早期糖尿病大鼠晶状体和视网膜水通道蛋白4表达的研究

目的观察早期糖尿病大鼠晶状体、视网膜水通道蛋白4(aquaporin 4,AQP-4)表达的变化,探讨糖尿病大鼠眼组织水代谢改变的机制。方法 SD大鼠分为正常对照组和糖尿病组。制作糖尿病大鼠模型,于第4、8周取材,用免疫组化法和计算机图像分析系统半定量分析各组大鼠晶状体、视网膜AQP-4表达的变化。结果正常及糖尿病大鼠AQP-4在晶状体上皮均无表达。AQP-4在视网膜上有表达,正常大鼠主要表达于视网膜视杆视锥层、节细胞层和神经纤维层,糖尿病大鼠从内界膜延伸至视细胞层整个视网膜厚度均可见AQP-4阳性表达,特别是在神经节细胞层毛细血管内皮和神经纤维层阳性表达更明显。糖尿病大鼠视网膜组织AQP-4阳性表达标随周龄的延长而增强。结论糖尿病大鼠视网膜AQP-4的表达较正常组增强,提示水通道蛋白的表达增加是糖尿病早期发生视网膜水肿的机制之一。     

早期糖尿病大鼠视网膜中血管生成素-1、血管内皮生长因子的表达

目的:研究血管生成素-1(angiopoietin-1,Ang-1)和血管内皮生长因子(vascular endothelial growth factor,VEGF)在早期糖尿病大鼠视网膜中的表达及其意义。方法:制备类似人类Ⅰ型糖尿病大鼠动物模型,取40只SD雄性大鼠分糖尿病组28只及正常对照组12只,糖尿病组28只SD雄性大鼠腹腔注射STZ65mg.Kg-1,建模成功后分别在1周、2周、3周、4周各取糖尿病组7只及正常对照组3只摘取大鼠眼球,应用免疫组化法检测Ang-1、VEGF在视网膜中的表达。结果:Ang-1、VEGF在正常大鼠视网膜的染色均为阴性,Ang-1在1、2、3周糖尿病大鼠视网膜内界膜、内核层及散在血管旁周细胞及Mǔller细胞中呈阳性表达,4周时呈阴性染色反应,Ang-1在糖尿病大鼠视网膜中表达1周阳性率约为39.5%,2周阳性率约为59.7%,3周阳性率约为78.9%,4周阳性率约为19.0%。VEGF在糖尿病大鼠视网膜中表达1周阳性率约为38.6%,2周阳性率约为54.3%,3周阳性率为78.9%,4周阳性率约为42%。结论:Ang-1、VEGF可能在糖尿病视网膜病变早期形成中起到重要...     

川芎嗪联合前列地尔治疗背景性糖尿病视网膜病变疗效的临床观察

目的:探讨川芎嗪(Ligustrazine)联合前列地尔(Alprostadil)治疗背景性糖尿病视网膜病变(Diabetic retinopathy,DR)的作用与疗效。方法:收集近年我院糖尿病伴背景性DR共73例(112只眼),分别按治疗中应用川芎嗪联合前列地尔或拜阿司匹林分为治疗组与对照组,比较两组治疗前后患者眼底病变改善情况。结果:川芎嗪联合前列地尔的治疗组疗效比拜阿司匹林的对照组好;眼底荧光检查表明,治疗组眼底病变改善明显好于对照组,差异显著(P<0.05)。结论:川芎嗪联合前列地尔对DR有较好的疗效。     

牛磺酸抑制糖尿病大鼠视网膜Muller细胞VEGF表达的研究

目的:进一步了解糖尿病引起视网膜受损的分子机制、探讨牛磺酸保护糖尿病大鼠视网膜损伤的可能机制.方法用链脲佐茵素诱导SD大鼠患糖尿病,分为正常对照组、糖尿病组、1%牛磺酸干预糖尿病组、5%%牛磺酸干预糖尿病组、胰岛素治疗糖尿病组.正常对照组、糖尿病组、胰岛素治疗组饲以基础饲料,牛磺酸干预组饲以基础饲料分别添加1%、5% 牛磺酸的饲料喂养,胰岛素治疗组每天皮下注射20U/kg胰岛素.在第2周、1月、2月、3月取视网膜,用RT-PCR、免疫组织荧光化学、Western-blotting检测视网膜Muller细胞VEGFmRNA和蛋白表达情况.结果:经链脲佐菌素诱导惠糖尿病2周后,SD大鼠视网膜Muller细胞VEGFmRNA和蛋白表达增加,且随病程的延长表达量有持续增加趋势(P＜0.05).患糖尿病3月后,整个视网膜中VEGF免疫染色明显增强,尤以外网状层(OPL)、内网状层(IPL)和视网膜外段变化最明显.牛磺酸干预糖尿病1月后.大鼠视网膜Muller细胞VEGFmRNA和蛋白的表达下调(P＜0.05).结论:牛磺酸抑制糖尿病患者视网膜Muller细胞VEGF的表达,减轻糖尿病引起的视网膜损害.     

肾上腺髓质素在糖尿病视网膜细胞高表达的研究

探讨肾上腺髓质素(Adrenomedullin,AM)在糖尿病视网膜病变(Diabeticretinopathy,DR)发病中的作用。Sprague-Dawley雄性大鼠尾静脉注射链脲佐菌素造模,以血糖测定和尿糖水平测定进行筛选,正常对照组尾静脉注射等量枸橼酸钠缓冲液。成模后继续饲养4周,取出眼球视网膜组织,连续冰冻切片,用免疫组织化学SABC法染色观察各组大鼠视网膜RPE细胞AM的表达情况。糖尿病大鼠成模前,两组动物的体重、血糖和尿糖检测结果间无显著性差异(P>0.05)。成模后4周,糖尿病组与正常组大鼠体重、血糖和尿糖数值差异有显著性意义(P<0.01)。AM在正常组大鼠视网膜节细胞层及内核层均有表达,正常组大鼠视网膜AM的光密度值为76.3±5.3,单位面积AM阳性细胞数为(4.5±1.1)×103/mm2。糖尿病大鼠视网膜内RPE细胞肾上腺髓质素表达显著增强,糖尿病大鼠视网膜RPE细胞AM的光密度值为105.7±11.9,单位面积AM阳性细胞数为(17.9±2.3)×103/mm2。两组相比,差异具有显著性(P<0.01)。AM在糖尿病大鼠视网膜RPE细胞表达量增加很可能是DR发生、发展的重要因素。     

HbA1c和FPG水平与2型糖尿病患者视网膜病变的相关性分析

目的：探讨糖化血红蛋（HbA1c）、空腹血糖（FPG）与2型糖尿病患者视网膜病变（DR）的相关性。方法：对我院2010年1月-2012年12月住院或门诊的336例2型糖尿病（T2DM）患者进行眼底检查或眼底血管荧光素造影检测,按有无视网膜病变分为视网膜病变（DR）组和无视网膜病变（NDR）组,并对HbA1c、FBG水平进行检测。结果：DR组较NDR组HbA1c水平高,差异有统计学意义（P〈0．05）,HbA1c水平和DR分期呈正相关（I=0．526,P〈0．001）,而DR组和NDR组FPG水平差异无无统计学意义（P〉0．05）。结论：可将HbA1c作为2型糖尿病患者视网膜病变发生和发展的监测指标之一。     

苯那普利对糖尿病大鼠肾小球MMP-9影响的实验研究

目的探讨苯那普利对糖尿病大鼠肾小球基质金属蛋白酶-9（MMP-9）表达的影响。方法将雄性SD大鼠30只,随机抽出10只动物作为正常对照组（C组）,其余20只采用腹腔注射STZ制备糖尿病大鼠模型。将这20只造模成功的糖尿病大鼠模型随机分为2组：糖尿病组（DM组）和药物处理组（DB组）。其中药物处理组即DB组大鼠每天应用苯那普利进行治疗性灌胃,C组和DM组用等量自来水灌胃。分别于治疗4、8周后,测定皿糖和内生肌苷清除率,用代谢笼收集24h尿量。利用免疫组织化学染色及图像分析方法,对MMP-9在肾组织不同时间点的含量变化进行定性分析。结果苯那普利治疗组血糖水平和内生肌苷清除率明显降低,与糖尿病模型组相比有显著性差异。免疫组织化学染色显示：MMP-9在正常肾小球有阳性表达;DM组大鼠病程第8周时,MMP-9在肾小球表达明显减弱;DB组大鼠病程第8周时,经苯那普利治疗后,与同期糖尿病组比较表达明显上调。图像分析方法定量分析免疫组织化学染色阳性面积百分比,苯那普利治疗后第4周和第8周糖尿病肾病的大鼠肾小球MMP-9的阳性面积百分比分别为25．55％±3．44％和20．10％±2．11％,与糖尿病组对比有明显的提高（P〈0．01）。结论MMP-9在糖尿病肾小球中的表达随病程进展逐渐减弱,苯那普利可能部分通过上调MMP-9在肾小球中的表达起到保护肾脏的作用。     

醛糖还原酶基因启动区C（-106）T多态性与糖尿病视网膜

目的：探讨醛糖还原酶（AR）基因启动区C（-106）T多态性与糖尿病视网膜病变（DR）的关系。方法：235例江苏汉族人群,其中2型糖尿病无视网膜病变组（NDR）63例,2型糖尿病伴视网膜病变组（DR）82例,正常对照组（YC）90例,用PCR-RFLP方法检测AR基因C（-106）T基因型,比较各组等位基因及基因型分布频率。结果：未发现NDR组和NC组之间AR基因C（-106）T各等位基因及基因型频率有显著差异（P分别为0．4505,0．7279）;DR组中CT及TT基因型频率均高于NC组,CC基因型频率低于NC组（P=0．0239）,DR组T等位基因频率显著高于NC组,C等位基因频率显著低于NC组（P=0．0038）。结论：AR基因启动区C（-106）T多态性与江苏汉族人群DR相关,T等位基因可能是DR的遗传危险因子。     

糖尿病大鼠血糖控制前后肾小管上皮细胞VEGF、TGF-β1及Smad蛋白表达的动态研究

目的动态观察链脲佐菌素（STZ）诱导的糖尿病大鼠血糖控制前后肾小管上皮细胞（TEC）中血管内皮生长因子（VEGF）、转化生长因子β1（TGF-β1）、Smad2/3、Smad4的表达情况,探讨四者在糖尿病大鼠TEC表型转变和肾间质纤维化中可能发挥的作用及相互关系。方法实验动物随机分为5组,依病程长短分为①A组（2周组）,②B组（4周组）,③C组（8周组）,④D组（16周组）,⑤E组（24周组）,每组分别设有正常对照组（N组）和糖尿病组（a组）;另外,16周、24周两组加设胰岛素治疗组（b组）。采用尾静脉注射STZ法复制糖尿病大鼠模型;免疫组织化学方法检测肾小管VEGF、TGF-β1、Smad2/3、Smad4及α-平滑肌肌动蛋白（-αSMA）和纤连蛋白（FN）的表达;Western blot检测肾皮质VEGF和TGF-β1蛋白;PAS染色光镜观察肾小管基底膜变化及细胞外基质沉积情况等形态学改变;生化方法测定血糖、血肌酐及24小时尿蛋白量。结果正常对照组VEGF、TGF-β1及Smad2/3、Smad4在肾小管均有少量表达,-αSMA在肾小管无表达;糖尿病组肾小管前述四者的表达均显著高于正常对照组,且从16周开始肾小管上皮细胞可见α-SMA蛋白阳性表达;糖尿病16周时肾小管VEGF、TGF-β1、Smad2/3、Smad4两两之间呈正相关;随糖尿病进展,α-SMA及FN在肾小管表达增多,24h尿蛋白增多,肾脏肥大指数增大,而VEGF、TGF-β1二者都分别和-αSMA、FN、24h尿蛋白及肾脏肥大指数呈正相关性;胰岛素治疗后,VEGF、TGF-β1、Smad2/3、Smad4及FN的表达都比糖尿病组明显下降,且各指标之间的正相关性依然存在,-αSMA蛋白则呈阴性表达。结论糖尿病肾病大鼠肾小管上皮细胞表达的VEGF、TGF-β1及Smad2/3、Smad4参与了TEC表型转变和肾间质纤维化的发生,并且VEGF和TGF-β1相互作用,共同促进了肾脏损害。胰岛素对DN大鼠TEMT和肾间质纤维化的影响可能部分是通过间接阻断VEGF、TGF-β1和Smad2/3、Smad4在TEC中的合成来实现的。     

转化生长因子β1和snail1参与糖尿病大鼠肾小管上皮细胞向间充质细胞转变

本文旨在观察转化生长因子β1（transforming growth factor-β1,TGF-β1）和锌指转录因子Snail1在糖尿病（diabetes mellitus,DM）大鼠肾组织中的表达,并初步探讨它们与肾小管上皮细胞向间充质细胞转变的关系。链脲佐菌素（streptozotocin,STZ）诱发大鼠DM,按病程分为2、4、8、12、16、20、24周、16周胰岛素治疗（16wA）、20,周胰岛素治疗（20wA）和24周胰岛素治疗（24wA）组（n=6）。其中胰岛素治疗组动物从第13周起用胰岛素控制血糖至正常水平,每一时点均设鼠龄匹配的正常对照组。测定各组血糖、24h尿蛋白、血肌酐（serum creatinine,Scr）、肾脏指数。PAS染色光镜观察肾脏病理学改变。免疫组织化学检测肾脏Snail1、TGF-β1、α-平滑肌肌动蛋白（α-smooth muscle actin,α-SMA）、E-钙黏素和纤连蛋白（fibronectin,FN）的表达;Western blot检测肾皮质Snail1、TGF-β1和E-钙黏素蛋白表达。RT-PCR检测肾皮质Snail1和E-钙黏素mRNA表达。结果显示：（1）DM各组大鼠的血糖、24h尿蛋白、Scr、肾脏指数均较正常对照组明显升高（P〈0．05,P〈0．01）,胰岛素治疗组大鼠上述指标均较DM组显著降低（P〈0．01）。（2）TGF-β1和Snail1免疫组织化学阳性染色见于DM各组大鼠肾小管,正常对照组未见阳性表达,胰岛素治疗组大鼠弱阳性表达,并随治疗时间延长而减少。从16周开始在DM大鼠肾小管上皮细胞可见α-SMA蛋白阳性表达,胰岛素治疗组大鼠未见α-SMA蛋白表达;DM组大鼠E-钙黏素蛋白阳性染色明显少于正常对照组。（3）DM组大鼠肾皮质TGF-β1和Snail1蛋白以及Snail1 mRNA表达较正常对照组显著增高（P〈0．01）,胰岛素治疗组大鼠则显著低于DM组（P〈0．01）;DM组E-钙黏素mRNA和蛋白表达与TGF-β1和Snail1呈相反变化。结果提示,TGF-β1和Snail1可能参与DM大鼠肾小管上皮细胞向间充质细胞转变,胰岛素治疗可抑制两者表达并阻断肾小管上皮细胞向间充质细胞转变。     

Effect of endothelin dual receptor antagonist on VEGF levels in streptozotocin-induced diabetic rat retina

Diabetic retinopathy (DR), one of the most serious causes of blindness, is often associated with the upregulation of vascular endothelial growth factor (VEGF) in retina. Recently, leukocyte adhesion (leukostasis) is blamed for the occlusion of retinal capillary vascularity, which ultimately contributes to the progression of diabetic retinopathy. In addition, intercellular adhesion molecule-1 (ICAM-1), a representative factor for leukostasis, is increased in the diabetic retina. Endothelin (ET)-1, a potent vasoconstrictor peptide, is deeply linked to the pathogenesis of diabetic retinopathy. Different therapeutic interventions concerning VEGF have already been proposed to prevent diabetic retinopathy. However, no study yet has reported whether ET-1 dual receptor antagonist could alter the upregulated VEGF and ICAM-1 levels in the diabetic retina. The present study investigated the effect of ET(A/B) dual receptor antagonist (SB209670; 1 mg/rat/day) on the expression of VEGF and ICAM-1 in the diabetic rat retina. Diabetes was induced by intraperitoneal injection of streptozotocin (STZ; 65 mg/kg) in Sprague-Dawley rats, whereas control rats (non-DM control) received only citrate buffer. After 1 week, the STZ-administered rats were randomly divided into two groups: one group (DM+SB209670) received ET(A/B) dual receptor antagonist for 2 weeks, and a vehicle group (DM+vehicle) was treated only with saline. After the treatment period, the retinas were removed from the eyeballs. In DM+vehicle group, the VEGF expression of the retinas was significantly increased (32.8 pg/mg) in comparison to that in the non-DM control group (26.2 pg/mg); this upregulation of VEGF was reversed in the DM+SB209670 group (28.6 pg/mg). The expression of retinal ICAM-1 was increased in the DM+vehicle group (152.2 pg/mg) compared with the non-DM control group (121.6 pg/mg). However, SB209670 treatment did not alter the expression of retinal ICAM-1 level (154.8 pg/ml) in DM rats. Thus we conclude that an ET(A/B) dual receptor antagonist could reverse the expression level of VEGF in the diabetic retina while failing to normalize the upregulated ICAM-1 expression.     

康柏西普在糖尿病大鼠早期视网膜病变中的作用及对VEGF、ICAM-1及CRP的影响

摘要 目的：探讨康柏西普在糖尿病大鼠早期视网膜病变中的作用及对血管内皮生长因子 (vascular endothelial growth factor,VEGF)和细胞间粘附分子-1(Intercellular adhesion molecule-1,ICAM-1)及C-反应蛋白(C-reactive protein,CRP)的影响。方法：糖尿病大鼠早期视网膜病变模型(n=27)随机平分为三组-模型组、替米沙坦组与康柏西普组,造模成功后当天三组分别给予注射生理盐水、替米沙坦、康柏西普治疗,1次/w,持续4 w,检测VEGF、ICAM-1及CRP表达情况。结果：大鼠造模成功后均出现食欲增多、饮水、尿量、体重减轻的现象。替米沙坦组与康柏西普组治疗第1 w与第4 w的体重高于模型组(P<0.05),康柏西普组高于替米沙坦组(P<0.05)。替米沙坦组与康柏西普组治疗第1 w与第4 w的空腹血糖低于模型组(P<0.05),康柏西普组低于替米沙坦组(P<0.05)。替米沙坦组与康柏西普组治疗第4 w的视网膜VEGF、ICAM-1、CRP蛋白相对表达水平低于模型组(P<0.05),康柏西普组低于替米沙坦组(P<0.05)。康柏西普组视网膜厚度变薄不明显,内、外核层细胞排列整齐,神经纤维层未见明显空泡样变性。结论：康柏西普在糖尿病大鼠早期视网膜病变中的应用能抑制VEGF、ICAM-1及CRP的表达,能促进降低血糖,增加大鼠体重。     

VEGF165b对糖尿病大鼠视网膜神经节细胞保护作用的研究

目的:VEGF165b是新发现的血管内皮生长因子的变构体之一,本研究将观察其对糖尿病大鼠视网膜神经节细胞的抗凋亡作用.方法:采用四氧嘧啶诱发糖尿病大鼠模型,分为正常对照组(CON),糖尿病组(DM),糖尿病VEGF165b低剂量治疗组(DMT1)、中剂量治疗组(DMT2),糖尿病高剂量治疗组(DMT3),糖尿病单纯胰岛素治疗组(DMT4),所有治疗组在糖尿病成模后1个月开始治疗.2个月后处死各组大鼠,摘取眼球进行光镜形态学观察、核苷酸末端转移酶介导的dUTP缺口翻译法(TUNEL法)视网膜神经节细胞凋亡检测.结果:VEGF165b治疗使糖尿病大鼠视网膜光镜形态学改变减轻,能有效的抑制视网膜神经节细胞凋亡.VEGF165b治疗组视网膜神经节凋亡细胞数较DM组明显减少(P＜0.01),与糖尿病大鼠单纯胰岛素治疗组相比差异也有统计学意义.随着VEGF165b浓度的增加视网膜神经节细胞凋亡个数减少,但1ng/μL组与10ng/μL组相比差异无统计学意义.结论:VEGF165b对视网膜神经节细胞有保护作用,可能对糖尿病视网膜病变具有治疗有意义.     

Dietary Taurine Supplementation Prevents Glial Alterations in Retina of Diabetic Rats

The preventive effect of dietary taurine supplementation on glial alterations in retina of streptozotocin-induced diabetic rats was examined in this study. Blood glucose content, content of taurine, glutamate and <gamma>-amino butyric acid (GABA) and expression of glial fibrillary acid protein (GFAP), vascular endothelial growth factor (VEGF), glutamate transporter (GLAST), glutamine synthetase (GS) and glutamate decarboxylase (GAD) in retina were determined in diabetic rats fed without or with 5% taurine in a controlled trial lasting 12 weeks, with normal rats fed without or with 5% taurine served as controls. Dietary taurine supplementation could not lower glucose concentration in blood (P > 0.05), but caused an elevation of taurine content and a decline in levels of glutamate and GABA in retina of diabetic rats (P < 0.05). The content of GABA in normal control group was not altered by taurine supplementation. With supplementation of taurine in diet, lower expression of GFAP and VEGF while higher expression of GLAST, GS and GAD in retina of diabetic rats were determinated by RT-PCR, Western-blotting and immunofluorescence (P < 0.05). GFAP, VEGF, GLAST, GS and GAD expressions in normal controls were not altered by taurine treatment. This may have prospective implications of using taurine to treat complications in diabetic retinopathy.     

吡格列酮治疗STZ诱导糖尿病大鼠视网膜中TSP-1的变化

观察吡格列酮在治疗STZ诱导糖尿病大鼠视网膜中血小板反应素Ⅰ(thmmbospondin-1,TSP-1)的变化,探讨噻唑烷二酮类药物在糖尿病视网膜病变中的影响和可能作用机制。用链脲佐菌素(STZ)腹腔注射建立大鼠糖尿病模型,随机分正常对照组(C组),糖尿病安慰剂组(D组)和糖尿病吡格列酮治疗组(DP组),8周后取视网膜组织行免疫组织化学、RT-PCR半定量检测TSP- 1mRNA水平的变化,结果显示在糖尿病早期视网膜神经节细胞层、内核层中均有明显的TSP1表达,对比于糖尿病对照组,糖尿病治疗组视网膜单位面积表达TSP1的细胞数量要较二组对照组高;糖尿病治疗组、糖尿病对照组和正常对照组间TSP-1的荧光灰度值分别为528.2±47.78、493.4±39.36和417.7±28.74:内参照GAPDH的荧光灰度值分别为796.4±21.18、811.2±36.80和832.4±43.18,各组间TSP-1 mRNA表达有显著性差异,提示吡格列酮对糖尿病早期视网膜中TSP-1的表达变化有影响,吡格列酮可能在延缓糖尿病视网膜病变的发生发展中起一定的作用。     

Diabetes-induced nitrative stress in the retina, and correction by aminoguanidine

Aminoguanidine inhibits the development of retinopathy in diabetic animals, but the mechanism remains unclear. Inasmuch as aminoguanidine is a relatively selective inhibitor of the inducible isoform of nitric oxide synthase (iNOS), we have investigated the effects of hyperglycemia on the retinal nitric oxide (NO) pathway in the presence and absence of aminoguanidine. In vivo studies utilized retinas from experimentally diabetic rats treated or without aminoguanidine for 2 months, and in vitro studies used bovine retinal endothelial cells and a transformed retinal glial cell line (rMC-1) incubated in 5 mm and 25 mm glucose with and without aminoguanidine (100 microg/mL). NO was detected as nitrite and nitrate, and nitrotyrosine and iNOS were detected using immunochemical methods. Retinal homogenates from diabetic animals had greater than normal levels of NO and iNOS (p < 0.05), and nitrotyrosine was greater than normal, especially in one band immunoprecipitated from retinal homogenates. Oral aminoguanidine significantly inhibited all of these increases. Nitrotyrosine was detected immunohistochemically only in the retinal vasculature of non-diabetic and diabetic animals. Retinal endothelial and rMC-1 cells cultured in high glucose increased NO and NT, and aminoguanidine inhibited both increases in rMC-1 cells, but only NT in endothelial cells. Hyperglycemia increases NO production in retinal cells, and aminoguanidine can inhibit this abnormality. Inhibition of diabetic retinopathy by aminoguanidine might be mediated in part by inhibition of sequelae of NO production.     

Interaction between NO and COX pathways in retinal cells exposed to elevated glucose and retina of diabetic rats

A nonselective inhibitor of cyclooxygenase (COX; high-dose aspirin) and a relatively selective inhibitor of inducible nitric oxide synthase (iNOS; aminoguanidine) have been found to inhibit development of diabetic retinopathy in animals, raising a possibility that NOS and COX play important roles in the development of retinopathy. In this study, the effects of hyperglycemia on retinal nitric oxide (NO) production and the COX-2 pathway, and the interrelationship of the NOS and COX-2 pathways in retina and retinal cells, were investigated using a general inhibitor of NOS [N(G)-nitro-l-arginine methyl ester (l-NAME)], specific inhibitors of iNOS [l-N(6)-(1-iminoethyl)lysine (l-NIL)] and COX-2 (NS-398), and aspirin and aminoguanidine. In vitro studies used a transformed retinal Müller (glial) cell line (rMC-1) and primary bovine retinal endothelial cells (BREC) incubated in 5 and 25 mM glucose with and without these inhibitors, and in vivo studies utilized retinas from experimentally diabetic rats (2 mo) treated or without aminoguanidine or aspirin. Retinal rMC-1 cells cultured in high glucose increased production of NO and prostaglandin E(2) (PGE(2)) and expression of iNOS and COX-2. Inhibition of NO production with l-NAME or l-NIL inhibited all of these abnormalities, as did aminoguanidine and aspirin. In contrast, inhibition of COX-2 with NS-398 blocked PGE(2) production but had no effect on NO or iNOS. In BREC, elevated glucose increased NO and PGE(2) significantly, whereas expression of iNOS and COX-2 was unchanged. Viability of rMC-1 cells or BREC in 25 mM glucose was significantly less than at 5 mM glucose, and this cell death was inhibited by l-NAME or NS-398 in both cell types and also by l-NIL in rMC-1 cells. Retinal homogenates from diabetic animals produced significantly greater than normal amounts of NO and PGE(2) and of iNOS and COX-2. Oral aminoguanidine and aspirin significantly inhibited all of these increases. The in vitro results suggest that the hyperglycemia-induced increase in NO in retinal Müller cells and endothelial cells increases production of cytotoxic prostaglandins via COX-2. iNOS seems to account for the increased production of NO in Müller cells but not in endothelial cells. We postulate that NOS and COX-2 act together to contribute to retinal cell death in diabetes and to the development of diabetic retinopathy and that inhibition of retinopathy by aminoguanidine or aspirin is due at least in part to inhibition of this NO/COX-2 axis.