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。     

氨基胍对缺血-再灌注损伤视网膜形态和功能的影响

视网膜缺血-再灌注损伤（retina ischemia reperfusion injury,RIRI）是眼科临床常见的病理过程,可导致视网膜结构和功能的严重损伤,其损伤机制复杂,治疗效果不理想,影响患者视功能的恢复。研究发现NO在RIRI中发挥着重要作用,一氧化氮合酶（nitric oxide synthase,NOS）是NO合成的限速酶,应用NOS抑制剂氨基胍（aminoguanidine,AG）对大鼠青光眼视网膜神经节细胞具有明显的保护作用,而AG对RIRI有无保护作用尚不清楚。     

川芎嗪对大鼠缺血再灌注视网膜SOD，MDA和NO水平及细胞凋亡的影响

目的 观察川芎嗪对大鼠视网膜缺血再灌注后视网超氧化物歧化酶（SOD）,丙二醛（MDA）,一氧化氮（NO）水平及细胞凋亡的影响。方法 采用大鼠视网膜压力缺血再灌注模型,分光光度法测定SOD,MDA和NO,琼脂糖凝胶电泳分析DNA断裂。结果 川芎嗪能显著对抗视膜膜缺血再灌注后视网膜SOD水平的下降及MDA和NO水平的升高;同时能阻断缺血再灌注12h后细胞DNSA凋亡样断裂。结论 川芎嗪可能通过抑制自由基的产生和提高抗氧化能力来对抗大鼠视网缺血再灌注诱导的细胞凋亡。     

一氧化氮与眼病的关系

一氧化氮（nitric oxide,NO）是近年来眼科疾病的研究热点.其在体内一氧化氮合酶（nitric oxide synthase,NOS）的催化下生成,作为一种小分子介质调节眼部血流.诸多脉络膜视网膜疾病如葡萄膜炎、青光眼、缺血-再灌注损伤、前部缺血性视神经病变、糖尿病视网膜病变以及视网膜色素变性等发病机制中均发现NO的异常,与NO相拮抗的内皮素-1（Endothelin-1,ET-1）也日益受到关注.对NO合成途径的干预有望成为一种新的眼科治疗手段.     

3'-大豆苷元磺酸钠在缺血再灌注损伤时对视网膜抗氧化能力及NOS活性的影响

目的 研究3'-大豆苷元磺酸钠(DSS)在大鼠视网膜缺血再灌注损伤时对视网膜抗氧化能力及一氧化氮合酶(NOS)活性的影响.方法 24只SD大鼠按随机数字表法分成4组:对照组、视网膜缺血再灌注模型组、低剂量(1 mg/kg)及高剂量(2 mg/kg)DSS组.颈总动脉夹闭法制作视网膜缺血再灌注模型,比色法测定血清丙二醛(MDA)、一氧化氮(NO)浓度及NOS、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)活性.结果 与对照组相比,模型组血清MDA浓度升高(P＜0.05),NO浓度降低、eNOS活性降低(P＜0.01).与模型组比较,低剂量DSS组MDA浓度降低、SOD活性升高(P＜0.05);高剂量DSS组GSH-Px活性升高(P＜0.05),tNOS活性升高(P＜0.05);DSS低剂量组(P＜0.05)及高剂量组(P＜0.01)NO浓度与模型组比较均升高,低剂量及高剂量DSS组eNOS活性均升高(P＜0.05).结论 DSS可通过提高eNOS、SOD及GSH-Px的活性,从而提高视网膜缺血再灌注损伤时的抗氧化能力及NO的释放,降低组织细胞损伤,保护眼功能.     

自由基与视网膜缺血-再灌注损伤

自由基在视网膜缺血-再灌注损伤中占有重要地位。视网膜缺血-再灌注时黄嘌呤氧化酶（XO）增加，花生四烯酸代谢系统环氧化旁路，线粒体功能障碍，一氧化氮合成酶（NOS）激活及中性粒细胞系统被激活，使自由基生成大大增加。体内清除自由基的酶系统和抗氧化剂不足以清除生成的自由基而引起脂质，蛋白质和核酸等生物大分子的氧化损伤。给予外源性的自由基清除剂和抗氧化酶等通过加速自由基清除或抑制自由基生成而减轻视网膜缺血-再灌注损伤。     

α-硫辛酸对视网膜缺血-再灌注损伤大鼠视网膜中血管内皮生长因子和基质金属蛋白酶表达的影响

目的探讨α-硫辛酸对大鼠视网膜缺血-再灌注损伤中血管内皮生长因子（VEGF）和基质金属蛋白酶-9（MMP-9）表达的影响及作用机制。方法按随机数字表法将78只SPF级健康成年Wistar大鼠分为正常对照组6只及α-硫辛酸组和缺血-再灌注组各36只。α-硫辛酸组和缺血-再灌注组根据再灌注时间的不同分为6、12、24、48h,3d和7d组。采用前房灌注生理盐水升高眼压的方法建立视网膜缺血-再灌注动物模型,其中α-硫辛酸组大鼠自造模前3d开始腹腔注射α-硫辛酸100mg/（kg.d）,缺血-再灌注组大鼠以同样的方法注射等体积生理盐水。在上述时间点分别处死大鼠并摘除眼球,行苏木精-伊红染色评估各组大鼠在各时间点视网膜组织的结构变化;应用免疫组织化学法检测大鼠视网膜缺血-再灌注损伤后不同时间点视网膜组织中VEGF和MMP-9的表达,并对各组VEGF和MMP-9的表达量进行比较。结果缺血-再灌注组大鼠在再灌注后6h出现视网膜水肿和视网膜神经节细胞（RGCs）的轻度改变,随着时间的延长,RGCs的结构改变明显加重。α-硫辛酸组视网膜水肿和RGCs结构的异常变化过程同缺血-再灌注组,但程度较轻。实验前后正常对照组大鼠视网膜中未见VEGF的表达;缺血-再灌注组于再灌注后12h开始出现VEGF的表达,随着时间的延长VEGF表达逐渐增加,到再灌注后48h达到高峰。各时间点缺血-再灌注组和α-硫辛酸组VEGF在视网膜的表达水平明显高于正常对照组,差异均有统计学意义（P〈0.05）,但各时间点α-硫辛酸组VEGF在视网膜的表达水平明显低于缺血-再灌注组,差异均有统计学意义（P〈0.05）。正常对照组大鼠视网膜未检测到MMP-9的表达;缺血-再灌注组在再灌注后6h可检测到MMP-9在视网膜中的表达,24h后其表达水平达到高峰。各时间点缺血-再灌注组MMP-9的表达明显高于正常对照组（P均〈0.05）,α-硫辛酸组视网膜中MMP-9的表达与缺血-再灌注组比较明显下降,差异均有统计学意义（P〈0.05）。缺血-再灌注组视网膜VEGF和MMP-9的表达呈正相关（r=0.834,P〈0.05）。结论视网膜缺血-再灌注损伤可诱导VEGF及MMP-9的表达在视网膜中过度表达,α-硫辛酸可通过抑制视网膜缺血-再灌注损伤中VEGF及MMP-9的表达而对视网膜起保护作用。     

选择性一氧化氮合酶抑制剂对鼠视网膜缺血再灌注损伤影响的光镜观察

目的:观察选择性一氧化氮合酶(nitric oxide synthase,NOS)抑制剂7-硝基-吲唑(7-nitro-indazole,7-NI)、氨基胍(aminoguanidine,AG)对视网膜缺血再灌注损伤影响的组织学变化,探讨NOS亚型神经原型和诱导型及其所产生的一氧化氮(nitric oxide,NO)在视网膜缺血再灌注损伤中的作用.方法:56只Sprague Dawley大鼠随机分为正常对照组、阴性对照组、缺血再灌注非处理组、7-NI缺血前处理组、7-NI再灌注前处理组、AG缺血前处理组、AG再灌注前处理组及7-NI+AG处理组计八组.采用升高眼内压的方法诱导视网膜缺血,100分钟后,缓慢降压至正常眼内压,使视网膜再灌注.用7-NI或/和AG处理动物(腹膜腔内注射),光镜观察视网膜组织学变化,图像分析仪测量视网膜内层(IRL)、内网层(IPL)厚度及神经节细胞(RGCs)的数目.结果:缺血再灌注非处理鼠IRL和IPL厚度比正常对照鼠明显变薄(P＜0.0001),RGCs层和内核层神经细胞比正常对照鼠明显减少,残留者排列紊乱,空泡形成及核固缩现象多见.7-NI缺血前处理鼠和AG再灌注前处理鼠IRL和IPL分别比缺血再灌注非处理鼠明显增厚(P＜0.001),RGCs层和内核层神经细胞少数丢失,排列稍乱,偶见空泡形成.7-NI+AG处理鼠IRL和IPL分别比7-NI缺血前处理组和AG再灌注前处理鼠都增厚(P＜0.001),RGCs层和内核层神经细胞排列基本整齐,偶见核固缩现象.结论:神经原型和诱导型NOS及其产生的NO在视网膜缺血再灌注损伤中起重要作用;选择性NOS抑制剂7-NI和AG能够选择性抑制神经原型和诱导型NOS的活性,从而抑制NO的生成,对视网膜缺血再灌注损伤有保护作用.     

选择性一氧化氮合酶抑制剂对大鼠视网膜缺血再灌注损伤影响的光镜观察

目的观察选择性一氧化氮合酶(nitric oxide synthase,NOS)抑制剂7-硝基-吲唑(7-nitro-indazole,7-NI)、氨基胍(aminoguanidine,AG)对视网膜缺血再灌注损伤影响的组织学变化,探讨NOS亚型神经原型和诱导型及其所产生的一氧化氮(nitric oxide,NO)在视网膜缺血再灌注损伤中的作用.方法56只Sprague Dawley大鼠随机分为正常对照组、阴性对照组、缺血再灌注非处理组、7-NI缺血前处理组、7-NI再灌注前处理组、AG缺血前处理组、AG再灌注前处理组及7-NI+AG处理组计八组.采用升高眼内压的方法诱导视网膜缺血,100分钟后,缓慢降压至正常眼内压,使视网膜再灌注.用7-NI或/和AG处理动物(腹膜腔内注射),光镜观察视网膜组织学变化,图像分析仪测量视网膜内层(IRL)、内网层(IPL)厚度及神经节细胞(RGCs)的数目.结果缺血再灌注非处理鼠IRL和IPL厚度比正常对照鼠明显变薄(P＜0.0001),RGCs层和内核层神经细胞比正常对照鼠明显减少,残留者排列紊乱,空泡形成及核固缩现象多见.7-NI缺血前处理鼠和AG再灌注前处理鼠IRL和IPL分别比缺血再灌注非处理鼠明显增厚(P＜0.001),RGCs层和内核层神经细胞少数丢失,排列稍乱,偶见空泡形成.7-NI+AG处理鼠IRL和IPL分别比7-NI缺血前处理组和AG再灌注前处理鼠都增厚(P＜0.001),RGCs层和内核层神经细胞排列基本整齐,偶见核固缩现象.结论神经原型和诱导型NOS及其产生的NO在视网膜缺血再灌注损伤中起重要作用;选择性NOS抑制剂7-NI和AG能够选择性抑制神经原型和诱导型NOS的活性,从而抑制NO的生成,对视网膜缺血再灌注损伤有保护作用.     

缺血-再灌注大鼠视网膜TPA变化与视网膜水肿的关系

目的　探讨缺血-再灌注对大鼠视网膜分泌组织型纤溶酶原激活物(TPA)的影响及其与视网膜水肿的关系。方法　采用提高眼压法造成视网膜缺血后,恢复眼压形成血流再灌注。实验分正常对照组、缺血 1h再灌注 1h组、缺血 1h再灌注 2h组、缺血 2h再灌注 1h组和缺血 2h再灌注 2h组。每组各取 10例测试视网膜组织TPA的活性和含水量。结果　缺血-再灌注后,大鼠视网膜组织TPA的活性和含水量随缺血和再灌注时间的延长而升高(P<0 01)。 结论　缺血-再灌注可引起视网膜组织TPA的活性升高和视网膜水肿,是视网膜组织结构和功能损伤的因素之一。     

高眼压缺血-再灌注中视网膜NOS的表达及L-NAME对其表达的影响

目的探讨高眼压缺血－再灌注损伤时视网膜一氧化氮合酶（ＮＯＳ）的表达、Ｌ－ＮＡＭＥ对ＮＯＳ表达的影响及视网膜ＮＯＳ的作用。方法用免疫组织化学ＳＡＢＣ法检测ｎＮＯＳ，ｅＮＯＳ和ｉＮＯＳ在高眼压缺血－再灌注模型视网膜中的表达及ＮＯＳ抑制剂Ｌ－ＮＡＭＥ对其表达的影响。结果在高眼压缺血－再灌注下，视网膜神经节细胞、神经胶质细胞ｎＮＯＳ，ｅＮＯＳ，ｉＮＯＳ均呈阳性，对照组ｎＮＯＳ呈弱阳性；注射Ｌ－ＮＡＭＥ后，在高眼压缺血－再灌注中，视网膜神经节细胞、神经胶质细胞均呈ｉＮＯＳ强阳性，ｎＮＯＳ，ｅＮＯＳ呈阴性。结论在高眼压缺血－再灌注中ｎＮＯＳ，ｅＮＯＳ，ｉＮＯＳ合成的一氧化氮（ＮＯ）可能对视网膜细胞等有细胞毒性作用；Ｌ－ＮＡＭＥ通过抑制ｎＮＯＳ，ｅＮＯＳ的活性，对高眼压缺血－再灌注视网膜损伤产生保护作用。     

急性高眼压状态大鼠视网膜一氧化氮 及其合酶变化的研究

目的通过对急性高眼压下大鼠视网膜一氧化氮(nitric oxid e,NO)及其合酶(nitric oxide synthase,NOS)变化的分析,探讨一氧化氮在高眼压视网膜损伤中的作用。方法Wistar大鼠60只,随机分成为高眼压30 min组;高眼压60 min组;高眼压90 min组;高眼压后12 h组和高眼压后24 h 组。前房加压灌注成高眼压模型。利用镀铜镉还原法测定视网膜中NO₂^－/NO₃^－ 的 含量从而间接反映视网膜组织中NO的含量。利用免疫组织化学法研究视网膜内神经结构型一氧化氮合酶(neuronal constitutive nitric oxide synthase,ncNOS)的分布及其变化。结果正常及缺血大鼠视网膜神经结构型一氧化氮合酶(ncNOS)主要位于大鼠视网膜内核层内侧,节细胞层,内丛状层。急性高眼压30min,60min ,90min大鼠视网膜NO的含量逐渐下降(P<0.01),ncNOS阳性 细胞数也逐渐减少(P<0.05),阳性物质表达减弱;急性高眼压 90min后再灌注过程中,NO的含量比90min时明显升高(P<0.05),但与正常比较仍显著下降(P<0.01)。ncNOS阳性细胞数继续减少(P <0.01)。结论一氧化氮参与了急性高 眼压下视网膜损伤过程;通过ncNOS催化的途径合成的NO对缺血以及缺血再灌注的视网膜可能具有重要的作用。(中华眼底病杂志,2001,17:230-233)     

Activation of the cGMP/nitric oxide signal transduction system by nicotine in the retina

Acetylcholine is one of the primary excitatory neurotransmitters/neuromodulators in the retina, but little is known about the downstream signaling pathways it can activate. The present study immunocytochemically examines the potential sources of acetylcholine and the location of the nicotinic cholinergic receptors in the turtle retina. It also examines how activation of these receptors can influence the nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) signal-transduction pathways. Photoreceptors, amacrine cells, and potentially ganglion cells contain choline acetyltransferase-like immunoreactivity (LI). Nicotinic acetylcholine receptors are immunocytochemically localized on photoreceptors, horizontal, bipolar, and ganglion cells. Nitric oxide imaging indicates that stimulation with nicotine increases NO production primarily in photoreceptors, horizontal, Muller, bipolar, and ganglion cells. In turn, very select populations of amacrine cells respond to this NO with increased levels of cGMP-LI. Selective inhibitors reveal that nitric oxide synthase is involved in most, but not all, of these increases in cGMP-LI. These results show that acetylcholine can activate the NO/cGMP signal-transduction pathways in both the inner and outer retina. This indicates that both of the major excitatory retinal transmitters, glutamate and acetylcholine, can stimulate NO production that increases levels of cGMP-LI in overlapping populations of retinal cells.     

Imaging of nitric oxide in the retina

Nitric oxide (NO) is the most widespread signaling molecule found in the retina in that it can be made by every retinal cell type. NO is able to influence a wide variety of synaptic mechanisms ranging from increasing or decreasing neurotransmitter release to the modulation of gap junction conductivity. Although biochemical methods can analyze overall levels of NO, such methods cannot indicate the specific cell types involved. In the last few years, fluorescent imaging methods utilizing diaminofluorescein have allowed the real-time visualization of neurochemically or light stimulated NO-induced fluorescence (NO-IF) in specific retinal cells. Recent experiments have shown that this NO-IF can be stabilized using paraformaldehyde fixation. This aldehyde stabilization has allowed the imaging of NO production in the dark and in response to light, as well as the neurochemical modulation of light stimulated NO production. The results of these studies indicate that NO is not always freely diffusible and that NO is largely retained in many cells which make it. The NO production in retina is highly damped in that in the absence of stimulation, the endogenous levels of NO production are extremely low. Finally, different neurochemical or light stimulation protocols activate NO production in specific cells and subcellular compartments. Therefore, although the NO signaling is widespread in retina, it is very selectively activated and has different functions in specific retinal cell types. The use of NO imaging will continue to play a critical role in future studies of the function of NO in retina and other neural systems.     

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.     

电刺激大鼠小脑顶核对视网膜缺血再灌注损伤的保护作用

目的　探讨电刺激大鼠小脑顶核对视网膜缺血再灌注损伤的保护作用。方法　大鼠随机分为缺血再灌注组、电刺激组和假手术组。观察视网膜形态学改变 ;用NADPH黄递酶组织化学染色法 (NADPH NDP)观察视网膜内诱导型一氧化氮合酶 (iNOS)的表达 ;采用TUNEL法检测视网膜细胞凋亡情况。结果　 (1)缺血再灌注组的内视网膜层 (包括内核层、内丛状层、节细胞 )、神经纤维层和内界膜厚度增加 ,尤其是内丛状层厚度明显高于假手术组 (t=3 6 80 ,P <0 0 1) ;电刺激组的内视网膜厚度与假手术组相比 ,差异无显著意义 (t=1 0 6 4 ,P >0 0 5 ) ;(2 )光镜观察可见缺血再灌注组有明显的细胞核染色质致密浓缩、核碎裂等改变 ,电刺激组仅见少量核浓缩及碎裂 ;(3)电刺激组的iNOS阳性的神经节细胞数明显低于缺血再灌注组 ,其差异有显著意义 (t=3 32 6 ,P <0 0 1) ;(4)电刺激组大鼠发生凋亡的视网膜细胞数明显低于缺血再灌注组 ,其差异有显著意义 (t=4 0 38,P <0 0 1)。结论　电刺激大鼠小脑顶核对缺血再灌注所导致的视网膜组织损伤具有保护作用。     

Nitric oxide: ocular blood flow, glaucoma, and diabetic retinopathy

Nitric oxide (NO) is widely recognized to be quite an important intercellular messenger in the cardiovascular and nervous systems or immunological reactions, including that in the eye. This molecule formed by constitutive NO synthase (NOS), endothelial (eNOS) and neuronal (nNOS), contributes to physiologically regulate ocular hemodynamics and cell viability and protects vascular endothelial cells and nerve cells or fibers against pathogenic factors associated with glaucoma, ischemia, and diabetes mellitus. Ocular blood flow is regulated by NO derived from the endothelium and efferent nitrergic neurons. Endothelial dysfunction impairs ocular hemodynamics by reducing the bioavailability of NO and increasing the production of reactive oxygen species (ROS). On the other hand, NO formed by inducible NOS (iNOS) expressed under influences of inflammatory mediators evokes neurodegeneration and cell apoptosis, leading to serious ocular diseases. NO over-produced by nNOS in the retina stimulated by excitotoxic amino acids or exposed to ischemia also mediates retinal injury. Because of these dichotomous roles of NO, which has both beneficial and pathogenic actions, one may face difficulties in constructing therapeutic strategies with NO supplementation or NOS inhibition. Up-to-date information concerning physiological roles of NO produced by the different NOS isoforms in the eye and interactions between NO and glaucoma, retinal ischemia, or diabetic retinopathy would help clinicians to select a valid pharmacological therapy that would be appropriate for a specific ocular disease.     

Activation of the aldosterone/mineralocorticoid receptor system and protective effects of mineralocorticoid receptor antagonism in retinal ischemia-reperfusion injury

The purpose of this project was to investigate the effects of the mineralocorticoid receptor antagonist against retinal ischemia-reperfusion injury and identify the aldosterone/mineralocorticoid receptor (MR) system in the rat retina. Retinal ischemia was induced by increasing intraocular pressure to 130?mmHg. Rats were treated with the angiotensin II type 1 receptor (AT1-R) antagonist (candesartan), MR antagonist (spironolactone), or aldosterone. Retinal damage was evaluated at 7 days after the ischemia by measuring the retinal thickness and the number of retinal ganglion cells. Pretreatment with candesartan, spironolactone, or candesartan and spironolactone significantly inhibited retinal ischemic injury. However, there was no protective effect against retinal ischemia-reperfusion injury provided by the combined aldosterone with candesartan treatment. Additionally, pretreatment with aldosterone alone also did not provide any neuroprotective effects against retinal ischemia-reperfusion injury. When rats were treated via local administration of aldosterone in the absence of ischemia, the number of retinal ganglion cells decreased while the retinal thickness remained unchanged. The present findings demonstrated the existence of a local aldosterone/MR system in the retina. Our results also demonstrated that an MR antagonist can attenuate subsequent ischemic damage in the rat retina.     

川芎嗪对大鼠缺血再灌注视网膜SOD,MD和NO水平及细胞凋亡的影响

目的 :观察川芎嗪对大鼠视网膜缺血再灌注后视网膜超氧化物歧化酶 (superoxidedismutase ,SOD)、丙二醛 (malondialdehyde ,MDA)、一氧化氮 (nitricoxide ,NO)水平及视网膜细胞凋亡的影响。方法 :采用大鼠视网膜压力缺血再灌注模型 ,分光光度法测定SOD、MDA和NO ,琼脂糖凝胶电泳分析DNA断裂。结果 :视网膜缺血 6 0min再灌注后SOD水平下降 ,而MDA和NO水平则升高 ;视网膜缺血 6 0min再灌注 12h ,提取DNA进行琼脂糖凝胶电泳可见凋亡样DNA断裂 (ApoptoticDNAfragmentation)。川芎嗪能显著对抗视网膜缺血再灌注时视网膜SOD水平的下降、MDA和NO水平的升高 ;同时能阻断大鼠视网膜缺血再灌注 12h后视网膜细胞DNA凋亡样断裂。结论 :川芎嗪可能通过抑制自由基的产生和提高抗氧化能力来对抗大鼠视网膜缺血再灌注诱导的细胞凋亡