographic Atrophy Identified by Deep Learning-Based Analysis of OCT

PurposeTo quantify morphological changes of the photoreceptors (PR) and retinal pigment epithelium (RPE) layers under pegcetacoplan therapy in geographic atrophy (GA) using deep learning-based analysis of optical coherence tomography (OCT) images. DesignPost-hoc longitudinal image analysis SubjectsPatients with GA due to age-related macular degeneration from two prospective randomized phase III clinical trials (OAKS and DERBY) MethodsDeep learning-based segmentation of RPE loss and PR degeneration, defined as loss of the ellipsoid zone (EZ) layer on OCT, over 24 months on SD-OCT images Main outcome measuresChange in the mean area of RPE loss and EZ loss over time in the pooled sham arms and the monthly (PM)/every other month (PEOM) treatment arms Results897 eyes of 897 patients were included. There was a therapeutic reduction of RPE loss growth by 22%/20% in OAKS and 27%/21% in DERBY for PM/PEOM compared to sham, respectively, at 24 months. The reduction on the EZ level was significantly higher with 53%/46% in OAKS and 47%/46% in DERBY for PM/PEOM compared to sham at 24 months. The baseline EZ-RPE difference had an impact on disease activity and therapeutic response. The therapeutic benefit for RPE loss growth increased with larger EZ-RPE difference quartiles from 21.9%, 23.1%, 23.9% to 33.6% for PM vs. sham (all p<0.01) and from 13.6% (p=0.11), 23.8%, 23.8% to 20.0% for PEOM vs. sham (p<0.01) in quartiles 1,2,3 and 4, respectively, at 24 months. Regarding EZ layer maintenance, the therapeutic reduction of loss increased from 14.8% (p=0.09), 33.3%, 46.6% to 77.8% (p<0.0001) between PM and sham and from 15.9% (p=0.08), 33.8%, 52.0% to 64.9% (p<0.0001) between PEOM and sham for quartiles 1-4 at 24 months. ConclusionOCT-based AI analysis objectively identifies and quantifies PR and RPE degeneration in GA. Reductions in further PR degeneration consistent with EZ loss on OCT are even higher than the effect on RPE loss in phase 3 trials of pegcetacoplan treatment. The EZ-RPE difference has a strong impact on disease progression and therapeutic response. Identification of patients with higher EZ-RPE loss difference may become an important criterion for the management of GA secondary to AMD.

Sodium-iodate injection can replicate retinal and choroid degeneration in pigmented mice: Using multimodal imaging and label-free quantitative proteomics analysis

Age-related macular degeneration (AMD) is the leading cause of irreversible visual loss in the elderly population. Sodium iodate (NaIO3), a stable oxidizing agent, has been injected to establish a reproducible model of oxidative stress-induced RPE and photoreceptor death. The aim of our study was to evaluate the morphological and molecular changes of retina and retinal pigment epithelium (RPE)-choroid in NaIO3-treated mouse using multimodal fundus imaging and label-free quantitative proteomics analysis. Here, we found that following NaIO3 injection, retinal degeneration was evident. Fundus photographs showed numerous scattered yellow-white speckled deposits. Optical coherence tomography (OCT) images indicated disruption of the retinal layers, damage of the RPE layer and accumulation of hyper-reflective matter in multiple layers of the outer retina. Widespread foci of a high fundus autofluorescence (FAF) signal were noticed. Fundus fluorescein angiography (FFA) revealed diffuse intense transmitted fluorescence mixed with scattered spot-like blocked fluorescence. Indocyanine green angiography (ICGA) presented punctate hyperfluorescence. Due to the atrophy of the RPE and Bruch's membrane and choroidal capillary complex, the larger choroidal vessels become more prominent in ICGA and optical coherence tomography angiography (OCTA). Transmission electron microscope (TEM) illustrated abnormal material accumulation and damaged mitochondria. Bioinformatics analysis of proteomics revealed that the differentially expressed proteins participated in diverse biological processes, encompassing phototransduction, NOD-like receptor signaling pathway, phagosome, necroptosis, and cell adhesion molecules. In conclusion, by multimodal imaging, we described the phenotype of NaIO3-treated mouse model mimicking oxidative stress-induced RPE and photoreceptor death in detail. In addition, proteomics analysis identified differentially expressed proteins and significant enrichment pathways, providing insights for future research, although the exact mechanism of oxidative stress-induced RPE and photoreceptor death remains incompletely understood.

Vitelliform subretinal deposits: shared pathogenesis and clinical diversity

Since its initial description, Best vitelliform macular dystrophy has been strongly linked to characteristic retinal lesions—subretinal yellowish accumulations, with a hyperreflective structure on optical coherence tomography, situated above the retinal pigment epithelium layer. However, research on this topic remains scarce. It is now recognized that, within the common pathophysiological mechanism of vitelliform lesion formation, this process may contribute to the pathogenesis of a broad spectrum of both genetic and acquired conditions.The purpose of this review is to update our understanding of genetically determined and acquired retinal diseases associated with the formation of such lesions.Materials and methods. analysis of scientific publications from databases including PubMed, ScienceDirect, and Cyberleninka was conducted. The review encompasses 16 studies, predominantly from the past decade.Results. Advances in instrumental and genetic diagnostics have revealed that vitelliform subretinal changes are not exclusive to Best disease but serve as another biomarker, indicating disruption in the metabolism of outer retinal layers. It is hypothesized that, irrespective of etiology, the primary triggering factor for lesion formation is the disjunction of outer segments of photoreceptors and impairment of phagocytic function in retinal pigment epithelial cells. This article presents a contemporary perspective on the presumed etiology and pathophysiology of vitelliform lesions, alongside clinical characteristics, manifestations, and prognosis of diseases associated with this phenomenon. Diseases with identified genetic mutations in the BEST1 gene (Bestrophinopathies) and PRPH2, as well as common acquired conditions like age-related macular degeneration and vitreoretinal traction syndrome, are discussed.Conclusions. The provided clinical characteristics, supported by multimodal visualization, are expected to aid in differential diagnosis and prognostication of the disease course.

Sequential Fabrication of a Three-Layer Retina-like Structure.

Tissue engineering is considered a promising approach to treating advanced degenerative maculopathies such as nonexudative age-related macular degeneration (AMD), the leading cause of blindness worldwide. The retina consists of several hierarchical tissue layers, each of which is supported by a layer underneath. Each of these layers has a different morphology and requires distinct conditions for proper assembly. In fact, a prerequisite step for the assembly of each of these layers is the organization of the layer underneath. Advanced retinal degeneration includes degeneration of the other retina layers, including the choroid, the retinal pigmented epithelium (RPE), and the photoreceptors. Here, we report a step-by-step fabrication process of a three-layer retina-like structure. The process included the 3D printing of a choroid-like structure in an extracellular matrix (ECM) hydrogel, followed by deposition of the RPE monolayer. After the formation of the blood vessel-RPE interface, the photoreceptor cells were deposited to interact with the RPE layer. At the end of the fabrication process, each layer was characterized for its morphology and expression of specific markers, and the integration of the three-layer retina was evaluated. We envision that such a retina-like structure may be able to attenuate the deterioration of a degenerated retina and improve engraftment and regeneration. This retinal implant may potentially be suitable for a spectrum of macular degenerative diseases for which there are currently no cures and may save millions from complete blindness.

Exploring ocular fibulin-3 (EFEMP1): Anatomical, age-related, and species perspectives

Fibulin-3 (FBLN3, aka EFEMP1) is a secreted extracellular matrix (ECM) glycoprotein implicated in ocular diseases including glaucoma and age-related macular degeneration. Yet surprisingly, little is known about its native biology, expression patterns, and localization in the eye. To overcome these shortcomings, we conducted gene expression analysis and immunohistochemistry for FBLN3 in ocular tissues from mice, pigs, non-human primates, and humans. Moreover, we evaluated age-related changes in FBLN3 and FBLN3-related ECM remodeling enzymes/inhibitors in aging mice. We found that FBLN3 displayed distinct staining patterns consistent across the mouse retina, particularly in the ganglion cell layer and inner nuclear layer (INL). In contrast, human retinas exhibited a unique staining pattern, with enrichment of FBLN3 in the retinal pigment epithelium (RPE), INL, and outer nuclear layer (ONL) in the peripheral retina. This staining transitioned to the outer plexiform layer (OPL) in the central retina/macula, and was accompanied by reduced RPE immunoreactivity approaching the fovea. Surprisingly, we found significant age-related increases in FBLN3 expression and protein abundance in the mouse retina which was paralleled by reduced transcript levels of FBLN3-degrading enzymes (i.e., Mmp2 and Htra1). Our findings highlight important species-dependent, retinal region-specific, and age-related expression and localization patterns of FBLN3 which favor its accumulation during aging. These findings contribute to a better understanding of FBLN3's role in ocular pathology and provide valuable insights for future FBLN3 research.

Characteristics and Treatment Outcomes of Hypotony Maculopathy Post-Glaucoma Filtering Surgery: An Optical Coherence Tomography-Based Study.

This study assesses morphological characteristics using SD-OCT in patients with hypotony maculopathy (HM) following glaucoma filtering surgery and evaluates the results of its treatment. Retrospective analysis of all HM patients between January 2019 and March 2023. Inclusion criteria consisted of both pre-operative and post-revision SD-OCT images of the macula and the presence of HM as observed on OCT images preoperatively. HM was graded according to its appearance in OCT both pre- and post-revision surgery. Change in visual acuity and IOP were assessed. 45 eyes of 45 patients were included. 21 eyes had HM limited to retinal pigment epithelium (RPE), 18 eyes had involvement of RPE and photoreceptor layers and 6 eyes had additional intra- or subretinal edema. After revision surgery with IOP elevation, 64% of eyes had complete HM regression with no HM signs in OCT imaging. 80% of patients achieved at least one grade improvement in HM. Preoperative visual acuity increased from 0.7±0.4 (logMAR) to 0.4±0.4 at 2 weeks postoperatively, over the course of an increase of IOP from 3.5±1.8mmHg to 17.1±10.6mmHg at day one. Eyes with complete HM regression had higher IOP at day 1 compared to those without improvement (P=0.04). The median time between HM onset and revision was 10.0 days for those with complete regression and 27 days for those without improvement (P=0.04). Bleb revision procedures for HM following glaucoma filtering surgery show promising outcomes, including notable improvements in visual acuity and IOP. The timing of revision surgery appears to influence outcome. In our study earlier intervention was associated with better results. Even delayed surgeries can lead to an improvement, although complete morphological restoration may not be achieved in advanced grades of HM.

Quantitative investigation on the effects of eyeball geometry, blood perfusion and natural convection in retinal laser surgery

The photothermal effects of laser on tissue are widely used in ophthalmology to treat retinal diseases. The accurate modelling of retinal laser surgery will prove valuable guidance for clinic treatment. There are several important issues should be carefully evaluated during retinal laser surgery modelling, including the eye geometry (whole eye or fundus only, retinal pigment epithelium layer incorporated or not), blood perfusion and natural convection. In previous publications, the existence of the anterior part of eyeball and retinal pigment epithelium layer is neglected to simply the eye geometry due to low laser light absorption (anterior part of eyeball) or small volume occupation (retinal pigment epithelium layer), respectively. However, the justification of this treatment has not been conducted. In this study, we will quantitatively investigate these factors systemically in a large pulse duration range that covers all the retinal laser surgery used in clinic. To do this, we present a bioheat transfer model for the simulation of the thermal response of the eye to laser irradiation. In the simulation, the 3D whole-eye geometry is considered. The Pennes bioheat transfer equation is adopted for the whole-eye temperature modelling. Simultaneously, we treat the fundus as a multilayered medium that all primary components, including RPE layer, are considered for first time in whole eye modelling. Our model calculates the thermal responses of the eye during laser retinal surgery with the pulse duration from nanosecond to tens of second. The effects of eye geometry, blood perfusion and natural convection are clearly clarified based on the laser pulse duration and incident energy used. The results show that neglecting the anterior part of eyeball will introduce the computational error more than 27 %. The existence of the retinal pigment epithelium layer should always be considered in retinal laser surgery modelling in all pulse durations used in clinic, otherwise more than 20 % computational error will be generated. When the pulse duration is longer than 10s, the effect of blood perfusion and natural convection in vitreous body should both be considered.

Reduced contrast sensitivity function is correlated with changes to cone photoreceptors in simple high myopia.

To investigate the contrast sensitivity function (CSF) changes in simple high myopia (SHM) and evaluate the correlations between these changes with the early changes in the retinal microstructure. This prospective study comprised 81 subjects, 20 with emmetropia (EM), 26 with low myopia and moderate myopia (LM/MM), and 35 with SHM. The area under the log CSF curve (AULCSF) and the cut-off spatial frequency (Cut-off SF) were employed as measures of CSF. Adaptive optics (AO) was employed to quantify the cone density, spacing, and regularity. The thickness and blood flow of the retinal sublayers were determined from vertical and horizontal optical coherence tomography angiography (OCTA) A-scans. Swept-source optical coherence tomography (SS-OCT) was employed to analyze the choroidal thickness (CT) and choroidal vascularity using a custom algorithm. Differences in the retinal and choroidal parameters, cone distribution, AULCSF, and Cut-off SF were compared among the three groups. Multivariate linear mixed models were used to elucidate the associations between photoreceptor morphological alterations, retinal and choroidal parameters, and AULCSF. The AULCSF and Cut-off SF were significantly lower in the SHM group compared to the EM and LM groups (p < 0.05). The SHM group had less cone density, larger cone spacing, and lower cone regularity than the EM and LM/MM groups (p < 0.05). Moreover, the thickness of the inner segment of photoreceptors (IS), retinal pigment epithelium (RPE) layer and choroid were reduced, and the outer segment of photoreceptors (OS) was thicker in the SHM group compared to the EM and LM/MM groups (all p < 0.05). A longer axial length (AL) was correlated with decreased AULCSF, cone density, and cone spacing (r = -0.800 to 0.752, all p < 0.050). Additionally, decreased CSF was correlated with lower cone density (r = 0.338, p = 0.035). Decreased contrast sensitivity was observed in patients with SHM and cone density was significantly correlated with reduced AUCSF.

Clinically compliant cryopreservation of differentiated retinal pigment epithelial cells

Background aimsAge-related macular degeneration (AMD) is the most common cause of blindness in elderly patients within developed countries, affecting more than 190 million worldwide. In AMD, the retinal pigment epithelial (RPE) cell layer progressively degenerates, resulting in subsequent loss of photoreceptors and ultimately vision. There is currently no cure for AMD, but therapeutic strategies targeting the complement system are being developed to slow the progression of the disease. MethodsReplacement therapy with pluripotent stem cell-derived (hPSC) RPEs is an alternative treatment strategy. A cell therapy product must be produced in accordance with Good Manufacturing Practices at a sufficient scale to facilitate extensive pre-clinical and clinical testing. Cryopreservation of the final cell product is therefore highly beneficial, as the manufacturing, pre-clinical and clinical testing can be separated in time and location. ResultsWe found that mature hPSC-RPE cells do not survive conventional cryopreservation techniques. However, replating the cells 2–5 days before cryopreservation facilitates freezing. The replated and cryopreserved hPSC-RPE cells maintained their identity, purity and functionality as characteristic RPEs, shown by cobblestone morphology, pigmentation, transcriptional profile, RPE markers, transepithelial resistance and pigment epithelium–derived factor secretion. Finally, we showed that the optimal replating time window can be tracked noninvasively by following the change in cobblestone morphology. ConclusionsThe possibility of cryopreserving the hPSC-RPE product has been instrumental in our efforts in manufacturing and performing pre-clinical testing with the aim for clinical translation.

Elovl4b knockout zebrafish as a model for ocular very-long-chain PUFA deficiency

Very-long-chain PUFAs (VLC-PUFAs) are a group of lipids with chain lengths >24 carbons, and the ELOVL4 (elongation of very-long-chain FA-4) enzyme is responsible for vertebrate VLC-PUFA biosynthesis. Studies on the role of VLC-PUFAs in vision have been hindered because of the need for adequate animal models to capture the global loss of VLC-PUFAs. Since homozygous Elovl4 ablation is lethal in neonatal mice because of catastrophic drying from the loss of their protective skin barrier, we established a zebrafish (Danio rerio) model of Elovl4 ablation. We generated Elovl4b KO zebrafish by creating a 56-bp deletion mutation in exon 2 of the Elovl4b gene using CRISPR-Cas9. We used GC-MS and LC-MS/MS to analyze the VLC-PUFA and lipid profiles from wild-type and Elovl4b KO fish eyes. We also performed histology and visual-behavioral tests. We found that heterozygous and homozygous Elovl4b KO zebrafish eyes had altered lipid profiles and a significantly lower C30 to C36 VLC-PUFA abundance than wild-type fish. Moreover, Elovl4b+/− and Elovl4b−/− KO larvae had significantly lower motor activity in response to light-dark cycles than their age-matched controls. Elovl4b−/− adult fish showed no obvious differences in gross retinal morphology and lamination compared with wild type, except for the presence of lipid droplets within the retinal pigment epithelial cell layer of Elovl4b−/− fish. Our data indicate that the loss of Elovl4b in zebrafish changes ocular lipid profiles and leads to visual abnormalities and subtle retinal changes. These findings highlight the use of zebrafish as a model for VLC-PUFA depletion and ELOVL4-related dysfunction.

Choriocapillaris, photoreceptor and inner retinal layers in spatial relationship to parapapillary alpha, beta, gamma and delta zone

Aims/Purpose: To examine the spatial relationships between the choriocapillaris, retinal layers and retinal pigment epithelium (RPE) in the parapapillary region.Methods: Human eyes enucleated due to uveal melanomas or secondary angle‐closure glaucoma were histomorphometrically examined.Results: The investigation consisted of 100 globes. In non‐highly myopic non‐glaucomatous eyes, the photoreceptor layer, choriocapillaris and retinal inner nuclear layer (INL) ended approximately at the end of the RPE layer, with no significant (all p ≥ 0.10) difference between the layers. In the non‐highly myopic, glaucomatous eyes and the highly myopic eyes, the photoreceptor layer, choriocapillaris and inner nuclear layer ended in the majority of eyes beyond the RPE‐end in direction to the optic disc border, with the INL extending beyond the RPE‐end significantly more than the photoreceptor layer (non‐highly myopic non‐glaucomatous group: 133 ± 183 μm versus 66.5 ± 96.8 μm; p = 0.001; highly myopic glaucomatous eyes: (266 ± 395 μm vs. 211 ± 286 μm; p = 0.03). A larger extension of the photoreceptor layer, choriocapillaris and INL beyond the RPE‐end correlated with longer axial length (beta: 0.50; B: 32.2; 95% CI: 21.6, 42.8) and wider beta zone (beta: 0.28; B: 0.10; 95% CI: 0.04, 0.16), with longer axial length (beta: 0.24; B: 23.0; 95% CI: 1.6, 44.5) and longer beta zone (beta: 0.59; B: 0.32; 95% CI: 0.22, 0.41; p &lt; 0.001), and with longer axial length (beta: 0.34; B: 43.7; 95% CI: 11.6, 75.7), longer gamma zone (beta: 0.52; B: 0.28; 5% CI: 0.15, 0.41) and diagnosis of non‐highly myopic glaucoma (beta: 0.28; B: 267; 95% CI: 80.8, 454), respectively.Conclusions: Non‐glaucomatous non‐highly myopic eyes differ from highly myopic eyes and glaucomatous eyes in the spatial relationship of the parapapillary tissue layers. The findings support the notion of an axial elongation‐associated shifting of the parapapillary RPE and Bruch's membrane for development of myopic beta zone, and an RPE‐cell loss for development of glaucomatous beta zone.

Determination of modes for safe application of selective micro-pulse individual retinal therapy of the macular zone using the modeling based on the theory of activated complex

Purpose: to make modeling of various modes of laser radiation, to assess the degree of damage to the retinal pigment epithelium and adjacent structures, and to determine modes and safety conditions of selective micro-pulse individual retinal therapy based on the theory of activated complex. Materials and methods. The authors have modeled the degree of damage to the chorioretinal complex and adjacent structures using the activated complex theory with the Eyring equation. The following definitions were used to determine the regime quality and safety conditions: micro-pulse mode is a mode with fixed values of micro-pulse duration, duty cycle and the number of pulses in the packet; power is a variable parameter. Efficiency is the ratio of the amount of denatured protein inside the retinal pigment epithelium layer to the total amount of protein in it. Selectivity is the ratio of the amount of denatured protein inside the retinal pigment epithelium layer to the total amount of denatured protein. Relative harmfulness is the ratio of denatured protein volume outside the retinal pigment epithelium to the volume of protein in it. Results and discussion. The quality of micro-pulse mode was found as depending on the selectivity value with efficiency equal to 90 %. With the efficiency value less than 50 %, the mode is non-selective, with more than 67 % – selective. Safety conditions for selective micro-pulse modes have been established, taking into account safety of the neurosensory retina and choroid in terms of relative harmfulness which should not exceed 1. Parameters at which micro-pulse modes have high values (more than 90 – 99) of efficiency and selectivity at the same time have been determined as well. It requires single pulse mode, micro pulse duration of 25 microseconds and power from 6 to 8 W. To expand the combination of parameters, it is necessary to be able to freely change them in increments of 1 pulse, 5 microseconds, 100 mW, with long interval and short duty cycle. These technical capabilities can be realized and implemented into clinical practice by manufacturers of laser devices. Conclusion. For selective micro-pulse individual retinal therapy of the macular zone with existing lasers, the following recommendation are to be observed: micro-pulse modes with pulse duration no more than 200 microseconds, short duty cycle less than 5 % and number of pulses in an envelope no more than 5; relative harmfulness less than one, radiation power selection, personalized patient’s characteristics.