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Purpose To assess the prevalence of autoimmune disease (AiD) in patients with primary open-angle glaucoma (POAG) undergoing ophthalmic surgery. Design Retrospective, cross-sectional study. Participants Patients with POAG undergoing any ophthalmic surgery and control subjects undergoing cataract surgery at the Massachusetts Eye and Ear from March 2019 to April 2020. Methods All available medical records with patient demographics, ocular, and medical conditions were reviewed. Differences in AiD prevalence were assessed and adjusted for covariates using multiple logistic regression. Additionally, a subgroup analysis comparing the POAG patients with and without AiD was performed. Main Outcome Measures To assess the prevalence of AiD based on the American Autoimmune Related Diseases Association list. Results A total of 172 patients with POAG and 179 controls were included. The overall prevalence of AiD was 17.4% in the POAG group and 10.1% in the controls (P = 0.044); 6.4% of POAG patients and 3.4% of controls had more than 1 AiD (P = 0.18). The most prevalent AiDs in POAG group were rheumatoid arthritis (4.6%) and psoriasis (4.1%), which were also the most common in controls (2.8% each). In a fully adjusted multiple logistic regression analysis accounting for steroid use, having an AiD was associated with 2.62-fold increased odds of POAG relative to controls (95% confidence interval, 1.27–5.36, P = 0.009); other risk factors for POAG derived from the analysis included age (odds ratio [OR], 1.04, P = 0.006), diabetes mellitus (OR, 2.31, P = 0.008), and non-White ethnicity (OR, 4.75, P 0.13, for both). Conclusions A higher prevalence of AiD was found in POAG patients compared with control patients undergoing ophthalmic surgery. The presence of AiD was associated with increased risk for POAG after adjusting for covariates. Additional factors may have prevented a difference in RNFL thickness in POAG patients with and without AiD. Autoimmunity should be explored further in the pathogenesis of POAG.

Muller cells (MC) are considered dormant retinal progenitor cells in mammals. Previous studies demonstrated ephrin-As act as negative regulators of neural progenitor cells in the retina and brain. It remains unclear whether the lack of ephrin-A2/A3 is sufficient to promote the neurogenic potential of MC. Here we investigated whether the MC is the primary retinal cell type expressing ephrin-A2/A3 and their role on the neurogenic potential of Muller cells. In this study, we showed that ephrin-A2/A3 and their receptor EphA4 were expressed in retina and especially enriched in MC. The level of ephrinAs/EphA4 expression increased as the retina matured that is correlated with the reduced proliferative and progenitor cell potential of MC. Next, we investigated the proliferation in primary MC cultures isolated from wild-type and A2-/- A3-/- mice by 5-ethynyl-2'-deoxyuridine (EdU) incorporation. We detected a significant increase of EdU+ cells in MC derived from A2-/- A3-/- mice. Next, we investigated the role of ephrin-A2/A3 in mice undergoing photoreceptor degeneration such as Rhodopsin knockout (Rho-/-) mice. To further evaluate the role of ephrin-A2/A3 in MC proliferation in vivo, EdU was injected intraperitoneally to adult wild-type, A2-/- A3-/- , Rho-/- and Rho-/- A2-/- A3-/- mice and the numbers of EdU+ cells distributed among different layers of the retina. EphrinAs/EphA4 expression was upregulated in the retina of Rho-/- mice compared to the wild-type mice. In addition, cultured MC derived from ephrin-A2-/- A3-/- mice also expressed higher levels of progenitor cell markers and exhibited higher proliferation potential than those from wild-type mice. Interestingly, we detected a significant increase of EdU+ cells in the retinas of adult ephrin-A2-/- A3-/- mice mainly in the inner nuclear layer; and these EdU+ cells were co-localized with MC marker, cellular retinaldehyde-binding protein, suggesting some proliferating cells are from MC. In Rhodopsin knockout mice (Rho-/- A2-/- A3-/- mice), a significantly greater amount of EdU+ cells were located in the ciliary body, retina and RPE than that of Rho-/- mice. Comparing between 6 and 12 weeks old Rho-/- A2-/- A3-/- mice, we recorded more EdU+ cells in the outer nuclear layer in the 12-week-old mice undergoing severe retinal degeneration. Taken together, Ephrin-A2/A3 are negative regulators of the proliferative and neurogenic potentials of MC. Absence of ephrin-A2/A3 promotes the migration of proliferating cells into the outer nuclear layer and may lead to retinal cell regeneration. All experimental procedures were approved by the Animal Care and Use Committee at Schepens Eye Research Institute, USA (approval No. S-353-0715) on October 24, 2012.

Retinal ischemic events, which result from occlusion of the ocular vasculature share similar causes as those for central nervous system stroke and are among the most common cause of acute and irreversible vision loss in elderly patients. Currently, there is no established treatment, and the condition often leaves patients with seriously impaired vision or blindness. The immune system, particularly T-cell- mediated responses, is thought to be intricately involved, but the exact roles remain elusive. We found that acute ischemia-reperfusion injury to the retina induced a prolonged phase of retinal ganglion cell loss that continued to progress during 8 weeks after the procedure. This phase was accompanied by microglial activation and CD4+ T-cell infiltration into the retina. Adoptive transfer of CD4+ T cells isolated from diseased mice exacerbated retinal ganglion cell loss in mice with retinal reperfusion damage. On the other hand, T-cell deficiency or administration of T-cell or interferon-gamma-neutralizing antibody attenuated retinal ganglion cell degeneration and retinal function loss after injury. These findings demonstrate a crucial role for T-cell-mediated responses in the pathogenesis of neural ischemia. These findings point to novel therapeutic targets of limiting or preventing neuron and function loss for currently untreatable conditions of optic neuropathy and/or central nervous system ischemic stroke.

Purpose Transcorneal electrical stimulation (TcES) is increasingly applied as a therapy for preserving and improving vision in retinal neurodegenerative and ischemic disorders. However, a common complaint about TcES is its induction of eye pain and dryness in the clinic, while the mechanisms remain unknown. Method TcES or transpalpebral ES (TpES) was conducted in C57BL6j mice for 14 days. The contralateral eyes were used as non-stimulated controls. Levels of intracellular [Ca2+] ([Ca2+]i) were assessed by Fura-2AM. The conductance resistances of the eye under various ES conditions were measured in vivo by an oscilloscope. Results Although TcES did not affect tear production, it significantly induced damage to the ocular surface, as revealed by corneal fluorescein staining that was accompanied by significantly decreased mucin (MUC) 4 expression compared to the control. Similar effects of ES were detected in cultured primary corneal epithelium cells, showing decreased MUC4 and ZO-1 levels after the ES in vitro. In addition, TcES decreased secretion of MUC5AC from the conjunctiva in vivo, which was also corroborated in goblet cell cultures, where ES significantly attenuated carbachol-induced [Ca2+]i increase. In contrast to TcES, transpalpebral ES (TpES) did not induce corneal fluorescein staining while significantly increasing tear production. Importantly, the conductive resistance from orbital skin to the TpES was significantly smaller than that from the cornea to the retina in TcES. Conclusion TcES, but not TpES, induces corneal epithelial damage in mice by disrupting mucin homeostasis. TpES thus may represent a safer and more effective ES approach for treating retinal neurodegeneration clinically.

Purpose Clinical manifestations of photoreceptor degeneration include gradual thinning of the outer nuclear layer (ONL) and progressive reduction of electroretinogram (ERG) amplitudes and vision loss. Although preclinical evaluations of treatment strategies greatly depend on rodent models, the courses of these changes in mice remain unclear. We thus sought to investigate the temporal correlations in changes of spatial vision, ERG response, and ONL thickness in mice with progressive photoreceptor degeneration. Methods Adult wild-type (WT) mice and mice carrying rhodopsin deficiency (Rho-/-), a frequently used mouse model of human retinitis pigmentosa, were selected for investigation. Mouse spatial vision, including visual acuity (VA) and contrast sensitivity (CS), was determined using optomotor response (OMR) assays; ONL thickness was quantified by spectral-domain optical coherence tomography (SD-OCT), and ERG was performed to evaluate retinal functions. The mice were killed when they were 14 weeks old, and the cone photoreceptors in retinal sections were counted. Results Spatial vision, ONL thickness, and ERG amplitudes remained stable in WT mice at all examined time points. While 6-week-old Rho-/- mice had VA, CS, as well as ERG responses similar to those of WT mice, progressive reductions in the spatial vision and retinal functions were recorded thereafter. Most tested 12-week-old Rho-/- mice had no visual-evoked OMR and ERG responses. Moreover, CS, but not VA, displayed a linear decline that was closely associated with ONL thinning, reduction of ERG amplitudes, and loss of cones. Conclusions We presented a comprehensive study of the relation between the changes of spatial vision, retinal function, and ONL thickness in postnatal week (PW)6 to PW12 Rho-/- mice. CS is a more sensitive indicator of spatial vision compared to VA, although both are required as separate parameters for monitoring the visual changes in retina undergoing photoreceptor degeneration.

Ischemia is a common pathological condition present in many neurodegenerative diseases, including ischemic stroke, retinal vascular occlusion, diabetic retinopathy, and glaucoma, threatening the sight and lives of millions of people globally. Ischemia can trigger excessive oxidative stress, inflammation, and vascular dysfunction, leading to the disruption of tissue homeostasis and, ultimately, cell death. Current therapies are very limited and have a narrow time window for effective treatment. Thus, there is an urgent need to develop more effective therapeutic options for ischemia-induced neural injuries. With emerging reports on the pharmacological properties of natural flavonoids, these compounds present potent antioxidative, anti-inflammatory, and antiapoptotic agents for the treatment of ischemic insults. Three major active flavonoids, baicalein, baicalin, and wogonin, have been extracted from Scutellaria baicalensis Georgi (S. baicalensis); all of which are reported to have low cytotoxicity. They have been demonstrated to exert promising pharmacological capabilities in preventing cell and tissue damage. This review focuses on the therapeutic potentials of these flavonoids against ischemia-induced neurotoxicity and damage in the brain and retina. The bioactivity and bioavailability of baicalein, baicalin, and wogonin are also discussed. It is with hope that the therapeutic potential of these flavonoids can be utilized and developed as natural treatments for ischemia-induced injuries of the central nervous system (CNS).

Non-invasive electrical stimulation (ES) is increasingly applied to improve vision in untreatable eye conditions, such as retinitis pigmentosa and age-related macular degeneration. Our previous study suggested that ES promoted retinal function and the proliferation of progenitor-like glial cells in mice with inherited photoreceptor degeneration, however, the underlying mechanism remains obscure. M&uuml, ller cells (MCs) are thought to be dormant residential progenitor cells that possess a high potential for retinal neuron repair and functional plasticity. Here, we showed that ES with a ramp waveform of 20 Hz and 300 &micro, A of current was effective at inducing mouse MC proliferation and enhancing their expression of progenitor cell markers, such as Crx (cone&ndash, rod homeobox) and Wnt7, as well as their production of trophic factors, including ciliary neurotrophic factor. RNA sequencing revealed that calcium signaling pathway activation was a key event, with a false discovery rate of 5.33 &times, 10&minus, 8 (P = 1.78 &times, 10) in ES-mediated gene profiling changes. Moreover, the calcium channel blocker, nifedipine, abolished the observed effects of ES on MC proliferation and progenitor cell gene induction, supporting a central role of ES-induced Ca2+ signaling in the MC changes. Our results suggest that low-current ES may present a convenient tool for manipulating MC behavior toward neuroregeneration and repair.

As the general population ages, more people are affected by eye diseases, such as retinopathies. It is therefore critical to improve imaging of eye disease mouse models. Here, we demonstrate that 1) rapid, quantitative 3D and 4D (time lapse) imaging of cellular and subcellular processes in the mouse eye is feasible, with and without tissue clearing, using light-sheet fluorescent microscopy (LSFM); 2) flat-mounting retinas for confocal microscopy significantly distorts tissue morphology, confirmed by quantitative correlative LSFM-Confocal imaging of vessels; 3) LSFM readily reveals new features of even well-studied eye disease mouse models, such as the oxygen-induced retinopathy (OIR) model, including a previously unappreciated ‘knotted’ morphology to pathological vascular tufts, abnormal cell motility and altered filopodia dynamics when live-imaged. We conclude that quantitative 3D/4D LSFM imaging and analysis has the potential to advance our understanding of the eye, in particular pathological, neurovascular, degenerative processes., eLife digest Eye diseases affect millions of people worldwide and can have devasting effects on people’s lives. To find new treatments, scientists need to understand more about how these diseases arise and how they progress. This is challenging and progress has been held back by limitations in current techniques for looking at the eye. Currently, the most commonly used method is called confocal imaging, which is slow and distorts the tissue. Distortion happens because confocal imaging requires that thin slices of eye tissue from mice used in experiments are flattened on slides; this makes it hard to accurately visualize three-dimensional structures in the eye. New methods are emerging that may help. One promising method is called light-sheet fluorescent microscopy (or LSFM for short). This method captures three-dimensional images of the blood vessels and cells in the eye. It is much faster than confocal imaging and allows scientists to image tissues without slicing or flattening them. This could lead to more accurate three-dimensional images of eye disease. Now, Prahst et al. show that LSFM can quickly produce highly detailed, three-dimensional images of mouse retinas, from the smallest parts of cells to the entire eye. The technique also identified new features in a well-studied model of retina damage caused by excessive oxygen exposure in young mice. Previous studies of this model suggested the disease caused blood vessels in the eye to balloon, hinting that drugs that shrink blood vessels would help. But using LSFM, Prahst et al. revealed that these blood vessels actually take on a twisted and knotted shape. This suggests that treatments that untangle the vessels rather than shrink them are needed. The experiments show that LSFM is a valuable tool for studying eye diseases, that may help scientists learn more about how these diseases arise and develop. These new insights may one day lead to better tests and treatments for eye diseases.

Glaucoma is the most prevalent neurodegenerative disease and a leading cause of blindness worldwide. The mechanisms causing glaucomatous neurodegeneration are not fully understood. Here we show, using mice deficient in T and/or B cells and adoptive cell transfer, that transient elevation of intraocular pressure (IOP) is sufficient to induce T-cell infiltration into the retina. This T-cell infiltration leads to a prolonged phase of retinal ganglion cell degeneration that persists after IOP returns to a normal level. Heat shock proteins (HSP) are identified as target antigens of T-cell responses in glaucomatous mice and human glaucoma patients. Furthermore, retina-infiltrating T cells cross-react with human and bacterial HSPs; mice raised in the absence of commensal microflora do not develop glaucomatous T-cell responses or the associated neurodegeneration. These results provide compelling evidence that glaucomatous neurodegeneration is mediated in part by T cells that are pre-sensitized by exposure to commensal microflora., National Institutes of Health (U.S.) (Grant EY025913), National Institutes of Health (U.S.) (Grant EY027067), National Institutes of Health (U.S.) (Grant EY025259), National Institutes of Health (U.S.) (Grant NS038253), National Institutes of Health (U.S.) (Grant AI69208)

Glaucoma is a complex neurodegenerative disease with many clinical subtypes. Some of its rare forms include pigmentary glaucoma, uveitic glaucoma and congenital glaucoma. While they all share common features of progressive retinal ganglion cell (RGC) loss, optic nerve damage and corresponding visual field loss, the exact mechanisms underlying glaucomatous neuron loss are not clear. This has largely hindered the development of a real cure for this disease. Elevated intraocular pressure (IOP) is a known major risk factor of glaucoma; however, progressive degeneration of RGCs and axons can also be found in patients with a normal IOP, i.e., normal tension glaucoma (NTG). Interestingly, patients who carry the gain-of-function mutation of the pro-inflammatory gene TBK1 - tumor necrosis factor (TNF) receptor associated factor NF-κB activator (TANK) binding kinase 1 - are at increased risk to develop NTG. This finding suggests a causal link between neuroinflammatory processes and glaucoma. Various studies have reported the presence of neuroinflammatory responses by microglia, astrocytes and other blood-born immune cells in the optic nerve head (ONH) at early stages of experimental glaucoma. Inhibition of certain pro-inflammatory pathways, particularly those associated with microglial activation, appears to be neuroprotective. In this review, we will focus on the inflammatory responses, in particular the proposed roles of microglia, in the pathogenesis of glaucoma.

Previous studies have demonstrated that activation of P2X7 receptors (P2X7R) results in the proliferation and migration of some types of tumor. Here, we asked whether and how the activated P2X7R contribute to proliferation and migration of human glioma cells. Results showed that the number of P2X7R positive cells was increasing with grade of tumor. In U87 and U251 human glioma cell lines, both expressed P2X7R and the expression was enhanced by 3′-O-(4-benzoylbenzoyl) ATP (BzATP), the agonist of P2X7R, and siRNA. Our results also showed that 10 μM BzATP was sufficient to induce the proliferation of glioma cell significantly, while the cell proliferation reached the peak with 100 μM BzATP. Also, the migration of U87 and U251 cells was significantly increased upon BzATP treatment. However, the number of apoptotic cells of U87 and U251 was not significantly changed by BzATP. In addition, the expression of ERK, p-ERK, and proliferating cell nuclear antigen (PCNA) protein was increased in BzATP-treated U87 and U251 glioma cells. PD98059, an inhibitor of the MEK/ERK pathway, blocked the increased proliferation and migration of glioma cells activated by BzATP. These results suggest that ERK pathway is involved in the proliferation and migration of glioma cells induced by P2X7R activation.

// Na Liu 1 , Shiqi Li 2 , Nan Wu 1 and Kin-Sang Cho 3 1 Department of Ophthalmology, Southwest Eye Hospital, Southwest Hospital, Third Military Medical University, Chongqing, China 2 Center of biotherapy, Southwest Hospital, Third Military Medical University, Chongqing, China 3 Schepens Eye Research Institute, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, Massachusetts, USA Correspondence to: Kin-Sang Cho, email: kinsang_cho@meei.harvard.edu Nan Wu, email: nanwu1122@163.com Keywords: cancer stem cell, acetylation, deacetylation, HDAC, HAT Received: March 14, 2017 Accepted: June 27, 2017 Published: July 11, 2017 ABSTRACT Cancer stem-like cell (CSC) model has been established to investigate the underlying mechanisms of tumor initiation and progression. The imbalance between acetylation and deacetylation of histone or non-histone proteins, one of the important epigenetic modification processes, is closely associated with a wide variety of diseases including cancer. Acetylation and deacetylation are involved in various stemness-related signal pathways and drive the regulation of self-renewal and differentiation in normal developmental processes. Therefore, it is critical to explore their role in the maintenance of cancer stem-like cell traits. Here, we will review the extensive dysregulations of acetylation found in cancers and summarize their functional roles in sustaining CSC-like properties. Additionally, the use of deacetyltransferase inhibitors as an effective therapeutic strategy against CSCs is also discussed.

As the general population ages and the incidence of diabetes increases epidemically, more people are affected by eye diseases, such as retinopathies. It is therefore critical to improve imaging of eye disease mouse models. Here, we demonstrate that 1) rapid, quantitative 3D and 4D (time lapse) imaging of cellular and subcellular processes in the murine eye is feasible, with and without tissue clearing, using light-sheet fluorescent microscopy (LSFM) and 2) LSFM readily reveals new features of even well studied eye disease mouse models, such as the Oxygen-Induced Retinopathy (OIR) model. Through correlative LSFM-Confocal imaging we find that flat-mounting retinas for confocal microscopy significantly distorts tissue morphology. The minimized distortion with LSFM dramatically improved analysis of pathological vascular tufts in the OIR model revealing “knotted” morphologies, leading to a proposed new tuft nomenclature. Furthermore, live-imaging of OIR tuft formation revealed abnormal cell motility and altered filopodia dynamics. We conclude that quantitative 3D/4D LSFM imaging and analysis has the potential to advance our understanding of pathological processes in the eye, in particular neuro-vascular degenerative processes.

Retinal ischemic events, which result from occlusion of the ocular vasculature share similar causes as those for central nervous system stroke and are among the most common cause of acute and irreversible vision loss in elderly patients. Currently, there is no established treatment, and the condition often leaves patients with seriously impaired vision or blindness. The immune system, particularly T-cell–mediated responses, is thought to be intricately involved, but the exact roles remain elusive. We found that acute ischemia-reperfusion injury to the retina induced a prolonged phase of retinal ganglion cell loss that continued to progress during 8 weeks after the procedure. This phase was accompanied by microglial activation and CD4(+) T-cell infiltration into the retina. Adoptive transfer of CD4(+) T cells isolated from diseased mice exacerbated retinal ganglion cell loss in mice with retinal reperfusion damage. On the other hand, T-cell deficiency or administration of T-cell or interferon-γ–neutralizing antibody attenuated retinal ganglion cell degeneration and retinal function loss after injury. These findings demonstrate a crucial role for T-cell–mediated responses in the pathogenesis of neural ischemia. These findings point to novel therapeutic targets of limiting or preventing neuron and function loss for currently untreatable conditions of optic neuropathy and/or central nervous system ischemic stroke.

Evolving research has provided evidence that noninvasive electrical stimulation (ES) of the eye may be a promising therapy for either preserving or restoring vision in several retinal and optic nerve diseases. In this review, we focus on minimally invasive strategies for the delivery of ES and accordingly summarize the current literature on transcorneal, transorbital, and transpalpebral ES in both animal experiments and clinical studies. Various mechanisms are believed to underlie the effects of ES, including increased production of neurotrophic agents, improved chorioretinal blood circulation, and inhibition of proinflammatory cytokines. Different animal models have demonstrated favorable effects of ES on both the retina and the optic nerve. Promising effects of ES have also been demonstrated in clinical studies; however, all current studies have a lack of randomization and/or a control group (sham). There is thus a pressing need for a deeper understanding of the underlying mechanisms that govern clinical success and optimization of stimulation parameters in animal studies. In addition, such research should be followed by large, prospective, clinical studies to explore the full potential of ES. Through this review, we aim to provide insight to guide future research on ES as a potential therapy for improving vision.

Purpose: Neurons carry electrical signals and communicate via electrical activities. The therapeutic potential of electrical stimulation (ES) for the nervous system, including the retina, through improvement of cell survival and function has been noted. Here we investigated the neuroprotective and regenerative potential of ES in a mouse model of inherited retinal degeneration. Methods: Rhodopsin-deficient (Rho−/−) mice received one or two sessions of transpalpebral ES or sham treatments for 7 consecutive days. Intraperitoneal injection of 5-ethynyl-2′-deoxyuridine was used to label proliferating cells. Weekly electroretinograms were performed to monitor retinal function. Retinal morphology, photoreceptor survival, and regeneration were evaluated in vivo using immunohistochemistry and genetic fate-mapping techniques. Müller cell (MC) cultures were employed to further define the optimal conditions of ES application. Results: Noninvasive transpalpebral ES in Rho−/− mice improved photoreceptor survival and electroretinography function in vivo. ES also triggered residential retinal progenitor-like cells such as MCs to reenter the cell cycle, possibly producing new photoreceptors, as shown by immunohistochemistry and genetic fate-mapping techniques. ES directly stimulated cell proliferation and the expression of progenitor cell markers in MC cultures, at least partially through bFGF signaling. Conclusions: Our study showed that transpalpebral ES improved photoreceptor survival and retinal function and induced the proliferation, probably photoreceptor regeneration, of MCs; this occurs via stimulation of the bFGF pathways. These results suggest the exciting possibility of applying noninvasive ES as a versatile tool for preventing photoreceptor loss and mobilizing endogenous progenitors for reversing vision loss in patients with photoreceptor degeneration.

Activation of axonal growth program is a critical step in successful optic nerve regeneration following injury. Yet the molecular mechanisms that orchestrate this developmental transition are not fully understood. Here we identified a novel regulator, insulin-like growth factor binding protein-like 1 (IGFBPL1), for the growth of retinal ganglion cell (RGC) axons. Expression of IGFBPL1 correlates with RGC axon growth in development, and acute knockdown of IGFBPL1 with shRNA or IGFBPL1 knockout in vivo impaired RGC axon growth. In contrast, administration of IGFBPL1 promoted axon growth. Moreover, IGFBPL1 bound to insulin-like growth factor 1 (IGF-1) and subsequently induced calcium signaling and mammalian target of rapamycin (mTOR) phosphorylation to stimulate axon elongation. Blockage of IGF-1 signaling abolished IGFBPL1-mediated axon growth, and vice versa, IGF-1 required the presence of IGFBPL1 to promote RGC axon growth. These data reveal a novel element in the control of RGC axon growth and suggest an unknown signaling loop in the regulation of the pleiotropic functions of IGF-1. They suggest new therapeutic target for promoting optic nerve and axon regeneration and repair of the central nervous system.

Epigenetic predisposition is thought to critically contribute to adult-onset disorders, such as retinal neurodegeneration. The histone methyltransferase, enhancer of zeste homolog 2 (Ezh2), is transiently expressed in the perinatal retina, particularly enriched in retinal ganglion cells (RGCs). We previously showed that embryonic deletion of Ezh2 from retinal progenitors led to progressive photoreceptor degeneration throughout life, demonstrating a role for embryonic predisposition of Ezh2-mediated repressive mark in maintaining the survival and function of photoreceptors in the adult. Enrichment of Ezh2 in RGCs leads to the question if Ezh2 also mediates gene expression and function in postnatal RGCs, and if its deficiency changes RGC susceptibility to cell death under injury or disease in the adult. To test this, we generated mice carrying targeted deletion of Ezh2 from RGC progenitors driven by Math5-Cre (mKO). mKO mice showed no detectable defect in RGC development, survival, or cell homeostasis as determined by physiological analysis, live imaging, histology, and immunohistochemistry. Moreover, RGCs of Ezh2 deficient mice revealed similar susceptibility against glaucomatous and acute optic nerve trauma-induced neurodegeneration compared to littermate floxed or wild-type control mice. In agreement with the above findings, analysis of RNA sequencing of RGCs purified from Ezh2 deficient mice revealed few gene changes that were related to RGC development, survival and function. These results, together with our previous report, support a cell lineage-specific mechanism of Ezh2-mediated gene repression, especially those critically involved in cellular function and homeostasis.

Optomotor response/reflex (OMR) assays are emerging as a powerful and versatile tool for phenotypic study and new drug discovery for eye and brain disorders. Yet efficient OMR assessment for visual performance in mice remains a challenge. Existing OMR testing devices for mice require a lengthy procedure and may be subject to bias due to use of artificial criteria. We developed an optimized staircase protocol that utilizes mouse head pausing behavior as a novel indicator for the absence of OMR, to allow rapid and unambiguous vision assessment. It provided a highly sensitive and reliable method that can be easily implemented into automated or manual OMR systems to allow quick and unbiased assessment for visual acuity and contrast sensitivity in mice. The sensitivity and quantitative capacity of the protocol were validated using wild type mice and an inherited mouse model of retinal degeneration – mice carrying rhodopsin deficiency and exhibiting progressive loss of photoreceptors. Our OMR system with this protocol was capable of detecting progressive visual function decline that was closely correlated with the loss of photoreceptors in rhodopsin deficient mice. It provides significant advances over the existing methods in the currently available OMR devices in terms of sensitivity, accuracy and efficiency.

AIM To characterize whether a glaucoma model with chronic elevation of the intraocular pressure (IOP) was able to be induced by anterior chamber injection of microbeads in rabbits. METHODS In order to screen the optimal dose of microbead injection, IOP was measured every 3d for 4wk using handheld applanation tonometer after a single intracameral injection of 10 µL, 25 µL, 50 µL or 100 µL microbeads (5×106 beads/mL; n=6/group) in New Zealand White rabbits. To prolong IOP elevation, two intracameral injections of 50 µL microbeads or phosphate buffer saline (PBS) were made respectively at days 0 and 21 (n=24/group). The fellow eye was not treated. At 5wk after the second injection of microbeads or PBS, bright-field microscopy and transmission electron microscopy (TEM) were used to assess the changes in the retina. The expression of glial fibrillary acidic protein (GFAP) in the retina was evaluated by immunofluorescence, quantitative real-time polymerase chain reaction and Western blot at 5wk after the second injection of microbeads. RESULTS Following a single intracameral injection of 10 µL, 25 µL, 50 µL or 100 µL microbead, IOP levels showed a gradual increase and a later decrease over a 4wk period after a single injection of microbead into the anterior chamber of rabbits. A peak IOP was observed at day 15 after injection. No significant difference in peak value of IOP was found between 10 µL and 25 µL groups (17.13±1.25 mm Hg vs 17.63±0.74 mm Hg; P=0.346). The peak value of IOP from 50 µL group (23.25±1.16 mm Hg) was significantly higher than 10 µL and 25 µL groups (all P

Optic neuropathy is one of the leading causes of irreversible blindness caused by retinal ganglion cell (RGC) degeneration. The development of induced pluripotent stem cell (iPSC)-based therapy opens a therapeutic window for RGC degeneration, and tissue engineering may further promote the efficiency of differentiation process of iPSCs. The present study was designed to evaluate the effects of a novel biomimetic polybenzyl glutamate (PBG) scaffold on culturing iPSC-derived RGC progenitors. The iPSC-derived neural spheres cultured on PBG scaffold increased the differentiated retinal neurons and promoted the neurite outgrowth in the RGC progenitor layer. Additionally, iPSCs cultured on PBG scaffold formed the organoid-like structures compared to that of iPSCs cultured on cover glass within the same culture period. With RNA-seq, we found that cells of the PBG group were differentiated toward retinal lineage and may be related to the glutamate signaling pathway. Further ontological analysis and the gene network analysis showed that the differentially expressed genes between cells of the PBG group and the control group were mainly associated with neuronal differentiation, neuronal maturation, and more specifically, retinal differentiation and maturation. The novel electrospinning PBG scaffold is beneficial for culturing iPSC-derived RGC progenitors as well as retinal organoids. Cells cultured on PBG scaffold differentiate effectively and shorten the process of RGC differentiation compared to that of cells cultured on coverslip. The new culture system may be helpful in future disease modeling, pharmacological screening, autologous transplantation, as well as narrowing the gap to clinical application.

The ciliary epithelium (CE) of adult mammals has been reported to provide a source of retinal stem cells (RSCs) that can give rise to all retinal cell types in vitro. A recent study, however, suggests that CE-derived cells possess properties of pigmented ciliary epithelial cells and display little neurogenic potential. Here we show that the neurogenic potential of CE-derived cells is negatively regulated by ephrin-A3, which is upregulated in the CE of postnatal mice and presents a strong prohibitory niche for adult RSCs. Addition of ephrin-A3 inhibits proliferation of CE-derived RSCs and increases pigment epithelial cell fate. In contrast, absence of ephrin-A3 promotes proliferation and increases expression of neural progenitor cell markers and photoreceptor progeny. The negative effects of ephrin-A3 on CE-derived RSCs are mediated through activation of an EphA4 receptor and suppression of Wnt3a/β-catenin signaling. Together, our data suggest that CE-derived RSCs contain the intrinsic machinery to generate photoreceptors and other retinal neurons, while the CE of adult mice expresses negative regulators that prohibit the proliferation and neural differentiation of RSCs. Manipulating ephrin and Wnt/β-catenin signaling may, thus, represents a viable approach to activating the endogenous neurogenic potential of CE-derived RSCs for treating photoreceptor damage and retinal degenerative disorders.

Some adult-onset disorders may be linked to dysregulated embryonic development, yet the mechanisms underlying this association remain poorly understood. Congenital retinal degenerative diseases are blinding disorders characterized by postnatal degeneration of photoreceptors, and affect nearly 2 million individuals worldwide, but ∼50% do not have a known mutation, implicating contributions of epigenetic factors. We found that embryonic deletion of the histone methyltransferase (HMT) Ezh2 from all retinal progenitors resulted in progressive photoreceptor degeneration throughout postnatal life, via derepression of fetal expression of Six1 and its targets. Forced expression of Six1 in the postnatal retina was sufficient to induce photoreceptor degeneration. Ezh2, although enriched in the embryonic retina, was not present in the mature retina; these data reveal an Ezh2-mediated feed-forward pathway that is required for maintaining photoreceptor homeostasis in the adult and suggest novel targets for retinal degeneration therapy.

Retinal degenerative diseases such as age-related macular degeneration, retinitis pigmentosa, and glaucoma result in permanent loss of retinal neurons and vision. Stem cell therapy could be a novel treatment strategy to restore visual function. In an ideal situation, a homogenous population of stem cell–derived retinal neurons with high purity is used for replacement therapy. Thus, it is crucial to elucidate the molecular mechanisms that regulate the development of retinal progenitor cells and subsequent generation of specific retinal neurons. Here, recent findings concerning the intrinsic and extrinsic factors that regulate retinal progenitor cell maintenance and differentiation are summarized, especially transcriptional factors and extrinsic signals. Understanding these mechanisms is indispensable because they have potential clinical applications, chiefly the generation of specific retinal cells such as retinal ganglion cells to treat glaucoma and other optic neuropathy diseases.