Surgical management and outcomes of pediatric open globe injuries requiring vitrectomy.

PURPOSE: To describe surgical management and establish visual outcomes of open globe injury (OGI) in pediatric patients requiring vitrectomy. METHODS: Forty-eight eyes of 48 pediatric patients underwent vitrectomy for OGI with secondary vitreoretinal complications in the eye center of Jilin University were included. Characteristics of patients, details of ocular examination and operation, presenting and final visual acuity were recorded. RESULTS: Presenting visual acuity less than 20/400 was found in 44 eyes (91.7%), which included no light perception (NLP) in four eyes. At last visit, there was no eyes with visual acuity of NLP, and 19 eyes (39.6%) had a vision recovery to 20/400 or better. Mechanisms of injury, intraocular contents prolapse, presence of hyphema, intraocular foreign body, vitreous hemorrhage, retinal detachment, and total time from injury to PPV > 2 weeks were significant predictors of visual prognosis. Logistic regression analysis showed that hyphema was a significant predictive factor for poor visual outcome. CONCLUSION: Visual acuity was improved in most of the patients with OGI in this study. Hyphema is an important presenting ocular sign in estimating the post-vitrectomy visual outcome for OGI in children. Proper timing of vitrectomy is suggested, and in this study patients may benefit more with early vitrectomy as less proliferative vitreoretinopathy (PVR) was found together with a better visual acuity.

Case report of the first molecular diagnosis of Stickler syndrome with a pathogenic COL2A1 variant in a Mongolia family.

BACKGROUND: Stickler syndrome is a group of connective tissue disorders that can affect eye (myopia, cataract, and retinal detachment), skeleton (spondyloepiphyseal dysplasia and precocious arthritis), craniofacies (midfacial under development and cleft palate), and inner ear (conductive and sensorineural); with the degree of symptoms varying among patients. Mutations in the COL2A1, COL11A1, COL11A2, COL9A1, COL9A2, and COL9A3 procollagen genes cause Stickler syndrome. CASE PRESENTATION: A 16-year-old Mongolian girl approached our clinics with retinal detachment. The proband had vitreous degeneration in both eyes, rhegmatogenous retinal detachment in her right eye, a large area of retina degeneration in her left eye, and coupled with severe myopia. No obvious hearing disorder was found, no abnormalities in bones and joints, and her communication and learning capability were also normal. Further clinical examination showed that the patient's other five family members across three generations had vitreous and retina degenerations. Exome sequencing showed a heterozygous splicing variant in COL2A1 in all patients. CONCLUSIONS: In this case report, a pathogenic splicing variant in the COL2A1 gene was identified in a Mongolian family affected with Stickler syndrome type I by exome sequencing. This heterozygous splicing variant in COL2A1 (NM_001844.4:C.2518-1G>A) that may impair splicing, which was suggested by in silico prediction. Next-generation sequencing is helpful for the differential diagnosis of this clinically variable and genetically heterogeneous disorder.

Pars plana vitrectomy with air tamponade for the treatment of medium-large macular holes.

BACKGROUND: To evaluate the surgical outcomes of pars plana vitrectomy (PPV) with air tamponade in medium-large macular holes (MHs). METHODS: Data for 26 eyes of 26 consecutive patients with medium-large full-thickness MHs (> 400 µm) who underwent PPV, internal limiting membrane (ILM) peeling, and sterile air tamponade were studied retrospectively. Best-corrected visual acuity and the closure rate were noted. The follow-up period was one to 20 months (median, four months). RESULTS: The age range of the patients was 53-73 years (median, 65 years). The mean minimum diameter of the MHs was 550 ± 99 µm. Prior to surgery, 10 eyes (38 per cent) were stage three, and 16 eyes (62 per cent) were stage four. The pre-operative symptom duration ranged from one month to 24 months (median, four months). Twenty-four MHs (92.3 per cent) were successfully closed after a single operation. Two (7.7 per cent) patients had a persistent MH. The average visual acuity, calculated as the logarithm of the minimal angle of resolution, improved from 1.44 ± 0.45 pre-operatively to 0.54 ± 0.29 at the end of the follow-up period (p < 0.001, paired t-test). CONCLUSION: Vitrectomy with ILM peeling and sterile air tamponade is an effective and safe surgical technique for managing medium-large MHs with a shorter history and does not require intravitreal long-acting gas tamponade while maintaining a long-term, face-down position.

Bone marrow mesenchymal stem cells enhance autophagy and help protect cells under hypoxic and retinal detachment conditions.

Our study aimed to evaluate the protective role and mechanisms of bone marrow mesenchymal stem cells (BMSCs) in hypoxic photoreceptors and experimental retinal detachment. The cellular morphology, viability, apoptosis and autophagy of hypoxic 661w cells and cells cocultured with BMSCs were analysed. In retinal detachment model, BMSCs were intraocularly transplanted, and then, the retinal morphology, outer nuclear layer (ONL) thickness and rhodopsin expression were studied as well as apoptosis and autophagy of the retinal cells. The hypoxia-induced apoptosis of 661w cells obviously increased together with autophagy levels increasing and peaking at 8 hours after hypoxia. Upon coculturing with BMSCs, hypoxic 661w cells had a better morphology and fewer apoptosis. After autophagy was inhibited, the apoptotic 661w cells under the hypoxia increased, and the cell viability was reduced, even in the presence of transplanted BMSCs. In retina-detached eyes transplanted with BMSCs, the retinal ONL thickness was closer to that of the normal retina. After transplantation, apoptosis decreased significantly and retinal autophagy was activated in the BMSC-treated retinas. Increased autophagy in the early stage could facilitate the survival of 661w cells under hypoxic stress. Coculturing with BMSCs protects 661w cells from hypoxic damage, possibly due to autophagy activation. In retinal detachment models, BMSC transplantation can significantly reduce photoreceptor cell death and preserve retinal structure. The capacity of BMSCs to reduce retinal cell apoptosis and to initiate autophagy shortly after transplantation may facilitate the survival of retinal cells in the low-oxygen and nutrition-restricted milieu after retinal detachment.

Induced Expression of VEGFC, ANGPT, and EFNB2 and Their Receptors Characterizes Neovascularization in Proliferative Diabetic Retinopathy.

Purpose: To investigate whole transcriptional differences between proliferative diabetic retinopathy (PDR) neovascular membranes (NVMs) and retinas, and the regulatory genes participating in retinal neovascularization in PDR. Methods: We used high-throughput sequencing technology to capture the whole-genome gene expression levels of all participants, including 23 patients with PDR or branch retinal vein occlusion (BRVO), 3 normal retinal samples, and 2 retinal samples from type II diabetic (T2D) eyes by donation, followed by analyses of expression patterns using bioinformatics methods, then validation of the data by in situ hybridization and Western blotting. Results: We showed that transcriptional profiles of the NVMs were distinct from those of the retinas. Angiogenesis growth factors VEGFC, ANGPT1, ANGPT2, and EFNB2, and their receptors FLT4, TIE1, TIE2, and EPHB4, respectively, were overexpressed. Expression of VEGFA was highly upregulated in T2D retina, but low in the NVMs, while angiogenesis transcription factors, including ETS1 and ERG, were coordinately upregulated in NVMs. Conclusions: This study described a PDR neovascularization model in which pathological retina-secreted vascular endothelial growth factor A (VEGFA) enhanced the expression of a set of angiogenesis transcription factors and growth factors, to cooperatively induce the retinal neovascularization. Based on these results, novel potential therapeutic targets and biomarkers for PDR treatment and diagnosis are suggested.

Idiopathic Macular Hole: A Comprehensive Review of Its Pathogenesis and of Advanced Studies on Metamorphopsia.

Vitreous anteroposterior traction is an important factor that affects macular hole (MH) formation at the early stage, and vitreous tangential traction can lead to further hole expansion after hole formation. Recent studies have highlighted the significance of Müller cells for the pathogenesis of MH. Since the advent of MH treatment, success rates for MH closure have significantly improved, as has postoperative visual acuity. However, metamorphopsia, an initial and common symptom of MH, still exists. Metamorphopsia is significantly related to the deterioration of visual quality of life and can be used as an independent index to evaluate visual function before and after surgery. In MH patients, metamorphopsia has different manifestations representing different clinical implications. M-CHARTS, as a new means of inspection, can quantify the degrees of metamorphopsia, and with the development of optical coherence tomography (OCT), layer-by-layer scanning of the retinal structure has become possible. These methods enable detailed analysis of the connections between the degree of metamorphopsia and relevant OCT parameters. Preoperative OCT parameters can be used to evaluate the prognosis of the postoperative visual function of MH patients and are therefore of great significance in guiding the treatment of MH patients.

Interaction between XRCC 1 gene polymorphisms and diabetes on susceptibility to primary open-angle glaucoma.

IMPACT STATEMENT: Some studies have suggested that diabetes and XRCC gene may be risk factors for glaucoma; however, no studies have focused on the interaction between the XRCC gene and T2DM with respect to POAG risk. Therefore, the present study evaluated the initiative gene-environment interactions in POAG.

3,5-Dimethoxy-4-hydroxy myricanol ameliorates photoreceptor cell degeneration in Pde6brd10 mouse model.

INTRODUCTION: Retinitis pigmentosa (RP) caused by the photoreceptor cell degeneration is currently incurable and leads to partial or complete blindness eventually. 3,5-dimethoxy-4-hydroxy myricanol (DM) is a novel compound isolated from the leaves of Micromelum integerrimum, with proliferative activities on NIH3T3 cells. This study was to investigate whether DM could mitigate retinal degeneration of rd10 mice, a well-characterized mouse model of RP. MATERIALS AND METHOD: Rd10 mice were treated with DM daily by intraperitoneal injection from postnatal day 12 (P12) to P26. Electroretinography (ERG) reflects the mass response of photoreceptor cells and was used to test the outer retinal function after DM treatment. Haematoxylin and Eosin staining was used to show the retinal morphology and evaluate the rod photoreceptor cell loss. TUNEL assay was used to detect the apoptosis-positive cells. Inflammatory factors were measured by ELISA to show the inflammatory response. Real-time PCR and western blot were applied to measure the gene and protein change to explore the underlying mechanisms. RESULTS: Results showed that DM significantly improved the retinal function by increasing the ERG amplitude, preserving the retinal morphology, reducing photoreceptor cell apoptosis, decreasing inflammatory response, and inhibiting endoplasmic reticulum stress in rd10 mice. CONCLUSION: This is the first time when the protective effects of DM against photoreceptor cell degeneration of rd10 mice have been demonstrated, providing scientific rationale to develop DM as a potential agent to treat RP.

Transcription factor SP1 mediates hyperglycemia-induced upregulation of roundabout4 in retinal microvascular endothelial cells.

Roundabout4 (Robo4) is a gene that is expressed specifically in vasculature and is involved in the angiogenesis and integrity of blood vessels. The expression level of Robo4 increases gradually along with the development of diabetic retinopathy (DR). In this study, we explored the mechanism of transcriptional regulation of Robo4 in retinal endothelial cells, and investigated the effects of this regulation on cellular functions under hyperglycemic conditions. Human retinal endothelial cells (HREC) exposed to hyperglycemia were used to detect the expression levels of specificity protein 1 (SP1) and Robo4 by RT-qPCR and western blotting. Small interfering RNA (SiRNA) transfection technology was used to analyze the regulatory relationship between SP1 and Robo4. The effect of transcription factor SP1 on Robo4 promoter activity and the location of SP1 binding sites were investigated using chromatin immunoprecipitation (ChIP) and luciferase assay. Cell migration, monolayer permeability and tube formation assays were performed to demonstrate the role of SP1/Robo4 in regulating HREC functions in hyperglycemic conditions. The results showed that hyperglycemia upregulated the mRNA and protein levels of SP1 and Robo4 in HREC. Depletion of SP1 by siRNA transfection inhibited the hyperglycemia induced overexpression of Robo4. ChIP combined with luciferase assay showed that under hyperglycemic conditions, SP1 significantly increased the transcriptional level of Robo4 via an additional SP1 binding site at -1912/-1908 in the Robo4 promoter. Repressing the SP1/Robo4 pathway effectively mitigated the abnormity in HREC migration, permeability and angiogenesis induced by hyperglycemia. All these findings indicate that hyperglycemia-induced upregulation of Robo4 is mediated by enhanced transcription of SP1. The SP1/Robo4 signaling pathway can regulate the migratory ability, monolayer permeability and angiogenesis of HREC under hyperglycemic conditions, suggesting that it may play an important role in microvascular dysfunction during DR.

Comparative Analysis of KnockOut™ Serum with Fetal Bovine Serum for the In Vitro Long-Term Culture of Human Limbal Epithelial Cells.

The limbal epithelial cells can be maintained on 3T3 feeder layer with fetal bovine serum supplemented culture medium, and these cells have been used to successfully treat limbal stem cell deficiency. However, fetal bovine serum contains unknown components and displays quantitative and qualitative lot-to-lot variations. To improve the culture condition, the defined KnockOut serum replacement was investigated to replace fetal bovine serum for culturing human limbal epithelial cell. Human primary limbal epithelial cells were cultured in KnockOut serum and fetal bovine serum supplemented medium, respectively. The cell growth rate, gene expression, and maintenance of limbal epithelial stem cells were studied and compared between these two groups. Human primary limbal epithelial cells were isolated and successfully serially cultivated in this novel KnockOut serum supplemented medium; the cell proliferation and stem cell maintenance were similar to those of cells grown in fetal bovine serum supplemented medium. These data suggests that this KnockOut serum supplemented medium is an efficient replacement to traditional fetal bovine serum supplemented medium for limbal epithelial cell culture, and this medium has great potential for long term maintenance of limbal epithelial cells, limbal epithelial stem cells transplantation, and tissue regeneration.

Identification and validation of reference genes for quantitative RT-PCR analysis of retinal pigment epithelium cells under hypoxia and/or hyperglycemia.

Retinal pigment epithelium (RPE) cell-based gene expression studies performed under hypoxia and/or hyperglycemia show huge potential for modeling cell responses in diabetic retinopathy, retinopathy of prematurity and other retinal diseases. However, normalization of gene expression on RPE cells under those conditions has commonly been done using either GAPDH or ß-actin as reference genes without any validation of their expression stability. Therefore, we aimed to establish a suitable set of reference genes for studies on RPE cells cultured under both normal culturing glucose and atmospheric oxygen tension (normoxia, 21%), under a low oxygen tension (hypoxia, 1%), under a high glucose growth medium (25 mmol/l) and under the combination of the two changed conditions above for distinct time points taking together from 24h to 7 days. Quantitative real-time PCR (qRT-PCR) was applied on RNA obtained from a cell line, ARPE-19. Stability of 14 commonly used reference genes was assessed and ranked according to their stability values using the geNorm and NormFinder softwares with the aim to find the most stable expressed gene under all conditions. Our findings confirm that HPRT1, GUSB and PPIA are the most suitable reference genes for RPE cell gene expression experiments subjected to hypoxia and/or hyperglycemia. To emphasize the importance of selecting the most stably expressed reference genes for obtaining reliable results, mRNA expression levels of hypoxia induced factor-1α were analyzed vs the best reference genes, the worst ones and the most commonly used ones. These reference genes gave the most reliable normalization for comparative analyses of gene transcription under those conditions.

A simple LC-MS method for determination of cyasterone in rat plasma: application to a pilot pharmacokinetic study.

A simple, specific, and sensitive liquid chromatography-mass spectrometry (LC-MS) method for determination of cyasterone in rat plasma was developed in our laboratory. Cucurbitacin B was used as an internal standard (IS). After protein precipitation with twofold volume of acetonitrile, the analyte and IS were separated on a Luna C18 column (100 × 4.6 mm, i.d., 3.0 µm; Phenomenex) by isocratic elution with acetonitrile-water (80:20, v/v) as the mobile phase at a flow rate of 0.4 mL/min. An electrospray ionization source was applied and operated in the positive ion mode; selected ion monitoring scan mode was used for quantification, and the target ions m/z 543.3 for cyasterone and m/z 581.3 for IS were chosen. Good linearity was observed in the concentration range of 0.40-400 ng/mL for cyasterone in rat plasma. Intra-day and inter-day precision were both <7.4%. This method was proved to be suitable for pharmacokinetic studies after oral (5.0 mg/kg) or intravenous (0.5 mg/kg) administration of cyasterone in rats. Copyright © 2015 John Wiley & Sons, Ltd.

A Comparative Treatment Study of Intravitreal Voriconazole and Liposomal Amphotericin B in an Aspergillus fumigatus Endophthalmitis Model.

PURPOSE: To compare the effects of voriconazole (VCZ) and liposomal amphotericin B (Amp-B) in an experimental model of Aspergillus fumigatus endophthalmitis. METHODS: Thirty guinea pigs received an intravitreal injection of A. fumigatus to induce endophthalmitis. The animals were randomly divided into three groups, including control (0.02 mL balanced salt solution intravitreal injection) and experimental (20 µg VCZ/0.02 mL or 20 µg liposomal Amp-B/0.02 mL intravitreal injection) groups. Corneal opacity, aqueous flare, and vitreous opacity were graded, and electroretinographic examinations were performed at multiple time points. At 28 days post treatment, histopathology was performed to examine the retinal architecture. RESULTS: The inflammation in the VCZ and liposomal Amp-B groups was milder than that in the control group. Corneal opacity, aqueous flare, and vitreous opacity scores, as well as electroretinographic recording, showed significantly less inflammation in the VCZ group compared with the liposomal Amp-B group during the early and middle stages of endophthalmitis (P < 0.05). Normal histologic structure of the retina was observed in eyes treated with VCZ and liposomal Amp-B. CONCLUSIONS: Both intravitreal VCZ and liposomal Amp-B were effective treatments for A. fumigatus-induced endophthalmitis in guinea pigs. Voriconazole was superior to liposomal Amp-B at doses similar to the initial therapy for acute infections. Further experimental and clinical studies are required to confirm the efficacy of these two antifungal drugs. Chinese Abstract.

Lentiviral-mediated growth-associated protein-43 modification of bone marrow mesenchymal stem cells improves traumatic optic neuropathy in rats.

The aim of the present study was to examine the effect of growth-associated protein-43 (GAP-43) on bone marrow mesenchymal stem cell (BMSC) differentiation in a rat model of traumatic optic neuropathy (TON). GAP­43 and short hairpin (sh)RNA­GAP­43 were inserted into pGLV5 and pGLV3 lentiviral vectors, respectively. The stable control, GAP­43­overexpression and GAP­43­knockdown cell lines (GFP/BMSCs, GAP­43/BMSCs and shGAP­43/BMSCs, respectively) were established. The expression of GAP­43, neuron­specific enolase (NSE), nestin, neurofilament (NF), neuron­specific nuclear­binding protein (NeuN) and ßIII­tubulin were detected in the GAP­43/BMSCs and shGAP­43/BMSCs with retinal cell­conditioned differentiation medium using semi­quantitative polymerase chain reaction (PCR), western blotting and cell immunofluorescence. In addition, the BMSCs were observed under fluorescence microscopy. The Sprague­Dawley rat models of TON were established and identified by retrograde labeling of retinal ganglion cells (RGCs) with fluoroGold (FG). The lentiviral­mediated GAP­43­modified BMSCs were then transplanted into the rat model of TON. The expression of GAP­43 was detected in the retinal tissues using qPCR and western blotting. The histopathology of the retinal tissues was observed using hematoxylin and eosin (H&E) staining. The GAP­43/BMSCs exhibited positive expression of NSE, NF, nestin and ßIII­tubulin, and exhibited a neuronal phenotype. The shGAP­43/BMSCs markedly inhibited expression of NeuN, NSE, NF, nestin and ßIII­tubulin induced by retinal cell­conditioned differentiation medium. The FG staining revealed that the number of labeled RGCs were significantly decreased in the TON model rats, compared with normal rats (P<0.05). The H&E staining revealed that the degree of pathological changes was improved in the GAP­43/BMSC group, compared with the GFP/BMSC and shGAP­43/BMSC groups. In conclusion, GAP­43 promoted BMSC differentiation into neuron-like cells, and intravitreally injected GAP-43/BMSCs promoted the process of nerve repair in a rat model of TON.

Investigation of the Regulation of Roundabout4 by Hypoxia-Inducible Factor-1α in Microvascular Endothelial Cells.

PURPOSE: We determined if hypoxia-inducible factor-1α (HIF-1α) and Roundabout4 (Robo4) colocalized in fibrovascular membranes (FVM) from patients with proliferative diabetic retinopathy (PDR), and investigated the regulation of HIF-1α on Robo4 in microvascular endothelial cells under normoxic and hypoxic conditions in vitro. METHODS: Immunofluorescence and confocal laser scanning microscopy were done to analyze the colocalization of HIF-1α and Robo4 in the FVM. Expression of HIF-1α was knocked down by small interfering RNA (siRNA) technology to study its effects on Robo4 expression of human retinal endothelial cells (HREC) and human dermal microvascular endothelial cells (HDMEC) under normoxic and/or hypoxic conditions. Full-length human HIF-1α gene was transfected into HREC and HDMEC using GFP lentivirus vectors to overexpress HIF-1α under normoxic conditions. The HIF-1α and Robo4 mRNA and protein expressions were quantified by real-time PCR and Western blot. A cell proliferation, migration assay, and flow cytometry were used to analyze the effect of HIF-1α regulation on Robo4 in HREC under hypoxic conditions. RESULTS: Colocalization of HIF-1α and Robo4 in vessels of FVM was confirmed by immunofluorescence staining. Knockdown of HIF-1α expression by siRNA in the HREC and HDMEC inhibited Robo4 expression in mRNA and protein level, while overexpressed HIF-1α increased Robo4 mRNA and protein expression. Silencing HIF-1α in endothelial cells under hypoxic conditions inhibited cell invasion and proliferation, which showed that HIF-1α and Robo4 overexpression due to hypoxic conditions correlated with HREC migration and proliferation. CONCLUSIONS: Both HIF-1α and Robo4 may have a vital role during the formation of FVM. The increased or decreased expression of Robo4 by stimulation or knockdown of HIF-1α suggesting that Robo4 is positively regulated by HIF-1α under normoxic and hypoxic conditions in microvascular endothelial cells in vitro. The HIF-1α gene promotes HREC invasion and proliferation by transcriptionally upregulating Robo4 under hypoxic conditions.

Effect of Robo4 on retinal endothelial permeability.

PURPOSE: To examine the role of Roundabout 4 (Robo4) in retinal endothelial permeability and analyze the structural events that lead to barrier disruption. MATERIALS AND METHODS: Small interfering RNA (siRNA) technology was used to knockdown Robo4 expression to study its effects on the permeability of human retinal vascular endothelial cells (HRVECs) in vitro. The endothelial cell permeability was detected by measuring the flux of rhodamine B isothiocyanate (RITC)-dextran across the HRVEC monolayers. The impact of Robo4 siRNA on the expression of tight junction (TJ) proteins and the activation of LIM-kinase (LIMK)/cofilin pathway were measured by western blotting. The change of actin cytoskeleton was detected using indirect immunofluorescence. RESULTS: Robo4 siRNA increased the permeability of HRVEC monolayers. The expression levels of TJ-associated proteins occludin and zonula occludens (ZO)-1 were suppressed in Robo4-depleted cells. In addition, there was rearrangement of F-actin in HRVECs. These processes were induced through increased activity in the LIMK/cofilin pathway which coincided with a disruption in the barrier properties of retinal endothelial monolayers. CONCLUSIONS: Knockdown of Robo4 expression in HRVECs induced endothelial hyperpermeability associated with the downregulation of ZO-1, occludin, and the rearrangement of F-actin and that LIM-kinase 1 (LIMK1)/cofilin signal transduction system may be involved in the modulating process.