Nitric Oxide Participates in the Brain Ischemic Tolerance Induced by Intermittent Hypobaric Hypoxia in the Hippocampal CA1 Subfield in Rats

Previous studies have shown that intermittent hypobaric hypoxia (IH) preconditioning protected neurons survival from brain ischemia. However, the mechanism remains to be elucidated. The present study explored the role of nitric oxide (NO) in the process by measuring the expression of NO synthase (NOS) and NO levels. Male Wistar rats (100) were randomly assigned into four groups: sham group, IH?+?sham group, ischemia group and IH?+?ischemia group. Rats for IH preconditioning were exposed to hypobaric hypoxia mimicking 5000 m high-altitude (P_B?=?404 mmHg, PO₂?=?84 mmHg) 6 h/day, once daily for 28 days. Global brain ischemia was established by four-vessel occlusion that has been created by Pulsinelli. Rats were sacrificed at 7th day after the ischemia for neuropathological evaluation by thionin stain. In addition, the expression of neuronal NOS (nNOS), inducible NOS (iNOS), and NO content in the hippocampal CA1 subfield were measured at 2nd day and 7th day after the ischemia. Results revealed that global brain ischemia engendered delayed neuronal death (DND), both nNOS and iNOS expression up-regulated, and NO content increased in the hippocampal CA1 subfield. IH preconditioning reduced neuronal injury induced by the ischemia, and prevented the up-regulation of NOS expression and NO production. In addition, l-NAME?+?ischemia group was designed to detect whether depressing NO production could alleviate the DND. Pre-administration of l-NAME alleviated DND induced by the ischemia. These results suggest that IH preconditioning plays a protective role by inhibiting the over expression of NOS and NO content after brain ischemia.     

一氧化氮、内皮素-1对大鼠肢体缺血/再灌注后脑损伤的影响

目的: 研究一氧化氮(NO)和内皮素-1(ET-1)在大鼠肢体缺血/再灌注(LI/R)后脑损伤中的作用,探讨NO/ET-1平衡关系的变化对脑损伤的影响.方法: 在大鼠LI/R损伤模型上,应用NO合成前体物质L-精氨酸(L-Arg)、一氧化氮合酶(NOS)抑制剂氨基胍(AG)、ETA受体阻断剂BQl23进行干预,观察血浆 NO、ET-1、MDA、XOD、SOD、LDH及脑组织tNOS、iNOS、cNOS、NO、ET-1、MDA、XOD、MPO、 SOD的变化.结果: 与对照组比较,I/R组血浆MDA、XOD、LDH及脑组织MDA、XOD、MPO升高,SOD活性降低(P<0.01),脑组织tNOS和iNOS明显升高,而cNOS明显降低(P<0.01),I/R组血浆及脑组织NO、ET-1增加,NO/ET-1比值降低,脑损伤加重.应用L-Arg及BQ123后,血浆及脑组织NO/ET-1比值较I/R组升高,脑损伤减轻,应用AG后,NO/ET-1比值降低,脑损伤进一步加重.结论: 肢体缺血/再灌注后,一氧化氮与内皮素-l的比值降低时脑损伤加重.     

一氧化氮合酶抑制剂L-NAME对大鼠脑缺血耐受诱导的影响

采用大鼠四血管闭塞全脑缺血耐受模型和脑组织切片形态学方法,观察应用一氧化氮合酶(NOS)抑制剂L—NAME对大鼠海马CAl区脑缺血耐受(BIT)诱导的影响,在整体水平探讨一氧化氮(NO)在BIT诱导中的作用。54只Wistar大鼠凝闭双侧推动脉后分为6组：(1)假手术组(n＝6);分离双侧颈总动脉,但不阻断脑血流;(2)损伤性缺血组(n＝6)：全脑缺血10min;(3)预缺血 损伤性缺血组(n＝6)：脑缺血预处理(CIP)3min,再灌注72h后行全脑缺血10min;(4)L—NAME组;分别于CIP前1h和后1、12及36h腹腔注射L—NAME(5mg／kg),每个时间点6只动物,其余步骤同预缺血 损伤性缺血组;(5)L—NAME L—精氨酸组(n＝6)：于CIP前1h腹腔注射L—NAME(5mg／kg)和L—精氨酸(300mg／kg),其它步骤同L—NAME组;(6)L—NAME 损伤性缺血组(n＝6)：于腹腔注射L—NAME(5mg／kg)72h后行全脑缺血10min。实验结果表明,(1)单纯10min全脑缺血可使海马CAl区组织学分级增加(表明损伤加重),神经元密度降低(P＜0．01);(2)预缺血 损伤性缺血组的海马CAl区组织学分级、神经元密度与假手术组相比,无显著性差别(P＞0．05);(3)L—NAME组中,应用L—NAME后海马CAl区组织学分级增加,神经元密度降低,与预缺血 损伤性缺血组相比有显著性差异(P＜0．05),表明L—NAME可阻断CIP对神经元的保护作用;(4)L—NAME L—精氨酸组与L—NAME组相比,海马CAl区组织损伤明显减轻(P＜0．05),但与预缺血 损伤性缺血组相比仍有显著性差别(P＜0．05),提示L-精氨酸可部分逆转L—NAME的作用;(5)L—NAME 损伤性缺血组的组织学表现与损伤性缺血组相同(P＞0．05)。这些结果表明,在整体情况下N0参与BIT的诱导。与CIP前1h及后1、12h给予L—NAME组相比,CIP后36h给予L—NAME对CIP保护作用的阻断效应明显减弱,提示N0在CIP后较早阶段即开始参与BIT的诱导。     

NO produced by endothelial NO synthase is a mediator of delayed preconditioning-induced endothelial protection

Preconditioning with brief periods of ischemia-reperfusion (I/R) induces a delayed protection of coronary endothelial cells against reperfusion injury. We assessed the possible role of nitric oxide (NO) produced during prolonged I/R as a mediator of this endothelial protection. Anesthetized rats were subjected to 20-min cardiac ischemia/60-min reperfusion, 24 h after sham surgery or cardiac preconditioning (1 x 2-min ischemia/5-min reperfusion and 2 x 5-min ischemia/5-min reperfusion). The nonselective NO synthase (NOS) inhibitor l-NAME, the selective inhibitors of neuronal (7-nitroindazole) or inducible (1400W) NOS, or the peroxynitrite scavenger seleno-l-methionine were administered 10 min before prolonged ischemia. Preconditioning prevented the reperfusion-induced impairment of coronary endothelium-dependent relaxations to acetylcholine (maximal relaxation: sham 77 +/- 3; I/R 44 +/- 6; PC 74 +/- 5%). This protective effect was abolished by l-NAME (41 +/- 7%), whereas 7-NI, 1400W or seleno-l-methionine had no effect. The abolition of preconditioning by l-NAME, but not by selective nNOS or iNOS inhibition, suggests that NO produced by eNOS is a mediator of delayed endothelial preconditioning.     

缺血预适应对大鼠肢体缺血/再灌注后肺损伤的影响

目的:观察肢体缺血预适应对大鼠肢体缺血/再灌注(I/R)后肺损伤的影响并探讨其机制。方法:将雄性Wistar大鼠随机分为4组(n=8):对照组(C),肢体缺血/再灌注组(LI/R),缺血预适应组(IPC)和L-NAME组。各组大鼠均于肢体缺血4h再灌注4h处死,分别测定其动脉血氧分压(PaO2)和二氧化碳分压(PaCO2),血浆及肺组织丙二醛(MDA)、一氧化氮(NO)、内皮素(ET)含量,计算血浆NO/ET比值;以及肺湿干比(W/D)、肺系数(LI),肺组织髓过氧化物酶(MPO)含量。结果:大鼠LI/R后4h,PaO2明显降低;W/D、LI、血浆及肺组织的MDA、NO、ET和肺组织MPO活性均明显增加,而血浆NO/ET比值明显减小。与LI/R组比较,IPC组各项损伤指标明显减轻,NO水平升高,血浆NO/ET比值明显增大。与对照组和IPC组比较,L-NAME处理组,各项损伤指标数值明显增加,NO水平降低;血浆NO/ET比值明显减小,差异均具有显著性。各组大鼠PaCO2的变化无显著性。结论:缺血预适应对肢体缺血/再灌注后肺损伤具有保护作用,其机制可能与内源性NO合成增加有关。     

肢体缺血预适应的肝保护作用与一氧化氮／内皮素-1系统关系的研究

目的：观察肢体缺血／再灌注（I／R）后一氧化氮／内皮素-1（NO／ET-1）失衡与肝损伤的关系以及缺血预适应（1pc）对NO／ET-1系统的调节作用。方法：实验用雄性Wistar大鼠18只,随机分为3组（n=6）：对照组（control）、缺血／再灌注组（I／R）和缺血预适应组（IPC＋I／R）,分别测定血浆谷草转氨酶（ALT）、谷丙转氨酶（AST）;血浆和肝组织一氧化氮（NO）、内皮素-1（ET-I）的含量变化,一氧化氮／内皮素-1（NO／ET-1）比值及肝组织的总一氧化氮合酶（tNOS）、诱导型一氧化氮合酶（iNOS）、结构型一氧化氮合酶（cNOS）的水平;免疫组化法检测肝组织的诱导型一氧化氮舍酶（iNOS）、内皮型一氧化氮合酶（eNOS）的表达;HE染色,在光学显微镜下观察肝组织的形态学改变。结果：发现肢体再灌注期血浆和肝组织NO、ET-1均明显增加,而NO／ET-1的比值却明显降低,同时血浆ALT、AST升高,光学显微镜下肝细胞、内皮细胞肿胀,肝细胞变性及肝窦淤血,炎性细胞浸润,肝损伤加重,肢体I／R后肝组织iNOS的表达增强,而eNOS（主要为eNOS）的表达减少,伴有总NOS活性增强。说明肢体缺血再灌注后肝组织内皮源的NO产生减少,而非内皮源的NO产生增多;IPC减轻了肢体I／R后引起的NO／ET-1失衡。结论：肢体I／R后肝组织损伤与NO／ET-1失衡有关,IPC对肢体I／R继发的肝组织损伤的保护作用可能是通过对NO／ET-1系统的调节作用而介导的,此时内皮源的NO产生增加,非内皮源的NO产生减少。     

一氧化氮和线粒体ATP敏感性钾通道介导血管紧张素转换酶抑制剂加强阈下预处理的作用

实验采用离体大鼠心脏Langendorff灌流模型,观察含巯基（卡托普利）和不含巯基（培哚普利拉）的两种血管紧张素转换酶抑制剂（angiotensin-converting enzyme inhibitors,ACEI）对抗心肌缺血的作用,并探讨一氧化氮（nitric oxide,NO）和线粒体ATP敏感性钾通道（mimchondrial ATP-sensitive potassium channel,mitoKATP channel）是否参与ACEI的心肌保护作用。结果表明：（1）给予大鼠心脏2min全心停灌和10min复灌作为闽下缺血预处理（subthreshold preconditioning,sPC）、卡托普利或培哚普利拉单独使用,均不能改善长时间缺血复灌（缺血30min＋复灌120min）引起的心肌损伤。（2）当两种ACEI分别和sPC联合使用时,与sPC组相比,缺血心脏在长时间缺血后的复灌期问左室舒张末压（left ventricular end-diastolic pressure,LVEDP）明显降低,左宦发展压（left ventricular developed pressure,LVDP）和冠脉流量明显增高,乳酸脱氢酶（lactate dehydrogenase,LDH）的释放量和心肌梗死面积明显低于sPC组。（3）利用NOS抑制剂L-NAME和mitoKATP通道的抑制剂5-HD灌流10min后,可明显抑制卡托普利／培哚普利拉和sPC联合使用引起的LVEDP降低,并使LVDP和冠脉流量降低,LDH的释放量和心肌梗死面积明显增高（P〈0．05）。（4）sPC、卡托普利或培哚普利拉单独使用,心脏NO的产生增加。ACEI和sPC联合使用,与三者单独使用相比NO的浓度亦明显增高（P〈0．05）。结果提示：含与不含巯基的ACEI与闽下缺血预处理联合使用均可使大鼠心脏功能明显改善,其心肌保护作用的机制可能通过NO途径,并和mitoKATP通道的激活有关。     

大鼠肢体缺血/再灌注后肺损伤及一氧化氮合酶的变化与意义

目的：研究大鼠肢体缺血／再灌注后急性肺损伤时,内皮型一氧化氮合酶（eNOS）和诱导型一氧化氮合酶（i-NOS）的表达及其在急性肺损伤发生中的作用。方法：雄性Wistar大鼠于后肢根部阻断血流后松解（4h／4h）,分别给予L-Arg和氨基胍（AG）预先干预,分为control、IR、L-Arg和AG组,免疫组织化学方法检测肺组织中iNOS和eNOS的表达,同时检测肺组织中MDA、MPO、W／D和NO2^-／NO3^-值,肺组织形态学观察以评价肺损伤的程度。结果：与control组比较,I／R组eNOS表达降低,iNOS表达增强,MDA、MPO、W／D和NO2^-／NO3^-值增加。肺组织充血、炎细胞浸润,肺泡腔渗液;与I／R组比较,L-Arg组eNOS、iNOS表达无明显变化,NO2^-／NO3^-增加。MDA、MPO、W／D降低,肺组织损伤有减轻趋势,AG组eNOS表达无明显变化,iNOS活性降低,NO2^-／NO3^-减少,MDA、MPO、W／D增加,肺组织损伤有加重趋势。结论：肢体缺血／再灌注急性肺损伤过程中,iNOS表达增加,NO生成增多,在肺损伤发生中有一定的保护作用。     

Role of neuronal and endothelial nitric oxide synthase in nitric oxide generation in the brain following cerebral ischemia.

Nitric oxide (NO) plays an important role in the pathogenesis of neuronal injury during cerebral ischemia. The endothelial and neuronal isoforms of nitric oxide synthase (eNOS, nNOS) generate NO, but NO generation from these two isoforms can have opposing roles in the process of ischemic injury. While increased NO production from nNOS in neurons can cause neuronal injury, endothelial NO production from eNOS can decrease ischemic injury by inducing vasodilation. However, the relative magnitude and time course of NO generation from each isoform during cerebral ischemia has not been previously determined. Therefore, electron paramagnetic resonance spectroscopy was applied to directly detect NO in the brain of mice in the basal state and following global cerebral ischemia induced by cardiac arrest. The relative amount of NO derived from eNOS and nNOS was accessed using transgenic eNOS(-/-) or nNOS(-/-) mice and matched wild-type control mice. NO was trapped using Fe(II)-diethyldithiocarbamate. In wild-type mice, only small NO signals were seen prior to ischemia, but after 10 to 20 min of ischemia the signals increased more than 4-fold. This NO generation was inhibited more than 70% by NOS inhibition. In either nNOS(-/-) or eNOS(-/-) mice before ischemia, NO generation was decreased about 50% compared to that in wild-type mice. Following the onset of ischemia a rapid increase in NO occurred in nNOS(-/-) mice peaking after only 10 min. The production of NO in the eNOS(-/-) mice paralleled that in the wild type with a progressive increase over 20 min, suggesting progressive accumulation of NO from nNOS following the onset of ischemia. NOS activity measurements demonstrated that eNOS(-/-) and nNOS(-/-) brains had 90% and < 10%, respectively, of the activity measured in wild type. Thus, while eNOS contributes only a fraction of total brain NOS activity, during the early minutes of cerebral ischemia prominent NO generation from this isoform occurs, confirming its importance in modulating the process of ischemic injury.     

Effect of total flavonoids of Radix Ilicis pubescentis on cerebral ischemia reperfusion model

This paper aims to observe the effects of total flavonoids of Radix Ilicis pubescentis on mouse model of cerebral ischemia reperfusion. Mice were orally given different doses of total flavonoids of Radix Ilicis pubescentis 10 d, and were administered once daily. On the tenth day after the administration of 1 h in mice after anesthesia, we used needle to hook the bilateral common carotid artery (CCA) for 10 min, with 10 min ischemia reperfusion, 10 min ischemia. Then we restored their blood supply, copy the model of cerebral ischemia reperfusion; We then had all mice reperfused for 24 h, and then took their orbital blood samples and measured blood rheology. We quickly removed the brain, with half of the brain having sagittal incision. Then we fixed the brains and sectioned them to observe the pathological changes of brain cells in the hippocampus and cortex. We also measured the other half sample which was made of brain homogenate of NO, NOS, Na⁺-K⁺-, ATP enzyme Mg²⁺-ATPase and Ca²⁺-ATPase. Acupuncture needle hook occlusion of bilateral common carotid arteries can successfully establish the model of cerebral ischemia reperfusion. After comparing with the model mice, we concluded that Ilex pubescens flavonoids not only reduce damage to the brain nerve cells in the hippocampus and cortex, but also significantly reduce the content of NO in brain homogenate, the activity of nitric oxide synthase (NOS) and increases ATP enzyme activity (P < 0.05, P < 0.01). In this way, cerebral ischemia reperfusion injury is improved. Different dosages of Ilex pubescens flavonoids on mouse cerebral ischemia reperfusion model have good effects.     

The relation of oxidative stress and apoptosis to histopathologic alterations in the lungs as a result of global cerebral ischemia

ABSTRACT

Heart attack and oxygen deficiency may cause necrosis in the brain and other tissues. We investigated the histopathological effects of nitric oxide (NO) on ischemia/reperfusion in lung and hippocampus using a rat brain bilateral occlusion ischemia model. Male rats were assigned to sham (SH), ischemic preconditioning (PC), global ischemia (GI) and ischemic reperfusion (IR) groups. Before ischemia was induced, blood was drawn to induce hypovolemic hypotension and for blood gas testing. After sacrifice, samples of hippocampus were harvested. Sections were examined using hematoxylin and eosin (H & E) staining and immunostaining using primary antibodies for GFAP, S100β, iNOS, eNOS and the TUNEL method. Following ischemia, we found evidence of gliosis induced oxidative stress and apoptosis in the hippocampus. No significant differences were detected between the SH and PC groups. In the GI and IR groups, apoptosis and necrosis were observed in the hippocampus. Lung sections were stained with H & E and Masson’s trichrome (MT) and immunostained for iNOS and eNOS. The TUNEL method was used to detect apoptosis. Interstitial edema, vascular congestion, intra-alveolar hemorrhage, perivascular edema, neutrophil infiltration and disruption of alveoli were observed after global ischemia and ischemic reperfusion. Inflammatory cells were detected in the connective tissue. The IR and GI groups exhibited significantly more apoptotic cells than the SH or PC groups. Free radicals, such as nitric oxide (NO), that appear following ischemia and reperfusion in the brain may also injure the lungs. Increased NO in both lung and brain tissue suggests that apoptosis in these organs can be induced by reactive nitrogen species.     

Protection of lung tissue against ischemia/reperfusion injury by preconditioning with inhaled nitric oxide in an in situ pig model of normothermic pulmonary ischemia

Topical administration of nitric oxide (NO) by inhalation is currently used as therapy in various pulmonary diseases, but preconditioning with NO to ameliorate lung ischemia/reperfusion (I/R) injury has not been fully evaluated. In this study, we investigated the effects of NO inhalation on functional pulmonary parameters using an in situ porcine model of normothermic pulmonary ischemia. After left lateral thoracotomy, left lung ischemia was maintained for 90 min, followed by a 5h reperfusion period (group I, n = 7). In group II (n = 6), I/R was preceded by inhalation of NO (10 min, 15 ppm). Animals in group III (n = 7) underwent sham surgery without NO inhalation or ischemia. In order to evaluate the effects of NO preconditioning, lung functional and hemodynamic parameters were measured, and the zymosan-stimulated release of reactive oxygen species in arterial blood was determined. Animals in group I developed significant pulmonary I/R injury, including pulmonary hypertension, a decreased pO(2) level in pulmonary venous blood of the ischemic lung, and a significant increase of the stimulated release of reactive oxygen species. All these effects were prevented, or the onset (release of reactive oxygen species) was delayed, by NO inhalation. These results indicate that preconditioning by NO inhalation before lung ischemia is protective against I/R injury in the porcine lung.     

缺血再灌注对小鼠肠神经丛nNOS 和iNOS表达的影响

目的观察缺血再灌注后小鼠回肠神经型一氧化氮合酶(neuron alnitric oxide synthase,nNOS)和诱导型一氧化氮合酶(induciblenitric oxide synthase,iNOS)的表达,探讨肠缺血再灌注损伤(ischemia-reperfusion injury,IRI)的发生机制。方法采用小鼠肠系膜上动脉缺血再灌注模型,根据不同再灌注时间对小鼠随机分1d组、3d组、5d组、7d组、对照组和假手术组,用SP法检测小鼠回肠nNOS和iNOS的表达情况。结果与对照组和假手术组相比较,nNOS在再灌注1d后开始在肌间神经丛持续高表达(P<0.01);而iNOS在再灌注3d后开始在肌间神经丛持续高表达(P<0.05)。结论nNOS和iNOS在肠缺血再灌注后的表达增强,提示一氧化氮及一氧化氮合酶与肠神经节细胞在缺血再灌注中的损伤有着密切关系。     

Attenuated nitric oxide synthase activity and protein expression accompany intestinal ischemia/reperfusion injury in rats

Intestinal ischemia/reperfusion (I/R) leads to bowel impairment via the release of reactive oxygen species (ROS) and neutrophil infiltration. In addition to modulating intestinal integrity, nitric oxide (NO(*)) inhibits neutrophil activation and scavenges ROS. Attenuated endogenous NO(*) formation may result in the accrual of these deleterious stimuli. Therefore, we determined nitric oxide synthase (NOS) activity in anesthetized rats subjected to 1 h of superior mesenteric ischemia or ischemia followed by reflow. NOS activity was measured in intestinal tissue homogenates as the conversion rate of (3)H-L-arginine to (3)H-L-citrulline. Our results demonstrate that intestinal ischemia leads to a decrease in NOS activity indicating lower NO(*) formation in the animal model. The attenuation in NOS activity was not reversed following 4 h of reperfusion. Western blot analysis revealed that the decline in enzyme activity was accompanied by reduced intestinal NOS III (endothelial constitutive NOS) expression. These findings provide biochemical evidence for impaired NO(*) formation machinery in intestinal I/R injury.     

白藜芦醇甙对大鼠心脏缺血/再灌注损伤的保护作用

本文利用冠脉结扎/放松方法和Langendorff灌注技术,建立在体和离体大鼠心脏缺血/再灌注(ischemia/reperfusion,I/R)损伤模型,探讨白藜芦醇甙(polydatin)对大鼠I/R心肌损伤的保护作用及其机制.观察白藜芦醇甙对缺血和再灌注心律失常、心肌梗死面积、心脏收缩功能、心肌超氧化物歧化酶(superoxide dismutase,SOD)活性、丙二醛(malondialdehyde,MDA)含量、NO含量以及一氧化氮合酶(nitric oxide synthase,NOS)活性的影响.结果显示:与对照组相比,白藜芦醇甙组大鼠缺血和再灌注心律失常明显降低(P<0.05,P<0.01);心肌梗死面积显著减少(P相似文献   

松龄血脉康胶囊对局灶性缺血损伤后大鼠脑组织TNF-α表达的影响

目的：探讨松龄血脉康预处理对大鼠急性局灶性脑缺血再灌注损伤脑组织TNF-α表达的影响。方法：36只雄性SD大鼠,随机分为松龄血脉康（SL-xmk）预处理组、假手术组、对照组,SL-xmk预处理组采用SL-xmk（937.5mg/kg）悬浮液对大鼠进行4w预防性灌胃处理,假手术组、对照组采用等容量生理盐水预防性灌胃处理,在预处理终点采用线栓法制作大鼠大脑中动脉阻塞（middlecerebralarteryocclusion,MCAO）模型。观察SL-xmk预处理后MCAO大鼠脑组织含水量、脑梗死体积变化的影响,运用免疫组织化学法检测大鼠缺血脑组织TNF-α免疫反应阳性细胞表达。结果：SL-xmk预处理后脑组织TNF-α表达显著下降,缺血脑组织含水量和脑梗死体积明显降低。结论：松龄血脉康可抑制急性局灶性脑缺血再灌注损伤后脑组织TNF-α的表达。     

NO对大鼠肢体缺血/再灌注后肾脏P-选择素表达的影响及意义

目的：探讨一氧化氮（NO）对大鼠LI／R后肾脏P-选择素表达的影响及意义。方法：采用本室常规方法复制大鼠LI／R模型。将大鼠随机分为四组：对照组,LI／R组,L—Arg组和L-NAME组。观察肢体缺血4h再灌注4h后各组动物血浆NO及肌酐（Cr）、尿素氮（BUN）的变化;观察肾组织NO、髓过氧化物酶（MPO）、总一氧化氮合酶（tNOS）、诱导型一氧化氮合酶（iNOS）、结构型一氧化氮合酶（cNOS）的改变;测定尿蛋白含量、利用聚丙烯酰胺凝胶电泳法检测尿蛋白性质;免疫组织化学方法检测肾组织P-选择素（P—selectin）的蛋白表达,结合自动图象分析系统对其结果进行定量分析;肾组织切片经六氨银染色在光镜下观察其形态学改变。结果：与control组比较．LI／R组大鼠血浆NO、BUN、Cr均明显升高;肾组织MPO、NO、tNOS、iNOS均明显增加,而cNOS明显下降;尿蛋白含量增多,电泳显示,有大分子量蛋白排出;光镜下可见肾小管上皮细胞水肿,有炎细胞浸润：免疫组化结果显示：P-selectin蛋白表达明显较control组增强。与LI／R组相比,L-Arg组各项损伤指标明显减轻;肾组织P—selectin蛋白表达明显减弱;L-NAME组血浆和肾组织各项损伤指标明显加重;肾组织P—selectin蛋白表达明显增加。结论：大鼠LI／R后急性肾损伤的发生可能与P—selectin的表达有关;NO可能通过减弱P-selectin的表达及中性粒细胞的浸润,减轻LI／R后肾脏组织形态学及肾功能的损伤性变化。     

高压氧预处理对大鼠局灶性脑缺血再灌注损伤的保护作用

目的:研究高压氧预处理对大鼠脑缺血再灌注损伤的保护作用。方法:36只SD大鼠随机分为假手术组、模型组及高压氧预处理组,每组12只。高压氧预处理组大鼠在造模前5天给予高压氧预处理。采用线栓法建立大鼠脑缺血再灌注模型,观察高压氧预处理对脑缺血再灌注损伤大鼠神经功能缺损评分、脑梗死面积的影响,检测大鼠缺血脑组织COX-2 mRNA和蛋白的表达以及IL-1β、TNF-α、MDA的含量。结果:高压氧预处理可明显改善脑缺血再灌注大鼠神经功能缺损评分,减少脑梗死面积,降低COX-2m RNA和蛋白表达量,抑制IL-1β、TNF-α的表达,降低MDA水平。结论:高压氧预处理对大鼠脑缺血再灌注损伤具有明显的保护作用,其机制可能与抑制IL-1β、TNF-α、COX-2的表达以及减弱脂质过氧化反应有关。     

Pharmacological preconditioning with erythropoietin reduces ischemia–reperfusion injury in the small intestine of rats

AimsConsidering the implications that arose from several recent experimental studies using recombinant human erythropoietin in rodents, erythropoietin has been regarded as a pharmacological preconditioning agent. The purpose of the present study was to evaluate whether erythropoietin has a preconditioning effect against ischemia and reperfusion injury in the small intestine of the rat.Main methodsIntestinal ischemia was induced in male Wistar rats by clamping the superior mesenteric artery for 30 min, followed by reperfusion for 180 min. Recombinant human erythropoietin (1000 or 3000 U/kg) or vehicle was administered intraperitoneally 24 h prior to ischemia. After collection of ileal tissue, evaluation of damage was based on measurements of the accumulation of polymorphonuclear neutrophils by technetium-99m-labeled leukocyte uptake, content of malondialdehyde, reduced glutathione, contractile responses to agonists, and an evaluation of histopathological features in intestinal tissue.Key findingsTreatment with erythropoietin 24 h before ischemia significantly reduced the tissue content of malondialdehyde and increased that of reduced glutathione. Pretreatment also significantly suppressed leukocyte infiltration into the postischemic tissue, as evidenced by the lower content of myeloperoxidase and technetium-99m-labeled leukocytes. Physiological and histopathological improvements were also significant with the rHuEpo treatment.SignificanceResults of the present study indicate that rHuEpo is an effective preconditioning agent in ischemic injury of the small intestine. Protection provided by recombinant human erythropoietin is closely related to the inhibition of oxidative stress and leukocyte infiltration, which might be among the possible protective mechanisms of erythropoietin in intestinal ischemia and reperfusion.     

The role of NO in ischemia/reperfusion injury in isolated rat heart

Nitric oxide (NO) is an important regulator of myocardial function and vascular tone under physiological conditions. However, its role in the pathological situations, such as myocardial ischemia is not unequivocal, and both positive and negative effects have been demonstrated in different experimental settings including human pathology. The aim of the study was to investigate the role of NO in the rat hearts adapted and non-adapted to ischemia. Isolated Langendorff-perfused hearts were subjected to test ischemic (TI) challenge induced by 25 min global ischemia followed by 35 min reperfusion. Short-term adaptation to ischemia (ischemic preconditioning, IP) was evoked by 2 cycles of 5 min ischemia and 5 min reperfusion, before TI. Recovery of function at the end of reperfusion and reperfusion-induced arrhythmias served as the end-points of injury. Coronary flow (CF), left ventricular developed pressure (LVDP), and dP/dt(max) (index of contraction) were measured at the end of stabilization and throughout the remainder of the protocol until the end of reperfusion. The role of NO was investigated by subjecting the hearts to 15 min perfusion with NO synthase (NOS) inhibitor L-NAME (100 mmol/l), prior to sustained ischemia. At the end of reperfusion, LVDP in the controls recovered to 29.0 +/- 3.9 % of baseline value, whereas preconditioned hearts showed a significantly increased recovery (LVDP 66.4 +/- 5.7 %, p < 0.05). Recovery of both CF and dP/dt(max) after TI was also significantly higher in the adapted hearts (101.5 +/- 5.8 % and 83.64 +/- 3.92 % ) as compared with the controls (71.9 +/- 6.3 % and 35.7 +/- 4.87 %, respectively, p < 0.05). NOS inhibition improved contractile recovery in the non-adapted group (LVDP 53.8 +/- 3.1 %; dP/dt(max) 67.5 +/- 5.92 %) and increased CF to 82.4 +/- 5.2 %. In contrast, in the adapted group, it abolished the protective effect of IP (LVDP 31.8 +/- 3.1 %; CF 70.3 +/- 3.4 % and dP/dt(max) 43.25 +/- 2.19 %). Control group exhibited 100 % occurrence of ventricular tachycardia (VT), 57 % incidence of ventricular fibrillation (VF) - 21 % of them was sustained VF (SVF); application of L-NAME attenuated reperfusion arrhythmias (VT 70 %, VF 20 %, SVF 0 %). Adaptation by IP also reduced arrhythmias, however, L-NAME in the preconditioned hearts increased the incidence of arrhythmias (VT 100 %, VF 58 %, SVF 17 %). In conclusion: our results indicate that administration of L-NAME might be cardioprotective in the normal hearts exposed to ischemia/reperfusion (I/R) alone, suggesting that NO contributes to low ischemic tolerance in the non-adapted hearts. On the other hand, blockade of cardioprotective effect of IP by L-NAME points out to a dual role of NO in the heart: a negative role in the non-adapted myocardium subjected to I/R, and a positive one, due to its involvement in the mechanisms of protection triggered by short-term cardiac adaptation by preconditioning.