视网膜光损伤机制中NOS作用的实验研究

目的　探讨一氧化氮合酶(nitricoxidesynthase ,NOS)活性异常变化在视网膜光损伤发生机制中的作用。方法　60只SD大鼠随机分为5组,1组为正常对照,其它4组以持续强光照射6小时造成视网膜光损伤,并按光照后1天和6天两个观察点各分为实验组与空白对照组,实验组大鼠给予左旋硝基精氨酸治疗,并分别测定比较各组大鼠视网膜外核层厚度以及NOS和超氧化物歧化酶(superoxidedismutase ,SOD)的活性。结果　光损伤后第1天和第6天空白对照组大鼠视网膜内NOS的活性均异常升高,SOD活性均显著下降,视网膜外核层厚度进行性减小;实验组大鼠视网膜内NOS的活性低于正常水平,SOD活性和视网膜外核层厚度均显著高于相应的空白对照组。结论　过度光照后视网膜内NOS活性持续异常升高是视网膜氧化损伤的重要原因并与感光细胞退行性变性的发生密切相关。     

过度光照后诱导型一氧化氮合酶在大鼠视网膜色素上皮的表达

目的探讨过度光照诱导大鼠视网膜色素上皮（retinal pigment epithelium．RPE）细胞表达诱导型一氧化氮合酶（inducible nitric oxide synthase，iNOS）的作用及其病理学意义。方法应用免疫组织化学的方法分别检测处于自然光照环境中的正常大鼠及接受强光过度照射后大鼠的视网膜RPE细胞中iNOS的表达情况。结果正常大鼠RPE细胞中未见iNOS的表达，而过度光照后大鼠RPE细胞的胞浆中表达高水平的iNOS，同时视网膜外核层感光细胞发生结构损伤。结论过度光照可诱导RPE细胞表达iNOS．这一异常改变可能是视网膜感光细胞结构损伤的重要原因。     

模拟强日光照射导致兔眼视网膜光损伤的实验研究

目的:探讨短时间模拟强日光照射对兔视网膜组织结构的影响及光损伤的致伤机制。方法:健康新西兰白兔25只随机分为5组,30000lux模拟日光照射兔眼15min和30min。照射后不同时间取视网膜进行光镜、透射电镜观察并对视网膜感光细胞凋亡率进行检测。结果:15min光照组2d时观察;光感受器细胞外节盘膜结构紊乱,板层结构离散,内节线粒体肿胀,部分嵴断裂;外颗粒层细胞胞浆有空泡样改变;感光细胞凋亡率为3.26%±0.98%。30min光照组2d时观察,外节盘膜结构紊乱,板层结构重度离散,内节线粒体肿胀,嵴断裂;外颗粒层细胞胞浆有空泡样改变;感光细胞凋亡率为3.63%±1.25%。30min光照组7d时观察,外节盘膜组织结构紊乱,板层结构消失,空泡化,内节线粒体肿胀,嵴断裂;外颗粒层细胞胞浆肿胀,大量空泡样改变,排列紊乱,部分胞膜破坏;感光细胞凋亡率为19.63%±1.32%。30min光照组14d时观察,外节盘膜组织结构恢复较规则,板层结构较致密,内节线粒体肿胀,嵴断裂;外颗粒层细胞胞浆肿胀减轻,排列较整齐;感光细胞凋亡率为18.98%±1.13%。结论:30000lux模拟强日光照射15min可导致兔视网膜急性光损伤,模拟强日光照射可导致视网膜感光细胞发生退行性变性,感光细胞凋亡是损伤发生的重要机制。     

氩绿激光致兔视网膜感光细胞凋亡的实验研究

目的:观察氩绿激光对兔视网膜损伤及修复的组织学改变及对兔视网膜感光细胞凋亡的影响。方法:将8只有色兔(16眼)中每只眼的上、下方视网膜随机分配为激光光凝区及空白对照区,光凝后24h、4wk通过光镜和电镜及TUNEL技术观察视网膜改变及感光细胞凋亡。结果:(1)光镜观察:光凝处视网膜损伤不明显但脉络膜小静脉充血,4wk后消退。(2)电镜观察:光凝处外节膜盘排列稀疏,神经节细胞轻微肿胀,外核层细胞异染色质增多,4wk后色素增殖,胶原增生。(3)TUNEL染色观察:光凝后24h,光凝处外颗粒层可见较多阳性细胞,内颗粒层偶见,4wk后接近正常。(4)感光细胞凋亡率:光凝后24h,感光细胞凋亡率较正常对照组增加,差异有统计学意义(P<0.01)。光凝后4wk较24h凋亡率显著降低,差异有统计学意义(P<0.01)。结论:轻度氩绿激光光凝视网膜损伤轻微。     

慢性光诱导建立微型猪视网膜变性模型的研究

目的 通过持续光照建立大型动物的慢性视网膜变性模型,用于视网膜移植实验研究.方法 利用可见光持续照射12只2月龄猪达3个月以上,慢性诱导微型猪视网膜变性.采用多焦视网膜电图检测正常猪及光性视网膜变性猪N1和P1波振幅和潜伏期,比较各观察值的变化;用光镜、电镜观察其视网膜外核层厚度变化及细胞器超微结构状况,从形态和功能两方面对光损伤模型进行鉴定.结果 同正常猪比较,光性视网膜变性猪N1、P1波振幅在1～6环内均明显降低,而在视网膜内传导时间明显延长;苏木精-伊红染色光镜下见感光细胞内外节变短,外核层细胞核减少至4～6排,外核层厚度减少了20.98%;电镜下可见后极部感光细胞线粒体肿胀明显、嵴断裂、数量减少.结论 微型猪光性视网膜变性模型经功能和形态两方面鉴定光损伤部位是在光感受器,建立用于视网膜移植实验的大型动物模型获得成功.     

兔眼经瞳孔温热疗法阈值能量治疗的组织病理反应和细胞凋亡

目的:观察兔眼视网膜经瞳孔温热疗法(transpupillary thermotherapy,TTT)阈值能量照射后组织病理反应和细胞凋亡情况.方法:健康青紫兰兔20只,采用阈值能量对实验眼采用1.2mm光斑的810nm激光照射60s.采用眼底镜和眼底照相对光斑进行形态学研究,采用光镜和电镜的方法研究光斑的组织病理和超微结构的改变,采用TUNEL法、荧光素标记Annexin Ⅴ-FITC/PI双染色流式细胞测定法观察视网膜细胞的凋亡.结果:TTT后1d可见视网膜轻度灰白色水肿,后视网膜逐渐出现色素沉着.组织病理学切片显示神经节细胞无显著性破坏.TUNEL染色可见视网膜全层均有细胞凋亡的发生,以内颗粒层为主,流式细胞双染测定显示以凋亡为主.结论:阈能级TTT照射未引起神经节细胞严重损伤,较安全,其作用机制以细胞凋亡为主.     

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。     

中药复方制剂对rds小鼠感光细胞凋亡的干预作用研究

目的观察中药复方制剂对先天性视网膜变性小鼠视网膜感光细胞凋亡的影响。方法以黄芪等药组成复方制剂1。对先天性视网膜变性动物模型rds小鼠,采用原位末端转移酶标记(TUNEL)方法及组织病理学技术,观察复方制剂1作用后,小鼠视网膜感光细胞数量及感光细胞凋亡的变化。结果仔鼠2周时,中药组小鼠视网膜感光细胞核数与对照组相比无明显差别,两组感光细胞凋亡率分别为1.6%及6.5%,组间差异有显著性(P<0.01);4周时中药组感光细胞核数目较对照组多,差异有显著统计学意义(P<0.01),两组感光细胞凋亡率分别为3.3%及8.5%,组间差异有显著统计学意义(P<0.01)。结论中药复方制剂1可以延缓rds小鼠视网膜色素变性过程中感光细胞凋亡的发展。     

光损伤对大鼠血-视网膜屏障功能的影响

目的：探讨强光对大鼠血－视网膜屏障功能的影响。方法：大鼠随机分为光照组及对照组,光照组大鼠经散瞳后进行10000lx强光照射（12h光照,12h避光,连续1～14d）,对照组只接受自然光线照射。分别于强光照射后第1、3、7、14 d 摘除相应的光照组和对照组大鼠双侧眼球;并用HE染色观察视网膜各层结构变化,用电镜观察视网膜超微结构变化,用伊凡思蓝（Evans blue,EB）灌注后激光共聚焦显微镜下微循环成像及分光光度法定量检测视网膜微循环通透性变化,来评估血－视网膜屏障变化。结果：大鼠在强光照射1d后就出现视网膜光感受器细胞变性、外节膜盘脱落、外核层厚度变薄等超微结构改变,并随着强光照射持续而逐渐加重,3 d后出现光感受器细胞凋亡,至14 d时外核层厚度已明显变薄、细胞数也明显减少。大鼠在强光照射1 d后视网膜血管就出现EB染料渗漏,至14 d时EB染料渗漏最明显。结论：强光照射可导致大鼠视网膜外核层光感受器细胞变性、凋亡,外核层厚度变薄、细胞数减少,血－视网膜屏障结构、功能破坏。     

视网膜光损伤的防治研究进展

光对视网膜的损伤能诱发活性氧自由基产生,使视网膜细胞处于氧化应激状态,从而造成细胞一系列损伤、凋亡、生物膜溶解和细胞坏死,导致感光细胞的凋亡和视网膜变性,引发眼病,甚至导致视力丧失。目前国内外对视网膜光损伤的防治有一些研究和报道。我们就从视网膜光损伤的机制和多方面的防治进行归纳研究,现综述如下。     

Protective effect of halothane anesthesia on retinal light damage: inhibition of metabolic rhodopsin regeneration

PURPOSE: To determine whether the volatile anesthetic halothane protects against light-induced photoreceptor degeneration in the rodent retina. METHODS: Albino mice and rats were anesthetized with halothane and exposed to high levels of white or blue light. Nonanesthetized animals served as controls. Retinal morphology was assessed by light microscopy, and apoptosis of photoreceptor cells was verified by detection of fragmented genomic DNA and in situ staining of apoptotic nuclei (TUNEL assay). Rhodopsin regeneration after bleaching was determined by measuring rhodopsin levels in retinas of mice or rats at different time points in darkness. RESULTS: Halothane anesthesia reversibly inhibited metabolic rhodopsin regeneration and thus prevented rhodopsin from absorbing high numbers of photons during light exposure. Consequently, photoreceptors of mice and rats anesthetized with halothane were completely protected against degeneration induced by white light. In remarkable contrast, however, halothane anesthesia did not protect against blue-light-induced photoreceptor cell death. CONCLUSIONS: After the initial bleach, halothane impeded photon absorption by rhodopsin by inhibiting metabolic rhodopsin regeneration. Apparently, the rhodopsin-mediated uptake of the critical number of photons to initiate white light-induced retinal degeneration was prevented. In contrast, halothane did not protect the retina against blue light. Blue light can efficiently restore functional rhodopsin from bleaching intermediates through a process termed photoreversal of bleaching. This process does not depend on the visual cycle via the pigment epithelium but nevertheless enables rhodopsin molecules to absorb the critical number of photons required to induce retinal degeneration.     

Minocycline partially inhibits caspase-3 activation and photoreceptor degeneration after photic injury

PURPOSE: To evaluate the possible role of caspase-3 in retinal photic injury, and to investigate whether minocycline can ameliorate light-induced photoreceptor degeneration. METHODS: Retinal photic injury was induced in rats by exposure to intense light. Expression of caspase-3 was studied using Western blot analysis, immunohistochemical staining and enzyme activity assay. Apoptotic photoreceptor cells were detected by the TdT-dUTP terminal nick-end labeling (TUNEL) method. Minocycline (15, 30 or 45 mg/kg) was administered before or after photic injury in rats randomly assigned to pretreatment and posttreatment groups. Minocycline and vehicle-treated retinas subjected to photic injury were compared with respect to Western blotting, enzyme activity assay, quantitative counts of TUNEL stains, morphometry of the outer nuclear layer (ONL) thickness and histopathological examination. RESULTS: After light exposure, active caspase-3 and poly-adenosine diphosphate-ribose-polymerase were upregulated in the retinas and increased caspase-3 immunoreactivity was observed in the ONL. Caspase-3 enzyme activity increased in the retinas that underwent photic injury, and this increase was significantly reduced in minocycline pretreated (30 and 45 mg/kg) and posttreated (45 mg/kg) groups. Intraperitoneal administration of minocycline before or after photic injury in rats also resulted in less TUNEL-positive photoreceptors, as assessed by the quantitative TUNEL counts. The degree of retinal degeneration, measured by the ONL thickness 14 days after photic injury, was significantly improved in minocycline pretreatment (45 mg/kg) rats. CONCLUSIONS: We demonstrate that increased caspase-3 activities localize specifically within the ONL after photic injury, and that minocycline partially inhibits caspase-3 activation and photoreceptor degeneration in this animal model.     

Increased sensitivity to light-induced damage in a mouse model of autosomal dominant retinal disease

PURPOSE: To describe a sensitivity to light-induced damage associated with expression of a T17M mutant human rhodopsin (hT17M) transgene in mice, with the goal of minimizing retinal injury during the subretinal delivery of rAAV-mediated gene therapy. METHODS: Mice were bred to express the hT17M rhodopsin transgene in a line that was hemizygous null for wild-type mouse rhodopsin (mrho(+/-)), and the eyes of transgenic mice and nontransgenic littermates were exposed for 2.5 minutes to unilateral illumination with fiber-optic light ranging from 5,000 to 10,000 lux. Funduscopic images were made with a handheld camera (Genesis; Kowa Company, Ltd., Tokyo, Japan). Full-field scotopic electroretinographic analysis (ERG) was performed to measure loss of retinal function. Morphometry in the light microscope was used to measure loss of rod photoreceptors. TUNEL staining and a nucleosome release assay were used to measure levels of apoptosis in retinal specimens. RESULTS: mrho(+/-);hT17M mice exhibited a sensitivity to light-induced damage that caused severe loss of a- and b-wave ERG responses. hT17M transgenic mice on the mrho(+/+) background were equally sensitive to light-induced damage. Histologic analysis showed a concomitant loss of photoreceptors and TUNEL labeling of fragmented DNA in rod photoreceptor cells, demonstrating that the damage occurred via an apoptotic pathway. Nontransgenic littermate mice were not affected by this exposure to light. Mice expressing an hP23H mutant human rhodopsin transgene were minimally sensitive to light-induced damage at these intensities, in comparison to hT17M mice. Treating the hT17M mice with an equivalent regimen of exposure to red light was less damaging to the retina, as measured by ERG and histology. CONCLUSIONS: Expression of a human hT17M mutant rhodopsin transgene in mice is associated with photoreceptor apoptosis in response to moderate exposure to light. This phenotype was not observed in nontransgenic littermates or in mice expressing an hP23H mutant human rhodopsin transgene. The results suggest that elimination of the glycosylation site at N15 is associated with increased sensitivity to light-induced damage.     

Neuroprotection in the juvenile rat model of light-induced retinopathy: evidence suggesting a role for FGF-2 and CNTF

PURPOSE: In a former study, it was demonstrated that the retina of juvenile Sprague-Dawley (SD) rat has a remarkable intrinsic resistance to light-induced retinopathy compared with the adult retina. The purpose of the present study was to investigate the cellular and molecular mechanisms underlying this endogenous resistance to light-induced damage. METHODS: Juvenile SD rats were exposed for 6 (from P14 to P20) or 14 (from P14 to P28) days to a bright, cyclic, luminous environment of 10,000 lux. Retinal histology was examined immediately after exposure to light or at 2 months of age, and photoreceptor cell death was quantified by measuring the thickness of the outer nuclear layer (ONL) and by TUNEL assays. Changes in protein levels and cellular localization of fibroblast growth factor (FGF)-2, ciliary neurotrophic factor (CNTF), and brain-derived neurotrophic factor (BDNF) were determined by Western blot analysis and retinal immunohistochemistry, respectively. RESULTS: The data demonstrate that although the rate of photoreceptor loss was different after 6 and 14 days of exposure to light, similar ONL thickness was reached at 2 months of age--that is, 4 to 5 weeks after exposure to light. A large number of TUNEL-positive photoreceptors was visualized immediately after 6 and 14 days of exposure to light, reflecting the intense cell death that was occurring in the ONL. Western blot analysis showed that exposure to light induced a strong upregulation of the neurotrophic factors FGF-2 and CNTF in juvenile retinas, whereas no change in BDNF protein expression was noted. Of interest, after exposure to light, endogenous FGF-2 and CNTF were selectively upregulated in Müller cells. CONCLUSIONS: The results show that endogenous expression of FGF-2 and CNTF by Müller glia may play a role in protecting the juvenile retina from light-induced damage.     

短暂清除小胶质细胞减缓光诱导损伤小鼠视网膜视功能退变

目的：探讨小胶质细胞在急性光诱导小鼠视网膜损伤中的保护作用。方法：实验研究。将30 只雄性ICR白化小鼠随机均分为对照组、光损伤组和小胶质细胞清除组。对照组不进行任何处理;光损伤组在15 000 lx白光下照射20 h;小胶质细胞清除组在白光照射前先持续5 d腹腔注射小分子抑制剂PLX5622,随后经15 000 lx白光照射20 h,继续注射3 d PLX5622后于次日取材。所有动物在取材当日,先运用视网膜电图（ERG）检测视网膜功能,随后摘取眼球,OCT包埋眼杯,进行冷冻切片,或者剥离视网膜。运用免疫组织化学和TUNEL染色等方法观察小鼠视网膜损伤程度及小胶质细胞的形态和功能。实验数据采用单因素方差分析进行比较。结果：运用15 000 lx白光照射白化小鼠20 h,可获得视网膜功能损伤和光感受器凋亡的稳定模型。ERG反应的a、b波振幅统计结果显示,光损伤组光感受器和双极细胞出现功能障碍,且TUNEL阳性细胞较对照组多（P=0.035）,激活的小胶质细胞亦明显较多（P<0.001）。免疫组织化学结果显示,清除小胶质细胞组外核层和外网状层的小胶质细胞较光损伤组少（P=0.027）,CtBP2信号和PKC-α信号较强,外节长度维持在正常水平（P<0.001）,但ERG反应b波振幅和TUNEL阳性细胞差异无统计学意义。结论：清除小胶质细胞可以在短期内减少光感受器细胞凋亡,并挽救视网膜功能。     

Neuroprotective effects of naloxone against light-induced photoreceptor degeneration through inhibiting retinal microglial activation

PURPOSE: To determine the role of microglial activation in light-induced photoreceptor degeneration and the neuroprotective effects of naloxone as a novel microglial inhibitor. METHODS: Sprague-Dawley rats were exposed to intense blue light for 24 hours. Daily intraperitoneal injection of naloxone or PBS as a control was given 2 days before exposure to light and was continued for 2 weeks. Apoptotic cells were detected by the TUNEL assay, and anti-OX42 antibody was used to label retinal microglia. Western blot was applied to evaluate the retinal interleukin (IL)-1beta protein levels. Retinal histologic examination and electroretinography (ERG) were also performed to evaluate the effects of naloxone on light-induced photoreceptor degeneration. RESULTS: TUNEL-positive cells were noted in the outer nuclear layer (ONL) of the retina as early as 2 hours and peaked at 24 hours after exposure to light. OX42-positive microglia occurred in the ONL and subretinal space at 6 hours, peaked at 3 days, and changed morphologically from the resting ramified to the activated amoeboid. Expression of IL-1beta protein was also significantly increased at 3 days. Compared with the control, the number of microglia in the outer retina was significantly decreased in the naloxone-treated group at 3 days, and the thickness of ONL and the amplitudes of dark-adapted a- and b-waves were also well preserved at 14 days. CONCLUSIONS: The activation and migration of microglia and the expression of neurotoxic factor (IL-1beta) coincide with photoreceptor apoptosis, suggesting that activated microglia play a major role in light-induced photoreceptor degeneration. Inhibiting microglial activation by naloxone significantly reduces this degeneration.     

Intense light exposure changes the crystallin content in retina

Toward a better understanding of light-induced photoreceptor damage, the crystallin content of rat retina was examined following intense light exposure. Nine crystallin species were identified by mass spectrometric analysis of rat retina fractionated by 2D gel electrophoresis. The Coomassie blue staining intensity of all crystallin 2D gel components was 2- to 3-fold greater in light exposed than in control retinas. Following light exposure, anti-alphaB-crystallin immunoreactivity was increased in rod outer segments and retinal pigment epithelium. These findings support a possible role for crystallins in protecting photoreceptors from light damage.     

Blue light-induced generation of reactive oxygen species in photoreceptor ellipsoids requires mitochondrial electron transport

PURPOSE: To investigate whether photoreceptor ellipsoids generate reactive oxygen species (rOx) after blue light illumination. METHODS: Cultured salamander photoreceptors were exposed to blue light (480 +/- 10 nm; 10 mW/cm(2)). The light-induced catalytic redox activity in the culture was monitored with the use of 3,3'-diaminobenzidine (DAB). Tetramethylrhodamine ethyl ester (TMRE) and 2',7'-dichlorodihydro-fluorescein acetate (DHF-DA) were used as probes to measure the mitochondrial membrane potential and intracellular rOx, respectively. RESULTS: A significant deposit of DAB polymers was found in the culture after exposure to blue light. Basal levels of rOx were observed in photoreceptor ellipsoids when cells were stained with DHF-DA. This staining colocalized with TMRE. After exposure to blue light, a sharp increase of rOx immediately occurred in the ellipsoids of most photoreceptors. When the light intensity was reduced, the response kinetics of rOx generation were slowed down; however, comparable amounts of rOx were generated after a standard time of exposure to light. The production of rOx in photoreceptors was markedly decreased when an antioxidant mixture was included in the medium during exposure to light. Rotenone or antimycin A, the respiratory electron transport blockers at complex I and III, respectively, significantly suppressed the light-evoked generation of rOx. CONCLUSIONS: A robust amount of rOx is produced in the ellipsoid when photoreceptors are exposed to blue light. This light-induced effect is antioxidant sensitive and strongly coupled to mitochondrial electron transport. The cumulative effect of light on rOx generation over time may implicate a role for mitochondria in light-induced oxidative damage of photoreceptors.     

Characterization of multiple light damage paradigms reveals regional differences in photoreceptor loss

Zebrafish provide an attractive model to study the retinal response to photoreceptor apoptosis due to its remarkable ability to spontaneously regenerate retinal neurons following damage. There are currently two widely-used light-induced retinal degeneration models to damage photoreceptors in the adult zebrafish. One model uses constant bright light, whereas the other uses a short exposure to extremely intense ultraviolet light. Although both models are currently used, it is unclear whether they differ in regard to the extent of photoreceptor damage or the subsequent regeneration response. Here we report a thorough analysis of the photoreceptor damage and subsequent proliferation response elicited by each individual treatment, as well as by the concomitant use of both treatments. We show a differential loss of rod and cone photoreceptors with each treatment. Additionally, we show that the extent of proliferation observed in the retina directly correlates with the severity of photoreceptor loss. We also demonstrate that both the ventral and posterior regions of the retina are partially protected from light damage. Finally, we show that combining a short ultraviolet exposure followed by a constant bright light treatment largely eliminates the neuroprotected regions, resulting in widespread loss of rod and cone photoreceptors and a robust regenerative response throughout the retina.