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Abstract Objectives To explore the mechanism underlying Müller Cell Pyroptosis (MCP) and its role in the development of Proliferative Vitreoretinopathy (PVR). Method The expression of pyroptosis-related factors, namely, cysteinyl aspartate-specific proteinase (caspase-1), interleukin (IL)-1β, IL-18, and Gasdermin D (GSDMD), was detected by quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) and western blotting at the mRNA and protein levels, respectively, in retinal tissues. Müller and spontaneously Arising Retinal Pigment Epithelia (ARPE)-19 primary cells with GSDMD overexpression or knockdown were cultivated. Western blotting was used to detect the levels of the following pyroptosis-related factors in retinal tissues: caspase-1, IL-1β, IL-18, and GSDMD. Through Cell Adhesion (CA) experiments, the changes in ARPE-19 CA in each group were observed. The migration and invasion of ARPE-19 cells were measured using the Transwell assay. The proliferation of ARPE-19 cells was measured with a Cell Counting Kit 8 (CCK-8) assay. Finally, the expression of the cytokines IL-1β and IL-18 in the ARPE-19 cell culture medium was detected using the Enzyme-Linked Immunosorbent Assay (ELISA). Results Compared with the surrounding normal tissues, the expression of caspase-1, IL-1β, IL-18, and GSDMD at the protein and mRNA levels in the retinal proliferative membrane samples of the patients decreased significantly (p < 0.05). MCP significantly enhanced ARPE-19 CA, migration and invasion, proliferation, and cytokine expression (p < 0.05). Conclusions MCP can promote the development of PVR lesions.
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AIM: To investigate the effects of antitumor drug paclitaxel(PTX)on the proliferation, apoptosis, cell cycle, cell morphology, and related protein expression of Müller cells, and to evaluate its potential toxicity to the retina.METHODS:Müller cells were cultured in vitro and divided into two groups: control group(normal medium)and PTX group. Retinal Müller cells were treated with different concentrations of PTX(0.005, 0.05, 0.5 and 5mg/L)for varying durations(12, 24, 36, 48 and 72h). The CCK8 method was used to assess the effects of different concentrations of PTX and treatment duration on the proliferation Müller cells. Flow cytometry was employed to investigate the impact of different concentrations of PTX on Müller cells apoptosis and cell cycle arrest. Immunofluorescence was used to observe morphological changes in Müller cells. The effects of PTX on the expression of apoptosis-related proteins and aquaporins were analyzed by Western blot and qRT-PCR.RESULTS: PTX exhibits the ability to inhibit the proliferation of Müller cells when cultured in vitro. The efficacy of this inhibition was found to be dependent on both the concentration of the drug and the duration of the stimulation. Higher concentrations of the drug and longer stimulation times resulted in a weaker ability of the cells to proliferate. Additionally, PTX also induces apoptosis in Müller cells, with increased drug concentrations and longer stimulation times leading to higher apoptosis rates. Flow cytometry analysis demonstrates that PTX arrests Müller cells in the G2-M phase of the cell cycle. Moreover, there is a distinct change in cell morphology, with a shift from the typical appearance characterized by clear and slender fibrous structures to a rounder morphology, accompanied by a significant decrease in cell numbers. Further, our findings reveal that there is a transient increase in the expression of cytoinflammatory factors following drug treatment compared to the control group. However, discontinuation of drug stimulation can alleviate this heightened expression. In treated cells, the expression of the CA XIV protein is upregulated compared to the control group, while the expression of vascular endothelial growth factor(VEGF)is downregulated(P&#x003C;0.05). Additionally, the levels of inflammatory factors in the PTX group are significantly higher than those in the control group(P&#x003C;0.05), suggesting that PTX has the potential to disrupt the retinal barrier function.CONCLUSION: PTX affects the proliferation and apoptosis of Müller cells, with the effects dependent on stimulation duration and drug concentration. In addition, PTX blocks the Müller cell cycle at the G2-M phase and alters cell morphology, leading to a transient upregulation of inflammatory factors and affecting the integrity of the retinal barrier. These findings indicate the potential toxicity of the antitumor drug PTX to the retina.
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Purpose: To evaluate the absence of external limiting membrane (ELM) and ellipsoid zone (indistinct retinal outer layers, I?ROL) in the walls of idiopathic full?thickness macular holes (FTMHs) circumferentially on optical coherence tomography (OCT) and its correlation with surgical outcome. Methods: In this retrospective observational study, OCT images of patients undergoing vitrectomy for FTMHs with at least 3?months of postoperative follow?up were analyzed for preoperative circumferential extent of I?ROL. Derived macular hole indices such as hole form factor (HFF), macular hole index (MHI), tractional hole index (THI), and hole diameter ratio (HDR) were also calculated. The circumferential extent of I?ROL was correlated with derived hole indices as well as anatomical closure, foveal architecture, and restoration of ELM following surgery. Results: All nine eyes (eight patients) with FTMH (mean size: 610.11 ± 122.95 microns) in the study showed I?ROL in ?1 quadrant. The mean HFF, MHI, THI, and HDR values were 0.72 ± 0.09, 0.35 ± 0.05, 0.71 ± 0.24, and 0.53 ± 0.14, respectively. All eyes achieved type?1 hole closure with improvement in best?corrected visual acuity to 0.58 ± 0.32 LogMAR from 0.81 ± 0.26 LogMAR. Regular foveal architecture was achieved in six eyes. Out of these, five eyes had I?ROL in ?2 quadrants, and one eye had I?ROL in <2 quadrants (P = 0.0476). Restoration of ELM was seen in aforementioned six eyes (complete = 5, partial = 1). Out of the five eyes with complete ELM restoration, four had a circumferential extent of I?ROL in ?2 quadrants (P = 0.0476). Complete restoration of ELM was associated with the complete restoration of the ellipsoid zone in three eyes. Conclusion: Preoperative circumferential extent of I?ROL in FTMH walls can be a potential predictive OCT marker for the type of closure, postoperative foveal architecture, and ELM restoration.
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Objective To expore the impact of teniary butyl hydroquinone (tBHQ) on Müller cells in SD rats retina under high glucose condition,and discuss the mechanism of tBHQ.Methods The Müller cells of SD rats were cultured in vitro and the experiment was divided into normal control group,high glucose group and tBHQ intervention group.Western blot and quantitative real time PCR were used to determine the expression of nuclear factor erythroid 2-related factor 2 (Nrf2),hemeoxygenase-1 (HO-1),hypoxia inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) protein and mRNA in each group.Results Müller cells cultured in vitro were flat with irregularly sharp.The nucleus was oval,while the cytoplasm was abundant.Adjacent cells were interwoven to a network.Western blot assay showed the overall expression of Nrf2,HO-1,HIF-1α,and VEGF in Müller cells of normal control group,high glucose group,and tBHQ intervention group were significantly different (F =73.831,148.618,152.269,91.217,all at P＜0.001);Among them,the relative expressions of Nrf2,HO-1,HIF-1α and VEGF proteins in the high glucose group were 0.17±0.02,0.47±0.02,0.67±0.07,and 0.6±0.05,which were increased in comparion with 0.06±0.01,0.19±0.03,0.06±0.00 and 0.07±0.02 in the normal control group,with statistically significant differences (t =4.114,9.275,16.479,13.353,all at P ＜ 0.001);the relative expressions of Nrf2 and HO-1 proteins in the tBHQ intervention group were 0.40±0.06 and 0.72±0.05,which were increased by comparion with those in the higher glucose group,with statistically significant differences (t =7.847,7.947,both at P＜0.001);the relative expressions of HIF-1α and VEGF proteins in the tBHQ intervention group were 0.18±0.04,0.26±0.07,which were decreased in comparion with those in the higher glucose group,but were increased in comparion with those in normal control group,with statistically significant differences (t =13.215,8.444,both at P＜ 0.001).Quantitative real time PCR showed that the relative mRNA expressions of Nrf2,HO-1,HIF-1α,and VEGF in Müller cells of normal control group,high glucose group,and tBHQ intervention group were significantly different (F =340.317,1 582.911,488.852,185.699,all at P＜0.001);the relative mRNA expressions of Nrf2,HO-1,HIF-1α,and VEGF proteins in the high-glucose group were 1.53 ± 0.06,1.50 ± 0.04,2.56 ± 0.09,and 3.04 ± 0.19,which were increased in comparion with 1.07±0.07,0.95±0.05,0.99±0.02,and 1.09±0.08 in the normal control group,with statistically significant differences (t =7.292,15.014,30.550,18.573,all at P ＜ 0.001);The relative mRNA expressions of Nrf2 and HO-1 proteins in the tBHQ intervention group were 2.68±0.09 and 2.94±0.05,which were increased in comparion with those in the high-glucose group,with statistically significant differences (t =18.046,39.458,both at P＜0.001);The relative mRNA expression of HIF-1α and VEGF protein in the tBHQ intervention group were 1.48±0.05 and 1.6±0.08,which were decreased by comparion with those in the higher glucose group were increased in comparion with those in normal control group,with statistically significant differences (t =21.036,13.739,both at P＜0.001).Conclusions tBHQ protects Müller cells from damage in high glucose condition by activating anti-oxidative stress signaling pathway of Nrf2/ARE.
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Objective@#To expore the impact of teniary butyl hydroquinone (tBHQ) on Müller cells in SD rats retina under high glucose condition, and discuss the mechanism of tBHQ.@*Methods@#The Müller cells of SD rats were cultured in vitro and the experiment was divided into normal control group, high glucose group and tBHQ intervention group.Western blot and quantitative real time PCR were used to determine the expression of nuclear factor erythroid 2-related factor 2 (Nrf2), hemeoxygenase-1 (HO-1), hypoxia inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) protein and mRNA in each group.@*Results@#Müller cells cultured in vitro were flat with irregularly sharp.The nucleus was oval, while the cytoplasm was abundant.Adjacent cells were interwoven to a network.Western blot assay showed the overall expression of Nrf2, HO-1, HIF-1α, and VEGF in Müller cells of normal control group, high glucose group, and tBHQ intervention group were significantly different (F=73.831, 148.618, 152.269, 91.217, all at P<0.001); Among them, the relative expressions of Nrf2, HO-1, HIF-1α and VEGF proteins in the high glucose group were 0.17±0.02, 0.47±0.02, 0.67±0.07, and 0.6±0.05, which were increased in comparion with 0.06±0.01, 0.19±0.03, 0.06±0.00 and 0.07±0.02 in the normal control group, with statistically significant differences (t=4.114, 9.275, 16.479, 13.353, all at P<0.001); the relative expressions of Nrf2 and HO-1 proteins in the tBHQ intervention group were 0.40±0.06 and 0.72±0.05, which were increased by comparion with those in the higher glucose group, with statistically significant differences (t=7.847, 7.947, both at P<0.001); the relative expressions of HIF-1α and VEGF proteins in the tBHQ intervention group were 0.18±0.04, 0.26±0.07, which were decreased in comparion with those in the higher glucose group, but were increased in comparion with those in normal control group, with statistically significant differences (t=13.215, 8.444, both at P<0.001). Quantitative real time PCR showed that the relative mRNA expressions of Nrf2, HO-1, HIF-1α, and VEGF in Müller cells of normal control group, high glucose group, and tBHQ intervention group were significantly different (F=340.317, 1 582.911, 488.852, 185.699, all at P<0.001); the relative mRNA expressions of Nrf2, HO-1, HIF-1α, and VEGF proteins in the high-glucose group were 1.53±0.06, 1.50±0.04, 2.56±0.09, and 3.04±0.19, which were increased in comparion with 1.07±0.07, 0.95±0.05, 0.99±0.02, and 1.09±0.08 in the normal control group, with statistically significant differences (t=7.292, 15.014, 30.550, 18.573, all at P<0.001); The relative mRNA expressions of Nrf2 and HO-1 proteins in the tBHQ intervention group were 2.68±0.09 and 2.94±0.05, which were increased in comparion with those in the high-glucose group, with statistically significant differences (t=18.046, 39.458, both at P<0.001); The relative mRNA expression of HIF-1α and VEGF protein in the tBHQ intervention group were 1.48±0.05 and 1.6±0.08, which were decreased by comparion with those in the higher glucose group were increased in comparion with those in normal control group, with statistically significant differences (t=21.036, 13.739, both at P<0.001).@*Conclusions@#tBHQ protects Müller cells from damage in high glucose condition by activating anti-oxidative stress signaling pathway of Nrf2/ARE.
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@#Hyaluronic acid(HA)is one of the main components of the extracellular matrix(ECM), and it is participated in many cells physiology and pathological processes, such as tissue reconstruction, expansion of cell gap, inflammation and tumorigenesis and so on. CD44 is a cell surface receptor for HA and widely distributed cell surface glycoprotein, which paticipate in specific adhesion of cell to cell and cell to matrix. CD44 is the most important hyaluronic acid receptor on the cell surface. Besides, CD44 is the main site of binding to HA. In this paper, we will elaborate from three aspects: the binding of HA and CD44 and its molecular basis, the expression and significance of HA/CD44 in glial cells(including Müller cells)and the expression and significance of HA/CD44 in the optic nerve, which makes readers have an understanding of the role of HA and CD44.
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@#AIM: To investigate the effect of aflibercept on the expression of signal transducers and activators of transcription 3(STAT3)in rat retinal Müller cells. <p>METHODS: The rat retinal Müller cells treated with different concentrations of Aflibercept 100 μL(diluted concentrations of 400, 200, 100 pg/mL, respectively). MTT assay were used to detect cell proliferation, flow cytometry. Apoptosis were detected by the instrument, cell invasion were detected by transwell chamber method, and protein(AKT, STAT3, GAPDH)expression were detected by Western-blot method.<p>RESULTS: The proliferation activity of Müller cells were decreased with the increased of aflibercept concentration, and compared the difference were statistically significant(<i>P</i><0.05). After treatment for 48h, the apoptotic rate of Müller cells were gradually increased with the increased of aflibercept concentration, and the invasion and penetration index of Müller cells gradually were decreased, and compared the difference were statistically significant(<i>P</i><0.05). After 48 h of transfection, the relative expression of AKT protein in Müller cells were not change significantly with the increased of Aflibercept concentration(<i>P</i>>0.05), and the relative expression of STAT3 protein decreased gradually, and compared the difference were statistically significant(<i>P</i><0.05).<p>CONCLUSION: Aflibercept can inhibit the expression of STAT3 protein in rat retinal Müller cells, thereby inhibit cell proliferation and invasion and promote apoptosis.
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Neural stem cell is a kind of stem cells that can differentiate into neural and glial cells. While Müller cells, the main endogenous neural stem cell in retina,have the features to reentry into the cell cycle and differentiate into neural cells after retinal damage. Although it is highly effective for retinal Müller cell differentiation spontaneously after retinal injury in vertebrates, this feature is rigorous restricted in mammals. Recently, some transcription factors,such as Ascl1, Sox2, Lin28, Atoh7, are sufficient to drive quiescent Müller cells back in proliferation to generate new retinal neurons. Moreover, combining Ascl1 expression with a histone deacetylase inhibitor can bypass the limitation and increase the generation of new neurons in the adult retina. These regenerated neurons integrate the existing neuronal network and are able to respond to light, indicating that they can likely be used to restore vision. While these results are extremely promising, the regenerative response is still limited, likely because the proliferative capacity of mammalian Müller cells is low compared to their zebrafish counterparts. It is indeed necessary to identify new factors increasing the efficiency of the regenerative response.
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Objective To observe the effect ofpolypyramidine tract binding protein-associated splicing factor (PSF) towards advanced glycation end products (AGEs) induced the apoptosis of Müller cells in vitro.Methods Experimental study.Müller cells were cultured and divided into groups according to the project design,plasmid enhanced green fluorescent protein-PSF were transfected into the cells to achieve the overexpression of PSF Müller cells in vitro,then cells were exposed to AGEs and the Morphological changes were observed by HE staining and Hoechst 33258 staining while the survival rate of cells were detected by MTT assay.The effects of PSF on AGEs-induced Müller apoptosis was measured by Cell Death Detection ELISA kit.Meanwhile,2',7'-diehlorofluorescin diaeetate staining was performed to monitor the protective effects of PSF on AGEs-induced Müller cells ROS.Results The morphology of cells in normal group was full and the cytoplasm staining was uniform.In N+AGEs group and Vec+AGEs group,cell volume decreased,cytoplasm was dense and concentrated,and eosinophilic staining was enhanced.The cell morphology of PSF+AGEs group was still full,with uniform cytoplasm staining and uniform nucleus staining.The viability of N+AGEs group,Vec+AGEs group and PSF+AGEs group were 0.42±0.11,0.35±0.12 and 0.68±0.12.The apoptosis values were 1.08 ± 0.16,0.96± 0.20 and 0.44± 0.08.The intracellular ROS levels were 28 833.67± 3 550.06,28 356.67±4 854.81,186 163.00±382.54.Compared with N+AGEs group and Vec+AGEs group,the cell viability of PSF+AGEs group was significantly improved (F=20.65,P=0.000),ce11 apoptosis value (F=43.43,P=0.000) and intracellular ROS level (F=1 8.86,P=0.000).Conclusion PSF overexpression play a protective role in AGEs-induced apoptosis by inhibiting the production of ROS in Müiller cells.
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@#Müller cells are the most important glial cells in the vertebrate retina. They extend from the inner limiting membrane to the outer membrane through the entire retina, participate in the blood-retinal barrier, and actively participate in retinal development and promote the maintenance of retinal homeostasis through many intracellular mechanisms. Müller cells play an important role in the development of diabetic retinopathy. The pathophysiological changes in diabetic retinopathy remain to be further studied. This article reviews the pathophysiological changes of Müller cells in diabetic retinopathy and the recent research progress.
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@#Idiopathic macular hole(IMH)refers to the defect of retinal nerve epidermis in macular area with unknown pathogenesis. As present, pars plana vitrectomy combined with internal limiting membrane peeling has become a normal operation for the treatment of IMH, which enables most IMH patients to achieve anatomic healing, however, it is still unclear about the healing mechanism. The role of the glial cell activation in the pathological process of nervous system injury and disease has paid more and more attention. Nearly all the injuries and diseases of nervous system(including retina)are accompanied by the activation of glial cells; and Müller cells are the main type of glial cells in the human retina. In terms of anatomy and function, they are widely related to the cell bodies and processes of neurons in all layers of the retina, and play a supporting, nutritional and information transmission role in neurons. Numerous studies have shown that the activation and proliferation of Müller cells play a leading role in closed MH. It is reviewed in this paper that the role and its related mechanisms of Müller cells in the formation and healing of IMH.
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PURPOSE: To determine the origin of epiretinal proliferation (EP), a condition that is occasionally observed in lamellar hole and macular hole cases, and EP outcomes after vitrectomy. METHODS: This is a retrospective observational case review of 17 eyes with EP that underwent vitrectomy, EP dissection, and internal limiting membrane peeling between January 2013 and December 2016. Surgical specimens of EP tissue were successfully obtained from 5 cases and they were analyzed after immunohistochemical staining. Postoperative outcomes, including best-corrected visual acuity (BCVA) and macular configuration in spectral domain-optical coherence tomography, were reviewed. RESULTS: Mean BCVA improved from 0.54 ± 0.36 logarithms of the minimum angle of resolution preoperatively to 0.32 ± 0.38 logarithms of the minimum angle of resolution postoperatively (p = 0.002). BCVA improved in 13 eyes and remained unchanged in four eyes. No cases experienced vision decline after surgery. All 17 patients' lamellar hole or macular hole were successfully closed. Despite hole closure, ellipsoid zone defects were not corrected in 11 of the 17 patients. In immunohistochemical analyses, anti-glial fibrillary acidic protein and pan-keratin (AE1/AE3) were positive, but synaptophysin, anti-α-smooth muscle actin, and anti-CD68 were negative. CONCLUSIONS: The epiretinal proliferative membrane seems to originate from Müller cells, not from the vitreous. It is unclear whether retinal pigment epithelia also contribute to EP formation. Gentle handling and preservation of the epiretinal proliferative tissue is crucial for successful surgical outcomes.
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Humans , Actins , Membranes , Prognosis , Retinal Perforations , Retinaldehyde , Retrospective Studies , Synaptophysin , Visual Acuity , VitrectomyABSTRACT
@#AIM: To investigate the effects of Aflibercept on the K+ channel of retinal Müller cell membrane cultured <i>in vitro</i>.<p>METHODS: Human Müller cells were divided into 3 groups(control group, high glucose group and experimental group). The control group were cultured in conventional DMEM medium; the high glucose group were cultured in high glucose DMEM medium; the experimental group were cultured with high glucose DMEM medium and 100μmol/L Aflibercept, and the K+ concentration of the cells were detected by MQAE, and the cell survival were detected by MTT assay, the flow cytometry were used to detect apoptosis rates, Western blot analysis were used to detect the Müller cell caspase-3 protein levels.<p>RESULTS: The Müller cells were positive for glutamine synthetase(GS)after 48h of culture, and the purification degree were above 90%. The relative concentrations of K+ in the control group, high glucose group and experimental group were(2.14±0.44)%,(23.11±4.39)%,(5.20±0.92)%, and cell viability were(100.00±0.00)%, respectively(73.24±4.13)%,(85.22±5.33)%, the apoptosis rates were(5.03±1.91)%,(26.73±3.14)%,(16.63±2.73)%, and compared the differences between two groups were statistically significant(<i>P</i><0.05). Compared with the control group, the level of caspase-3 protein in the high glucose group Müller cells were increased significantly(<i>P</i><0.05); compared with the high glucose group Müller cells, the caspase-3 protein level in the experimental group Müller cells were decreased significantly(<i>P</i><0.05).<p>CONCLUSION: Aflibercept can inhibit the K+ channel of retinal Müller cells <i>in vitro</i>, inhibit the apoptosis of Müller cells induced by high glucose, decrease the expression of caspase-3 protein, and promote cell proliferation.
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Objective To observe the protective effect of dl-3-n-Butylphthalide (NBP) on apoptosis of retinal Müller cells induced by hydrogen peroxide (H2O2).Methods Human retinal Müller cells cultured in vitro were divided into normal control group,model group (H2O2 group) and experimental group (H2O2+NBP group).The cells in the H2O2 group and H2O2+NBP group were cultured with 200 μ mol/L H2O2 for 2 h.Then the culture solution of the H2O2 group replace with complete medium and the H2O2+NBP group replace with complete medium containing 1 tmol/L NBP.The normal control group was a conventional cultured cells.Müller cells were identified by immunofluorescence staining.Hematoxylin-eosin (HE) staining was used to observe the apoptosis morphological changes.MTT assay was used to detect the activity of of retinal Müller cells after after 24 h and 48 h of NBP intervention.Hoechst33258 staining was used to observe the apoptosis.LIVE/DEAD (R)cell activity/cytotoxicity kit was used to detect cell viability.Dichlorofluorescein diacetate (DCFH-DA) + endoplasmic reticulum (ER) red fluorescent probe (ER-Tracker Red) double staining was used to observe the expression level of reactive oxygen species (ROS) in ER of cells.One-way ANOVA combined with Dunnett statistical method were used for data analysis.Results HE staining showed that the number of cells in H2O2+NBP group was higher than that in H2O2 group.MTT assay showed that after 24 h and 48 h of NBP intervention,the differences in cell viability between the normal control group and the H2O2 group,the H2O2 group and the H2O2+NBP group were statistically significant (t=28.96,3.658,47.58,20.33;P＜0.001,0.022).The results of Hoechst33258 showed that the nuclear nucleus of a few cells in the H2O2+NBP group was crescent-shaped and the nuclear fragmentation was reduced,and the blue fluorescence of the remaining cells was uniform.The LIVE/DEAD ~ cell activity/cytotoxicity kit showed that the number of dead cells with red fluorescence in the H2O2 group increased significantly,and the number of viable cells with green fluorescence decreased significantly.In the H2O2+NBP group,the number of viable cells with green fluorescence increased,and the number of dead cells with red fluorescence decreased.The double staining results of DCFH-DA+ER-Tracker Red showed that the green fluorescence intensity of H2O2 group was significantly enhanced;the green fluorescence intensity of H2O2+NBP group was lower than that of H2O2 group.Conclusion NBP alleviates H2O2-induced apoptosis of human retinal Müller cells by inhibiting ROS production.
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Retinal degeneration is an incurable and irreversible blinding disease caused by the retinal neural cell death. An effective and safe strategy to substitute these injured cells is necessary for retinal recovery. Neural stem cells (NSCs) can differentiate into neural and glial cells. While Müller cells,the main endogenous NSCs in retina, have the features to reentry into the cell cycle and differentiate into neural cells after retinal damage. Although it is highly effective for retinal Müller cell differentiation spontaneously after retinal injury in vertebrates,this feature is rigorously restricted in mammals. Recently,some transcription factors,such as Ascl1,sox2,lin28 and atoh7,can drive quiescent Müller cells back into proliferation to generate new retinal neurons. Moreover,combining Ascl1 expression with a histone deacetylase inhibitor can bypass the limitation and increase the generation of new neurons in adult retina. These regenerated neurons integrate the existing neuronal network and are able to respond to light,indicating that they can likely be used to restore vision. In addition,transplantation of exogenous stem cells can induce Müller cell reprogramming. While these results are extremely promising, the regenerative response is still limited, likely because the proliferative capacity of mammalian Müller cells is low in comparison to their zebrafish counterparts. There may be some kinds of unclear reverse mechanism that suppresses the reprogramming of Müller cells. It is indeed necessary to identify new factors increasing the efficiency of regenerative response.
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Background Retinal Müller cells are important gliocytcs and the source of retinal stem cells.Researching the biological behavior of Müller cells is of important significance to the study on retinal physiopathological process and stem cell therapy of retinal diseases.To establish a stable culture method of Müller cells is a solid basis of relative basic research.Objective This study was to establish a simple and stable method of isolation and culture of human retinal Müller cells and provide sufficient and high-quality Müller cell source.Methods Human retinal Müller cells were isolated from healthy human donor eyes.The mixture solution of hyaluronidase (100 U) and 0.25％ trypsin were used to digest chopped retinal tissue.The DMEM/F12 medium with 20％ fetal bovine serum (FBS) was added to stop the digestion process.RPMI1640 medium with 20％ FBS was used to culture the cell for 72 hours and then replaced the half medium.The cells were passaged by the RPMI1640 medium with 20％ FBS.The morphology of the cells were examied under the optical microscope,and the expressions of glial fibrillary acidic protein (GFAP),a marker of gliocytes,and glutamine synthetase (GS),a special marker of retinal Müller cells,were detected by immunochemistry and immunofluorescence technology.Results Human retinal Müller cells were successfully isolated by enzyme mixture solution of hyaluronidase (100 U) and O.25％ trypsin.The cells were adherent to walls 24 hours after primary culture and completely merged 9-10 days after culture.The cells showed oval in shape with abundant cytoplasm,and a part of cells presented with cone-shaped bulge bilaterally and ectasia in the posterior containing large nuclei.After cells passage,the cells were enlarged and grew toward polygonal shape.The positive expression of GFAP was observed in more than 95％ cells and strongly positive expression of GS was observed in more than 90％ cells by immunohistochemstry and immunofluorescent staining.Conclusions Human retinal Müller cells can be successfully isolated by hyaluronidase combined with trypsin digestion.Abundent and pure human retinal Müller cells can be obtained by successively using RPMI1640 medium with 20％ FBS and 10％ FBS.
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Objective: To investigate spectrum-effect relationship of the extract from Salvia miltiorrhiza on HIF-1α of retinal Müller cells in the advanced glycationend products (AGEs) or hypoxia conditions, regarding tanshinone IIA as reference to explore the relationship between pharmacological effects and chemical substances, and to reveal the material basis of S. miltiorrhiza treating diabetic retinopathy (DR). Methods: Fingerprints of different S. miltiorrhiza extract were established by HPLC and characteristic peak's area was recorded. After that, the expression quantity of HIF-1α of retinal Müller cells were tested in the AGEs or hypoxia conditions under the two different conductions: S. miltiorrhiza extract or only tanshinone IIA. Gray relational analysis and partial least squares regression analysis (PLSR) were combined to build spectrum-effect relationship. Results: Seventeen characteristic peaks were marked out. Compared with the model set, 10 batches of extracts and tanshinone IIA could lower the expression quantity of HIF-1α. The statistical results showed that the peaks 1, 5, 6, 9, 10, 11, 12, 13, 14, 15, and 16 would inhibit HIF-1α expression, which made great contribution. Conclusion: The 15,16-dihydrotanshinone I, cryptotanshinone, tanshinone I, tanshinone IIA, and other six components might be the main effective components in the ethanol extract from S. miltiorrhiza.
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Objective To investigate the protective effect of brain derived neurotrophic factor (BDNF) on Müller cells in the retina of diabetic rats.Methods A total of 54 healthy male SD rats were recruited and randomly divided into control group,diabetic group and BDNF group.Then a diabetic model was established by intraperitoneal injection of streptozotocin in rats of diabetic and BDNF groups.Preparation of BDNF injection was performed using PBS balanced salt solution containing 0.1 g · L 1 BSA.The BDNF group was given BDNF injection,while the control and diabetic group were injected with equal dose of PBS balanced salt solution 4 weeks after successful modeling.And after 8 weeks,the expression of L-glutamate/L-aspartate transporter (GLAST),glutamine synthetase (GS) and synaptophysin (SYN) were detected by immunofluorescence technique and Western blot,and the content of glutamic acid in retina was determined by glutamic acid determination kit.Results Compared with the control group,the expression of GLAST,GS and SYN were significantly decreased in diabetic group,and the content of glutamic acid was increased significantly (all P ＜ 0.01).Compared with the diabetic group,the expression of GLAST,GS and SYN were increased in BDNF group,and the glutamate level was decreased significantly (all P ＜0.01).Conclusion In the early stage of diabetic retinopathy,administration of exogenous BDNF can unregulated the expression of GLAST,SYN and GS and improve the function of Müller cells to protect RGC against damage,suggesting that BDNF has neuroprotective effects on Müller cells in retina of rats with diabetic retinopathy.
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PURPOSE: To evaluate the effects of valproic acid (VPA), a histone deacetylase inhibitor (HDACI), on the expression of hypoxia-inducible factor-1 alpha (HIF-1α) and vascular endothelial growth factor (VEGF) in human retinal Müller cells under hypoxic conditions. METHODS: Chemical hypoxia was induced in human retinal Müller cells (MIO-M1) by treatment with increasing concentrations of cobalt(II) chloride (CoCl₂). Müller cells were also treated with a set concentration of CoCl₂, along with various concentrations of VPA. The expression of HIF-1α and VEGF in the treated Müller cells was determined by enzyme-linked immunosorbent assay. RESULTS: Exposure of human retinal Müller cells to increasing concentrations of CoCl₂ produced a dose-dependent increase in HIF-1α expression. The addition of increasing concentrations of VPA lead to a dose-dependent decrease in expression of HIF-1α and VEGF in Müller cells exposed to a set concentration of CoCl₂. CONCLUSIONS: HDACI VPA downregulated the expressions of HIF-1α and VEGF in human retinal Müller cells under hypoxic conditions. Using HDACI to target HIF-1α expression in Müller cells could be a new therapeutic strategy for the treatment of retinal vascular diseases.
Subject(s)
Humans , Hypoxia , Enzyme-Linked Immunosorbent Assay , Ependymoglial Cells , Histone Deacetylase Inhibitors , Histone Deacetylases , Histones , Retinaldehyde , Valproic Acid , Vascular Diseases , Vascular Endothelial Growth Factor AABSTRACT
Glaucoma is a degenerative disease of the retina and the optic nerve.The apoptosis of retinal ganglion cells is the pathological basis of glaucoma,but its mechanism has not been fully elucidated.Currently,a large number of studies in vivo and in vitro have demonstrated that retinal glial cells (including Müller cells,astrocytes and microglial cells) are closely related to the development of glaucoma.Retinal glial cells not only release active factors but also express various neurotransmitter receptors,ion channels,surface markers and effector molecules.Therefore,they are actively involved in neuronal information transmission in addition to the traditional view of the role of nutritional support for neurons.In glaucoma,retinal glial cells are activated to undergo a great number of changes in physiology,biochemistry and morphological features.These activated cells initiate different signaling cascades that may serve as a protective role.Meanwhile,they may release toxic factors to start or aggravate the damaging effects on retinal neurons.Usually,both effects occur in glaucoma in parallel,but the underlying mechanisms and signaling pathways are still not clear.More research is needed to understand that how the retinal glial cells are able to rebuild the neuronal function after injury or to promote the neurodamage.With all these unresolved issues in mind,the progress in the study of the mechanism of Müller glial cells,microglial cells and astrocytes participating in the glaucomatous disease were reviewed.