特异性阻断剂Nec-1对视网膜缺血再灌注损伤模型小鼠坏死性凋亡的影响及作用

目的 探讨特异性阻断剂Nec-1对视网膜缺血再灌注损伤(retinal ischemia reperfusion injury,RIRI)模型中坏死性凋亡的影响及作用.方法 取60只野生型C57小鼠随机分为实验组、对照组及空白组.每组20只.在进行Nec-1对坏死性凋亡影响研究中,空白组15只小鼠不做任何处理,实验组15只小鼠给予玻璃体内注射2 μL Nec-1 (2 mol·L-1)预处理,对照组15只小鼠无预处理;预处理4h后实验组及对照组通过前房灌注法建立RIRI模型;再灌注损伤后3d,每种检测方法各取5只小鼠分别于取材后行Western blot、免疫荧光定量PCR、免疫荧光染色,检测以下基因mRNA和蛋白的表达变化:IL-13、IL-6、TNF-α、RIP3、RIP1及Caspase-8.在进行Nec-1对视网膜神经节细胞(retinal ganglion cell,RGC)的影响研究中,实验组、对照组及空白组各5只小鼠纳入荧光金标记.空白组小鼠荧光金标记后不做任何处理.荧光金标记7d后,实验组小鼠给予玻璃体内注射2μL Nec-1(2 mol·L-1)预处理,对照组无预处理.预处理4h后实验组及对照组通过前房灌注法建立RIRI模型.再灌注损伤后3d,收集视网膜组织行铺片RGC计数研究.结果 与对照组相比,实验组RIP3、RIPI的mRNA表达明显降低,差异均有统计学意义(均为P ＜0.001);IL-1β、IL-6、TNF-α的mRNA表达亦均明显降低,差异均有统计学意义(均为P＜0.001);Caspase-8的表达变化不明显,与对照组相比差异无统计学意义(P =0.654 8).通过Western blot检测发现,实验组RIP3蛋白表达较对照组明显降低,其变化趋势与RIP3 mRNA水平的变化基本一致.通过视网膜切片免疫荧光染色检测亦发现,实验组RIP3蛋白表达较对照组明显降低.实验组全视网膜铺片中每个视野下荧光金逆行标记RGC数(197.3±3.6)较对照组(107.5±6.1)明显增多,差异有统计学意义(P＜0.001).结论 实验性RIRI模型中,Nec-1能阻断坏死性凋亡,并显著增加RGC数.     

视网膜缺血--再灌注损伤的保护

视网膜缺血-再灌注损伤是目前研究较多的一个课题,其损伤机制复杂,目前通过各种实验研究探索其损伤机制以及减轻或防止缺血-再灌注损伤的药物和方法很多.现就视网膜缺血-再灌注损伤的保护机制进行总结归纳.     

大鼠视网膜缺血-再灌注损伤超微结构观察及机制探讨

目的研究大鼠视网膜缺血-再灌注损伤中的超微结构改变以及凋亡相关基因表达的变化,探讨其损伤机制。方法将28只大鼠随机分为正常组和手术组,手术组按照再灌注后不同时间段分为1h、6h、12h、24h、48h、72h组。前房加压法制作大鼠视网膜缺血-再灌注损伤模型,透射电镜检测视网膜超微结构改变,免疫组织化学法检测视网膜组织中bcl-2、bax、Fas的表达。结果(1)正常组视网膜神经纤维中微管及线粒体清晰可见;视网膜神经节细胞(retinal ganglion cells,RGCs)核大,电子密度低,核仁明显,细胞器丰富;再灌注损伤后RGCs核膜肿胀,线粒体嵴模糊不清,可见凋亡小体,神经纤维中微管模糊、减少甚至消失,以再灌注后24h为甚;(2)再灌注后6h,bax表达逐渐递增,24h达到高峰,48h开始下降,72h不明显;(3)bcl-2在视网膜神经节细胞层、纤维层及内核层有微弱表达,各个时间段变化不明显;(4)Fas表达改变与bax基本一致。结论视网膜缺血-再灌注损伤中,细胞凋亡是引起视网膜内核层和神经节细胞层细胞死亡的主要方式,其损伤机制与凋亡相关基因bcl-2、bax、Fas的表达变化有关。     

核转录因子-κB在大鼠视网膜缺血再灌注损伤中的表达

目的建立大鼠视网膜缺血再灌注(retinal ischemical reperfusion injury,RIRI)模型,观察不同程度损伤的视网膜组织病理改变,检测凋亡的存在,探讨核转录因子-κB(nuclear factor-kappa B,NF-κB)在大鼠RIRI中的表达。方法SD大鼠随机分为角膜损伤组和缺血再灌注组,分别建立角膜损伤模型和建立15kPa、60min高眼压视网膜缺血模型,光镜观察视网膜损伤后的组织病理改变,原位细胞凋亡检测,免疫组织化学检测NF-κB的表达情况。结果角膜损伤组视网膜无改变,缺血再灌注组出现组织病理改变,包括视网膜水肿,空泡变性,核固缩、溶解,结构紊乱等,随再灌注时间的延长,视网膜损害加重。原位细胞凋亡检测中,缺血再灌注组的凋亡细胞阳性表达均分布于神经节细胞层及内核层细胞,24h时表达最强。NF-κB在再灌注6h开始表达,24h表达最强。结论RIRI主要导致神经节细胞层及内核层细胞损伤,NF-κB的表达和凋亡可能是损伤的重要机制,且二者有着密切联系,表现出一致的规律性。     

缺血再灌注损伤诱导大鼠视网膜细胞凋亡

目的 观察缺血再灌注大鼠视网膜损伤及细胞凋亡情况。 方法 采用升高大鼠眼压到109.725 mm Hg(1 mm Hg=0.133 kPa)持续1 h的方法制作视网膜缺血再灌注模型，采用常规眼球切片观察不同缺血和再灌注时间的视网膜损伤的组织病理改变；采用DNA琼脂糖凝胶电泳法检测视网膜神经元凋亡情况；采用DNA原位末端标记(terminal dUTP nick end labelling, TUNEL)法定位凋亡的视网膜细胞。 结果 缺血30 min 再灌注24、48 h的大鼠视网膜无明显的病理改变；缺血60 min再灌注24、48 h的大鼠视网膜神经节细胞层和内核层细胞明显变薄；缺血60 min再灌注12、24 h的大鼠视网膜有梯状条带。而正常对照组、缺血30 min再灌注24、48 h组及缺血60 min再灌注48 h组大鼠视网膜均无类似表现。TUNEL法显示视网膜内的细胞凋亡主要发生在节细胞和内核层光感受细胞。 结论 大鼠视网膜缺血再灌注主要是导致视网膜神经节细胞层和内核层细胞损伤，细胞凋亡可能是损伤的重要机制。 (中华眼底病杂志, 2002, 18: 296-298)     

视网膜缺血-再灌注损伤干预治疗新进展

视网膜缺血-再灌注损伤(RIRI)是眼科常见的病理生理损伤性眼病,常发生于视网膜动静脉阻塞、糖尿病视网膜病变、急性闭角型青光眼等与缺血相关的眼病.表现为缺血性患眼在血液再灌注后,细胞功能发生代谢性障碍,视网膜组织结构损伤,视功能下降.缺血-再灌注损伤是由多种因素共同作用的结果,目前公认的假说主要包括氧自由基的损伤、细胞内的钙超载、白细胞介素介导作用和细胞凋亡等.但目前对于RIRI的保护及治疗研究有限,本文就国内外有关RIRI的干预治疗新进展综述如下.     

家兔视网膜缺血-再灌注损伤后形态学的动态变化

目的　观察家兔视网膜缺血 -再灌注后视网膜结构的动态变化。方法　通过前房灌注加压至 16 .7k Pa,维持 1h,建立缺血模型 ,观察再灌注 2～ 14d内其结构变化及内层视网膜平均厚度的变化。结果　家兔视网膜在缺血 -再灌注后 2～ 14d内表现为神经细胞持续丢失 ,视网膜层次逐渐不清 ,萎缩、变薄。其中神经节细胞、神经纤维及视锥、视杆对缺血最敏感 ,外颗粒层次之 ,内颗粒层最能耐受缺血 -再灌注后的损伤。结论　视网膜缺血 -再灌注损伤是个持续性、进行性的损伤过程 ,与功能变化相一致     

Caspase-7与视网膜缺血-再灌注损伤

视网膜缺血-再灌注损伤是细胞凋亡、炎症反应等多种机制参与的病理生理过程。半胱氨酸蛋白酶７（Ｃａｓｐａｓｅ-７）是Ｃａｓｐａｓｅ家族中的凋亡效应分子,近期研究表明,它在调控炎症细胞因子方面也起着重要作用。本文就Ｃａｓｐａｓｅ-７生物学特性及其与细胞凋亡、炎症反应在视网膜缺血-再灌注损伤的分子机制进行综述。     

一氧化氮与视网膜缺血-再灌注损伤

一氧化氮（NO）在视网膜缺血-再灌注损伤中占重要地位。视网膜缺血-再灌注时,一氧化氮合成酶（NOS）被多种炎性介质和细胞因子激活,使NO大量生成。NO是一种活性很强的自由基,具有广泛的生物学活性。在缺血-再灌注早期,少量NO可降低缺血缺氧对视网膜的损伤程度;晚期过多的NO可通过多种途径对视网膜造成损害。就目前有关NO在视网膜缺血-再灌注损伤中的研究进展进行综述。     

糖尿病视网膜病变缺血再灌注损伤中相关调控因子的研究进展

糖尿病视网膜病变（DR）是一种常见的缺血性眼病。DR视网膜缺血再灌注（RIR）损伤的缺血过程、再灌注损伤过程及其结局3个阶段中氧化应激、钙超载、一氧化氮（NO）、兴奋性氨基酸（EAA）等因素引起多种炎性介质和基因过度表达,进而造成炎症反应、损伤、凋亡和坏死等组织改变和功能障碍。这些过程受凋亡基因、促红细胞生成素、单核细胞趋化蛋白1、血管内皮生长因子（VEGF）、碱性成纤维细胞生长因子（bFGF）、基质细胞衍生因子-1、NO、一氧化氮合酶（NOS）等多种相关因子的调控。就其中主要调控因子的特点及临床应用方面的研究进展进行综述。     

Expression of matrix metalloproteinases and their inhibitors in experimental retinal ischemia-reperfusion injury in rats

Matrix metalloproteinases (MMPs) are endopeptidases that degrade the extracellular matrix (ECM) and are involved in the pathogenesis of retinal degeneration along with tissue inhibitors of metalloproteinases (TIMPs). The present study examined the expression and activation of two specific members of MMPs (MMP-2 and MMP-9) and their related inhibitors (TIMP-1 and TIMP-2) in an experimental retinal ischemia-reperfusion injury. Retinal ischemia-reperfusion injury (RIRI) was induced in adult rats with a ligation method. After one hour of ischemia and a varied reperfusion time (0, 3, 6, 12, 24, 48 and 76 hr), the rat eyes were enucleated. Retinal extracts underwent zymographic analysis to measure the activity of MMP-2/9. The activity of TIMP-1 and TIMP-2 was measured by reverse zymography. The protein level was examined by Western blot. Immunohistochemistry analysis was undertaken to assess the anatomical distribution of MMP-9 in the retina after RIRI. The gelatinolytic activity of ProMMP-2 (72 kDa) was increased markedly at 6 hr after RIRI. ProMMP-9 (92 kDa) was not detected in the control specimens, while it appeared at 3 hr, increased markedly at 6 hr, and reached maximal levels at 24 hr after RIRI. The gelatinolytic activity found ian retinal extracts was shown to be inhibited by 10 m M EDTA and activated in vitro by a known metalloproteinase activator (4-aminophenylmercuric acetate (APMA)), indicating that these enzymes were of the metalloproteinase class. By western blot, MMP-2/9 levels increased parallel to protein activity level in zymography. No corresponding increase in TIMP-1 and TIMP-2 protein activity and protein level was detected by reverse zymography and western blot. Elevated levels of MMP-9 and its distribution in retina were confirmed by immunohistochemistry. Expression of MMP-9 was detected in the inner and outer segments of rat retina, and the level becomes stronger at 24 hr after RIRI. In this study, ProMMP-2 and ProMMP-9 were expressed and increased significantly, but their inhibitors (TIMP-1 and TIMP-2) remained relatively unaltered in ischemic retina after RIRI in rats. These results suggest that MMP-2 and MMP-9 may play an important role in the pathomechanism of retinal ischemic injury.     

复方五花血藤对鼠视网膜缺血再灌注损伤的保护作用

目的　探讨复方五花血藤对大鼠视网膜缺血再灌注损伤的保护作用。方法　采用健康成年ＳＤ大鼠９０只,随机分为正常对照组、模型组和治疗组,其中模型组和治疗组建立视网膜缺血再灌注模型,治疗组于造模前连续３ｄ给予复方五花血藤灌胃,每天２次（总剂量为１０ｇ?ｋｇ－１?ｄ－１）,模型组和正常对照组灌服相同剂量的生理盐水。通过ＨＥ染色、透射电镜观察再灌注６ｈ、１２ｈ、２４ｈ、４８ｈ与７２ｈ各组大鼠视网膜的形态学变化,原位杂交方法检测各组视网膜凋亡相关因子ｓｕｒｖｉｖｉｎ的表达情况。结果　与正常对照组相比,模型组再灌注后６ｈ视网膜神经节细胞层高度水肿,内丛状层明显增厚,部分视网膜神经节细胞发生空泡变性,但随时间延长病变逐渐减轻。治疗组各时间点视网膜损伤均较模型组轻。与正常对照组相比,模型组ｓｕｒｖｉｖｉｎ在再灌注后６ｈ即开始增加,２４ｈ达到高峰,以后逐渐下降,但各时间点均高于正常对照组（均为Ｐ＜０．０５）。与模型组相比,治疗组ｓｕｒｖｉｖｉｎ各时间点表达均较高（均为Ｐ＜００５）。结论　初步证实复方五花血藤对于大鼠视网膜缺血再灌注损伤有较为明显的保护作用,此研究对于临床药物的开发和利用具有积极意义。     

缺血预处理对大鼠视网膜缺血再灌注损伤保护作用

目的：探讨缺血预处理是否对视网膜缺血再灌注损伤有保护作用及其机理,方法：利用前房灌注生理盐水形成高眼压的视网膜缺血再灌注损伤的动物模型,视网膜缺血时间为1h分别于缺血前30min、24h或72h对大鼠一只眼5min短暂缺血即预处理,24h或72h后行视网膜电图（ERG）、电镜、光镜、丙二醛（MDA）及热休克蛋白70（HSP70）检测,或者一侧眼行5min假处理,24h后行1h缺血,24h或72h再行上述检测,所有对侧眼不作处理作对照,结果：与假处理相相比,缺血前24、72h进行预处理后的大鼠视网膜光镜、电镜表现损害明显减轻,ERGb波明显恢复（P＜0．01）,MDA含量降低（P＜0．01）,缺血前30min预处理的视网膜表现严重的损害,ERGb波几安全消失,结论：缺血预处理对视网膜缺血再灌注损伤有保护作用,且有一定时限性。     

视网膜缺血再灌注损伤和细胞因子的研究进展

视网膜缺血再灌注（retinal ischemia reperfusion,RIR)损伤是由多种因素，如自由基，炎性细胞因子，白细胞等介导的复杂病理生理过程，RIR损伤和细胞因子之间存在密切联系，一方面，RIR损伤可以诱导多种炎性细胞因子，如白细胞介素1和6的表达，另一方面，多种外源性神经营养因子，如碱性成纤维细胞生长因子，重组人肝细胞生长因子，脑源性神经生长因子等，具有保护RIR损伤的作用，研究RIR损伤和细胞因子关系具有重要意义，因此我们报告了国外有关RIR损伤和细胞因子的最新研究进展。     

AQP-1和VEGF在大鼠视网膜缺血再灌注损伤中的表达

目的:研究大鼠视网膜缺血再灌注损伤后水通道蛋白-1和血管内皮生长因子的表达情况并探讨二者在视网膜缺血再灌注损伤中表达的意义。方法:通过提高眼内压的方法建立大鼠视网膜缺血再灌注模型,于术后6,12,24,48h;3,7d用免疫组化的方法检测水通道蛋白-1和血管内皮生长因子在大鼠视网膜的表达情况,并以正常大鼠视网膜作为对照。结果:正常对照组大鼠视网膜未能检测到血管内皮生长因子的阳性表达,缺血再灌注12h后开始出现表达,48h达高峰,且差异具有统计学意义(P<0.01)。水通道蛋白-1在正常对照组可见到阳性表达,缺血再灌注组随时间增长表达不断增强,7d在外层视网膜也出现表达,差异有统计学意义(P<0.01)。结论:水通道蛋白-1和血管内皮生长因子在视网膜缺血性疾病中起着重要的作用,二者可能共同影响了视网膜的水代谢过程。     

Protective effects of catalase on retinal ischemia/reperfusion injury in rats

Retinal ischemia/reperfusion (I/R) injury causes profound tissue damage, especially retinal ganglion cell (RGC) death. The aims of the study were to investigate whether catalase (CAT) has a neuroprotective effect on RGC after I/R injury in rats, and to determine the possible antioxidant mechanism. Wistar female rats were randonmized into four groups: normal control group (Control group), retinal I/R with vehicle group (I/R with vehicle group), retinal I/R with AAV-CAT group (I/R with AAV-CAT group), and normal retina with AAV-CAT group (normal with AAV-CAT group). One eye of each rat was pretreated with recombinant adeno-associated virus containing catalase gene (I/R with AAV-CAT group or normal with AAV-CAT group) and recombinant adeno-associated virus containing GFP gene (I/R with vehicle group) by intravitreal injection 21 days before initiation of I/R injury. Retinal I/R injury was induced by elevating intraocular pressure to 100 mmHg for 1 h. The number of RGC and inner plexiform layer (IPL) thickness were measured by fluorogold retrograde labeling and hematoxylin and eosin staining at 6 h, 24 h, 72 h and 5d after injury. Hydrogen peroxide (H₂O₂), the number of RGC, IPL thickness, malondialdehyde(MDA), 8-hydroxy-2-deoxyguanosine (8-OHdG), CAT activity and nitrotyrosine were measured by fluorescence staining, immunohistochemistry and enzyme-linked immunosorbent assay analysis at 5 days after injury. Electroretinographic (ERG) evaluation was also used. Pretreatment of AAV-CAT significantly decreased the levels of H₂O₂, MDA, 8-OHdG and nitrotyrosine, increased the catalase activity, and prevented the reduction of a- and b- waves in the I/R with AAV-CAT group compare with the I/R with vehicle group (p < 0.01). Catalase attenuated the I/R-induced damage of RGC and IPL and retinal function. Therefore, catalase can protect the rat retina from I/R-induced injury by enhancing the antioxidative ability and reducing oxidative stress, which suggests that catalase may be relevant for the neuroprotection of inner retina from I/R-related diseases.     

Activation of autophagy in photoreceptor necroptosis after experimental retinal detachment

AIM:To investigate whether photoreceptor necroptosis induced by z-VAD-FMK (pan caspase inhibitor) was involved the activation of autophagy and whether Necrostatin-1, a specific necroptosis inhibitor, could inhibit this induction of autophagy after experimental retinal detachment.METHODS:Experimental retinal detachment models were created in Sprague-Dawley rats by subretinal injection of sodium hyaluronate and subretinal injections of z-VAD-FMK, vehicle or z-VAD-FMK plus Necrostatin-1. Three days after retinal detachment, morphologic changes were observed by transmission electron microscopy. In other animals, retinas were subjected to immunoprecipitation and Western Blotting, then probed with anti-RIP1, phosphoserine, LC-3II or caspase 8 antibody.RESULTS:It was proved by immunoprecipitation and western blotting, that photoreceptor necroptosis was mediated by caspase-8 inhibition and receptor interacting protein kinase (RIP1) phosphorylation activation. Transmission electron microscope and western blotting results indicated that photoreceptor necroptosis was involved the LC-3II and autophagosomes induction. We also discovered Necrostatin-1 could inhibit RIP1 phosphorylation and LC-3II induction.CONCLUSION:These data firstly indicate photoreceptor necroptosis is associated with the activation of autophagy. Necrostatin-1 protects photoreceptors from necroptosis and autophagy by down-regulation of RIP1 phosphorylation and LC-3II.     

促红细胞生成素对大鼠急性缺血视网膜节细胞的保护作用

目的:探讨重组人促红细胞生成素(recombinant human erythropoietin,rhEPO)对大鼠视网膜缺血再灌注损伤(retina ischemia reperfusion injury,RIR)中视网膜神经节细胞(retinal ganglion cell,RGC)的保护作用。方法:成年雌性SD大鼠20只,采用夹闭视网膜动脉30min造成大鼠双眼缺血再灌注模型。所有大鼠均于建模前1h给予左眼rhEPO10U(6μL),右眼给予同等剂量眼用平衡盐液。按照建模后眼球取材时间不同(1,4,7,14d)分为4组,每组5只,均于取材前4d利用荧光金(fluorogold,FG)逆行标记大鼠RGC,视网膜铺片RGC计数,比较双眼存活RGC数量。结果:rhEPO治疗眼RGC存活数多于平衡盐液对照眼。结论:rhEPO对大鼠视网膜急性缺血后RGC具有保护作用。     

尼莫地平对兔视网膜缺血再灌注损伤保护作用的实验研究

目的　探讨尼莫地平对实验性视网膜缺血再灌注损伤的保护作用及其机理。方法　 72只兔随机分为阴性对照组、模型组、尼莫地平组 ,经前房恒压 ( 110mmHg) ( 1kPa =7.5mmHg)灌注生理盐水 6 0min ,建立视网膜缺血损伤的动物模型。并于造模前 6h及 1h尼莫地平组行尼莫地平溶液灌胃 ,模型组及对照组予等量生理盐水灌胃。观察缺血再灌注损伤后 1、3、7、15d各组视网膜组织学及超微结构变化 ,计数视网膜神经节细胞 (retinalganglioncells,RGC) ,视网膜内层厚度 (thicknessofinnerretinallayers ,TIRL) ,测定丙二醛(malondialdehyde,MDA)、超氧化物歧化酶 (superoxidedismutase,SOD)含量。 结果　模型组再灌注后 1d ,视网膜各层结构即有不同程度的损伤 ,3、7、15d出现节细胞数目减少 ,视网膜内层厚度变薄 ,伴随MDA升高和SOD降低 ,上述变化随时间延长而加重。尼莫地平治疗组RGC和TIRL均从第 3天开始较模型组有显著性增加 ,RGC计数 7d时增加最显著 (P <0 .0 1) ;IRL厚度 15d时增加最显著 (P <0 .0 1)。尼莫地平组各时间点MDA含量均低于模型组 ,而SOD活性都高于模型组 ,差异有显著性。各时间点RGC计数减少和IRL厚度降低呈显著正相关 (r =0 .90 5 ,F =2 7.2 7,P <0 .0 1) ;视网膜MDA含量的降低和SOD活性的升高呈显著