缺血再灌后血管内皮空泡的形成与内皮的损伤

目的：在活体上探讨缺血再灌后血灌内上细胞损伤及白细胞、血小板与内皮之间粘附的变化。方法：用失血及与再回输血液造成缺血再灌流模型,在高倍显微镜下观察肠系膜微血管内皮损伤及血细胞粘附的变化。结果：缺血再灌后1－3h细静脉、集合毛细血管内出现白细胞、血小板的粘附,血管内皮水肿、管壁增厚,有的血管内皮细胞的胞浆形成圆丘形的空泡,空泡从血管内皮突入管胺、空泡直径10－30μm多出现的细动脉内,在同一根血管内可同时出现几个空泡,大的空泡几科占据血管腔的2／3。结论：缺血再灌后血管内皮水肿及空泡形成,显示内皮细胞的严重损伤。     

淋浆对内毒素休克大鼠微循环障碍的干预作用

目的：探讨淋浆对内毒素休克的干预作用及其机制。方法：Wistar雄性大鼠60只,随机分为对照组、模型组和淋浆组,以颈静脉注射LPS（15 mg/kg）复制内毒素休克模型,造模15 min后,淋浆组自颈静脉注射正常淋浆（占全血量1/15）,观察对平均动脉血压（MAP）、回肠下段肠系膜微循环、细静脉壁白细胞粘附数、血浆P-选择素和细胞间粘附分子（ICAM-1）含量的影响。结果：正常淋浆可防止内毒素休克的MAP进行性下降,解除肠系膜微血管的病理性缩窄,减少白细胞在细静脉壁的粘附,改善微循环的流态,降低血浆P-选择素和ICAM-1的水平。结论：小量正常淋浆对LPS攻击导致内毒素休克的微循环障碍和低血压均有良好的干预作用,其机制与减少细胞粘附分子生成有关。     

板蓝根对内毒素致HL-60细胞释放TNF-α和IL-8的影响

目的:探讨板蓝根抗内毒素活性部位对内毒素(LPS)致HL-60细胞释放肿瘤坏死因子(TNF-α)和白细胞介素8(IL-8)的影响.方法:MTT法确定最适细胞浓度和供试液浓度;ELISA法定量检测LPS预刺激、板蓝根预刺激、LPS与板蓝根同时刺激等三种给药方式作用后TNF-α和IL-8的浓度,观察三种给药方式对内毒素诱导TNF-α、IL-8释放的抑制强度.结果:细胞密度在0.25～2×105cell·mL-1范围时,吸光度与细胞数呈良好的线性关系;供试液无毒界限为7.812mg·mL-1;三种给药方式均能显著抑制LPS诱导HL-60细胞释放TNF-α和IL-8,其中供试液与LPS共育时抑制作用最强.结论:板蓝根可能通过抑制炎性因子过度释放发挥其抗内毒素作用.     

黄芪多糖对内毒素诱导人单核/巨噬细胞炎症因子表达的影响

目的：研究黄芪多糖对内毒素诱导的人单核/巨噬细胞中多种炎症因子表达的影响。方法：采用体外培养的人单核/巨噬细胞,以100ng/ml内毒素作为刺激因子,应用人炎症因子抗体芯片检测1mg/ml黄芪多糖处理后人单核/巨噬细胞中IL-1β、IL-6、IL-8和金属蛋白酶组织抑制剂-2（TIMP-2）等多种炎症因子表达的变化情况。结果：黄芪多糖可显著抑制内毒素诱导单核细胞分泌IL-1β和IL-6的活性,同时,促进TIMP-2蛋白表达。结论：黄芪多糖对内毒素诱导的人单核/巨噬细胞多种炎症因子表达有调节作用。     

骶神经电刺激对脊髓损伤大鼠肠黏膜机械屏障的保护作用

目的：观察骶神经电刺激对脊髓损伤大鼠肠黏膜机械屏障的保护作用。方法：56只Wistar大鼠分7组（n=8）：正常组、急性完全性脊髓损伤（SCI）组和骶神经电刺激组（按24、48、72h各8只）。进行内毒素测定;肠系膜淋巴结、肝脏、脾脏菌培养;肠道形态学观察;紧密连接蛋白zo-1的蛋白表达测定。结果：对照组肠黏膜不同程度损伤;肠道上皮细胞及细胞间连接破坏;内毒素血症和细菌移位明显。实验组肠黏膜得到改善,内毒素水平下降且细菌移位减少。ZO-1蛋白表达无统计学差异。对照组ZO-1的分布出现不同程度的散乱、排列不规则,实验组分布得到改善。结论：骶神经电刺激可促肠蠕动、排肠内容物、减少肠道菌群数量,保护肠黏膜上皮细胞及紧密连接的机械屏障,减少细菌移位和内毒素血症。     

粒细胞集落刺激因子动员内皮祖细胞预防再狭窄的研究

目的:观察粒细胞集落刺激因子是否能通过加速内皮修复预防再狭窄,并探讨该作用与其动员效应即EPCs数量和功能变化的相关性.方法:30只SD大鼠随机均分为假手术组、损伤未干预组和G-CSF组,后两组行颈总动脉球囊损造模,G-CSF组连续给药7天后观察各组外周血内皮祖细胞的数量和增值、粘附、迁移功能,4周后观察再狭窄和再内皮化程度.结果:G-CSF组再内皮化率高于对照组,再狭窄率低于对照组,再内皮化率和再狭窄率呈线性负相关;G-CSF组内皮祖细胞数量明显增加,内皮祖细胞增值、粘附、迁移功能也明显提高.结论:G-CSF通过加速内皮修复能预防再狭窄,该作用与其动员效应即内皮祖细胞数量的增加和增值、粘附、迁移功能的提高有关.     

内毒素血症大鼠肠系膜微循环中血管内皮细胞与白细胞的粘附性变化

本实验采用中文吖啶橙荧光标记技术,结合微循环观察用显微超高速摄录像装置,观察了内毒素对微血管内白细胞与微静脉血管内皮细胞的粘附性的影响。结果表明,内毒素对大鼠的血压、微血管口径和微动脉血流速度影响不大,微静脉血流速度在滴注内毒素后４５和６０ｍｉｎ下降了１６．６７％和１７．９５％（Ｐ＜０．０５）;但内毒素能迅速改变微静脉内的白细胞流态,明显增加附壁滚动的白细胞数和粘附白细胞密度指数,经测量同一微静脉内的白细胞和红细胞流速,求得白细胞与微静脉内皮细胞之间的破裂力在５ｍｉｎ和１５ｍｉｎ时下降了２５．９６％和４２．８８％（Ｐ＜０．０１）,下降趋势持续整个实验过程;说明内毒素能明显地增加白细胞与微静脉血管内皮细胞之间的粘附力。由此提示,研究白细胞与微静脉血管内皮细胞之间粘附力增强机制及寻找其抑制因素对改善微循环紊乱、抢救休克具有重要的临床意义。     

血塞通体内中和内毒素作用的试验研究

目的:探讨血塞通体内中和内毒素的作用.方法:健康大耳白兔24只,随机分为正常对照组、内毒素组、血塞通干预组,每组8只.内毒素组经耳缘静脉静推LPS(1mg/kg),12h后再由耳缘静脉注射一次LPS(1mg/kg),复制SIRS/MODS动物模型;对照组耳缘静脉静推等量生理盐水;血塞通干预组于Oh,12h经耳缘静脉推注LPS(Img/kg)并于第一次注入内毒素后6h,12h,18h静推血塞通(50mg/kg).于24h时间点测定各组动物血清肿瘤坏死因子-α(TNF-α)、白细胞介素-(IL-1)、白细胞介素-8(IL-8)、血小板活化因子(PAF).光镜下观察各组动物脏器病理学变化.结果:血塞通干预组与内毒素组相比,IL-1、IL-8、PAF、TNF-α均较内毒素组有不同程度降低.血塞通干预组脏器损伤组织学改变较内毒素组明显减轻.结论:血塞通在体内能明显中和内毒素的作用,为防治SIRS/MODS提供了实验依据.     

大鼠脑缺血—再灌注损伤细胞间粘附分子—1表达的研究

本文用暂时性脑缺血的大鼠模型对血管内皮细胞间粘附分子１（ＩＣＡＭ１）的表达进行了研究。免疫组化显示,ＩＣＡＭ１的表达于缺血１ｈ再灌６ｈ后明显升高;激光共聚焦扫描显微镜定量检测说明再灌后其表达量比单纯缺血增加了４７％。脑组织过氧化物酶ＭＰＯ（μ／ｇ）含量检测及光镜观察均证实再灌后白细胞在缺血区聚集增多。局部脑组织ＩＬ１含量（ＯＤ值／ｇ）在灌流３ｈ后明显升高。结果说明：①脑缺血再灌注损伤时血管内皮细胞ＩＣＡＭ１的表达为时间依赖性增加;②ＩＣＡＭ１的调节表达与脑损伤时局部细胞因子ＩＬ１的分泌有关;③再灌注后脑缺血区有白细胞的大量聚集;④ＩＣＡＭ１表达量的增加是白细胞在缺血区粘附到血管壁、游出血管损伤周围脑组织的前提条件。     

炎性体与器官移植

炎性体是指存在于细胞质内能够激活半胱天冬酶-1（caspase-1）的大分子复合物,活化的caspase-1通过酶切白细胞介素-1β（IL-1β）和白细胞介素-18（IL-18）前体分子而生成具有生物学活性的IL-1β和IL-18.近来研究发现,炎性体分子NLRP1、NLRP2、NLRP5、CARD8、CASP5的基因型与移植的临床结果相关,心脏移植排斥反应时ASC和IL-1β的表达升高,缺血再灌注损伤中NLRP3炎性体激活增加IL-1β分泌,表明炎性体的激活与移植排斥反应和缺血再灌注损伤密切相关,但诱导炎性体活化的配体和参与的炎性体分子有待进一步研究.     

Inflammatory responses to ischemia,and reperfusion in skeletal muscle

Skeletal muscle ischemia and reperfusion is now recognized as one form of acute inflammation in which activated leukocytes play a key role. Although restoration of flow is essential in alleviating ischemic injury, reperfusion initiates a complex series of reactions which lead to neutrophil accumulation, microvascular barrier disruption, and edema formation. A large body of evidence exists which suggests that leukocyte adhesion to and emigration across postcapillary venules plays a crucial role in the genesis of reperfusion injury in skeletal muscle. Reactive oxygen species generated by xanthine oxidase and other enzymes promote the formation of proinflammatory stimuli, modify the expression of adhesion molecules on the surface of leukocytes and endothelial cells, and reduce the bioavailability of the potent antiadhesive agent nitric oxide. As a consequence of these events, leukocytes begin to form loose adhesive interactions with postcapillary venular endothelium (leukocyte rolling). If the proinflammatory stimulus is sufficient, leukocytes may become firmly adherent (stationary adhesion) to the venular endothelium. Those leukocytes which become firmly adherent may then diapedese into the perivascular space. The emigrated leukocytes induce parenchymal cell injury via a directed release of oxidants and hydrolytic enzymes. In addition, the emigrating leukocytes also exacerbate ischemic injury by disrupting the microvascular barrier during their egress across the vasculature. As a consequence of this increase in microvascular permeability, transcapillary fluid filtration is enhanced and edema results. The resultant increase in interstitial tissue pressure physically compresses the capillaries, thereby preventing microvascular perfusion and thus promoting the development of the no-reflow phenomenon. The purpose of this review is to summarize the available information regarding these mechanisms of skeletal muscle ischemia/reperfusion injury.     

Substance P induces degranulation of mast cells and leukocyte adhesion to venular endothelium

Substance P (SP), one of the established neurotransmitters, evokes an immunoinflammatory response involving leukocyte adhesion to venular endothelium and the degranulation of mast cells. The pathogenetic relationship between these responses, however, remains unresolved. In this study, we propose to examine the changes associated with the activation of mast cells, as well as leukocyte adhesion to venular endothelium by in vivo observation of the rat mesentery. The use of an in vitro assay for intracellular Ca²⁺ mobilization and the degranulation of mast cells demonstrated the significant upper shift of concentration response to SP (10⁻⁴–10⁻⁵ M). In vivo experiments on the mesenteric microcirculation also showed that SP induced a significant increase in the number of degranulated mast cells as well as in the number of leukocytes adherent to the venular wall. Tranilast, a mast cell stabilizer, as well as SP antagonist (CP-96,345) significantly attenuated the extent of mast cell degranulation and leukocyte adhesion elicited by SP. Although an immunoneutralization against CD18 by WT-3 significantly attenuated the leukocyte adhesion, it had no influence on the mast cell degranulation after SP superfusion. These separate in vivo observations show that SP induces leukocyte adhesion to the venular endothelium, possibly through the degranulation of mast cells.     

Enhancement of reperfusion injury by elevation of microvascular pressures

Elevated venous pressure can be associated with severe tissue injury. Few links, however, between venous hypertension and tissue damage have been established. We examined here the effects of micropressure elevation on the outcome of venular occlusion/reperfusion in the mesenteric microvasculature of male Wistar rats. One hour of venular occlusion (diameter approximately 50 microm) by micropipette occlusion followed by reperfusion were carried out with sham surgery without occlusion as control. Leukocyte rolling, adhesion, and migration, oxygen radicals detected by dichlorofluorescein (DCF), and parenchymal cell death detected by propidium iodide (PI) were recorded simultaneously in the same vessel at a location upstream of the occlusion site with elevated micropressure and at a downstream location with low micropressure. The number of rolling, adhering, and migrating leukocytes increased on the upstream side of the occlusion to a higher level than downstream of the occlusion site. During occlusion, DCF intensity on the venular endothelium was greater on the upstream side than in the downstream side, but there were no differences during reperfusion. The number of PI-positive cells adjacent to the venules increased significantly compared with controls, and it remained greater on the upstream higher-pressure side than the downstream side. Leukocyte adhesion and transvascular migration in postcapillary venules as well as parenchymal cell death could be significantly reduced by the hydroxyl radical scavenger dimethylthiourea. Microhemorrhages of blood cells into the mesentery interstitium were observed only on the upstream side of the occlusion. These results indicate that an elevation of the venular blood pressure during occlusion/reperfusion exacerbates the inflammatory cascade and tissue injury. Venous occlusion may constitute an important mechanism for tissue injury.     

Modification of postischemic increase of leukocyte adhesion and vascular permeability in the hamster by Iloprost.

This study of the postischemic events in the hamster cheek pouch showed that there is an increase in number of leukocytes adhering to the venular endothelium after reperfusion. It also showed that the stable prostacyclin analogue Iloprost could counteract both the postischemic increase in leukocyte adhesion and the postischemic increase in vascular permeability to macromolecules. The hamsters were anesthetized and the cheek pouch was everted and prepared for intravital microscopy. Temporary ischemia (30 min) was obtained using an expandable cuff placed around the proximal part of the cheek pouch. Fluorescein labelled dextran (FITC-dextran, Mw 150,000) was used as a tracer of macromolecular leakage from the postcapillary venules. Iloprost, given either topically (0.1 nM) or as an intravenous infusion (40 ng/kg/min), reduced the postischemic permeability increase (P less than 0.05) but did not affect the hemodynamics or the permeability response induced by histamine. It is proposed that these effects could be due to inhibition of leukocyte activation by Iloprost, indicating that these cells could play a role in the permeability increase during reperfusion after ischemia.     

Resveratrol,a red wine constituent polyphenol,prevents superoxide-dependent inflammatory responses induced by ischemia/reperfusion,platelet-activating factor,or oxidants

Moderate consumption of red wine has been shown to exert cardioprotection against ischemia/reperfusion. Because oxidant-dependent leukocyte infiltration plays a critical role in ischemia/reperfusion-induced tissue injury, we hypothesized that resveratrol, a red wine constituent polyphenol would attenuate postischemic leukocyte recruitment and subsequent endothelial dysfunction. Intravital microscopic approaches were used to quantify leukocyte/endothelial cell interactions and venular protein leakage in rat mesenteries exposed to either 20 min ischemia and 60 min reperfusion (I/R), oxidants generated by the reaction of hypoxanthine and xanthine oxidase (HX/XO), platelet-activating factor (PAF), or leukotriene B4 (LTB4). I/R or HX/HX produced marked increases in the number of adherent (LA) and emigrated (LE) leukocytes, which were associated with significant increases in venular albumin leakage (VAL). Intravenous administration of resveratrol or superoxide dismutase (SOD) attenuated these increases in LA, LE, and VAL. Superfusion of the mesentery with PAF or LTB4 also markedly increased LA, LE, and VAL. While resveratrol attenuated the proinflammatory effects of PAF, LTB4-induced changes were not affected by resveratrol. Resveratrol prevents leukocyte recruitment and endothelial barrier disruption induced by a number of superoxide-dependent proinflammatory stimuli, including I/R, HX/XO, or PAF. These salutary effects appear to be related to the antioxidant properties of resveratrol and contribute to the cardioprotective actions associated with consumption of red wine.     

Inhaled NO reduces leukocyte-endothelial cell interactions and myocardial dysfunction in endotoxemic rats

Inhaled nitric oxide (NO) has been shown to have some protective effect in the peripheral distal inflamed vasculature. The objective of the study was to determine whether inhaled NO would reduce endotoxin-induced leukocyte activation and myocardial contractile dysfunction. Rats were treated with either saline or endotoxin (10 mg/kg iv) and then allowed to breathe (4 h) either air or air plus NO (10 ppm). In endotoxemic rats, mesenteric venular endothelium leukocyte firm adhesion increased compared with control rats (1.15 +/- 0.32 vs. 4.08 +/- 0.96 leukocytes/100 microm; P < 0.05). Inhaled NO significantly attenuated endotoxin-induced venular endothelium leukocyte adhesion (4.08 +/- 0.96 vs. 1.86 +/- 0.76 leukocytes/100 microm; P < 0.05) and FITC-conjugated anti-intercellular adhesion molecule-1 fluorescence intensity. Endotoxin-induced myocardial dysfunction and leukocyte content increases were reduced in inhaled NO-treated rats. These observations suggest that inhaled NO reduces the degree of cardiovascular dysfunction and inflammation in endotoxemic rats.     

Paradoxical attenuation of leukocyte rolling in response to ischemia- reperfusion and extracorporeal blood circulation in inflamed tissue

In contrast to acute preparations such as the exteriorized mesentery or the cremaster muscle, chronically instrumented chamber models allow one to study the microcirculation under "physiological" conditions, i.e., in the absence of trauma-induced leukocyte rolling along the venular endothelium. To underscore the importance of studying the naive microcirculation, we implanted titanium dorsal skinfold chambers in hamsters and used intravital fluorescence microscopy to study venular leukocyte rolling in response to ischemia-reperfusion injury or extracorporeal blood circulation. The experiments were performed in chambers that fulfilled all well-established criteria for a physiological microcirculation as well as in chambers that showed various extents of leukocyte rolling due to trauma, hemorrhage, or inflammation. In ideal chambers with a physiological microcirculation (<30 rolling leukocytes/mm vessel circumference in 30 s), ischemia-reperfusion injury and extracorporeal blood circulation significantly stimulated leukocyte rolling along the venular endothelium and, subsequently, firm leukocyte adhesion. In contrast, both stimuli failed to elicit leukocyte rolling in borderline chambers (30-100 leukocytes/mm), and in blatantly inflamed chambers with yet higher numbers of rolling leukocytes at baseline (>100 leukocytes/mm), we observed a paradoxical reduction of leukocyte rolling after ischemia-reperfusion injury or extracorporeal blood circulation. A similar effect was observed when we superfused leukotriene B4 (LTB4) onto the chamber tissue. The initial increase in leukocyte rolling in response to an LTB4 challenge was reversed by a second superfusion 90 min later. These observations underscore 1) the benefit of studying leukocyte-endothelial cell interaction in chronically instrumented chamber models and 2) the necessity to strictly adhere to well-established criteria of a physiological microcirculation.