Blood to brain transport after newborn cerebral ischemia/reperfusion injury

These experiments examine the transfer of urea, sodium, and sucrose from blood to brain in an animal model of newborn cerebral ischemia-reperfusion injury. Cerebral ischemia (20 min) was produced in anesthetized, ventilated piglets by increasing intracranial pressure above mean arterial blood pressure, thereby reducing cerebral perfusion pressure to zero. The blood to brain transfer of urea, sodium, and sucrose was then measured in sham control piglets and at 30 min and 2 hr of reperfusion following ischemia. At 30 min of reperfusion, urea and sodium transfer were increased while sucrose transfer was unchanged. However, at 2 hr of reperfusion, transfer of all three tracers was elevated. The difference in the time course of increased transport of these substances into the brain following ischemia cannot be explained by size differences, indicating that changes in the blood-brain barrier following ischemia are more complex than merely opening junctions between cells and creating leaky sites. Alterations in blood-brain barrier transport could participate in altered neuronal function after ischemia-reperfusion injury.     

中风胶囊对脑缺血/再灌注损伤模型鼠脑组织自噬相关蛋白表达的影响*

目的: 观察中风胶囊对脑缺血/再灌注损伤(CIRI)模型鼠脑组织自噬相关蛋白表达的影响,初步揭示其对神经元损伤保护的分子机制。方法: 采用改良线栓法构建大鼠脑缺血/再灌注损伤模型,随机将60只雄性SD大鼠分为假手术组、模型组、丁苯酞组(0.054 g/kg)、中风胶囊高剂量组(1.08 g/kg)、中风胶囊中剂量组(0.54 g/kg)、中风胶囊低剂量组(0.27 g/kg),每组10只。造模结束后灌胃给药10 d,每天1次,实验结束后处死各组大鼠,摘取脑组织。各组大鼠末次给药24 h后进行神经功能评分;HE染色法观察各组大鼠脑组织病理形态;ELISA法检测各组大鼠血清雌二醇(E2)和卵泡刺激素(FSH);RT-PCR法与Western blot法分别测定各组大鼠脑组织PI3K/Akt/Beclin1信号通路关键基因及蛋白的表达。结果: 与假手术组比较,模型组大鼠体重及脑组织中p-PI3K、p-Akt等蛋白表达均显著降低,脑指数、神经功能缺损评分及脑组织Beclin1、LC3基因和蛋白表达均显著升高(P＜0.05或P＜0.01),脑组织结构排列疏松,间质水肿,神经细胞呈三角形,核固缩深染。与模型组相比,中风胶囊高剂量组大鼠体重显著升高,神经功能缺损评分显著下降(P＜0.05),脑组织病理损伤较模型组明显改善;中风胶囊各剂量组的脑指数及脑组织Beclin1、LC3的基因和蛋白表达均显著降低,脑组织中p-PI3K、p-Akt等蛋白表达均显著升高(P＜0.05或P＜0.01)。结论: 中风胶囊通过调控PI3K/Akt/Beclin1信号通路中Beclin1和LC3的表达来抑制CIRI模型鼠的自噬反应,从而发挥保护其脑神经元损伤的作用。     

Effects of molsidomine on retinal ischemia/reperfusion injury in rabbits

We investigated the effect of molsidomine (MOL) on ischemia/reperfusion (I/R) injury. Rabbits were assigned to four groups: group 1, sham; group 2, I/R; group 3, MOL treatment for 4 days after I/R; group 4, MOL treatment for 1 day before I/R and 3 days after I/R. Retinal I/R was produced by elevating the intraocular pressure to 150 mm Hg for 60 min. Seven days after I/R, the eyes were enucleated. Retinal changes were examined using histochemistry. The levels of malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT) also were measured. We found a significant increase in the thickness of the outer nuclear layer of group 3 compared to the other groups. In groups 3 and 4, caspase-3 stained cells in the ganglion cell layer were decreased compared to group 2. We found a significant increase in caspase-3 stained cells in the inner nuclear layer (INL) of group 2 compared to the other groups. We found a significant increase in caspase-3 stained cells in group 3 compared to group 4 in the INL. The MDA level in group 2 was significantly higher than group 1 and MOL significantly decreased MDA levels in groups 3 and 4. We found that MOL protected the retina from I/R injury by enhancing antioxidative effects and inhibiting apoptosis of retinal cells.     

The hemodynamic effects of acute myocardial ischemia and reperfusion in Clawn miniature pigs.

Acute myocardial ischemia was induced by occluding the LAD in Clawn miniature pigs. Eight pigs (group 1) were subjected to 6 h ischemia and nine pigs (group 2) were subjected to 20 min ischemia, followed by reperfusion for 340 min. Three animals of the group 1 died due to ventricular fibrillation after occlusion and in group 2, four animals died due to the arrhythmia after reperfusion. Though the ischemic area of group 2 (15.6% of the ventricle) was narrower than that of group 1 (21.7%), the survival rate was lower. We supposed that ischemia-reperfusion injuries were strongly connected with the hemodynamics of group 2. Clawn miniature pigs are useful experimental animals for myocardial ischemic researches.     

下肢骨骼肌缺血后处理对兔缺血/再灌注心肌坏死和凋亡的影响

目的:探讨在体情况下,骨骼肌缺血后处理对兔缺血/再灌注心肌坏死和凋亡的影响。方法:新西兰大白兔36只,随机分成3组(每组随机选取6只进行梗死范围的测定,另外6只进行凋亡测定):①假手术组(Sham组);②缺血/再灌注组(I/R组);③远端后处理组(RPostC组)。在缺血前、后及再灌注60 min、120 min分别抽血测定肌酸激酶(CK),乳酸脱氢酶(LDH)的活性。采用伊文思兰(evans blue)和三苯基氯化四氮唑(TTC)染色方法确定心肌缺血区范围以及心肌坏死区范围。用Tunel法检测兔心肌缺血区细胞凋亡情况,免疫组织化学方法检测心肌缺血区蛋白caspase-3、Bcl-2及Bax的表达。结果:RPostC组心肌坏死程度、再灌注末CK活性较I/R组明显减低。RPostC组缺血区心肌Tunel阳性指数显著低于I/R组(21.79%±1.07%vs35.81%±1.10%,P<0.05)。而RPostC组缺血区心肌细胞caspase-3阳性指数显著低于I/R组(25.03%±1.16%vs39%±2.43%,P<0.05)。与Sham组比较,I/R组及RPostC组Bax蛋白表达指数、Bcl-2蛋白表达指数均升高;但RPostC组的Bax/Bcl-2比值降低,而I/R组的Bax/Bcl-2比值升高。与I/R组相比较,RPostC组Bax蛋白表达指数及Bax/Bcl-2比值显著降低,Bcl-2表达指数显著升高,差异均有统计学意义。结论:远端后处理能够明显的减少缺血/再灌注心肌细胞的坏死和凋亡,其减轻心肌细胞凋亡的机制可能与抑制促凋亡基因caspase-3的活化及Bcl-2表达的上调有关。     

脑缺血／再灌对海马磷酸酪氨酸蛋白含量的影响及其机制的研究

目的和方法：采用蒙古沙土鼠双侧颈总动脉结扎（BCAO）前脑缺血模型,研究N－甲基D－门冬氨酸（NMDA）受体（NR）非竞争性拮抗剂氯胺酮（ketamine,KT)、L－型电压门控钙离子通道（L－type voltage gatd calcium channel,L型VGCC）拮抗剂硝苯吡啶（Nifedipine ND）及非NR拮抗剂6,7－二硝基喹恶啉上卫四（6,7－dinitroquinoxaline-2,3dione DNQX)对沙土鼠脑缺血及缺血／再灌海马可溶性（S3）、突触体（P2）和颗粒性（P3）部分中磷酸酪氨酸蛋白（p-tyr-pr）含量变化的影响。结果：①缺血15min,三部分（P2,P3,S3）p-tyr-pr含量均下降,而S3部分p-tyr-pr含量下降更明显;随着脑缺血再灌时间的延长,三部分P-tyr-Pr含量均逐渐升高。S3部分恢复快,P2与P3部分相比,升高的速度较慢,但升高的幅度较大,且再灌后期变化不大;②脑缺血前腹腔注射KT或ND,均可部分地拮抗缺血再灌引起的p-tyr-pr含量的升高,而DNQX对此无影响。结论：缺血／再灌引起的p-tyr-pr变化与NR通道及L型VGCC有关,而与非NR无关。     

Oxygen radical mechanisms of brain injury following ischemia and reperfusion.

This review addresses current understanding of oxygen radical mechanisms as they relate to the brain during ischemia and reperfusion. The mechanism for radical production remains speculative in large part because of the difficulty of measuring radical species in vivo. Breakdown of lipid membranes during ischemia leads to accumulation of free fatty acids. Decreased energy stores during ischemia result in the accumulation of adenine nucleotides. During reperfusion, metabolism of free fatty acids via the cyclooxygenase pathway and metabolism of adenine nucleotides via the xanthine oxidase pathway are the most likely sources of oxygen radicals. Although leukocytes have been found to accumulate in some models of ischemia and reperfusion, their mechanistic role remains in question. Therapeutic strategies aimed at decreasing brain injury have included administration of radical scavengers at the time of reperfusion. Efficacy of traditional oxygen radical scavengers such as superoxide dismutase and catalase may be limited by their inability to cross the blood-brain barrier. Lipid-soluble antioxidants appear more efficacious because of their ability to cross the blood-brain barrier and because of their presence in membrane structures where peroxidative reactions can be halted.     

川芎嗪对脑缺血/再灌注后所致肺损伤的影响

目的：观察川芎嗪对脑缺血／再灌注后肺损伤的影响。方法：采用Pulsinelli等的方法建立大鼠急性全脑缺血／再灌注模型。将Wistar大鼠随机分为三组,即：对照组（Control）、缺血／再灌注组（I／R）、川芎嗪＋缺血／再灌注组（TEP＋I／R）,分别测定各组肺功能（PaO2、PaCO2）,肺系数（LI％）、血浆和肺组织中与自由基有关物质的含量。结果：川芎嗪可有效改善脑缺血／再灌注后肺功能,减轻肺水肿,减少胞浆酶的漏出,增加自由基清除醇的活性,抑制组织脂质过氧化的发生。结论：川芎嗪对脑缺血／再灌注后肺损伤具有保护作用,其机制可能与其抗氧自由基和膜保护作用有关。     

Glucose-insulin-potassium preserves systolic and diastolic function in ischemia and reperfusion in pigs

Clinical and experimental studies have suggested benefit of treatment with intravenous glucose-insulin-potassium (GIK) in acute myocardial infarction. However, patients hospitalized with acute coronary syndromes often experience recurrent myocardial ischemia without infarction that may cause progressive left ventricular (LV) dysfunction. This study tested the hypothesis that anticipatory treatment with GIK attenuates both systolic and diastolic LV dysfunction resulting from ischemia and reperfusion without infarction in vivo. Open-chest, anesthetized pigs underwent 90 min of moderate regional ischemia (mean subendocardial blood flow 0.3 ml x g(-1) x min(-1)) and 90 min reperfusion. Eight pigs were treated with GIK (300 g/l glucose, 50 U/l insulin, and 80 meq/l KCl; infused at 2 ml x kg(-1) x h(-1)) beginning 30 min before ischemia and continuing through reperfusion. Eight untreated pigs comprised the control group. Regional LV wall area was measured with orthogonal pairs of sonomicrometry crystals. GIK significantly increased myocardial glucose uptake and lactate release during ischemia. After reperfusion, indexes of regional systolic function (external work and fractional systolic wall area reduction), regional diastolic function (maximum rate of diastolic wall area expansion), and global LV function (LV positive and negative maximum rate of change in pressure with respect to time) recovered to a significantly greater extent in GIK-treated pigs than in control pigs (all P < 0.05). The findings suggest that the clinical utility of GIK may extend beyond treatment of acute myocardial infarction to anticipatory metabolic protection of myocardium in patients at risk for recurrent episodes of ischemia.     

红花对兔肺缺血/再灌注损伤及环氧酶表达的影响

目的：探讨红花（safflor injection,SI）抗肺缺血/再灌注损伤作用及其机制。方法：复制在体兔肺缺血/再灌注损伤模型。30只日本大耳兔,随机均分为三组：假手术组（S组）,缺血/再灌注组（I/R组）和缺血/再灌注＋红花注射液组（SI组）。实验结束时,自颈动脉抽血检测丙二醛（MDA）含量、超氧化物歧化酶（SOD）和黄嘌呤氧化酶（XO）活力。取肺组织测湿干重比（W/D）,计算肺泡损伤率（IAR）,电镜观察细胞超微结构改变。免疫组化法检测肺组织COX-1、COX-2蛋白表达的变化 组织原位杂交法检测肺组织COX-1mRNA、COX-2mRNA表达的变化。结果：I/R组血清MDA、XO均显著高于S组,SOD明显低于S组（P〈0.01） I/R组和SI组的W/D与IAR均高于S组（均P〈0.05和P〈0.01）,SI组显著低于I/R组（P〈0.01） I/R组肺组织的超微结构损伤严重,SI组损伤程度明显较轻 免疫组化和原位杂交发现I/R组肺组织COX-2蛋白和COX-2mRNA表达皆显著高于SI组（均P〈0.01）,肺组织COX-1蛋白和COX-1mRNA表达三组间无明显变化。结论：肺缺血/再灌注损伤可诱导肺组织COX-2的表达,SI可能通过抗氧化应激和下调肺组织COX-2蛋白及其基因的表达而减轻肺缺血/再灌注损伤。     

促红细胞生成素对脑缺血/再灌注损伤的保护作用

目的:探讨促红细胞生成素(Epo)对大鼠脑缺血/再灌注损伤的保护作用。方法:32只SD大鼠,采用夹闭双侧颈总动脉30min再灌注24h制作脑缺血/再灌注模型。随机分为4组(n=8):假手术组、脑缺血/再灌注组、Epo组及阳性对照组(尼莫地平),观察缺血/再灌注后血清一氧化氮(NO)和脑组织匀浆中超氧化歧化酶(SOD)活性、丙二醛(MDA)含量及脑组织含水量的变化。结果:Epo组血清NO和脑组织匀浆中MDA含量显著下降,SOD活性显著升高,脑组织含水量显著下降,与缺血/再灌注组相比有显著性差异。结论:大鼠脑缺血/再灌注后,Epo能减轻脑组织的含水量,减少自由基的生成,减轻脂质过氧化反应,对脑缺血/再灌注损伤有保护作用。     

牛磺酸对家兔缺血/再灌注心肌细胞凋亡的影响

目的:研究牛磺酸(Tau)对家兔缺血/再灌注损伤心肌细胞凋亡的影响.方法:阻断家兔心脏左冠状动脉前降支45 min,再灌注180 min引起心肌缺血/再灌注损伤,在心肌缺血前5 min耳缘静脉注射牛磺酸(200mg/kg),应用DNA片段原位末端标记法 (TUNEL染色),DNA凝胶电泳和流式细胞仪(FCM)观测心肌细胞凋亡.结果:琼脂糖凝胶电泳显示损伤对照组(I/R) 心肌DNA呈云梯状改变,而Tau I/R组无此改变.与损伤对照组(I/R) 比较,Tau I/R组缺血心肌凋亡细胞明显减少(TUNEL染色).流式细胞仪测定I/R组及Tau I/R组缺血心肌凋亡率分别为17.66%±1.54%和4.86%±1.23%.I/R组的缺血心肌Fas和Bax蛋白表达较非缺血心肌高 (P<0.01),Bcl-2/Bax比例较非缺血心肌低(P<0.01);而在Tau I/R组,Fas和Bax蛋白表达较I/R组的低 (P<0.01),Bcl-2/Bax比例较I/R组高(P<0.01).结论:牛磺酸可减少I/R家兔心肌细胞凋亡,其机制与调控凋亡相关基因 Fas,Bax和Bcl-2的蛋白表达有关.     

Determination of plasma leukotrienes in antigen-induced bronchoconstrictive guinea pigs.

Convenient extraction and radioimmunoassay methods for measurement of leukotrienes C4 and D4 (LTC4 and LTD4) in biological fluids are described. LTC4 or LTD4 in plasma was extracted with acetonitrile, and the extract was washed with dichloromethane then adjusted to pH 3.5 or 6.0, respectively. Each leukotriene was partially purified by using a C18-bonded silica cartridge and quantitated by radioimmunoassay. Amounts of LTC4 and LTD4 in the range of 0.025-1.6 ng could be assayed in plasma. This procedure was employed to examine the increase in plasma LTC4 (0.249 +/- 0.036 ng/ml) and LTD4 (1.399 +/- 0.235 ng/ml) of guinea pigs during intravenous challenge-induced anaphylactic bronchoconstriction, and the suppression of the increase of bronchoconstriction and leukotrienes by the administration of 5-lipoxygenase inhibitors such as E6080 (6-hydroxy-2-(4-sulfamoylbenzyl-amino)- 4,5,7-trimethylbenzothiazole hydrochloride), AA861 (2,3,5-trimethyl-6-(12-hydroxy-5,10-dodecadiynyl)-1,4-benzoquinone ) and phenidone. On the other hand, LTC4 and LTD4 were not detected in plasma after an inhaled challenge, though significant bronchoconstriction was provoked. It was concluded that the present study validates a new technique for quantitating plasma leukotrienes on the basis of pH and a suitable method for evaluating the pharmacological efficacy of 5-lipoxygenase inhibitors.     

Effects of vagotomy on respiratory mechanics in newborn and adult pigs

We have examined breathing patterns and respiratory mechanics in anesthetized tracheostomized newborn piglets and adult pigs and the changes determined by cervical bilateral vagotomy. Piglets had a respiratory system compliance and resistance, on a per kilogram basis, respectively, higher and smaller than the adults. After vagotomy neither variable changed in the newborn, but resistance dropped in the adult. This may suggest that efferent vagal control of bronchomotor tone is more pronounced in the adult. Respiratory system time constant was longer in newborns both before and after vagotomy. The distortion of the chest wall, examined as the ratio between the volume inhaled spontaneously and the passive volume for the same abdominal motion, was more marked in newborns, reflecting their higher chest wall compliance. The work per minute, computed from the pressure and volume changes, was larger in piglets. After vagotomy the external work per minute was not different; however, the larger tidal volumes were accompanied by a larger chest distortion. This may indicate that vagal control of the breathing pattern, by limiting the depth of inspiration and hence the amount of chest distortion, has implications on the energetics of breathing.     

ZFP580对大鼠心肌缺血/再灌注后心室重塑的影响

目的：探究锌指转录因子（ZFP580）与心肌缺血/再灌注损伤后心室重塑的关系。方法：72只SD大鼠随机分为假手术（sham）组（n=8）和心肌缺血/再灌注（I/R）组（n=64）,其中I/R组分别在再灌注后的0.5 h、1 h、2 h、4h、1 d,7 d,14 d,28 d处死后取材,观察心肌组织中ZFP580的表达。培养大鼠H9C2心肌细胞,每组设3个复孔,分别在转化生长因子β1（TGF-β1）刺激0 h、8 h、16 h、24 h后观察心肌细胞肥大情况,并检测心肌细胞中β-MHC、心房利钠肽（ANP）以及ZFP580 mRNA的表达。利用慢病毒介导的基因转染获得高表达ZFP580的H9C2心肌细胞,转染72h后,检测心肌细胞中基质金属蛋白酶3（MMP-3）的表达。结果：成功建立心肌缺血/再灌注损伤模型,大鼠心肌I/R损伤后第14天,心肌组织大面积梗死,心肌细胞呈嗜酸性变。大鼠心肌组织中ZFP580及TGF-β1表达上调。TGF-β1（5 ng/ml）刺激H9C2心肌细胞后诱导心肌细胞肥大,心肌细胞肥大标志蛋白β-MHC、ANP表达上调,且心肌细胞中ZFP580mRNA表达上调（P < 0.05）。高表达ZFP580的H9c2心肌细胞中MMP-3表达下调（P < 0.05）。结论：锌指转录因子ZFP580可能参与了心肌缺血/再灌注后心室重塑的过程,其作用可能与参与TGF-β1诱导的心肌细胞肥大过程以及抑制心肌细胞产生MMP-3有关。     

PPAR-gamma activation fails to provide myocardial protection in ischemia and reperfusion in pigs

Peroxisome proliferator-activated receptor (PPAR)-gamma modulates substrate metabolism and inflammatory responses. In experimental rats subjected to myocardial ischemia-reperfusion (I/R), thiazolidinedione PPAR-gamma activators reduce infarct size and preserve left ventricular function. Troglitazone is the only PPAR-gamma activator that has been shown to be protective in I/R in large animals. However, because troglitazone contains both alpha-tocopherol and thiazolidinedione moieties, whether PPAR-gamma activation per se is protective in myocardial I/R in large animals remains uncertain. To address this question, 56 pigs were treated orally for 8 wk with troglitazone (75 mg x kg(-1) x day(-1)), rosiglitazone (3 mg x kg(-1) x day(-1)), or alpha-tocopherol (73 mg x kg(-1) x day(-1), equimolar to troglitazone dose) or received no treatment. Pigs were then anesthetized and subjected to 90 min of low-flow regional myocardial ischemia and 90 min of reperfusion. Myocardial expression of PPAR-gamma, determined by ribonuclease protection assay, increased with troglitazone and rosiglitazone compared with no treatment. Rosiglitazone had no significant effect on myocardial contractile function (Frank-Starling relations), substrate uptake, or expression of proinflammatory cytokines during I/R compared with untreated pigs. In contrast, preservation of myocardial contractile function and lactate uptake were greater and cytokine expression was attenuated in pigs treated with troglitazone or alpha-tocopherol compared with untreated pigs. Multivariate analysis indicated that presence of an alpha-tocopherol, but not a thiazolidinedione, moiety in the test compound was significantly related to greater contractile function and lactate uptake and lower cytokine expression during I/R. We conclude that PPAR-gamma activation is not protective in a porcine model of myocardial I/R. Protective effects of troglitazone are attributable to its alpha-tocopherol moiety. These findings, in conjunction with prior rat studies, suggest interspecies differences in the response to PPAR-gamma activation in the heart.