Effect of nitric oxide synthase inhibitor N^G-nitro-L-arginine on the content of amino acid in ischemic brain tissues of rats

BACKGROUND： Nitric oxide synthase （NOS） inhibrtors have been widely used to investigate the role of NO on cerebral ischemic injury, but the results are controversial. Moreover, it has been considered to aggravate the ischemic neuronal damage with the release of excessively excitatory amino acids （EAA） during cerebral ischemia. On the other hand, some inhibitory amino acid is suggested to be important for the neuronal protection against ischemic brain damage. Our study has recently showed that treatment with the NOS inhibitor NG-nitro-L-arginine （L-NA） reduced focal cerebral ischemic damage. The effect of L-NA on the contents of excitatory and inhibitory amino acid in the rat brain following cerebral ischemia is still unclear. OBJECTIVE： By evaluating the effect of NOS inhibitor, L-NA on the contents of aspartate, glutamate, glycine and γ-aminobutyric acid （GABA） in striatum, hippocampus and cortex in the rat brain following cerebral ischemia respectively, to investigate the beneficial effect of L-NA on cerebral ischemic injury and the possible mechanism. DESIGN： A randomized and controlled experiment SETTING ： Department of Pharmacology, Hebei Academy of Medical Sciences MATERIALS： A total of 42 male healthy SD rats （grade Ⅱ, weighting 250-300 g） were provided by the Experimental Animal Center of Hebei Province （Certification： 04036）. Aspartate, glutamate, glycine, GABA, L-NA and 2,3,5-triphenyltetrazolium chloride （TTC） were obtained from Sigma Chemicals Co, St Louis, MO, USA. HPLC-ultraviolet detector system consisted of Agilent 1100 HPLC. METHODS： The experiment was carried out in Department of Pharmrcology, Hebei Academy of Medical Sciences from June 2005 to June 2006. Rats were randomly divided into three groups： sham-operated group （n = 6）, ischemic group （n = 18）, L-NA group （n = 18）. The model of focal cerebral ischemia in rat was prepared with intraluminal line occlusion methods. In sham-operated rats, the external carotid artery was surgically prepared, but the filament was not inserted. Each group was further divided into 3 subgroups （n = 6 for each）： drugs were administrated at 2, 6 and 12 hours after the middle cerebral artery occlusion （MCAO） respectively. L-NA （20 mg/kg, ip） was administrated, twice a day, for 3 consecutive days. Same volume of normal saline was administrated in ischemic and sham operation groups. The changes of infarcted volume and the contents of amino acids were respectively assayed. Image analysis software was used for the measurement of the infarcted area. The results were expressed as a percentage of the infarcted volume of cerebral/volume of whole brain （IV%） in order to control for edema formation. The contents of aspartate, glutamate, glycine and GABA in striatum, hippocampus and cortex in the rat brain following cerebral ischemia were respectively measured by HPLC method. All data were analyzed with one-way ANOVA and Dunnett＇s test. MAIN OUTCOME MEASURES： （1） The volume of cerebral infarction; （2） The contents of aspartate, glutamate glycine and GABA in brain tissue after cerebral ischemia. RESULTS ： All 42 rats were involved in the final analysis. （1） Infarcted volume： Volume was 0 in sham-operated group. When L-NA was administrated at 2 and 6 hours after MCAO, the infarcted volume was （20.13±3.59）% and （23.12±5.84）% in L-NA group, which was not similar to that in ischemic group [（22.10±3.98）%, （25.38± 5.37）%, P〉 0.05]. However, the infarcted volume was markedly decreased compared with that of ischemic group when L-NA was administrated at 12 hours after MCAO [（26.11±3.55）% and （37.15±3.58）%, P 〈 0.01]. Changes of amino acid content： At 2 and 6 hours after ischemia, the contents of aspartate, glutamate, glycine and GABA in striatum, hippocampus and cortex in ischemic group were significantly increased compared with those in sham-operated group （ P〈 0.05-0.01）. However, contents in L-NA group were similar to those in ischemic group （P 〉 0.05）. At 12 hours after ischemia, the contents of aspartate [（0.21 ±0.06）, （0.36±0.05）, （0.29±0.12） mg/g] and glutamate [（0.55±0.06）, （0.78±0.10）, （0.52±0.10） mg/g] in striatum, hippocampus and cortex in L-NA group were significantly decreased compared with those in ischemic group [（0.49±0.17）, （0.63± 0.03）, （0.51±0.15） mg/g; （0.98±0.30）, （1.15±0.15）, （0.93±0.15） mg/g, P〈 0.05-0.01]. Glycine in hippocampus was （0.40±0.07） mg/g, which was higher than that in ischemic group [（0.21±0.07） mg/g, P 〈 0.05]. GABA in striatum, hippocampus and cortex was （0.93±0.10）, （0.62±0.12） and （0.81 ±0.10） mg/g, respectively, which was higher than that in ischemic group [（0.60±0.08）, （0.37±0.17）, （0.59±0.10） mg/g, P 〈 0.05-0.01]. CONCLUSION ： It may be concluded that L-NA have beneficial effect on ischemic cerebral injury in ischemic later stage in rats. The possible mechanism is that L-NA can decrease the contents of aspartate and glutamate, increase the contents of glycine and GABA.

Remote ischemic preconditioning protects liver ischemia-reperfusion injury by regulating eNOS-NO pathway and liver microRNA expressions in fatty liver rats

BACKGROUND: Ischemic preconditioning (IPC) is a strategy to reduce ischemia-reperfusion (I/R) injury. The protective effect of remote ischemic preconditioning (RIPC) on liver I/R injury is not clear. This study aimed to investigate the roles of RIPC in liver I/R in fatty liver rats and the involvement of endothelial nitric oxide synthase-nitric oxide (eNOS-NO) pathway and microRNA expressions in this process. METHODS: A total of 32 fatty rats were randomly divided into the sham group, I/R group, RIPC group and RIPC+I/R group. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and nitric oxide (NO) were measured. Hematoxylin-eosin staining was used to observe histological changes of liver tissues, TUNEL to detect hepatocyte apoptosis, and immunohistochemistry assay to detect heat shock protein 70 (HSP70) expression. Western blotting was used to detect liver inducible NOS (iNOS) and eNOS protein levels and realtime quantitative polymerase chain reaction to detect miR-34a, miR-122 and miR-27b expressions. RESULTS: Compared with the sham and RIPC groups, serum ALT, AST and iNOS in liver tissue were significantly higher in other two groups, while serum NO and eNOS in liver tissue were lower, and varying degrees of edema, degeneration and inflammatory cell infiltration were found. Cell apoptosis number was slightly lower in the RIPC+I/R group than that in I/R group. Compared with the sham group, HSP70 expressions were significantly increased in other three groups (all P<0.05). Compared with the sham and RIPC groups, elevated miR-34a expressions were found in I/R and RIPC+I/R groups (P<0.05). MiR-122 and miR-27b were found significantly decreased in I/R and RIPC+I/R groups compared with the sham and RIPC groups (all P<0.05). CONCLUSION: RIPC can reduce fatty liver I/R injury by affecting the eNOS-NO pathway and liver microRNA expressions.

Changes of Gastric Nitric Oxide in Neonatal Rats with Hypoxic-Ischemic Encephalopathy

In order to study the changes of nitric oxide (NO) in gastric wall in hy-poxic neonatal rats, the activity of nitric oxide synthase (NOS) of gastric wall was measured and the NADPH-diaphorase biochemistry test was taken to show the distribution of NOS in the gastric wall of neonatal rats. The results showed that compared with normal rats, NOS had no significant changes in the acute hy-poxic rats (P>0. 05). However, in hypoxic-ischemic encephalopathy (HIE) neonatal rats, the activity of NOS was markedly increased (P<0. 01), and the NOS positive reactive products were markedly increased in the gastric muscle, but no changes in the mucosal and submucosal layers were observed. The results suggest that the decreased gastric motivation and the gastric mucosal lesions caused by asphyxia are associated with the changes of NO in gastric wall.

Remote ischemic perconditioning prevents liver transplantation-induced ischemia/reperfusion injury in rats: Role of ROS/RNS and e NOS

AIM To investigate the underlying mechanisms of the protective role of remote ischemic perconditioning (RIPerC) in rat liver transplantation. METHODS Sprague-Dawley rats were subjected to sham, orthotopic liver transplantation (OLT), ischemic postconditioning (IPostC) or RIPerC. After 3 h reperfusion, blood samples were taken for measurement of alanine aminotransferase, aspartate aminotransferase, creatinine (Cr) and creatinine kinase-myocardial band (CK-MB). The liver lobes were harvested for the following measurements: reactive oxygen species (ROS), H2O2, mitochondrial membrane potential (Delta psi m) and total nitric oxide (NO). These measurements were determined using an ROS/H2O2, JC1 and Total NOx Assay Kit, respectively. Endothelial NO synthase (eNOS) was analyzed by reverse transcription-polymerase chain reaction (RTPCR) and western blotting, and peroxynitrite was semiquantified by western blotting of 3-nitrotyrosine. RESULTS Compared with the OLT group, the grafts subjected to RIPerC showed significantly improved liver and remote organ functions (P < 0.05). ROS (P < 0.001) including H2O2 (P < 0.05) were largely elevated in the OLT group as compared with the sham group, and RIPerC (P < 0.05) reversed this trend. The collapse of Delta psi m induced by OLT ischemia/reperfusion (I/R) injury was significantly attenuated in the RIPerC group (P < 0.001). A marked increase of NO content and phosphoserine eNOS, both in protein and mRNA levels, was observed in liver graft of the RIPerC group as compared with the OLT group (P < 0.05). I/R-induced 3-nitrotyrosine content was significantly reduced in the RIPerC group as compared with the OLT group (P < 0.05). There were no significant differences between the RIPerC and IPostC groups for all the results except Cr. The Cr level was lower in the RIPerC group than in the IPostC group (P < 0.01). CONCLUSION Liver graft protection by RIPerC is similar to or better than that of IPostC, and involves inhibition of oxidative stress and up-regulation of the PI3K/Akt/eNOS/NO pathway.

How to protect liver graft with nitric oxide

Organ preservation and ischemia reperfusion injury associated with liver transplantation play an important role in the induction of graft injury. One of the earliest events associated with the reperfusion injury is endothelial cell dysfunction. It is generally accepted that endothelial nitric oxide synthase （e-NOS） is cell-pro- tective by mediating vasodilatation, whereas inducible nitric oxide synthase mediates liver graft injury after transplantation. We conducted a critical review of the literature evaluating the potential applications of regulating and promoting e-NOS activity in liver preservation and transplantation, showing the most current evidence to support the concept that enhanced bioavailability of NO derived from e-NOS is detrimental to ameliorate graft liver preservation, as well as preventing subse- quent graft reperfusion injury. This review deals mainly with the beneficial effects of promoting ＂endogenous＂ pathways for NO generation, via e-NOS inducer drugs in cold preservation solution, surgical strategies such as ischemic preconditioning, and alternative ＂exogenous＂ pathways that focus on the enrichment of cold storage liquid with NO donors. Finally, we also provide a basic bench-to-bed side summary of the liver physiology and cell signalling mechanisms that account for explaining the e-NOS protective effects in liver preservation and transplantation.

Conventional, but not remote ischemic preconditioning, reduces iNOS transcription in liver ischemia/reperfusion

AIM:To study the effects of preconditioning on inducible nitric oxide synthase(iNOS)and interleukin 1(IL-1)receptor transcription in rat liver ischemia/reperfusion injury(IRI).METHODS:Seventy-two male rats were randomized into 3 groups:the one-hour segmental ischemia(IRI,n=24)group,the ischemic preconditioning(IPC,n=24)group or the remote ischemic preconditioning(RIPC,n=24)group.The IPC and R-IPC were performed as 10 min of ischemia and 10 min of reperfusion.The iNOS and the IL-1 receptor mRNA in the liver tissue was analyzed with real time PCR.The total Nitrite and Nitrate(NOx)in continuously sampled microdialysate(MD)from the liver was analyzed.In addition,the NOx levels in the serum were analyzed.RESULTS:After 4 h of reperfusion,the iNOS mRNA was significantly higher in the R-IPC(ΔCt:3.44±0.57)group than in the IPC(ΔCt:5.86±0.82)group(P=0.025).The IL-1 receptor transcription activity was reduced in the IPC group(ΔCt:1.88±0.53 to 4.81±0.21),but not in the R-IPC group,during reperfusion(P=0.027).In the MD,a significant drop in the NOx levels was noted in the R-IPC group(12.3±2.2 to 4.7±1.2μmol/L)at the end of ischemia compared with the levels in early ischemia(P=0.008).A similar trend was observed in the IPC group(11.8±2.1 to 6.4±1.5μmol/L),although this difference was not statistically significant.The levels of NOx rose quickly during reperfusion in both groups.CONCLUSION:IPC,but not R-IPC,reduces iNOS and IL-1 receptor transcription during early reperfusion,indicating a lower inflammatory reaction.NOx is consumed in the ischemic liver lobe.

Curcumin protects against ischemic spinal cord injury The pathway effect

Inducible nitric oxide synthase and N-methyI-D-aspartate receptors have been shown to participate in nerve cell injury during spinal cord ischemia. This study observed a protective effect of curcumin on ischemic spinal cord injury. Models of spinal cord ischemia were established by ligating the lumbar artery from the left renal artery to the bifurcation of the abdominal aorta. At 24 hours after model establishment, the rats were intraperitoneally injected with curcumin, Reverse transcrip- tion-polymerase chain reaction and immunohistochemical results demonstrated that after spinal cord ischemia, inducible nitric oxide synthase and N-methyI-D-aspartate receptor mRNA and protein expression significantly increased. However, curcumin significantly decreased inducible nitric oxide synthase and N-methyI-D-aspartate receptor mRNA and protein expression in the ischemic spinal cord. Tadov scale results showed that curcumin significantly improved motor function of the rat hind limb after spinal cord ischemia. The results demonstrate that curcumin exerts a neuroprotective ef- fect against ischemic spinal cord injury by decreasing inducible nitric oxide synthase and N-methyI-D-aspartate receptor expression.

Estrogen inhibits lipid peroxidation after hypoxic-ischemic brain damage in neonatal rats

Sprague-Dawley neonatal rats within 7 days after birth were used in this study. The left common carotid artery was occluded and rats were housed in an 8% O2 environment for 2 hours to establish a hypoxic-ischemic brain damage model. 17β-estradiol （1 × 10-5 M） was injected into the rat abdominal cavity after the model was successfully established. The left hemisphere was obtained at 12, 24, 48, 72 hours after operation. Results showed that malondialdehyde content in the left brain of neonatal rats gradually increased as modeling time prolonged, while malondialdehyde content of 17β-estrodial-treated rats significantly declined by 24 hours, reached lowest levels at 48 hours, and then peaked at 72 hours after injury. Nicotinamide-adenine dinucleotide phosphate histochemical staining showed the nitric oxide synthase-positive cells and fibers dyed blue/violet and were mainly distributed in the cortex, hippocampus and medial septal nuclei. The number of nitric oxide synthase-positive cells peaked at 48 hours and significantly decreased after 17β-estrodial treatment. Our experimental findings indicate that estrogen plays a protective role following hypoxic-ischemic brain damage by alleviating lipid peroxidation through reducing the expression of nitric oxide synthase and the content of malondialdehyde.

Role of nitric oxide during early phase myocardial ischemic preconditioning in rats

Background To date, there have been no reports on altered nitric oxide （NO） content in ischemia/reperfusion with regard to in vivo preconditioning procedures. These studies are important for understanding the mechanisms of NO during early myocardial ischemic preconditioning. The aim of the present study was to investigate the mechanisms of NO during early myocardial ischemic preconditioning by measuring levels of NO and cyclic guanosine monophosphate （cGMP）, as well as activity of nitric oxide synthase （NOS） in ischemia/reperfusion with respect to preconditioning in rats. Methods Sixty-six female Sprague-Dawley rats were randomly divided into four groups： ischemic preconditioning group （IP）, ischemia/reperfusion group （I/R）, control group （CON）, and preconditioning procedure group （PC）. In the PC group, rats were further divided into PC1-, PC1＋, PC2-, PC2＋, PC3-, and PC3＋ subgroups. Rats underwent left coronary artery occlusion and reperfusion, and subsequently, NOS activity and levels were assessed with spectrophotometric analysis. cGMP contents were measured with radioimmunoassay. Results The level of NO and cGMP, as well as the activity of NOS, were significantly higher in the IP group compared to the I/R and CON groups （P 〈0.05）. During preconditioning prior to prolonged ischemia, NO and cGMP levels varied markedly with ischemia and reperfusion. The levels of NO repeatedly increased when the heart was exposed to three episodes of 5-minute ischemia, and were almost completely reversed during each reperfusion period. NO and cGMP levels were significantly different between the 5-minute period of ischemia and the same period of reperfusion during preconditioning. Conclusions NO plays an important role during early phase myocardial ischemic preconditioning in rats. NO and cGMP could be triggers and mediators of early phase myocardial ischemic preconditioning. Altered NOS activity following ischemic stress could be the primary inducer of higher NO levels detected. NO and cGMP fluctuations might be the trigger for protection during early phase myocardial ischemic preconditioning.

缺血后处置缓解离体大鼠心肌缺血/再灌注损伤

目的观察缺血后处置对大鼠心肌缺血/再灌注损伤的影响,并初步探讨其作用机制。方法复制离体大鼠心肌缺血/再灌注损伤模型及缺血后处置模型。记录±dp/dtmax和LVEDP。用比色法检测灌流液中乳酸脱氢酶(LDH)、丙二醛(MDA)水平及超氧化物歧化酶(SOD)活性,Western blot方法检测心脏eNOS及ERK1/2的表达,RT-PCR方法测定心脏细胞色素P4502J3(CYP2J3)mRNA表达。结果与缺血/再灌注组(IR)相比,缺血后处置组(IPo)±dp/dtmax均增高(P<0.01),而LVEDP、LDH降低(P<0.05);IR组MDA水平高于对照组(CON)及IPo组(P<0.01),SOD活性低于IPo组(P<0.01);IR组大鼠心肌eNOS及磷酸化ERK1/2的表达均高于CON组和IPo组;IPo组大鼠心脏CYP2J3 mRNA的表达明显高于CON组和IR组。结论缺血后处置可能通过提高再灌注心肌SOD活性,增加氧自由基清除,而改善缺血/再灌注引起的心功能障碍及细胞损伤;CYP2J3/EET系统有可能参与其保护作用。

大鼠局灶性脑缺血后─氧化氮合酶活性与细胞凋亡关系

目的：了解局灶性脑缺血再灌注过程中一氧化氮合酶（ＮＯＳ ）变化与脑细胞凋亡的关系，探索阻断脑细胞凋亡的时间窗。方法：用线栓 法建立局灶性脑缺血模型，大脑中动脉阻塞（ＭＣＡＯ）的时间分别为１５、３０、６０、９０、１２０ ｍｉｎ ，再灌注２４ ｈ。用还原型尼克酰胺腺嘌呤二核苷酸脱氢酶（ＮＡＤＰＨ－ｄ）组化法，检测局灶性脑 缺血再灌注后缺血中心区（顶叶皮层和尾壳核）ＮＯＳ活性变化。用苏木精－伊红和ＴＵＮＥＬ染色法 检测缺血中心区脑细胞凋亡情况。结果：ＮＯＳ阳性神经元数于３０ ｍｉｎ时增多 最显著，９０～１２０ ｍｉｎ时急剧减少。各组凋亡细胞数随缺血时间延长而增多。结 论：局灶性脑缺血再灌注过程中神经型ＮＯＳ（ｎＮＯＳ）对缺血早期的脑损伤尤其是细胞 凋亡起主要作用。

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45 min的部分肝门阻断及60 min的再灌注。③L-精氨酸(L-Arg)组,缺血前20 min经阴茎背静脉注射L-Arg(300 mg/kg),余同②组。实验结束后,取下腔静脉血2 ml,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)和一氧化氮合成酶(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NOe、NOS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。

葛根素对大鼠局灶性缺血脑组织内海马神经元型一氧化氮合酶表达的影响

目的观察葛根素对大鼠局灶性脑缺血海马神经元神经型一氧化氮合酶(nNOS)表达的影响。方法局灶性脑缺血模型由线栓法阻塞大鼠大脑中动脉制成,葛根素腹腔注射干预治疗,采用氯化三苯基四氮唑染色方法测定脑梗死体积,免疫组织化学方法测定大鼠脑缺血后不同时间点前后海马区nNOS阳性神经元表达的变化。结果 2 h和12 h干预组脑梗死体积明显小于对照组(P<0.05),24 h干预组与对照组比较无统计学差异(P>0.05)。缺血2 h组海马nNOS阳性细胞开始增加,12 h组最高,24 h组较前有所下降;干预组大鼠海马nNOS阳性细胞数与对照组比较降低明显,有统计学差异(P<0.01)。结论 nNOS参与早期脑缺血损伤,葛根素通过抑制nNOS表达对大鼠脑神经元损伤起保护作用。

瓜蒌皮注射液干预急性心肌梗塞药效与舒张血管机制的研究

目的:研究瓜蒌皮注射液干预急性心肌梗塞的作用效果,并探讨其机制。方法:结扎大鼠冠脉动脉,制备冠心病急性心肌梗死模型。丹参注射液为阳性对照药。大鼠腹腔注射给药。氯化三苯四氮唑染色(TTC),测定心肌梗死率;生化试剂盒检测血浆中肌酸激酶(CK)、肌酸激酶同工酶(CK-MB)、谷草转氨酶(AST)、羟基丁酸脱氢酶(α-HBDH)和乳酸脱氢酶(LDH)5项心肌酶活性,确定心肌细胞坏死程度;生化试剂盒检测血浆中内皮素(ET)、一氧化氮(NO)含量及内皮型一氧化氮合酶(eNOS)的活性,明确瓜蒌皮注射液调节血管舒张的能力。结果:对急性心肌缺血大鼠,瓜蒌皮注射液可显著降低其心肌梗死率及血浆中5项心肌酶活性,提高eNOS活性及NO分泌量,降低ET含量,维护缺血心肌正常生理功能,作用效果与丹参注射液无显著性差异。结论:瓜蒌皮注射液可能通过抑制ET的合成、激活eNOS活性,提高NO分泌量,增强冠状动脉的扩张,增加冠脉流量;从而实现对大鼠急性心肌梗塞的保护作用。

亚低温对大鼠局灶性脑缺血再灌注后不同脑区iNOS表达的影响

目的探讨亚低温对大鼠局灶性脑缺血再灌注后不同脑区诱导型一氧化氮合酶(iNOS)表达的影响。方法雄性SD大鼠,随机分为假手术组、常温缺血组和亚低温组。采用线栓法制作大脑中动脉闭塞再灌注模型,于缺血后48h,观察不同组间组织形态学变化,检测不同脑区iNOS蛋白表达、iNOS活性和产物NO含量。结果常温缺血后48h,纹状体和皮质均检测到iNOS活性升高和免疫阳性反应,且皮质缺血半暗带区iNOS免疫反应明显强于纹状体和皮质缺血核心区。亚低温明显缩小梗死面积,抑制皮质和纹状体iNOS活性,明显下调半暗带区iNOS蛋白表达,减少NO产生。结论亚低温可能通过减少半暗带区iNOS蛋白表达,抑制iNOS活性,减少NO产生而起到脑保护作用。

远隔缺血预适应对大鼠局灶性脑缺血后诱导型一氧化氮合酶的影响

目的研究远隔缺血预适应(remote ischemic preconditioning,RIPC)对大鼠脑缺血再灌注后诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)表达的影响,以探讨RIPC保护脑缺血损伤的相关机制。方法应用线栓法制作大脑中动脉栓塞(middle cerebral artery occlusion,MCAO)模型,将21只健康雄性SD大鼠采用数字表法随机分为3组:假手术组(sham,n=7),MCAO组(n=7),RIPC+MCAO组(n=7)。在MCAO手术前连续3 d,进行远隔肢体缺血预适应处理(双侧股动脉夹闭10 min/放开10 min,每天3次)。在缺血2 h-再灌注24 h后进行神经功能评分,TTC染色检测脑梗死体积,采用Western blotting方法检测梗死侧脑组织的iNOS蛋白表达水平。结果 1)在MCAO手术过程中,3组实验动物的生理指标均在正常范围内,且组间差异无统计学意义。与sham组相比,MCAO组大鼠再灌注24 h时神经功能缺损评分显著升高、脑梗死体积明显增大(P<0.05)。而RIPC+MCAO组大鼠神经功能较MCAO组大鼠得到明显改善、脑梗死体积显著减少(P<0.05)。2)再灌注24 h时,与sham组相比,MCAO组大鼠脑梗死侧iNOS蛋白表达显著增加(P<0.05)。与MCAO组相比,RIPC+MCAO组大鼠脑梗死侧iNOS蛋白表达显著降低(P<0.05)。结论 RIPC处理对大鼠脑缺血损伤具有保护作用,其机制与降低脑缺血大鼠脑内iNOS蛋白表达有关。

脑缺血预处理对大鼠海马CA1区一氧化氮合酶活性和一氧化氮含量的影响

目的:观察脑缺血预处理(CIP)后大鼠海马一氧化氮合酶(NOS)活性和一氧化氮(NO)含量的变化,探讨NO在脑缺血耐受(BIT)诱导中的作用。方法:将140只凝闭双侧椎动脉的Wistar大鼠分为sham、CIP组、损伤性缺血组和CIP+损伤性缺血组。夹闭双侧颈总动脉致全脑缺血3min作为CIP,10min作为损伤性缺血,第4组中CIP与损伤性缺血之间间隔3d。所有动物均于末次脑缺血恢复再灌注后0h、2h、16h、24h、36h、72h和7d(每个时点n=5)取海马CA1区脑组织,分光光度法检测NOS活性,硝酸还原酶法检测NO2-/NO3-含量。结果:CIP组NOS活性和NO2-/NO3-含量于再灌注后16h开始升高,24h达高峰,接近sham组的1.5倍,36h降至基础水平,其升高的持续时间短于BIT诱导的时程(1-7d);损伤性缺血组NOS活性和NO2-/NO3-含量的变化趋势与CIP组类似,但其峰值(24h)超过sham组的2倍,显著大于CIP组(P<0.05);CIP+损伤性缺血组NOS活性和NO2-/NO3-含量亦有一定程度的升高,但其峰值(24h)明显低于损伤性缺血组(P<0.05)。结论:CIP引起NOS活性及NO2-/NO3-含量的适度增加,参与BIT的诱导;同时CIP阻止损伤性缺血后NO的过量生成所致的细胞毒性作用可能是其诱导BIT的另一途径。

神经节苷脂GM_1对新生鼠缺血缺氧后NOS表达的影响

目的 :探讨神经节苷脂GM1对新生儿缺氧缺血性脑病的保护作用及可能机理。方法 :通过建立新生鼠缺氧缺血性脑病动物模型 ,观察缺血缺氧后不同时期脑组织的病理变化和一氧化氮合酶 (NOS)表达 ,以及GM1对其影响。结果 :GM1给药组脑组织损伤明显减轻 ,缺血缺氧可诱导脑组织中NOS表达水平上调 ,GM1部分地抑制了缺血缺氧后NOS的表达水平。结论 :GM1对新生儿缺氧缺血性脑损伤具有一定程度的保护作用 ,其作用可能是通过部分抑制NOS的表达。

一氧化氮合酶在缺血预处理对大鼠肾脏缺血-再灌注损伤保护中的作用

目的 :探讨一氧化氮合酶 (NOS)在缺血预处理对大鼠肾脏缺血再灌注损伤保护中的作用。方法 :将大鼠随机分为对照组 (CON)、缺血再灌注组 (IR)和缺血预处理后缺血再灌注组 (IPC)。光镜下观察并行肾小管评分 ,用免疫组化法检测各组中不同灌注时间的 3种 NOS的变化。结果 :不同缺血再灌注时间点病理组织学肾小管评分 IPC组均低于 IR组 (P均 <0 .0 5 ) ,而 IPC组和 CON组之间无显著性差异 (P均 >0 .0 5 ) ;诱导型NOS(i NOS)在 IR组中的表达明显高于 IPC组和 CON组 (P<0 .0 1或 P<0 .0 5 ) ;而内皮型 NOS(e NOS)和神经元型 NOS(n NOS)在各组中的表达无显著性差异 (P均 >0 .0 5 )。结论 :缺血预处理对大鼠肾脏缺血再灌注损伤有保护作用 ,其保护机制与 i NOS的生成减少有关 ;e NOS和 i NOS在缺血预处理过程中无明显变化。

心肌缺血再灌注时氧化亚氮与氧化亚氮合酶的变化及L-精氨酸对其的影响

目的:观察大鼠心肌缺血再灌注时氧化亚氮(n itric oxide,NO)和氧化亚氮合酶(n itric oxide synthase,NOS)的变化及L-精氨酸对其的影响,探讨心肌缺血再灌注损伤的可能机制。方法:大鼠80只,分为假手术对照(sham)组,心肌缺血再灌注组,肾脏缺血预处理+心肌缺血再灌注组和L-精氨酸治疗+心肌缺血再灌注组,检测血清NO及NOS水平,光镜下进行中性粒细胞计数。结果:缺血再灌注(M IR)组缺血30 m in时相点与对照组相比NO,NOS无差异,再灌注后NO,NOS开始下降,随时间延长,NO,NOS逐渐减少。与M IR组再灌注对应时相比较,肾脏缺血预处理+M IR组及精氨酸治疗+M IR组NO及NOS均显著增加。缺血后再灌注时随时间延长,中性粒细胞数量逐渐增加,给予精氨酸干预或肾脏缺血预处理后中性粒细胞数量显著下降。结论:心肌缺血后再灌注时有大量中性粒细胞浸润,与心肌损伤关系密切。缺血预处理可通过增加NO水平减轻随后的缺血再灌注损伤,L-精氨酸可通过增加NO,减少中性粒细胞浸润起心肌保护作用。