Bioactivity and gene expression profiles of hiPSC-generated retinal ganglion cells in MT-ND4 mutated Leber's hereditary optic neuropathy.

Leber's hereditary optic neuropathy (LHON) is the maternally inherited mitochondrial disease caused by homoplasmic mutations in mitochondrial electron transport chain Complex I subunit genes. The mechanism of its incomplete penetrance is still largely unclear. In this study, we created the patient-specific human induced pluripotent stem cells (hiPSCs) from MT-ND4 mutated LHON-affected patient, asymptomatic mutation carrier and healthy control, and differentiated them into retinal ganglion cells (RGCs). We found the defective neurite outgrowth in affected RGCs, but not in the carrier RGCs which had significant expression of SNCG gene. We observed enhanced mitochondrial biogenesis in affected and carrier derived RGCs. Surprisingly, we observed increased NADH dehydrogenase enzymatic activity of Complex I in hiPSC-derived RGCs of asymptomatic carrier, but not of the affected patient. LHON mutation substantially decreased basal respiration in both affected and unaffected carrier hiPSCs, and had the same effect on spare respiratory capacity, which ensures normal function of mitochondria in conditions of increased energy demand or environmental stress. The expression of antioxidant enzyme catalase was decreased in affected and carrier patient hiPSC-derived RGCs as compared to the healthy control, which might indicate to higher oxidative stress-enriched environment in the LHON-specific RGCs. Microarray profiling demonstrated enhanced expression of cell cycle machinery and downregulation of neuronal specific genes.

Establishing Liposome-Immobilized Dexamethasone-Releasing PDMS Membrane for the Cultivation of Retinal Pigment Epithelial Cells and Suppression of Neovascularization.

Age-related macular degeneration (AMD) is the eye disease with the highest epidemic incidence, and has great impact on the aged population. Wet-type AMD commonly has the feature of neovascularization, which destroys the normal retinal structure and visual function. So far, effective therapy options for rescuing visual function in advanced AMD patients are highly limited, especially in wet-type AMD, in which the retinal pigmented epithelium and Bruch's membrane structure (RPE-BM) are destroyed by abnormal angiogenesis. Anti-VEGF treatment is an effective remedy for the latter type of AMD; however, it is not a curative therapy. Therefore, reconstruction of the complex structure of RPE-BM and controlled release of angiogenesis inhibitors are strongly required for sustained therapy. The major purpose of this study was to develop a dual function biomimetic material, which could mimic the RPE-BM structure and ensure slow release of angiogenesis inhibitor as a novel therapeutic strategy for wet AMD. We herein utilized plasma-modified polydimethylsiloxane (PDMS) sheet to create a biomimetic scaffold mimicking subretinal BM. This dual-surface biomimetic scaffold was coated with laminin and dexamethasone-loaded liposomes. The top surface of PDMS was covalently grafted with laminin and used for cultivation of the retinal pigment epithelial cells differentiated from human induced pluripotent stem cells (hiPSC-RPE). To reach the objective of inhibiting angiogenesis required for treatment of wet AMD, the bottom surface of modified PDMS membrane was further loaded with dexamethasone-containing liposomes via biotin-streptavidin linkage. We demonstrated that hiPSC-RPE cells could proliferate, express normal RPE-specific genes and maintain their phenotype on laminin-coated PDMS membrane, including phagocytosis ability, and secretion of anti-angiogenesis factor PEDF. By using in vitro HUVEC angiogenesis assay, we showed that application of our membrane could suppress oxidative stress-induced angiogenesis, which was manifested in decreased secretion of VEGF by RPE cells and suppression of vascularization. In conclusion, we propose modified biomimetic material for dual delivery of RPE cells and liposome-enveloped dexamethasone, which can be potentially applied for AMD therapy.

P3HT:Bebq2-Based Photovoltaic Device Enhances Differentiation of hiPSC-Derived Retinal Ganglion Cells.

Electric field stimulation is known to affect various cellular processes, including cell fate specification and differentiation, particularly towards neuronal lineages. This makes it a promising therapeutic strategy to stimulate regeneration of neuronal tissues. Retinal ganglion cells (RGCs) is a type of neural cells of the retina responsible for transduction of visual signals from the retina to the brain cortex, and is often degenerated in various blindness-causing retinal diseases. The organic photovoltaic materials such as poly-3-hexylthiophene (P3HT) can generate electric current upon illumination with light of the visible spectrum, and possesses several advantageous properties, including light weight, flexibility and high biocompatibility, which makes them a highly promising tool for electric stimulation of cells in vitro and in vivo. In this study, we tested the ability to generate photocurrent by several formulations of blend (bulk heterojunction) of P3HT (which is electron donor material) with several electron acceptor materials, including Alq3 and bis(10-hydroxybenzo[h]quinolinato)beryllium (Bebq2). We found that the photovoltaic device based on bulk heterojunction of P3HT with Bebq2 could generate photocurrent when illuminated by both green laser and visible spectrum light. We tested the growth and differentiation capacity of human induced pluripotent stem cells (hiPSC)-derived RGCs when grown in interface with such photostimulated device, and found that they were significantly increased. The application of P3HT:Bebq2-formulation of photovoltaic device has a great potential for developments in retinal transplantation, nerve repair and tissue engineering approaches of treatment of retinal degeneration.

COMPARISON OF PHOTODYNAMIC THERAPY USING HALF-DOSE OF VERTEPORFIN OR HALF-FLUENCE OF LASER LIGHT FOR THE TREATMENT OF CHRONIC CENTRAL SEROUS CHORIORETINOPATHY.

PURPOSE: To compare the efficacy and the detrimental effects of half-drug dose and half-laser light fluence of photodynamic therapy (PDT) for the treatment of chronic central serous chorioretinopathy. DESIGN: We conducted a prospective randomized, observer-masked comparison study. METHODS: Forty eyes (40 patients) with chronic central serous chorioretinopathy were enrolled in this study and were equally divided into 2 groups. The first (half-dose) group received only half the standard dose of verteporfin infusion (3 mg/m) and were irradiated by the standard 83 seconds of laser light (50 J/cm) for the PDT treatment; the second (half-fluence) group received the standard dose of verteporfin infusion (6 mg/m) and were irradiated by only 42 seconds of laser light (25 J/cm). Patients were examined at baseline and 1 week, 1 month, 3 months, and 6 months after PDT treatments with best-corrected visual acuity and optical coherence tomography. Fluorescein angiography and indocyanine green angiography (ICGA) were performed at baseline and at 1 month, 3 months, and 6 months after PDT treatment. Primary outcome measures were the changes in the best-corrected visual acuity and in central retinal thickness and subretinal fluid in optical coherence tomography. Secondary outcomes were the changes in the choroidal perfusion in the ICGA, which was measured as the fluorescein ratio of the PDT-treated area to a nontreated reference area in ICGA. RESULTS: Best-corrected visual acuity was significantly improved at post-PDT 1 month, 3 months, and 6 months (all P < 0.01) in both the half-dose and the half-fluence group. Central retinal thickness was significantly improved at all post-PDT time points in both groups (P < 0.05). All patients in the half-dose group and 19 patients (95%) in the half-fluence group had complete absorption of subretinal fluid at post-PDT 3 months and 6 months. The choroidal perfusion (as reflected by the decrease of the ratio of fluorescence) in ICGA was significantly decreased at all post-PDT follow-up time points in both groups (P < 0.01). However, there were no significant differences in all the measurements between the two groups, including best-corrected visual acuity, central retinal thickness, and hypofluorescence in ICGA at baseline and at each post-PDT follow-up time point. CONCLUSION: Both half-dose and half-fluence modifications of PDT were similarly effective in improving the visual acuity and subretinal fluid for chronic CSC. Both types of modification of PDT were also similar in causing postlaser choroidal hypoperfusion.

Elongation of Axon Extension for Human iPSC-Derived Retinal Ganglion Cells by a Nano-Imprinted Scaffold.

Optic neuropathies, such as glaucoma and Leber's hereditary optic neuropathy (LHON) lead to retinal ganglion cell (RGC) loss and therefore motivate the application of transplantation technique into disease therapy. However, it is a challenge to direct the transplanted optic nerve axons to the correct location of the retina. The use of appropriate scaffold can promote the proper axon growth. Recently, biocompatible materials have been integrated into the medical field, such as tissue engineering and reconstruction of damaged tissues or organs. We, herein, utilized nano-imprinting to create a scaffold mimicking the in vitro tissue microarchitecture, and guiding the axonal growth and orientation of the RGCs. We observed that the robust, long, and organized axons of human induced pluripotent stem cell (iPSC)-derived RGCs projected axially along the scaffold grooves. The RGCs grown on the scaffold expressed the specific neuronal biomarkers indicating their proper functionality. Thus, based on our in vitro culture system, this device can be useful for the neurophysiological analysis and transplantation for ophthalmic neuropathy treatment.

Correlation of microRNA-145 levels and clinical severity of pterygia.

PURPOSE: MicroRNA-145 (miR-145) has known anti-tumor properties and has been reported to be involved in regulating corneal epithelium differentiation. The exact role of miR-145 in ocular tissue remains unclear. In this study, we evaluate the effect of miR-145 expression levels on pterygium properties. SETTING: Ophthalmology department of a tertiary medical center. DESIGN: : Case series study. METHODS: Information regarding patient age, pterygium recurrence and pterygium severity (extension [E], vascularity [V] and thickness [T]) were gathered from records. Expression levels of miR-145 were obtained through examination of excised pterygium tissue. Correlations between age, pterygium classification, and miR-145 levels were evaluated. RESULTS: This study evaluated 253 patients (mean age 54.1±10.8 years). As pterygium severity increased, miR-145 levels decreased. Negative correlations were also found between miR-145 expression levels and pterygium extension (E) and vascularity (V). Thickness (T) had a weak negative correlation. There was only a mild negative correlation between patient age and miR-145 levels, which was only seen in patients with primary pterygium (not recurrent ones). Additionally, miR-145 expression was significantly higher in primary samples than in recurrent ones. CONCLUSIONS: We demonstrated an association between miR-145 and pterygium characteristics, consistent with its known tumor suppression effect. Because the management of pterygium is often difficult, we suggest that miR-145 should be further studied as a potential treatment.

Improvement of carbon tetrachloride-induced acute hepatic failure by transplantation of induced pluripotent stem cells without reprogramming factor c-Myc.

The only curative treatment for hepatic failure is liver transplantation. Unfortunately, this treatment has several major limitations, as for example donor organ shortage. A previous report demonstrated that transplantation of induced pluripotent stem cells without reprogramming factor c-Myc (3-genes iPSCs) attenuates thioacetamide-induced hepatic failure with minimal incidence of tumorigenicity. In this study, we investigated whether 3-genes iPSC transplantation is capable of rescuing carbon tetrachloride (CCl(4))-induced fulminant hepatic failure and hepatic encephalopathy in mice. Firstly, we demonstrated that 3-genes iPSCs possess the capacity to differentiate into hepatocyte-like cells (iPSC-Heps) that exhibit biological functions and express various hepatic specific markers. 3-genes iPSCs also exhibited several antioxidant enzymes that prevented CCl(4)-induced reactive oxygen species production and cell death. Intraperitoneal transplantation of either 3-genes iPSCs or 3-genes iPSC-Heps significantly reduced hepatic necrotic areas, improved hepatic functions, and survival rate in CCl(4)-treated mice. CCl(4)-induced hepatic encephalopathy was also improved by 3-genes iPSC transplantation. Hoechst staining confirmed the successful engraftment of both 3-genes iPSCs and 3-genes iPSC-Heps, indicating the homing properties of these cells. The most pronounced hepatoprotective effect of iPSCs appeared to originate from the highest antioxidant activity of 3-gene iPSCs among all transplanted cells. In summary, our findings demonstrated that 3-genes iPSCs serve as an available cell source for the treatment of an experimental model of acute liver diseases.

One-year outcomes of intravitreal bevacizumab (avastin) therapy for polypoidal choroidal vasculopathy.

PURPOSE: To report on 1-year visual, anatomical, and angiographic responses with intravitreal bevacizumab for the treatment of polypoidal choroidal vasculopathy. METHODS: Patients with macula-involved, symptomatic polypoidal choroidal vasculopathy with initial best-corrected visual acuity of 20/400 or better and a minimal follow-up period of 12 months were retrospectively enrolled. Eyes were treated with intravitreal bevacizumab (2.5 mg) at baseline and monitored monthly for best-corrected visual acuity and central retinal thickness (by optical coherence tomography). Indocyanine green angiography was evaluated on a 6-month basis. Eyes were retreated on an "as-needed" basis according to visual and anatomical changes. RESULTS: A total of 35 eyes of 33 patients were treated with a mean of 3.3 (range, 1-8) times of injection. Best-corrected visual acuity significantly improved from a mean logarithm of the minimum angle of resolution of 0.79 ± 0.42 at baseline (Snellen equivalent, 20/123) to 0.69 ± 0.47 (20/94), 0.66 ± 0.45 (20/87), 0.67 ± 0.44 (20/87), 0.67 ± 0.48 (20/87), and 0.67 ± 0.51 (20/87) at 1, 3, 6, 9, and 12 months, respectively (P = 0.002, 0.0003, 0.0008, 0.017, and 0.02, respectively; paired Student's t-test). Central retinal thickness also significantly improved from a mean of 297 ± 94 µm at baseline to 215 ± 58 µm, 214 ± 59 µm, 218 ± 79 µm, 213 ± 75 µm, and 221 ± 61 µm at 1, 3, 6, 9, and 12 months, respectively (all P < 0.0001, paired Student's t-test). Indocyanine green angiography showed 3 of 32 eyes (9.4%) and 5 of 31 eyes (16.1%) with completely resolved polyps and 11 of 32 eyes (34.4%) and 10 of 31 eyes (32.3%) with reduced polyps at 6 and 12 months, respectively. No systemic complication or severe local complication, such as endophthalmitis, was found. CONCLUSION: Intravitreal bevacizumab therapy has a favorable outcome in improving visual acuity and macular exudative changes in patients with polypoidal choroidal vasculopathy. It can also moderately reduce polypoidal lesions on indocyanine green angiography.

Enhanced differentiation of three-gene-reprogrammed induced pluripotent stem cells into adipocytes via adenoviral-mediated PGC-1α overexpression.

Induced pluripotent stem cells formed by the introduction of only three factors, Oct4/Sox2/Klf4 (3-gene iPSCs), may provide a safer option for stem cell-based therapy than iPSCs conventionally introduced with four-gene iPSCs. Peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) plays an important role during brown fat development. However, the potential roles of PGC-1α in regulating mitochondrial biogenesis and the differentiation of iPSCs are still unclear. Here, we investigated the effects of adenovirus-mediated PGC-1α overexpression in 3-gene iPSCs. PGC-1α overexpression resulted in increased mitochondrial mass, reactive oxygen species production, and oxygen consumption. Microarray-based bioinformatics showed that the gene expression pattern of PGC-1α-overexpressing 3-gene iPSCs resembled the expression pattern observed in adipocytes. Furthermore, PGC-1α overexpression enhanced adipogenic differentiation and the expression of several brown fat markers, including uncoupling protein-1, cytochrome C, and nuclear respiratory factor-1, whereas it inhibited the expression of the white fat marker uncoupling protein-2. Furthermore, PGC-1α overexpression significantly suppressed osteogenic differentiation. These data demonstrate that PGC-1α directs the differentiation of 3-gene iPSCs into adipocyte-like cells with features of brown fat cells. This may provide a therapeutic strategy for the treatment of mitochondrial disorders and obesity.

SirT1--a sensor for monitoring self-renewal and aging process in retinal stem cells.

Retinal stem cells bear potency of proliferation, self-renewal, and differentiation into many retinal cells. Utilizing appropriate sensors one can effectively detect the self-renewal and aging process abilities. Silencing information regulator (SirT1), a member of the sirtuin family, is a NAD-dependent histone deacetylase and an essential mediator for longevity in normal cells by calorie restriction. We firstly investigate the SirT1 mRNA expression in retinal stem cells from rats and 19 human eyes of different ages. Results revealed that SirT1 expression was significantly decreased in in vivo aged eyes, associated with poor self-renewal abilities. Additionally, SirT1 mRNA levels were dose-dependently increased in resveratrol- treated retinal stem cells. The expression of SirT1 on oxidative stress-induced damage was significantly decreased, negatively correlated with the level of intracellular reactive oxygen species production. Treatment with resveratrol could effectively further reduce oxidative stress induced by H(2)O(2) treatment in retinal stem cells. Importantly, the anti-oxidant effects of resveratrol in H(2)O(2)-treated retinal stem cells were significantly abolished by knockdown of SirT1 expression (sh-SirT1). SirT1 expression provides a feasible sensor in assessing self-renewal and aging process in retinal stem cells. Resveratrol can prevent reactive oxygen species-induced damages via increased retinal SirT1 expression.

Acute zonal occult outer retinopathy presenting as optic neuritis.

Acute zonal occult outer retinopathy (AZOOR) is an outer retinal disorder characterized by the acute loss of visual functions. Herein, we report a case of AZOOR presenting features mimicking optic neuritis. A 17-year-old healthy male reported fogginess in the right eye for 2 weeks. His best-corrected visual acuity was 20/20 in both eyes. Results of a color vision test and pupillary reaction were unremarkable. Funduscopic examination revealed a subtle hyperemic disc surrounded by hyperpigmentation in the right eye. Visual field examination confirmed an enlarged blind spot in the affected eye. Fundus autofluorescence imaging revealed zonal hyperautofluorescence around the optic disc. Fluorescein angiography showed optic disc staining and a window defect in the retinal pigment epithelium. Optical coherence tomography demonstrated loss of the ellipsoid line at the corresponding hyperautofluorescent region. All these characteristics indicated a diagnosis of AZOOR. However, the prolonged P100 wave observed through visual-evoked potential examination, hyperintensity T2 signal at the retrobulbar optic nerve through magnetic resonance imaging, and mild hyperemic optic disc along with optic disc staining through fluorescein angiography resemble the characteristics of optic neuritis. Because the clinical features of AZOOR are similar to those of optic neuritis, ophthalmologists should be able to differentiate between these two diseases to achieve a timely and correct diagnosis.

Changes in the Systemic Expression of Sirtuin-1 and Oxidative Stress after Intravitreal Anti-Vascular Endothelial Growth Factor in Patients with Retinal Vein Occlusion.

OBJECTIVES: Retinal vein occlusions (RVO) are associated with systemic risk factors. However, the ocular occlusive events might also influence a patient's systemic condition. This study tried to investigate serum biomarkers associated with oxidative stress, before and after intravitreal anti-vascular endothelial growth factor (aVEGF) therapy in patients with RVOs. METHODS: Newly-onset RVO patients were categorized into two groups: comorbid with macular edema requiring aVEGF therapy (treatment group) and no edema (observation group). Age and sex-matched patients (who received cataract surgery) were included as the control group. Intravitreal ranibizumab with a pro-re-nata regimen were administered. Serum samples were collected prior to treatment, at 6 and 12 months after therapy/observation and were collected once before controls who received cataract surgery. mRNA expression of sirtuin-1, its downstream genes, anti-oxidative biomarkers, and proinflammatory cytokines were measured. RESULTS: There were 32, 26, and 34 patients enrolled in the treatment, observation, and control groups, respectively. The expressions of sirtuin-1 and its downstream genes were significantly lower in patients with RVO compared with the control group. Sirtuin-1 gene expression increased after 1 year of aVEGF therapy in the treatment group but remained unchanged in the observation group. Biomarkers of oxidative stress and proinflammatory cytokines were reduced after 1 year of aVEGF therapy. These biomarkers remained with no changes in the observation group. CONCLUSIONS: Our study showed that the systemic oxidative stress increased in RVO patients. The aVEGF therapy could alter the gene expression of anti-oxidative proteins and reduce systemic oxidative stress in these patients.

Modulation of osmotic stress-induced TRPV1 expression rescues human iPSC-derived retinal ganglion cells through PKA.

BACKGROUND: Transient receptor potential vanilloid 1 (TRPV1), recognized as a hyperosmolarity sensor, is a crucial ion channel involved in the pathogenesis of neural and glial signaling. Recently, TRPV1 was determined to play a role in retinal physiology and visual transmission. In this study, we sought to clarify the role of TRPV1 and the downstream pathway in the osmotic stress-related retina ganglion cell (RGC) damage. METHODS: First, we modified the RGC differentiation protocol to obtain a homogeneous RGC population from human induced pluripotent stem cells (hiPSCs). Subsequently, we induced high osmotic pressure in the hiPSC-derived RGCs by administering NaCl solution and observed the behavior of the TRPV1 channel and its downstream cascade. RESULTS: We obtained a purified RGC population from the heterogeneous retina cell population using our modified method. Our findings revealed that TRPV1 was activated after 24 h of NaCl treatment. Upregulation of TRPV1 was noted with autophagy and apoptosis induction. Downstream protein expression analysis indicated increased phosphorylation of CREB and downregulated brain-derived neurotrophic factor (BDNF). However, hyperosmolarity-mediated defective morphological change and apoptosis of RGCs, CREB phosphorylation, and BDNF downregulation were abrogated after concomitant treatment with the PKA inhibitor H89. CONCLUSION: Collectively, our study results indicated that the TRPV1-PKA pathway contributed to cellular response under high levels of osmolarity stress; furthermore, the PKA inhibitor had a protective effect on RGCs exposed to this stress. Therefore, our findings may assist in the treatment of eye diseases involving RGC damage.

Nanomedicine-based Curcumin Approach Improved ROS Damage in Best Dystrophy-specific Induced Pluripotent Stem Cells.

Best dystrophy (BD), also termed best vitelliform macular dystrophy (BVMD), is a juvenile-onset form of macular degeneration and can cause central visual loss. Unfortunately, there is no clear definite therapy for BD or improving the visual function on this progressive disease. The human induced pluripotent stem cell (iPSC) system has been recently applied as an effective tool for genetic consultation and chemical drug screening. In this study, we developed patient-specific induced pluripotent stem cells (BD-iPSCs) from BD patient-derived dental pulp stromal cells and then differentiated BD-iPSCs into retinal pigment epithelial cells (BD-RPEs). BD-RPEs were used as an expandable platform for in vitro candidate drug screening. Compared with unaffected sibling-derived iPSC-derived RPE cells (Ctrl-RPEs), BD-RPEs exhibited typical RPE-specific markers with a lower expression of the tight junction protein ZO-1 and Bestrophin-1 (BEST1), as well as reduced phagocytic capabilities. Notably, among all candidate drugs, curcumin was the most effective for upregulating both the BEST1 and ZO-1 genes in BD-RPEs. Using the iPSC-based drug-screening platform, we further found that curcumin can significantly improve the mRNA expression levels of Best gene in BD-iPSC-derived RPEs. Importantly, we demonstrated that curcumin-loaded PLGA nanoparticles (Cur-NPs) were efficiently internalized by BD-RPEs. The Cur-NPs-based controlled release formulation further increased the expression of ZO-1 and Bestrophin-1, and promoted the function of phagocytosis and voltage-dependent calcium channels in BD-iPSC-derived RPEs. We further demonstrated that Cur-NPs enhanced the expression of antioxidant enzymes with a decrease in intracellular ROS production and hydrogen peroxide-induced oxidative stress. Collectively, these data supported that Cur-NPs provide a potential cytoprotective effect by regulating the anti-oxidative abilities of degenerated RPEs. In addition, the application of patient-specific iPSCs provides an effective platform for drug screening and personalized medicine in incurable diseases.

Elevation of serum oxidative stress in patients with retina vein occlusions.

PURPOSE: Retina vein occlusion (RVO) is a visual-threatening retinal disease that causes irreversible impaired quality of life. The contribution of oxidative stress behind clinical course of RVO was rarely investigated. The study aimed to measure the serum oxidative biomarker in patients with RVO to investigate further physical response. METHODS: We measured the serum levels of malondialdehyde (MDA), 8-hydroxy-2-deoxyguanosine (8OHdG), Sirutin 1 (SIRT1), peroxisome proliferator- activated receptor gamma (PPAR-r), Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), orkhead box protein O1 (FOXO1), orkhead box protein O3 (FOXO3), catalase, (SOD) and hydrogen peroxide (H2 O2 ) among 19 patients with cataract as control group and 36 patients with RVO, respectively. RESULTS: The mean MDA, 8OHdG and hydrogen peroxide in the serum were significantly higher in patients with RVO compared with the results in control group subjects. Whereas SIRT1, PPAR-r, PGC-1, FOXO1, FOXO3, catalase and SOD levels in serum were significantly decreased in patients with RVO compared with control group. CONCLUSION: We demonstrated that the serum level of MDA, 8OHdG and hydrogen peroxide is increased in patients with RVO. Among these, the elevation of MDA, 8OHdG and hydrogen peroxide suggests the increasing of serum oxidative stress in RVO patients. All enzymes related reactive oxygen species scavenge were decreased. Thus, focal RVO may increase systemic oxidative stress within serum.

Morphological and Molecular Defects in Human Three-Dimensional Retinal Organoid Model of X-Linked Juvenile Retinoschisis.

X-linked juvenile retinoschisis (XLRS), linked to mutations in the RS1 gene, is a degenerative retinopathy with a retinal splitting phenotype. We generated human induced pluripotent stem cells (hiPSCs) from patients to study XLRS in a 3D retinal organoid in vitro differentiation system. This model recapitulates key features of XLRS including retinal splitting, defective retinoschisin production, outer-segment defects, abnormal paxillin turnover, and impaired ER-Golgi transportation. RS1 mutation also affects the development of photoreceptor sensory cilia and results in altered expression of other retinopathy-associated genes. CRISPR/Cas9 correction of the disease-associated C625T mutation normalizes the splitting phenotype, outer-segment defects, paxillin dynamics, ciliary marker expression, and transcriptome profiles. Likewise, mutating RS1 in control hiPSCs produces the disease-associated phenotypes. Finally, we show that the C625T mutation can be repaired precisely and efficiently using a base-editing approach. Taken together, our data establish 3D organoids as a valid disease model.

Artificial intelligence-based decision-making for age-related macular degeneration.

Artificial intelligence (AI) based on convolutional neural networks (CNNs) has a great potential to enhance medical workflow and improve health care quality. Of particular interest is practical implementation of such AI-based software as a cloud-based tool aimed for telemedicine, the practice of providing medical care from a distance using electronic interfaces. Methods: In this study, we used a dataset of labeled 35,900 optical coherence tomography (OCT) images obtained from age-related macular degeneration (AMD) patients and used them to train three types of CNNs to perform AMD diagnosis. Results: Here, we present an AI- and cloud-based telemedicine interaction tool for diagnosis and proposed treatment of AMD. Through deep learning process based on the analysis of preprocessed optical coherence tomography (OCT) imaging data, our AI-based system achieved the same image discrimination rate as that of retinal specialists in our hospital. The AI platform's detection accuracy was generally higher than 90% and was significantly superior (p < 0.001) to that of medical students (69.4% and 68.9%) and equal (p = 0.99) to that of retinal specialists (92.73% and 91.90%). Furthermore, it provided appropriate treatment recommendations comparable to those of retinal specialists. Conclusions: We therefore developed a website for realistic cloud computing based on this AI platform, available at https://www.ym.edu.tw/~AI-OCT/. Patients can upload their OCT images to the website to verify whether they have AMD and require treatment. Using an AI-based cloud service represents a real solution for medical imaging diagnostics and telemedicine.

Glutamate Stimulation Dysregulates AMPA Receptors-Induced Signal Transduction Pathway in Leber's Inherited Optic Neuropathy Patient-Specific hiPSC-Derived Retinal Ganglion Cells.

The mitochondrial genetic disorder, Leber's hereditary optic neuropathy (LHON), is caused by a mutation in MT-ND4 gene, encoding NADH dehydrogenase subunit 4. It leads to the progressive death of retinal ganglion cells (RGCs) and causes visual impairment or even blindness. However, the precise mechanisms of LHON disease penetrance and progression are not completely elucidated. Human-induced pluripotent stem cells (hiPSCs) offer unique opportunities to investigate disease-relevant phenotypes and regulatory mechanisms underlying LHON pathogenesis at the cellular level. In this study, we successfully generated RGCs by differentiation of LHON patient-specific hiPSCs. We modified the protocol of differentiation to obtain a more enriched population of single-cell RGCs for LHON study. Based on assessing morphology, expression of specific markers and electrophysiological activity, we found that LHON-specific hiPSC-derived were more defective in comparison with normal wild-type RGCs. Based on our previous study, whereby by using microarray analysis we identified that the components of glutamatergic synapse signaling pathway were significantly downregulated in LHON-specific RGCs, we focused our study on glutamate-associated α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors. We found that the protein expression levels of the subunits of the AMPA receptor, GluR1 and GluR2, and their associated scaffold proteins were decreased in LHON-RGCs. By performing the co-immunoprecipitation assay, we found several differences in the efficiencies of interaction between AMPA subunits and scaffold proteins between normal and LHON-specific RGCs.

Neuroprotection by Imipramine against lipopolysaccharide-induced apoptosis in hippocampus-derived neural stem cells mediated by activation of BDNF and the MAPK pathway.

Depression is accompanied by the activation of the inflammatory-response system, and increased production of proinflammatory cytokines may play a role in the pathophysiology of depressive disorders. Imipramine (IM), a tricyclic antidepressant drug, has recently been shown to promote neurogenesis and improve the survival rate of neurons in the hippocampus. However, whether IM elicits a neuroprotective or anti-inflammatory effect, or promotes the differentiation of neural stem cells (NSCs) remains to be elucidated. In this study, we cultured NSCs derived from the hippocampal tissues of adult rats as an in vitro model to evaluate the NSCs drug-modulation effects of IM. Our results showed that 3 microM IM treatment significantly increased the survival rate of NSCs, and up-regulated the mRNA and protein expression of brain-derived neurotrophic factor (BDNF) and Bcl-2 in Day-7 IM-treated NSCs. Similar to BDNF-treated effect, incubation of NSCs with 3 microM IM increased Bcl-2 protein levels and further prevented lipopolysaccharide (LPS)-induced apoptosis through the activation of the mitogen-activated protein kinase (MAPK)/extracellular-regulated kinase (ERK) pathway. Inhibition of BDNF expression with small interfering RNA (siRNA), or blocking the MAPK pathway with U0126 further significantly decreased Bcl-2 protein levels and abrogated the neuroprotective effects of IM against LPS-induced apoptosis in NSCs. In addition, the percentages of serotonin and MAP-2-positive neuronal cells in the Day 7 culture of IM-treated NSCs were significantly increased. By using microdialysis with high performance liquid chromatography-electrochemical detection, the functional release of serotonin in the process of serotoninergic differentiation of IM-treated NSCs was concomitantly increasing and mediated by the activation of the BDNF/MAPK/ERK pathway/Bcl-2 cascades. In sum, the study results indicate that IM can increase the neuroprotective effects, suppress the LPS-induced inflammatory process, and promote serotoninergic differentiation in NSCs via the modulation of the BDNF/MAPK/ERK pathway/Bcl-2 cascades.

Acoustic waves improves retroviral transduction in human retinal stem cells.

BACKGROUNDS: The plasticity of retinal stem cells (RSCs), a type of cells that can differentiate into neuron cells and photoreceptor cells, endows them with potential therapeutic properties that can be applied to regenerative medicine. Gene modification of these stem cells before trans-differentiation and transplantation enhances their survival and increases their therapeutic function. The different ways to effectively deliver gene into RSCs are still discussed. This study aimed to use the acoustic waves to improve the efficacy of gene delivery for RSCs. METHODS: RSCs were obtained from non-fetal human ocular pigmented ciliary margin tissues. The enhanced green fluorescent protein-encoded murine stem cell retroviruses (MSCV) were prepared and used to infect RSCs. Glass chambers containing RSCs, retroviruses, and various concentrations of polybrene (0, 0.8, 2, 4 and 8 µg/mL) were exposed under 20 or 25 Vp-p ultrasonic standing wave fields (USWF) for 5 min. The percentage of green fluorescent protein positive cells in each sample was calculated and compared to test the efficacy of gene transduction. RESULTS: Our results showed that the efficiency of gene transduction by MSCV infection was enhanced following the concentration of polybrene and the energy of USWF. The percentage of green fluorescent protein positive cells was significantly higher in chambers that contained 8 µg/mL of polybrene and was exposed to 20Vp-p of USWF for 5 min. In addition, the percentage increased in chambers contained 2, 4 and 8 µg/mL of polybrene when they were exposed to 25Vp-p of USWF. Comparing to those did not treated with ultrasound, the efficiency of retroviral transduction to RSCs increased 4-fold after exposed to USWF for 5 min. CONCLUSION: We demonstrated the ability of ultrasound standing waves to improve retroviral transduction into RSCs. We believe that this may be applied to the experimental designs of future studies and may have possible therapeutic uses.