Influence of oxygen free radicals and free radical scavengers on the growth behaviour and oxidative tissue damage of bovine retinal pigment epithelium cells in vitro

Purpose: This investigation was carried out to ascertain whether oxygen free radicals can influence the growth behaviour and consecutive lipid peroxidation of retinal pigment epithelium (RPE) cells in vitro and whether scavengers can counteract these effects. Methods: The experimental model was based on calf RPE cells. Hypoxanthine/xanthine oxidase (HX/XO) and superoxide dismutase/catalase (SOD/CAT) served as the radical generating system and scavengers, respectively. The components were tested alone and in combination. Lipid peroxides were determined in culture supernatants by a thiobarbituric acid assay. Results: Concentrations of up to 100 mol/1 of HX alone and 500/1000 U of XO alone, as well as the application of the scavengers without the radical generating system (HX/XO), had no effect. Doserelated reduction of cell growth and increase of lipid peroxidation were found with HX/XO treatment (single dose of 500 and 1000 U/ml 24 h after seeding). After application of 500 or 1000 U/ml of XO, CAT, when given alone (1200 U/ ml), counteracted the effect of the radicals on cell growth and lipid peroxidation; SOD (300 U/ml) had no effect. A combination of SOD and CAT was no better than the effect of CAT alone. Conclusion: The prevention of radical-induced reduction of cell growth and lipid peroxidation by scavengers supports trials of therapy using antioxidants and/or free radical scavengers for various ocular syndromes with RPE involvement.     

Involvement of the endocannabinoid system in retinal damage after high intraocular pressure-induced ischemia in rats

PURPOSE: To evaluate whether high intraocular pressure (IOP)-induced ischemia is associated with modifications in the retinal endocannabinoid metabolism and to ascertain whether drugs that interfere with the endocannabinoid system may prevent retinal damage due to ischemic insult. METHODS: Anandamide (AEA) synthesis, transport, hydrolysis, and AEA endogenous levels were assessed by means of high-performance liquid chromatography in the retinas of rats undergoing 45 minutes of ischemia followed by 12 hours of reperfusion. Under these experimental conditions, binding to cannabinoid (CB1R) and vanilloid (TRPV1) receptor was assessed with rapid-filtration assays. AEA-hydrolase (FAAH, fatty acid amide hydrolase), CB1R and TRPV1 protein content was determined by enzyme-linked immunosorbent assay. Finally, to characterize the neuroprotective profile of drugs that interfere with the endocannabinoid system, cell counting in the retinal ganglion cell (RGC) layer and real-time polymerase chain reactions for Thy-1 mRNA expression were used. RESULTS: In rat retina, ischemic insult followed by reperfusion resulted in enhanced FAAH activity and protein expression paralleled by a significant decrease in the endogenous AEA tone, whereas the AEA-membrane transporter or the AEA-synthase NAPE-PLD (N-acyl-phosphatidylethanolamine-hydrolyzing-phospholipase-d) were not affected. Retinal ischemia-reperfusion decreased the expression of cannabinoid (CB1) and vanilloid (TRPV1) receptors. Systemic administration of a specific FAAH inhibitor (e.g., URB597) reduced enzyme activity and minimized the retinal damage observed in ischemic-reperfused samples. Similarly, intravitreal injection of the AEA stable analogue, R(+)-methanandamide, reduced cell loss in the RGC layer, and this was prevented by systemic administration of a CB1 or TRPV1 selective antagonist (e.g., SR141716 and capsazepine, respectively). CONCLUSIONS: The original observation that retinal ischemia-reperfusion reduces endogenous AEA via enhanced expression of FAAH supports the deduction that this is implicated in retinal cell loss caused by high IOP in the RGC layer.     

Increase of free oxygen radicals in aqueous humour induced by selective Nd:YAG laser trabeculoplasty in the rabbit

PURPOSE: To investigate the impact of selective Nd:YAG laser trabeculoplasty on free oxygen radicals and antioxidant enzymes of the aqueous humour in the rabbit. METHODS: One eye of 18 rabbits was subjected to 360 degrees selective laser trabeculoplasty (LT) with a frequency-doubled Nd:YAG laser (532 nm). The anterior chamber aqueous humour was aspirated 3, 12 hours and 1, 3, 7, 10 days after treatment. Lipid peroxide (LPO) and glutathione S transferase (GST) levels and superoxide dismutase (SOD) activities of aqueous humour were measured. RESULTS: Concentrations of LPO in the aqueous humour of the treated eyes were significantly higher than the untreated eyes until the 7th day. Aqueous SOD activity significantly decreased 3 hours after LT and remained low until day 7. Aqueous GST levels were significantly decreased between 12 hours and 7 days after the LT. CONCLUSIONS: Selective LT was followed by an immediate increase in the aqueous humour LPO concentration and decreases of SOD and GST in the rabbit, probably due to photovaporization and photodisruption caused by the frequency-doubled Nd:YAG laser. The increased aqueous LPO levels suggest that free oxygen radicals are formed in the pigmented trabecular meshwork during LT, and may be responsible for the inflammatory complications of this procedure.     

Role of oxygen radicals in experimental allergic uveitis

The experimental autoimmune disease elicited by a large dose of retinal S antigen in guinea pigs is characterized by massive necrotizing uveitis and retinitis. Treatment of these animals with the antioxidants superoxide dismutase, catalase, and sodium benzoate resulted in marked reduction of uveal inflammation. The attenuated inflammation was characterized by a relatively well-preserved retina and retinal pigment epithelium along with a reduction of subretinal exudate and vitreous inflammation. These findings suggest that reactive oxygen metabolites may play a role in the destruction of ocular tissue and amplification of the inflammatory process in experimental uveitis.     

Role of oxygen radicals in ocular inflammation and cellular damage

Oxyradicals probably play a major role in a number of specific pathological conditions of intraocular tissues, such as cataract formation and retinal degeneration. This paper reviews some of the mechanistic concepts relating to tissue damage by highly reactive oxidants derived from endogenous precursors, hydrogen peroxide and superoxide. Experimental generation of superoxide in the anterior chamber of the rabbit eye showed leucocyte infiltration to be the principal acute response occurring in 4 hr. This finding suggests that superoxide may play a significant role in the ocular inflammatory response, possibly by reacting with a precursor substance in the aqueous humor to produce a chemotactic factor as has been previously found for blood plasma.     

Functional recovery of retinal pigment epithelial damage in experimental retinal detachment

The integrity of the RPE barrier function in retinal detachment was studied in vitro. The retinal pigment epithelium (RPE)-choroid tissue was isolated from cynomolgus monkey eyes with acute (less than 1 hr), subacute (1-2 weeks), and chronic (8-20 months) retinal detachments, and clamped between Ussing-type chambers. Electrical characteristics and choroid-to-retina permeability to carboxyfluorescein were determined. In the HEPES-buffered bathing solution, transepithelial potential difference and resistance in eyes with acute retinal detachments (0.2 mV and 134 ohm-cm2, respectively) were significantly lower than subacute (7.9 and 350) and chronic (10.4 and 348) retinal detachments. Furthermore, the permeability was increased five-fold in acute retinal detachments with respect to subacute and chronic retinal detachments, indicating a breakdown of the RPE barrier in acute retinal detachment. No statistical difference was found between subacute and chronic retinal detachments. In this animal model, RPE barrier function is destroyed at the onset of retinal detachment, but recovers in a week or two, and is maintained in the chronic stage. Histological examination revealed that RPE recovery was accomplished by RPE proliferation and hyperplasia.     

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.     

Involvement of purinergic P2 receptors in experimental retinal neovascularization

PURPOSE: The present study was aimed to investigate the expression of purinergic P2 receptors in oxygen-induced retinal neovascularization. METHODS: Immunohistochemistry was used to study the expression of purinergic P2Y2 and P2X2 receptors in the neonatal mouse retina during normal vascular development and after oxygen-induced retinopathy (OIR). The effect of the P2 antagonists, suramin and PPADS, on the extent of oxygen-induced retinal neovascularization was analyzed. RESULTS: In normal mice, the expression of P2Y2 receptors was weak throughout the retina, whereas P2X2 receptor expression was detected in the outer plexiform layer. In mice treated with oxygen, P2Y2 expression was detected in the ganglion and in the nerve fiber layers, whereas P2X2 expression was found in the inner and outer plexiform layers. Oxygen-induced preretinal neovascularization was strongly inhibited by the P2 antagonists, suramin (p<0.05) and PPADS (p<0.05), and this was accompanied by a down-regulation of P2X2 receptor expression in the inner plexiform layer in suramin-treated mice. CONCLUSIONS: The data suggest that purinergic P2 receptors are involved in neovascularization associated with OIR.     

Effects of oxygen and bFGF on the vulnerability of photoreceptors to light damage

PURPOSE. To test whether tissue oxygen levels affect the vulnerability of photoreceptors to damage by bright continuous light (BCL). METHODS. Albino rats were raised in standard conditions of cyclic light (12-hour light, 12-hour darkness) with the light level at 5 to 10 lux or 40 to 65 lux. They were then exposed to BCL (1000-1400 lux), either continuously for 48 hours or for the day or night components of the 48-hour period. During BCL, some rats were kept in room air (normoxia, 21% oxygen), some in hypoxia (10%), and some in hyperoxia (70%). Their retinas were examined for cell death, for the expression of basic fibroblast growth factor (bFGF), and for response to light (electroretinogram, ERG). RESULTS. The death of retinal cells induced by BCL was confined to photoreceptors. Within the retina, the severity of death was inversely related to the level of bFGF immunolabeling in the somas of the outer nuclear layer (ONL) before exposure. The death of photoreceptors was accompanied by an upregulation of bFGF protein levels in the ONL and by a decline in the ERG. Both hypoxia and hyperoxia during BCL reduced the photoreceptor death, bFGF upregulation, and ERG decline caused by BCL. The protective effects of hyperoxia and hypoxia were evident during both the day and night halves of the daily cycle. Hypoxia or hyperoxia alone did not upregulate bFGF or ciliary neurotrophic factor (CNTF) expression in the retina. CONCLUSIONS. Photoreceptors are protected from light damage by hypoxia and hyperoxia during exposure. The protection provided by oxygen levels operates during both day and night. The protection is not mediated by an upregulation of bFGF or CNTF.     

The role of clusterin in retinal development and free radical damage

AIM: To assess the role of clusterin in retinal vascular development and in free radical damage in vivo and in vitro. METHODS: The expression of clusterin, von Willebrand factor (vWF), flk-1, heat shock protein 27 (Hsp27) and heat shock protein 70 (Hsp70) was examined in the retinas of developing mice and oxygen-induced retinopathy (OIR) mice by immunofluorescence staining and western blot analysis. Hydrogen peroxide (H(2)O(2))-pretreated human retinal endothelial cells (HREC) and astrocytes were cultured in the presence or absence of exogenous clusterin, and then the cell viability was measured using the MTT assay and DAPI staining. RESULTS: Clusterin was expressed mainly in the inner retina and co-localised with vWF, an endothelial cell marker. During the mouse developmental process, clusterin expression was decreased, which was similar to the expression of flk-1, vWF and Hsp27. Furthermore, in the OIR model, clusterin expression changed in a similar way to both vWF and Hsp27. Under hypoxic conditions, clusterin expression increased in HREC and astrocytes. In H(2)O(2)-pretreated HREC and astrocytes, clusterin protected against apoptotic cell death. CONCLUSIONS: These results suggest that clusterin is associated with protection from apoptotic retinal cell death in retinal development and in free radical damage.     

Pressure-induced retinal ischemia in rats: an experimental model for quantitative study.

The advent of treatment modalities with the potential to ameliorate retinal ischemic injury calls for methods allowing their quantitative assessment. We thus established a model of pressure-induced retinal ischemia/reperfusion injury in rats. The intraocular pressure (IOP) was raised to 110 mm Hg by cannulation of the anterior chamber for a duration of 0, 90 or 120 min. The eyes were reperfused for 3 or 7 days. Morphologically, retinal injury occurred in a pattern consistent with retinal and choroidal vascular occlusion. Damage increased in severity with prolonged durations of ischemia. Morphometric determination of the mean thickness of inner retinal layers (MTIRL) revealed significant differences between controls and the 90- or 120-min ischemia groups (p less than 0.05 and p less than 0.01, respectively). The difference in MTIRL between 3 and 7 days of reperfusion was not significant. Replacement of normal saline by a solution of 5% dextrose in the hydrostatic device used to increase the IOP led to a decrease in retinal injury after 120 min of ischemia (p less than 0.01). This model combines a relatively simple methodology, cost-effective execution and a fast, semicomputerized method of quantitation. Depletion of carbohydrates during ischemia may contribute to retinal injury in this model.     

谷氨酸对视网膜缺血的损害机制和相关治疗策略

视网膜缺血损害的机制复杂,大量的研究资料表明视网膜在缺血及再灌注时谷氨酸的释放量增加,谷氨酸的兴奋性毒性作用在缺血视网膜的病理发展中起到了重要的作用,是引起缺血视网膜神经元死亡的重要因素.通过减少谷氨酸的释放,应用谷氨酸受体拮抗剂限制谷氨酸的生物活性等,可以阻止或减轻缺血视网膜的损害.虽然到目前为止临床上还未出现治疗视网膜缺血的有效药物,但对谷氨酸的损害机制和相关治疗策略的实验性研究,为治疗该疾病提供了广阔的前景.     

Vitreous body oxygen tension following experimental branch retinal vein obstruction.

We obstructed either temporal or nasal superior and inferior retinal veins on one eye of rhesus monkeys with xenon photocoagulation. This resulted in large areas of nonperfused retina adjacent to normal retina in the same eye. Intraretinal neovascularization (new vessels in the retina) developed following the absorption of retinal hemorrhage and edema. We used microelectrodes to measure and compare vitreous body oxygen tensions over the nonperfused and over the normal retinal areas. There was no significant difference between the oxygen tension measurements in the same eye.     

Hydrogen peroxide-mediated corneal endothelial damage. Induction by oxygen free radical

Polymorphonuclear leukocytes and other inflammatory cells release superoxide anion and additional oxidant species following stimulation. Corneal endothelial cells were exposed to a flux of chemically generated superoxide anion (oxygen-free radical) produced by the combination of 1 mM hypoxanthine and 0.06 U/ml xanthine oxidase. Exposure of endothelial cells to the combination of hypoxanthine and xanthine oxidase resulted in anatomic disruption of the cells with interference in the function of endothelial water movement and resultant swelling of the corneal stroma. Catalase reduced the corneal swelling caused by exposure of endothelium to the oxygen-free radical generating system, whereas superoxide dismutase, ascorbic acid, D-mannitol, and ethanol did not prevent damage. The data suggest that hydrogen peroxide produced during the dismutation reaction of the superoxide anion is one of the toxic species, whereas the superoxide anion itself and the hydroxyl-free radical probably do not participate. The data suggest that corneal endothelial cells are susceptible to physiologic and anatomic damage induced by the products of reactive oxygen species, which, from previous studies, are known to be generated by inflammatory cells. The development of therapeutic modalities directed at the prevention of damage produced by hydrogen peroxide and other oxidant species may be of benefit in reducing corneal endothelial cell damage secondary to ocular inflammatory disease processes.     

实验性玻璃体腔内高氧对视网膜超微结构的影响

目的 通过研究玻璃体腔内高氧对视网膜超微结构的影响，观察氧对视网膜组织的毒性作用，为实现临床应用玻璃体腔内的氧疗提供基础实验依据。方法：以2个月龄猪为实验动物，模拟临床常规玻璃体切割手术后眼内充填混合气体，在混合气体中提高氧比例达40％，设置眼内充填常规混合气体组和空白对照组做为实验对照组，持续数天后观察视网膜全层超微结构的改变。结果 眼内高氧组7d即可引起视网膜神经节细胞核膜的明显溶解，双层膜结构消失，且导致胞浆内线粒体肿胀，双层膜结构模糊，嵴排列出现紊乱。结论 玻璃体腔内的氧含量增加到一定程度（40％）时，则可引起视网膜组织的氧化损害，具有一定的氧毒性。     

Sensitivity to retinal light damage and surgical blood oxygen levels

Increased oxygen levels decrease the threshold for photochemical retinal damage. We measured arterial oxygen levels in a group of ophthalmic surgical patients. As expected, levels exceeded unanesthetized measurements by one to two times. Based on experimental data, this could decrease the threshold for light-induced retinal damage during ophthalmic surgery by 40% to 50%. While the clinical implications of light-induced retinal damage in surgical eye patients are unclear, it is prudent to take steps to minimize light exposure during surgery.     

氧自由基在实验性角膜炎中的作用

目的探讨氧自由基在角膜炎症过程中的作用。方法将新西兰白兔用脂多糖介导建立动物角膜炎模型,分别用化学发光法和硫代巴比妥酸比色法测定角膜组织超氧化物歧化酶（ｓｕｐｅｒ－ｏｘｉｄｅｄｉｓｍｕｔａｓｅ,ＳＯＤ）活力和丙二醛（ｍａｌｏｎｄｉａｌｄｅｈｙｄｅ,ＭＤＡ）水平的变化,观察其临床和病理变化。结果炎症组角膜ＳＯＤ活力明显低于对照组（Ｐ＜０．００１）,ＭＤＡ水平高于对照组（Ｐ＝０．００１）,炎症组角膜ＭＤＡ水平的升高发生于ＳＯＤ活力下降之后。角膜炎症状的轻重程度与角膜ＳＯＤ的活力呈负相关（ｒ＝－０．９５４,Ｐ＜０．００１）,与角膜ＭＤＡ水平呈正相关（ｒ＝０．７３４,Ｐ＜０．００１）。结论氧自由基在角膜炎症过程中起着重要作用,并可能通过生物膜中多聚不饱和脂肪酸产生脂质过氧化而损害角膜。     

Protective role of excitatory amino acid antagonists in experimental retinal ischemia

Background: Excitatory amino acids and their analogues (NMDA, kainate and AMPA) are implicated in the pathogenesis of ischemic brain injury. In order to fully understand their involvement in the pathogenesis of retinal ischemic injury, we studied the electrophysiological and histopathological effects of two excitatory amino acid antagonists, cis-PDA and MK 801, in an experimental retinal ischemia model. Methods: The two antagonists were injected intravitreously 15 min before ischemia was induced by elevatory intraocular pressure caused by external compression. Electrophysiological and histopathological evaluation was made 48 h after 45 min transient ischemia. Results: The excitatory amino acid antagonists cis-PDA and MK 801 can partially protect against retinal ischemic injury; whereas the mean post-ischemic b-wave amplitude corresponded to 41% of the pre-ischemic value in the control group, it was 64% (P=0.003) and 59% (P=0.005) following administration of cis-PDA and MK 801 respectively. Histopathological study corroborated these data, showing significant differences for morphometric parameters (P=0.011 and P=0.007 respectively). Conclusion: These preliminary results suggest the possibility of limiting excito-toxicity, one of the lesion-forming mechanisms in ischemic retinal injury.