Search

Radiation retinopathy is a serious vision-impairing complication of radiation therapy used to treat ocular tumors. Characterized by retinal vasculopathy and subsequent retinal damage, the first sign of radiation retinopathy is the preferential loss of vascular endothelial cells. Ensuing ischemia leads to retinal degradation and late stage neovascularization. Despite the established disease progression, the pathophysiology and cellular mechanisms contributing to radiation retinopathy remain unclear. Clinical experience and basic research for other retinal vasculopathies, such as diabetic retinopathy and retinopathy of prematurity, can inform our understanding of radiation retinopathy; however, the literature investigating the fundamental mechanisms in radiation retinopathy is limited. Treatment trials have shown modest success but, ultimately, fail to address the cellular events that initiate radiation retinopathy. Animal models of radiation retinopathy could provide means to identify effective therapies. Here, we review the literature for all animal models of radiation retinopathy, summarize anatomical highlights pertaining to animal models, identify additional physiological factors to consider when investigating radiation retinopathy, and explore the use of clinically relevant tests for studying in vivo models of radiation retinopathy. We encourage further investigation into the mechanistic characterization of radiation retinopathy in the hope of discovering novel treatments., (Copyright © 2019. Published by Elsevier Ltd.)

Purpose: Human pluripotent stem cell (hPSC)-derived retinal organoids are a platform for investigating retinal development, pathophysiology, and cellular therapies. In contrast to histologic analysis in which multiple specimens fixed at different times are used to reconstruct developmental processes, repeated analysis of the same living organoids provides a more direct means to characterize changes. New live imaging modalities can provide insights into retinal organoid structure and metabolic function during in vitro growth. This study employed live tissue imaging to characterize retinal organoid development, including metabolic changes accompanying photoreceptor differentiation., Methods: Live hPSC-derived retinal organoids at different developmental stages were examined for microanatomic organization and metabolic function by phase contrast microscopy, optical coherence tomography (OCT), fluorescence lifetime imaging microscopy (FLIM), and hyperspectral imaging (HSpec). Features were compared to those revealed by histologic staining, immunostaining, and microcomputed tomography (micro-CT) of fixed organoid tissue., Results: We used FLIM and HSpec to detect changes in metabolic activity as organoids differentiated into organized lamellae. FLIM detected increased glycolytic activity and HSpec detected retinol and retinoic acid accumulation in the organoid outer layer, coinciding with photoreceptor genesis. OCT enabled imaging of lamellae formed during organoid maturation. Micro-CT revealed three-dimensional structure, but failed to detect lamellae., Conclusions: Live imaging modalities facilitate real-time and nondestructive imaging of retinal organoids as they organize into lamellar structures. FLIM and HSpec enable rapid detection of lamellar structure and photoreceptor metabolism. Live imaging techniques may aid in the continuous evaluation of retinal organoid development in diverse experimental and cell therapy settings.

Retinal degeneration (RD) is a significant cause of incurable blindness worldwide. Photoreceptors and retinal pigmented epithelium are irreversibly damaged in advanced RD. Functional replacement of photoreceptors and/or retinal pigmented epithelium cells is a promising approach to restoring vision. This paper reviews the current status and explores future prospects of the transplantation therapy provided by pluripotent stem cell-derived retinal organoids (ROs). This review summarizes the status of rodent RD disease models and discusses RO culture and analytical tools to evaluate RO quality and function. Finally, we review and discuss the studies in which RO-derived cells or sheets were transplanted. In conclusion, methods to derive ROs from pluripotent stem cells have significantly improved and become more efficient in recent years. Meanwhile, more novel technologies are applied to characterize and validate RO quality. However, opportunity remains to optimize tissue differentiation protocols and achieve better RO reproducibility. In order to screen high-quality ROs for downstream applications, approaches such as noninvasive and label-free imaging and electrophysiological functional testing are promising and worth further investigation. Lastly, transplanted RO-derived tissues have allowed improvements in visual function in several RD models, showing promises for clinical applications in the future.

Retinal degeneration is a leading cause of vision impairment and blindness worldwide and medical care for advanced disease does not exist. Stem cell-derived retinal organoids (RtOgs) became an emerging tool for tissue replacement therapy. However, existing RtOg production methods are highly heterogeneous. Controlled and predictable methodology and tools are needed to standardize RtOg production and maintenance. In this study, we designed a shear stress-free micro-millifluidic bioreactor for nearly labor-free retinal organoid maintenance. We used a stereolithography (SLA) 3D printer to fabricate a mold from which Polydimethylsiloxane (PDMS) was cast. We optimized the chip design using in silico simulations and in vitro evaluation to optimize mass transfer efficiency and concentration uniformity in each culture chamber. We successfully cultured RtOgs at three different differentiation stages (day 41, 88, and 128) on an optimized bioreactor chip for more than 1 month. We used different quantitative and qualitative techniques to fully characterize the RtOgs produced by static dish culture and bioreactor culture methods. By analyzing the results from phase contrast microscopy, single-cell RNA sequencing (scRNA seq), quantitative polymerase chain reaction (qPCR), immunohistology, and electron microscopy, we found that bioreactor-cultured RtOgs developed cell types and morphology comparable to static cultured ones and exhibited similar retinal genes expression levels. We also evaluated the metabolic activity of RtOgs in both groups using fluorescence lifetime imaging (FLIM), and found that the outer surface region of bioreactor cultured RtOgs had a comparable free/bound NADH ratio and overall lower long lifetime species (LLS) ratio than static cultured RtOgs during imaging. To summarize, we validated an automated micro-millifluidic device with significantly reduced shear stress to produce RtOgs of comparable quality to those maintained in conventional static culture.

PurposeCompare results obtained using infrared two-photon microperimetry (2PM-IR) with conventional visual function tests in healthy subjects of varying ages with and without simulated media opacities.MethodsSubjects from two separate cohort studies completed cone contrast threshold (CCT) testing, conventional microperimetry, visible light microperimetry from a novel device (2PM-Vis), and infrared two-photon microperimetry. The first cohort study, which consisted of six healthy volunteers (23 to 29 years of age), evaluated the effects of simulated media opacities on visual performance testing. Subjects underwent testing on four visual function devices nine separate times under the following conditions: no filter, red filter, green filter, blue filter, light brown filter, dark brown filter, polarized black filter (0° rotation), and polarized black filter (90° rotation). Subjects subsequently performed 2PM-IR and 2PM-Vis testing without a filter in the mydriatic state. The second cohort study evaluated the effect of age on visual test performance in 42 healthy subjects split between two groups (ages 20-40 years and 60-80 years).ResultsRetinal sensitivity measured by 2PM-IR demonstrated lower variability than all other devices relying on visible spectrum stimuli. Retinal sensitivity decreased proportionally with the transmittance of light through each filter. CCT scores and retinal sensitivity decreased with age in all testing modalities. Visible spectrum testing modalities demonstrated larger test result differences between young and old patient cohorts; this difference was inversely proportional to the wavelength of the visual function test.Conclusions2PM-IR mitigates media opacities that may mask small differences in retinal sensitivity when tested with conventional visual function testing devices.Translational relevanceConventional visual function tests that emit visible light may not detect differences in retinal function during the early stages of age-related diseases due to the confounding effects of cataracts. Infrared light, which has greater transmittance through ocular tissue, may reliably quantify retinal sensitivity and thereby detect degenerative changes early on.