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。     

氨基胍对兔缺血-再灌注损伤后视网膜形态学及一氧化氮和一氧化氮合酶表达的影响

背景临床研究表明,多种眼科疾病如青光眼、视网膜中央动脉阻塞、缺血性视神经病变等均可导致视网膜缺血-再灌注损伤（RIRI）,严重影响视功能,因而对治疗RIRI药物的研究是非常必要的。目的观察并探讨氨基胍对兔RIRI后形态学的变化,并研究其对一氧化氮（NO）及一氧化氮合酶（iNOS）在视网膜中表达的影响及其机制。方法清洁级日本大耳白兔66只,以随机数字表法分为正常组、RIRI模型组和氨基胍治疗组。用前房灌注生理盐水法升高眼压60min后恢复灌注建立兔RIRI模型,氨基胍治疗组每日在模型兔腹腔内注射氨基胍注射液80mg／kg,而RIRI模型组以同样的方法注射等量生理盐水。RIRI模型组和氨基胍治疗组分别于缺血即时、再灌注后6、24、72h各取2只兔活体行眼底彩色照相及荧光素眼底血管造影（FFA）。各组兔分别于再灌注后1、6、24、72h用空气栓塞法处死并摘除眼球以制备视网膜切片,用TUNEL法检测视网膜组织神经细胞的凋亡变化,通过硝酸还原酶法检测NO浓度,比色法检测iNOS活力。结果各时间点眼底彩色照相及FFA检查结果表明,与RIRI模型组比较,氨基胍治疗组视网膜水肿程度减轻,血管闭塞程度及比例降低,荧光素渗漏量及面积减轻并减少。TUNEL染色凋亡细胞计数检测表明,正常组兔视网膜未见TUNEL阳性细胞,而缺血-再灌注1、6、24、72h后RIRI模型组兔视网膜凋亡细胞计数均明显高于氨基胍治疗组（F分组=2762．37,P=0．00;F时间=894．24,P=0．00）。RIRI模型组和氨基胍治疗组各组内相邻时间点之间TUNEL阳性细胞的差异均有统计学意义（RIRI模型组：q=24．47、36．59、-20．37,P〈0．05;氨基胍治疗组：q=20．94、16．79、-6．92,P〈0．05）,再灌注后24h各组TUNEL阳性细胞数量达到高峰。各时间点RIRI模型组兔视网膜NO浓度明显高于氨基胍治疗组（q=3．84、4．01、8．91、3．75,P〈0．05）,各组内相邻时间点之间NO浓度的差异均有统计学意义（RIRI模型组：q=4．77、13．40、-10．29,P〈0．05;氨基胍治疗组：q=4．55、9．05、-5．08,P〈0．05）,各组24h视网膜中NO浓度达峰值。各时间点RIRI组视网膜iNOS活力均明显高于氨基胍治疗组（q=-3．74、-4．94、-6．53、-3．98,P〈0．05）;各组内相邻时间点间iNOS活力的差异均有统计学意义（RIRI模型组：q=8．43、6．71、-6．39,P〈0．05;氨基胍治疗组：q=4．16、5．08、-3．93,P〈0．05）,各组24h iNOS活力达峰值。结论氨基胍对维持RIRI后的视网膜形态和功能起保护作用,其作用机制可能为抑制iNOS的活性,减少NO的生成。     

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.     

一氧化氮合成酶在正常幼猫视觉系统的分布

目的 观察一氧化氮合成酶在正常幼猫视觉系统的分布,探讨一氧化氮在视觉系统中的作用。方法 用ＮＡＤＰＨ黄递酶（ＮＤＰ）组织化学染色法观察了一氧化氮合成酶在正常幼猫视觉系统视网膜、外侧膝状体、视皮 分布、结果 正常幼猫视网膜和视皮层１７区均可见ＮＯＳ阳性细胞和纤维;外太体（ＬＧＮ）各层无ＮＯＳ阳性细胞,但可见ＮＯＳＢ是性纤维,结论一氧化氮可能作为一种重要的神经递质参与视觉信息的形成、整合传递     

Effects of aminoguanidine on retinal apoptosis in mice with oxygen-induced retinopathy

AIM: To explore the protective effects of aminoguanidine (AG) on retinal apoptosis in mice with oxygen-induced retinopathy (OIR).METHODS:A total of 80 C57BL/6J mice, aged 7 days, were randomly divided into four groups:normal, high oxygen, high oxygen saline and high oxygen treated with AG. In the normal group, mice were housed in normoxic conditions from postnatal day P7 to P17. Mice in the other 3 groups were placed under hyperoxic conditions (75±2%O2) in an oxygen-regulated chamber for 5 days and subsequently placed in normoxic conditions for 5 days. Mice in the AG group were treated once daily, from P12 to P17, with AG hemisulfate (100mg/kg body weight, intraperitoneally) dissolved in physiological saline. An equivalent amount of 0.9% physiological saline was administered, as above, to mice in the high oxygen saline group. Ten mice were randomly selected from each group on P14 and on P17, euthanized and the retinas examined. Apoptotic cells in the retina were detected using the terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) method. The expression of nitric oxide synthase (iNOS) in the retina was detected by immunohistochemistry and changes in rod cells were observed using electron microscopy.RESULTS:TUNEL-positive cells and iNOS immunoreactive neurons were present in the inner nuclear and ganglion cell retinal layers of mice in the high oxygen group. The number of TUNEL-positive cells was significantly greater in the high oxygen group compared with the normal group (t=-20.81, P14d <0.05; t=-15.05, P17d<0.05). However, the number of TUNEL-positive cells in the AG treatment group was significantly lower (t=-13.21, P14d<0.05; t=-6.61,P17d <0.05) compared with the high oxygen group. The expression of iNOS was significantly higher in the high oxygen group compared with the normal group (t=-21.95, P14d<0.05; t=-17.30, P17d<0.05). However, the expression of iNOS in the AG treatment group was significantly lower (t=-12.17,P14d<0.05; t=-10.30,P17d<0.05) compared with the high oxygen group. The outer segments of the rods were disorganized and short in the high oxygen group. Rod morphology appeared to be slightly improved in the AG group.CONCLUSION:AG may protect retinal neurons in OIR by inhibiting apoptosis. The mechanism may be related to iNOS.     

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。     

过度光照后诱导型一氧化氮合酶在大鼠视网膜色素上皮的表达

目的探讨过度光照诱导大鼠视网膜色素上皮（retinal pigment epithelium．RPE）细胞表达诱导型一氧化氮合酶（inducible nitric oxide synthase，iNOS）的作用及其病理学意义。方法应用免疫组织化学的方法分别检测处于自然光照环境中的正常大鼠及接受强光过度照射后大鼠的视网膜RPE细胞中iNOS的表达情况。结果正常大鼠RPE细胞中未见iNOS的表达，而过度光照后大鼠RPE细胞的胞浆中表达高水平的iNOS，同时视网膜外核层感光细胞发生结构损伤。结论过度光照可诱导RPE细胞表达iNOS．这一异常改变可能是视网膜感光细胞结构损伤的重要原因。     

Regulation of nitric oxide synthase 2 in rabbit corneal cells

PURPOSE. The purpose of these studies was to investigate the role of interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), interleukin 1beta (IL-1beta), and transforming growth factor-beta (TGF-beta) in the regulation of inducible nitric oxide synthase (NOS2) activity in rabbit corneal cells. METHODS. Rabbit corneal epithelial, stromal, and endothelial cells were grown in culture and treated with cytokines and growth factors, alone or in combination. NOS activity was measured at times up to 72 hours after treatment by assaying the culture medium for nitrite using the Griess reaction. Cell lysates were analyzed by Western blot analysis for NOS2 protein. RNA was isolated and amplified with NOS1-, NOS2-, and NOS3-specific primers by RT-PCR. RESULTS. NOS2 expression was induced by combined cytokine treatment from nondetectable levels to abundant levels in low passage (<4) stromal cells and to low levels in corneal endothelial cells but not in corneal epithelial cells. In the absence of IFN-gamma, little or no nitrite accumulation was induced by TNF-alpha, IL-1beta, or lipopolysaccharide (LPS) treatment. The inductive effects of IFN-gamma were antagonized in a dose-dependent manner by the myxoma virus rabbit IFN-gamma receptor homolog, M-T7. rRaIFN-gamma, in combination with IL-1beta and TNF-alpha, induced the appearance of NOS2 mRNA within 24 hours but detectable nitrite did not accumulate in large amounts (>10 microM) until after 24 hours postinduction. NOS2 was identified as a 130 kDa protein on Western blot analysis using monoclonal antibody against murine NOS2. TGF-beta(1) and beta(2) inhibited the accumulation of cytokine-induced nitrite in a dose-dependent manner while not significantly reducing the steady state level of NOS2 mRNA. The activity of the induced NOS was inhibited by 1400W, a NOS2-selective inhibitor, but not 7-nitroindazole, a NOS1-selective inhibitor. CONCLUSIONS. In cultured corneal stromal cells, NOS2 expression was upregulated by IFN-gamma in combination with IL-1beta and TNF-alpha but not by any of these cytokines alone, while TGF-beta downregulated the activity. Cultures of corneal epithelial cells could not be induced to express NOS2, yet cultures of endothelial cells produced low amounts of NO in response to cytokines. The NOS1 and NOS3 isoforms were not detected in any of these corneal cells.     

Inducible nitric oxide synthase isoform is a key mediator of leukostasis and blood-retinal barrier breakdown in diabetic retinopathy

PURPOSE: Nitric oxide (NO) is involved in leukostasis and blood-retinal barrier (BRB) breakdown in the early stages of diabetic retinopathy (DR), but it is unclear which NO synthase (NOS) isoforms are primarily involved. In this study, the authors aimed to clarify the involvement of constitutive (eNOS, nNOS) and inducible NOS (iNOS) isoforms and the mechanisms underlying NO-mediated leukostasis and BRB breakdown. METHODS: Diabetes was induced with streptozotocin for 2 weeks. Mice were treated with a NOS inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME), which shows a preference for constitutive isoforms over iNOS. Vessel leakage was assessed with Evans blue. Leukostasis was quantified in flat-mounted retinas with confocal microscopy, in vivo with a scanning laser ophthalmoscope, and in vitro in a retinal endothelial cell line. ICAM-1, occludin, and ZO-1 levels were assessed by Western blot, flow cytometry, or immunohistochemistry. Nitrotyrosine content was assessed by immunohistochemistry. RESULTS: Diabetes increased leukostasis within retinal vessels and BRB permeability, which were reduced by L-NAME. Similar effects were observed in diabetic iNOS knockout mice. In diabetic mouse retinas, ICAM-1 protein levels increased, whereas the immunoreactivity of tight junction proteins, occludin and ZO-1 decreased, in correlation with increased protein levels of all NOS isoforms. Those effects were prevented by L-NAME and also in diabetic iNOS knockout mice. High glucose and nitrosative/oxidative stress also increased leukostasis caused by ICAM-1 upregulation. CONCLUSIONS: These results indicate that the iNOS isoform plays a predominant role in leukostasis and BRB breakdown. The mechanism involves ICAM-1 upregulation and tight junction protein downregulation.     

Inducible nitric oxide synthase mediates retinal DNA damage in Goto-Kakizaki rat retina

PURPOSE: To examine the nitrosative and oxidative DNA damage induced by 8-nitroguanine and 8-hydroxy-2-deoxy guanosine (8-OHdG), and to determine the role played by inducible nitric oxide synthase (iNOS) in damage to DNA in the retina of the Goto-Kakizaki (GK) rat. METHODS: Experiments were performed on GK rats, an animal model of spontaneous type 2 diabetes without obesity or visible diabetic vascular lesions. Immunohistochemistry was used to determine the retinal distribution of 8-nitroguanine, 8-OHdG, and iNOS in GK rats and control rats. The change in the expression of 8-nitroguanine and 8-OHdG in GK rats was also determined following an intravitreal injection of 1400W, an inhibitor of iNOS activity. RESULTS: Immunohistochemical analysis showed that 8-nitroguanine and 8-OHdG were expressed strongly in the inner nuclear layer of GK retinas but only weakly in control retinas. This expression was correlated with an increase in the expression of iNOS in GK retinas, which was confirmed by the inhibition of iNOS activity by 1400W. CONCLUSION: These findings demonstrate that iNOS plays a crucial role in nitrosative and oxidative DNA damage in GK rats, suggesting a retinal neurotoxic role of nitric oxide and superoxide in diabetic retinas.     

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     

Inhibition of nitric oxide synthesis in corneas in storage media

The nitrate/nitrite content in storage media was determined after nitric oxide synthase inhibition by adding 400 microl of 100 mm N(G)-monomethyl-l-arginine (LMMA) to four chambers of Optisol GS corneal storage media, each containing one viable human cornea. The companion corneas in storage media without LMMA served as controls. Four hundred microlitre aliquots obtained at baseline (day 0) and at one-day intervals for 20 more days for both groups were analyzed for nitrate and nitrite (breakdown products of nitric oxide) concentration levels using a spectrophotometric method based on the Greiss reaction. Average nitrate/nitrite concentrations, statistically analyzed using a polynomial random coefficients model, showed a statistically significant marked reduction in the levels of nitrate and nitrite accumulation in the study chambers as compared to control chambers for days 1-20(P < 0.001) There was also a reduction in the accumulation rate of nitrate and nitrite concentrations, as compared to controls (P < 0.05) until around day 8 when the differences in rates were no longer statistically significant. The progressive increase in nitrate and nitrite accumulation in corneal storage media can be blunted by the addition of a nitric oxide synthase inhibitor. Given the toxic free radical properties of nitric oxide, corneas in storage awaiting transplantation may benefit from having a nitric oxide synthase inhibitor added to storage media.     

川芎嗪联合氨基胍对糖尿病大鼠视网膜保护作用的机制

目的　探讨川芎嗪联合氨基胍对糖尿病视网膜病变防治作用及其机制。方法　用链尿佐菌素 (STZ)制作糖尿病大鼠动物模型 ,分为正常对照组、糖尿病对照组、糖尿病氨基胍治疗组、糖尿病川芎嗪 +氨基胍治疗组 ,于第 12周测定大鼠视网膜组织NO的含量、NOS活性和AR的活性。结果　糖尿病川芎嗪 +氨基胍治疗组、糖尿病氨基胍治疗组大鼠视网膜组织NOS及AR活性显著低于糖尿病对照组 (P <0 0 1) ,NO的含量升高 (P <0 0 1) ;糖尿病氨基胍治疗组仍高于正常对照组 (P <0 0 1) ,NO的含量降低 (P <0 0 1) ;糖尿病川芎嗪 +氨基胍治疗组与正常组无显著差异。结论　川芎嗪联合氨基胍能够抑制糖尿病大鼠视网膜组织NOS、AR活性 ,从而对糖尿病视网膜病变起一定保护作用     

Inhibited experimental corneal neovascularization by neutralizing anti-SDF-1α antibody

AIM: To explore the effect of SDF-1α on the development of experimental corneal neovascularization (CRNV). METHODS: CRNV was induced by alkali injury in mice. The expression of SDF-1α and CXCR4 in burned corneas was examined by Flow Cytometry. Neutralizing anti-mouse SDF-1α antibody was locally administrated after alkali injury and the formation of CRNV 2 weeks after injury was assessed by Immunohistochemistry. The expression of VEGF and C-Kit in burned corneas was detected by RT-PCR. RESULTS: The number of CRNV peaks at 2 weeks after alkali injury. Compared to control group, SDF-1α neutralizing antibody treatment significantly decreased the number of CRNV. RT-PCR confirmed that SDF-1α neutralizing antibody treatment resulted in decreased intracorneal VEGF and C-Kit expression. CONCLUSION: SDF-1α neutralizing antibody treated mice exhibited impaired experimental CRNV through down regulated VEGF and C-Kit expression.     

Endothelial nitric oxide synthase deficiency influences normal cell cycle progression and apoptosis in trabecular meshwork cells

AIM: To clarify how the endothelial nitric oxide synthase (eNOS, NOS3) make effect on outflow facility through the trabecular meshwork (TM). METHODS: Inhibition of NOS3 gene expression in human TM cells were conducted by three siRNAs. Then the mRNA and protein levels of NOS3 in siRNA-treated and negative control (NC) cells were determined, still were the collagen, type IV, alpha 1 (COL4A1) and fibronectin 1 by real-time PCR and Western blot analysis. In addition, NOS3 concentrations in culture supernatant fluids of TM cells were measured. Cell cycle and cell apoptosis analysis were performed using flow cytometry. RESULTS: The mRNA level of NOS3 was decreased by three different siRNA interference, similar results were obtained not only of the relative levels of NOS3 protein, but also the expression levels of COL4A1 and fibronectin 1. The number of cells in S phase was decreased, while contrary result was obtained in G2 phase. The number of apoptotic cells in siRNA-treated groups were significant increased compared to the NC samples. CONCLUSION: Abnormal NOS3 expression can make effects on the proteins levels of extracellular matrix component (e.g. fibronectin 1 and COL4A1). Reduced NOS3 restrains the TM cell cycle progression at the G2/M-phase transition and induced cell apoptosis.     

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.     

一氧化氮(NO)对角膜神经再生的影响作用

目的 探讨一氧化氮(NO)对角膜神经再生的影响作用。方法 本研究以亚硝酸钠(NaNO₂)作为外源性NO供体,在细胞实验中以小鼠神经母细胞瘤细胞(Neuro-2a)为研究对象。采用不同浓度的NaNO₂处理Neuro-2a细胞并筛选出NO的最佳神经营养浓度。在动物实验中,将30只SD大鼠随机分组,10只作为NC组,其余20只大鼠建立角膜碱烧伤模型,再随机分为PBS组和NO组,每组10只。从碱烧伤当天开始,PBS组给予PBS治疗,NO组给予10.00μmol·L^-1 NaNO₂与PBS混合治疗。用荧光素钠染色后观察并记录大鼠角膜上皮愈合情况,计算角膜上皮愈合率。用CCK-8检测细胞活性,流式细胞术检测细胞凋亡率,免疫荧光法检测细胞神经元标记物的表达。于大鼠角膜碱烧伤处理后7 d取大鼠角膜上皮组织,分别采用实时荧光定量PCR和Western blot法检测每组角膜上皮中神经元标志物βⅢ-微管蛋白和神经生长因子(NGF)、胶质细胞源性神经营养因子(GDNF)、睫状神经营养因子(CNTF)的表达水平。结果 10...