Expression of nitric oxide synthase during the development of RCS rat retinas

Nitric oxide (NO) has been reported to be both neurodestructive and neuroprotective in the central nervous system and could possibly play an important role in neurodegenerative disorders. On the assumption that NO synthesis may influence degenerative processes in the retina, we have examined the development and distribution of nitric-oxide-synthase(NOS)-immunoreactive cells in developing Royal College of Surgeons (RCS) rat retinas, which is an animal model for retinal degeneration. An antibody against constitutive neuronal NOS was used for immunocytochemistry on RCS rat retinas from postnatal (PN) days 3, 7, 10, 14, 35, 70 and 281 and compared with that in the normal rats of PN days 3, 7, 10, 14, 54 and adults. Immunoreactive cells were not seen in PN 3 retinas but were distinctly seen in the PN 7 retina along with a plexus in the inner plexiform layer. In both groups (normal and RCS rats) a distinct sublayering of the plexus in the inner plexiform layer could be seen at PN 10, which became more distinct at PN 14. The immunoreactive cells were detected also in the oldest retina examined, which was PN 281 in the case of RCS rats. In both groups, certain amacrine cells, certain bipolar cells and certain horizontal cells were found to be immunoreactive. In conclusion, the developmental timetable of the NOS immunoreactivity was identical in the normal and the RCS rat retinas. The NOS-immunoreactive cells persisted in the RCS retinas even when the retina had degenerated extensively. Abnormalities with the inducible isoforms of NOS cannot be ruled out from this study. We conclude that the chronological and qualitative development of the constitutive neuronal NOS immunoreactivity is normal in RCS rat retinas.     

Endothelial nitric oxide synthase is expressed in amacrine cells of developing human retinas

PURPOSE: To examine the expression and cellular distribution pattern of endothelial nitric oxide synthase (eNOS) in the developing human retina and to compare its expression with that in rats. METHODS: Expression of eNOS was examined by immunohistochemistry in retinas of humans ranging from 8.5 to 28 weeks of gestation (WG) and of rats. RESULTS: In the developing human retina, eNOS expression was first detected in the proximal margin of the neuroblastic layer in the incipient fovea-surrounding area at 12 WG. At 17 to 28 WG, eNOS-immunoreactive cells were located in the innermost part of the inner nuclear layer and in the ganglion cell layer, expanding to both temporal and nasal retinas and the processes projecting into the inner plexiform layer. These eNOS-positive cells coexpressed syntaxin and glutamate decarboxylase, and are probably GABAergic amacrine cells. The onset of eNOS expression in developing amacrine cells, however, preceded the invasion of retinal vasculature, long before vascular function involving these cells can be expected, suggesting that eNOS has a role not only in vasoregulation but also in retinal development. From 20 WG on, eNOS was also detected in the photoreceptors adjacent to the fovea. eNOS expression in amacrine cells and photoreceptors was observed in the central-to-peripheral and temporal-to-nasal gradients. However, in the developing rat retina, eNOS was expressed exclusively in the vascular endothelial cells. CONCLUSIONS: The results support that eNOS plays a role, not only in the regulation of vascular function but also in the process of retinal development in humans.     

脂联素调控糖尿病大鼠视网膜一氧化氮合酶的表达及意义

背景糖尿病视网膜病变（DR）是糖尿病最常见的微血管并发症,其发病机制尚未完全阐明。脂联素可以通过抑制血管内皮细胞的炎症反应、降低血管细胞间黏附能力等影响糖尿病微血管病变的发生,脂联素与一氧化氮合酶（NOS）的作用关系报道较少。目的观察脂联素对糖尿病大鼠视网膜NOS表达的影响。方法选择8～10周龄雌性SD大鼠40只,按随机数字表法将大鼠分为正常对照组10只、脂联素组及糖尿病对照组各15只。脂联素组及糖尿病对照组大鼠用一次性腹腔注射四氧嘧啶法建立糖尿病模型,注射24h后测空腹血糖值〉16mmol／L、尿糖〉＋＋＋为造模成功,纳入研究共24只糖尿病大鼠。脂联素组大鼠给予10μg／kg重组脂联素注射。采用Westernblot法检测脂联素蛋白在正常对照组、脂联素组及糖尿病对照组大鼠视网膜中的定量变化,采用免疫组织化学法观察NOS在各组大鼠视网膜中的表达和定位。结果正常对照组、脂联素组及糖尿病对照组大鼠视网膜中脂联素蛋白含量（脂联素／p—actin）分别为0．85±0．21、0．79±0．17和0．42±0．08,总体比较差异有统计学意义（,=4．236,P=0．000）;糖尿病对照组大鼠视网膜脂联素蛋白较正常对照组和脂联素组明显下降,差异均有统计学意义（q=6．615,P=0．000;q=6．026,P=0．000）。正常对照组、脂联素组、糖尿病对照组NOS阳性反应强度的4值分别为0．244±0．035、0．262±0．032和0．367±0．066,总体差异有统计学意义（F=3．752,P=0．001）;糖尿病对照组NOS含量较正常对照组、脂联素组明显升高,差异均有统计学意义（q=3．488,P=0．002;q=3．079,P=0．005）。NOS主要表达于视网膜内核层和视网膜神经节细胞（RGCs）层。结论脂联素可下调糖尿病大鼠视网膜中NOS的含量,减少NO在视网膜中的生成,减轻NO对视网膜血管内皮细胞结构和功能的损伤,从而延缓DR的发生。     

Nitric oxide synthase activity in tissues of the bovine eye

• Background: Nitric oxide synthase (NOS) is present in many ocular tissues where it may have different physiological functions. This warrants a thorough characterization of NOS activity in the eye. • Methods: NOS distribution and its biochemical properties were determined in the retina, choroid, ciliary processes (CP), and trabecular meshwork (TM). • Results: Retinal NOS required NADPH (diphenyleneiodonium, a flavoprotein inhibitor, which inhibited enzyme activity with an IC₅₀ of 0.36 μM, FAD (40 μM), FMN (40 μM), and BH₄ (4 μM) as cofactors for optimal activity. Ocular NOS appeared to be regulated by free divalent cations, since its activity was inhibited by EDTA (slopes >3.0 and IC₅₀ values of 12.8, 19.7, and 53 μM, respectively). Ocular NOS required calmodulin, since NOS activity was inhibited by trifluoperazine (calmodulin inhibitor, IC₅₀=41 μM). NOS activity is widely distributed in the eye, (choroid >reinta >CP >TM) and is mainly cytosolic (70–95%).l-Arginine analogs inhibited NOS in the retina, choroid, and TM. In all three tissues,N ^G-methyl-l,-arginine displayed the highest affinity for inhibition (IC₅₀=0.2–0.7 μM) followed by canavanine (IC₅₀=13–33 μM), while aminoguanidine only weakly inhibited NOS (IC₅₀=93–179 μM). • Conclusion: In all tissues, the order of potency of inhibition points to the presence of constitutive rather than inducible NOS. Moreover, it is possible that TM contains more than a single form of NOS.     

Inducible nitric oxide synthase inhibitors abolished histological protection by late ischemic preconditioning in rat retina

Brief ischemia was reported to protect retinal cells against injury induced by subsequent ischemia-reperfusion with de novo protein synthesis, and this phenomenon is known as late ischemic preconditioning. The aims of the present study were to determine whether nitric oxide synthase (NOS) was involved in the mechanism of late ischemic preconditioning in rat retina using pharmacological tools. Under anesthesia with pentobarbital sodium, male Sprague-Dawley rats were subjected to 60 min of retinal ischemia by raising intraocular pressure to 130 mm Hg. Ischemic preconditioning was achieved by applying 5 min of ischemia 24 hrs before 60 min of ischemia. Retinal sections sliced into 5 microm thick were examined 7 days after ischemia. Additional groups of rats received NG-nitro-L-arginine and NG-monomethyl-L-arginin, non-selective NO synthase inhibitors, 7-nitroindazole, a neuronal NOS inhibitor, and aminoguanidine and L-N6-(1-iminoethyl) lysine, inducible NO synthase (iNOS) inhibitors before preconditioning, and were subjected to 60 min of ischemia. In the non-preconditioned group, cell loss in the ganglion cell layer and thinning of the inner plexiform and inner nuclear layer were observed 7 days after 60 min of ischemia. Ischemic preconditioning for 5 min completely protected against the histological damage induced by 60 min of ischemia applied 24 hrs thereafter. Treatment of rats with aminoguanidine and L-N6-(1-iminoethyl) lysine, but not NG-nitro-L-arginine, NG-monomethyl-L-arginine or 7-nitroindazole, wiped off the protective effect of ischemic preconditioning. The inhibitory effect of aminoguanidine was abolished by L-arginine, but not D-arginine. The results in the present study suggest that NO synthesized by iNOS is involved in the histological protection by late ischemic preconditioning in rat retina.     

Nitric oxide amplifies the rat electroretinogram

It is well established that nitric oxide (NO) participates in retinal signal processing through stimulation of its receptor enzyme, soluble guanylyl cyclase (sGC). However, under pathological conditions such as uveoretinitis, diabetic or ischemic retinopathy, elevated NO concentrations may cause protein S-nitrosation and peroxynitrite formation in the retina, promoting cellular injury and apoptosis. Previous electroretinogram (ERG) studies demonstrated deleterious effects of NO on the retinal light response, but showed no evidence for a role in normal signal processing. To better understand the function of NO in ocular physiology, we investigated the effects of exogenous NO, produced by NO donors with different release kinetics, on the flash ERG of the rat. Within a limited concentration range, NO strongly amplified ERG a- and b-waves, oscillatory potentials, and the scotopic threshold response. Amplification exceeded 100% under dark adaptation, whereas the photopic ERG and the isolated cone response were increased by less than 50%. Blocking photoreceptor-bipolar cell synapses by AP-4 demonstrated a significant increase of the isolated a-wave by NO, and modeling the ERG generator PIII supported photoreceptors as primary NO targets. The sGC inhibitors ODQ and NS2028 did not reduce NO-dependent ERG amplification, ruling out an involvement of the classical NO effector cyclic GMP. Using immunohistochemistry, we show that illumination and exogenous NO altered the S-nitrosation level of the photoreceptor layer, suggesting that direct protein modifications caused by elevated levels of NO may be responsible for the observed phenomenon.     

Prediction and verification of miRNA expression in human and rat retinas

PURPOSE: MicroRNAs (miRNAs) play a global role in regulating gene expression and have important tissue-specific functions. Little is known about their role in the retina. The purpose of this study was to establish the retinal expression of those miRNAs predicted to target genes involved in vision. METHODS: miRNAs potentially targeting important "retinal" genes, as defined by expression pattern and implication in disease, were predicted using a published algorithm (TargetScan; Envisioneering Medical Technologies, St. Louis, MO). The presence of candidate miRNAs in human and rat retinal RNA was assessed by RT-PCR. cDNA levels for each miRNA were determined by quantitative PCR. The ability to discriminate between miRNAs varying by a single nucleotide was assessed. The activity of miR-124 and miR-29 against predicted target sites in Rdh10 and Impdh1 was tested by cotransfection of miRNA mimics and luciferase reporter plasmids. RESULTS: Sixty-seven miRNAs were predicted to target one or more of the 320 retinal genes listed herein. All 11 candidate miRNAs tested were expressed in the retina, including miR-7, miR-124, miR135a, and miR135b. Relative levels of individual miRNAs were similar between rats and humans. The Rdh10 3'UTR, which contains a predicted miR-124 target site, mediated the inhibition of luciferase activity by miR-124 mimics in cell culture. CONCLUSIONS: Many miRNAs likely to regulate genes important for retinal function are present in the retina. Conservation of miRNA retinal expression patterns from rats to humans supports evidence from other tissues that disruption of miRNAs is a likely cause of a range of visual abnormalities.     

光化学损伤下大鼠视网膜超氧化物歧化酶基因mRNA表达的实验研究

通过建立大鼠视网膜光化学损伤模型，应用原位杂交技术对正常及光化学损伤大鼠视风膜超氧化物歧化酶基因ｍＲＮＡ表达情况进行动态观察。结果表明，视网膜组织各层均可见超氧化物歧化酶基因ｍＲＮＡ的表达；连续光照可使其表达水平迅速下降，原位杂交为研究视网膜基因表达状况的一种良好方法。     

缺氧性视网膜病变患者血及玻璃体中NO和VEGF浓度的变化

目的:检测一氧化氮(nitric oxide,NO)在缺氧性视网膜病变患者的玻璃体和血中的浓度变化,并与血管内皮生长因子(vascular endothelial growth factor,VEGF)相比较,从而阐明两者的关系。方法:增生性糖尿病视网膜病变(proliferative diabetic reti-nopathy,PDR)16例,视网膜分支静脉阻塞5例,孔源性视网膜脱离11例和黄斑裂孔10例。抽取患者血液,并于玻璃体切除手术中抽取玻璃体。Griess反应检测NO的浓度,ELISA检测VEGF的浓度。结果:玻璃体中NO浓度在PDR组中明显升高(15.2,4.6~50.9μmol/L),与其他3组相比具有统计学意义(P=0.0046);血液中的NO浓度在4组中无明显差异。玻璃体中的VEGF浓度在PDR(1507.2,50.7~3722.0mg/L)和BRVO(838.8,212.7~850.6mg/L)组中均明显升高,与其他两组相比具有统计学意义(P=0.0008),而PDR组又高于BRVO组(P=0.001);各组病例血液中VEGF浓度无明显差异(P=0.76)。4组病例中,NO和VEGF间均未见相关性。结论:NO和VEGF在PDR组的玻璃体中均明显升高,表明二者参与了糖尿病视网膜新生血管生成的过程。     

Expression of the inducible isoform of nitric oxide synthase in the retinas of human subjects with diabetes mellitus

PURPOSE: Inducible nitric oxide synthase has been implicated in the pathogenesis of cerebral ischemic damage, in the angiogenic process and in diabetic vascular damage. This study was undertaken to determine whether inducible nitric oxide synthase is present in the retinas from human subjects with diabetes mellitus. METHODS: This was an experimental immunohistochemical prospective study. Ten postmortem eyes from five subjects with diabetes mellitus, 10 eyes from five subjects without diabetes and without known ocular disease, and two eyes from one subject with unilateral ocular ischemic syndrome secondary to severe carotid artery obstruction were examined. We used immunohistochemical techniques and antibodies directed against inducible nitric oxide synthase, glial fibrillary acidic protein, and vimentin. The main outcome measure was immunoreactivity for these antibodies. RESULTS: Immunoreactivity for inducible nitric oxide synthase was not observed in retinas from all subjects without diabetes and without ocular disease. Six retinas from three subjects with diabetes and nonproliferative retinopathy, and the retina from the eye with ocular ischemic syndrome showed immunoreactivity for inducible nitric oxide synthase in cells with elongated processes. Based on morphology and on glial fibrillary acidic protein and vimentin immunoreactivity, this inducible nitric oxide synthase immunoreactivity appeared to localize to retinal Müller glial cells. CONCLUSIONS: These observations suggest that Müller cells may be involved in the microvascular remodeling of the diseased retina and that high concentrations of nitric oxide produced by inducible nitric oxide synthase could contribute to neurotoxicity and angiogenesis that occur in diabetic retinopathy.     

Endothelial nitric oxide synthase gene polymorphisms in non-arteritic anterior ischemic optic neuropathy

Background To examine the association between the polymorphisms of the endothelial nitric oxide synthase (eNOS) gene and the occurrence of non-arteritic anterior ischemic optic neuropathy (NAION). Methods We studied 15 patients with NAION (mean age, 62 years; 60% male). We investigated two polymorphisms of the eNOS gene, Glu298Asp polymorphism of exon 7 and T(–786)C polymorphism of the promoter region. The genotype distribution in NAION was compared with the control (mean age, 63 years; 63% male) distribution. Results There was no significant difference in the genotype distribution of the Glu298Asp polymorphism between the NAION and control groups (P=1.000), whereas the genotype distribution of the T(–786)C polymorphism varied significantly between the patients with NAION and control subjects (P=0.002). After adjusting on covariates, individuals with the CC genotype of the T(–786)C polymorphism were more likely to develop NAION compared with those with TT genotype (odds ratio=0.09: 95% CI 0.01–0.86). Conclusions We found an increased prevalence of T(–786)C polymorphism of the eNOS gene in patients with NAION. Our data suggest that the T(–786)C polymorphism of the eNOS gene may be an important risk factor in the development of NAION in Japanese subjects.     

Inducible nitric oxide synthase and vascular endothelial growth factor are colocalized in the retinas of human subjects with diabetes

PURPOSE: Nitric oxide (NO) mediates vascular endothelial growth factor (VEGF)-induced angiogenesis and vascular hyperpermeability. This study was undertaken to study the cellular distribution of inducible nitric oxide synthase (iNOS) and VEGF in the retinas from human subjects with diabetes mellitus. In addition, glial reactivity and peroxynitrite generation were detected by immunolocalization of glial fibrillary acidic protein (GFAP) and nitrotyrosine, respectively. METHODS: Eight post-mortem eyes from four consecutive subjects with diabetes mellitus and eight eyes from four subjects without diabetes and without known ocular disease were prospectively collected and examined. We used immunohistochemical techniques and antibodies directed against iNOS, VEGF, GFAP, and nitrotyrosine. RESULTS: In retinas from all subjects without diabetes, weak GFAP immunoreactivity was confined to nerve fibre and ganglion cell layers. There was no immunoreactivity for iNOS, nitrotyrosine, and VEGF. All diabetic retinas showed GFAP induction in Müller cells and GFAP upregulation in nerve fibre and ganglion cell layers. All diabetic retinas showed cytoplasmic immunoreactivity for iNOS, and VEGF in ganglion cells, cells in the inner nuclear layer, and glial cells. In serial sections, ganglion cells and cells in the inner nuclear layer expressing VEGF were localized in the same area of iNOS-expressing ganglion cells and cells in the inner nuclear layer. Six retinas from three subjects with diabetes showed immunoreactivity for nitrotyrosine in vascular endothelial cells in inner retinal layer. CONCLUSIONS: iNOS and VEGF are colocalized in diabetic retinas. Increased GFAP immunoreactivity is a pathological event in the retina during diabetes.     

Effect of hypercholesterolemia on inducible nitric oxide synthase expression in a rat model of elevated intraocular pressure

PURPOSE: This study was performed to examine the effect of hypercholesterolemia on inducible nitric oxide synthase (NOS-2) expression and oxidative tissue injury in an experimental rat model of elevated IOP. METHODS: Wistar rats were maintained on either regular chow or a high-cholesterol diet for 24 weeks. Intraocular pressure (IOP) was elevated in hypercholesterolemic rats by unilaterally cauterizing three episcleral vessels. Rats were divided into four experimental groups as follows; hypercholesterolemia, hypercholesterolemia+elevated IOP, elevated IOP and control. NOS-2 distribution, lipid peroxidation and retinal nerve fiber layer (RNFL) thickness was evaluated in all experimental groups at the end of 24 weeks. RESULTS: Light microscopic evaluation of retinas in hypercholesterolemic rats revealed breaks and discontinuation in focal areas in the outer nuclear layer (ONL). NOS-2 positive staining was observed throughout the outer plexiform layer (OPL), inner plexiform layer (IPL) and ganglion cell layer (GCL) in rats with elevated IOP and/or hypercholesterolemia. Calculated values of RNFL thickness in hypercholesterolemic rats were significantly higher than those in the control and elevated IOP group. Vitreous malondialdehyde (MDA) levels detected in elevated IOP (3.51+/-0.31 nmol/mg protein) and hypercholesterolemia+elevated IOP (5.14+/-1.28 nmol/mg protein) groups were significantly higher than those detected in hypercholesterolemic (1.92+/-1.43 nmol/mg protein) and control (1.89+/-0.24 nmol/mg protein) groups. CONCLUSION: The presented data confirms hypercholesterolemia as a risk factor in the development of glaucomatous optic neuropathy (GON) and suggests that increased circulating cholesterol may exacerbate disease progression by inducing NOS-2 expression and elevating oxidant tissue injury.     

Differential expression of nitric oxide synthase in experimental uveoretinitis.

PURPOSE: To investigate the site and the cellular source of inducible nitric oxide synthase (iNOS) expression in human S-antigen peptide-induced experimental autoimmune uveoretinitis (EAU). METHODS: Twenty-one Lewis rats were sensitized with human S-antigen peptides. Three rats were killed each consecutive day from day 6 through day 12 after sensitization. Frozen sections of the enucleated eyes were analyzed for iNOS by the dual immunohistochemical method. Primary antibodies included rabbit anti-mouse iNOS combined with anti-human endothelium NOS, anti-rat lysosomal protein (ED1), or anti-rat major histocompatibility complex class II molecule (OX6) monoclonal antibodies. Secondary antibodies were fluorescein-conjugated anti-mouse IgG and streptavidin rhodamine-labeled anti-rabbit IgG. The adjacent sections were separately stained with ED1, iNOS, and glial fibrillary acidic protein (GFAP). The mouse macrophage cell line RAW 264.7 was exposed to either interferon (IFN)gamma/lipopolysaccharide (LPS) or S-antigen and to interphotoreceptor retinoid-binding protein (IRBP), myelin basic protein, and bovine serum albumin for 12 hours. Cells were harvested for detection of iNOS expression by northern blot analysis hybridization and detection of protein by immunohistochemistry. RESULTS: In the retina of eyes with EAU, ED1+/iNOS+ and OX6+/iNOS+ cells were first detected on day 9 after sensitization. These iNOS+ cells increased in number on subsequent days in parallel with the increasing severity of retinal damage. Most of the cells localized around the outer retina. In contrast, a large number of ED1+ and OX6+ cells that were localized in the uvea and conjunctiva were negative for iNOS. Retinal pigment epithelial cells did not stain for iNOS. Macrophages exposed to IFNgamma/LPS, S-antigen, and IRBP showed expression of iNOS mRNA and the protein. CONCLUSIONS: Macrophages are an important source of NO production in eyes with EAU. These macrophages preferentially express iNOS in the retina. Such a differential expression of iNOS by the macrophages appears to be related to retinal soluble proteins.     

Abnormal centrifugal axons in streptozotocin-diabetic rat retinas

PURPOSE: To characterize the effects of diabetes on the expression of histidine decarboxylase mRNA and on the morphology of the histaminergic centrifugal axons in the rat retina. METHODS: Rats were made diabetic by streptozotocin. After 3 months, retinal histidine decarboxylase expression was analyzed by in situ hybridization in radial sections. Flatmount retinas from a second group of rats were labeled with an antiserum to histamine or an antibody to phosphorylated neurofilament protein. RESULTS: Histidine decarboxylase mRNA was expressed in cells in the inner and outer nuclear layers of diabetic retinas, but not in normal retinas. However, immunoreactive (IR) histamine was not localized to perikarya in either the normal or the diabetic retinas. Instead, a population of centrifugal axons was labeled. These axons emerged from the optic disc and had varicose terminal branches in the inner plexiform layer (IPL) of the peripheral retina. Some branches ended on large retinal blood vessels and others in dense clusters in the IPL. In rats with streptozotocin-induced diabetes, the centrifugal axon terminals developed many large swellings that contained neurofilament immunoreactivity; these swellings were rare in normal retinas. CONCLUSIONS: Diabetes perturbs the retinal histaminergic system, causing increases in histidine decarboxylase mRNA expression in neurons or glia and abnormal focal swellings on the centrifugal axons.     

Differential gene expression pattern of diabetic rat retinas after intravitreal injection of erythropoietin

Background: To profile the pattern of gene expression in diabetic rat retinas with or without intravitreal injection of erythropoietin. Design: By using streptozotocin‐induced diabetic rats, after intravitreal injection of erythropoietin, neurosensory retinas were collected to determine the effect of erythropoietin on gene expression. Participants: Three groups of Sprague–Dawley rats were studied: normal control (15), diabetic rats with saline injection (15) and diabetic rats with intravitreal erythropoietin treatment (15). Methods: Diabetes was induced by intra‐peritoneal injection of streptozotocin. Intravitreal injection of erythropoietin was performed at the following time points: 0, 30 and 120 days after diabetes onset. Four days after each injection at above‐mentioned time points, the retinas were harvested for microarray assay. The real‐time PCR was used to evaluate the microarray data. Results: Genes encoding inflammatory factors, such as interleukin‐2 and interleukin‐11, which were upregulated in the diabetic retinas, were restored after erythropoietin treatment. Genes encoding pro‐apoptotic effectors, like Tnfrsf5, Bid3 and Bcl2l1, were also upregulated in diabetic rats and attenuated in erythropoietin‐treated group. In addition, real‐time PCR were employed to confirm the changes of the genes Trex2, G1P2, DHX58, RGD1311906 and LOC689064, which have not been reported in diabetic retinopathy. Conclusions: Intravitreal erythropoietin treatment is able to normalize the gene expression responsible for pro‐apoptotic and inflammatory responses noted in diabetic retinas.     

Constitutive nitric oxide synthase activity is required to trigger ischemic tolerance in mouse retina

Profound morphologic and functional protection against retinal ischemic injury can be achieved if the tissue is 'preconditioned' one day earlier with a brief, noninjurious ischemic challenge. To begin to address the mechanistic basis of this 'ischemic tolerance', we used genetic and pharmacologic approaches to test the hypothesis that nitric oxide (NO) derived from one of the three NO synthase (NOS) isoforms was responsible for triggering the adaptive response to brief preconditioning ischemia. Retinae of adult mice were preconditioned with 5-min preconditioning ischemia and subjected to 45-min injurious ischemia 24 hr later. Some animals were treated with the constitutive NOS inhibitor L-nitroarginine (5 mg/kg, i.p.) 1 hr before preconditioning. Retinal layer thicknesses and cell counts were determined one week postischemia in 5-mum thin sections, and flash electroretinograms were obtained at 1 and 7 days postischemia. We confirmed that ischemic preconditioning afforded morphologic and functional protection in the strains of wild-type mice studied. Histopathologic analyses of inducible NOS (iNOS) knockout mice revealed that ischemic preconditioning was completely effective, whereas ischemic tolerance was not achieved in the retinae of endothelial NOS (eNOS) and neuronal NOS (nNOS) knockout mice. The participation of the constitutive NOS enzymes in preconditioning-induced tolerance was confirmed by the finding that administration of the NOS inhibitor L-NA to wild-type mice prior to ischemic preconditioning blocked the development of ischemic tolerance. These cross-validating genetic and pharmacologic findings indicate that NO derived from both eNOS and nNOS is a required molecular signal in the adaptive response to ischemic preconditioning in the retina.     

Characterization and diabetes-induced impairment of nitric oxide synthase in rat choroid

PURPOSE: To examine the nitric oxide (NO) system in the choroid of normal rats and rats with streptozotocin (STZ)-induced diabetes. METHODS: We assayed NO synthase (NOS) activity by monitoring the conversion of L-[(14)C] arginine to L-[(14 )C] citrulline, identified the NOS isoforms by immunoblotting, and examined the effects of STZ-induced diabetes on NOS in the rat choroid. RESULTS: Calcium-independent NOS activity was insignificant in the choroid of normal and diabetic rats. Choroidal calcium-dependent NOS activity was high and comparable to that in the cerebellum. Neuronal (n) NOS protein in the choroid was found in both the membrane and cytosolic fractions and showed similar subcellular distribution as NOS activity, while endothelial (e) NOS protein in the choroid was present almost solely in the membrane fraction. Total NOS activity (nNOS + eNOS) and protein levels of nNOS and eNOS in the choroid were significantly reduced 6 weeks after the induction of diabetes. CONCLUSIONS: The high NOS activity in the choroid measured in vitro appears to come mostly from nNOS. Because choroidal nNOS exists in the parasympathetic perivascular nerve fibers, the decrease in choroidal nNOS in diabetic eyes suggests that the choroid undergoes a diabetes-induced neuronal disorder. Thus, diabetic choroidopathy encompasses diabetic neuropathy and microangiopathy.     

一氧化氮与形觉剥夺性近视眼

一氧化氮是近年来发展的视网膜神经递质。一氧化氮合酶是合成一氧化氮的关键酶，在视网膜分布广泛。一氧化氮不仅参与了视觉信息的形成，整合和传导，具有重要的生理功能，而且在形觉剥夺实验性近视眼的发生中起重要作用。其作用机制尚不清楚，可能与一氧化氮抑制细胞增殖，调节巩膜基质中金属蛋白酶的活性，影响视网膜中其他生物活性物质的合成和释放密切相关。