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.     

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.     

短时长吸入一氧化氮预处理对大鼠肺缺血再灌注损伤的保护作用

目的 探讨低浓度一氧化氮(NO)预处理及其吸入时长的不同对肺缺血再灌注损伤(IRI)的影响和机制.方法 成年雄性SD大鼠77只,分为空白组(Sham组)、缺血再灌注组(I/R组)、NO预处理10 min组(NO-10 min组)、NO预处理1 min组(NO-1 min组),NO预处理60 min组(NO-60 min组),分别于不同的再灌注时间点采集标本,检测动脉血氧分压(PaO2),肺组织干/湿重比(W/D),丙二醛(MDA)浓度,髓过氧化酶(MPO)活性,肺损伤组织学评价等指标,比较前3组再灌注后2、6、24 h结果,取IRI最严重时间点,比较各组肺功能指标以及血清和左肺组织NO浓度.结果 再灌注6h肺损伤最严重;与I/R组比较,NO-10 min组各时间点指标明显改善(P＜0.05),NO吸入具有肺保护作用;NO-1 min组肺损伤无改善(P＞0.05);NO-10 min组与NO-60 min组对肺损伤保护作用相似(P＞0.05).结论 短时长吸入低浓度NO预处理可以改善IRI,但是其保护作用不与NO预处理时长成正比.     

诱导性一氧化氮合酶在大鼠肝缺血再灌注损伤中的作用

目的 探讨诱导性一氧化氮合酶（iNOS）在大鼠部分肝缺血再灌注损伤中的作用.方法 选取雄性Sprague-Dawley大鼠30只,体重225～250 g,随机分成氨基胍（AG）组、脂多糖（LPS）组、对照组.每组均按相同方法建立70%的肝缺血再灌注损伤模型（缺血1 h,再灌注6 h）,取再灌注大鼠肝组织及血清样本.氨基胍（AG）组（n=10）：术前30 min尾静脉注射AG 100 mg/kg（质量浓度10 kg/L）;脂多糖（LPS）组（n=10）：术前30 min尾静脉注射LPS 10 mg/kg（质量浓度1 kg/L）;对照组（n=10）：术前30 min尾静脉注射生理盐水（10 μL/kg）.检测血清谷氨酸转氨酶（ALT）水平,实时荧光定量PCR测定肝组织iNOS mRNA的表达,Western blot测定肝组织iNOS蛋白的表达,考马斯法测定肝组织匀浆丙二醛（MDA）含量、超氧化物歧化酶（SOD）活性,HE染色光镜下组织学观察等.结果 AG组与对照组相比,肝组织中iNOS mRNA表达量明显下降（P〈0.05）,iNOS蛋白表达量明显下降（P〈0.05）;ALT和MDA明显下降（P〈0.05）;SOD明显升高（P〈0.05）;肝细胞水肿较轻,排列相对整齐.LPS组与对照组相比,肝组织中iNOS mRNA表达量明显升高（P〈0.05）,iNOS蛋白表达量明显升高（P〈0.01）;ALT和MDA明显升高（P〈0.05）;SOD则明显降低（P〈0.05）;肝细胞水肿,排列紊乱,并且出现水样变性.结论 iNOS 升高会加重缺血再灌注损伤,这一过程可能通过改变氧化还原状态实现.     

依布西林对大鼠无心跳供体肺的保护作用

目的 观察依布西林在大鼠无心跳供体(NHBD)肺保护中的作用.方法 将60只SD大白鼠随机分为A组:有心跳供体(HBD)组;B组:NHBD组;C组:NHBD+依布硒林(Ebselen)组.B组、C组供体处死后维持辅助呼吸,放置室温中30 min,再灌注低钾右旋糖苷(LPD)液.受体鼠行"原位左肺移植术".C组受体在肺移植前1 h给予Ebselen.结果 C组移植后肺顺应性为0.1740±0.0100,结扎右肺门后15、30 min动脉血氧分压分别为(93.97±5.94)、(92.30±6.57)mm-Hg,肺组织丙二醛(MDA)含量为(0.63±0.23)nmol/mg蛋白,肺组织能量代谢物总量为(821.51±29.70)mol/g,与B组比较差异均有统计学意义(P＜0.05).结论 给予受体一定浓度的Ebselen可改善NHBD肺保护作用.

Abstract：

Objective To evaluate the protective effect of ebselen on the rat lungs from non-heartbeating donors (NHBD). Methods Sixty Sprague-Dawley rats were randomly divided into 3 groups:group A, heart-beating donor; group B, NHBD with 30 min of warm ischemia time (WIT); group C, NHBD with 30 min of WIT and administration of ebselen. The donor lungs in groups B and C maintained ventilation at room temperature for 30 min after asystolia and then were flushed with LPD solution. The recipient rats underwent left lung transplantation. The recipients in group C were administered with ebselen 1 h before transplantation. Results All the recipients survived during the observation period. The pulmonary compliance of group C was 0. 1740 ±0. 0100. The PaO2 at 15 min and 30 min after the ligation of the right pulmonary hila was (93.97 ±5.94), (92. 30 ±6. 57) mmHg, respectively. Malondialdehyde (MDA) of the pulmonary tissue was (0. 63 ±0. 23) nmol/mg pro and the energy metabolism was (821.51 ±29.70)mol/g. The difference between group B and group C was significant (P ＜ 0. 05 ). Conclusion The administration of ebselen is a safe and effective treatment in the preservation of the rat lungs from NHBD.     

Endothelial nitric oxide synthase expression in ischemia-reperfusion injury after living related-donor renal transplantation

Ischemia-reperfusion injury during renal transplantation has been linked to early graft dysfunction and late graft failure. Nitric oxide (NO), produced by NO synthase (NOS), participates in the recovery from ischemia. We correlated the intensity of graft immunoreactivity for the endothelial NOS isoform (eNOS) during early reperfusion with graft function in 25 children receiving grafts from related donors. Renal allograft biopsy specimens were obtained before transplantation, 1 h after renal artery reperfusion, and 1 year after transplantation. Immunohistochemical staining for eNOS occurred mainly within the endothelium of glomerular capillaries and peritubular capillaries as well as in tubule cells. The mean intensity score for eNOS staining (0-9) was 3.0+/-1.4 before transplantation, 4.5+/-1.9 at 1 h, and 3.3+/-1.9 at 1 year (baseline vs 1 h, P<0.05). Creatinine clearance (ml/min) in patients with a 1-h eNOS score of below 5 and of at least 5, respectively, was 77.1+/-28.4 vs 104.3+/-25.3 at 1 month, 78.7+/-33.4 vs 105.2+/-24.4 at 3 months, 64.7+/-30.1 vs 100.1+/-25.3 at 1 year, 58.2+/-31.3 vs 84.7+/-18.8 at 3 years, and 71.2+/-19.7 vs 78.3+/-23.1 at 5 years ( P<0.05 for 1 month, 1 year, and 3 years). We concluded that elevated eNOS expression after reperfusion in living related-donor renal transplantation enhances the recovery from renal ischemia and, consequently, reduces late graft deterioration.     

Differential nitric oxide synthase expression during hepatic ischemia-reperfusion

BACKGROUND: In recent years the important role of nitric oxide in hepatic ischemia-reperfusion injury has been increasingly recognised. The prevailing consensus is that reperfusion injury may be partly the result of decreased production of nitric oxide from endothelial nitric oxide synthase and excessive production of nitric oxide from the inducible isoform. We therefore undertook this study to characterize the expression of different nitric oxide synthase isoforms during hepatic reperfusion. METHODS: Male Wistar rats (n = 6) were subjected to 45 minutes of partial hepatic ischemia (left lateral and median lobes) followed by 6 hours of reperfusion. Control animals (n = 6) were subjected to sham laparotomy. The expression of endothelial and inducible nitric oxide synthase was examined using immunohistochemistry and Western blotting. Liver sections were also stained with nitrotyrosine antibody, a specific marker of protein damage induced by peroxynitrite (a highly reactive free radical formed from nitric oxide). RESULTS: Liver sections from all the control animals showed normal expression of the endothelial isoform and no expression of inducible nitric oxide synthase. Livers from all the animals subjected to hepatic ischemia showed decreased expression of endothelial nitric oxide synthase, and all but one animal from this group showed expression of the inducible isoform both in inflammatory cells and in hepatocytes. Western blotting confirmed these findings. Staining with the antinitrotyrosine antibody was also confined to five liver sections from animals subjected to hepatic ischemia. CONCLUSIONS: During the reperfusion period after hepatic ischemia, endothelial nitric oxide synthase is downregulated while inducible nitric oxide synthase is expressed in both hepatocytes and inflammatory cells. The presence of nitrotyrosine in livers subjected to hepatic ischemia-reperfusion suggests that the expression of inducible nitric oxide synthase plays an important role in mediating reperfusion injury in this model.     

Propofol reduces nitric oxide-induced apoptosis in testicular ischemia-reperfusion injury by downregulating the expression of inducible nitric oxide synthase

Background: The aim of the present study was to investigate the underlying mechanisms in the preventive effects of intravenous anesthetics on testicular ischemia–reperfusion injury.
Methods: Forty male Wistar Albino rats were randomly assigned to four groups of 10 rats each. Anesthesia was induced and maintained with thiopental in groups 1 and 2 and with propofol in groups 3 and 4. Groups 2 and 4 received left testicular ischemia (torsion) for 1 h and reperfusion (detorsion) for 24 h. Groups 1 and 3 (control groups) had no testicular torsion and detorsion. At 24 h of reperfusion, animals were killed and ipsilateral testes were removed for determination of tissue nitric oxide (NO) levels and immunohistochemical evaluation of endothelial nitric oxide synthase (eNOS), inducible NOS (iNOS), and apoptosis protease-activating factor 1 (APAF-1).
Results: Between groups 1 and 3, there were no differences in tissue NO levels and eNOS, iNOS, and APAF-1 expressions. iNOS and APAF-1 expressions were markedly increased in group 2, but these parameters were at the mild to moderate level in group 4 at 24 h of reperfusion. Also, elevated expression of iNOS was accompanied by a high NO production in group 2 compared with group 4. Although eNOS expressions were increased in both the groups (groups 2 and 4), there were no significant differences between these groups.
Conclusions: Propofol as an anesthetic agent may attenuate germ cell-specific apoptosis and decrease NO biosynthases through downregulation of iNOS expression in an animal model of testicular torsion and detorsion.     

异氟醚保护离体大鼠肺缺血再灌注损伤

INTRODUCTION Ischemia reperfusion(IR)inducedlunginjuryis characterizedbysequestrationofneutrophilsinpulmonary vascularbedandincreasedlungmicrovascularpermeabili ty,whichcanresultinpulmonaryedemaandimpairegasexchange.Adhesionofneutrophilstoendothelialcells duringIRisincreasedbythereleaseofinflammatoryme diatorsandcytokines,suchastumornecrosisfactoralpha(TNFα).previousstudieshaveindicatedthatTNFαisan essentialcomponentofthecascadeofeventsthatleadto IR inducedlunginjury[1].TNFαinduce…     

Induction of inducible nitric oxide synthase in hepatocytes isolated from rats with ischemia-reperfusion injury

BACKGROUND: Recent evidence indicates that nitric oxide (NO) has a crucial role in hepatic ischemia-reperfusion (I/R) injury. However, little is known about how I/R influences the gene expression of inducible nitric oxide synthase (iNOS) in hepatocytes. Under inflammatory conditions, we compared the induction of iNOS in hepatocytes isolated from normal and I/R-treated rats. METHODS: Hepatocytes were isolated using the collagenase perfusion method from rats treated with I/R (30-minute ischemia of middle and left lobes, followed by 3-hour reperfusion) or sham operation (control): Primary cultures of rat hepatocytes were incubated with an inflammatory cytokine, interleukin-1beta (IL-1beta), to compare the iNOS induction/NO production between the 2 groups. RESULTS: Both control and I/R groups had no production of nitrite (a stable metabolite of NO) in the absence of IL-1beta. In the control group, IL-1beta stimulated dose- and time-dependent production of NO. The I/R group showed more than 2-fold increased levels of NO production. Western and Northern blot analyses revealed that the I/R group also showed increased levels of iNOS protein and its messenger RNA. CONCLUSION: These results suggest that I/R directly affects the inducibility of the iNOS gene in hepatocytes by IL-1beta. Increased NO may be associated with protective or toxic effects in hepatic I/R injury.     

Isoflurane protects the isolated rat lungs against ischemia-reperfusion injury

Objective To study the effects of isoflurane on ischemia-reperfusion （IR）-induced injury in isolated rat lungs. Methods This study was perforrned on an isolated buffer-perfused rat lung model. There were four groups： CON group （n = 6）： perfusion for 60 min without ischemia; IR group （n = 6） ： after perfusion for 30 min, then interruption of perfusion and ventilation for 45 rain followed by reperfusion for 30 rain; ISO1 group （n = 6） ： 1.38% isoflurane was administered for 30 min before 45 min ischemia, then followed by 30 ,nin reperfusion; ISO2 group （n= 6）： 1.38% isoflurane was administered during the period of reperfusion. The Myeloperoxidase （MPO） activity of lung, neutrophils counts, tumor necrosis factor-α（TNF-α）, superoxide dismu ＇tase （SOD） activity, malondialdehyde （MDA） in perfusate, and the Wet-to-Dry lung weight ratios were measured. Results IR caused significant increases in the wet-to-dry lung weight ratios, the activity of lung MPO, the contents of MDA and TNF-α in perfusate, but decreasess in the ,SOD activity aod neutrophils counts. Administration of isoflurane （both ISO1 and ISO2 groups） significantly protected against IR-induced injury the via inhibiting increases of MPO activity and contents of MDA and TNF-a in perfusate. Isoflurane significantly increased the SOD activity and neutrophils counts in peffusate. No difference was found between ISO1 and ISO2 groups. Conclusion Our resuhs suggest that isoflurane protects the lungs against IR-induced injury in isolated mt lung model.     

The nitric oxide synthase cofactor tetrahydrobiopterin reduces allograft ischemia-reperfusion injury after lung transplantation.

OBJECTIVE: Exogenous nitric oxide reduces ischemia-reperfusion injury after solid organ transplantation. Tetrahydrobiopterin, an essential cofactor for nitric oxide synthases, may restore impaired endothelium-dependent nitric oxide synthesis. We evaluated whether tetrahydrobiopterin administration to the recipient attenuates lung reperfusion injury after transplantation in swine. METHODS: Unilateral left lung transplantation was performed in 15 weight-matched pigs (24-31 kg). Donor lungs were flushed with 1.5 L cold (1 degrees C) low-potassium-dextran solution and preserved for 20 hours. Group I animals served as controls. Group II and III animals were treated with a bolus of tetrahydrobiopterin (20 mg/kg). In addition, in group III a continuous infusion of tetrahydrobiopterin (10 mg/kg per hour over 5 hours) was given. One hour after reperfusion, the recipient right lung was occluded. Cyclic guanosine monophosphate levels were measured in the pulmonary venous and central venous blood. Extravascular lung water index, hemodynamic variables, lipid peroxidation, and neutrophil migration to the allograft were assessed. RESULTS: In group III a significant reduction of extravascular lung water was noted in comparison with the controls (P =.0047). Lipid peroxidation in lung allograft tissue was significantly reduced in group II (P =.0021) and group III ( P =. 0077) in comparison with group I. Pulmonary venous levels of cyclic guanosine monophosphate increased up to 23 +/- 1 pmol/mL at 5 hours in group II and up to 40 +/- 1 pmol/mL in group III (group I, 4.1 +/- 0.5 pmol/mL [I vs III]; P <.001), whereas central venous levels of cyclic guanosine monophosphate were unchanged in all groups. CONCLUSION: Tetrahydrobiopterin administration during lung allograft reperfusion may reduce posttransplantation lung edema and oxygen-derived free radical injury in the graft. This effect is mediated by local enhancement of the nitric oxide/cyclic guanosine monophosphate pathway.     

Sildenafil attenuates intestinal injury in necrotizing enterocolitis independently of endothelial nitric oxide synthase

BackgroundNecrotizing enterocolitis (NEC) is a devastating disease that impacts the intestine of premature infants. Sildenafil has shown benefit in colitis and ischemia/reperfusion models but has not been adequately studied in NEC. Sildenafil's best studied mechanism involves augmenting nitric oxide induced vasodilation. We hypothesized that sildenafil would improve outcomes during experimental NEC in an eNOS dependent manner.MaterialsNEC was induced in five-day old mouse pups with gavage formula feeds plus intermittent hypoxia and hypothermia. Using wild type (WT) mice, the route of sildenafil administration was studied in the following groups: (1) breastfed controls, (2) NEC + oral (PO) sildenafil, (3) NEC + PO vehicle, (4) NEC + intraperitoneal (IP) sildenafil, (5) NEC + IP vehicle. The eNOS KO groups studied included: (1) breastfed controls, (2) NEC + PO sildenafil, (3) NEC + PO vehicle. Data were tested for normality and compared using t-tests or Mann-Whitney with a p-value <0.05 considered significant.ResultsIn WT mice, oral and IP sildenafil resulted in improved clinical outcomes compared to their respective vehicle group. Only orally administered sildenafil significantly improved perfusion to the intestine and protected it from macroscopic and histologic injury. When repeated in eNOS KO mice, oral sildenafil improved clinical scores and attenuated intestinal injury scores, despite no effect on intestinal perfusion.ConclusionsSildenafil, when administered orally, improves clinical outcomes and protects the intestine in a murine model of experimental necrotizing enterocolitis. While sildenafil requires eNOS to impact mesenteric perfusion, it does not appear to be dependent on eNOS to attenuate intestinal injury.