Müller cells are activated in response to retinal outer nuclear layer degeneration in rats subjected to simulated weightlessness conditions

A microgravity environment has been shown to cause ocular damage and affect visual acuity,but the underlying mechanisms remain unclear.Therefore,we established an animal model of weightlessness via tail suspension to examine the pathological changes and molecular mechanisms of retinal damage under microgravity.After 4 weeks of tail suspension,there were no notable alterations in retinal function and morphology,while after 8 weeks of tail suspension,significant reductions in retinal function were observed,and the outer nuclear layer was thinner,with abundant apoptotic cells.To investigate the mechanism underlying the degenerative changes that occurred in the outer nuclear layer of the retina,proteomics was used to analyze differentially expressed proteins in rat retinas after 8 weeks of tail suspension.The results showed that the expression levels of fibroblast growth factor 2(also known as basic fibroblast growth factor)and glial fibrillary acidic protein,which are closely related to Müller cell activation,were significantly upregulated.In addition,Müller cell regeneration and Müller cell gliosis were observed after 4 and 8 weeks,respectively,of simulated weightlessness.These findings indicate that Müller cells play an important regulatory role in retinal outer nuclear layer degeneration during weightlessness.

Overexpressing NeuroD1 reprograms Müller cells into various types of retinal neurons

The onset of retinal degenerative disease is often associated with neuronal loss. Therefore, how to regenerate new neurons to restore vision is an important issue. NeuroD1 is a neural transcription factor with the ability to reprogram brain astrocytes into neurons in vivo. Here, we demonstrate that in adult mice, NeuroD1 can reprogram Müller cells, the principal glial cell type in the retina, to become retinal neurons. Most strikingly, ectopic expression of NeuroD1 using two different viral vectors converted Müller cells into different cell types. Specifically, AAV7 m8 GFAP681::GFP-ND1 converted Müller cells into inner retinal neurons, including amacrine cells and ganglion cells. In contrast, AAV9 GFAP104::ND1-GFP converted Müller cells into outer retinal neurons such as photoreceptors and horizontal cells, with higher conversion efficiency. Furthermore, we demonstrate that Müller cell conversion induced by AAV9 GFAP104::ND1-GFP displayed clear dose-and time-dependence. These results indicate that Müller cells in adult mice are highly plastic and can be reprogrammed into various subtypes of retinal neurons.

miR-26a-5p调控PTEN/PI3K/Akt信号通路对高糖诱导视网膜Müller细胞活化及凋亡的影响

目的检测微小RNA-26a-5p(microRNA-26a-5p,miR-26a-5p)对PTEN/PI3K/Akt信号通路及高糖刺激的视网膜Müller细胞凋亡的影响,探讨糖尿病视网膜神经损伤的潜在机制。方法采用不同浓度的葡萄糖刺激视网膜Müller细胞构建糖尿病视网膜神经损伤模型,采用CCK-8及流式细胞仪观察细胞增殖及凋亡情况,细胞转染过表达miR-26a-5p模拟物,Real-time PCR检测miR-26a-5p、PTEN、PI3K、Akt的表达情况,ELISA测定细胞上清液中IL-1β及IL-6的水平,采用Graphpad 8.0软件处理数据。结果高糖刺激视网膜Müller细胞生长活跃,与对照组相比,50 mmol/L高糖刺激Müller细胞活力在12 h、24 h时逐渐增加,48 h时活力减弱,细胞凋亡增加,组间差异有统计学意义(P<0.05)。高糖刺激后视网膜Müller细胞中miR-26a-5p水平下降,PTEN的表达显著升高,PI3K及Akt水平下降,IL-1β及IL-6的水平明显升高,过表达miR-26a-5p后,PTEN水平降低,PI3K/Akt及炎性因子降低,细胞凋亡减少(P<0.01)。结论miR-26a-5p通过调控PTEN/PI3K/Akt的表达,影响炎症因子释放,减轻高糖诱导的视网膜Müller细胞凋亡。

Investigating Müller glia reprogramming in mice: a retrospective of the last decade, and a look to the future

Müller glia,as prominent glial cells within the retina,plays a significant role in maintaining retinal homeostasis in both healthy and diseased states.In lower vertebrates like zebrafish,these cells assume responsibility for spontaneous retinal regeneration,wherein endogenous Müller glia undergo proliferation,transform into Müller glia-derived progenitor cells,and subsequently regenerate the entire retina with restored functionality.Conversely,Müller glia in the mouse and human retina exhibit limited neural reprogramming.Müller glia reprogramming is thus a promising strategy for treating neurodegenerative ocular disorders.Müller glia reprogramming in mice has been accomplished with remarkable success,through various technologies.Advancements in molecular,genetic,epigenetic,morphological,and physiological evaluations have made it easier to document and investigate the Müller glia programming process in mice.Nevertheless,there remain issues that hinder improving reprogramming efficiency and maturity.Thus,understanding the reprogramming mechanism is crucial toward exploring factors that will improve Müller glia reprogramming efficiency,and for developing novel Müller glia reprogramming strategies.This review describes recent progress in relatively successful Müller glia reprogramming strategies.It also provides a basis for developing new Müller glia reprogramming strategies in mice,including epigenetic remodeling,metabolic modulation,immune regulation,chemical small-molecules regulation,extracellular matrix remodeling,and cell-cell fusion,to achieve Müller glia reprogramming in mice.

视网膜再生中调控Müller细胞重编程的表观遗传修饰和微环境因素研究进展

[背景]Müller细胞(Müller glia,MG)重编程是视网膜再生领域的研究热点.与斑马鱼(Danio rerio)不同,哺乳动物视网膜损伤后MG丧失再生视网膜能力,发生反应性胶质化产生胶质瘢痕,导致哺乳动物MG重编程再生视网膜能力丧失的分子机制仍存在许多不清楚之处.[进展]随着干细胞理论和研究方法的不断进步,越来越多研究利用单细胞测序等技术从转录、表观遗传修饰、细胞外微环境等多层次深入挖掘视网膜再生中MG重编程的调控机制,以期实现哺乳动物的视网膜再生.本文总结了不同物种中已发现的影响MG重编程能力差异的关键分子,着重关注表观遗传修饰和微环境因素.[展望]视网膜再生中MG重编程是受细胞内外多种因素协同调控的复杂过程.对斑马鱼和哺乳动物视网膜再生中MG重编程影响因素进行详细的对比分析,将有助于突破哺乳动物视网膜再生瓶颈,为开发有临床转化潜力的疗法提供新的途径.

Müller细胞在常见眼底疾病中的研究进展

神经胶质细胞在维持中枢神经系统稳态方面发挥关键作用,Müller细胞作为视网膜中主要神经胶质细胞,在维持视网膜结构和功能正常方面有重要作用。Müller细胞的损伤会导致视网膜功能障碍。近年来许多研究表明视网膜功能是否正常很大程度上取决于Müller细胞。本文综述了近五年来Müller细胞在常见的眼底疾病中的作用及相关机制,以期深入了解Müller细胞在常见的眼底疾病中的影响与价值,为眼底疾病发病机制的研究、筛查、诊断、及预防提供新思路。

Impaired pericyte-Müller glia interaction via PDGFRβsuppression aggravates photoreceptor loss in a rodent model of light-induced retinal injury

AIM:To investigate the involvement of pericyte-Müller glia interaction in retinal damage repair and assess the influence of suppressing the platelet-derived growth factor receptorβ(PDGFRβ)signaling pathway in retinal pericytes on photoreceptor loss and Müller glial response.METHODS:Sprague-Dawley rats were exposed to intense light to induce retinal injury.Neutralizing antibody against PDGFRβwere deployed to block the signaling pathway in retinal pericytes through intravitreal injection.Retinal histology and Müller glial reaction were assessed following light injury.In vitro,normal and PDGFRβ-blocked retinal pericytes were cocultured with Müller cell line(rMC-1)to examine morphological and protein expression changes upon supplementation with light-injured supernatants of homogenized retinas(SHRs).RESULTS:PDGFRβblockage 24h prior to intense light exposure resulted in a significant exacerbation of photoreceptor loss.The upregulation of GFAP and p-STAT3,observed after intense light exposure,was significantly inhibited in the PDGFRβblockage group.Fur ther upregulation of cytokines monocyte chemoattractant protein 1(MCP-1)and interleukin-1β(IL-1β)was also observed following PDGFRβinhibition.In the in vitro coculture system,the addition of light-injured SHRs induced pericyte deformation and upregulation of proliferating cell nuclear antigen(PCNA)expression,while Müller cells exhibited neuron-like morphology and expressed Nestin.However,PDGFRβblockage in retinal pericytes abolished these cellular responses to light-induced damage,consistent with the in vivo PDGFRβblockage findings.CONCLUSION:Pericyte-Müller glia interaction plays a potential role in the endogenous repair process of retinal injury.Impairment of this interaction exacerbates photoreceptor degeneration in light-induced retinal injury.

Müller细胞胶质间质转化在视网膜纤维化疾病中的作用

胶质细胞间质转化(GMT)指胶质细胞在各种因素刺激下逐渐获得间质细胞表型和特征的生物学过程,与视网膜纤维化疾病密切相关。Müller细胞是视网膜上最主要的大胶质细胞,在受到各种病理因素刺激时被激活并发生转分化。目前,已有研究表明,Müller细胞GMT与糖尿病视网膜病变、特发性黄斑前膜(iERM)、年龄相关性黄斑变性(ARMD)、增生性玻璃体视网膜病变(PVR)等多种疾病密切相关。尽管GMT相关调控机制尚不完全明确,但其作为一个潜在的干预靶点,具有良好的研究前景。了解Müller细胞GMT在视网膜疾病中的研究进展,能够为多种视网膜疾病的早期诊断和治疗提供新思路,对于全面揭示细胞转型家族在视网膜疾病中的交互作用具有重要的临床和科学意义。

股骨远端前外侧切口锁定钢板、前侧附加重建钢板在股骨远端Müller分型C2、C3型骨折治疗中的应用分析

目的 探究股骨远端前外侧切口锁定钢板、前侧附加重建钢板在股骨远端Müller分型C2、C3型骨折治疗中的应用效果。方法 回顾性选取2018年3月至2022年6月在海安市人民医院接受治疗的120例股骨远端Müller分型C2、C3型骨折患者作为本研究的研究对象,按照治疗方法不同分为对照组和研究组,每组各60例。对照组患者接受前外侧切口锁定钢板治疗,研究组患者接受前外侧切口锁定钢板+前侧附加重建钢板治疗。门诊随访12个月,比较两组患者临床指标(手术时间、术中出血量、术中C型壁透视次数、术后下地康复时间、骨折愈合时间及骨折愈合率),治疗效果,膝关节功能,术后1、2、3、6个月的膝关节活动度,术后6个月患者的下肢最大伸膝肌力矩、最大内屈角及术后并发症发生情况。结果 两组手术时间、术中出血量、术中C型壁透视次数及骨折愈合率比较,差异均无统计学意义(P>0.05);研究组术后下地康复时间、骨折愈合时间分别为(4.89±0.98)周、(6.61±1.06)个月,均短于对照组[(6.04±1.55)周、(8.45±1.02)个月],差异均有统计学意义(P<0.05)。研究组的治疗优良率为91.67%,高于对照组(78.33%),差异有统计学意义(P<0.05)。术后6个月,两组膝关节功能比较,差异无统计学意义(P>0.05)。术后1、2个月,两组膝关节活动度比较,差异均无统计学意义(P>0.05);术后3、6个月,研究组患者的膝关节活动度分别为(103.56±2.38)°、(115.68±2.77)°,均高于对照组[(92.45±5.46)°、(95.88±3.49)°],差异均有统计学意义(P<0.05)。术后6个月,研究组在触底即时及支撑末期时最大伸膝肌力矩分别为(22.66±1.26)、(18.99±2.15) Nm,均高于对照组[(19.56±2.01)、(17.58±1.87) Nm],最大内屈角分别为(3.56±0.26)°、(6.55±0.51)°,均小于对照组[(4.55±0.31)°、(7.66±0.46)°],差异均有统计学意义(P<0.05)。两组随访期内并发症发生率比较,差异无统计学意义(P>0.05)。结论 使用前侧附加重建钢板治疗股骨远端Müller分型C2、C3型骨折,相比前外侧切口锁定钢板治疗,能够提高治疗的优良率,缩短术后下地康复时间和骨折愈合时间,改善远期膝关节活动度、最大伸膝肌力矩和最大内屈角。

Vaginal clear cell adenocarcinoma in Herlyn-Werner-Wunderlich syndrome:A case report

BACKGROUND Herlyn-Werner-Wunderlich(HWW)syndrome is a rare Müllerian duct anomaly,characterized by a combination of urogenital abnormalities.The occurrence of primary cervico-vaginal carcinomas in patients with HWW syndrome is excep-tionally rare,posing significant challenges for screening,early diagnosis,and effective management.CASE SUMMARY We report a rare case of primary clear cell carcinoma of the vagina complicated in a 40-year-old woman with HWW syndrome.The patient presented with irregular vaginal bleeding for 4 years.On gynecological examination,an oblique vaginal septum was suspected.Surgical resection of the vaginal septum revealed a com-municating fistula and a tumor on the left vagina and the left side of the septum,which was confirmed as clear cell carcinoma.One month later,she underwent a radical hysterectomy,vaginectomy,bilateral salpingo-oophorectomy,and pelvic lymph node dissection.Due to significant side effects,she completed only one course of chemotherapy.A year later,lung metastasis was detected and continued to grow.A thoracoscopic wedge resection of the right upper lobe was performed 4 years after the initial surgery.We also conducted a systemic review of the lite-rature on primary cervical or vaginal carcinoma in HWW syndrome to explore this rare entity.CONCLUSION Cervico-vaginal adenocarcinomas in patients with HWW syndrome are occult,and require early surgical intervention and regular imaging surveillance.

Modulation of TGFβ_2 and dopamine by PKC in retinal Müller cells of guinea pig myopic eye

AIM: To investigate the effect of protein kinase C (PKC) on transforming growth factor-β2 (TGFβ2) and dopamine in retinal Müller cells of guinea pig myopic eye. METHODS: Myopia was induced by translucent goggles in guinea pig, whose retinal Müller cells were cultured using the enzyme-digesting method. Retinal Müller cells were divided into 5 groups: normal control, myopia, myopia plus GF109203X, myopia plus PMA, myopia plus DMSO. PKC activities were detected by the non-radioactive methods. TGFβ2 and tyrosine hydroxylase (TH) proteins were analyzed by Western Blotting in retinal Müller cells. Dopamine was determined by the high-performance liquid chromatography- electrochemical detection in suspensions. RESULTS: After 14 days deprived, the occluded eyes became myopic with ocular axle elongating. Müller cells of guinea pigs were obtained using enzyme digestion. Compared with normal control group, the increase in PKC activity and the up-regulation in TGFβ2 expression were found in retinal Müller cells of myopic eyes, with the decrease of TH and dopamine content (P <0.05). After PKC activated by PMA, TGFβ2 and TH content were up-regulated with the increase of dopamine content (P <0.05). While the PKC activities was inhibited by GF109203X, proteins of TGFβ2 and TH were down-regulated in the myopic eyes, with the decrease of dopamine content (P <0.05). CONCLUSION: TGFβ2 and dopamine are modulated by PKC in Müller cells of the myopic eyes in guinea pig.

Effects of 530 nm monochromatic light on basic fibroblast growth factor and transforming growth factor-β1 expression in Müller cells

AIMTo expose rat retinal M&#x000fc;ller cells to 530 nm monochromatic light and investigate the influence of varying light illumination times on basic fibroblast growth factor (bFGF) and transforming growth factor-&#x003b2;1 (TGF-&#x003b2;1) expression.METHODSThree groups of rat retinal M&#x000fc;ller cells cultured in vitro under a 530 nm monochromatic light were divided into 6, 12 and 24h experimental groups, while cells incubated under dark conditions served as the control group. The bFGF and TGF-&#x003b2;1 mRNA expression, protein levels and fluorescence intensity of the M&#x000fc;ller cells were analyzed.RESULTSThe bFGF mRNA expression and protein levels were significantly upregulated in M&#x000fc;ller cells in all three experimental groups compared with the control group (P&#x0003c;0.05), while that of TGF-&#x003b2;1 was downregulated (P&#x0003c;0.05). Also, bFGF expression was positively correlated, but TGF-&#x003b2;1 expression was negatively correlated with illumination time. The largest changes for both cytokines were seen in the 24h group. The changes in bFGF and TGF-&#x003b2;1 fluorescence intensity were highest in the 24h group, and significant differences were observed among the experimental groups (P&#x0003c;0.05).CONCLUSIONThe expressions of bFGF and TGF-&#x003b2;1 changed in a time-dependent manner in M&#x000fc;ller cells exposed to 530 nm monochromatic light with 250 lx illumination intensity. M&#x000fc;ller cells might play a role in the development of myopia by increasing bFGF expression or decreasing TGF-&#x003b2;1 expression. Changes in cytokine expression in retinal M&#x000fc;ller cells may affect monochromatic light-induced myopia.

Pirfenidone suppresses the abnormal activation of human Müller cells after platelet-derived growth factor-BB stimulation

AIM: To determine the effect of pirfenidone on the activated human Müller cells by platelet-derived growth factor-BB(PDGF-BB). METHODS: The primary human Müller cells were separated from retinal tissues and established the pathogenic model by stimulated with PDGF-BB. The Müller cells behaviour of normal group and the model group was measured by MTT assay, Trypan blue assay, cell migration assay, and collagen contraction assay. The expression of transforming growth factor(TGF)-β1,-β2, and pigment epithelium-derived factor(PEDF) was estimated with realtime polymerase chain reaction(PCR), Western blot and immunofluorescence analyses. RESULTS: A pathogenic/proliferative model of Müller cells was established by stimulating normal cultured Müller cells with 10 ng/mL PDGF-BB for 48 h. After treated with 0.2 and 0.3 mg/mL pirfenidone, the proliferation, migration and collagen contraction was statistically significantly depressed in the model group compared with the normal groups. The expression levels of TGF-β1 and TGF-β2 were significantly down-regulated, while the PEDF expression was significantly up-regulated after treated with 0.2 and 0.3 mg/mL pirfenidone in the model group. CONCLUSION: Pirfenidone effectively suppress the proliferation, migration and collagen contraction of the human Müller cells stimulated with PDGF-BB through down-regulation of TGF-β1/TGF-β2 and up-regulation of PEDF.

Enrichment of retinal ganglion and Müller glia progenitors from retinal organoids derived from human induced pluripotent stem cells-possibilities and current limitations

BACKGROUND Retinal organoids serve as excellent human-specific disease models for conditions affecting otherwise inaccessible retinal tissue from patients.They permit the isolation of key cell types affected in various eye diseases including retinal ganglion cells(RGCs)and Müller glia.AIM To refine human-induced pluripotent stem cells(hiPSCs)differentiated into threedimensional(3D)retinal organoids to generate sufficient numbers of RGCs and Müller glia progenitors for downstream analyses.METHODS In this study we described,evaluated,and refined methods with which to generate Müller glia and RGC progenitors,isolated them via magnetic-activated cell sorting,and assessed their lineage stability after prolonged 2D culture.Putative progenitor populations were characterized via quantitative PCR and immunocytochemistry,and the ultrastructural composition of retinal organoid cells was investigated.RESULTS Our study confirms the feasibility of generating marker-characterized Müller glia and RGC progenitors within retinal organoids.Such retinal organoids can be dissociated and the Müller glia and RGC progenitor-like cells isolated via magnetic-activated cell sorting and propagated as monolayers.CONCLUSION Enrichment of Müller glia and RGC progenitors from retinal organoids is a feasible method with which to study cell type-specific disease phenotypes and to potentially generate specific retinal populations for cell replacement therapies.

The ROCK pathway inhibitor Y-27632 mitigates hypoxia and oxidative stress-induced injury to retinal Müller cells

Rho kinase （ROCK） was the first downstream Rho effector found to mediate RhoA-induced actin cytoskeletal changes through effects on myosin light chain phosphorylation. There is abundant evidence that the ROCK pathway participates in the pathogenesis of retinal endothelial injury and proliferative epiretinal membrane traction. In this study, we investigated the effect of the ROCK pathway inhibitor Y-27632 on retinal Müller cells subjected to hypoxia or oxidative stress. Müller cells were subjected to hypoxia or oxidative stress by exposure to CoCl2 or H2O2. After a 24-hour treatment with Y-27632, the 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyl tetrazolium bromide assay was used to assess the survival of Müller cells. Hoechst 33258 was used to detect apoptosis, while 2′,7′-dichlorodihydrofluorescein diacetate was used to measure reactive oxygen species generation. A transwell chamber system was used to examine the migration ability of Müller cells. Western blot assay was used to detect the expression levels of α-smooth muscle actin, glutamine synthetase and vimentin. After treatment with Y-27632, Müller cells subjected to hypoxia or oxidative stress exhibited a morphology similar to control cells. Y-27632 reduced apoptosis, α-smooth muscle actin expression and reactive oxygen species generation under oxidative stress, and it reduced cell migration under hypoxia. Y-27632 also upregulated glutamine synthetase expression under hypoxia but did not impact vimentin expression. These findings suggest that Y-27632 protects Müller cells against cellular injury caused by oxidative stress and hypoxia by inhibiting the ROCK pathway.

Potential role of Müller cells in the pathogenesis of macropsia associated with epiretinal membrane:a hypothesis revisited

Pathophysiological explanations for metamorphopsia associated with retinal pathologies generally focus on photoreceptor organization disruption. However, the retinal microarchitecture is complicated, and we hypothesize that other retinal cells may also be involved. Metamorphopsia has been widely studied in eyes with epiretinal membranes and we revisit the idea that Müller cell displacement causes retinal macropsia. A Pub Med query and related article search for the macula ultrastructure under normal and pathological conditions revealed an enormous amount of information, particularly ultrahigh definition optical coherence tomography and other retinal imaging modality studies. Findings of these imaging studies support our hypothesis that Müller cells, and not cone photoreceptors, are primarily responsible for macropsia in eyes with epiretinal membranes. More specifically, we conclude that displacement of Müller cell endfeet, and not photoreceptor cones, is a more likely the explanation for retinal macropsia associated with epiretinal membranes.

Effect of taurine on GFAP and TauT expressions in rat retinal Müller cells in high glucose culture

Objective： To detect the expression of glial fibrillary acid protein （GFAP） and taurine transporter （TauT） in the retinal Müller cells in high glucose culture with taurine and to explore the influence of glucose on the taurine transporting, and the possible protective effects of taurine on MUller cells in early diabetic retinopathy. Methods： The Müller cells from the rat retina were cultured in high glucose, and GFAP and Taut expressions were detected in the cells treated with different doses of taurine by immuocytochemical fluorescein staining and Western blotting. Results： High glucose enhanced the expression of GFAP and decreased the expression of TauT in Müller cells. Taurine decreased the up-regulation of GFAP in the cells which was induced by high glucose; 0. 1-10 mmol/L taurine increased the expression of TauT in Müller cells. Conclusion： Taurine can inhibit the changes in Müller cell resulted from high glucose.

pigment epithelium-derived factor protects the morphological structure of retinal Müller cells in diabetic rats

AIM: To investigate if pigment epithelium-derived factor(PEDF) has any protective effect on the retinal Müller cells of Sprague-Dawley rats suffering from diabetes mellitus.METHODS: Sixty Sprague-Dawley rats were randomly divided into a negative control group, a group receiving0.1 μg/μL PEDF, another group receiving 0.2 μg/μL PEDF,and a group receiving balanced salt solution(BSS). Rats in both the PEDF and BSS groups were treated intravitreally based on previously established diabetic models. After 4wk of treatment, morphological alterations of Müller cells and protein expression of glutamine synthase(GS) and glial fibrillary acidic protein(GFAP)were analyzed.RESULTS:PEDFateither0.1μg/μLor0.2μg/μLsignificantly improved the structures of both nuclei and organelles of Müller cells compared to the BSS-treated group.Expression of GS was significantly higher in the 0.2 μg/μL PEDF group than that in the BSS group(P =0.012), but expression of GFAP was significantly lower in the 0.2 μg/μL PEDF group than that in the BSS group(P =0.000);however, there were no significant differences in expression of these proteins between the 0.1 μg/μL PEDF group and the BSS group(P =0.608, P =0.152). CONCLUSION: PEDF protects the morphological ultrastructure of Müller cells, improves the expression of glutamate synthase and prevents cell gliosis.

Absence of ephrin-A2/A3 increases retinal regenerative potential for Müller cells in Rhodopsin knockout mice

Müller cells(MC) are considered dormant retinal progenitor cells in mammals.Previous studies demonstrated ephrin-As act as negative regulators of neural progenitor cells in the retina and brain.It remains unclear whether the lack of ephrin-A2/A3 is sufficient to promote the neurogenic potential of MC.Here we investigated whether the MC is the primary retinal cell type expressing ephrin-A2/A3 and their role on the neurogenic potential of Müller cells.In this study, we showed that ephrin-A2/A3 and their receptor EphA4 were expressed in retina and especially enriched in MC.The level of ephrin As/EphA4 expression increased as the retina matured that is correlated with the reduced proliferative and progenitor cell potential of MC.Next, we investigated the proliferation in primary MC cultures isolated from wild-type and A2~(–/–) A3~(–/–) mice by 5-ethynyl-2′-deoxyuridine(EdU) incorporation.We detected a significant increase of EdU~+ cells in MC derived from A2~(–/–) A3~(–/–) mice.Next, we investigated the role of ephrin-A2/A3 in mice undergoing photoreceptor degeneration such as Rhodopsin knockout(Rho~(–/–)) mice.To further evaluate the role of ephrin-A2/A3 in MC proliferation in vivo, EdU was injected intraperitoneally to adult wild-type, A2~(–/–) A3~(–/–), Rho~(–/–) and Rho~(–/–) A2~(–/–) A3~(–/–) mice and the numbers of EdU~+ cells distributed among different layers of the retina.Ephrin As/EphA4 expression was upregulated in the retina of Rho~(–/–) mice compared to the wild-type mice.In addition, cultured MC derived from ephrin-A2~(–/–) A3~(–/–) mice also expressed higher levels of progenitor cell markers and exhibited higher proliferation potential than those from wild-type mice.Interestingly, we detected a significant increase of EdU~+ cells in the retinas of adult ephrin-A2~(–/–) A3~(–/–) mice mainly in the inner nuclear layer;and these EdU~+ cells were co-localized with MC marker, cellular retinaldehyde-binding protein, suggesting some proliferating cells are from MC.In Rhodopsin knockout mice(Rho~(–/–) A2~(–/–) A3~(–/–) mice), a significantly greater amount of EdU~+ cells were located in the ciliary body, retina and RPE than that of Rho~(–/–) mice.Comparing between 6 and 12 weeks old Rho~(–/–) A2~(–/–) A3~(–/–) mice, we recorded more EdU~+ cells in the outer nuclear layer in the 12-week-old mice undergoing severe retinal degeneration.Taken together, Ephrin-A2/A3 are negative regulators of the proliferative and neurogenic potentials of MC.Absence of ephrin-A2/A3 promotes the migration of proliferating cells into the outer nuclear layer and may lead to retinal cell regeneration.All experimental procedures were approved by the Animal Care and Use Committee at Schepens Eye Research Institute, USA(approval No.S-353-0715) on October 24, 2012.

Effects of Avastin on Expression of AQP4 in Müller Cells under Hypoxia

The aim of this study was to investigate the effects of Avastin on aquaporin4(AQP4) expression in human retinal Müller cells in vitro under hypoxia,so as to explore the mechanism of Avastin treating retinal edema.The human Müller cells were cultured using the enzymatic digestion method.Müller cells were identified under the transmission electron microscopy and by using immunofluorescence staining.By using semi-quantitative reverse transcription polymerase chain reaction(RT-PCR),the expression of AQP4 mRNA and VEGF mRNA in Müller cells cultured with 500 μmol/L CoCl 2 for 0,3,6,12 and 24 h,and with 0,100,300,500 and 700 μmol/L CoCl 2 for 24 h was detected.The expression of AQP4 mRNA in Müller cells cultured with 50 ng/mL exogenous vascular endothelial growth factor(VEGF) for 0,0.5,1,2 and 4 h,and with 0,25,50 and 75 ng/mL VEGF for 24 h was detected.Amplified cDNA products of AQP4 mRNA in Müller cells cultured with 500 μmol/L CoCl 2 and 200 μg/mL Avastin for 24 h were detected.The results showed that more than 95% cells displayed positive immunofluorescence reaction.Characteristic 8-10 nm intracellular filaments could be seen in the cytoplasm under the transmission electron microscopy.In the CoCl 2 experimental groups,the expression of AQP4 mRNA and VEGF mRNA in Müller cells was increased as compared with the control group.Alteration of AQP4 mRNA and VEGF mRNA levels showed a significantly positive correlation(r 2 =0.822,P<0.05).The expression of AQP4 mRNA in Müller cells was increased by VEGF.The expression of AQP4 mRNA was significantly decreased by Avastin as compared with the control group.It is suggested that Avastin can decrease the expression of AQP4 mRNA in human Müller cells under chemical hypoxic conditions partially via VEGF path,which may be one of the mechanisms of Avastin treating retinal edema.