小胶质细胞活化与rd小鼠遗传性视网膜变性

目的研究小胶质细胞活化与rd小鼠遗传性视网膜变性的关系。方法对出生后8、10、12、14、16及18d的rd小鼠及对照小鼠视网膜进行感光细胞凋亡TUNEL法检测及形态计量学分析。CD11b免疫组织化学染色标记视网膜小胶质细胞。结果rd小鼠出生后10d视网膜感光细胞层开始出现TUNEL染色阳性细胞,第16d达到高峰。视网膜小胶质细胞在rd小鼠出生后10d开始活化,第14d达到高峰。小胶质细胞向感光细胞层的迁移与感光细胞凋亡之间存在紧密的时间和空间关系。结论rd小鼠视网膜变性以感光细胞凋亡为主。小胶质细胞活化可能在视网膜变性过程中发挥重要作用。     

光诱导感光细胞凋亡的信号级联

视网膜光化学损伤的实质是感光细胞凋亡。近年来随着分子生物学技术的应用,对光诱导感光细胞凋亡的研究已由视网膜的形态及功能方面逐渐深入到诱导凋亡的信号级联方面并取得了很大进展,本就此作一综述。     

光损伤导致视网膜感光细胞凋亡的实验研究

目的探讨视网膜光损伤后感光细胞病理学改变的特征及其发生机制。方法以白色强光持续照射的方法制成大鼠视网膜光损伤模型并采用常规HE染色与TUNEL技术对光损伤后视网膜感光细胞的病理学改变进行动态观察研究。结果白色强光照射后视网膜感光细胞发生进行性的变性,TUNEL标记结果显示光损伤后视网膜外核层出现大量阳性着色细胞。结论持续高强度白光照射可选择性地导致视网膜感光细胞发生进行性的变性而凋亡是感光细胞退行性变性的重要发生机制。     

去小胶质细胞化对糖尿病小鼠视网膜光感受器细胞的影响

目的观察去小胶质细胞化对早期糖尿病小鼠视网膜光感受器细胞的影响。方法选取6～8周龄的SPF级雄性C57BL/6J小鼠作为实验动物,未经处理的5只作为空白对照组(A组),10只采用链脲佐菌素(STZ)腹腔注射法成功诱导出糖尿病后的小鼠随机等分为B、C两组。A、B组继续喂养标准实验饲料4周,C组在喂养1周标准实验饲料后添加含290 mg·kg^-1 PLX3397(集落刺激因子1受体拮抗剂)的AIN-76A(标准饮食配制的啮齿类实验动物纯化饲料)3周以去除小胶质细胞。4周后于同一时间点处死各组动物,眼球标本均于处死后即刻获取并固定。制备视网膜石蜡切片,HE染色后光学显微镜下观察视网膜结构;小胶质细胞特异性抗体P2ry12免疫荧光化学法检测小胶质细胞在视网膜上的分布,并测算平均吸光度(D)值以间接代表小胶质细胞的数量;TUNEL法测定光感受器细胞的凋亡情况;将上述观察指标在三组间进行比较。结果光镜观察视网膜结构显示,与A组相比,B组神经纤维层变水肿,内丛状层、内核层及外核层排列变疏松;C组也出现上述改变,但程度较轻。小胶质细胞的免疫荧光化学检测结果显示:A组可在视网膜内层...     

Activation of multiple pathways during photoreceptor apoptosis in the rd mouse

PURPOSE: The primary purpose of this study was to characterize photoreceptor apoptosis in the rd mouse. Given that apoptosis is the final common pathway in many cases of retinal degeneration, the ability to retard or even arrest this process may ameliorate retinal disorders such as retinitis pigmentosa (RP). The absence of any recognized therapy emphasizes the fact that a detailed knowledge of the molecular events involved is necessary to identify rational targets for therapeutic intervention. METHODS: Flow cytometry was used to measure physical and chemical characteristics in the photoreceptor population. Individual cells flow in suspension past one or more lasers, scattering light and emitting fluorescence. Western blot techniques demonstrated cleavage of calpain-specific substrates. Retinal explant cultures were used for inhibitor studies. Postnatal day 10 (P(10)) rd retinas were cultured without retinal pigment epithelium (RPE) attached up to P(17). RESULTS: This study demonstrated calcium overload in the cytosol and subsequently in mitochondria. Mitochondrial membrane depolarization and reactive oxygen species (ROS) were detected later, during the peak of cell death. Analysis of downstream events indicated early activation of calcium-activated calpains. Treatment of rd retinal explants with the calpain inhibitor N-acetyl-Leu-Leu-Nle-CHO (ALLN) successfully inhibited calpain-induced alpha-fodrin cleavage, yet it did not protect against photoreceptor degeneration. Finally, the results demonstrate an increase in the levels of both precursor and processed forms of the aspartate protease cathepsin D. CONCLUSIONS: Excessive calcium influx is an early event that initiates the activation of calcium-activated proteases. However, these proteases are not singularly the cause of death, because their inhibition does not prevent apoptosis. Indeed, the results presented herein suggest that multiple pathways are involved and that each of these components may have to be addressed for cell death to be successfully inhibited.     

蓝光诱导的光感受器细胞凋亡与c-Fos蛋白的表达

目的:观察宽谱蓝光诱导Lewis大鼠视网膜光损伤后光感受器细胞的凋亡及c-Fos蛋白的表达。方法:8～10wk龄雌性Lewis大鼠24只,在循环光环境下饲养并随机分为6组。暗适应24h后,5组接受3012×115Lux的宽谱蓝光(400～500nm)照射1h,光照后予暗适应并于0,6,12,24及48h颈椎脱位法处死大鼠,摘除眼球;另1组为正常对照组,不予光照。采用透射电镜及TUNEL试剂盒检测视网膜细胞凋亡,免疫组化法检测视网膜内c-Fos蛋白的表达。结果:蓝光可特异性引起大鼠光感受器细胞凋亡和光感受器细胞内c-Fos蛋白的表达上调。光照后外核层细胞开始出现凋亡,24h达峰值,大量光感受器细胞出现核固缩并可见凋亡小体形成。c-Fos蛋白的表达在时间与空间分布上与TUNEL阳性细胞基本一致,在同一时间点,二者呈正相关(r =0.905,P <0.05)。结论:蓝光可诱导大鼠光感受器细胞凋亡,视网膜外核层中c-Fos蛋白的表达上调对视网膜蓝光损伤后光感受器细胞凋亡可能具有重要作用。     

Absence of voltage-dependent calcium channels delays photoreceptor degeneration in rd mice

Retinal degeneration results from the apoptotic cell death of photoreceptors. While mutations in a large number of genes give rise to retinal degeneration, the specific mechanisms are not well understood. One hypothesis involves mediation of apoptosis by high concentrations of intracellular Ca(2+). We used a mouse line that carries the rd mutation but also lacks the major L-type voltage-dependent Ca(2+) channel at the photoreceptor synapse to examine whether this route of Ca(2+) entry plays a role in photoreceptor degeneration. In both experimental and control mice, the photoreceptors degenerate. However, at postnatal days 16, 18, and 21 there is a delay in photoreceptor cell loss in the experimental mice, which lack L-type voltage-dependent Ca(2+) channels, compared to controls. These data indicate that Ca(2+) entry via the L-type voltage-dependent Ca(2+) channel contributes to the mechanisms responsible for photoreceptor cell death in this mouse model of retinitis pigmentosa.     

Human neural progenitor cells promote photoreceptor survival in retinal explants

Different types of progenitor and stem cells have been shown to provide neuroprotection in animal models of photoreceptor degeneration. The present study was conducted to investigate whether human neural progenitor cells (HNPCs) have neuroprotective properties on retinal explants models with calpain- and caspase-3-dependent photoreceptor cell death. In the first experiments, HNPCs in a feeder layer were co-cultured for 6 days either with postnatal rd1 mouse or normal rat retinas. Retinal histological sections were used to determine outer nuclear layer (ONL) thickness, and to detect the number of photoreceptors with labeling for calpain activity, cleaved caspase-3 and TUNEL. The ONL thickness of co-cultured rat and rd1 retinas was found to be almost 10% and 40% thicker, respectively, compared to controls. Cell counts of calpain activity, cleaved caspase-3 and TUNEL labeled photoreceptors in both models revealed a 30-50% decrease when co-cultured with HNPCs. The results represent significant increases of photoreceptor survival in the co-cultured retinas. In the second experiments, for an identification of putative survival factors, or a combination of them, a growth factor profile was performed on conditioned medium. The relative levels of various growth factors were analyzed by densitometric measurements of growth factor array membranes. Following growth factors were identified as most potential survival factors; granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GMCSF), insulin-like growth factor II (IGF-II), neurotrophic factor 3 (NT-3), placental growth factor (PIGF), transforming growth factors (TGF-β1 and TGF-β2) and vascular endothelial growth factor (VEGF-D). HNPCs protect both against calpain- and caspase-3-dependent photoreceptor cell death in the rd1 mouse and against caspase-3-dependent photoreceptor cell death in normal rat retinas in vitro. The protective effect is possibly achieved by a variety of growth factors secreted from the HNPCs.     

The zinc-binding domain of Nna1 is required to prevent retinal photoreceptor loss and cerebellar ataxia in Purkinje cell degeneration (pcd) mice

The Purkinje cell degeneration (pcd) mouse undergoes retinal photoreceptor degeneration and Purkinje cell loss. Nna1 is postulated to be the causal gene for pcd. We show that a BAC containing the Nna1 gene rescues retinal photoreceptor loss and Purkinje cell degeneration, confirming that Nna1 loss-of-function is responsible for these phenotypes. Mutation of the zinc-binding domain within the transgene destroyed its ability to rescue neuronal loss in pcd(5J) homozygous mice. In conclusion, Nna1 is required for survival of retinal photoreceptors and other neuron populations that degenerate in pcd mice. A functional zinc-binding domain is crucial for Nna1 to support neuron survival.     

Latanoprost protects rat retinal ganglion cells from apoptosis in vitro and in vivo

We investigated whether latanoprost has a direct anti-apoptotic effect in retinal ganglion cell (RGC) line and RGCs in the rat. RGC-5 cells were induced to undergo apoptosis by serum deprivation and exogenous glutamate. The level of cell death with or without latanoprost acid was monitored by an XTT assay and by immunocytochemistry with activated caspase-3. Changes in the level of intracellular calcium ([Ca²⁺]i) were measured with fluo-4 fluorescence. The XTT assay revealed that latanoprost acid increased RGC-5 cell viability. Latanoprost acid significantly reduced caspase-3 positive cells and suppressed [Ca²⁺]i evoked by glutamate. U0126, a mitogen-activated protein/extracellular signal-regulated kinase 1 and 2 inhibitor, partially blocked the rescue effect of latnanoprost acid (p = 0.013). In vivo, rat RGCs were degenerated by optic nerve crush. After topical instillation of latanoprost for 7 days, RGCs labeled with fluorogold were significantly. Retinal flatmounts were subjected to terminal dUTP nick end labeling (TUNEL) staining to detect apoptotic cells. TUNEL-positive cells were significantly decreased in eyes with topically instilled latanoprost (p = 0.015). These data suggest that latanoprost has an neuroprotective ability in RGCs.     

Rhodopsin和recoverin在MNU诱导的光感受器损伤中的表达变化

目的：观察N-甲基-N-亚硝基脲( MNU)诱导的大鼠视网膜光感受器损伤过程中Rhodopsin 和recoverin表达变化与损伤的时效关系。
　　方法：将36只SPF级7周龄大鼠随机分为正常对照组, MNU模型组(6h组,12h组,24h组,3d组,7d组),每组各6只。模型组一次性腹腔注射60mg/kg MNU,正常对照组腹腔注射等量PBS。右眼行HE,TUNEL,透射电镜评估视网膜组织损伤的超微结构变化及细胞凋亡程度,左眼取视网膜组织通过Western blot和免疫荧光观察视网膜组织中Rhodopsin和recoverin的mRNA表达变化。
　　结果：透射电镜观察到MNU注射12 h 后出现凋亡小体,24 h后外核层大部分细胞呈阳性反应;TUNEL 检测发现MNU注射24 h 光感受器细胞凋亡指数最高,达(29.7±2.3)%,与电镜结果吻合。 Western blot 结果表明, MNU注射12 h后表达有极显著性差异( P<0.01),而Recoverin的表达从注射后24h有极显著性差异(P<0.01)。
　　结论：一次性腹腔注射60 mg/kg MNU能特异性诱导SD大鼠视网膜光感受器细胞凋亡, Rhodopsin和recoverin表达下调与MNU诱导光感受器细胞的选择性凋亡有关。     

Remodeling of cone photoreceptor cells after rod degeneration in rd mice

We studied the survival of cone photoreceptors following the degeneration of rods in the rd mouse. Cones were visualized by selective expression of green fluorescent protein (GFP) following transduction with an adeno-associated virus (AAV) vector. As previously reported, many cones survive after the initial degeneration of the rods. Soon after the initial degeneration, they lose their outer segments and all but a vestigial inner segment; and they partially retract or lose their axon and synaptic pedicle. However, they retain many fundamental features of the cone phenotype, and for many weeks show a polarized morphology indicative of substantial regrowth of processes. The cells retain their laminar position, forming a cell row just distal to a much thinned outer plexiform layer. The somata subsequently enlarge. Most of the cells extend bipolar processes, recreating the original bipolar morphology of a photoreceptor cell - though now turned on its side relative to the native position. The cells express short- or middle-wavelength opsins, recoverin and connexin36. One or more of the polarized processes could often be shown to contain synaptic ribbons, as visualized by antibodies against RIBEYE. The cones do not express protein kinase C alpha, Go alpha, ChX10 or calbindin, markers of bipolar or horizontal cells. The partially differentiated cone morphology persists for at least several months, after which the processes begin to retract and there is slow loss of the cells. Thus, during the time following the loss of their rod-dominated microenvironment, the cones achieve a semi-stable state in which much of their normal phenotype is preserved. Cone photoreceptors in retinas of human RP donors appear from their morphology to undergo a similar progression. The therapeutic window for rescue of cone photoreceptors may be longer than would have been thought.     

Deletion of prominin-1 in mice results in disrupted photoreceptor outer segment protein homeostasis

AIM: To illustrate the underlying mechanism how prominin-1 (also known as Prom1) mutation contribute to progressive photoreceptor degeneration. METHODS: A CRISPR-mediated Prom1 knockout (Prom1-KO) mice model in the C57BL/6 was generated and the photoreceptor degeneration phenotypes by means of structural and functional tests were demonstrated. Immunohistochemistry and immunoblot analysis were performed to reveal the localization and quantity of related outer segment (OS) proteins. RESULTS: The Prom1-KO mice developed the photoreceptor degeneration phenotype including the decreased outer nuclear layer (ONL) thickness and compromised electroretinogram amplitude. Immunohistochemistry analysis revealed impaired trafficking of photoreceptor OS proteins. Immunoblot data demonstrated decreased photoreceptor OS proteins. CONCLUSION: Prom1 deprivation causes progressive photoreceptor degeneration. Prom1 is essential for maintaining normal trafficking and normal quantity of photoreceptor OS proteins. The new light is shed on the pathogenic mechanism underlying photoreceptor degeneration caused by Prom1 mutation.     

新出生期视网膜感光细胞纤毛结构超微形态学分析

目的了解新出生期视网膜感光细胞纤毛结构特点.方法以新出生期小牛视网膜为研究对象,行透射电镜分析.结果新出生期感光细胞纤毛平均长度为(0.7243±0.0115) μm,直径为(0.2771±0.0104)μm.纤毛顶部与感光细胞外节段基部相连,纤毛表面质膜与外节段表面质膜延连,纤毛基部伸入内节段,底端呈开放式与内节段胞浆连通,可见纤毛基板但未见"小根"等结构.纤毛内部可见周围微管,近纤毛顶部可见部分中央微管.纤毛下端位于内节段内部分电子密度明显增高,基板平均长度为(0.3337±0.0321)μm.其中所见两个点状高密度结构至胞浆膜面距离平均为(0.2863±0.0226)μm.结论新出生期小牛视网膜感光细胞纤毛具有较其他组织纤毛明显不同的结构特点,其对于维系感光细胞节段生长具有重要作用.     

NOX2基因缺陷对rd1小鼠感光细胞凋亡的保护作用

目的 观察NADPH氧化酶2(NOX2)基因缺陷对遗传性视网膜变性小鼠1(rd1)感光细胞的保护作用。设计 实验研究。研究对象 出生后14天的NOX2基因缺陷rd1小鼠(实验组)6只及同龄NOX2基因缺陷的C57BL/6N小鼠(对照组1)、无NOX2基因缺陷的rd1小鼠(对照组2)、C57BL/6N野生正常小鼠(对照组3)各6只(共24只)。方法 对照组1与对照组2小鼠多次交配获得实验组小鼠并进行基因型鉴定。取该实验鼠及对照组小鼠眼球,对视网膜进行HE染色并测量视网膜外核层厚度,TUNEL染色并计算凋亡细胞占外核层细胞总数百分比、免疫荧光法CD1 1b抗体标记小胶质细胞并检测NOX2主要亚单位gp91^pbox蛋白的表达。主要指标 视网膜外核层厚度,感光凋亡细胞百分比,gp91^pbox蛋白的表达量,小胶质细胞活化情况。结果 与同龄对照组2相比,实验组小鼠视网膜内外核层排列整齐,其外核层厚度(36.18±2.59)μm明显大于对照组2小鼠(21.45±1.33)μm(t=8.77,P=0.001)。实验组小鼠视网膜凋亡细胞主要出现于外核层,但数量...     

Nicotinamide prevents N-methyl-N-nitrosourea-induced photoreceptor cell apoptosis in Sprague-Dawley rats and C57BL mice

In previous studies, it was found that a single systemic administration of N-methyl-N-nitrosourea (MNU) to rats and mice resulted in the retinal degeneration in all treated animals over a 7 day period. Retinal degeneration was due to photoreceptor cell apoptosis that was identical to the apoptosis seen in human retinitis pigmentosa (RP). In the present study, nicotinamide (NAM), a water-soluble B-group vitamin (vitamin B(3)), suppressed photoreceptor cell loss in a dose-dependent manner when administered immediately after MNU treatment. In rats, a dose of NAM >or=25 mg kg(-1) completely suppressed photoreceptor cell loss, and 10 mg kg(-1) partially suppressed photoreceptor cell loss. In mice, doses of 1000 and >or=100 mg kg(-1) were needed for complete and partial suppression, respectively. Thus, rats were more responsive to NAM than mice. The retinoprotective effect of 1000 mg kg(-1) NAM lasted throughout the long-term (35 days) observation period, with no apparent toxicity. Also, in rats, 1000 mg kg(-1) NAM completely suppressed photoreceptor cell loss when administered up to 4 hr after MNU treatment, and partially suppressed photoreceptor cell loss when administered 6 hr after MNU treatment. In mice, administration of NAM 2-6 hr after MNU resulted in partial suppression. NAM did not reduce levels of 7-methyldeoxyguanosine DNA adduct, but did reduce photoreceptor cell apoptosis. Although the mechanism of action underlying this retinoprotection remains to be clarified, NAM may be a potential therapeutic agent for the treatment of retinal degeneration.     

Activation of retinal ganglion cells in wild-type and rd1 mice through electrical stimulation of the retinal neural network

We compared the thresholds and response properties of extracellularly recorded retinal ganglion cells (RGCs) in wild-type and rd1 mouse retinas to electrical stimulation of the retinal neural network. Retinas were stimulated in vitro with biphasic current pulses (1 ms/phase) applied with a 400-microm diameter, subretinal electrode. Three types of responses were observed in both wild-type and rd1 RGCs. Type I cells elicited a single burst of spikes within 20 ms following application of the electrical stimulus, type II cells elicited a single burst of spikes with a latency greater than 37 ms, and type III cells elicited two and occasionally three bursts of spikes. For all ages examined, ranging from postnatal day (P) 25 to P186, the thresholds of RGCs were overall consistently higher in rd1 mice. Median threshold values were 14 and 50 muA in wild-type and rd1 mice, respectively. We propose that photoreceptors lower the thresholds for activation of RGCs whereas postreceptoral neurons determine the response properties of RGCs to electrical stimuli.