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。     

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。     

视网膜缺血再灌注损伤后视网膜神经节细胞pax6的表达变化与意义

【摘要】 目的 探索视网膜缺血再灌注损伤后视网膜神经节细胞pax6的表达变化及意义。 设计 实验研究。研究对象 缺血再灌注损伤大鼠视网膜。方法 成年健康雄性Sprague-Dawley大鼠 30只,随机选取5只作为空白对照组,其余25只为视网膜缺血再灌注损伤组,采用升高右眼眼压的 方法制作视网膜缺血再灌注损伤模型。视网膜缺血再灌注后1、2、4、6、8周分5组,每组5只,不 同时间点取右眼行免疫荧光染色,观察视网膜神经节细胞中pax6表达情况。主要指标 pax6的表达 。结果 视网膜缺血再灌注损伤后随着时间推移视网膜各层逐渐出现pax6表达阳性的细胞,对照组 视网膜神经节细胞pax6表达阳性率为（1.28±1.41）%,损伤后1、2、4、6、8周分别为（0.99± 1.23）%、（14.45±2.72）%、（50.88±4.73）%、（71.00±4.72）%、（78.80±4.62）% （F=1.350,P＜0.0001）。各组与对照组两两比较,缺血后1周视网膜神经节细胞pax6表达阳性率 差异无统计学意义（P=0.835）,缺血再灌注损伤2、4、6、8周视网膜神经节细胞pax6表达阳性率 均明显升高（P均＜0.0001）。结论 视网膜缺血再灌注损伤后视网膜各层均出现pax6表达阳性细胞 ,视网膜缺血再灌注损伤能诱导视网膜内源性干细胞激活。（眼科, 2012, 21： 414-417）     

缺血再灌注损伤小鼠视网膜中铜蓝蛋白的表达

目的 探讨小鼠视网膜缺血再灌注(RIR)损伤后铜蓝蛋白在视网膜中的表达和意义.方法 采用前房灌注法建立RIR损伤动物模型,免疫组织化学方法 结合计算机图像分析系统定性和半定量分析不同时段视网膜组织中铜蓝蛋白的表达.结果 正常小鼠视网膜仅在神经节细胞层可见铜蓝蛋白表达,缺血再灌注损伤后6 h,铜蓝蛋白开始在内丛状层、内核层和外丛状层表达并逐渐增加,48 h达高峰,之后下降.结论 铜蓝蛋白参与了RIR损伤的早期过程,其作用可能是清除自由基和保护视网膜.     

视网膜缺血再灌注损伤后HSP70、NF-κB的表达和细胞凋亡的关系

目的探讨大鼠视网膜缺血再灌注损伤后热休克蛋白70（HSP70）、核蛋白因子-κB（NF-κB）表达和损伤神经细胞凋亡的关系。方法前房加压法制作大鼠视网膜缺血再灌注损伤模型，28只大鼠随机分为正常组和缺血再灌注组，后者又按照再灌注后不同时间段分为1、6、12、24、48、72h组。应用末端脱氧核酸转移酶介导的脱氧三磷酸尿苷缺口末端标记（TUNEL）法检测视网膜神经细胞凋亡指数（AI），过氧化物酶标记的链酶卵白素（SP）免疫组织化学法检测视网膜组织中HSP70、NF-κB的表达。结果视网膜神经细胞的凋亡出现于再灌注后6h，并逐渐递增，24h达到高峰．48h开始下降，72h不明显。HSP70在再灌注后1h即有表达，随时间段延长表达逐渐增加，至再灌注后24h达到高峰．之后渐下降。NF-κB的表达变化与凋亡细胞变化的规律基本一致。结论视网膜缺血再灌注损伤导致神经节细胞的凋亡：HSP70和NF-κB在视网膜缺血再灌注损伤中表达升高，对神经细胞的凋亡起着重要的调节作用。     

紫外线及缺血—再灌注诱导晶体和视网膜自由基产生的研究

晶体和视网膜紫外线照射及视网膜缺血分别是白内障及视网膜病变的主要原因。本文用电子自旋共振(ESR)方法研究了兔晶体和视网膜经紫外线照射及视网膜缺血-再灌注后自由基信号的变化。发现晶体在紫外线照射后超氧自由基信号出现。视网膜在紫外线照射及缺血-再灌注后有新的自由基信号产生,这种变化可能是晶体和视网膜损伤的重要原因。     

小鼠视网膜缺血-再灌注后核因子-κB的激活

目的 研究小鼠视网膜缺血-再灌注所致视网膜神经细胞凋亡中,核因子-κB的表达。方法 通过升高小鼠眼内压造成视网膜缺血,用计算机图像分析方法测量视网膜再灌注后神经细胞凋亡的比例和视网膜厚度的改变。免疫组化标记核因子-κB p65亚单位,并与原位缺口末端标记（TUNEL）做双重荧光标记,分析核因子-κB的表达与细胞凋亡之间的时相关系。结果 视网膜缺血-再灌注后最初24h,视网膜内层厚度增加,至168h,厚度显著减少。再灌注后6h,神经节细胞和内核细胞层中p65的免疫表达增强,至24h达到高峰,这一过程与TUNEL标记的时相一致。结论 视网膜缺血-再灌注损伤后,核因子-κB的激活对于视网膜神经细胞的凋亡有重要作用,对于其起促进凋亡还是抑制凋亡的作用,则有待于进一步研究。     

雌二醇对视网膜缺血-再灌注损伤后基质细胞衍生因子-1表达的影响

目的 通过观察视网膜缺血-再灌注损伤后基质细胞衍生因子-1(stromal cell-derived factor-1,SDF-1)的表达情况,以及雌二醇对SDF-1的表达及调控作用,研究雌二醇对大鼠视网膜缺血-再灌注损伤的保护作用机制.方法 通过升高大鼠眼压的方法建立视网膜缺血-再灌注损伤模型,应用RT-PCR和Western blot方法检测视网膜缺血-再灌注损伤后各时间点(6h、12h、24 h)视网膜SDF-1表达情况;腹腔注射雌二醇后观察雌二醇对视网膜缺血-再灌注损伤后SDF-1表达的影响;并且应用雌激素受体拮抗剂ICI 182-780研究雌激素受体对雌二醇诱导视网膜缺血-再灌注损伤后的SDF-1的影响.结果 SDF-1 mRNA和蛋白在缺血-再灌注6h组、缺血-再灌注12h组和缺血-再灌注24 h组表达均增加,与正常对照组比较差异均有统计学意义(均为P＜0.05),并且在缺血-再灌注12h组SDF-1表达达高峰.雌二醇预处理对视网膜缺血-再灌注具有一定的保护作用;缺血-再灌注+雌二醇组SDF-1 mRNA和蛋白表达增加,与缺血-再灌注对照组及缺血-再灌注+溶剂对照组比较差异均有统计学意义(均为P ＜0.05).与缺血-再灌注+雌二醇组相比较,缺血-再灌注+雌二醇+ICI182-780组SDF-1 mRNA和蛋白表达明显降低,差异均有统计学意义(均为P＜0.05).结论 雌二醇对视网膜缺血-再灌注损伤的保护作用是通过雌激素受体介导的SDF-1 mRNA和蛋白表达增强实现的.     

氟桂嗪对视网膜缺血损伤保护作用的实验研究

目的：观察钙离子通道阻滞剂氟桂嗪（flunarizine）在视网膜缺血损伤中的保护作用。方法：用TBA法测定兔眼视网膜缺血再灌注不同时期脂质过氧化代谢产物丙二醛（MDA）含量变化，比较实验组、氟桂嗪治疗组的差异。结果：实验组、治疗组在缺血再灌注30，60，90min时与正常对照组相比MDA含量均有显著增加（P＜0.01）；氟桂嗪治疗组MDA含量在缺血再灌注30，60，90min3个时间段均明显低于实验组（P＜0.01）。结论:细胞内Ca^2 超载参与了视网膜组织缺血损伤的病理生理过程。氟桂嗪对急性视网膜缺血损伤具有保护作用。     

Y27632对急性视网膜缺血再灌注大鼠视网膜组织形态学的影响

研究Rho激酶抑制剂Y27632 对视网膜缺血再灌注损伤大鼠视网膜组织形态学的影响。方法：实验研究。将60只SD大鼠随机分为4组，每组15只：正常对照组（正常组）、急性缺血再灌注损伤组（IRI组）、0.9%氯化钠溶液对照组（生理盐水组）、Y27632治疗组（Y27632组）。再灌注损伤后24 h（10只）和168 h（5只）处死各组动物，行HE染色、ADP酶染色检查，光镜下观察大鼠视网膜组织病理学变化及视网膜厚度变化。数据采用单因素方差分析。结果：正常组大鼠视网膜结构清晰，三层细胞结构排列整齐。IRI组于再灌注24 h后视网膜厚度增加，内外丛状层组织疏松，视网膜节细胞、内外核层细胞水肿明显、排列紊乱，视网膜节细胞减少。168 h后，视网膜水肿消退、厚度变薄、呈萎缩状，神经节细胞及内外核层细胞数量减少，在视网膜前和神经纤维层可见毛细血管。再灌注 24 h后，IRI组视网膜厚度较正常组增加（P=0.005），Y27632组视网膜厚度低于生理盐水组（P=0.032）。再灌注168 h后，IRI组视网膜厚度低于正常组（P<0.001），Y27632组视网膜厚度较生理盐水组增加（P=0.025）。正常组大鼠视网膜血管自视乳头发出，向四周呈放射状均匀分布，毛细血管网结构清晰。再灌注24 h后，IRI组视网膜血管管径变细，走行较僵直，分支减少，视乳头周围及中周部视网膜可见大片无灌注区，无灌注区周围可见新生血管芽渐成网状。Y27632组可见视乳头周围及中周部视网膜局部无灌注区形成，无灌注区周围可见新生血管。后极部4PD无灌注区面积明显小于IRI组及生理盐水组。结论：Y27632玻璃体腔注射可以减轻视网膜缺血再灌注早期的视网膜水肿，减少视网膜神经节细胞的凋亡，减少视网膜新生血管生成，减轻再灌注晚期的视网膜萎缩，具有视神经保护作用。     

Free radical-mediated effects in reperfusion injury: a histologic study with superoxide dismutase and EGB 761 in rat retina

In Sprague-Dawley rats, retinal ischemia was induced by occlusion of the central retinal artery, while reperfusion was initiated by unclamping and removing the occluder. Ninety minutes of regional ischemia followed by 24 h of reperfusion resulted in a development of retinal edema in the inner plexiform layer and a migration of neutrophils into the retinal tissue. Oxygen free radicals have been implicated as inducers of cell damage in different tissues. This finding has led us to speculate that, if oxygen free radicals play an important role in the development of reperfusion injury, superoxide dismutase (SOD) and EGB 761 (Tanakan, extract of Ginkgo biloba, IPSEN) should be protective against reperfusion-induced injury. Under our experimental conditions, SOD dose-dependently reduced the development of edema formation (which was expressed in micrometers, measuring the thickness of the inner plexiform layer). Thus, 3,750, 7,500 and 15,000 U/kg of SOD reduced the reperfusion-induced edema formation from its drug-free ischemic value of 112 +/- 4 to 107 +/- 7, 91 +/- 6 (p less than 0.05) and 85 + 4 microns (p less than 0.001), respectively. Furthermore, SOD significantly reduced the migration of neutrophils which can also contribute to the development of reperfusion-induced injury. The same protective effect was observed, concerning the edema formation and neutrophil migration, in the EGB 761-treated groups. Our results indicate that free radicals play an important role in the development of reperfusion-induced injury, and the treatment of ischemic and reperfused retina with free radical scavengers may reduce the severity of reperfusion damage.     

Involvement of oxygen free radicals in experimental retinal ischemia and the selective vulnerability of retinal damage.

Protective effects of CV-3611, a free radical scavenger, on retinal ischemic injury in the rat and on glutamate-induced cytotoxicity in a cell line were evaluated. Transient retinal ischemia was induced by raising intraocular pressure of rats to 110 mm Hg for 45 min, and the electroretinogram (ERG) was measured to evaluate retinal function. No ERG could be recorded immediately after reperfusion, and thereafter the ERG gradually recovered. Recovery of the a-wave latency and the amplitudes of the a and b waves in the CV-3611-treated (10 mg/kg, p.o.) group were significantly better than those in the control group up to 24 h after reperfusion. In both the control and CV-3611 group, the b wave showed better recovery than the a wave up to 6 h after reperfusion, while the relationship was reversed after 24-hour reperfusion. Glutamate (10 mM)-induced cytotoxicity in the N18-RE-105 cell, a neural retina-neuroblastoma hybridoma, was quantified by measuring lactate dehydrogenase. Three and 10 microM of CV-3611 significantly attenuated the glutamate-induced cytotoxicity in N18-RE-105 cells. Thus, the radical scavenger (CV-3611) promoted the recovery of retinal function after ischemia-reperfusion injury and ameliorated glutamate-induced cytotoxicity. These results suggest that oxygen free radicals play an important role in the early phase of retinal ischemic injury. Moreover, differential recovery processes of the a and b waves after ischemia suggest that the selective vulnerability of the retina to ischemia could change functionally during the period of reperfusion.     

Comparison of intraocular treatment of DMTU and SOD following retinal ischemia in rats.

The effect of intravitreal injections of DMTU (dimethylthiourea) and SOD (superoxide dismutase), two free radical scavengers, was evaluated in a rat model of retinal ischemia induced by elevated intraocular pressure. The drugs were administered just before or just after a 60 min ischemia. At days 2 and 7 after reperfusion, retinal recovery was evaluated by electroretinography. At day 7, layer thicknesses and cell rows were measured from histologic sections of paraffin-embedded retinas. In the vehicle-treated control group, we observed a decrease in the inner retinal layers and b-wave amplitude impairment. SOD injection (6 units/eye) protected the retina from ischemia/reperfusion injury. At day 2 after reperfusion, electroretinographic recovery was more efficient when SOD was administered just after ischemia (99%) than after pretreatment with SOD (81%) (p<0.03). In the DMTU-treated group (75 microg/eye), only the pretreatment induced significant electrophysiologic (40%) (p<0.001) and morphologic recovery.     

Alterations of energetic metabolite levels by free radicals during optic nerve ischemia.

An experimental model of optic nerve ischemia was designed in the rabbit to determine early biochemical alterations, i.e.--changes of high energy phosphate metabolites (ATP and phosphocreatine)--in occlusive and peri-occlusive areas. Vascular occlusion provoked a rapid fall of ATP and phosphocreatine in the optic nerve. Free radicals scavengers, superoxide dismutase plus catalase or dimethylthiourea were able to counteract the drop of phosphate metabolites in the peri-occlusive area. These results show that hypoxia leads to oxygen-derived free radical generation which can be responsible for cell damage and emphasize the role of free radicals in the pathogenesis of ocular diseases related to vascular dysfunction.     

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     

Ischaemia- and reperfusion-induced Na+, K+, Ca2+ and Mg2+ shifts in rat retina: effects of two free radical scavengers, SOD and EGB 761.

Using Sprague-Dawley rats with transient (90-min) regional ischaemia induced by retinal artery occlusion in the eye, we have shown that superoxide dismutase (SOD) and EGB 761 (IPSEN, France), two free radical scavengers, can dramatically reduce the reperfusion-induced sodium and calcium gains, and potassium loss in retinal tissue. Investigating whether this was a 'direct' protective effect, operating during reperfusion, or an 'indirect' effect arising from the action of SOD or EBG 761 on the tissue during ischaemia. SOD (15,000 U kg-1) and EGB 761 (100 mg kg-1) were added to the rats at the moment of reperfusion (after an ischaemic insult). Eyes were subjected to 90 min ischaemia followed by 4 and 24 hr of reperfusion, respectively. In the drug-free control group, 90 min of ischaemia resulted in an accumulation of retinal sodium (2-fold) and calcium (3-fold), and a loss of cell potassium (by 40%) and magnesium (by 40%). During the first 4 hr of reperfusion the ionic imbalance was unchanged, while after 24 hrs of reperfusion a normalization was observed and the ion content of the retina almost returned to their preischaemic values. SOD and EGB 761 treatment significantly reduced the reperfusion-induced ionic imbalance (magnesium was an exception) and improved the recovery of retinal ion contents. Our results indicate that the elimination of oxygen radicals by free radical scavengers may reduce the reperfusion-induced ionic imbalance and improve the ionic homeostasis in the injured retinal cells.     

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。     

Role of free radicals in uveitis

In acute inflammation, the tissue damage is generally mediated through polymorphonuclear leukocytes and macrophages. Recent studies on the mechanism of this leukocyte-mediated cytolysis have demonstrated that these cells, on stimulation, release a variety of reactive products. These metabolic products consist of superoxide and hydrogen peroxide, which in turn can form highly reactive species such as hydroxyl radicals. It is believed that these oxygen products play an important role in the tissue damage that occurs in inflammations such as uveitis. The ocular structures contain various antioxidant enzymes that normally protect against free radical mediated injury. In experimental animal models of uveitis, it has been shown that antioxidants and free radical scavengers can function as antiinflammatory agents and can protect the eye from inflammation-mediated tissue damage.     

Ischemia-reperfusion injury of retinal endothelium by cyclooxygenase- and xanthine oxidase-derived superoxide

The formation of reactive oxygen species (ROS) may be important in the pathogenesis of microvascular dysfunction and injury in ischemic retinopathies. The authors hypothesized that retinal endothelial cells can generate injurious levels of superoxide radical in response to ischemia/reperfusion, that endothelial xanthine oxidase and cyclooxygenase are important enzymatic sources of superoxide radical under these conditions, and that superoxide scavengers and inhibitors of these enzymes can protect endothelium from ischemic injury. The authors used confluent cultures of mouse retinal endothelial cells (MREC) subjected to exogenously generated superoxide or simulated ischemia-reperfusion to test these hypotheses. Cell injury was assessed biochemically by lactate dehydrogenase release into the culture medium. MREC were injured in a duration-dependent fashion by exposure to the superoxide-generating mix of hypoxanthine and xanthine oxidase. Increasing periods of oxygen and glucose deprivation (OGD) for 5-9 hr followed by replenishment of substrates for 2 hr led to progressive increases in endothelial cell injury; a significant proportion of the injury occurred during the period of substrate replenishment. Significant MREC protection was achieved by the superoxide scavengers SOD (1000 U ml(-1)) and a carboxylic acid derivative of carboxyfullerene (10 microM), the xanthine oxidase inhibitors oxypurinol (100 microM) and diphenyleneiodonium (DPI) (100 n M), and the cyclooxygenase inhibitors indomethacin (300 microM) and ibuprofen (300 microM). It is concluded that MREC are vulnerable to auto-oxidative injury by superoxide radical generated following a period of OGD. Both xanthine oxidase- and cyclooxygenase-dependent pathways are important enzymatic sources of superoxide formation in this setting. These enzymes and the ROS produced from their activity may be viable therapeutic targets to reduce microvascular dysfunction and injury in ischemic retinopathies.     

Quantitative analysis of hydroxyl radicals in the anterior optic nerve of the cat following transient ischemia

PURPOSE: Ischemic insults in the optic nerve are relatively common ocular disorders. This study provides quantitative data on the generation of hydroxyl radicals in the anterior optic nerve of the cat during the early reperfusion phase following 90 minutes of ischemia. MATERIALS AND METHODS: Cats were injected intravenously with sodium salicylate that by acting as an hydroxyl radical trap, forms 2,3-dihydroxybenzoic acid (2,3-DHBA). Ischemia was achieved by intraocular pressure (IOP) elevation via cannulation of the anterior chamber. Ischemia was maintained for 60 minutes in 6 eyes and 90 minutes in 12 eyes, followed by 5 minutes of reperfusion. In an additional 6 eyes, ischemia was maintained for 90 minutes without reperfusion. RESULTS: After 90 minutes of ischemia and reperfusion, mean normalized levels of 2,3-DHBA (ie, ng 2,3-DHBA/microg salicylate/mg protein) that represent the levels of hydroxyl radicals were 2.47 times (at least) higher in the anterior optic nerves than in the fellow sham-operated controls (P = 0.03). These levels were 3.9 times (at least) greater than in the corresponding levels after 90 minutes of ischemia without reperfusion (P= 0.005). CONCLUSION: This study provides a quantitative analysis and evidence for the generation of hydroxyl radicals in the optic nerve of the cat following transient ischemia. A quantitative analysis may provide an important tool to detect even relatively small levels of free radicals in the tissues, and to evaluate the relative efficacy of various therapeutic agents in the inhibition of free radical generation following ischemia and reperfusion.