Nitric oxide generated by corneas in corneal storage media

PURPOSE: The purpose of the study was to quantify nitric oxide release by human corneal buttons in storage media over time. METHODS: Group 1 consisted of six chambers of Optisol GS corneal storage media, each containing a viable human corneal button with an attached scleral rim (unsuitable for transplantation), sampled at 1-day intervals for at least 17 days (range, 17-28 days). Group 2 consisted of 34 chambers of Optisol GS media, each used to store a corneal button for penetrating keratoplasty, sampled immediately after each surgery. An unused vial of Optisol GS storage medium was sampled daily for 17 days to serve as a background medium control. The total amount of nitrite and nitrate in each sample was determined by a spectrophotometric method based on the Griess reaction. RESULTS: Data from the daily sampling in group 1 showed that nitrite and nitrate concentrations in storage media containing human corneas increase from a baseline level (beginning at the time the corneas are placed in the media) to an equilibrium concentration of 2.77 microM in a mean time of 6.15 days. Seventy-six percent of the data points from group 2 fell within the 80% predictive interval derived from group 1. No nitrite or nitrate was detected in background medium control samples. CONCLUSION: The progressive increase in nitrite and nitrate in corneal storage media over time suggests that nitric oxide is continuously released by corneas during storage before transplantation. Given the toxic free radical properties of nitric oxide, corneas in storage media may be subjected to the cumulative toxic effects of nitric oxide.     

Immunohistochemical detection and Western blot analysis of nitrated protein in stored human corneal epithelium

While the production of nitric oxide by human corneas in storage has recently been demonstrated, protein nitration as a result of this production has not been demonstrated. In this study, nitrated protein accumulation in the epithelium of stored human corneas was assessed. One half of five donor corneas maintained in storage media for 3 days were prepared for immunohistochemical studies. The other halves remained in storage media for 7 additional days and were also processed for immunohistochemistry. Mouse monoclonal antibody to nitrotyrosine adducts was used to define the localisation of these epitopes. The density of antibody staining was observed and quantified on a digital camera system and statistically analysed. Immunostaining in the epithelium was greater in tissues recovered after 10 days in storage compared to the intensity of staining after 3 days of storage (p<0.0001). No staining was evident in the epithelium in sections exposed to non-immune mouse IgG. Western blot analysis was performed on epithelial cells scraped from corneal surfaces of one-half of four donor corneas in storage for 3 days and from the other half at 10 days of storage. Nitrated BSA was used as a positive control. After extraction and homogenisation, identical protein concentrations of each sample were loaded per lane on 10% gels and subjected to SDS-PAGE. Proteins were blotted and probed with the anti-nitrotyrosine antibody. Western blot immunoreactivity was detected in epithelial samples at the 3 and 10 day recovery times with the latter samples showing greater staining intensity. Nitrated protein, thought to indicate toxic peroxynitrite formation, accumulates in the human corneal epithelium with time of storage. Our study shows that there is an association between increased nitrated protein and storage time.     

Effects of the COOH-terminal tripeptide alpha-MSH(11-13) on corneal epithelial wound healing: role of nitric oxide

It is known that alpha-melanocyte stimulating hormone (alpha-MSH) may exert anti-inflammatory effects and facilitate reparative processes in different tissues. The effective message sequence of alpha-MSH resides in the COOH-terminal tripeptide alpha-MSH(11-13). This study was undertaken to investigate the effects of topical administration of the COOH-terminal tripeptide sequence of alpha-MSH (alpha-MSH(11-13), KPV) on corneal epithelial wound healing in rabbits and the possible role of nitric oxide (NO) in these effects. The whole corneal epithelium was denuded in both eyes by mechanical abrasion. The area of the corneal epithelial defect was stained with fluorescein, photographed, and then measured before the treatment and every 12 h by a computerized software. The mean epithelial wound area and the mean percent of epithelial defect remaining at each follow-up control were compared between experimental groups. Rabbits were topically treated with KPV 1, 5 or 10 mg/ml (30 microl), two drops four times in a day, for 4 days, starting immediately after corneal abrasion, while control animals received topical phosphate-buffered saline as vehicle. In order to study the role of NO in corneal repair processes, the NO donor, sodium nitroprusside (SP, 10 mg/ml, 30 microl) was administered in both eyes, two drops four times in a day, for 4 days. The effects of KPV or SP were challenged by pre-treatment with the nitric oxide synthase inhibitor, N omega-nitro-L-arginine methyl ester (L-NAME, 10 mg/ml, 30 microl) 30 min prior to KPV or SP instillation. The mean percent epithelial defect remaining each time was significantly smaller in animals treated with KPV or SP in comparison to controls. Sixty hours later, eight out of eight (100%) corneas treated with KPV or SP were completely re-epithelized (P<0.05) while none of the corneas treated with placebo were re-epithelized. Pre-treatment with L-NAME inhibited the facilitating effect of KPV on corneal epithelial wound healing process and totally prevented the effect of SP. Rabbit corneal epithelial cells (RCE) in culture were exposed for 1, 6 and 24 h to different KPV concentrations (0.1, 1 and 10 microM) in medium containing 15% foetal bovine serum (FBS). Cell viability was stimulated by 1 and 10 microM concentrations of the substance. Thus, KPV may facilitate corneal epithelial wound healing in rabbits with a mechanism that may involve NO disposition in corneal tissue. However, it is not known whether this mechanism is likely to depend on a direct stimulating repairing activity shared by the entire molecule of alpha-MSH.     

The Role of Nitric Oxide in Resistance to P. aeruginosa Ocular Infection

Purpose: This study determined the role of inducible nitric oxide synthase (iNOS) and nitric oxide (NO) in the resistance response of BALB/c mice to P. aeruginosa-induced keratitis. Methods: RT-PCR, nitrite detection, iNOS inhibition, ELISA, and immunohistochemistry were used. Results: Early after infection, iNOS mRNA expression and nitrite levels in cornea were elevated compared to levels in the uninfected cornea. Treatment with aminoguanidine sulfate (AG), an inhibitor of iNOS, resulted in extensive corneal destruction, reduced nitrite levels, and reduced nitrotyrosine staining. Infected mice also had increased bacterial burden and elevated levels of MIP-1α, IL-1β, and MIP-2 in the cornea. Dual-labeling immunohistochemistry established the macrophage as the major source of iNOS in the infected cornea. Conclusions: These data provide evidence that iNOS is constitutively expressed in the BALB/c cornea; that iNOS-derived NO is required for bacterial killing/stasis; and that the macrophage is the major cell source of NO.     

Advanced glycation end products induce death of retinal neurons via activation of nitric oxide synthase

The purpose of this study was to determine whether advanced glycation end products (AGEs) are neurotoxic for cultured retinal neurons consisting mainly of amacrine cells, and to determine whether endogenous nitric oxide (NO) is involved in the toxicity. Cultured retinal neurons obtained from fetal Wistar rats (gestational age 19 days) were maintained in culture for 10 days, and then exposed to different concentrations of AGEs (0.02, 0.1, and 0.5 mg ml(-1)) in cultured media for different lengths of time. Both trypan blue exclusion and TUNEL assay were used to determine whether AGEs were neurotoxic, and NG-nitro-L-arginine methyl ester (L-NAME, 500 microM), a nitric oxide synthase (NOS) inhibitor, was used to determine whether NO was involved. Immunohistochemical analyses were performed to determine whether specific receptors of AGEs (RAGE) are present on cultured retinal neurons; caspase-3 was activated, and 3-nitrotyrosine was expressed on neurons treated with AGEs. Nitrite levels were measured in the supernatants of the media where neurons were incubated with AGEs. AGEs induced cell death in a time- and dose-dependent manner. TUNEL-positive cells and immunoreactivity to cleaved caspase-3 were enhanced on neurons following exposure to AGEs. L-NAME significantly suppressed the AGEs-induced neurotoxicity as assessed by both trypan blue exclusion and TUNEL assays. Activation of NOS was suggested by enhanced immunoreactivity to 3-nitrotyrosine on neurons and increased nitrite levels in the media incubated with AGEs. These results indicate that AGEs are neurotoxic to retinal neurons in culture through the activation of NOS. Apoptotic pathways may be in part involved in the death of the neurons.     

Regulation of lens aldose reductase activity by nitric oxide

To examine the regulation of aldose reductase (AR) activity by nitric oxide (NO) in human lens epithelial cells (HLEC), cultured rat lens, and normal and diabetic rat lens, we have incubated HLEC or cultured rat lenses with 1 mm of the NO donors S-nitroso-N-acetylpenicillamine (SNAP) or S-nitrosoglutathione (GSNO), and the AR activity and sorbitol content were measured. Non-diabetic and diabetic (treated with streptozotocin 65 mg kg(-1) body wt, i.p.) rats were injected with the nitric oxide synthase (NOS) inhibitor, L-NAME (50 mg kg(-1) body wt day(-1), x 10 days i.p.) or NOS substrate, L-arginine (200 mg kg(-1) body wt day(-1), x 10 days i.p.). In a separate group of rats, a nitroglycerin (NG)-patch that releases 200 ng min(-1) NO was applied to the dorsal neck region. After 10 days of treatment, the lenses were removed and their AR activity and sorbitol content were measured. Incubation of HLEC with SNAP or GSNO reduced AR activity. A similar reduction in AR activity and sorbitol accumulation was observed when diabetic and non-diabetic rat lenses were cultured in the presence of SNAP and GSNO. Total protein-SSG in diabetic lens was lower compared to normal lens. Treatment of diabetic and non-diabetic rats with L-NAME enhanced AR activity and sorbitol accumulation, whereas NG patch and L-arginine significantly decreased AR activity and sorbitol accumulation in diabetic lenses compared to non-diabetic. Increased S-glutathiolation of AR was observed in the presence of SNAP. These results suggest that decreased glutathiolation of cellular proteins in diabetic rat lens compared to non-diabetic lens is related to decreased NO availability in diabetic rats which would decrease GSNO. Restoring the NO levels in diabetic animals increases glutathiolation of cellular proteins, inhibits AR activity and prevents sorbitol accumulation. Exogenous delivery of NO may represent a potentially useful strategy for preventing or delaying diabetic cataractogenesis and the development of other diabetic complications.     

May nitric oxide molecule have a role in the pathogenesis of human cataract?

Recent studies have shown that nitric oxide molecule may have a role in the development of cataract. In this study, we measured the levels of a nitric oxide metabolite (nitrite) in the cataractous and normal human lenses. A modified Griess assay was used to determine the nitrite levels in the lenses as a measure of nitric oxide, based on the spectrophotometric method. Nitrite was detected in 26 (44.1%) cataractous lenses and was found below the detection limit in 33 (55.9%) cataractous lenses. Mean nitrite levels in cataractous lenses (2.77+/-5.26nmol/100mg) was found higher than the normal lenses (0.77+/-0.79nmol/100mg) but this increase was not statistically significant. Comparison of nitrite levels among lenses with various types of cataracts revealed higher levels in lenses with posterior subcapsular cataracts. Hypertensive patients had also significantly higher nitrite levels in their cataractous lenses. The increased levels in the cataractous lenses display a possible role of nitric oxide in the pathogenesis of cataract in human eyes.     

Lysosomal enzyme levels in corneal storage media. K-Sol versus McCarey-Kaufman

Evidence indicates that lysosomal enzymes can carry out corneal autolysis during corneal storage and that they are damaging to the corneal endothelium. The authors investigated the release of lysosomal enzymes into two corneal storage media (K-Sol and McCarey-Kaufman [M-K]) by paired human donor corneas during 4 degrees C storage. The authors also studied the interaction of these media with lysosomal enzymes from human cornea. K-Sol and M-K stimulated (P less than 0.01) both beta-glucuronidase and alpha-galactosidase about equally. beta-N-Acetyl-glucosaminidase, a major catabolic enzyme of the cornea, was inhibited by the chondroitin sulfate in K-Sol by over 90% (P less than 0.01). Corneas stored in M-K released more lysosomal enzymes than corneas stored in K-Sol. At 4 days, the values approached significance (P less than 0.06) and by day 10 significantly higher values were found in the M-K media (P less than 0.01). Both storage methods showed a linear release. Individual corneas were found to vary in their release rates. Whether corneas that release more enzyme will show higher endothelial cell loss or produce less successful penetrating keratoplasty grafts deserves further study.     

遗传性视网膜谱性视细胞凋亡与视网膜诱生型一氧化氮合酶活性的时程变化

目的 研究遗传性视网膜变性视细胞凋亡与诱生型一氧化氮合酶活性之间的关系。方法 对生后不同鼠龄RCS大鼠和Wistar大鼠的视网膜进行凋亡细胞的TUNEL检测、计算机自动图像分析及诱生型一氧化氮合酶活性的测定。结果 RCS大鼠视网膜视细胞自生后第25天出现凋亡,第30-35天达高峰;视细胞凋亡初期,即生后第25天,视网膜诱生型一氧化氮合酶活性达高峰。结论 在遗传性视网膜变性的视网膜。诱生型一氧化合酶激活可能在视细胞凋亡中起诱导作用。     

大鼠角膜重度碱烧伤后视网膜一氧化氮合酶变化与细胞凋亡的研究

目的研究大鼠角膜重度碱烧伤后视网膜诱导型一氧化氮合酶(iNOS)变化与细胞凋亡的关系。方法制作SD大鼠角膜重度碱烧伤模型76只(152眼)。在烧伤后不同时间点以免疫组化检测iNOS在视网膜的表达,以酶法检测视网膜组织中iNOS含量的变化,并以末端脱氧核苷酸转移酶介导的dUTP缺口末端标记(TUNEL)法检测视网膜凋亡细胞,并与正常大鼠相对照。结果正常组视网膜中未测到iNOS的表达和含量,重度碱烧伤后大鼠视网膜中iNOS的表达和含量从伤后1d开始升高,于7d达最高,30d恢复正常,与正常组比较差异有统计学意义(P<0.05)。伤后3d、7d、15d在视网膜神经节细胞可见不同程度的TUNEL阳性细胞,7d组凋亡阳性表达最强,与其他各组比较差异有统计学意义(P<0.01)。结论角膜重度碱烧伤后视网膜组织细胞存在凋亡,凋亡可能与iNOS表达有关。     

人脐带血清器官培养液保存猪角膜的实验研究

目的 观察胎牛血清器官培养液和人脐带血清器官培养液对猪角膜内皮细胞形态学、组织学、超微结构、酶活性及代谢等的影响。方法 器官培养方法：4周以内31℃密闭培养,之后脱水24h。选择猪眼113只,其中100只角膜分为两组进行配对器官培养保存。第1组(50只角膜)：应用培养液Ⅰ(含10％胎牛血清);第2组(50只角膜,第1组的对侧角膜)：应用培养液Ⅱ(含10％人脐带血清)。13只角膜作为正常对照。器官培养每周每组各取出12只角膜进行内皮细胞形态学分析、HE染色、酶组织化学染色。保存2周、4周时行扫描电镜检查。检测保存前后培养液的pH值和葡萄糖、乳酸浓度。器官培养过程中行微生物学检测。结果 器官培养期间角膜内皮细胞单层保持完整,多形性增加,两组之间角膜内皮细胞密度、细胞面积的变异系数和六边形细胞比例在保存4周内差异无显著意义。保存4周的猪角膜与正常猪角膜相比,平均内皮细胞丢失率第1组为10．98％,第2组为10．85％。两组角膜器官培养后的组织学、超微结构、酶活性无明显区别。扫描电镜显示内皮细胞层完整,细胞形态改变。酶组织化学染色显示上皮、内皮细胞酶活性旺盛,基质细胞的酶活性随保存时间的延长而降低。角膜代谢状态良好。器官培养液污染率为6％。结论 两种器官培养液均可以保持角膜内皮活性达4周,推测人脐带血清能够替代胎牛血清作为角膜器官培养液的成分。(中华眼科杂志,2004,40：533-538)     

Improved corneal storage: Penetrating keratoplasties in rabbits

In the present study, rabbit corneas were stored in McCarey-Kaufman corneal bathing media (M-K media) at 4°C for up to 14 days and used as donor material in penetrating keratoplasties. These results were compared to penetrating keratoplasties with corneas stored in refrigerated moist chambers. Evaluation of the graft clarity shows that all the M-K media stored corneas were clear from the day of surgery. This contrasted with the 14-day moist chamber stored grafts which were hazy for the first week then either became opaque or remained hazy. Electron microscopy of postoperative grafts of 14-day M-K stored donor corneas, show the endothelial ultrastructure to be normal by the third day postoperative.     

Comparison of Chen Medium and Optisol-GS for human corneal preservation at 4 degrees C: results of transplantation

PURPOSE: To compare results after transplantation of donor corneas stored in Chen Medium (containing beta-hydroxybutyrate without sodium bicarbonate or chondroitin sulfate) to corneas stored in Optisol-GS medium (containing sodium bicarbonate and 2.5% chondroitin sulfate). METHODS: We performed 32 consecutive penetrating keratoplasties with donor corneas stored at 4 degrees C in either Chen Medium or Optisol-GS by random assignment. Corneal thickness measurements were made at 1 day, 1 week, 3 weeks, 2 months, and 1 year postkeratoplasty. Specular microscopic images of the donor endothelium were obtained at the beginning of storage and 2 months and 1 year postkeratoplasty. The percentage of intact epithelium 1 day after keratoplasty and the graft epithelialization time were estimated by the surgeons. Donor rim cultures were performed. RESULTS: No statistically significant differences in corneal thickness or endothelial cell loss between the corneas stored in the two media were found at any time, although differences of less than 12% cell loss or 0.09-mm thickness at 2 months or less than 25% cell loss or 0.10-mm thickness at 1 year could not be excluded with 90% certainty in this small series. The mean percentages of intact graft epithelium on day 1, 64% for Chen Medium and 65% for Optisol-GS, were not significantly different. Endothelial cell density 2 months postkeratoplasty was significantly decreased for corneas stored in both media. Endothelial cell loss at 2 months was directly correlated with storage time in both media. CONCLUSIONS: After keratoplasty, no statistically significant differences in corneal thickness, epithelial survival, and endothelial cell loss were found between corneas stored in Chen Medium and Optisol-GS. Endothelial cell loss at 2 months was significantly correlated with storage time in both media.     

Possible role for nitric oxide releasing nerves in the regulation of ocular blood flow in the rat

AIM—To investigate the role of nitrergic nerves in the regulation of ocular blood flow.
METHODS—Conscious, lightly restrained rats were treated with either the neuronal nitric oxide synthase inhibitor 7-nitroindazole (7-NI), or the non-selective inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), and ocular blood flow was measured ex vivo from tissue samples, using the fully quantitative [14C]-iodoantipyrine technique.
RESULTS—In the peripheral circulation, L-NAME produced an increase in arterial blood pressure (+22%) while 7-NI had no effect. In contrast, both 7-NI and L-NAME produced significant decreases in ocular blood flow (−31% and −59% respectively). The ocular vascular resistance calculated from ocular blood flow and mean arterial blood pressure increased by 29% following 7-NI, but by 130% following L-NAME.
CONCLUSIONS—Nitric oxide releasing neurons may play an important contributory role in regulating ocular blood flow.

Keywords: nitric oxide; neuronal nitric oxide synthase; 7-nitroindazole; NG-nitro-L-arginine methyl ester; ocular blood flow     

Reduction of nitrite production by endothelin-1 in isolated porcine ciliary processes

It has been postulated that endothelin-1 and nitric oxide (NO) could participate in the modulation of aqueous humor dynamic in the eye. This study investigates whether endothelin-1 can reduce the production of nitrite (a stable metabolite of NO) in isolated porcine ciliary processes. Nitrite production was measured (Griess reaction) in the medium surrounding isolated ciliary processes before and after exposure to different drugs. Results are expressed in percent of basal nitrite production. In a concentration-dependent manner (0.1 nM to 1 microM), endothelin-1 significantly decreased basal nitrite production (1 microM: 81.7+/-3.5%; P<0.001). This effect was prevented by the endothelin type A (ETA)-receptor antagonist BQ123 (1 microM: 95.6+/-4.3%; P<0.05), but not by the endothelin type B (ETB)-receptor antagonist BQ788 (1 microM: 83.3+/-4.4%; P=0.86) or by the prostaglandin F2alpha analog unoprostone (30 microM: 86.7+/-3.7%; P=0.74). The adenylcyclase activator forskolin significantly increased basal nitrite production (1 microM: 143.4+/-3.9%, P<0.001). This effect was prevented both by endothelin-1 (0.1 microM; 113.9+/-6.7%: P<0.01) or the nitric oxide synthase inhibitor L-NAME (0.5 mM: 103.3+/-8.4%: P<0.001). The inhibitory effect of endothelin-1 on forskolin-induced nitrite production was significantly reversed by BQ123 (1 microM: 132.4+/-5.1%; P<0.01), but not by BQ788 (1 microM: 112.7+/-4.1%; P=0.64) or unoprostone (30 microM: 109.3+/-4.8%; P=0.98). These results suggest that endothelin-1, through an ETA receptor activation, can reduce both basal and forskolin-induced nitrite production in isolated porcine ciliary processes.     

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.     

Morphologic changes of K-Sol preserved human corneas

Supplementation of tissue culture medium with chondroitin sulfate has been shown to enhance donor corneal preservation. We assessed the efficacy of one of these chondroitin-supplemented media (K-Sol) in comparison with McCarey-Kaufman (MK) medium in maintaining corneal cellular morphology. Thirty-six human corneas, obtained within 8.6 h after death, were placed into K-Sol medium for up to 20 days preservation, and five paired control corneas were placed into MK medium for up to 6 days preservation. Specular photomicrographs were obtained every second to third day for a predetermined storage interval, then studied morphologically in a masked protocol by light microscopy, transmission electron microscopy, and scanning electron microscopy. Endothelial cell loss by specular microscopy averaged 5.8% after 1 week (6 to 8 days) and 7.4% after 13 days in K-Sol medium. Epithelial changes were erratic throughout the 20 day K-Sol preservation period. However, substantial keratocyte changes occurred after 10 days, and endothelial morphology uniformly deteriorated after 17 days. The morphologic data suggest that human corneas may be able to be preserved in K-Sol medium at 4 degrees C for up to 10 days but should be cautiously used thereafter.     

一氧化氮与血管生成的研究进展

一氧化氮(NO)是一种重要的信使分子和生物活性物质,参与调节机体的一系列生理活动以及新生血管生成等病理过程.本文就NO及其合成酶(NOS)在血管生成中的作用做一简要综述.     

Corneal temperature reversal after storage in Chen medium compared with Optisol GS.

PURPOSE: To compare corneal endothelial cell function by measuring corneal thickness during temperature reversal between corneas stored in two different storage media, Optisol GS and Chen Medium (CM). METHODS: Twenty paired corneas from 10 human donors were randomly assigned for storage at 4 degrees C in Optisol GS (10 corneas) or CM (10 corneas). The storage media were masked, and measurements were done in a masked fashion. After storage for 48 hours, corneal thickness was measured by ultrasonic pachymetry at 2-hour intervals for 12 hours, during which time the corneas were perfused with BSS (balanced salt solution) Plus at 37 degrees C. Scanning electron microscopy of two pairs of corneas from two donors was performed to assess ultrastructural change after 12 hours of warming. RESULTS: Corneal thickness decreased during the first 4 hours of the warming period and then increased during the 6-to 12-hour warming period. These changes in corneal thickness over time were similar for the two storage media (p = 0.212). Scanning electron microscopy showed greater amounts of endothelial cell disruption in Optisol GS-stored corneas than those stored in CM after 12 hours of warming and perfusion. CONCLUSIONS: The endothelial pump of corneas stored in CM appear to be as well-preserved as those stored in Optisol GS, although greater endothelial disruption may be present with Optisol GS by scanning electron microscopy. Further studies are required to compare the clinical effectiveness of these two media.