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  • Circular RNA-ZNF609 regulates corneal neovascularization by acting as a sponge of miR-184
    Exp. Eye Res. (IF 2.998) Pub Date : 2020-01-16
    Pengcheng Wu; Dongyan Zhang; Yuanyuan Geng; Rui Li; Yanan Zhang

    Corneal neovascularization can cause abnormal blood vessels to grow in the normally transparent and translucent cornea leading to various sight-threatening eye diseases. microRNAs and circular RNAs are known to play essential roles in the regulation of numerous biological functions. It is urgently needed to understand the molecular mechanism of miRNAs and circular RNAs in the corneal neovascularization. We aimed to elucidate the role of a specific a circular RNA, cZNF609, in the corneal neovascularization. cZNF609 and miR-184 levels were determined by RT-qPCR. Luciferase reporter assay and RNA immunoprecipitation assay were conducted to verify the target of cZNF609. The biological function of cZNF609 and miR-184 were assessed via cell proliferation, migration, and tube formation assays in vitro as well as the corneal suture model in vivo. The up-regulation of cZNF609 and down-regulation of miR-184 were observed during corneal neovascularization. cZNF609 acted as a miR-184 sponge to block miR-184 activity. Overexpression of miR-184 suppressed HCEKs cell proliferation, migration in vitro, and angiogenesis in vivo. The miR-184-mediated inhibition effect can be rescued through the re-introduction of cZNF609. Mechanically, cZNF609/miR-184 interaction regulated the downstream Akt and VEGF signaling pathway. Intervention of cZNF609 and miR-184 may serve as a potential strategy for pathological corneal neovascularization treatment.

    更新日期:2020-01-16
  • Two-year observation of morphologic and histopathologic changes in the monkey cornea following small incision allogenic lenticule implantation
    Exp. Eye Res. (IF 2.998) Pub Date : 2020-01-16
    Jing Zhao; Rui Liu; Yang Shen; Xiaoyu Zhang; Yu Zhao; Haipeng Xu; Ye Xu; Xingtao Zhou

    Purpose To observe the morphologic and histopathologic changes of femtosecond laser assisted small incision allogenic intrastromal lenticule implantation (AILI) in monkey corneas. Methods 6 healthy adult monkeys were included. One eye of two monkeys and both eyes of one monkey received femtosecond lenticule extraction with a −4.0 diopter (D) correction. Each extracted refractive donor lenticule was immediately allogeneically transplanted into a corneal stromal pocket created by a femtosecond laser in another monkey's eye. A postoperative two-year follow-up was performed with slit lamp microscopy, corneal topography, anterior segment optical coherence tomography and in vivo confocal microscopy. All eyes were enucleated for Hematoxylin-Eosin staining and transmission electron microscopy (TEM) observation. Results No complications were observed in the follow-up period. At postoperative 2 years, the corneas remained clear and the lenticules were integrated with the surrounding tissue under slit lamp microscopy. Nerve fiber regeneration was detected in the lenticule layer as observed through confocal microscopy. Corneal power was increased by 1.83 ± 1.36 D after 2 years, which was less than at 6 months (3.27 ± 1.2 D). Disordered fibers and decreased keratocytes in the implanted lenticules could be detected under light microscopy and TEM, with a clear boundary between the lenticules and the surrounding tissue. Conclusions Small incision AILI is feasible and safe for reshaping the cornea. Corneal healing remained stable while refraction showed a moderate regression within postoperative 2 years.

    更新日期:2020-01-16
  • Requirement for the collagen receptor Endo180 in collagen gel contraction mediated by corneal fibroblasts
    Exp. Eye Res. (IF 2.998) Pub Date : 2020-01-11
    Koichi Nishida; Koji Sugioka; Junko Murakami; Aya Kodama-Takahashi; Isamu Nanri; Hiroshi Mishima; Teruo Nishida; Shunji Kusaka

    The interaction of keratocytes with extracellular matrix components plays an important role in the maintenance of corneal transparency and shape as well as in the healing of corneal wounds. In particular, the interaction of these cells with collagen and cell-mediated collagen contraction contribute to wound closure. Endo180 is a receptor for collagen that mediates its cellular internalization. We have now examined the role of Endo180 in collagen contraction mediated by corneal fibroblasts (activated keratocytes). Antibodies to Endo180 inhibited the contractile activity of mouse corneal fibroblasts embedded in a three-dimensional collagen gel and cultured in the presence of serum, with this effect being both concentration and time dependent and essentially complete at an antibody concentration of 0.2 μg/ml. Whereas corneal fibroblasts cultured in a collagen gel manifested a flattened morphology with prominent stress fibers under control conditions, they showed a spindlelike shape with few stress fibers in the presence of antibodies to Endo180. Antibodies to Endo180 had no effect on the expression of α–smooth muscle actin or the extent of collagen degradation in collagen gel cultures of corneal fibroblasts. Immunohistofluorescence analysis did not detect the expression of Endo180 in the unwounded mouse cornea. However, Endo180 expression was detected in keratocytes migrating into the wound area at 3 days after a corneal incisional injury. Together, our results suggest that Endo180 is required for the contraction of collagen matrix mediated by corneal fibroblasts and that its expression in these cells may contribute to the healing of corneal stromal wounds.

    更新日期:2020-01-13
  • A single Asp isomer substitution in an αA-crystallin-derived peptide induces a large change in peptide properties
    Exp. Eye Res. (IF 2.998) Pub Date : 2020-01-10
    Kosuke Magami; Ingu Kim; Noriko Fujii

    The eye lens is mainly composed of crystallins, which undergo modifications such as oxidation, deamidation and isomerization with aging. Asp58, Asp76, Asp84, and Asp151 residues of αA-crystallin are site-specifically isomerized to L-iso, D-, and D-iso isomers in aged-related cataract lenses. In addition, an αA66–80 peptide, corresponding to the 66–80 (66SDRDKFVIFLDVKHF80) fragment of human αA-crystallin, is detected in aged lens. This peptide induces protein aggregation and causes loss of the chaperone function of α-crystallin. The αA66–80 peptide contains Asp76, but it is not known whether isomerization of Asp76 in αA66–80 specifically induces protein aggregation or affects α-crystallin function. Using Fmoc-based solid-phase synthesis, here we synthesized four αA66–80 peptides, each containing L-, L-iso, D-, or D-isoAsp at position 76, and compared their structures and properties. Normal αA66–80 peptide containing the L-Asp76 isomer increased the EDTA-induced aggregation of ADH protein, DTT-induced aggregation of insulin, and heat-induced aggregation of βL-crystallin. αA66–80 peptide containing D- or D-isoAsp76 had similar or no effects on the aggregation of these proteins. By contrast, αA66–80 peptide containing L-isoAsp76 inhibited the aggregation of all three proteins, indicating that it has chaperone activity. With regard to secondary structure, αA66–80 peptide containing the L-, D-, or D-isoAsp76 isomer had random-coil structure, whereas αA66–80 peptide containing L-isoAsp76 had β-sheet like structure. A Thioflavin T (ThT) assay indicated that only the L-isoAsp-containing αA66–80 peptide has β-sheet structure and generates amyloid fibrils. Collectively, these observations indicate that isomerization of Aps76 to the Lβ isomer endows β-sheet structure and chaperone function on this peptide.

    更新日期:2020-01-11
  • In-vivo longitudinal changes in thickness of the canine postnatal retina
    Exp. Eye Res. (IF 2.998) Pub Date : 2020-01-10
    Valérie L. Dufour; Yinxi Yu; Wei Pan; Gui-Shuang Ying; Gustavo D. Aguirre; William A. Beltran

    The objectives of the present work were to assess by spectral domain optical coherence tomography (OCT) the changes in thickness of the outer nuclear layer (ONL), the ONL + photoreceptor inner segment (IS), and the retinal thickness, as a function of age in the normal canine retina. OCT retinal scans extending from the edge of the optic nerve head (ONH) along the superior and inferior meridians were captured in both eyes of 17 normal dogs at age ranging from 4 to 119 weeks. The different parameters along the superior and the inferior regions were determined following manual segmentation using the Heidelberg Eye Explorer software. Changes in thickness with age were modeled using one-phase exponential decay models. In vivo OCT imaging results showed no interocular statistically significant differences in ONL, ONL + IS, and retinal thickness at any age. All three parameters were however found to be statistically significantly thicker in the superior vs inferior retina. A rapid thinning of the three layers occurs in both the superior and inferior retina between 4 and 12 weeks of age, before reaching a plateau at around 20 weeks of age. In conclusion, the ONL, ONL + IS, and retinal thickness of the normal canine retina decrease significantly during the first three postnatal months, and is likely attributed to an overall increase in the eye volume and tangential dispersion of the photoreceptor since early photoreceptor developmental cell death is very limited at that age. Establishment of the natural history of ONL, ONL + IS, and retinal thinning will allow a more accurate assessment of the progression of a retinal degenerative condition as well as facilitate the detection of positive rescue effect of novel retinal therapies evaluated in this large animal model.

    更新日期:2020-01-11
  • Gypenosides mediate cholesterol efflux and suppress oxidized LDL induced inflammation in retinal pigment epithelium cells
    Exp. Eye Res. (IF 2.998) Pub Date : 2020-01-10
    Lincoln Biswas; Zhihong Zeng; Annette Graham; Xinhua Shu

    Age-related macular degeneration (AMD) is a predominant cause of visual deficit in aged population. Abnormal accumulation of cholesterol, including oxidized low-density lipoprotein (oxLDL), underneath the retinal pigment epithelium (RPE) cells contributes to the development of AMD. Gypenosides (Gyp) are glycosides extracted from Gynostemma pentaphyllum and have demonstrated protective effects against inflammation and oxidative stress. To determine the therapeutic potential of Gyp for AMD, we investigated its effect on cholesterol trafficking and metabolism and assessed the protective function of Gyp against oxLDL-induced damage in RPE cells. Cholesterol efflux to high-density lipoprotein (HDL) and human serum was significantly increased in RPE cells treated with Gyp when compared to untreated control cells. Expression of cholesterol metabolism (CYP27A1, CYP46A1) and trafficking (TSPO, ABCA1 and ABCG1) genes was also markedly increased in Gyp-treated RPE cells. OxLDL-treated RPE cells had significantly increased cholesterol accumulation and lipid droplet formation. There were marked increases in reactive oxygen species (ROS) generation and proinflammatory cytokines via NF-κB activation in RPE cells treated with oxLDL, while incubation with Gyp rectified these changes. These findings provide pharmacological evidence that Gyp has the potential to treat patients with early onset AMD by promoting cellular cholesterol removal from RPE cells and inhibiting inflammation and oxidative stress.

    更新日期:2020-01-11
  • The effects of negative periocular pressure on intraocular pressure
    Exp. Eye Res. (IF 2.998) Pub Date : 2020-01-09
    C. Ross Ethier; Paul Yoo; John P. Berdahl

    Glaucoma is a major cause of blindness, and IOP reduction remains the only clinically-validated therapy. In this study, we analyze a novel IOP-lowering strategy that uses a modest negative pressure (vacuum) applied locally to the periorbital region by a pair of goggles with each lens individually connected to a programmable pump. Motivated by clinical data showing an IOP reduction, we used an existing validated lumped-parameter model of the eye to understand the putative mechanism of this treatment. The model considers aqueous humor dynamics, episcleral venous pressure, and changes in ocular blood volume to describe how IOP changes with time in response to an external perturbation. We find that clinical data are qualitatively and quantitatively consistent with model predictions if we include two primary mechanisms in the model: first, negative pressure application causes a relatively rapid increase in globe volume accompanied by increased blood volume in the eye. Second, negative pressure application reduces episcleral venous pressure, causing a slower adjustment of IOP due to altered aqueous humor dynamics. These results provide testable hypotheses that hopefully will lead to a fuller experimentally-driven understanding of how negative periocular pressure influences IOP. Evaluating the long-term effects of such treatments on glaucoma patients requires further clinical study.

    更新日期:2020-01-09
  • Protective effect inhibiting the expression of miR-181a on the diabetic corneal nerve in a mouse model
    Exp. Eye Res. (IF 2.998) Pub Date : 2020-01-09
    Jianzhang Hu; Yurong Huang; Yi Lin; Jingqiong Lin

    To investigate the protective effect of inhibiting miR-181a on diabetic corneal nerve in mice, we chose male C57BL/6 mice with streptozotocin (STZ) -induced diabetes as animal models. The expression of miR-181a in trigeminal ganglion tissue (TG) of diabetic mice was detected by real-time PCR. In vitro, we cultured mouse trigeminal ganglion neurons and measured the neuronal axon growth when treated under miR-181a antagomir and negative conditions (NTC). Immunofluorescence showed a significant increase in neuronal axon length in trigeminal ganglion cells treated with miR-181a antagomir. In animal models, we performed epithelial scraping and subconjunctival injection of the miR-181a antagomir and miRNA antagomir NTC to observe the corneal nerve repair by corneal nerve staining. miR-181a antagomir subconjunctival injection significantly increased the corneal epithelium healing of diabetic mice compared with that of the NTC group. Meanwhile, corneal nerve staining showed that the repair of corneal nerve endings was significantly promoted. As the targets of the 181a, ATG5 and BCL-2 were previously identified. The results of Western blot showed that the expression of autophagy associated protein ATG5 and LC3B-II and the expression of anti-apoptotic protein Bcl-2 were decreased in the high-glucose cell culture environment and the diabetic TG tissue. The expression of ATG5, LC3B-II and Bcl-2 were significantly increased after miR-181a antagomir treatment compared with negative control group. This study showed that inhibition of miR-181a expression in diabetic mice could increase ATG5-mediated autophagic activation, BCL-2-mediated inhibition of apoptosis, and promote the growth of trigeminal sensory neurons and the regeneration of corneal nerve fibers. It has a protective effect on diabetic corneal neuropathy.

    更新日期:2020-01-09
  • High-throughput transcriptional profiling combined with angiogenesis antibody array analysis in an orbital venous malformation cohort
    Exp. Eye Res. (IF 2.998) Pub Date : 2020-01-08
    Peiwei Chai; Jie Yu; Yongyun Li; Yingyun Shi; Xianqun Fan; Renbing Jia

    Orbital venous malformations (OVMs) are the most common benign orbital vascular disorders in adults and are characterized as enlarging encapsulated vascular neoplasms. These painless lesions grow slowly and become symptomatic with proptosis or visual disturbance. However, the pathogenic mechanism and diagnostic markers of OVMs remain poorly understood. To identify potential pathways involved in OVM formation, a cDNA microarray analysis was conducted with OVM samples and normal vascular tissues. These data were deposited in the National Omics Data Encyclopedia (NODE) database (accession number: OER033009). These pathway expression data were further confirmed by reverse transcription qPCR (RT-qPCR) in an OVM cohort. To explore the diagnostic markers in OVM, an angiogenesis antibody array was analyzed. The altered factors were further validated by enzyme-linked immunosorbent assay (ELISA) in the OVM cohort. Transcriptome screening revealed upregulated autophagy and VEGF pathways and downregulated Hippo, Wnt, hedgehog and vascular smooth muscle contraction signaling pathways in OVM samples. Furthermore, plasma EGF (p < 0.001) and Leptin (p < 0.01) levels were significantly elevated in OVM patients. Here, for the first time, we revealed the transcriptional background and plasma diagnostic markers in OVM, providing a novel understanding of OVM pathogenesis and facilitating the early diagnosis of OVM.

    更新日期:2020-01-09
  • Nuclear factor-kappa beta signaling is required for transforming growth factor Beta-2 induced ocular hypertension
    Exp. Eye Res. (IF 2.998) Pub Date : 2020-01-08
    Humberto Hernandez; Amanda L. Roberts; Colleen M. McDowell

    A major risk for the development of primary open-angle glaucoma (POAG) is elevated intraocular pressure (IOP). Elevated IOP is caused by increased outflow resistance due in part to excessive extracellular matrix (ECM) deposition in the trabecular meshwork (TM). The role of transforming growth factor beta 2 (TGFβ2) in inducing ECM production is well understood. Recent studies suggest that toll-like receptor 4 (TLR4) plays an important role in fibrogenesis. We have previously described a crosstalk between TGFβ2 and TLR4 in the development of ocular hypertension and glaucomatous TM damage. Nuclear factor-kappa beta (NF-κB) is critical for TLR4 signaling. To determine the transactivation of NF-κB, TM cells were stimulated with cellular fibronectin containing the EDA isoform (cFN-EDA), TGFβ2, or lipopolysaccharide (LPS) in combination with a selective TLR4 inhibitor. cFN-EDA, TGFβ2, and LPS all induced transactivation of NF-κB and inhibition of TLR4 blocked the effect of each treatment paradigm. To evaluate the role of NF-κB in IOP regulation, we utilized our inducible mouse model of ocular hypertension by injection of Ad5.TGFβ2 in mice harboring a mutation in NF-κB and wild-type controls. IOP was measured over time and eyes accessed by immunohistochemistry for the ECM protein FN and the specific FN-EDA isoform. Ad5.TGFβ2 induced ocular hypertension and expression of FN and FN-EDA in wild-type mice, but mutation in NF-κB blocked the effect. These data suggest that NF-κB is necessary for TGFβ2-induced ECM production and ocular hypertension and the transactivation of NF-κB is dependent on both TGFβ2 and TLR4.

    更新日期:2020-01-08
  • Nox4-mediated ROS production is involved, but not essential for TGFβ-induced lens EMT leading to cataract
    Exp. Eye Res. (IF 2.998) Pub Date : 2020-01-08
    S.J. Das; T. Wishart; K. Jandeleit-Dahm; F.J. Lovicu

    The reactive oxygen species (ROS) producing enzyme, NADPH oxidase 4 (Nox4),is upregulated in response to TGFβ in lens epithelial cells in vitro, and its selective inhibition was shown to block aspects of TGFβ-induced epithelial-mesenchymal transition (EMT). In the present in situ study we validate the role(s) of Nox4 in TGFβ-induced lens EMT leading to anterior subcapsular cataract (ASC) formation. Mice overexpressing TGFβ in the lens, that develop ASC, were crossed to Nox4-deficient mice. When comparing mice overexpressing TGFβ in lens, to mice that were also deficient for Nox4, we see the delayed onset of cataract, along with a delay in EMT protein markers normally associated with TGFβ-induced fibrotic cataracts. In the absence of Nox4, we also see elevated levels of ERK1/2 activity that was shown to be required for TGFβ/Smad2/3-signaling. qRT-PCR revealed upregulation of Nox2 and its regulatory subunit in TGFβ-overexpressing lens epithelial cells devoid of Nox4. Taken together, these findings provide an improved platform to delineate putative Nox4 (and ROS) interactions with Smad2/3 and/or ERK1/2, in particular in the development of fibrotic diseases, such as specific forms of cataract.

    更新日期:2020-01-08
  • Quantitative analysis of metabolites in glucose metabolism in the aqueous humor of patients with central retinal vein occlusion
    Exp. Eye Res. (IF 2.998) Pub Date : 2020-01-08
    Pinghui Wei; Meiqin He; He Teng; Guoge Han

    Quantitative analysis of aqueous humor (AH) was performed to investigate glucose metabolism in patients with central retinal vein occlusion (CRVO), and to explore metabolic changes after anti-vascular endothelial growth factor (VEGF) treatment. AH samples were collected from 35 patients. Participants diagnosed with CRVO (n = 15) were compared to participants who underwent cataract surgery (n = 20). Thirteen of the participants with CRVO received second-round anti-VEGF treatments. Ultra-high performance liquid chromatography tandem-mass spectrometry (UHPLC-MS/MS) was used to quantify metabolites of the AH. Central macular thickness (CMT) and retinal ganglion cell layer (RGC) thickness were measured using spectral-domain optical coherence tomography. Thirteen metabolites involved in glucose metabolism were identified. Among these metabolites, succinate, glutamate, and glutamine were significantly decreased for the CRVO group (p = 0.028, 0.009, and 0.017, respectively). The α-ketoglutarate/citrate (K/C) ratio had a significant positive correlation with glutamine levels for both control (r = 0.922, p < 0.001) and CRVO groups (r = 0.674, p = 0.006). A significant increase in lactate was observed after intravitreal anti-VEGF administration (t = 2.273, p = 0.045); the change in CMT was negatively correlated with this increase (r = −0.745, p = 0.003). The alteration of RGC thickness was negatively correlated with increases in both glutamine (r = −0.619, p = 0.024) and glucose (r = −0.754, p = 0.003). These results indicate that, compared to glucose metabolism, glutamine was significantly decreased in the AH of patients with CRVO, and may therefore serve as a potential target for CRVO therapy. The glycolytic pathway might be enhanced after intravitreal anti-VEGF injection, which is an important insight into CRVO pathophysiology.

    更新日期:2020-01-08
  • The negative regulatory Spred1 and Spred2 proteins are required for lens and eye morphogenesis
    Exp. Eye Res. (IF 2.998) Pub Date : 2020-01-07
    Fatima Wazin; Frank J. Lovicu

    The transparent and refractive properties of the ocular lens are dependent on its precise cellular structure, supported by the regulation of lens cellular processes of proliferation and differentiation that are essential throughout life. The ERK/MAPK-signalling pathway plays a crucial role in regulating lens cell proliferation and differentiation, and in turn is regulated by inhibitory molecules including the Spred family of proteins to modulate and attenuate the impact of growth factor stimulation. Given Spreds are strongly and distinctly expressed in lens, along with their established inhibitory role in a range of different tissues, we investigated the role these antagonists play in regulating lens cell proliferation and differentiation, and their contribution to lens structure and growth. Using established mice lines deficient for either or both Spred 1 and Spred 2, we demonstrate their role in regulating lens development by negatively regulating ERK1/2 activity. Mice deficient for both Spred 1 and Spred 2 have impaired lens and eye development, displaying irregular lens epithelial and fibre cell activity as a result of increased levels of phosphorylated ERK1/2. While Spred 1 and Spred 2 do not appear to be necessary for induction and early stages of lens morphogenesis (prior to E11.5), nor for the formation of the primary fibre cells, they are required for the continuous embryonic growth and differentiation of the lens.

    更新日期:2020-01-07
  • Involvement of regulated necrosis in blinding diseases: Focus on necroptosis and ferroptosis
    Exp. Eye Res. (IF 2.998) Pub Date : 2020-01-07
    Jing-Jie Peng; Wei-Tao Song; Fei Yao; Xuan Zhang; Jun Peng; Xiu-Ju Luo; Xiao-Bo Xia

    Besides apoptosis, necrosis can also occur in a highly regulated and genetically controlled manner, defined as regulated necrosis, which is characterized by a loss of cell membrane integrity and release of cytoplasmic content. Depending on the involvement of its signal pathway, regulated necrosis can be further classified as necroptosis, ferroptosis, pyroptosis and parthanatos. Numerous studies have demonstrated that regulated necrosis is involved in the pathogenesis of many diseases covering almost all organs including the brain, heart, liver, kidney, intestine, blood vessel, eye and skin, particularly myocardial infarction and stroke. Most recently, growing evidence suggests that multiple types of regulated necrosis contribute to the degeneration of retinal ganglion cells, retinal pigment epithelial cells or photoreceptor cells, which are the main pathologic features for glaucoma, age-related macular degeneration or retinitis pigmentosa, respectively. This review focuses on the involvement of necroptosis and ferroptosis in these blinding diseases.

    更新日期:2020-01-07
  • Gut microbiota compositional profile and serum metabolic phenotype in patients with primary open-angle glaucoma
    Exp. Eye Res. (IF 2.998) Pub Date : 2020-01-07
    Haijun Gong; Simin Zhang; Qiguan Li; Chengguo Zuo; Xinbo Gao; Bingru Zheng; Mingkai Lin

    The gut microbiota (GM) and its influence on host metabolism are considered to be an environmental factor that contributes to the progression of many immune and neurodegenerative diseases. However, the features of the GM and serum metabolites in Primary open-angle glaucoma (POAG) patients have not been clearly elucidated. The purpose of this research is to explore the gut microbial composition and serum metabolic phenotype in POAG patients. 16S rRNA V4 genes of bacteria from the fecal samples of 30 POAG patients and 30 healthy subjects were sequenced by the Illumina MiSeq platform and then analyzed by QIIME. Their serum samples were analyzed by gas chromatography/mass spectrometry (GC-MS)-based metabolomics. The association between gut microbial species and host circulating metabolites and clinical phenotypes was also analyzed. Compared with controls, f Prevotellaceae, g unidentified Enterobacteriaceae, and s Escherichia coli increased the most in POAG patients, whereas g Megamonas and s Bacteroides plebeius significantly decreased in POAG patients. The alteration of the endogenous metabolomic profile in POAG patients included five amino acids or dipeptides, two hormone derivates, one purine derivative, one bile acid derivative and one organic acid. It also showed that citric acid was positively correlated with Megamonas, whereas L-γ-Glutamyl-L-alanine, MHPG, cholic acid glucuronide and hypoxanthine were negatively correlated with Megamonas. Mean visual acuity was negatively correlated with Blautia, mean VF-MD was negatively correlated with Faecalibacterium, and average RNFL thickness was positively correlated with Streptococcus. Our results revealed that there was a distinct difference in GM composition and serum metabolic phenotype between POAG patients and healthy individuals. This finding suggests the potential correlations between the GM and serum metabolites in the pathogenesis of glaucoma and thus provides new insight into the GM-targeted interventions of this disease.

    更新日期:2020-01-07
  • Dynamic spatiotemporal expression pattern of limbal stem cell putative biomarkers during mouse development
    Exp. Eye Res. (IF 2.998) Pub Date : 2020-01-03
    Zhi Hou Guo; Yi Ming Zeng; Jun Sheng Lin

    Limbal stem cells (LSCs), a subpopulation of limbal epithelial basal cells, are crucial to the homeostasis and wound healing of corneal epithelium. The identification and isolation of LSCs remains a challenge due to lack of specific LSCs biomarkers. In this study, Haematoxylin-eosin (HE), 4′, 6-diamidino-2-phenylindole (DAPI), and immunohistochemistry (IHC) stains were performed on the pre- and post-natal limbus tissues of mice which has the advantage of more controllable in term of sampling age relative to human origin. By morphological analysis, we supported that there is an absence of the Palisades of Vogt (POV) in the mouse. The development of prenatal and neonatal cornea was dominated by its stroma, whereas after eyelids opened at P14, the corneal epithelial cells (CECs) quickly go stratification in response to the liquid-air interface. Based on IHC staining, we found that the expression of LSCs putative biomarkers in limbal epithelial basal cells appeared in chronological order as follows: Vim = p63 > CK14 > CK15 (where = represents same time; > represents earlier), and in corneal epithelial basal cells were weakened in chronological order as follows: Vim > p63 > CK15 > CK14, which might also represent the stemness degree. Furthermore, the dynamic spatial expression of the examined LSCs putative biomarkers during mouse development also implied a temporal restriction. The expression of Vim in epithelial cells of mouse ocular surface occurred during E12-E19 only. The expression of CK15 was completely undetectable in CECs after P14, whereas the others putative molecular markers of LSCs, such as p63 and CK14, still remained weak expression, suggesting that CK15 was suitable to serve as the mouse LSCs biomarkers after P14. In this study, our data demonstrated the dynamic spatiotemporal expression pattern of LSCs putative biomarkers in mouse was age-related and revealed the time spectrum of the expression of LSCs in mouse, which adds in our knowledge by understanding the dynamic expression pattern of biomarkers of stem cells relate to maintenance of their stemness.

    更新日期:2020-01-04
  • Differential sensitivity of the On and Off visual responses to retinal ischemia
    Exp. Eye Res. (IF 2.998) Pub Date : 2019-12-31
    Maria A. Bermudez; Francisco Gonzalez

    Retinal ischemia is a common condition that may lead into vision impairment and blindness. In this study, we evaluated changes separately in On and Off visual responses induced by retinal ischemia. To do this, reversible retinal ischemia was induced in anaesthetized rats by increasing the intraocular pressure until the eye fundus became whitish for either 30 or 60 min. Both electroretinogram (ERG) and multiunit neuronal activity in the superior colliculus (SC) were recorded simultaneously for at least 20 min before, during, and after ischemia. In addition, in normal eyes, intravitreal glycine (Gly) injections were performed to further investigate the mechanisms involved in this process. We found that collicular Off responses were more sensitive to ischemia than On responses. The Off response was the first one to decay at the time ischemia was induced and the last to recover after blood reperfusion. The duration of ischemia also differentially affected both responses. After 30 min of ischemia, 14% of SC recordings failed to recover Off responses. After 1 h of ischemia, the percentage of recordings that failed to recover Off responses increased to 50%. Post-ischemic ERGs remained unaltered in all cases. Intravitreal Gly injections caused suppression of Off responses in the SC. Higher doses caused suppression of both On and Off responses in the SC but with no effect on the ERG at the doses tested. In summary, Off responses were more sensitive than On responses to ischemia suggesting that different mechanisms drive the two types of responses. The recovery of transitory ischemia was not complete in the SC responses whereas the ERG remained unaltered, suggesting that retinal damage produced by ischemia is more prominent in ganglion cells. Our results provide critical information for understanding ischemia repercussions and visual processing in the early visual system.

    更新日期:2019-12-31
  • COL2A1 protective variant reduces sporadic rhegmatogenous retinal detachment severity
    Exp. Eye Res. (IF 2.998) Pub Date : 2019-12-31
    Tsz Kin Ng; Wanghao Chen; Qianwen Chen; Yuqian Zheng; Yanxuan Xu; Weiqi Chen; Guihua Zhang; Jianhuan Chen; Chi Pui Pang; Haoyu Chen

    Rhegmatogenous retinal detachment (RRD) is the most common type of RD, the separation of neurosensory retina from the underlying retinal pigment epithelium. The RRD patients can be benefited from appropriate treatment if detected early, especially for the people predicted at high risk. In this study, we aimed to investigate the genetic association and clinical correlation of collagen type II alpha 1 (COL2A1) variants with sporadic RRD in a southern Chinese population. Totally 156 RRD patients and 254 control subjects were recruited, and 12 COL2A1 tag single nucleotide polymorphisms were genotyped by the TaqMan assay. The RRD patients had poorer visual acuity (P < 0.001) and lower intraocular pressure (IOP; P < 0.001) in their surgical eyes compared to the fellow eyes. The COL2A1 rs1793958 variant was significantly associated with RRD in the genotypic (P = 0.024), allelic (P = 0.011, odds ratio (OR) = 0.669), recessive (P = 0.011, OR = 0.384) and homozygous models (P = 0.007, OR = 0.348). RRD patients carrying the rs1793958 G allele had smaller retinal detachment area (P = 0.041) and smaller IOP differences (P = 0.046) between the surgical and fellow eyes compared to those carrying the wildtype AA genotype. In summary, this study revealed that the COL2A1 rs1793958 variant is associated with reduced risk of sporadic RRD, and patients carrying rs1793958 G allele have lower RRD severity.

    更新日期:2019-12-31
  • Human umbilical cord mesenchymal stem cells alleviate ongoing autoimmune dacryoadenitis in rabbits via polarizing macrophages into an anti-inflammatory phenotype
    Exp. Eye Res. (IF 2.998) Pub Date : 2019-12-28
    Xiaoxiao Lu; Na Li; Lu zhao; Di Guo; Huanfa Yi; Liyuan Yang; Xun Liu; Deming Sun; Hong Nian; Ruihua Wei

    Mesenchymal stem cells (MSCs) exhibit beneficial effects on autoimmune dacryoadenitis. However, the underlying mechanisms are not fully understood. In this study, we investigated the therapeutic effect of human umbilical cord mesenchymal stem cells (hUC-MSCs) on rabbit autoimmune dacryoadenitis, an animal model of Sjögren's syndrome (SS) dry eye, and explored whether the effects of MSCs were related to their modulation on macrophage polarization. We have showed that systemic infusion of hUC-MSCs after disease onset efficiently diminished the chronic inflammation in diseased LGs and improved the clinical symptoms. Further analysis revealed that hUC-MSC treatment significantly inhibited the expression of pro-inflammatory M1 macrophage markers iNOS, TNF-α and IL-6, and promoted the expression of anti-inflammatory M2 macrophage markers Arg1, CD206, IL-10, IL-4 and TGF-β in LGs. Mechanistically, hUC-MSCs activated AKT pathway in macrophages, resulting in upregulation of M2-associated molecule Arg1, which was partly abolished by PI3K inhibitor, LY294002. Together, our data indicated that hUC-MSCs can skew macrophages into an M2 phenotype via affecting AKT pathway. These data may provide a new insight into the mechanisms of hUC-MSCs in the therapy of SS dry eye.

    更新日期:2019-12-29
  • Angiotensin II and aldosterone activate retinal microglia
    Exp. Eye Res. (IF 2.998) Pub Date : 2019-12-26
    Indrajeetsinh Rana; Varaporn Suphapimol; Jack R. Jerome; Dean M. Talia; Devy Deliyanti; Jennifer L. Wilkinson-Berka

    Microglial cells are important contributors to the neuroinflammation and blood vessel damage that occurs in ischemic retinopathies. We hypothesized that key effectors of the renin-angiotensin aldosterone system, angiotensin II (Ang II) and aldosterone, increase the density of microglia in the retina and stimulate their production of reactive oxygen species (ROS) as well as pro-angiogenic and pro-inflammatory factors. Two animal models were studied that featured up-regulation of Ang II or aldosterone and included transgenic Ren-2 rats which overexpress renin and Ang II in tissues including the retina, and Sprague Dawley rats with ischemic retinopathy and infused with aldosterone. Complementary studies were performed in primary cultures of retinal microglia from neonatal Sprague Dawley rats exposed to hypoxia (0.5% O2) and inhibitors of the angiotensin type 1 receptor (valsartan), the mineralocorticoid receptor (spironolactone) or aldosterone synthase (FAD286). In both in vivo models, the density of ionized calcium-binding adaptor protein-1 labelled microglia/macrophages was increased in retina compared to genetic or vehicle controls. In primary cultures of retinal microglia, hypoxia increased ROS (superoxide) levels as well as the expression of the NADPH oxidase (NOX) isoforms, NOX1, NOX2 and NOX4. The elevated levels of ROS as well as NOX2 and NOX4 were reduced by all of the treatments, and valsartan and FAD286 also reduced NOX1 mRNA levels. A protein cytokine array of retinal microglia revealed that valsartan, spironolactone and FAD286 reduced the hypoxia-induced increase in the potent pro-angiogenic and pro-inflammatory agent, vascular endothelial growth factor as well as the inflammatory factors, CCL5 and interferon γ. Valsartan also reduced the hypoxia-induced increase in IL-6 and TIMP-1 as well as the chemoattractants, CXCL2, CXCL3, CXCL5 and CXCL10. Spironolactone and FAD286 reduced the levels of CXCL2 and CXCL10, respectively. In conclusion, our findings that both Ang II and aldosterone influence the activation of retinal microglia implicates the renin-angiotensin aldosterone system in the pathogenesis of ischemic retinopathies.

    更新日期:2019-12-27
  • Biomechanical contribution of the sclera to dynamic corneal response in air-puff induced deformation in human donor eyes
    Exp. Eye Res. (IF 2.998) Pub Date : 2019-12-25
    B. Audrey Nguyen; Matthew A. Reilly; Cynthia J. Roberts

    This study was conducted to evaluate the impact of varying scleral material properties on the biomechanical response of the cornea under air-puff induced deformation. Twenty pairs of human donor eyes were obtained for this study. One eye from each pair had its sclera stiffened using 4% glutaraldehyde, while the fellow eye served as control for uniaxial strip testing. The whole globes were mounted in a rigid holder and intraocular pressure (IOP) was set using a saline column. Dynamic corneal response parameters were measured before and after scleral stiffening using the CorVis ST, a dynamic Scheimpflug analyzer. IOP was set to 10, 20, 30, and 40 mmHg, with at least 3 examinations performed at each pressure step. Uniaxial tensile testing data were fit to a neo-Hookean model to estimate the Young's modulus of treated and untreated sclera. Scleral Young's modulus was found to be significantly correlated with several response parameters, including Highest Concavity Deformation Amplitude, Peak Distance, Highest Concavity Radius, and Stiffness Parameter-Highest Concavity (SP-HC). The interaction between IOP and scleral Young's modulus was significantly correlated only to HC Deformation Amplitude. There were significant increases in SP-HC after scleral stiffening at multiple levels of IOP, while no significant difference was observed in the corneal Stiffness Parameter – Applanation 1 (SP-A1) at any level of IOP. Scleral mechanical properties significantly influenced the corneal deformation response to an air-puff. The stiffer the sclera, the greater the constraining effect on corneal deformation resulting in lower displaced amplitude. This may have important clinical implications and suggests that both corneal and scleral material properties contribute to the observed corneal response in air-puff induced deformation.

    更新日期:2019-12-26
  • Features of ectopic lymphoid-like structures in human uveitis
    Exp. Eye Res. (IF 2.998) Pub Date : 2019-12-23
    Simon J. Epps; Natalie Coplin; Philip J. Luthert; Andrew D. Dick; Sarah E. Coupland; Lindsay B. Nicholson

    Persistent non-infectious uveitis has a significant morbidity, but the extent to which this is accompanied by inflammation driven remodelling of the tissue is unclear. To address this question, we studied a series of samples selected from two ocular tissue repositories and identified 15 samples with focal infiltration. Eleven of fifteen contained lymphocytes, both B cells (CD20 positive) and T cells (CD3 positive). In 20% of the samples there was evidence of ectopic lymphoid like structures with focal aggregations of B cells and T cells, segregated into anatomically different adjacent zones. To investigate inflammation in the tissue, an analysis of 520 immune relevant transcripts was carried out and 24 genes were differentially upregulated, compared with control tissue. Two of these (CD14 and fibronectin) were increased in ocular inflammation compared to control immune tissue (tonsil). We demonstrate that in a significant minority of patients, chronic persistent uveitis leads to dysregulation of ocular immune surveillance, characterized by the development of areas of local ectopic lymphoid like structures, which may be a target for therapeutic intervention directed at antibody producing cells.

    更新日期:2019-12-23
  • Piezo channel plays a part in retinal ganglion cell damage
    Exp. Eye Res. (IF 2.998) Pub Date : 2019-12-23
    Wataru Morozumi; Satoshi Inagaki; Yuki Iwata; Shinsuke Nakamura; Hideaki Hara; Masamitsu Shimazawa

    Piezo channel is one of the mechanosensitive channels that senses pressure and shearing stress. Previous reports show that Piezo channel is expressed in many tissues such as skin and lung and they have many important roles. In addition, the mRNA of Piezo has been detected in astrocytes in the optic nerve head of mice. However, it is not yet clear where Piezo channel localize in eye and what kind of effects it have. Thus, the purpose of this study was to determine the expression sites of Piezo channel in mouse eyes and effect of Piezo channel on retinal ganglion cells. Immunostaining analysis showed that the Piezo 1/2 were expressed in the cornea, trabecular meshwork of the anterior ocular segment, lens epithelial cells, and on the retinal ganglion cell layer. The expression of retinal Piezo 2 was increased in retinal disorder model mouse caused by high IOP. Piezo 1 agonist Yoda 1 suppressed neurite outgrowth in retinal ganglion cells. On the other hand, Piezo antagonist GsMTx4 promoted neurite outgrowth in retinal ganglion cells. These findings indicate that Piezo channel may contribute to diseases relating the IOP such as glaucoma.

    更新日期:2019-12-23
  • Therapeutic effects of mesenchymal stem cell-derived exosomes on retinal detachment
    Exp. Eye Res. (IF 2.998) Pub Date : 2019-12-19
    Mingming Ma; Bing Li; Mingliang Zhang; Lei Zhou; Fuhua Yang; Feifei Ma; Hui Shao; Qiutang Li; Xiaorong Li; Xiaomin Zhang

    Retinal detachment (RD) induces ischemia and oxygen deficiency in the retina and results in multiple pathological events; photoreceptor cell degeneration and death is the eventual cause of vision decline. In this study, we investigated the therapeutic effects of mesenchymal stem cell-derived exosomes (MSC-Exos) in a rat retinal detachment (RD) model. The model was developed using a subretinal injection of 1% hyaluronic acid in male Sprague-Dawley rats. MSC-Exos were sub-retinally injected at the time of retinal separation to study their therapeutic function. The retinal expression levels of inflammatory cytokines TNF-α, IL-1β, and MCP-1 were detected by RT-PCR, the autophagy-related protein 5 (Atg5) and microtubule-associated protein 1 light chain 3 beta (LC3) were detected by Western blot, and apoptosis was examined using TUNEL assays at 3 days following RD. Retinal structure was observed at 7 days post-RD. Proteomic analysis was also performed to detect proteins carried by MSC-Exos using iTRAQ-based technology combined with one-dimensional nano LC-nano-ESI- MS/MS. We found that expression of TNF-α and IL-1β were significantly reduced, the LC3-II to LC3-I ratio was enhanced and cleavage of Atg5 was decreased after MSC-Exo treatment. Treatment with MSC-Exos also suppressed photoreceptor cell apoptosis and maintained normal retinal structure when compared to control groups. Proteomic analysis revealed that MSC-Exos contained proteins with anti-inflammatory, neuroprotective and anti-apoptotic effects. These results suggest that MSC-Exos have therapeutic effects on RD-induced retinal injury and can be used to reduce effects of retinal detachment on photoreceptor cell degeneration in patients.

    更新日期:2019-12-19
  • Effects of topical erythropoietin on healing experimentally-induced avascular scleral damage in a rabbit model
    Exp. Eye Res. (IF 2.998) Pub Date : 2019-12-19
    Sepehr Feizi; Mozhgan Rezaei Kanavi; Sahar Safari; Hadi Ebrahimi; Mohammad Ali Javadi

    The present study was designed to investigate the effect of topical erythropoietin on the healing process of induced necrotizing scleritis and to evaluate the ocular side effects of this treatment modality in a rabbit model. Necrotizing scleritis was induced in 8 New Zealand albino rabbits. The animals were then randomly divided into one of two groups: a treated group administered a topical erythropoietin-containing cellulose-based gel every 8 h or a control group treated with a cellulose-based gel without erythropoietin every 8 h. The sizes of the lesions measured at different time points were compared between the groups. After three months, the rabbits’ eyes were enucleated and histologically and immunohistochemically evaluated for angiogenesis and apoptosis. The lesions were completely vascularized in all eyes of the treated group and 50% of eyes of the control group. The mean interval from the induction of scleral necrosis to a complete improvement was 28 days in the treated group and 62.5 days in the control group (P = 0.04). Histological examination revealed that erythropoietin enhanced the improvement of necrotizing scleritis by stimulating angiogenesis and reducing apoptosis. Neovascularization of the cornea, iris, or retina was not observed in the treated group. We observed a significantly faster recovery to complete improvement of necrotizing scleritis in rabbit eyes treated with erythropoietin compared to those of the control group. Treated eyes had a higher rate of complete healing and had no ocular safety concerns. This therapeutic modality represents a promising treatment for scleral necrosis following various types of ocular surgery.

    更新日期:2019-12-19
  • Predictive genetics for AMD: Hype and hopes for genetics-based strategies for treatment and prevention
    Exp. Eye Res. (IF 2.998) Pub Date : 2019-12-17
    Michael B. Gorin; Michael J. DaSilva

    Age-related macular degeneration (AMD) is a complex disease with multiple genetic and environmental risk factors. In the age of molecular genetics, many investigators have established a link between genes and development or progression of the disease. This later evolved to determine whether phenotypic features of AMD have distinct genetic profiles. Molecular genetics have subsequently been introduced as factors in risk assessment models, increasing the predictive value of these tools. Models seek to predict either development or progression of disease, and different AMD-related genes aid our understanding of these respective features. Several investigators have attempted to link molecular genetics with treatment response, but results and their clinical significance vary. Ocular and systemic biomarkers may interact with established genes, promising future routes of ongoing clinical assessment. Our understanding of AMD molecular genetics is not yet sufficient to recommend routine testing, despite its utility in the research setting. Clinicians must be wary of misusing population-based risk models from genetic and biomarker associations, as they are not necessarily relevant for individual counseling. This review addresses the known uses of predictive genetics, and suggests future directions.

    更新日期:2019-12-18
  • Childhood glaucoma genes and phenotypes: Focus on FOXC1 mutations causing anterior segment dysgenesis and hearing loss
    Exp. Eye Res. (IF 2.998) Pub Date : 2019-12-11
    Angela C. Gauthier, Janey L. Wiggs

    Childhood glaucoma is an important cause of blindness world-wide. Eleven genes are currently known to cause inherited forms of glaucoma with onset before age 20. While all the early-onset glaucoma genes cause severe disease, considerable phenotypic variability is observed among mutations carriers. In particular, FOXC1 genetic variants are associated with a broad range of phenotypes including multiple forms of glaucoma and also systemic abnormalities, especially hearing loss. FOXC1 is a member of the forkhead family of transcription factors and is involved in neural crest development necessary for formation of anterior eye structures and also pharyngeal arches that form the middle ear bones. In this study we review the clinical phenotypes reported for known FOXC1 mutations and show that mutations in patients with reported ocular anterior segment abnormalities and hearing loss primarily disrupt the critically important forkhead domain. These results suggest that optimal care for patients affected with anterior segment dysgenesis should include screening for FOXC1 mutations and also testing for hearing loss.

    更新日期:2019-12-11
  • Oxidative stress markers dynamics in keratoconus patients’ tears before and after corneal collagen crosslinking procedure
    Exp. Eye Res. (IF 2.998) Pub Date : 2019-12-10
    Ioana-Miruna Balmus, Anisia Iuliana Alexa, Roxana-Elena Ciuntu, Ciprian Danielescu, Bogdan Stoica, Sabina Ioana Cojocaru, Alin Ciobica, Alina Cantemir

    Keratoconus (KC) is a controversial ophthalmological disease, often considered both multifactorial and multigenic with poor or not entirely understood etiopathogenesis. Corneal collagen crosslinking (CXL) procedure is the most common surgical therapy for KC which both slows corneal thinning and halts disease progression. While extensive studies provide consistent evidence on systemic oxidative stress in KC patients and animal models, little is known on the tear fluid oxidative stress markers such as antioxidant enzymes activity or lipid peroxidation markers. Also, little is known considering the oxidative status dynamics following CXL. In this way, we aimed to evaluate three oxidative stress markers in the tears of KC patients before and after CXL procedure. Total superoxide dismutase (SOD) and glutathione peroxidase (GPx) enzymatic activity and malondiladehyde (MDA) levels were assessed from the tears of 20 kC patients who received the recommendation for CXL procedure. Significantly decreased SOD activity (p = 0.0014) was observed in KC patients tears, as compared to age and sex-matched controls which could lead to significant lipid peroxidation boost (p < 0.001). Significantly higher GPx enzyme activity was observed in KC patients, as compared to control (p < 0.001), suggesting a compensatory response to intense lipid peroxidation. Following CXL, SOD activity significantly decreases and GPx activity extensively increases, as compared to baseline KC levels and controls (p < 0.001). This work provides additional evidence on oxidative stress status in the tears of KC considering general oxidative stress markers dynamics both before and after the CXL procedure. We also demonstrated that the CXL procedure could have further relevance in the management of this disorder.

    更新日期:2019-12-11
  • Correlation of pterygium severity with IQ-domain GTPase-activating protein 1 (IQGAP1) and mast cells
    Exp. Eye Res. (IF 2.998) Pub Date : 2019-12-10
    Tzu-Ting Lai, Yi-Chen Sun, Wei-Cherng Hsu, Yi-Ting Hsieh, Elizabeth P. Shen

    IQ-domain GTPase-activating protein 1 (IQGAP1) is a multidomain scaffold protein that is involved in cytoskeleton dynamics and tumor metastasis. Although the role of IQGAP1 in various cancers had been reported, the function of IQGAP1 in pterygium has not been studied. In this study, surgically excised pterygium and control conjunctival tissue from cataract patients were analysed by immunohistochemistry, confocal microscopy, and Western blot for IQGAP1 expression, mast cell counts, and microvascular count. Pterygium was clinically divided into mild and severe types according to Tan's classification and Kim's criteria based on translucency and vascularity of the tissue. Greater clinical severity of pterygium was associated with higher expression of IQGAP1 expression. Compared to normal conjunctival tissue, severe pterygium had significantly higher IQGAP1 expression (P = 0.005), which strongly correlated to the number of microvessels (P = 0.003) and mast cells (P = 0.01). Confocal microscopy revealed IQGAP1 colocalization with mast cell and CD31. IQGAP1 expression was higher in the pterygium body compared to the head. In conclusion, the level of IQGAP1 expression was found to be correlated to the clinical severity of pterygium. Mast cells were identified in pterygium and is suspected to be involved in promoting fibrovascular invasion.

    更新日期:2019-12-11
  • Dynamic in vivo quantification of rod photoreceptor degeneration using fluorescent reporter mouse models of retinitis pigmentosa
    Exp. Eye Res. (IF 2.998) Pub Date : 2019-12-06
    Harry O. Orlans, Alun R. Barnard, Robert E. MacLaren

    Imaging techniques have revolutionised the assessment of retinal disease in humans and animal models. Here we describe a novel technique for the in vivo visualisation of rod photoreceptors which permits semiquantitative assessment of outer retinal degeneration, and validate this approach in two mouse models of retinitis pigmentosa (RP). Transgenic mice carrying an Nrl-EGFP allele and homozygous for either knock-out of rhodopsin (Nrl-EGFP, Rho−/−) or heterozygous for knock-in of P23H mutant rhodopsin (Nrl-EGFP, RhoP23H/+) were used in this study. These novel strains have green fluorescent rods which undergo a progressive degeneration. Fundus imaging was performed at three-weekly intervals by near infrared reflectance (NIR) and blue light autofluorescence (BAF) confocal scanning laser ophthalmoscopy (cSLO). Mean grey values (mGV), which quantify fluorescence levels within such images, were compared for degenerate and age-matched non-degenerate (Nrl-EGFP, Rho+/+) controls. Mean grey value significantly decreased over time in the Rho−/− and RhoP23H/+ groups but was maintained in Rho+/+ mice (P < 0.001, two-way ANOVA). This corresponded to outer nuclear layer (ONL) thinning as observed by histology. The mGV of superior retina was significantly greater than that of inferior retina in RhoP23H/+ (P = 0.0024) but not in age-matched Rho+/+ (P = 0.45) or Rho−/- (P = 0.65) mice reflecting histological findings. Focal loss of rods could be visualised and mapped in vivo with this technique following a toxic insult, with thinning of the ONL being confirmed in hypofluorescent regions by spectral domain ocular coherence tomography (OCT). Fluorescence labelling of rods permits in vivo characterisation of models of RP and may provide new insights into patterns of degeneration, or rescue effect after treatment. mGV can be used in such cases as a semiquantitative metric of ONL degeneration, and can be used to identify regional variations in photoreceptor loss.

    更新日期:2019-12-06
  • An experimental study to test the efficacy of Mesenchymal Stem Cells in reducing corneal scarring in an ex-vivo organ culture model
    Exp. Eye Res. (IF 2.998) Pub Date : 2019-12-05
    Jeyanth Suresh Rose, Sharmili Lalgudi, Aarwin Joshua, Joshua Paul, Augustine Thambaiah, Syrpailyne Wankhar, Geeta Chacko, Thomas Kuriakose, Sanita Korah

    In this study, we evaluated the effect of placenta-derived Mesenchymal Stem Cells (MSCs) versus placebo in improving corneal transparency following experimental injury in an ex-vivo organ culture model of post-mortem human corneas. We also compared the influence of MSCs on the basic histopathology of the cornea and the immunohistochemistry markers of fibrotic corneal scarring. Mesenchymal Stem Cells extracted from the placenta were isolated and expanded in-vitro. Five pairs of post-mortem human corneas harvested for the corneal transplant of equal grade were included in the study. Corneas of the same pair were randomly assigned to either the case arm or the control arm. All corneas underwent a standardized superficial keratectomy, 4 mm in diameter. The case and control arm corneas received an intrastromal injection of MSCs and placebo respectively. The corneal button was maintained in an organ culture system for 28 days under the standard protocol. Laser light was passed through the corneas mounted on a self-styled modified artificial anterior chamber. Image analysis was used to quantify corneal transparency. Haematoxylin & Eosin staining and Immunohistochemistry was done for Alpha SMA (Smooth Muscle Actin). Laser scatter measurements were measured using Image Analysis (Image J Software). The difference in the mean of Full-Width Half Maximum (FWHM), Max intensity and Red pixel intensity between the cases and the controls was 101.5, 16.3 and 11.4 respectively which was found to be statistically significant (P < 0.05). Histopathology showed a disorganized Bowman's layer in the controls as compared to the cases. Alpha Smooth Muscle Actin at the injury site stained 3 + in all controls as compared to 1 + in the cases, showing a statistically significant difference (p = 0.005). Based on our findings, we consider that placenta-derived Mesenchymal Stem Cells can alter evolving corneal scarring into a more favourable outcome with better corneal transparency and lesser fibrotic corneal scarring.

    更新日期:2019-12-05
  • Local synthesis of hepcidin in the anterior segment of the eye: A novel observation with physiological and pathological implications
    Exp. Eye Res. (IF 2.998) Pub Date : 2019-12-04
    Ajay Ashok, Suman Chaudhary, Dallas McDonald, Alexander Kritikos, Disha Bhargava, Neena Singh

    Purpose The avascular cornea, trabecular meshwork (TM), and lens obtain iron, an essential biometal, from the aqueous humor (AH). The mechanism by which this exchange is regulated, however, is unclear. Recently we reported that non-pigmented ciliary epithelial cells express ferroportin (Fpn) (Ashok, 2018), an iron export protein modulated by hepcidin, the master regulator of iron homeostasis secreted mainly by the liver. Here, we explored whether ciliary epithelial and other cells in the anterior segment synthesize hepcidin, suggesting local regulation of iron exchange at this site. Methods Human and bovine eyes were dissected to isolate the ciliary body (CB), corneal endothelial (CE), TM, lens epithelial (LE), and outer epithelial cell layer of the iris. Total mRNA and protein lysates were processed to evaluate the synthesis and expression of hepcidin, the iron regulatory peptide hormone, ferroportin (Fpn), the only known iron export protein, ceruloplasmin (Cp), a ferroxidase necessary for iron export, transferrin receptor (TfR), a major iron uptake protein, and ferritin, a major iron storage protein. A combination of techniques including reverse transcription polymerase chain reaction (RT-PCR) of total mRNA, Western blotting of protein lysates, and immunofluorescence of fixed tissue sections were used to accomplish these goals. Results RT-PCR of isolated tissue samples revealed hepcidin-specific mRNA in the CB, TM, CE, and LE of the bovine eye. Western blotting of protein lysates from these tissues showed reactivity for hepcidin, Fpn, ferritin, and TfR. Western blotting and immunohistochemistry of similar tissues isolated from cadaveric human eyes showed expression of hepcidin, Fpn, and Cp in these samples. Notably, Fpn and Cp were expressed on the basolateral membrane of non-pigmented ciliary epithelial cells, facing the AH. Conclusions Synthesis and expression of hepcidin and Fpn in the ciliary epithelium suggests local regulation of iron transport from choroidal plexus in the ciliary body to the AH across the blood-aqueous barrier. Expression of hepcidin and Fpn in CE, TM, and LE cells indicates additional regulation of iron exchange between the AH and cornea, TM, and lens, suggesting autonomous regulation of iron homeostasis in the anterior segment. Physiological and pathological implications of these observations are discussed.

    更新日期:2019-12-04
  • Neuroprotective effects of DAAO are mediated via the ERK1/2 signaling pathway in a glaucomatous animal model
    Exp. Eye Res. (IF 2.998) Pub Date : 2019-12-04
    Xuejin Zhang, Rong Zhang, Jihong Wu, Junyi Chen

    Neuronal excitotoxicity caused by over activation of N -Methyl-D-Aspartate (NMDA) receptors is an important risk factor for the retinal ganglion cells (RGCs) death in glaucoma. D-serine played a role as a key co-agonist for NMDA receptor activity and neurotoxicity. Our previous studies have demonstrated that increased D-serine and serine racemase (SR) expression in the retina of the chronic intraocular hypertension (COH) model were detected. D-amino acid oxidase (DAAO) treatment significantly increased RGCs survival in the glaucomatous eyes. However, the molecular mechanism remains unclear. In the present study, we investigated the extracellular signal-regulated protein kinase1/2 (ERK1/2) signaling pathway involved in DAAO neuroprotective effects on RGC survival and explore the effect of inhibited ERK1/2 phosphorylation on RGC survival and Müller cell activation in a COH rat model. We found that ERK1/2 phosphorylation and p38 kinase (p38) phosphorylation increased in the COH model, while c-Jun N-terminal kinase (JNK) phosphorylation didn't change. DAAO treatment induced ERK-1/2 MAP kinase phosphorylation and its upstream regulator, p-MEK increased in the COH model. The increased p-ERK was mainly located in retinal Müller cells. In contrast, p-JNK and p-p38 protein expression was not significantly different under these conditions. Quantitative analysis of RGC survival by fluorescent labeling and TdT-mediated dUTP nick-end labeling (TUNEL) assays confirmed that p-ERK1/2 inhibition by PD98059 attenuates DAAO-mediated reductions in RGC apoptosis. Additionally, p-ERK1/2 inhibition induced elevated glial fibrillary acidic protein (GFAP) expression in Müller cells in the COH model. Together, these results suggest that the ERK1/2 signaling pathway is involved in DAAO's neuroprotective effects on RGC survival.

    更新日期:2019-12-04
  • Minocycline induces apoptosis of photoreceptor cells by regulating ER stress
    Exp. Eye Res. (IF 2.998) Pub Date : 2019-12-01
    Wenqin Xu, Mengzhang Tao, Zhicha Hu, Tianfang Chang, Yusheng Wang

    Our previous work reported that minocycline induced inhibition of microglial activation aggravated visual injury in an oxygen induced retinopathy animal model. We hypothesized that minocycline might have aggravated injury to the photoreceptor. Some patients who use minocycline to treat acne complain of visual impairment; however, no studies have addressed minocycline toxicity to photoreceptors. Here, we identified mechanistic effect of minocycline on photoreceptor apoptosis. The results of Cell Counting Kit-8 and Ki67 staining demonstrated that minocycline inhibited the proliferation of 661W cells, and flow cytometry and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) demonstrated that minocycline promoted cell apoptosis. Additionally, minocycline administration activated signaling associated with endoplasmic reticulum stress, the pancreatic ER kinase-like ER kinase (PERK)–eukaryotic translation initiation factor 2α (eIF2α)–CCAAT/enhancer-binding protein homologous protein (CHOP) cascade, which represented the key mechanism underlying the initiation of apoptosis. Moreover, we observed downregulated nuclear factor erythroid 2-related factor 2 (Nrf2) after administration of minocycline for 12 h (12 h) and Nrf2 transferred from nuclear to cytoplasm after 6 h, indicating that Nrf2 in nuclear may alleviated the pro-apoptotic effect of minocycline on photoreceptor cells. Upregulating Nrf2 inhibited apoptosis in minocycline treated 661W cells. These represent the first data demonstrating minocycline toxicity to photoreceptors via its pro-apoptotic effects through the regulation of ER stress pathways.

    更新日期:2019-12-02
  • Occupational effect of sugarcane biomass burning on the conjunctival mucin profile of harvest workers and residents of an adjacent town - A Brazilian panel study
    Exp. Eye Res. (IF 2.998) Pub Date : 2019-12-01
    Monique Matsuda, Alfésio L.F. Braga, Mônica Valeria Marquezini, Mário L.R. Monteiro, Paulo H.N. Saldiva, Ubiratan de Santos

    Pre-harvest burning of sugarcane fields produces large amounts of air pollutants which are known to cause health problems, including ocular surface abnormalities. In this study, we evaluated the effect of biomass burning on mucus quality and mucin gene expression (MUC1, MUC5AC, MUC16) in the conjunctiva of sugarcane workers (SWs) and residents of an adjacent town (RTs). Impression cytology samples of the inferior tarsal and bulbar conjunctiva of 78 SWs and 32 RTs were collected before (T1) and immediately after (T2) a 6-month harvest period. The neutral, acid and total mucus content of goblet cells was determined by PAS and AB staining. The levels of MUC5AC, MUC1 and MUC16 mRNA in the conjunctiva were measured by real-time PCR. Compared to RTs, SWs had higher levels of bulbar acid mucus and MUC16 mRNA and tarsal MUC5AC mRNA at T2 and lower levels of neutral mucus at T1 and T2. In the SW group, MUC1 mRNA levels were higher at T2 than at T1, but the levels of neutral and acid mucus were similar. In the RT group, acid mucus decreased and neutral mucus increased in the bulbar and tarsal conjunctiva at T2. In conclusion, our findings show that sugarcane harvesting is associated with abnormalities in mucus quality and content and changes in mucin mRNA levels on the ocular surface. This may help explain the ocular inflammatory signs and symptoms observed in subjects exposed to air pollutants and high temperatures from sugarcane biomass burning.

    更新日期:2019-12-02
  • Idelalisib inhibits vitreous-induced Akt activation and proliferation of retinal pigment epithelial cells from epiretinal membranes
    Exp. Eye Res. (IF 2.998) Pub Date : 2019-11-29
    Tianyi Xin, Haote Han, Wenyi Wu, Xionggao Huang, Jing Cui, Joanne Aiko Matsubara, Jingyuan Song, Fang Wang, Marcus Colyer, Hetian Lei

    Proliferative vitreoretinopathy (PVR) is a blinding fibrotic eye disease that develops in 8–10% of patients who undergo primary retinal detachment-reparative surgery and in 40–60% of patients with open-globe injury. At present, there is no pharmacological treatment for this devastating disease. Vitreal growth factors activate their respective receptors of cells in the vitreous, trigger their downstream signaling transduction (e.g. phosphoinositide 3 kinases (PI3Ks)/Akt), and drive cellular responses intrinsic to the pathogenesis of PVR. PI3Ks play a central role in experimental PVR. However, which isoform(s) are involved in PVR pathogenesis remain unknown. Herein, we show that p110δ, a catalytic subunit of receptor-regulated PI3K isoform δ, is highly expressed in epiretinal membranes from patients with PVR, and that idelalisib, a specific inhibitor of PI3Kδ, effectively inhibits vitreous-induced Akt activation, proliferation, migration and contraction of retinal pigment epithelial cells derived from an epiretinal membrane of a PVR patient. Small molecules of kinase inhibitors have shown great promise as a class of therapeutics for a variety of human diseases. The data herein suggest that idelalisib is a promising PVR prophylactic.

    更新日期:2019-11-29
  • Elevated pressure influences relative distribution of segmental regions of the trabecular meshwork
    Exp. Eye Res. (IF 2.998) Pub Date : 2019-11-29
    Janice A. Vranka, Julia A. Staverosky, VijayKrishna Raghunathan, Ted S. Acott

    Elevated intraocular pressure (IOP) is the primary risk factor for glaucoma and is the only treatable feature of the disease. There is a correlation between elevated pressure and homeostatic reductions in the aqueous humor outflow resistance via changes in the extracellular matrix of the trabecular meshwork. It is unclear how these extracellular matrix changes affect segmental patterns of aqueous humor outflow, nor do we understand their causal relationship. The goal of this study was to determine whether there are changes in the segmental outflow regions with perfusion in normal eyes, and whether these regions change during the IOP homeostatic response to elevated pressure. Using human anterior segment perfusion organ culture, we measured the amount of high flow (HF), intermediate flow (MF), and low flow (LF) regions before and after 7 days of perfusion at either physiologic pressure (”1x”) or at elevated pressure (”2x”). We found a small but significant decrease in the amount of HF regions over 7 days perfusion at 1x pressure, and a twofold increase in the amount of MF regions over 7 days perfusion at 2x pressure. Small positional differences, or shifts in the specific location of HF, MF, or LF, occurred on a per eye basis and were not found to be statistically significant across biological replicates. Differences in the amount of segmental flow regions of contralateral eyes flowed at 1x pressure for 7 days were small and not statistically significant. These results demonstrate that perfusion at physiologic pressure had little effect on the distribution and amount of HF, MF and LF regions. However, the overall amount of MF regions is significantly increased in response to perfusion at elevated pressure during IOP homeostatic resistance adjustment. The amount of both HF and LF regions was decreased accordingly suggesting a coordinated response in the TM to elevated pressure.

    更新日期:2019-11-29
  • Molecular genetics of congenital cataracts
    Exp. Eye Res. (IF 2.998) Pub Date : 2019-11-23
    JinYu Li, Xiangjun chen, YongBin Yan, Ke Yao

    Congenital cataracts, the most common cause of visual impairment and blindness in children worldwide, have diverse etiologies. According to statistics analysis, about one quarter of congenital cataracts caused by genetic defects. Various mutations of more than one hundred genes have been identified in hereditary cataracts so far. In this review, we briefly summarize recent developments about the genetics, molecular mechanisms, and treatment of congenital cataracts. The progressing studies of these pathogenic mutations and molecular genetics is making it possible for us to comprehend the underlying mechanisms of cataractogenesis and providing new insights into the preventive, diagnostic and therapeutic approaches of cataracts.

    更新日期:2019-11-26
  • Repression of microRNA-21 inhibits retinal vascular endothelial cell growth and angiogenesis via PTEN dependent-PI3K/Akt/VEGF signaling pathway in diabetic retinopathy
    Exp. Eye Res. (IF 2.998) Pub Date : 2019-11-21
    Jian-Min Lu, Zhen-Zhen Zhang, Xiang Ma, Shi-Feng Fang, Xiu-Hong Qin

    Diabetic retinopathy (DR) is a microvascular complication of diabetes and one of the most common causes of blindness in active stage. This study is performed to explore the effects of microRNA-21 (miR-21) on retinal vascular endothelial cell (RVEC) viability and angiogenesis in rats with DR via the phosphatidylinositiol 3-kinase/protein kinase B (PI3K/Akt)/vascular endothelial growth factor (VEGF) signaling pathway by binding to phosphatase and tensin homolog (PTEN). Sprague Dawley (SD) rats were used for establishment of DR models. Target relationship between miR-21 and PTEN was assessed by bioinformatics prediction in combination with dual-luciferase reporter gene assay. Identification of expression of miR-21, PTEN and PI3K/Akt/VEGF signaling pathway-related genes in the retinal tissues was then conducted. In order to assess the contributory role of miR-21 in DR, the RVECs were transfected with mimic or inhibitor of miR-21, or siRNA-PTEN, followed by the detection of expression of PTEN and PI3K/Akt/VEGF-related genes, as well as the measurement of cell viability, cell cycle and apoptosis. Increased expression of miR-21 and PI3K/Akt/VEGF related genes, along with a reduced expression of PTEN was observed in the retinal tissues of DR rats. PTEN was targeted and negatively regulated by miR-21, while the PI3K/Akt/VEGF signaling pathway was activated by miR-21. RVECs transfected with miR-21 inhibitor exhibited promoted viability and angiogenesis, and inhibited apoptosis. To conclude, our results indicated that miR-21 overexpression could potentially stimulate RVEC viability and angiogenesis in rats with DR through activation of the PI3K/Akt/VEGF signaling pathway via repressing PTEN expression, highlighting the potential of miR-21 as a target for DR treatment.

    更新日期:2019-11-22
  • The peroxisome proliferator-activated receptor-β/δ antagonist GSK0660 mitigates retinal cell inflammation and leukostasis
    Exp. Eye Res. (IF 2.998) Pub Date : 2019-11-20
    Megan E. Capozzi, Sara R. Savage, Gary W. McCollum, Sandra S. Hammer, Carla J. Ramos, Rong Yang, Colin A. Bretz, John S. Penn

    Diabetic retinopathy (DR) is triggered by retinal cell damage stimulated by the diabetic milieu, including increased levels of intraocular free fatty acids. Free fatty acids may serve as an initiator of inflammatory cytokine release from Müller cells, and the resulting cytokines are potent stimulators of retinal endothelial pathology, such as leukostasis, vascular permeability, and basement membrane thickening. Our previous studies have elucidated a role for peroxisome proliferator-activated receptor-β/δ (PPARβ/δ) in promoting several steps in the pathologic cascade in DR, including angiogenesis and expression of inflammatory mediators. Furthermore, PPARβ/δ is a known target of lipid signaling, suggesting a potential role for this transcription factor in fatty acid-induced retinal inflammation. Therefore, we hypothesized that PPARβ/δ stimulates both the induction of inflammatory mediators by Müller cells as well the paracrine induction of leukostasis in endothelial cells (EC) by Müller cell inflammatory products. To test this, we used the PPARβ/δ inhibitor, GSK0660, in primary human Müller cells (HMC), human retinal microvascular endothelial cells (HRMEC) and mouse retina. We found that palmitic acid (PA) activation of PPARβ/δ in HMC leads to the production of pro-angiogenic and/or inflammatory cytokines that may constitute DR-relevant upstream paracrine inflammatory signals to EC and other retinal cells. Downstream, EC transduce these signals and increase their synthesis and release of chemokines such as CCL8 and CXCL10 that regulate leukostasis and other cellular events related to vascular inflammation in DR. Our results indicate that PPARβ/δ inhibition mitigates these upstream (MC) as well as downstream (EC) inflammatory signaling events elicited by metabolic stimuli and inflammatory cytokines. Therefore, our data suggest that PPARβ/δ inhibition is a potential therapeutic strategy against early DR pathology.

    更新日期:2019-11-21
  • MicroRNA profiling reveals important functions of miR-125b and let-7a during human retinal pigment epithelial cell differentiation
    Exp. Eye Res. (IF 2.998) Pub Date : 2019-11-20
    Fatemeh Shahriari, Leila Satarian, Sharif Moradi, Ali Sharifi Zarchi, Stefan Günther, Arian Kamal, Mehdi Totonchi, Seyed-Javad Mowla, Thomas Braun, Hossein Baharvand

    Retinal pigment epithelial (RPE) cells are indispensable for eye organogenesis and vision. To realize the therapeutic potential of in vitro-generated RPE cells for cell-replacement therapy of RPE-related retinopathies, molecular mechanisms of RPE specification and maturation need to be investigated. So far, many attempts have been made to decipher the regulatory networks involved in the differentiation of human pluripotent stem cells into RPE cells. Here, we exploited a highly-efficient RPE differentiation protocol to determine global expression patterns of microRNAs (miRNAs) during human embryonic stem cell (hESC) differentiation into RPE using small RNA sequencing. Our results revealed a significant downregulation of pluripotency-associated miRNAs along with a significant upregulation of RPE-associated miRNAs in differentiating cells. Our functional analyses indicated that two RPE-enriched miRNAs (i.e. miR-125b and let-7a) could promote RPE fate at the expense of neural fate during RPE differentiation. Taken together, these mechanistic interrogations might shed light on a better understanding of RPE cell development and provide insights for the future application of these cells in regenerative medicine.

    更新日期:2019-11-21
  • Spectral domain - Optical coherence tomography (SD-OCT) as a monitoring tool for alterations in mouse lenses
    Exp. Eye Res. (IF 2.998) Pub Date : 2019-11-18
    Daniel Pawliczek, Claudia Dalke, Helmut Fuchs, Valerie Gailus-Durner, Martin Hrabě de Angelis, Jochen Graw, Oana Veronica Amarie

    The eye lens displayes a variety of phenotypes in the wake of genetical modifications or environmental influences. Therefore, a high-resolution in vivo imaging method for the lens is desirable. Optical coherence tomography (OCT) has become a powerful imaging tool in ophthalmology, especially for retinal imaging in small animal models such as mice. Here, we demonstrate an optimized approach specifically for anterior eye segment imaging with spectral domain OCT (SD-OCT) on several known murine lens cataract mutants. Scheimpflug and histological section images on the same eye were used in parallel to assess the observed pathologies. With SD-OCT images, we obtained detailed information about the different alterations from the anterior to the posterior pole of the lens. This capability makes OCT a valuable high-resolution imaging modality for the anterior eye segment in mouse.

    更新日期:2019-11-18
  • Elevated ocular pressure reduces voltage-gated sodium channel NaV1.2 protein expression in retinal ganglion cell axons
    Exp. Eye Res. (IF 2.998) Pub Date : 2019-11-14
    Michael L. Risner, Nolan R. McGrady, Silvia Pasini, Wendi S. Lambert, David J. Calkins

    Glaucoma is an age-related neurodegenerative disease that is commonly associated with sensitivity to intraocular pressure. The disease selectively targets retinal ganglion cells (RGCs) and constituent axons. RGC axons are rich in voltage-gated sodium channels, which are essential for action potential initiation and regeneration. Here, we identified voltage-dependent sodium channel, NaV1.2, in the retina, examined how this channel contributes to RGC light responses, and monitored NaV1.2 mRNA and protein expression in the retina during progression of modeled glaucoma. We found NaV1.2 is predominately localized in ganglion cell intraretinal axons with dispersed expression in the outer and inner plexiform layers. We showed Phrixotoxin-3, a potent NaV1.2 channel blocker, significantly decreased RGC electrical activity in a dose-dependent manner with an Ic50 of 40 nM. Finally, we found four weeks of raised intraocular pressure (30% above baseline) significantly increased NaV1.2 mRNA expression but reduced NaV1.2 protein level in the retina up to 57% (p < 0.001). Following prolonged intraocular pressure elevation, NaV1.2 protein expression particularly diminished at distal sections of ganglion cell intraretinal axons (p ≤ 0.01). Our results suggest NaV1.2 might be a therapeutic target during disease progression to maintain RGC excitability, preserving presynaptic connections through action potential backpropagation.

    更新日期:2019-11-14
  • Spatiotemporal distribution of chondroitin sulfate proteoglycans after optic nerve injury in rodents
    Exp. Eye Res. (IF 2.998) Pub Date : 2019-11-06
    Craig S. Pearson, Andrea G. Solano, Sharada M. Tilve, Caitlin P. Mencio, Keith R. Martin, Herbert M. Geller

    The accumulation of chondroitin sulfate proteoglycans (CSPGs) in the glial scar following acute damage to the central nervous system (CNS) limits the regeneration of injured axons. Given the rich diversity of CSPG core proteins and patterns of GAG sulfation, identifying the composition of these CSPGs is essential for understanding their roles in injury and repair. Differential expression of core proteins and sulfation patterns have been characterized in the brain and spinal cord of mice and rats, but a comprehensive study of these changes following optic nerve injury has not yet been performed. Here, we show evidence that the composition of CSPGs in the optic nerve and retina following optic nerve crush (ONC) in mice and rats exhibits an increase in aggrecan, brevican, phosphacan, neurocan and versican, similar to changes following spinal cord injury. We also observe an increase in inhibitory 4-sulfated (4S) GAG chains, which suggests that the persistence of CSPGs in the glial scar opposes the growth of CNS axons, thereby contributing to the failure of regeneration and recovery of function.

    更新日期:2019-11-07
  • Retinal differentiation in an altricial bird species, Taeniopygia guttata: An immunohistochemical study
    Exp. Eye Res. (IF 2.998) Pub Date : 2019-11-06
    Guadalupe Álvarez-Hernán, Ismael Hernández-Núñez, Eva María Rico-Leo, Alfonso Marzal, José Antonio de Mera-Rodríguez, Joaquín Rodríguez-León, Gervasio Martín-Partido, Javier Francisco-Morcillo

    The bird retina offers an excellent model to investigate the mechanisms that coordinate the morphogenesis, histogenesis, and differentiation of neuron and glial cells. Although these developmental features have been intensively studied in the chicken (Gallus gallus, Linnaeus 1758), a precocial bird species, little is known about retinogenesis in altricial birds. The purpose of this study was to examine the differentiation of retinal cells in the altricial zebra finch (Taeniopygia guttata, Vieillot, 1817) and compare the results with those from previous studies in G. gallus. By using immunohistochemical techniques, the first differentiated TUJ1-/Isl1-positive neuroblasts were detected in the vitreal surface of the neuroblastic layer at later incubation times in T. guttata than in G. gallus (108 h vs 55 h). The immunoreactivity of these early differentiation markers coincided temporo-spatially with the appearance of the first PCNA-negative nuclei. Furthermore, the first visinin-positive photoreceptors (132 h vs 120 h) and the first Prox-1-immunoreactive neuroblasts (embryonic day 7.25 (E7.25) vs E6.5) were also detected at later embryonic stages in the retina of T. guttata than in the retina of G. gallus. At E13, one day before hatching, abundant PCNA- and pHisH3-immunoreactivities were detected in the T. guttata retina, while proliferation was almost absent in the G. gallus retina at perinatal stages. Therefore, these results suggest that cell differentiation in the retina is delayed in the altricial bird compared to precocial birds. Furthermore, the T. guttata retina was not completely developed at hatching, and abundant mitotically active precursor cells of retinal neurons were found, suggesting that retinal neurogenesis was intense at perinatal stages.

    更新日期:2019-11-07
  • The seasonal changes of the heme oxygenase in the retina pig
    Exp. Eye Res. (IF 2.998) Pub Date : 2019-11-06
    S. Nowak, M. Romerowicz-Misielak, K. Kozioł, M. Koziorowski

    The eye is a very important organ in the human body which is affected by various external factors. One of these factors is the sunlight which can cause the visual impairment and as well as the increase in the oxidative stress. The heme oxygenase I (HO-1) plays a very important role in the fight against the oxidative stress. The HO enzyme catalyses the degradation of the heme to the ferrous iron, the biliverdin and the carbon monoxide (CO). The HO-2 is the isoform HO-1 and is mainly constitutively expressed. We have studied the changes in the HO-1 and the HO-2 in the retina on the level of the RNA and the protein in the summer and in the winter season (the biggest difference is in the length of the day light). The retina of the eye was obtained from the breeding pigs in concern (Sus scrofa f. domestica) posthumously. The expression of the HO-1 genes in the retina cells is higher in the winter and the amount of protein decreases. However, the HO enzyme concentration definitely increases in the summer, when the production of the free radicals (the oxidative stress) related to the exposition to the sunlight is greater. The obtained results suggest that various factors have the influence on the protein synthesis. One of the factors, can be the miRNA which blocks the synthesis of the HO. Another factors, influencing the HO are the biological clock, the sunlight and the UV radiation associated with it.

    更新日期:2019-11-06
  • Oxidative stress in corneal injuries of different origin: Utilization of 3D human corneal epithelial tissue model
    Exp. Eye Res. (IF 2.998) Pub Date : 2019-11-06
    Yulia Kaluzhny, Miriam W. Kinuthia, Allison M. Lapointe, Thoa Truong, Mitchell Klausner, Patrick Hayden

    The purpose of the current work was to utilize a three dimensional corneal epithelial tissue model to study dry eye disease and oxidative stress related corneal epithelial injuries for the advancement of ocular therapeutics. Air-liquid interface cultures of normal human corneal epithelial cells were used to produce 3D corneal epithelial tissues appropriate for physiologically relevant exposure to environmental factors. Oxidative stress was generated by exposing the tissues to non-toxic doses of ultraviolet radiation (UV), hydrogen peroxide, vesicating agent nitrogen mustard, or to desiccating conditions that stimulated morphological, cellular, and molecular changes relevant to dry eye disease. Corneal specific responses, including barrier function, tissue viability, reactive oxygen species (ROS) accumulation, lipid peroxidation, cytokine release, histology, and gene expression were evaluated. Three dimensional (3D) corneal epithelial tissue model structurally and functionally reproduced key features of molecular responses of various types of oxidative stress-induced ocular damage. The most pronounced effects for different treatments were: UV irradiation - intracellular ROS accumulation; hydrogen peroxide exposure - barrier impairment and IL-8 release; nitrogen mustard exposure - lipid peroxidation and IL-8 release; desiccating conditions - tissue thinning, decline in mucin expression, increased lipid peroxidation and IL-8 release. Utilizing a PCR gene array, we compared effects of corneal epithelial damage on the expression of 84 oxidative stress-responsive genes and found specific molecular responses for each type of the damage. Topical application of lubricant eye drops improved tissue morphology while decreasing lipid peroxidation and IL-8 release from tissues incubated at desiccating conditions. This model is anticipated to be a valuable tool to study molecular mechanisms of corneal epithelial damage and aid in the development of therapies against dry eye disease, oxidative stress- and vesicant-induced ocular injuries.

    更新日期:2019-11-06
  • Topography and pachymetry maps for mouse corneas using optical coherence tomography
    Exp. Eye Res. (IF 2.998) Pub Date : 2019-11-05
    Alice S. Liu, Dillon M. Brown, Rachel E. Conn, Ryan P. McNabb, Machelle T. Pardue, Anthony N. Kuo

    The majority of the eye's refractive power lies in the cornea, and pathological changes in its shape can affect vision. Small animal models offer an unparalleled degree of control over genetic and environmental factors that can help elucidate mechanisms of diseases affecting corneal shape. However, there is not currently a method to characterize the corneal shape of small animal eyes with topography or pachymetry maps, as is done clinically for humans. We bridge this gap by demonstrating methods using optical coherence tomography (OCT) to generate the first topography and pachymetry (thickness) maps of mouse corneas. Radii of curvature acquired using OCT were validated using calibration spheres as well as in vivo mouse corneas with a mouse keratometer. The resulting topography and pachymetry maps are analogous to those used diagnostically in clinic and potentially allow for characterization of genetically modified mice that replicate key features of human corneal disease.

    更新日期:2019-11-06
  • Chromatic pupillometry for the characterization of the pupillary light reflex in Octodon degus
    Exp. Eye Res. (IF 2.998) Pub Date : 2019-11-02
    Nicolas Palanca-Castan, Paloma A. Harcha, David Neira, Adrian G. Palacios

    The common degu (Octodon degus) is an emerging model in biomedical science research due to its longevity and propensity to develop human-like conditions. However, there is a lack of standardized techniques for this non-traditional laboratory animal. In an effort to characterize the model, we developed a chromatic pupillometry setup and analysis protocol to characterize the pupillary light reflex (PLR) in our animals. The PLR is a biomarker to detect early signs for central nervous system deterioration. Chromatic pupillometry is a non-invasive and anesthesia-free method that can evaluate different aspects of the PLR, including the response of intrinsically photosensitive retinal ganglion cells (ipRGCs), the disfunction of which has been linked to various disorders. We studied the PLR of 12 degus between 6 and 48 months of age to characterize responses to LEDs of 390, 450, 500, 525 and 605 nm, and used 5 with overall better responses to establish a benchmark for healthy PLR (PLR+) and deteriorated PLR (PLR-). Degu pupils contracted up to 65% of their horizontal resting size before reaching saturation. The highest sensitivity was found at 500 nm, with similar sensitivities at lower tested intensities for 390 nm, coinciding with the medium wavelength and short wavelength cones of the degu. We also tested the post-illumination pupillary response (PIPR), which is driven exclusively by ipRGCs. PIPR was largest in response to 450 nm light, with the pupil preserving 48% of its maximum constriction 9 s after the stimulus, in contrast with 24% preserved in response to 525 nm, response driven mainly by cones. PLR-animals showed maximum constriction between 40% and 50% smaller than PLR+, and their PIPR almost disappeared, pointing to a disfunction of the iPRGCs rather than the retinal photoreceptors. Our method thus allows us to non-invasively estimate the condition of experimental animals before attempting other procedures.

    更新日期:2019-11-04
  • Effects of intravitreal injection of human CD34+ bone marrow stem cells in a murine model of diabetic retinopathy
    Exp. Eye Res. (IF 2.998) Pub Date : 2019-11-01
    Amirfarbod Yazdanyar, Pengfei Zhang, Christian Dolf, Zeljka Smit-McBride, Whitney Cary, Jan A. Nolta, Robert J. Zawadzki, Nicholas Marsh-Armstrong, Susanna S. Park

    Human CD34 + stem cells are mobilized from bone marrow to sites of tissue ischemia and play an important role in tissue revascularization. This study used a murine model to test the hypothesis that intravitreal injection of human CD34 + stem cells harvested from bone marrow (BMSCs) can have protective effects in eyes with diabetic retinopathy. Streptozotocin-induced diabetic mice (C57BL/6J) were used as a model for diabetic retinopathy. Subcutaneous implantation of Alzet pump, loaded with Tacrolimus and Rapamycin, 5 days prior to intravitreal injection provided continuous systemic immunosuppression for the study duration to avoid rejection of human cells. Human CD34 + BMSCs were harvested from the mononuclear cell fraction of bone marrow from a healthy donor using magnetic beads. The CD34 + cells were labeled with enhanced green fluorescent protein (EGFP) using a lentiviral vector. The right eye of each mouse received an intravitreal injection of 50,000 EGFP-labeled CD34 + BMSCs or phosphate buffered saline (PBS). Simultaneous multimodal in vivo retinal imaging system consisting of fluorescent scanning laser ophthalmoscopy (enabling fluorescein angiography), optical coherence tomography (OCT) and OCT angiography was used to confirm the development of diabetic retinopathy and study the in vivo migration of the EGFP-labeled CD34 + BMSCs in the vitreous and retina following intravitreal injection. After imaging, the mice were euthanized, and the eyes were removed for immunohistochemistry. In addition, microarray analysis of the retina and retinal flat mount analysis of retinal vasculature were performed. The development of retinal microvascular changes consistent with diabetic retinopathy was visualized using fluorescein angiography and OCT angiography between 5 and 6 months after induction of diabetes in all diabetic mice. These retinal microvascular changes include areas of capillary nonperfusion and late leakage of fluorescein dye. Multimodal in vivo imaging and immunohistochemistry identified EGFP-labeled cells in the superficial retina and along retinal vasculature at 1 and 4 weeks following intravitreal cell injection. Microarray analysis showed changes in expression of 162 murine retinal genes following intravitreal CD34 + BMSC injection when compared to PBS-injected control. The major molecular pathways affected by intravitreal CD34 + BMSC injection in the murine retina included pathways implicated in the pathogenesis of diabetic retinopathy including Toll-like receptor, MAP kinase, oxidative stress, cellular development, assembly and organization pathways. At 4 weeks following intravitreal injection, retinal flat mount analysis showed preservation of the retinal vasculature in eyes injected with CD34 + BMSCs when compared to PBS-injected control. The study findings support the hypothesis that intravitreal injection of human CD34 + BMSCs results in retinal homing and integration of these human cells with preservation of the retinal vasculature in murine eyes with diabetic retinopathy.

    更新日期:2019-11-01
  • Lysine malonylation and propionylation are prevalent in human lens proteins
    Exp. Eye Res. (IF 2.998) Pub Date : 2019-10-31
    Rooban B. Nahomi, Sandip K. Nandi, Stefan Rakete, Cole Michel, Kristofer S. Fritz, Ram H. Nagaraj

    Acylated lysine residues represent major chemical modifications in proteins. We investigated the malonylation and propionylation of lysine residues (MalK, PropK) in the proteins of aging human lenses. Western blot results showed that the two modifications are present in human lens proteins. Liquid chromatography-mass spectrometry (LC-MS/MS) results showed 4–18 and 4–32 pmol/mg protein of MalK and PropK, respectively, in human lens proteins with no apparent changes related to aging. Mass spectrometry results revealed that MalK- and PropK-modified lysine residues are present in all major crystallins, other cytosolic proteins, and membrane and cytoskeletal proteins of the lens. Several mitochondrial and cytosolic proteins in cultured human lens epithelial cells showed MalK and PropK modifications. Sirtuin 3 (SIRT3) and sirtuin 5 (SIRT5) were present in human lens epithelial and fiber cells. Moreover, lens epithelial cell lysate deacylated propionylated and malonylated lysozyme. The absence of SIRT3 and SIRT5 led to higher PropK and MalK levels in mouse lenses. Together, these data suggest that MalK and PropK are widespread modifications in lens and SIRT3 and SIRT5 could regulate their levels in lens epithelial cells.

    更新日期:2019-11-01
  • The circadian clock regulates RPE-mediated lactate transport via SLC16A1 (MCT1)
    Exp. Eye Res. (IF 2.998) Pub Date : 2019-10-31
    Nemanja Milićević, Jacoline B. ten Brink, Anneloor L.M.A. ten Asbroek, Arthur A. Bergen, Marie-Paule Felder-Schmittbuhl

    Multiple retinal cells were shown to harbor a circadian oscillator, including retinal pigment epithelial cells (RPE). However, little is known about the functions that are regulated by the RPE clock. The aim of this study was to investigate whether the circadian clock in the RPE regulates the transport of glucose and its glycolytic metabolic by-product - lactate. To that end, we first characterized the mRNA expression profile of glucose and monocarboxylate transporters in ARPE-19 cells. We found that SLC2A1 and SLC16A1 were, respectively, the most abundantly expressed glucose and lactate (monocarboxylate) transporters. We further observed that the protein products of SLC2A1 (encoding GLUT1) and SLC16A1 (encoding MCT1) localize on the apical membrane of ARPE-19 monolayers. In a subsequent time-course experiment, we found that SLC2A1 and SLC16A1 mRNA oscillated in ARPE-19 monolayers, but not in dispersed cells, suggesting that monolayer cellular organization is necessary for rhythmic regulation of these transporters. In these monolayers, we found that MCT1 proteins varied over time in contrast to GLUT1 proteins which did not vary over time. Spectrophotometric measurements of supernatants sampled from ARPE-19 monolayer cultures revealed that glucose concentrations did not significantly differ between apical (Api) supernatants and basolateral (BL) ones. In addition, we did not find rhythms in Api or BL glucose concentrations. Conversely, we found higher lactate concentrations in Api supernatants than BL ones. Further, we found that Api lactate concentrations were rhythmic. Pearson's r revealed that the concentration gradients (Api - BL) of glucose and lactate correlated with the gene expression of respective SLC2A1 and SLC16A1 transporters. Incubation with photoreceptor outer segments (P.OS) affected the mRNA expression of SLC16A1 and SLC2A1 in ARPE-19 monolayers in a time-dependent manner, thus suggesting that the retina might modulate the RPE clock-controlled expression of transporters via interactions with POS. In conclusion, this work provides evidence that the transport of lactate is regulated by the circadian clock in the RPE.

    更新日期:2019-11-01
  • Local targeting of the CD200-CD200R axis does not promote corneal graft survival.
    Exp. Eye Res. (IF 2.998) Pub Date : 2014-12-03
    Susan M Nicholls,David A Copland,Andrea Vitova,Lucia Kuffova,John V Forrester,Andrew D Dick

    Corneal graft rejection is primarily a CD4(+) T cell-mediated mechanism in which macrophages may play an important inflammatory role. CD200Fc fusion protein is an artificial agonist of CD200R1, a receptor expressed predominantly on myeloid cells, engagement of which is known to down-regulate macrophage function. We therefore wished to test whether CD200Fc could be used as a therapeutic agent to prolong corneal graft survival. The distribution of CD200R1 and CD200, its natural ligand, was examined by immunohistology in the cornea and conjunctiva of unoperated rats and rats that had received corneal allografts. Mouse CD200Fc was injected subconjunctivally into transplanted rats on six occasions from the day of surgery until day 10 after transplantation. Control groups received injections of mouse IgG or diluent PBS. Allo-transplants were also performed in CD200(-/-) and control mice. The ability of CD200Fc to bind rat macrophages in vitro and to inhibit nitric oxide production was tested. Mean day of rejection in CD200Fc, IgG and PBS-treated rats was 12, 10 and 9 respectively (p=0.24). Mean day of rejection in CD200(-/-) and wild type mice was 17.5 and 16.0 respectively (p=0.07). Mouse CD200Fc bound to rat macrophages in a dose-dependent manner, but was unable to inhibit nitric oxide production. The fact that treatment with CD200Fc did not inhibit graft rejection and the failure of CD200 deficiency to affect graft survival suggests that local targeting of the CD200-CD200R axis to suppress macrophage activation is not a useful therapeutic strategy in corneal graft rejection.

    更新日期:2019-11-01
  • High affinity nuclear and nongenomic estradiol binding sites in the human and mouse lens.
    Exp. Eye Res. (IF 2.998) Pub Date : 2013-04-20
    M Rachel Kirker,Katie M Gallagher,Paula A Witt-Enderby,Vicki L Davis

    Estrogen is reported to be protective against cataracts in women and animal models. Immunodetection methods have identified the classic estrogen receptors (ER), ERα and ERβ, in human lens epithelial cells and their RNAs have been detected in the rat and human lens. To verify that estrogen binding occurs in the lens, sensitive [(125)I]-17β-estradiol binding analyses were performed on subcellular lens fractions from women (ages 39-78 years). The presence of high affinity estradiol binding sites in the nuclear, cytoplasmic, and membrane fractions indicate the lens is able to respond to estrogens, even up to age 78, although fewer binding sites were detected in the postmenopausal women. Additionally, due to the importance of mouse models in estrogen action and lens research, lenses from intact female mice were also analyzed. Both the C57BL/6 and FVB/N mouse strains also possessed high affinity binding sites in all three lens fractions. Furthermore, transcripts for ERα, ERβ, and G protein-coupled estrogen receptor (GPER; previously called GPR30) that bind estradiol with high affinity were expressed in the human and mouse lenses. These data provide the first evidence of GPER expression in the lens. Its role, functions, and subcellular location are currently unknown, but a G-shift assay in the membrane fractions of human and mouse lenses did not show evidence that estradiol induced classic G protein-coupled receptor activation. All three receptor transcripts were also detected in the lens capsule region isolated from female C57BL/6 mice, which is mainly comprised of epithelial cells. In contrast, only ERα and GPER were expressed in the cortex/nuclear region, which is primarily composed of differentiating and organelle-free fiber cells. No significant differences in specific estradiol binding and receptor RNA expression were observed in the lenses between male and female C57BL/6 mice. These findings indicate that the lens is an estrogen target tissue in both sexes. The identification of GPER, in addition to ERα and ERβ, in the lens also adds to the complexity of possible estrogen responses in the lens. Accordingly, the protective effects of estrogen in women and animals may be mediated by all three estrogen receptors in the lens. In addition, the similarities in binding and receptor RNA expression in the lenses of both species suggest that mice can be used to model estrogen action in the human lens.

    更新日期:2019-11-01
  • Involvement of the CD200 receptor complex in microglia activation in experimental glaucoma.
    Exp. Eye Res. (IF 2.998) Pub Date : 2011-02-08
    Sarah Taylor,Claudia J Calder,Julie Albon,Jonathan T Erichsen,Micheal E Boulton,James E Morgan

    The interaction of the myeloid restricted molecule CD200R with its widely expressed ligand CD200 is involved in the down-regulation of microglia activation. In the present study, we examined the involvement of CD200R in microglia activation in experimental ocular hypertension to determine the role of microglia activation in retinal ganglion cell (RGC) death, the key pathological event in glaucoma. Experimental glaucoma was induced in adult Brown Norway rats by sclerosis of the episcleral veins with the injection of hypertonic saline. Immunohistochemical methods were used to determine the involvement of microglia using GFAP, CD45, OX42 and OX41 and the involvement of CD200 and CD200R in the optic nerve head. Our data demonstrate the increased presence of microglia within the optic nerve head during ocular hypertension, identified by positive staining with OX42 and OX41. The peak of microglia correlates with peak in RGC death at days 20-27 (T3) post OHT induction. In addition, CD200 and CD200R positive cells were increased in ocular hypertensive eyes. Increased expression of CD200 was detected in the early phase (days 1-7; T1) of OHT and decreased over time, whilst the expression of CD200R was detected in the middle phase (days 20-27; T3) of OHT, correlating with the increase in microglia markers. Changes in the expression of CD200R/CD200 occur early in experimental glaucoma and precede the peak in microglia infiltration and RGC death, suggesting that CD200R-positive microglia play an important role in the initiation of RGC death during OHT, indicating a potential area for therapeutic intervention in treating glaucoma.

    更新日期:2019-11-01
  • Significant upregulation of small heat shock protein αA-crystallin in retinal detachment.
    Exp. Eye Res. (IF 2.998) Pub Date : 2019-09-25
    Sumaya Hamadmad,Mohd Hussain Shah,Rania Kusibati,Bongsu Kim,Brandon Erickson,Tyler Heisler-Taylor,Sanjoy K Bhattacharya,Mohamed H Abdel-Rahman,,Colleen M Cebulla

    更新日期:2019-11-01
  • 更新日期:2019-11-01
  • Corrigendum to "Nerve fibre layer degeneration and retinal ganglion cell loss long term after optic nerve crush or transection in adult mice" [Exp Eye Res. (2018) 170:40-50. doi: 10.1016/j.exer.2018.02.010. Epub 2018 Feb 13].
    Exp. Eye Res. (IF 2.998) Pub Date : null
    M C Sánchez-Migallón,F J Valiente-Soriano,M Salinas-Navarro,F M Nadal-Nicolás,M Jiménez-López,M Vidal-Sanz,M Agudo-Barriuso

    更新日期:2019-11-01
  • Inhibition of high glucose-induced VEGF release in retinal ganglion cells by RNA interference targeting G protein-coupled receptor 91.
    Exp. Eye Res. (IF 2.998) Pub Date : 2013-02-06
    Jianyan Hu,Qiang Wu,Tingting Li,Yongdong Chen,Shuai Wang

    Recent research using a rat oxygen-induced retinopathy model has demonstrated that the G protein-coupled receptor 91 (GPR91) of retinal ganglion neurons is the principal respondent to succinate and consequently induces the release of angiogenic factor vascular endothelial growth factor (VEGF). The aim of this study was to determine whether GPR91 modulate the release of VEGF from retinal ganglion cells in a high-glucose model in vitro and to dissect the role of GPR91 in the pathogenesis of diabetic retinopathy. We constructed a lentiviral small hairpin RNA (shRNA) expression vector targeting GPR91 (LV.shGPR91) and infected the retinal ganglion cell line RGC-5 to obtain stably transduction system. The knockdown effect of GPR91 was detected by Western blotting. After incubation with succinate and various concentrations of glucose, the expression of VEGF in RGC-5 cells was evaluated by real-time PCR and Western blotting, and the release of VEGF protein was measured using an ELISA assay. Conditioned media were also collected, and the effects of proliferation and migration of RF/6A cells, a vascular endothelial cell line, were evaluated by CCK-8 and Transwell assays. The phosphorylation levels of extracellular signal-regulated kinase 1/2 (ERK1/2), p38 mitogen-activated protein kinase (p38 MAPK), and c-Jun N-terminal kinase (JNK) in RGC-5 cells after exposure to high glucose were evaluated by Western blotting. Following a single exposure of RGC-5 cells to the encoding lentivirus, more than 80% of infected cells expressed GFP at 72 h, and the level of GPR91 protein was significantly downregulated. GPR91 shRNA inhibited the cell survival rates of RGC-5 cells incubated with high glucose (F = 21.36, P = 0.002). The mRNA and protein expression of VEGF in LV.shGPR91 RGC-5 cells decreased markedly compared to that of LV.shScrambled or untransduced control cells incubated with different concentrations of glucose or succinate (P < 0.01). The VEGF protein level in medium from RGC-5 cells treated with high glucose (F = 57.43, P = 0.000) or succinate (F = 241.91, P = 0.000) was also downregulated when transduced with GPR91 shRNA. The siRNA-mediated knockdown of GPR91 was also found to inhibit the proliferation of RF/6A cells in high glucose-stimulated (t = 8.21, P = 0.001) or succinate-stimulated (t = 3.36, P = 0.028) conditioned media. However, the siRNA-mediated knockdown of GPR91 suppressed the migration of RF/6A cells incubated with moderate levels of glucose (t = 2.97, P = 0.018). The exposure of RGC-5 cells to high glucose activated ERK1/2 and JNK MAPK signaling blocking by GPR91 shRNA (P < 0.01). These results indicate that GPR91 modulates the high glucose-induced VEGF release of RGC-5 cells, possibly by inhibiting ERK1/2 and JNK MAPK signaling.

    更新日期:2019-11-01
  • Controlled microenvironments to evaluate chemotactic properties of cultured Müller glia.
    Exp. Eye Res. (IF 2.998) Pub Date : 2018-05-14
    Juan Pena,Nihan Dulger,Tanya Singh,Jing Zhou,Robert Majeska,Stephen Redenti,Maribel Vazquez

    Emerging therapies have begun to evaluate the abilities of Müller glial cells (MGCs) to protect and/or regenerate neurons following retina injury. The migration of donor cells is central to many reparative strategies, where cells must achieve appropriate positioning to facilitate localized repair. Although chemical cues have been implicated in the MGC migratory responses of numerous retinopathies, MGC-based therapies have yet to explore the extent to which external biochemical stimuli can direct MGC behavior. The current study uses a microfluidics-based assay to evaluate the migration of cultured rMC-1 cells (as model MGC) in response to quantitatively-controlled microenvironments of signaling factors implicated in retinal regeneration: basic Fibroblast Growth factor (bFGF or FGF2); Fibroblast Growth factor 8 (FGF8); Vascular Endothelial Growth Factor (VEGF); and Epidermal Growth Factor (EGF). Findings indicate that rMC-1 cells exhibited minimal motility in response to FGF2, FGF8 and VEGF, but highly-directional migration in response to EGF. Further, the responses were blocked by inhibitors of EGF-R and of the MAPK signaling pathway. Significantly, microfluidics data demonstrate that changes in the EGF gradient (i.e. change in EGF concentration over distance) resulted in the directional chemotactic migration of the cells. By contrast, small increases in EGF concentration, alone, resulted in non-directional cell motility, or chemokinesis. This microfluidics-enhanced approach, incorporating the ability both to modulate and asses the responses of motile donor cells to a range of potential chemotactic stimuli, can be applied to potential donor cell populations obtained directly from human specimens, and readily expanded to incorporate drug-eluting biomaterials and combinations of desired ligands.

    更新日期:2019-11-01
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