Protective effects of naringenin eye drops on N-methylN-nitrosourea-induced photoreceptor cell death in rats

AIM:To investigate the effects of naringenin eye drops on N-methyl-N-nitrosourea (MNU)-induced photoreceptor cell death in rats.METHODS:Photoreceptor cell death was induced by single intraperitoneal injection of MNU(60 mg/kg)in rats.Both eyes of all animals were instilled with one drop of vehicle,0.5% or 1.0% naringenin eye drops three times per day from 7d before to 17d after MNU injection.Effects of naringenin on MNU-induced photoreceptor cell death were evaluated by electrophysiological and histological analysis.RESULTS:Flash electroretinography (FERG)and oscillatory potentials (OPs) recordings showed that the vehicle control group had remarkable reduction of amplitudes and prolongation of latency times.FERG and OPs responses were significantly reversed in MNUinduced rats treated with 0.5%or 1.0% naringenin eye drops compared with the vehicle control.The retinal morphological results showed that naringenin dosedependently preserved the outer nuclear layer,outer retina and total retina.CONCLUSION:These results indicate that topical treatment with naringenin eye drops prevented retinal neurons from MNU-induced structural and functional damages.

Role of glycolysis in retinal vascular endothelium, glia, pigment epithelium, and photoreceptor cells and as therapeutic targets for related retinal diseases

Glycolysis produces large amounts of adenosine triphosphate(ATP)in a short time.The retinal vascular endothelium feeds itself primarily through aerobic glycolysis with less ATP.But when it generates new vessels,aerobic glycolysis provides rapid and abundant ATP support for angiogenesis,and thus inhibition of glycolysis in endothelial cells can be a target for the treatment of neovascularization.Aerobic glycolysis has a protective effect on Müller cells,and it can provide with a target for visual protection and maintenance of the blood-retinal barrier.Under physiological conditions,the mitochondria of RPE can use lactic acid produced by photoreceptor cells as an energy source to provide ATP for survival.In pathological conditions,because RPE cells avoid their oxidative damage by increasing glycolysis,a large number of glycolysis products accumulate,which in turn has a toxic effect on photoreceptor cells.This shows that stabilizing the function of RPE mitochondria may become a target for the treatment of diseases such as retinal degeneration.The decrease of aerobic glycolysis leads to the decline of photoreceptor cell function and impaired vision;therefore,aerobic glycolysis of stable photoreceptor cells provides a reliable target for delaying vision loss.It is of great significance to study the role of glycolysis in various retinal cells for the targeted treatment of ocular fundus diseases.

Cytotoxic effect of specific T cells from mice with experimental autoimmune uveitis on murine photoreceptor cells

AIM:To investigate the cytotoxic effect of specific T cells from mice with experimental autoimmune uveitis(EAU)as well as their secreted interferon(IFN)-γand interleukin(IL)-17A on murine photoreceptor(661 W)cells.METHODS:An EAU model was established in female mice by injection of interphotoreceptor retinoid binding protein(IRBP)emulsion supplemented with complete Freund’s adjuvant(CFA)and Mycobacterium tuberculosis(TB).On day 12 after induction of EAU,specific T cells from spleen and lymph node tissues were isolated and cultured for 4 d and the levels of IFN-γand IL-17A in the supernatants were determined by enzyme-linked immunosorbent assays(ELISAs).T cells and their supernatants were added to 661 W cells to observe the alteration of cell morphology;IFN-γand IL-17A were separately added to 661 W cells to observe the effect of IFN-γand IL-17A on cell proliferation.RESULTS:The levels of IFN-γand IL-17A in the T cell supernatants were 1568.64±38.79 pg/m L and 1456.57±46.98 pg/mL,respectively.The supernatants apparently inhibited 661 W cell proliferation(P<0.05).T cells could also attach to the surface of 661 W cells,and IFN-γshowed a more serious cytotoxic effect on 661 W cells than IL-17A,inhibiting cell proliferation(P<0.01).CONCLUSION:IFN-γand IL-17A from T cells of EAU mice model can exert cytotoxic effects on murine photoreceptor cell proliferation,and IFN-γshows more serious cytotoxic effects on murine photoreceptor cells than IL-17A.

HSP70 cleavage-induced photoreceptor cell death caused by N-methyl-N-nitrosourea

Retinal degenerative diseases （RDs） such as retinitis pigmentosa （RP） are characterized by slowly progressive photoreceptor cell death, but the molecular mechanism underlying RP remains unclear. Animal models for RP have led to a better understand- ing of the disease pathological mechanisms, yet it remains difficult to identify an appropriate genetic model for RDs in general because there are many causative genes （Rossmiller et al., 2012）.

Activation of the TRAAK two-pore domain potassium channels in rd1 mice protects photoreceptor cells from apoptosis

AIM: To investigate the expression of TWIK-related arachidonic acid-stimulated K+ channel(TRAAK) in retinal degeneration mice(rd1) and further evaluate how TRAAK affect photoreceptor cell apoptosis.METHODS: The rd1 mice were distributed into blank(no treatment), control(1.4% DMSO, intraperitoneal injection) and riluzole groups(4 mg/kg·d, intraperitoneal injection) from postnatal 7 d to 10, 14 and 18 d;C57 group(no treatment), as age-matched wild-type control. The thickness of the outer nuclear layer(ONL) of retina was detected by paraffin section hematoxylin and eosin staining. The expression of TRAAK and the apoptosis of the ONL cells were detected by immunostaining, Western blotting, and real-time polymerase chain reaction. RESULTS: The channel agonist riluzole activated TRAAK and delayed the apoptosis of photoreceptor cells in ONL layer of rd1 mice. Both at mRNA and protein levels, after riluzole treatment, TRAAK expression was significantly upregulated, when compared with the control and blank group. Then we detected a series of apoptosis related mRNA and protein. The anti-apoptotic factor Bcl-2 downregulated and the pro-apoptotic factors Bax and cleaved-caspase-3 upregulated significantly. CONCLUSION: Riluzole elevates the expression of TRAAK and inhibits the development of apoptosis. Activation of TRAAK may have some potential effects to put off photoreceptor apoptosis.

Single-cell RNA sequencing analysis of the retina under acute high intraocular pressure

High intraocular pressure causes retinal ganglion cell injury in primary and secondary glaucoma diseases,yet the molecular landscape characteristics of retinal cells under high intraocular pressure remain unknown.Rat models of acute hypertension ocular pressure were established by injection of cross-linked hyaluronic acid hydrogel(Healaflow■).Single-cell RNA sequencing was then used to describe the cellular composition and molecular profile of the retina following high intraocular pressure.Our results identified a total of 12 cell types,namely retinal pigment epithelial cells,rod-photoreceptor cells,bipolar cells,Müller cells,microglia,cone-photoreceptor cells,retinal ganglion cells,endothelial cells,retinal progenitor cells,oligodendrocytes,pericytes,and fibroblasts.The single-cell RNA sequencing analysis of the retina under acute high intraocular pressure revealed obvious changes in the proportions of various retinal cells,with ganglion cells decreased by 23%.Hematoxylin and eosin staining and TUNEL staining confirmed the damage to retinal ganglion cells under high intraocular pressure.We extracted data from retinal ganglion cells and analyzed the retinal ganglion cell cluster with the most distinct expression.We found upregulation of the B3gat2 gene,which is associated with neuronal migration and adhesion,and downregulation of the Tsc22d gene,which participates in inhibition of inflammation.This study is the first to reveal molecular changes and intercellular interactions in the retina under high intraocular pressure.These data contribute to understanding of the molecular mechanism of retinal injury induced by high intraocular pressure and will benefit the development of novel therapies.

Differentiation potential of human adipose tissue derived stem cells into photoreceptors through explants culture and enzyme methods

AIM： To investigate the retinal photoreceptor differentiation potential of human orbital adipose tissue-derived stem cells （ADSCs） generated by enzyme （EN） and explant （EX） culture methods.METHODS： We investigated potentials of human orbital ADSCs to differentiate into photoreceptors through EN and EX culture methods. EN and EX orbital ADSCs were obtained from the same donor during rehabilitative orbital decompression, and then were subject to a 3-step induction using Noggin, DKK-1, IGF-1 and b-FGF at different time points for 38d. Stem cell, eye-field and photoreceptor-related gene and protein markers were measured by reverse transcription-polymerase chain reaction （RT-PCR） and immunofluorescent （IMF） staining.RESULTS： Both EX and EN orbital ADSCs expressed CD133, a marker of cell differentiation. Moreover, PAX6 and rhodopsin, markers of the retinal progenitor cells, were detected from EX and EN orbital ADSCs. In EX orbital ADSCs, PAX6 mRNA was detected on the 17th day and then the rhodopsin mRNA was detected on the 24th day. In contrast, the EN orbital ADSCs expressed PAX6 and rhodopsin mRNA on the 31st day. EX orbital ADSCs expressed rhodopsin protein on the 24th day, while EN orbital ADSCs expressed rhodopsin protein on the 31st day. CONCLUSION： Orbital ADSCs isolated by direct explants culture show earlier and stronger expressions of markers towards eye field and retinal photoreceptor differentiation than those generated by conventional EN method.

Roles of Optineurin and Extracellular Vesicles in Glaucomatous Retinal Cell Loss

Objective To explore the role of extracellular vesicles(EVs)in the pathogenesis of glaucoma caused by E50K mutation.Methods A photoreceptor cell line,RGC-5,was transfected with empty plasmids and plasmids expressing wild-type(WT)optineurin(OPTN)or E50K OPTN to investigate the effects of OPTN glaucoma as well as to identify the role of EVs in glaucoma pathology.The RGC-5 cells were also stimulated with glutamate,and their viability was evaluated using flow cytometry or CCK-8 assay.EVs were extracted,labeled with PKH-26,and added into the medium for normal RGC-5 culture,and the status of the cells was observed thereafter.Results WT OPTN overexpression,E50K OPTN,and glutamate stimulation induced apoptosis of RGC-5 cells.However,when glutamate stimulation was used as an add-on treatment,the degree of apoptosis in WT OPTN-overexpressing RGC-5 cells was significantly lower than that in E50K OPTN-expressing and normal RGC-5 cells.The viability of normal RGC-5 cells was reduced when co-cultured with WT OPTN-overexpressing RGC-5 or E50K OPTN-overexpressing RGC-5.EVs released by the latter two transfected lines similarly reduced normal RGC-5 survival.Conclusion Our results indicate that WT OPTN overexpression may lead to photoreceptor apoptosis.However,overexpression also confers a degree of protection against high concentrations of extracellular glutamate.Additionally,EVs released by transfected RGC-5 cells may regulate the cell state.These findings may improve our understanding of the mechanisms of cell-cell interactions in pathological conditions,providing a basis for the use of EVs as novel targets for early diagnosis and treatment of glaucoma.

Proteome changes during bone mesenchymal stem cell differentiation into photoreceptor-like cells in vitro

Human bone marrow stem cell (BMSC) may be directed to differentiate into multiple cell types, including adipocyte, chondrocyte, osteocyte and photoreceptor, among others. At present, little is known about the features of the BMSC and the protein control mechanism underlying their differentiation into photoreceptor-like cells. In the present study, BMSCs are induced to differentiate into photoreceptor-like cells in an in vitro model simulating the in vivo microenvironment. Up to 32 proteins are identified and differentially expressed through two-dimensional difference gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to establish a differential protein database for photoreceptor-like cells from BMSC-induced differentiation. Western blot analysis further confirms the expression of some of the identified proteins. The present study proposes the total protein expression and possible molecular mechanism during the differentiation of BMSCs into photoreceptor cells.

Intravitreal stem cell paracrine properties as a potential neuroprotective therapy for retinal photoreceptor neurodegenerative diseases

Retinal degenerations are the leading causes of irreversible visual loss worldwide. Many pathologies included under this umbrella involve progressive degeneration and ultimate loss of the photoreceptor cells, with age-related macular degeneration and inherited and ischemic retinal diseases the most relevant. These diseases greatly impact patients' daily lives, with accompanying marked social and economic consequences. However, the currently available treatments only delay the onset or slow progression of visual impairment, and there are no cures for these photoreceptor diseases. Therefore, new therapeutic strategies are being investigated, such as gene therapy, optogenetics, cell replacement, or cell-based neuroprotection. Specifically, stem cells can secrete neurotrophic, immunomodulatory, and anti-angiogenic factors that potentially protect and preserve retinal cells from neurodegeneration. Further, neuroprotection can be used in different types of retinal degenerative diseases and at different disease stages, unlike other potential therapies. This review summarizes stem cell-based paracrine neuroprotective strategies for photoreceptor degeneration, which are under study in clinical trials, and the latest preclinical studies. Effective retinal neuroprotection could be the next frontier in photoreceptor diseases, and the development of novel neuroprotective strategies will address the unmet therapeutic needs.

提高移植光感受器功能性整合的策略

视网膜退行性疾病的最终结局是光感受器的大量丢失,造成视力不可逆的损害,目前基本上无有效治疗措施。光感受器移植为一种潜在的细胞治疗手段,旨在通过替换丢失的光感受器,重建视网膜回路,在一定程度上帮助恢复视网膜功能。然而,物质交换机制的发现揭示了既往研究结果中移植光感受器的整合比例低、外段形成不足及突触形成不够等问题,显示了该疗法临床转化的难度。本文通过多个维度综述了提高移植光感受器功能性整合的策略,探究相关内容的可行性,具体包括选择最佳发育时间窗的移植细胞群,增强与宿主视网膜间的相互作用;破坏宿主外界膜,减轻视网膜重塑,提高移植细胞的迁移及整合;利用免疫调节,减少小胶质细胞活化,改善宿主的移植微环境;通过视网膜片或生物支架的移植形式,提高移植光感受器的组织性;合理地开发及使用生物材料,优化移植细胞的生理微环境;充分评估手术参数,降低手术本身对移植细胞及宿主视网膜的影响。

滋阴明目方通过Akt/FoxO1/FasL通路抑制感光细胞凋亡治疗视网膜色素变性的机制研究

目的观察滋阴明目方对视网膜色素变性(retinitis pigmentosa,RP)小鼠视网膜组织中磷酸化蛋白激酶B(phosphorylated protein kinase B,p-Akt)、叉头框蛋白1(forkhead box transcription factor O1,FoxO1)以及凋亡相关因子配体(Fas ligand,FasL)表达的影响,探讨滋阴明目方抑制感光细胞凋亡的机制。方法将60只rd10小鼠随机分为模型组、滋阴明目方低剂量组[10 g/(kg·d)]、滋阴明目方中剂量组[20 g/(kg·d)]、滋阴明目方高剂量组[40 g/(kg·d)]、维生素A组[5 g/(kg·d)],每组12只;选取12只C57小鼠作为空白对照组(等量生理盐水),每组连续干预28 d。通过眼底照相观察小鼠眼底形态改变;进行视网膜电图检查并记录A波和B波振幅;HE染色观察病理形态学变化并测定外核层厚度;Western blot法检测小鼠视网膜组织p-Akt、FoxO1、FasL、半胱氨酸天冬氨酸特异性蛋白(cysteine aspartate-specific protease,Caspase)-3和Caspase-8蛋白表达。结果与空白对照组比较,模型组小鼠视盘苍白、变形,血管萎缩,视网膜电图的A波与B波振幅均降低(P<0.01),视网膜结构模糊,各层界限不清,感光细胞大量丧失,外核层明显变薄(P<0.01),视网膜组织中p-Akt蛋白表达水平明显降低(P<0.01),FoxO1、FasL、Caspase-3和Caspase-8蛋白表达水平明显升高(P<0.01)。与模型组比较,滋阴明目方中、高剂量组及维生素A组小鼠眼底血管较清晰,无视盘苍白表现;视网膜各层结构清晰,细胞排列相对整齐。与模型组、滋阴明目方低剂量组比较,滋阴明目方中、高剂量组的A波与B波振幅、视网膜外核层厚度均明显升高(P<0.01),维生素A组的B波振幅、视网膜外核层厚度明显升高(P<0.01);与滋阴明目方中剂量组比较,滋阴明目方高剂量组的A波与B波振幅、视网膜外核层厚度均明显升高(P<0.01),维生素A组的A波与B波振幅均明显降低(P<0.01);与滋阴明目方高剂量组比较,维生素A组的A波与B波振幅、视网膜外核层厚度均明显降低(P<0.01)。与模型组比较,滋阴明目方低、中、高剂量组和维生素A组p-Akt蛋白表达水平明显升高(P<0.01),FoxO1、FasL和Caspase-3蛋白表达水平降低(P<0.05,P<0.01),滋阴明目方中、高剂量组Caspase-8蛋白表达水平明显降低(P<0.01)。与滋阴明目方低剂量组比较,滋阴明目方高剂量组和维生素A组p-Akt蛋白表达水平明显升高(P<0.01),滋阴明目方中、高剂量组FoxO1、FasL、Caspase-3和Caspase-8蛋白表达水平明显降低(P<0.01)。与滋阴明目方中剂量组比较,滋阴明目方高剂量组和维生素A组p-Akt蛋白表达水平明显升高(P<0.01),滋阴明目方高剂量组FoxO1、Caspase-3和Caspase-8蛋白表达水平明显降低(P<0.01),维生素A组FoxO1、FasL、Caspase-3和Caspase-8蛋白表达水平明显升高(P<0.01)。与滋阴明目方高剂量组比较,维生素A组p-Akt蛋白表达水平明显降低(P<0.01),FoxO1、FasL、Caspase-3和Caspase-8蛋白表达水平明显升高(P<0.01)。结论滋阴明目方可能通过调控Akt/FoxO1/FasL通路,增强p-Akt表达,抑制FoxO1及下游基因FasL、Caspase-3和Caspase-8的蛋白表达,从而减少rd10小鼠视网膜细胞的凋亡,保护视网膜结构和功能,延缓RP的进展。

磷酸戊糖途径抑制感光细胞凋亡及分子机制的研究进展

感光细胞是一种特殊的神经上皮细胞,在视觉信号的产生和传导中具有重要作用,其主要通过大量摄取葡萄糖进行糖代谢以满足生理需求。而感光细胞凋亡,则是视网膜疾病导致患者盲的共同原因,该过程伴有氧化应激和合成代谢的改变。近期研究发现,糖代谢的重要分支——磷酸戊糖途径,在上述病理发展中起重要作用。其产物烟酰胺腺嘌呤二核苷酸磷酸(NADPH)作为感光细胞中重要的供氢体,参与物质合成代谢,对抗氧化应激,进而抑制感光细胞的凋亡。本文将从代谢活动和分子通路的角度,针对磷酸戊糖途径抑制感光细胞凋亡的作用机制进行综述,以期为临床科研工作提供参考。

Correlation between photoreceptor injury-regeneration and behavior in a zebrafish model

Direct exposure to intensive visible light can lead to solar retinopathy, including macular injury. The signs and symptoms include central scotoma, metamorphopsia, and decreased vision. However, there have been few studies examining retinal injury due to intensive light stimulation at the cellular level. Neural network arrangements and gene expression patterns in zebrafish photoreceptors are similar to those observed in humans, and photoreceptor injury in zebrafish can induce stem cell-based cellular regeneration. Therefore, the zebrafish retina is considered a useful model for studying photoreceptor injury in humans. In the current study, the central retinal photoreceptors of zebrafish were selectively ablated by stimulation with high-intensity light. Retinal injury, cell proliferation and regeneration of cones and rods were assessed at 1, 3 and 7 days post lesion with immunohistochemistry and in situ hybridization. Additionally, a light/dark box test was used to assess zebrafish behavior. The results revealed that photoreceptors were regenerated by 7 days after the light-induced injury. However, the regenerated cells showed a disrupted arrangement at the lesion site. During the injury-regeneration process, the zebrafish exhibited reduced locomotor capacity, weakened phototaxis and increased movement angular velocity. These behaviors matched the morphological changes of retinal injury and regeneration in a number of ways. This study demonstrates that the zebrafish retina has a robust capacity for regeneration. Visual impairment and stress responses following high-intensity light stimulation appear to contribute to the alteration of behaviors.

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.

光诱导视网膜损伤的研究进展

视网膜是眼组织中最容易受到光损伤的部位,过度暴露在强光下会造成不可逆的损伤。目前对光损伤视网膜的机制并没有一个明确的说明,可能是由于一系列的反应所堆积与微环境变化有关联。本文在视网膜内细胞层次,从视锥与视杆细胞、视网膜色素细胞以及神经节细胞三方面对光诱导视网膜的损伤进行综述。

蓝光致棕色挪威大鼠慢性视网膜光损伤的实验研究

目的通过构建蓝光致棕色挪威(Brown Norway,BN)大鼠慢性视网膜光损伤的模型,探究大鼠光感受器细胞及视网膜色素上皮细胞(RPEc)损伤的特点及可能损伤机制。方法根据随机数字表法将大鼠分组为光照0 d(正常对照组)和光照1、3、7及14 d组,每组8只。正常对照组不进行光照;余各组每日暴露于光照强度为(1000±100)Lux的LED蓝光光源环境中3 h,连续1、3、7及14 d,观察大鼠的行为活动;视网膜电流图(ERG)记录最大混合反应的a、b波振幅和潜伏期;进行眼底照相;HE染色观察视网膜组织;酶联免疫吸附试验(ELISA)检测大鼠视网膜组织的活性氧(ROS)含量。结果与正常对照组比较,光照3 d组对光声刺激的反应迟钝,光照7 d及14 d组精神较萎靡,行动稍迟缓,对光声刺激反应更为迟钝;光照3 d组偶见视网膜出血点,RPEc层基底部色素颗粒增多,外核层可见轻度细胞核固缩;光照7 d组视网膜上见散在点状出血点、黄白色点状颗粒物,视网膜静脉迂曲扩张;光照14 d组视网膜上见大量黄白色点状渗出,视网膜动脉呈铜丝样甚至银丝样外观,视网膜静脉迂曲扩张;光照7、14 d组视网膜光感受器内节/外节结构排列紊乱,外核层细胞核固缩,RPEc层基底部色素颗粒沉积。与正常对照组比较,光照3、7、14 d组大鼠视网膜变薄(P<0.05);光照3、7及14 d组ERG b波潜伏期逐渐延长、a波、b波振幅逐渐降低,视网膜组织ROS逐渐升高(P<0.05)。结论蓝光持续照射BN大鼠可产生氧化应激反应,导致慢性视网膜光损伤,且照射时间越长,视网膜光损伤越重。

杆体锥体分离概念及特征

弓形虫脉络膜视网膜炎患者中存在的外层视网膜光感受器分离首次以杆体锥体分离(BALAD)来命名,表现为光学相关断层扫描(OCT)上光感受器内节在肌样体水平上分离,形成独特的视网膜囊腔。随后许多研究相继报道了不同疾病中存在的BALAD。外层视网膜中,光感受器内节的肌样体区结构相对薄弱,当促进光感受器外节附着在视网膜色素上皮(RPE)上的外向力超过光感受器内节肌样体的抗拉伸强度时,肌样体带分裂,形成BALAD。BALAD具有其独特的多模态影像特征,识别BALAD可以为临床诊断、鉴别以及治疗眼部疾病提供新的思路。本文就BALAD命名的发展过程、解剖结构特征、病理生理机制、多模态影像特征等方面进行综述。

蓝光对豚鼠离焦性近视进展的抑制作用及其视锥细胞密度变化机制

目的观察蓝光干预对光学离焦性近视豚鼠屈光发育的影响及其作用机制。方法选取普通级2周龄三色豚鼠48只,采用抛硬币法随机分成蓝光组和白光组,每组各24只。所有豚鼠右眼佩戴-5.00 D镜片建立光学离焦模型,为实验眼;左眼为自身对照,不予遮盖。实验前及实验开始后8周,采用带状光检影镜测量豚鼠屈光度,A型超声测量前房深度、晶状体厚度及眼轴长度,角膜曲率计测量角膜曲率半径。实验开始后8周,采用过量麻醉法处死豚鼠,取右眼眼球并分离视网膜,采用视网膜铺片免疫荧光染色观察豚鼠视网膜S及M视锥细胞密度;采用高效液相色谱分析法检测视网膜视黄酸表达;采用实时荧光定量PCR检测视网膜视黄酸受体(RAR-β)和巩膜中基质金属蛋白酶2(MMP-2)、组织金属蛋白酶抑制剂2(TIMP-2)及Ⅰ型胶原的表达;采用苏木精-伊红染色观察巩膜厚度变化。结果实验开始后8周,蓝光组实验眼较白光组实验眼出现(0.63±0.12)D相对远视,眼轴增长延缓(0.08±0.00)mm;蓝光组对照眼较白光组对照眼出现(0.42±0.09)D相对远视,眼轴增长延缓(0.08±0.00)mm;蓝光组实验眼较蓝光组对照眼近视加深(1.52±0.09)D,眼轴增长(0.06±0.00)mm;白光组实验眼较白光组对照眼近视加深(1.66±0.07)D,眼轴增长(0.13±0.00)mm,差异均有统计学意义(均P<0.05)。蓝光组豚鼠视网膜背侧和腹侧M视锥细胞密度小于白光组,背侧和腹侧S视锥细胞密度大于白光组,差异均有统计学意义(t=32.33、52.23、42.09、25.02,均P<0.05)。蓝光干预后近视延缓与腹侧S视锥细胞密度增加呈强正相关(r=0.95,P<0.01)。蓝光组视黄酸含量、RAR-β和MMP-2相对表达量较白光组减少,TIMP-2和Ⅰ型胶原相对表达量较白光组增加,差异均有统计学意义(t=18.73、7.45、3.72、6.19、9.03,均P<0.05)。蓝光组巩膜厚度为(125.0±7.8)μm,较白光组的(102.0±6.3)μm明显增厚,差异有统计学意义(t=26.93,P<0.05)。结论蓝光可抑制豚鼠离焦性近视进展;豚鼠屈光度的改变可能通过视网膜视锥细胞密度变化影响视网膜视黄酸及巩膜胶原的表达来实现。

长链非编码RNA Neat1通过RD3导致氧诱导视网膜病感光细胞变性

目的探讨长链非编码RNA(lncRNA)Neat1导致氧诱导视网膜病(OIR)感光细胞变性的机制,以期为早产儿视网膜病(ROP)的防治提供新的思路。方法高氧环境下制造ROP模型OIR小鼠,以同日龄正常SPF新生小鼠作为对照。应用二代测序技术(NGS)及分析软件,筛选与OIR小鼠视网膜发育异常相关的差异lncRNA。对芯片检测到的差异表达基因及同时又被预测为可能是调控靶标的编码基因,取交集进行GO分析、Pathway分析。采用qPCR法对差异表达的靶基因进行验证。通过UCSC基因组浏览工具分析Neat1基因。采用qPCR法检测ROP患儿血液、OIR小鼠视网膜组织中Neat1、视网膜变性蛋白3(RD3)的表达水平。采用放射免疫法测定ROP患儿血液、OIR小鼠视网膜组织中环磷酸鸟苷(cGMP)含量。结果NGS结果显示,OIR小鼠视网膜中Neat1呈差异性表达。qPCR验证结果与NGS结果一致。生物信息学预测RD3可能为Neat1调控靶标,在小鼠Neat1序列下游发现RD3基因。与对照组相比,ROP患儿血液和OIR小鼠视网膜组织中Neat1的mRNA水平升高,RD3低表达,cGMP高表达。结论lncRNA Neat1可能在RD3介导下通过Neat1/RD3/cGMP通路导致视网膜感光细胞发生变性。