Inhaled nitric oxide affects endogenous surfactant in experimental lung transplantation

BACKGROUND: Inhalation of nitric oxide (NO) has been proposed as a therapy to improve lung transplantation outcome. We investigated the effect that inhaled NO has on the surfactant system in the context of ischemia-reperfusion injury. METHODS: Single left-lung transplantation was performed in weight-matched pairs of Landrace pigs. A double-lung block from the donor animal was flushed with University of Wisconsin solution at 4 degrees C followed by immersion in cold University of Wisconsin solution for 22 hr. The left donor lung was transplanted into the recipient. Recipients were divided into two groups: (1) treated with inhaled NO (40 ppm) (n=6) immediately after initiating lung reperfusion and (2) without treatment (n=6). Lung function was measured during 2 hr of reperfusion. Surfactant components in small and large aggregates, isolated from cell-free bronchoalveolar lavages, and surfactant function were measured. RESULTS: NO inhalation significantly decreased arterial oxygenation. With respect to the surfactant system, NO inhalation worsened the surfactant adsorption rate to an air-liquid interface and affected levels of hydrophobic surfactant proteins (SPs), SP-B and SP-C, and phospholipids, which decreased in large surfactant aggregates but not in small surfactant aggregates. SP-A was reduced in large surfactant aggregates of transplanted lungs from both untreated and NO-treated groups. CONCLUSION: A decreased level of SP-A, SP-B, and SP-C in large surfactant aggregates of transplanted lungs treated with NO is a marker of lung injury. We conclude that treatment with inhaled NO after lung transplantation is deleterious for the surfactant system and causes a parallel worsening of arterial oxygenation.     

卡托普利对缺血-再灌注鼠心肌一氧化氮及其合酶活性的影响

目的　研究一氧化氮 (NO)和一氧化氮合酶 (NOS)在心肌再灌注损伤中的作用 ,探讨卡托普利(captopril)对缺血 -再灌注鼠心肌保护的机制。　方法　采用 L angendorff离体鼠心灌流模型 ,将 18只 SD大白鼠随机分为 3组 (每组 6只 ) ,对照组、缺血 -再灌注组、卡托普利组。观察心肌 NOS同工酶活性、过氧化物歧化酶活性、丙二醛含量、肌酸激酶含量和冠脉流出液 NO的变化。　结果　缺血 -再灌注组与对照组比较心肌诱导型 NOS(i NOS)活性增高 (P<0 .0 0 1) ,而心肌原生型 NOS(c NOS)活性及总 NOS活性显著降低 (P<0 .0 0 1,0 .0 5 ) ,冠脉流出液 NO含量下降(P<0 .0 1)。卡托普利组再灌注 30分钟 ,心肌 i NOS活性低于缺血 -再灌注组 (P<0 .0 1) ,c NOS活性和总 NOS活性高于缺血 -再灌注组 (P<0 .0 1,0 .0 5 ) ,再灌注期间冠脉流出液 NO水平高于缺血 -再灌注组 (P<0 .0 1) ,心肌损伤较缺血 -再灌注组减轻。　结论　心肌 NOS同工酶活性及 NO产生的失常是心肌再灌注损伤的重要机制之一 ,卡托普利可通过调节心肌 NOS同工酶活性 ,维持正常的 NO水平 ,起到心肌保护作用。     

一氧化氮及其合酶在心肌缺血再灌注损伤中作用的研究

目的：用鼠的离体工作心脏研究心肌缺血再灌注损伤一氧化氮（ＮＯ）、ＮＯ合酶（ＮＯＳ）的作用。方法：ＲＴ－ＰＣＲ定量检测心肌组织结构型ＮＯＳ（ｃＮＯＳ）的ｍＲＮＡ表达,测定心肌组织的ｃＮＯＳ、诱导型ＮＯＳ（ｉＮＯＳ）及冠状动脉动脉冠脉）流出液的ＮＯ,同时检测心脏缺血再灌注前后的心功能变化。并分别于次序停跳液中加用缓激肽（ＢＫ）、Ｌ－精氨酸及ＢＫ加Ｌ－精氨酸,观察其对心肌缺血再灌注损伤的影响。结果：心肌     

The role of nitric oxide after a short period of liver ischemia-reperfusion

BACKGROUND: Liver ischemia-reperfusion injury is a serious problem during liver resection and transplantation. Nitric oxide (NO) has been suggested to have a cytoprotective effect for microcirculation, while the interaction of active oxygen species and NO produces peroxynitrite anion. The present study attempts to clarify the role of NO in liver ischemia-reperfusion injury. METHODS: Wistar male rats were subjected to 30 min of hepatic ischemia followed by reperfusion. The model rats were divided into the three following groups: a control group that was not administered NO synthase inhibitors, and two experimental groups that were administered either N(omega)-nitro-L-arginine methyl ester (L-NAME) or aminoguanidine. In each group, we examined active oxygen species and nitric oxide production, and investigated liver function by measuring serum transaminase levels. In addition, we conducted histopathologic examinations and microcirculation examinations using intravital videomicroscopy. RESULTS: In the control group, NO concentrations in the plasma increased with time after reperfusion. A decrease in NO production was detected in the groups administered NO synthase inhibitors. Elevated serum transaminase levels became more prominent after L-NAME administration, while aminoguanidine administration reduced its level. The degree of microcirculation failure was found to be more prominent in the L-NAME-administered group over both the control group and the aminoguanidine-administered group. A significantly lower survival rate was observed at 6 h after reperfusion in the L-NAME-administered group over that of the other groups. CONCLUSIONS: A reduction of the ischemia-reperfusion injury is important in inhibiting the production of high-output NO and peroxynitrite, and in maintaining NO levels necessary for maintenance of microcirculation.     

Short-term "preconditioning" with inhaled nitric oxide protects rabbit lungs against ischemia-reperfusion injury.

BACKGROUND: Pulmonary edema, owing to an impairment of microvascular barrier function, is an important feature in lung ischemia/reperfusion (IR) injury. Inhalation of nitric oxide (NO) during the period of reperfusion has previously been shown to reduce this leakage response. METHODS: We investigated the impact of short-term (30 min) low-dose (10 ppm) pre-ischemic NO inhalation on IR injury in buffer-perfused rabbit lungs, subsequently undergoing 210 min of warm, anoxic-ventilated ischemia. RESULTS: Far-reaching suppression of the leakage response, reflected by manifold increased capillary filtration coefficients and edema formation, was noted in lungs with pre-ischemic NO administration, corresponding to the beneficial effect of NO inhalation during reperfusion. The effect of NO pre-exposure was not related to vasodilation, because microvascular pressures were unchanged, and was mimicked by pre-ischemic intravascular administration of sodium nitroprusside with subsequent washout of this agent. NO inhalation during reperfusion, but not pre-ischemic, short-term NO administration, provoked a manifold increase in the accumulation of guanosine 3',5'-cyclic monophosphate (cGMP) in the perfusate. The cGMP-analogue, 8-Br-cGMP, mimicked the anti-edematous effect of NO when present during reperfusion, but pre-ischemic, short-term administration of 8-Br-cGMP provided only limited protection. The guanylate cyclase-inhibitor, 1H-[1, 2, 4]-Oxadiazolo-[4,3-a]-quinoxalin-1-one (ODQ), largely antagonized the beneficial effects of NO inhalation during reperfusion but had only minor influence on the effect of NO pre-exposure. CONCLUSIONS: "Preconditioning" of the lung vasculature with short-term NO administration maintains endothelial integrity in a subsequent ischemia/reperfusion maneuver, with nonvasodilatory and non-cGMP-related mechanisms suggested to be largely responsible. This finding may offer interesting perspectives for donor management in clinical lung transplantation.     

Pulmonary nitric oxide metabolism following infrarenal aortic cross-clamp-induced ischaemia-reperfusion injury.

OBJECTIVES: to investigate endogenous pulmonary nitric oxide metabolism following infrarenal aortic cross-clamp-induced ischaemia-reperfusion injury. METHODS: groups of male Wistar rats (n=6) were subjected to 60 minutes of infrarenal aortic cross-clamping under general anaesthesia. Rats were culled after 0, 60 and 120 minutes> reperfusion, following release of the aortic clamp. A sham-operated control group was also studied. Acute lung injury (ALI) was quantified by measuring the protein concentration in lung bronchoalveolar lavage (BAL) fluid. Pulmonary myeloperoxidase activity (MPO) was measured as an index of neutrophil infiltration and degranulation in the lung. Plasma tumour-necrosis factor-alpha (TNF-alpha) was measured as an index of the pro-inflammatory cytokine response and pulmonary nitric oxide synthase (NOS) activity was determined by measuring conversion of(3)H L-arginine to(3)H L-citrulline in tissue homogenates. RESULTS: these data show significant ALI with increased pulmonary microvascular permeability and MPO activity in animals subject to 60 minutes> ischaemia and 60 minutes or 120 minutes of reperfusion compared to control animals (p<0.01). Plasma TNF-alpha levels were significantly increased following 60 minutes of ischaemia compared to controls (p<0.01) and remained significantly increased in animals subject to reperfusion (p<0.01). Pulmonary NOS activity was significantly increased in animals subject to reperfusion (p<0.01). CONCLUSIONS: the reperfusion phase of infrarenal aortic cross-clamping provokes a significant increase in pulmonary NOS metabolism. The increase in plasma TNF-alpha and MPO activity suggests that this response may be secondary to inducible NOS expression. Manipulation of this response may benefit patients at risk of acute injury following infrarenal aortic reconstruction.     

A prospective trial of inhaled nitric oxide in clinical lung transplantation

BACKGROUND: Reperfusion injury (RI) is a major cause of mortality and morbidity among lung transplant recipients. We sought to determine if prophylactic administration of inhaled nitric oxide (NO) to lung transplant recipients at reperfusion would prevent RI. We also hypothesized that if prophylactic NO proves ineffective in preventing RI then it may improve pulmonary hemodynamics and gas exchange in the subset of patients who develop RI. METHODS: After informed consent, 28 consecutive, adult lung transplant recipients received NO at 20 ppm at reperfusion. NO was withdrawn for 15 min at 6 and 12 hr after reperfusion, and gas exchange and hemodynamics were measured. RESULTS: Five of the 28 lung transplant recipients (18%) developed RI. Withdrawal of NO for 15 min in this subset of patients resulted in a significant rise in mean pulmonary artery pressure and a reduction in oxygenation index. All five patients with RI were kept on inhaled NO until full functional recovery of the allograft and were then weaned from mechanical ventilation. None required extracorporeal membrane oxygenation support; the early mortality in this group was 20% (1/5). The remaining 23 patients without RI had uneventful early postoperative course and were weaned from NO and mechanical ventilation within 36 hr of transplantation. CONCLUSIONS: Prophylactic-inhaled NO does not prevent RI in human lung transplantation. However, inhaled NO, started at reperfusion, improves gas exchange and reduces pulmonary artery pressure in those patients who develop RI.     

Nitric oxide pathway after long-term cold storage and reperfusion in a heterotopic rat heart transplantation model

Recent studies have suggested the involvement of the nitric oxide (NO) pathway in ischemia-reperfusion injury related to cardiac transplantation. Herein, we assessed the NO pathway by quantifying endothelial (e) and inducible (i) nitric oxide synthase (NOS) expression and total NOS activity in a rat heart transplant model during cold ischemia with Celsior cardioplegia and reperfusion. Experiments were performed using a modified Lewis-Lewis heterotopic abdominal heart transplantation with 3 or 6 hours of ischemia with or without 1 hour of blood reperfusion. NOS expression and activity were determined using Western blotting and colorimetric assays, respectively, on freeze-clamped hearts after ischemia without (n = 10) or with reflow (n = 12) compared with basal values. Hearts submitted to 3 hours of ischemia and 1 hour of reperfusion showed a postischemic rate pressure product of 5190 +/- 3047 mm Hg/min (reversible ischemia), but no contractility was observed after 6 hours of ischemia. eNOS protein levels were lower after 3 hours of ischemia compared with the basal value (P = .0005) and were further decreased after 6 hours of ischemia (P < .0001 versus basal value and P = .0018 versus 3 hours of ischemia). Reperfusion did not further decrease eNOS protein levels. iNOS protein was not detected in any condition. NOS activity was increased after 3 hours of ischemia versus basal value (P = .0065) but not after 6 hours of ischemia without any effect of reperfusion. We concluded that eNOS expression was altered during ischemia and the amplitude of the alteration depended on the duration of ischemia. Reversible ischemia was associated with increased NOS activity at the end of ischemia with no variation at reperfusion.     

Inhaled nitric oxide attenuates apoptosis in ischemia-reperfusion injury of the rabbit lung

Background

Lung ischemia-reperfusion injury occurs after lung transplantation and various clinical procedures. Recently, apoptosis was reported to be induced after ischemia-reperfusion. We investigated the effects of inhaled nitric oxide (NO) on lung ischemia-reperfusion and apoptosis after ischemia-reperfusion.

Methods

As a control group, the left pulmonary hilum of Japanese white rabbits (n = 10) was occluded for 120 minutes and reperfused for 120 minutes. In the inhaled NO group (n = 10), 20 parts per million nitric oxide was inhaled during reperfusion. The sham-operated group was ligated at the right hilum and perfused by the left lung only for 120 minutes. The mean pulmonary arterial pressures and Pao₂ were measured during reperfusion. The wet-to-dry weight ratio of the left lower lobe of the lung was calculated. The number of apoptotic cells was estimated using the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) technique. The TUNEL staining for a time course study was done using 15 control animals that were killed by exsanguination at 15, 30, and 60 minutes after reperfusion.

Results

After 120 minutes of reperfusion, the mean pulmonary arterial pressures in the control group and in the inhaled NO group were 23.0 ± 3.2 mm Hg and 13.6 ± 2.4 mm Hg, respectively (p < 0.01). At the same time point, the Pao₂ in the control group and in the inhaled NO group were 46.1 ± 15.9 mm Hg and 88.1 ± 14.7 mm Hg, respectively (p < 0.01). The wet-to-dry weight ratios in the control group and in the inhaled NO group were 0.856 ± 0.024 and 0.808 ± 0.006, respectively (p < 0.01). Apoptotic cells appeared in the early phase of reperfusion (after 15 minutes' reperfusion). The number of apoptotic cells was significantly lower in the inhaled group than in the control group after 120 minutes' reperfusion (1.76% versus 2.87%, p < 0.01).

Conclusions

Our results suggest that the inhaled NO prevents lung ischemia-reperfusion injury and attenuates apoptosis after reperfusion in the rabbit lung.     

Combined exogenous surfactant and inhaled nitric oxide therapy for lung ischemia-reperfusion injury in minipigs

BACKGROUND: The combined application of exogenous surfactant and inhaled nitric oxide was evaluated for prevention of ischemia-reperfusion injury of the lung. METHODS: Left lungs were selectively perfused in 18 minipigs in situ with cold preservation solution. After 90 min of warm ischemia, the lungs were reperfused and the right pulmonary artery and bronchus were ligated (control group, n=6). Exogenous surfactant was instilled via bronchoscopy during ischemia (surfactant group, n=6). In a third group, surfactant was applied, followed by administration of inhaled nitric oxide (surfactant+NO group, n=6). Hemodynamic and respiratory parameters were recorded for 7 hr, and bronchoalveolar lavage fluid (BALF) was obtained before and after reperfusion for measurement of surface tension, small aggregate/large aggregate ratio, protein and phospholipid contents, and a differential cell count. RESULTS: Control group animals survived for 3.7+/-1.4 hr. In both surfactant-treated groups, five out of six animals survived the observation period (P<0.001). Dynamic compliance of the lung was decreased in control animals (P<0.001). In the surfactant+NO group, arterial PO2 was higher than in both other groups (P<0.001). BALF cell count and histology showed reduced neutrophil infiltration in surfactant+NO-treated lungs. Surface tension assessed in BALF with a pulsating bubble surfactometer was severely impaired in control animals (gammamin, 14.82+/-9.95 mN/m), but maintained in surfactant-treated (gammamin, 1.11+/-0.56 mN/m) and surfactant+NO-treated animals (gammamin, 3.90+/-2.35 mN/m, P=0.02). CONCLUSIONS: Administration of exogenous surfactant in lung reperfusion injury results in improved lung compliance. The addition of inhaled NO improves arterial oxygenation and reduces neutrophil extravasation compared with surfactant treatment alone.     

Continuous infusion of nitroglycerin improves pulmonary graft function of non-heart-beating donor lungs

BACKGROUND: The warm ischemic period of lungs harvested from a non-heart-beating donor (NHBD) results in an increased ischemia-reperfusion injury after transplantation. The intravenous application of nitroglycerin (NTG), a nitric oxide (NO) donor, proved to be beneficial during reperfusion of lung grafts from heart-beating donors. The objective of the present study was to investigate the effect of nitroglycerin on ischemia-reperfusion injury after transplantation of long-term preserved NHBD-lungs. METHODS: Sixteen pigs (body weight, 20-30 kg) underwent left lung transplantation. In the control group (n=5), lungs were flushed (Perfadex, 60 mL/kg) and harvested immediately after cardiac arrest. In the NHBD group (n=5) and the NHBD-NTG group (n=6), lungs were flushed 90 min (warm ischemia) after cardiac arrest. After a total ischemia time of 19 hr, lungs were reperfused and graft function was observed for 5 hr. Recipient animals in the NHBD-NTG group received 2 microg/kg/min of NTG administered intravenously during the observation period starting 5 min before reperfusion. Tissue specimens and bronchoalveolar lavage fluid (BALF) were obtained at the end of the observation period. RESULTS: Compared with the control group, pulmonary gas exchange was significantly impaired in the NHBD group, whereas graft function in the NHBD-NTG group did not change. Leukocyte fraction and protein concentration in the BALF and histologic alteration of the NHBD-NTG group were not different from controls. CONCLUSIONS: Continuous infusion of NTG in the early reperfusion period improves pulmonary graft function of NHBD lungs after long-term preservation. The administration of an NO donor during reperfusion may favor the use of NHBD lungs to alleviate the critical organ shortage in lung transplantation.     

Pulmonary vasoconstriction during regional nitric oxide inhalation: evidence of a blood-borne regulator of nitric oxide synthase activity

BACKGROUND: Inhaled nitric oxide (INO) is thought to cause selective pulmonary vasodilation of ventilated areas. The authors previously showed that INO to a hyperoxic lung increases the perfusion to this lung by redistribution of blood flow, but only if the opposite lung is hypoxic, indicating a more complex mechanism of action for NO. The authors hypothesized that regional hypoxia increases NO production and that INO to hyperoxic lung regions (HL) can inhibit this production by distant effect. METHODS: Nitric oxide concentration was measured in exhaled air (NO(E)), NO synthase (NOS) activity in lung tissue, and regional pulmonary blood flow in anesthetized pigs with regional left lower lobar (LLL) hypoxia (fraction of inspired oxygen [FIO2] = 0.05), with and without INO to HL (FIO2 = 0.8), and during cross-circulation of blood from pigs with and without INO. RESULTS: Left lower lobar hypoxia increased exhaled NO from the LLL (NO(E)LLL) from a mean (SD) of 1.3 (0.6) to 2.2 (0.9) parts per billion (ppb) (P < 0.001), and Ca2+-dependent NOS activity was higher in hypoxic than in hyperoxic lung tissue (197 [86] vs. 162 [96] pmol x g(-1) x min(-1), P < 0.05). INO to HL decreased the Ca2+-dependent NOS activity in hypoxic tissue to 49 [56] pmol x g(-1) x min(-1) (P < 0.01), and NO(E)LLL to 2.0 [0.8] ppb (P < 0.05). When open-chest pigs with LLL hypoxia received blood from closed-chest pigs with INO, NO(E)LLL decreased from 2.0 (0.6) to 1.5 (0.4) ppb (P < 0.001), and the Ca2+-dependent NOS activity in hypoxic tissue decreased from 152 (55) to 98 (34) pmol x g(-1) x min(-1) (P = 0.07). Pulmonary vascular resistance increased by 32 (21)% (P < 0.05), but more so in hypoxic (P < 0.01) than in hyperoxic (P < 0.05) lung regions, resulting in a further redistribution (P < 0.05) of pulmonary blood flow away from hypoxic to hyperoxic lung regions. CONCLUSIONS: Inhaled nitric oxide downregulates endogenous NO production in other, predominantly hypoxic, lung regions. This distant effect is blood-mediated and causes vasoconstriction in lung regions that do not receive INO.     

Dual effects of nitric oxide in acute colon obstruction

Nitric oxide (NO) plays central role in the pathophysiology of large bowel diseases. In the gastrointestinal tract the predominant form of nitric oxide synthase (NOS) isoenzymes is neuronal NOS (nNOS). The aims were to investigate the role of NO and the activation of NOS isoforms during acute colonic obstruction. Haemodynamic changes, large bowel motility and plasma levels of nitrate-nitrite (NOx) were observed for 7 hrs in anaesthetized dogs. Group 1 (n=6) served as sham-operated control. In groups 2 (n=8), 3 (n=6), and 4 (n=6) colon obstruction was initiated. Groups 3 and 4 were treated with non-selective NOS inhibitor N-nitro-L-arginine (NNA, 4 mg/kg) or with the selective nNOS inhibitor 7-nitroindazol (7-NI, 5 mg/kg) 3 hr after the obstruction. At the end of the experiments, tissue biopsies were taken from the oral and aboral parts of the colon to determine the constitutive and inducible NOS (cNOS and iNOS, respectively) activities. RESULTS: The cNOS activity of the colon was significantly higher orally then aborally in each group. After obstruction the characteristic features of hyperdynamic sepsis were observed. The obstruction caused significant increase in iNOS activity, which was significantly reduced by the NOS inhibitors. The obstruction increased the motility on both parts of the colon. The administration of NNA transiently inhibited, but later significantly increased the motility of the colon segments. Inhibition of nNOS by 7-NI treatment did not influence the hemodynamic parameters but decreased the motility. CONCLUSION: Neuronal NO increases colon motility at the early stage of large bowel obstruction, however, during a concomitant sepsis the excess of inducible NO will moderate this effect.     

异丙酚对大鼠肺一氧化氮合酶活性的影响

目的 了解异丙酚对大鼠肺一氧化氮合酶(NOS)活性的影响,探讨异丙酚对肺血管、支气管扩张作用的机理。方法40只SD大鼠,随机分为异丙酚组(n=20)、对照组(n=20),分别腹腔注射等容积异丙酚(1ml·kg-1,即100mg·kg-1)和生理盐水(10ml·kg-1)。异丙酚组待鼠翻正反射消失后,经尾缘静脉泵以异丙酚10mg·kg-1·h-1,20min后处死,对照组鼠腹腔注射20min后处死。检测支气管肺泡灌洗液NO水平、肺组织匀浆中NOS酶活性、NO水平及内皮型NOS(eNOS)、神经型NOS(nNOS)在肺内的表达与分布(免疫组化法)。结果 异丙酚组支气管灌洗液和肺组织匀浆中NO水平均明显高于对照组(P<0.01),肺组织匀浆中NOS酶活性也明显大于对照组(P<0.01)。异丙酚组肺血管内皮细胞nNOS和eNOS、支气管粘膜上皮细胞nNOS染色表达强阳性。结论 异丙酚可以刺激肺中NOS活性,升高肺内内源性NO水平,在异丙酚的扩张肺血管、支气管中发挥一定的作用。     

In vivo adenovirus-mediated endothelial nitric oxide synthase gene transfer ameliorates lung allograft ischemia-reperfusion injury

OBJECTIVE: Nitric oxide regulates vascular tone, inhibits platelet aggregation, and inhibits leukocyte adhesion, all of which are important modulators of ischemia-reperfusion injury. This study aimed to determine the effects of endothelial constitutive nitric oxide synthase gene transfer on ischemia-reperfusion injury in a rat lung transplant model. METHODS: In group I, donor animals were injected intravenously with 5 x 10(9) pfu of adenovirus-encoding endothelial constitutive nitric oxide synthase. Groups II and III served as controls, whereby donor animals were injected with either 5 x 10(9) pfu of adenovirus encoding beta-galactosidase or saline solution, respectively. Twenty-four hours after injection, left lungs were harvested and preserved for 18 hours at 4 degrees C, then implanted into isogeneic recipients, which were put to death 24 hours later. Recombinant endothelial constitutive nitric oxide synthase gene expression was evaluated by Western blotting and immunohistochemistry. Lung grafts were assessed by measuring arterial oxygenation, myeloperoxidase activity, and wet/dry weight ratios. RESULTS: Western blotting confirmed the overexpression of endothelial constitutive nitric oxide synthase in lungs so transfected compared with controls. Twenty-four hours after reperfusion, mean arterial oxygenation was significantly improved in group I compared with group II and III controls (189.4 +/- 47.1 mm Hg vs 71.7 +/- 8.9 mm Hg and 67.8 +/- 12.2 mm Hg, P =.02, P =.01, respectively). Myeloperoxidase activity, a reflection of tissue neutrophil sequestration, was also significantly reduced in group I compared with groups II and III (0.136 +/- 0.038 DeltaOD/mg/min vs 0. 587 +/- 0.077 and 0.489 +/- 0.126 DeltaOD/mg/min, P =.001, P =.01, respectively). CONCLUSION: Adenovirus-mediated gene transfer with endothelial constitutive nitric oxide synthase ameliorates ischemia-reperfusion injury as manifested by significantly improved oxygenation and decreased neutrophil sequestration in transplanted lung isografts. Endothelial constitutive nitric oxide synthase gene transfer may reduce acute lung dysfunction after lung transplantation.     

Congenital NOS2 deficiency protects mice from LPS-induced hyporesponsiveness to inhaled nitric oxide

BACKGROUND: In animal models, endotoxin (lipopolysaccharide) challenge impairs the pulmonary vasodilator response to inhaled nitric oxide (NO). This impairment is prevented by treatment with inhibitors of NO synthase 2 (NOS2), including glucocorticoids and L-arginine analogs. However, because these inhibitors are not specific for NOS2, the role of this enzyme in the impairment of NO responsiveness by lipopolysaccharide remains incompletely defined. METHODS: To investigate the role of NOS2 in the development of lipopolysaccharide-induced impairment of NO responsiveness, the authors measured the vasodilator response to inhalation of 0.4, 4, and 40 ppm NO in isolated, perfused, and ventilated lungs obtained from lipopolysaccharide-pretreated (50 mg/kg intraperitoneally 16 h before lung perfusion) and untreated wild-type and NOS2-deficient mice. The authors also evaluated the effects of breathing NO for 16 h on pulmonary vascular responsiveness during subsequent ventilation with NO. RESULTS: In wild-type mice, lipopolysaccharide challenge impaired the pulmonary vasodilator response to 0.4 and 4 ppm NO (reduced 79% and 45%, respectively, P < 0.001), but not to 40 ppm. In contrast, lipopolysaccharide administration did not impair the vasodilator response to inhaled NO in NOS2-deficient mice. Breathing 20 ppm NO for 16 h decreased the vasodilator response to subsequent ventilation with NO in lipopolysaccharide-pretreated NOS2-deficient mice, but not in lipopolysaccharide-pretreated wild-type, untreated NOS2-deficient or untreated wild-type mice. CONCLUSIONS: In response to endotoxin challenge, NO, either endogenously produced by NOS2 in wild-type mice or added to the air inhaled by NOS2-deficient mice, is necessary to impair vascular responsiveness to inhaled NO. Prolonged NO breathing, without endotoxin, does not impair vasodilation in response to subsequent NO inhalation. These results suggest that NO, plus other lipopolysaccharide-induced products, are necessary to impair responsiveness to inhaled NO in a murine sepsis model.     

大鼠肝缺血／再灌注后肝组织一氧化氮和不同亚型一氧化氮合酶的变化

目的探讨一氧化氮（NO）和一氧化氮合成酶（NOS）在肝缺血／再灌注（I／R）过程中的变化和作用。方法健康雄性SD大鼠24只，随机分为3组（每组8只）：①正常对照组，术中只分离肝周围韧带，不做肝门阻断及再灌注。②I/R组，进行45min的部分肝门阻断及60min的再灌注。③L-精氨酸（L—Arg）组，缺血前20min经阴茎背静脉注射L—Arg（300mg／kg），余同②组。实验结束后，取下腔静脉血2ml，并迅速切取缺血肝组织。检测血清丙氨酸转氨酶（ALT）、门冬氨酸转氨酶（AST）、乳酸脱氢酶（LDH）；测定肝组织中超氧化物歧化酶（SOD）、丙二醛（MDA）、黄嘌呤氧化酶（XOD）、一氧化氮（NO）和一氧化氯合成酶（NOS）等指标；观察光镜和电镜下肝组织学变化。结果与正常对照组相比，I／R组iNOS升高，NO降低；L-Arg组NO、eNOS均高于I／R组。2、3组比1组大鼠的肝组织病理损害重、肝功能差，L—Arg组病理损害较I／R组明显减轻、肝功能改善。结论NO对大鼠肝I／R损伤具有保护作用．不同亚型NOS的变化参与其中。     

Short-term inhaled nitric oxide in canine lung transplantation from non-heart-beating donor

BACKGROUND: Use of lungs harvested from non-heart-beating donors (NHBDs) would increase the pulmonary donor pool; however, this strategy would have higher risk of early postoperative graft dysfunction due to unavoidable warm ischemic time. We evaluated the effects of short-term inhaled nitric oxide (NO) during reperfusion in canine left single-lung allotransplantation from a non-heart-beating donor. METHODS: The donor dogs were sacrificed without heparinization and left at room temperature for 3 hours. Then, recipient dogs received a left single-lung allotransplantation. After implantation, the right bronchus and pulmonary artery were ligated. In group 1 (n = 6), NO gas was administered continuously at a concentration of 40 parts per million throughout a 6-hour assessment period. In group 2 (n = 6), NO gas was administered for the initial 1 hour during reperfusion. In group 3 (n = 6), nitrogen gas was administered for control. RESULTS: Groups treated with NO exhibited lower pulmonary vascular resistance, as well as improved survival and oxygenation. There was no significant difference in these parameters between group 1 and group 2. Myeloperoxidase activity was significantly lower in NO-treated groups. CONCLUSIONS: Inhaled NO during reperfusion is beneficial in lung transplantation from non-heart beating donors. The beneficial effect is obtained mainly during the first hour of reperfusion.     

Endogenous nitric oxide and exogenous nitric oxide supplementation in hepatic ischemia-reperfusion injury in the rat

BACKGROUND: Although nitric oxide (NO) is thought to be beneficial in hepatic ischemia-reperfusion (I/R), the mechanisms for this effect are not well established. METHODS: To investigate the effects of endogenous NO and exogenous NO supplementation on hepatic I/R injury and their pathogenic mechanisms, serum ALT and hyaluronic acid (endothelial cell damage), and hepatic malondialdehyde and H2O2 (oxidative stress), myeloperoxidase activity (leukocyte accumulation), and endothelin (vasoconstrictor peptide opposite to NO) were determined at different reperfusion periods in untreated rats and rats receiving L-NAME, L-NAME+L-arginine, and spermine NONOate (exogenous NO donor). RESULTS: After reperfusion every parameter increased in untreated animals. Endogenous NO synthesis inhibition by L-NAME increased hepatocyte and endothelial damage as compared to untreated rats, which was reverted and even improved by the addition of L-arginine. Spermine NONOate also improved this damage. However, different mechanisms account for the beneficial effect of endogenous and exogenous NO. Oxidative stress decreased by both L-NAME and L-NAME+L-arginine, but remained unmodified by spermine NONOate. Myeloperoxidase increased by L-NAME and this effect was reverted by the addition of L-arginine, whereas no change was observed with spermine NONOate. Endothelin levels were not modified by L-NAME and L-NAME+L-arginine, but decreased with spermine NONOate. CONCLUSIONS: These results suggest that, although both endogenous and exogenous NO exert a protective role in experimental hepatic I/R injury, the mechanisms of the beneficial effect of the two sources of NO are different.     

The role of nitric oxide pathway in the renal ischemia-reperfusion injury in rats

INTRODUCTION: Nitric oxide (NO), synthesized from L-arginine by the enzyme nitric oxide synthase (NOS), seems to play an ambiguous role during tissue ischemia-reperfusion injury. Our objective was to evaluate the effects of L-arginine, a NO donor, and N(G)-nitro-L-arginine-methylester (L-NAME), a NOS inhibitor, on oxidative stress, renal dysfunction, histologic alterations and surgical mortality rate induced by renal ischemia-reperfusion (RIR) in uninephrectomized rats. MATERIALS AND METHODS: One-hundred and ninety-seven Wistar rats were randomized into five experimental groups. Group 1: sham operation; group 2: right uninephrectomy (UNI); group 3: UNI + RIR in the contralateral kidney; group 4: UNI + L-NAME (20 mg/kg; intraperitoneally) + RIR; and group 5: UNI + L-arginine + RIR. The effect of the drugs was evaluated by lipid peroxidation measured by the renal malondialdehyde (MD) content and chemiluminescence (CL) levels, serum creatinine (Cr) levels, urinary volume, tubular necrosis and athrophy, inflammatory infiltrate, interstitial fibrosis as histologic evaluation and surgical mortality rate after the procedures. A P value less than 0.05 was considered significant. RESULTS: Right uninephrectomy did not alter the renal parameters. RIR increased Cr levels (at 24 and 96 h of reperfusion), index of lipid peroxidation (both MD and QL levels), and worsened the histologic aspects. Pretreatment with L-arginine reduced the kidney levels of QL when compared with the non-treated group (5574 +/- 909 vs. 13 660 +/- 1104 cps/mg of protein; P < 0.05) but increased the MD levels (0.97 +/- 0.24 vs. 0.79 +/- 0.06 nmol/mg of protein; P < 0.05). Moreover, L-arginine attenuated the increment of Cr levels, inflammatory infiltrate and tubular athrophy in rats subjected to RIR (P < 0.05). On the other hand, pretreatment with L-NAME increased both CL (17 482 +/- 4397 vs. 13 660 +/- 1104 cps/mg of protein; P < 0.05) and MD levels (1.16 +/- 0.11 vs. 0.79 +/- 0.06 nmol/mg of protein; P < 0.05). Furthermore, L-NAME worsened the renal dysfunction (P < 0.05) at 192 h after the RIR, and surgical mortality rates were similar (P > 0.05). CONCLUSION: L-arginine has a tendency to exert a beneficial effect on renal damage during RIR in rats. Moreover, L-NAME seems to worsen the renal damage by increasing the kidney-levels of CL and impairment of renal function probably due to reduction of NO production.