Deciphering the connection between microvascular damage and neurodegeneration in early diabetic retinopathy.

Diabetic retinopathy (DR), a common diabetes complication leading to vision loss, presents early clinical signs linked to retinal vasculature damage, affecting the neural retina at advanced stages. However, vascular changes and potential effects on neural cells before clinical diagnosis of DR are less well understood. To study the earliest stages of DR we performed histological phenotyping and quantitative analysis on postmortem retinas from 10 donors with diabetes and without signs of DR (such as microaneurysms and haemorrhages), plus 3 controls and 1 DR case, focusing on capillary loss in the deeper (DVP) and superficial vascular plexuses (SVP) and neural retina effects. The advanced DR case exhibited profound vascular and neural damage, whereas the ten randomly selected donors with diabetes appeared superficially normal. The SVP was indistinguishable from the controls. In contrast, over half of the retinas from donors with diabetes showed capillary dropout in the DVP and increased capillary diameter. However, we could not detect any localised neural cell loss in the vicinity of dropout capillaries. Instead, we observed a subtle pan-retinal loss of inner nuclear layer (INL) cells in all diabetes cases (p<0.05), independent of microvascular damage. In conclusion, our findings demonstrate a novel histological biomarker for early-stage diabetes-related damage in human postmortem retina, common in people with diabetes before clinical DR diagnosis. Furthermore, the mismatch between capillary dropout and neural loss questions the notion of microvascular loss directly causing neurodegeneration at the earliest stages of DR, so diabetes may affect the two readouts independently.

Laboratory exploration of the use of ripasudil in descemetorhexis with a human ex vivo model.

The aim of the study was to investigate the effect of ripasudil on corneal endothelial cell survival and migration after two types of descemetorhexis on a human ex vivo model. Eleven human corneoscleral buttons were incubated in either 50 ml organ culture medium containing 10 µM ripasudil or 50 µl dimethyl sulfoxide (DMSO), the vehicle in ripasudil for 2 days prior to wound creation then for 14 days after. The wound was created with either full trephination scoring or by shallow trephination plus manual peeling. At day 14, immunohistochemistry with vimentin and Na+/K+/ATPase markers was conducted. Tissues were assessed at day 3, 7 and 14 for morphology, cell migration, cell viability and cell density. Full trephination scoring created more damage on tissues compared to shallow trephination with full Descemet membrane peeling. In the full trephination scoring group, no differences in cell viability were noted when ripasudil and DMSO were compared. With the peeling method, Ripasudil could protect the endothelial cell death and maintain the morphology compared to the control. At day 14, no differences in the peripheral cell viability and density were found between ripasudil and DMSO, although the ripasudil group presented significantly increased central cell count and cell viability. Increased cell migration was noted with ripasudil and the initial cell morphology of those migrated cells was similar to that of fibroblasts. In conclusion, ex vivo modelling suggested that peeling resulted in less cell damage than scoring and ripasudil maintained better morphology and promoted migration. These effects might be via transformation of endothelial cells into a more motile spindle-like phenotype.

Comparison of the structural integrity and quality of corneal endothelium stored in organ culture storage medium versus Eusol-C.

In this experimental study, we compared the structural integrity and cell quality of corneal endothelium stored in organ culture medium (OCS) and Eusol-C. The experiment included rabbit and human cornea experiments in vitro. Thirty rabbit corneas and thirty-two human corneas were collected and divided into two groups. All right corneas were allocated in experiment group and left corneas were placed in control group. The corneas in experimental group were stored in OCS at 34 °C, and the corneas in control group were stored in Eusol-C at 4 °C for 7, 14, 21, 28, and 35 days, respectively. Endothelial cell morphology, cell count, and trypan blue staining for viability were assessed before storage (Day 0) and at days 7, 14, 21, 28 and 35. The structural integrity of human corneal endothelial cell was analyzed using immunohistochemistry. The samples of storage solution for microbial culture were collected on the third day and at the end of storage. The results show that no bacterial and fungal infections were found in both groups. After 14 days of storage, the morphology of endothelial cell was better in the experimental group than in the control group. The endothelial cell stored in OCS were better than those stored in Eusol-C at the end of storage times, except human cornea 14 days storage group. The ZO-1 protein staining showed the typical polygonal morphology of endothelial cell stored in the OCS. Corneal endothelial cells stored in the OCS had better quality up to 28 days. It can be applied to Chinese eye banks as a method of corneal preservation.

Transfer RNA derived fragment, tRF-Glu-CTC, aggravates the development of neovascular age-related macular degeneration.

Rationale: Angiogenesis expedites tissue impairment in many diseases, including age-related macular degeneration (AMD), a leading cause of irreversible blindness in elderly. A substantial proportion of neovascular AMD patients, characterized by aberrant choroidal neovascularization (CNV), exhibit poor responses or adverse reactions to anti-VEGF therapy. Herein, we aimed to unveil the function of newly identified transfer RNA-derived small RNA, tRF-Glu-CTC, in the pathology of CNV and determine its potential in inhibiting angiogenesis. Methods: Small non-coding RNA sequencing and quantitative polymerase chain reaction were conducted to detect expression pattern of tRF-Glu-CTC in CNV development. Immunofluorescence staining, fundus fluorescein angiography and ex vivo choroidal sprouting assays were employed for the evaluation of tRF-Glu-CTC's function in CNV development. The role of tRF-Glu-CTC in endothelial cells were determined by in vitro endothelial cell proliferation, migration and tube formation assays. Transcriptome sequencing, dual-luciferase reporter assay and in vitro experiments were conducted to investigate downstream mechanism of tRF-Glu-CTC mediated pathology. Results: tRF-Glu-CTC exhibited substantial up-regulation in AMD patients, laser-induced CNV model, and endothelial cells under hypoxia condition, which is a hallmark of CNV. Inhibiting tRF-Glu-CTC reduced angiogenesis and hypoxia stress in the neovascular region without neuroretina toxicity in laser-induced CNV model, showing an anti-angiogenic effect comparable to bevacizumab, while overexpression of tRF-Glu-CTC significantly augmented CNV. Mechanically, under hypoxia condition, angiogenin was involved in the production of tRF-Glu-CTC, which in turn triggered endothelial cell tubulogenesis, migration and promoted the secretion of inflammatory factors via the suppression of vasohibin 1 (VASH1). When downregulating VASH1 expression, the inhibition of tRF-Glu-CTC showed minimal suppression on angiogenesis. Conclusions: This study demonstrated the important role of tRF-Glu-CTC in the progression of angiogenesis. Targeting of tRF-Glu-CTC may be an alternative to current anti-VEGF therapy for CNV in AMD and other conditions with angiogenesis.

Inhibiting the activation of MAPK (ERK1/2) in stressed Müller cells prevents photoreceptor degeneration.

Rationale: Müller cells play an essential role in maintaining the health of retinal photoreceptors. Dysfunction of stressed Müller cells often results in photoreceptor degeneration. However, how these cells communicate under stress and the signalling pathways involved remain unclear. In this study, we inhibited the MAPK (ERK1/2) signalling, mainly activated in Müller cells, evaluated the protective effects on the photoreceptors and further explored the signalling communication between stressed Müller cells and degenerating photoreceptors. Methods: We evaluated the changes of MAPK (ERK1/2) signalling and its downstream targets in human retinal explants treated with PD98059, a specific phosphorylated ERK1/2 inhibitor, by western blot and immunostaining. We further assessed photoreceptor degeneration by TUNEL staining and outer nuclear layer thickness. We also injected PD98059 into the eyes of mice exposed to photo-oxidative stress. We evaluated the protective effects on photoreceptor degeneration by optical coherence tomography (OCT) and electroretinography (ERG). The crosstalk between Müller cells and photoreceptors was further dissected based on the changes of transcription factors by RNA sequencing and protein profiles of multiple signalling pathways. Results: We found that MAPK (ERK1/2) signalling was mainly activated in Müller cells under stress, both ex vivo and in vivo. PD98059 inhibited the phosphorylation of ERK1/2, reduced expression of the gliotic marker glial fibrillary acidic protein (GFAP) in Müller cells and increased levels of the neuroprotective factor, interphotoreceptor retinoid-binding protein (IRBP) in photoreceptors. Inhibition of pERK1/2 also reduced retinal photo-oxidative damage in mice retinas assessed by OCT and ERG. We also identified that the JAK/STAT3 signalling pathway might mediate signalling transduction from Müller cells to photoreceptors. Conclusion: MAPK (ERK1/2) deactivation through chemical inhibition, mainly in stressed Müller cells, can alleviate gliosis in Müller cells and restore the expression of IRBP in photoreceptors, which appears to prevent retinal degeneration. Our findings suggested a new way to prevent photoreceptor degeneration by manipulating the stress response in Müller cells.

Plasma Apolipoproteins Predicting the Occurrence and Severity of Diabetic Retinopathy in Patients With Type 2 Diabetes Mellitus.

Objective: Apolipoproteins are amphipathic molecules and the major components of plasma lipoproteins. This study aims to investigate the effects of dysregulated apolipoprotein (apo) profiles and their ratios on type 2 diabetes mellitus (T2DM) and diabetic retinopathy (DR) further to test the hypothesis that altered serum level of apolipoproteins is strong biomarkers for DR. Research Design and Methods: This case-control study consists of 157 patients with T2DM including DM without DR, non-proliferative DR (NPDR), and proliferative DR (PDR). Fifty-eight age- and sex-matched healthy subjects were enrolled as normal controls. Blood biochemistry profile including serum levels of glucose, glycated hemoglobin (HbA1c), lipid profile [total cholesterol (TC), Triglycerides (TG), high and low-density lipoprotein (HDL-C and LDL-C)] was estimated. Apolipoproteins (apos, A-I, A-II, B, C-II, C-III, and E) was evaluated by protein chips (Luminex technology). Apolipoprotein ratios and arteriosclerosis-associated plasma indices were calculated. The Kruskal-Wallis test, independent sample t-test or Mann-Whitney U test, and multivariate regression analysis were performed to investigate the association of serum lipid biomarkers and the DR severity. Results: Serum level of apoA-I was negatively correlated with TC-(HDL-C)/HDL-C (p < 0.001), fasting glucose (p < 0.001), HbA1c (p < 0.001), and (p<0.001), while apoE, apoC-II/apoC-III, apoA-II/apoA-I were positively correlated with above traditional biomarkers (p < 0.001). Single variable logistic analysis results showed that body mass index (BMI) (p = 0.023), DM duration (p < 0.001), apoE (p < 0.001), apoC-II/apo C-III (p < 0.001), apoE/apoC-II (p < 0.001), atherogenic index (p = 0.013), fasting glucose (p < 0.001), HbA1c (p < 0.001), LPA (p = 0.001), and LDL-C/HDL-C (p = 0.031) were risk factors for the occurrence and severity of DR. Multivariate logistic regression mode showed that apoC-II/apoC-III and apoB/non-HDL-C (p < 0.001) as well as apoE/apoC-II (p = 0.001) were the independent risk factors for the occurrence and severity of DR-apopA-I and apoA-II are protective factors for DR-after controlling for the duration of DM, HbA1c, fasting glucose, and LPA. Conclusions: apoE, apoC-II/apoC-III, apoE/apoC-II, and apoB/non-HDL-C could be used as novel biomarkers for occurrence and severity of DR, whereas apoA-I and apoA-II resulted as protective factors for DR.

Transketolase in human Müller cells is critical to resist light stress through the pentose phosphate and NRF2 pathways.

The Pentose Phosphate Pathway (PPP), a metabolic offshoot of the glycolytic pathway, provides protective metabolites and molecules essential for cell redox balance and survival. Transketolase (TKT) is the critical enzyme that controls the extent of "traffic flow" through the PPP. Here, we explored the role of TKT in maintaining the health of the human retina. We found that Müller cells were the primary retinal cell type expressing TKT in the human retina. We further explored the role of TKT in human Müller cells by knocking down its expression in primary cultured Müller cells (huPMCs), isolated from the human retina (11 human donors in total), under light-induced oxidative stress. TKT knockdown and light stress reduced TKT enzymatic activities and the overall metabolic activities of huPMCs with no detectable cell death. TKT knockdown restrained the PPP traffic flow, reduced the expression of NAD(P)H Quinone Dehydrogenase 1 (NQO1), impaired the antioxidative response of NRF2 to light stress and aggravated the endoplasmic reticulum (ER) stress. TKT knockdown also inhibited overall glucose intake, reduced expression of Dihydrolipoamide dehydrogenase (DLD) and impaired the energy supply of the huPMCs. In summary, Müller cell-mediated TKT activity plays a critical protective role in the stressed retina. Knockdown of TKT disrupted the PPP and impaired overall glucose utilisation by huPMCs and rendered huPMCs more vulnerable to light stress by impairing energy supply and antioxidative NRF2 responses.

Alleviation of Diabetic Tendon Injury via Activation of Tendon Fibroblasts Autophagy under Berberine Treatment.

Berberine (BBR) is an isoquinoline alkaloid isolated from Coptis chinensis and possesses valuable pharmacological activities, including anti-inflammatory, anti-tumor, and alleviating several complications of type 2 diabetes mellitus (T2DM). However, the role of BBR in regulating diabetic tendon injury remains poorly understood. In this study, a rat model of T2DM was constructed, and cell apoptosis and autophagy were assessed in tendon tissues after BBR treatment through TdT-Mediated dUTP nick-end labeling (TUNEL) assay and immunohistochemical analysis. Tendon fibroblasts were obtained from the rat Achilles tendon, and the role of BBR in regulating cell apoptosis, the production of inflammatory cytokines, and autophagy activation were assessed using flow cytometry, quantitative real-time PCR (qRT-PCR), and western blot analysis. We demonstrated that BBR treatment significantly increased autophagy activation and decreased cell apoptosis in tendon tissues of T2DM rats. In tendon fibroblasts, BBR repressed High glucose (HG)-induced cell apoptosis and production of proinflammatory cytokines. HG treatment resulted in a decrease of autophagy activation in tendon fibroblasts, whereas BBR restored autophagy activation. More important, pharmacological inhibition of autophagy by 3-MA weakened the protective effects of BBR against HG-induced tendon fibroblasts injury. Taken together, the current results demonstrate that BBR helps relieve diabetic tendon injury by activating autophagy of tendon fibroblasts.

High glucose represses the proliferation of tendon fibroblasts by inhibiting autophagy activation in tendon injury.

Diabetic foot ulcer (DFU) is a kind of common and disabling complication of Diabetes Mellitus (DM). Emerging studies have demonstrated that tendon fibroblasts play a crucial role in remodeling phase of wound healing. However, little is known about the mechanism underlying high glucose (HG)-induced decrease in tendon fibroblasts viability. In the present study, the rat models of DFU were established, and collagen deposition, autophagy activation and cell apoptosis in tendon tissues were assessed using Hematoxylin-Eosin (HE) staining, immunohistochemistry (IHC), and TdT-mediated dUTP Nick-End Labeling (TUNEL) assay, respectively. Tendon fibroblasts were isolated from Achilles tendon of the both limbs, and the effect of HG on autophagy activation in tendon fibroblasts was assessed using Western blot analysis, Cell Counting Kit-8 (CCK-8) assay, and flow cytometry. We found that cell apoptosis was increased significantly and autophagy activation was decreased in foot tendon tissues of DFU rats compared with normal tissues. The role of HG in regulating tendon fibroblasts viability was then investigated in vitro, and data showed that HG repressed cell viability and increased cell apoptosis. Furthermore, HG treatment reduced LC3-II expression and increased p62 expression, indicating that HG repressed autophagy activation of tendon fibroblasts. The autophagy activator rapamycin reversed the effect. More importantly, rapamycin alleviated the suppressive role of HG in tendon fibroblasts viability. Taken together, our data demonstrate that HG represses tendon fibroblasts proliferation by inhibiting autophagy activation in tendon injury.

Traditional chinese medicine syndromes classification associates with tumor cell and microenvironment heterogeneity in colorectal cancer: a single cell RNA sequencing analysis.

BACKGROUND: Colorectal cancer (CRC) is one of the common gastrointestinal malignancies, tumor heterogeneity is the main cause of refractory CRC. Syndrome differentiation is the premise of individualized treatment of traditional Chinese medicine (TCM), but TCM syndrome lacks objective identification in CRC. This study is to investigate the correlation and significance of tumor heterogeneity and TCM syndromes classification in CRC. METHODS: In this study, we using scRNA-seq technology, investigate the significance of tumor heterogeneity in TCM syndromes classification on CRC. RESULTS: The results showed that 662 cells isolated from 11 primary CRC tumors are divided into 14 different cell clusters, and each cell subtype and its genes have different functions and signal transduction pathways, indicating significant heterogeneity. CRC tumor cell clusters have different proportions in Excess, Deficiency and Deficiency-Excess syndromes, and have their own characteristic genes, gene co-expression networks, gene functional interpretations as well as monocle functional evolution. Moreover, there were significant differences between the high expressions of MUC2, REG4, COL1A2, POSTN, SDPR, GPX1, ELF3, KRT8, KRT18, KRT19, FN1, SERPINE1, TCF4 and ZEB1 genes in Excess and Deficiency syndrome classification in CRC (P < 0.01). CONCLUSIONS: The Excess and Deficiency syndromes classification may be related to tumor heterogeneity and its microenvironment in CRC.

Effects of human platelet lysate on the growth of cultured human corneal endothelial cells.

Human platelet lysate (hPL) as a replacement for foetal bovine serum (FBS) in culturing human corneal endothelium is an emerging area of interest, although there are limited studies evaluating the quality of the hPL being used. Our study aimed to evaluate variations between sources of hPL and to explore the efficacy of hPL (with and without heparin) as a replacement for FBS in culturing human corneal endothelial cells in vitro. Immortalized human corneal endothelial cells (B4G12) and primary human corneal endothelial cells (PHCEnCs, n = 11 donors, age from 36 to 85 years old) were cultured with 5% hPL or FBS. A full characterisation of the effects of hPL and FBS on cell growth was conducted using IncuCyte Zoom (percentage cell confluence and population doubling time, PDT) to analyse cell proliferation. AlamarBlue assays were used to measure cell viability. The concentration of fibrinogen, PDGF, hEGF, VEGF and bFGF in two sources of hPL were analyzed by Enzyme-linked immunosorbent assay. Expression and localization of Na+/K+-ATPase, ZO-1 and CD166 on PHCEnCs and B4G12 cells were assessed with immunofluorescence and immunoblotting. Our results showed that a significant difference in fibrinogen, hEGF and VEGF concentrations was found between two sources of hPL. Heparin impaired the positive effect of hPL on cell growth. PDT and alamarBlue showed that hPL significantly increased proliferation and viability of PHCEnCs in two of three donors, and immunostaining indicated that hPL increased ZO-1 and CD166 expression but not Na+/K+-ATPase on PHCEnCs. In addition, heterogeneities on immunopositivity of Na+/K+-ATPase and ZO-1 and morphology were found on PHCEnCs derived from an individual donor cultured with hPL medium. In conclusion, hPL showed positive effect on primary corneal endothelial cell growth, and maintenance of their cellular characteristics compared to FBS. hPL can be considered as a supplement to replace FBS in PHCEnC culture. However, the variation observed between different hPL sources suggests that a standard quality control monitoring system such as storage time and a minimal concentration of growth factors may need to be established.

Ameliorative effects of the traditional Chinese medicine formula Qing-Mai-Yin on arteriosclerosis obliterans in a rabbit model.

CONTEXT: Qing-Mai-Yin (QMY) is a clinically used herbal formula for treating arteriosclerosis obliterans (ASO). OBJECTIVE: To evaluate the chemical constituents and effects of QMY on ASO rabbit model. MATERIALS AND METHODS: Forty-eight New Zealand rabbits were divided into six groups (n = 8): normal (normal rabbits treated with 0.5% CMC-Na), vehicle (ASO rabbits treated with 0.5% CMC-Na), positive (simvastatin, 1.53 mg/kg), and QMY treatment (300, 600, and 1200 mg/kg). ASO rabbit model was prepared by high fatty feeding, roundly shortening artery, and bovine serum albumin immune injury. QMY (300, 600 and 1200 mg/kg) was orally administered for 8 weeks. The effects and possible mechanisms of QMY on ASO rabbits were evaluated by pathological examination, biochemical assays, and immunohistochemical assays. The compositions of QMY were analysed using HPLC-Q-TOF-MS/MS analysis. RESULTS: Compared to the vehicle rabbit, QMY treatment suppressed plaque formation and intima thickness in aorta, and decreased intima thickness, whereas increased lumen area of femoral artery. Additionally, QMY treatment decreased TC, TG and LDL, decreased CRP and ET, and increased NO and 6-K-PGF1α in serum. Furthermore, the potential mechanisms studied revealed that QMY treatment could suppress expression of TNF-α, IL-6, ICAM-1 and NF-κB in endothelial tissues, and increase IκB. In addition, HPLC analysis showed QMY had abundant anthraquinones, stilbenes, and flavonoids. CONCLUSION: QMY has ameliorative effects on ASO rabbit, and the potential mechanisms are correlated to reducing inflammation and down-regulating NF-κB. Our study provides a scientific basis for the future application and investigation of QMY.

Thermostable small-molecule inhibitor of angiogenesis and vascular permeability that suppresses a pERK-FosB/ΔFosB-VCAM-1 axis.

Vascular permeability and angiogenesis underpin neovascular age-related macular degeneration and diabetic retinopathy. While anti-VEGF therapies are widely used clinically, many patients do not respond optimally, or at all, and small-molecule therapies are lacking. Here, we identified a dibenzoxazepinone BT2 that inhibits endothelial cell proliferation, migration, wound repair in vitro, network formation, and angiogenesis in mice bearing Matrigel plugs. BT2 interacts with MEK1 and inhibits ERK phosphorylation and the expression of FosB/ΔFosB, VCAM-1, and many genes involved in proliferation, migration, angiogenesis, and inflammation. BT2 reduced retinal vascular leakage following rat choroidal laser trauma and rabbit intravitreal VEGF-A165 administration. BT2 suppressed retinal CD31, pERK, VCAM-1, and VEGF-A165 expression. BT2 reduced retinal leakage in rats at least as effectively as aflibercept, a first-line therapy for nAMD/DR. BT2 withstands boiling or autoclaving and several months' storage at 22°C. BT2 is a new small-molecule inhibitor of vascular permeability and angiogenesis.

TIMING INFLUENCE ON OUTCOMES OF VITRECTOMY FOR OPEN-GLOBE INJURY: A Prospective Randomized Comparative Study.

PURPOSE: To compare the impact of surgical timing on anatomical and functional outcomes of vitrectomy for open-globe injury. METHODS: Fifty-three patients were entered into this prospective open-label study, with 26 patients randomized into early surgery group (vitrectomy conducted within 4 days) and 27 into delayed surgery group (vitrectomy performed between 10-14 days after injury). Six-month data were available for 46 patients and 7 were lost to follow-up. The main outcome measures were incidence of traumatic proliferative vitreoretinopathy assessed intraoperatively and postoperatively, reattachment of retina, eye enucleation, improvement of the best-corrected visual acuity, and complications. RESULTS: Patient demographics and surgical intervention were similar in both groups. Final analysis of 46 patients demonstrated higher rates of traumatic proliferative vitreoretinopathy assessed both intraoperatively and postoperatively in the delayed group (P = 0.000; P = 0.054). In the early surgery group, 18 of 21 patients had retinal detachment, of which 15 patients (83%) achieved retinal reattachment by the first vitreoretinal surgery, 2 patients (11%) by a second surgery, and 1 (6%) received enucleation. In the delayed surgery group, 22 of 25 patients had retinal detachment. Retinal reattachment was achieved in 7 (32%) and 8 (36%) with the first and the second surgery, respectively, and 7 patients (32%) received enucleation (P = 0.005). In the early surgery group, best-corrected visual acuity improved significantly, moderately, and decreased in 8 patients (38%), 11 eyes (52%) and 2 eyes (10%), respectively. In the delayed surgery group, best-corrected visual acuity improved significantly, moderately, and worsened in 3 eyes (12%), 12 eyes (48%), and 10 eyes (40%) (P = 0.041), respectively. No statistically significant difference was observed in the rate of postoperative complications between the two groups. CONCLUSION: Early vitrectomy after open-globe injury leads to better anatomical and functional outcomes.

Factors Affecting Corneal Organ Culture Contamination: A 6-year Study at the New South Wales Tissue Bank.

PURPOSE: To report the rate of microbial contamination and analyze possible risk factors for contamination of banked corneas stored using the organ culture method. METHODS: Data from the New South Wales Tissue Banks incorporating the Lions NSW Eye Bank, between September 1, 2011, and November 30, 2017, were reviewed retrospectively. All corneas collected during this period and stored in organ culture storage media were tested for microbial contamination. The influence of potential factors on the rate of contamination was analyzed using the χ test and logistic regression using generalized estimating equations. RESULTS: A total of 4410 corneas were included in this study, of which 110 were medium culture positive, representing a microbial contamination rate of 2.5%. The main contaminants were Candida species followed by Staphylococcus species. Corneal tissue collected in summer and autumn had a significantly higher contamination rate (P = 0.006). All other factors studied were not shown to have a statistically significant association with contamination after accounting for within-pair correlation and confounders. CONCLUSIONS: A relatively low contamination rate of 2.5% observed in our study reflects the stringent laboratory protocols, strict donor selection criteria, and high level of experience among staff at the Lions NSW Eye Bank. Our study demonstrated that the season of collection had a strong association with the rate of organ culture contamination. Because Candida species contributed the largest percentage of contamination, specific measures to reduce and eliminate fungal proliferation should be considered by eye banks particularly in warm seasons.

Descemet's stripping without endothelial keratoplasty.

PURPOSE OF REVIEW: To summarize the recent literature regarding descemetorhexis stripping without endothelial keratoplasty (DWEK), increasingly referred to as Descemet's stripping only (DSO). To report the characteristic clinical, confocal and histologic findings associated with this procedure. RECENT FINDINGS: Reported clearance rates following DSO range from 63 to 100% in recent series, with variation between surgical techniques. Topical Rho-kinase inhibitor has been reported as successfully salvaging failing cases. Its use as an adjuvant to the surgery is gaining widespread adoption with the results of early series now arriving. Apart from a phenotype of central guttata with clear periphery, patient characteristics which determine success remain elusive. Surgical factors affecting success are increasingly well understood, with stromal injury felt to be a retardant to healing. Characteristic clinical signs have been observed and are described herein. Clinical, confocal and light microscopic images are obtained from patients in clinical trials of DSO with ripasudil. SUMMARY: DSO is gaining acceptance as a surgical option for a subset of patients with Fuchs' Dystrophy. The addition of Rho-associated kinase inhibitor appears to improve predictability but further results to this effect must be published and scrutinized.

Simvastatin protects photoreceptors from oxidative stress induced by all-trans-retinal, through the up-regulation of interphotoreceptor retinoid binding protein.

BACKGROUND AND PURPOSE: Simvastatin is a 3-hydroxy-3-methylglutaryl CoA reductase inhibitor with multiple targets and effects. It protects neurons in the brain, but its protective effects on photoreceptors are unclear. In this study, we evaluated the neuroprotective effect of simvastatin on photoreceptors exposed to stress induced by all-trans-retinal (atRAL). EXPERIMENTAL APPROACH: AlamarBlue and LDH assays were used to evaluate the viability and metabolic activity of Y79 cells (a retinoblastoma cell line) exposed to atRAL-induced stress with or without simvastatin pretreatment. Changes in cellular ROS were evaluated using flow cytometry and mitochondrial stress markers JC-1 and HSP60. Changes in levels of two photoreceptor-specific markers, cone-rod homeobox protein (CRX) and interphotoreceptor retinoid binding protein (IRBP), were evaluated with western blot. The results were validated in ex vivo human retinal explants and a mouse model of photoreceptor degeneration. KEY RESULTS: Simvastatin improved mitochondrial function, alleviated oxidative stress and up-regulated the photoreceptor-specific markers IRBP and its upstream regulator CRX in Y79 cells and ex vivo human retinal explants under atRAL-induced stress. Simvastatin attenuated photoreceptor degeneration in association with up-regulation of IRBP and CRX expression after knockdown of IRBP in a murine model. CONCLUSION AND IMPLICATIONS: Our findings suggest that simvastatin has a novel role in protecting photoreceptors from atRAL-induced stress. Simvastatin treatment resulted in up-regulation of IRBP and its upstream transcription factor CRX in Y79 cells, ex vivo human retinal explants, and murine retinas in vivo. Further studies of simvastatin to treat photoreceptor degeneration are warranted.

c-Jun-mediated microRNA-302d-3p induces RPE dedifferentiation by targeting p21Waf1/Cip1.

Dedifferentiation of retinal pigment epithelium (RPE) cells and choroidal neovascularization (CNV) contributes to the pathogenesis of age-related macular degeneration (AMD). MicroRNAs (miRNAs) have crucial roles in AMD onset and progression. We thus aim to investigate the effects of miRNAs on RPE dedifferentiation and endothelium cell (EC) behavior, and analyze its downstream pathways. We have previously identified miR-302d-3p as the most downregulated miRNA signature along with RPE differentiation. Herein, in vitro study supported that miR-302d-3p induces RPE dedifferentiation typified by reduction of RPE characteristic markers, interrupts its phagocytosis, and promotes its migration, proliferation, and cell-cycle progression. c-Jun was identified as a potential upstream transcript factor for MIR302D, which might modulate RPE function by regulating miR-302d-3p expression. P21Waf1/Cip1, a cyclin-dependent kinase inhibitor encoded by the CDKN1A gene, was identified as a downstream target of miR-302d-3p. Our data suggested that p21Waf1/Cip1 could promote RPE differentiation, and inhibit its proliferation, migration, and cell-cycle progression. We also demonstrated that miR-302d-3p suppresses RPE differentiation through directly targeting p21Waf1/Cip1. In addition, the miR-302d-3p/CDKN1A axis was also involved in regulating tube formation of ECs, indicating its potential involvement in CNV formation. Taken together, our study implies that miR-302d-3p, regulated by c-Jun, contributes to the pathogenesis of both atrophic and exudative AMD. MiR-302d-3p promotes RPE dedifferentiation, migration, proliferation and cell-cycle progression, inhibits RPE phagocytosis, and induces abnormal EC behavior by targeting p21Waf1/Cip1. Pharmacological miR-302d-3p inhibitors are prospective therapeutic options for prevention and treatment of AMD.

FUNDUS-WIDE SUBRETINAL AND PIGMENT EPITHELIAL ABNORMALITIES IN MACULAR TELANGIECTASIA TYPE 2.

PURPOSE: Macular telangiectasia Type 2 (MacTel) causes glial and photoreceptor cell death in a small, oval patch in the central retina. Beyond this oval area, no disease manifestations have been described so far. Here, we describe a novel pathological aspect of MacTel in the retinal pigment epithelium (RPE) that is not restricted to the clinically affected area but covers the entire retina. METHODS: We have studied postmortem eyes from four patients with MacTel by immunohistochemistry and electron microscopy. RESULTS: We found cellular debris in the subretinal space (between photoreceptor outer segments and RPE), consisting mainly of outer segments and RPE components. In healthy eyes, the RPE normally phagocytoses the tips of the continuously growing outer segments, a process considered to be essential for photoreceptor survival. However, in the patients with MacTel, we found no evidence of ongoing outer segment phagocytosis, and the apical surface of the RPE appeared abnormal throughout most of the retina. CONCLUSION: Reduced outer segment phagocytosis may explain the accumulating debris in the subretinal space but is a surprising finding because visual function in the peripheral retina is normal in patients with MacTel. Nevertheless, the subclinical pathology might induce a specific stress to which the central area is uniquely susceptible.

Increased Indoleamine 2,3-Dioxygenase and Quinolinic Acid Expression in Microglia and Müller Cells of Diabetic Human and Rodent Retina.

Purpose: We investigated the relationship between inflammation, neuronal loss, and expression of indoleamine 2, 3-dioxygenase (IDO) and quinolinic acid (QUIN) in the retina of subjects with type 1 diabetes (T1D) and type 2 diabetes (T2D) and in the retina of rats with T1D. Methods: Retinas from T1D (n = 7), T2D (n = 13), and 20 age-matched nondiabetic human donors and from T1D (n = 3) and control rats (n = 3) were examined using immunohistochemistry for IDO, QUIN, cluster of differentiation 39 (CD39), ionized calcium-binding adaptor molecule (Iba-1, for macrophages and microglia), Vimentin (VIM; for Müller cells), neuronal nuclei (NeuN; for neurons), and UEA1 lectin (for blood vessels). Results: Based on morphologic criteria, CD39+/ionized calcium binding adaptor molecule 1(Iba-1+) resident microglia and CD39-/Iba-1+ bone marrow-derived macrophages were present at higher density in T1D (13% increase) and T2D (26% increase) human retinas when compared with controls. The density and brightness of IDO+ microglia were increased in both T1D and T2D human retinas. The intensity of QUIN+ expression on CD39+ microglia and VIM+ Müller cells was greatly increased in both human T1D and T2D retinas. T1D retinas showed a 63% loss of NeuN+ neurons and T2D retinas lost approximately 43% when compared with nondiabetic human retinas. Few QUIN+ microglia-like cells were seen in nondiabetic retinas, but the numbers increased 18-fold in T1D and 7-fold in T2D in the central retina. In T1D rat retinas, the density of IDO+ microglia increased 2.8-fold and brightness increased 2.1-fold when compared with controls. Conclusions: Our findings suggest that IDO and QUIN expression in the retinas of diabetic rats and humans could contribute to the neuronal degeneration that is characteristic of diabetic retinopathy.