脑缺血再灌注后血管内血栓形成与血浆纤维蛋白原水平的变化

目的 :观察脑缺血再灌注后血管内血栓形成与血浆纤维蛋白原水平的变化。方法 :在动物活体模型上 ,连续观察脑缺血 /再灌注时脑软膜微血管内血栓形成的过程并测量了缺血 /再灌注不同时期血浆纤维蛋白原的变化。结果 :单纯缺血期 ,血流速度明显减慢 ,血管内血细胞呈粒流或泥沙流 ,细静脉内白细胞贴壁滚动逐渐增多。微血管管径缩小 ,有部分毛细血管血流停滞。再灌注后 ,血流速度较缺血期明显加快 ,但细静脉内白细胞粘附贴壁也明显增多 ,随着再灌时间的延长 ,白细胞粘附贴壁越来越多且牢固 ,血管内皮增厚 ,管腔内壁变得粗糙 ,纤维蛋白形成 ,呈丝…     

微血管内皮大空泡形成对微循环的影响

大鼠肠系膜微血管缺血再灌后内皮细胞出现明显改变,缺血再灌后３０ｍｉｎ～１ｈ细静脉、集合毛细血管内除有白细胞、血小板的粘附以及红细胞聚集、血管内皮水肿外,还发现血管内皮细胞的胞浆形成圆丘形的空泡。空泡从血管内壁内壁突入管腔,空泡直径１０～３０μｍ,多发生在直径２０～７０μｍ的细动脉,静脉内也可出现,但不如动脉多。在同一根血管内可同时出现几个空泡,大的空泡几乎堵塞血管腔。严重者几个空泡出现在同一管腔的周围,造成管腔的堵塞,使血细胞不能顺利通过。结果表明血管内皮大空泡的形成将阻碍血液的流动,加重微循环的障碍和血管周围组织细胞的损伤。     

002 脑缺血再灌注后血管内血栓形成与血浆纤维蛋白原水平的变化

目的: 观察脑缺血再灌注后血管内血栓形成与血浆纤维蛋白原水平的变化. 方法: 在动物活体模型上, 连续观察脑缺血/再灌注时脑软膜微血管内血栓形成的过程并测量了缺血/再灌注不同时期血浆纤维蛋白原的变化. 结果: 单纯缺血期, 血流速度明显减慢, 血管内血细胞呈粒流或泥沙流, 细静脉内白细胞贴壁滚动逐渐增多. 微血管管径缩小, 有部分毛细血管血流停滞. 再灌注后, 血流速度较缺血期明显加快, 但细静脉内白细胞粘附贴壁也明显增多, 随着再灌时间的延长, 白细胞粘附贴壁越来越多且牢固, 血管内皮增厚, 管腔内壁变得粗糙, 纤维蛋白形成, 呈丝状附着在血管腔内, 时而缠络血细胞及血小板, 呈絮状团块附着在血管壁上, 逐渐形成壁栓, 阻碍血细胞的流动. 时有絮状团块被血流冲动、脱落, 进入血流, 形成流动的血栓. 正常组、单纯缺血15 min、缺血30 min和缺血30 min后再灌30 min、 1 h、 6 h各组的血浆纤维蛋白(mg/dl)分别为31.3±5.4、 126.0±14.9、 90.7±10.3、 109.4±5.8、 124.0±11.5、 68.8±8.3. 由此可见, 缺血期与再灌后血浆纤维蛋白原均较正常组高, 在缺血15 min和再灌1 h时出现二个高峰值, 与正常组比较有显著性差异(P<0.01). 再灌6 h时又明显降低, 与单纯缺血30 min组比较有显著性差异(P<0.01). 结论: 再灌注后快速恢复的血流激活了多种细胞粘附分子, 加重了内皮细胞的损伤, 导致血栓形成, 微循环灌注障碍. 并认为血浆纤维蛋白的形成在此病理过程中起着至关重要的作用.     

缺血再灌脑血管内皮损伤及GP Ⅲ a表达的研究

研究沙土鼠缺血再灌注后微血管内皮损伤和GPⅢa的表达。方法用夹闭沙土鼠双侧颈总动脉、30min再通造成灌注模型 ,由颈动脉注入FITC标记血小板。荧光显微镜下观察活体软脑膜微血管中血小板对内皮细胞的粘附 ,用CD61抗体经免疫组化方法观察缺血再灌注沙土鼠脑组织血管内血小板和内皮细胞上GPⅢa糖蛋白的表达。结果再灌注后即刻 ,软脑膜细静脉内有白细胞和血小板粘附 ,呈星点状分布 ;再灌注(30～60)min ,可观察到小动脉和细动脉内皮形成的空泡和血栓形成。免疫组化显示缺血再灌注(1～6)h血小板和内皮细胞膜上GPⅢa表达增强。结论缺血再灌注可引起脑内皮细胞损伤和血小板粘附蛋白表达增强     

怡开对白细胞粘附和血液流变性影响的研究

目的 :深入探讨怡开改善微循环及血液流变学的作用机理。方法 :利用活体肠系膜微循环观察、血液粘度和红细胞变形性测定方法。结果 :对照组用内毒素后明显引起静脉内白细胞粘附、游出和血小板聚集 ,血流减慢 ,内皮水肿、内皮细胞间间隙增大、血管内皮损伤、出血等对血管内皮有明显的损伤作用。用PK预防的大鼠明显减轻内毒素引起的白细胞粘附、游出和血小板聚集 ,未见血管内皮水肿和血栓形成 ,出血比对照组轻。结论 :用PK预防的大鼠能明显减轻白细胞粘附、游出和血小板聚集等 ,减轻血管内皮水肿和血栓形成 ,对血管内皮的损伤有一定预防作用     

复方丹参液对细胞粘附和血管内皮损伤及改善微循环的作用

目的利用活体微循环观察内毒素引起大鼠肠系膜微血管内白细胞、血小板与内皮的粘附行为和血管内皮损伤的变化及复方丹参液预防白细胞、血小板粘附和对血管内皮损伤保护的作用。方法Wistar大鼠体重250～300g,雌雄均用。大鼠随机分为正常组、对照组、复方丹参液组 ,每组10只。正常组大鼠以常规方式喂养 ,不给任何药物。对照组以常规方式喂养外 ,每天以饮用水灌胃 ,2次/d ,以便与给药组有同样的刺激。复方丹参液大剂量组 ,每天用复方丹参液0.5g/2ml/次灌胃 ,2次/d ,约为日成人量的1/10。复方丹参液给药组、对照组由尾静脉注入1mg/Kg体重内毒素 ,2h后用20 %乌拉坦 ,按1ml/100g腹腔注射麻醉 ,正常组不给内素素。采用大鼠肠系膜微循环观察方法 ,观察红细胞聚集性 ,白细胞、血小板粘附。血栓形成的模型采用光化学反应原理 ,在血管内注入光敏剂 ,在强光照射下直接观察血小板粘附及血栓形成的过程 ,记录血栓出现的时间、血栓大小等。结果①正常微循环 :A、V、C为线流 ,无红细胞聚集及粘附 ,动、静脉壁薄 ,细胞间连接紧密 ,未见内皮水肿 ,内皮内外侧未见细胞粘附和游出。②内毒素引起细胞粘附和内皮的损伤 :大鼠静脉内注入内毒素后 ,血流稍有减慢 ,30min后细静脉血管内有明显白细胞粘附。部分集合毛细血管细静脉血     

一氧化氮抑制新内膜形成中血管平滑肌细胞活性

一氧化氮（ＮＯ）为由广泛存在于机体组织中的ＮＯ合成酶（ＮＯＳ）催化Ｌ－精氨酸（Ｌ－Ａｒｇ）和分子氧而合成的一种自由基气体，在血管结构中调节全身内环境，可舒张血管，调节血管张力，抑制血小板的粘附聚集，调节白细胞的活化。最新的研究表明，ＮＯ尚可抑制血管损伤诱导的新内膜形成（ＮＩＦ），而血管平滑肌细胞（ＶＳＭＣｓ）的增殖、迁移和表型转换在ＮＩＦ中有重要作用。作者就ＮＯ对ＮＩＦ过程中ＶＳＭＣｓ功能的影响作一综述。     

脑缺血再灌注后血栓形成与血浆纤维蛋白原水平的变化

目的 :在动物活体模型上 ,连续观察脑缺血 /再灌注时脑软膜微血管内血栓形成的过程并测量缺血 /再灌注不同时期血浆纤维蛋白原的变化。方法 :用夹闭沙土鼠双侧颈总动脉的方法复制脑缺血再灌注模型。颈动脉注入 0 .0 67%异硫氢酸荧光黄 (FITC) 0 .2ml后 ,在荧光显微镜下观察脑软膜微血管内血栓形成的过程 ,并在缺血 15min、30min、再灌注 30min、1h、6h时采血 ,用凝血酶凝固法测量血浆纤维蛋白原。结果 :单纯缺血期血流速度明显减慢 ,细动脉、细静脉管径缩小 ,有部分毛细血管内血流停滞。再灌注后 ,血流速度加快 ,随再灌时间的延长 ,白细胞粘附、贴壁明显增多 ,血管内皮增厚 ,纤维蛋白丝网络血细胞及血小板 ,呈絮状团块附着在血管内壁上 ,逐渐形成壁栓 ,阻碍血细胞的流过。缺血期和再灌注后血浆纤维蛋白原水平均高于正常对照组 ,以缺血 30min和再灌 1h时增高最为明显 (P <0 .0 1)。再灌 6h时又明显降低 ,与单纯缺血 30min组比较有显著性差异 (P <0 .0 1)。结论 :再灌注后快速恢复的血流加重了内皮细胞的损伤 ,白细胞、血小板粘附 ,导致血栓形成 ,并认为血浆纤维蛋白的形成在此病理过程中起着重要的作用     

心肌缺血／再灌注对球结膜微循环的影响以及一氧化氮的调节作用

对照观察兔急性心肌缺血／再灌注时球结膜微循环的变化，测定动物血浆中内皮舒张因子／一氧化氮（ＥＤＲＦ／ＮＯ）、血栓素Ｂ２（ＴＸＢ２）、６－酮－前列腺素Ｆ１α（６－Ｋｅｔｏ－ＰＧＦ１α）、超氧化物歧化酶（ＳＯＤ）、丙二醛（ＭＤＡ）的浓度变化，并用光镜和电镜对缺血／再灌注心肌组织作形态学观察。结果发现：（１）兔心肌缺血／再灌注后球结膜微循环异常；表现为Ａ３、Ａ４微动脉血管管径缩小，毛细血管交换距离增大。（２）血浆中ＥＤＲＦ／ＮＯ水平降低；（３）血浆ＴＸＢ２、ＭＤＡ水平升高，ＳＯＤ、６－Ｋｅｔｏ－ＰＧＦ１α下降；（４）光镜下：缺血区心肌组织细胞肿胀变性。电镜下：内皮细胞肿胀，部分毛细血管腔内有红细胞、白细胞附壁阻塞，线粒体肿胀，糖原颗粒减少。结果提示：心肌在较短时间缺血后再灌注（缺血３０ｍｉｎ再灌注３０ｍｉｎ）后就可产生组织损伤，这可能与血管内皮受损，ＥＤＲＦ／ＮＯ合成释放减少，缩血管物质ＴＸＡ２升高，血小板、白细胞粘附、聚集在血管内皮上，氧自由基产生过多等有关。上述因素共同作用导致缺血／再灌注心肌微循环功能障碍进而引起组织形态改变。     

不同流动方式的流体对血管内皮细胞粘附分子VCAM-1表达的影响

目的 :动脉粥样硬化 (ＡＳ)早期病变易发生于动脉分叉及动脉弯曲处 ,推测与血液流动方式有关 :此处血流呈低切应力摆动方式 ,血管其它部位的血流呈稳层流方式。单核细胞粘附于血管内膜 ,进而穿过内膜迁移到内膜下成为巨噬泡沫细胞 ,这是ＡＳ的病理基础的早期细胞行为 ,细胞粘附分子的表达是细胞粘附的分子基础。ＶＣＡＭ 1是参与内皮和单核粘附的主要粘附分子之一 ,了解不同血流方式对血管内皮细胞 (ＶＥＣ)粘附分子ＶＣＡＭ 1表达的影响 ,有助于ＡＳ发生机制的阐明。方法 :接种的第二代人脐静脉内皮细胞 (ＨＵＶＥＣ)玻片置于平板流动腔中 …     

Vacuole formation and endothelial damage in microvessels after ischemia reperfusion

Vacuole formation and endothelial damage in microvessels after ischemia reperfusion     

ICAM-1单抗对烧伤休克大鼠微循环的影响

目的:探讨运用血管内皮细胞粘附分子(ICAM-1)单克隆抗体减少白细胞粘附和防治烧伤休克的效应及改善微循环的作用机制。方法:复制大鼠烧伤休克模型,用红细胞跟踪相关仪和电视测微仪测量微静脉血液流速、口径、流量,镜下观察细静脉白细胞粘附数,并记录动物存活时间。结果:单抗能减缓烧伤休克大鼠平均动脉压和微静脉血流速度的下降趋势,明显减少微静脉中白细胞附壁粘着数,使动物的平均存活时间显著延长,但仍不足24h。结论:运用ICAM-1单抗能阻断白细胞与内皮细胞的粘附,减少白细胞嵌塞微静脉,达到改善烧伤休克微循环和保护组织细胞的作用,但重症休克需要综合治疗。     

Direct in vivo observations of P-selectin glycoprotein ligand-1-mediated leukocyte–endothelial cell interactions in the pulmonary microvasculature in abdominal sepsis in mice

Objective

P-selectin glycoprotein ligand-1 (PSGL-1) has been shown to play a significant role in septic lung injury. However, the detailed role of PSGL-1 in the pulmonary leukocyte recruitment remains elusive. We have developed a method based on intravital fluorescence microscopy of the lung microcirculation to examine the role of PSGL-1 in the extravasation process of leukocytes in septic lung damage.

Methods

Male C57BL/6 mice were treated with a control antibody or an anti-PSGL-1 antibody prior to cecal ligation and puncture (CLP). Leukocyte–endothelium interactions and microvascular hemodynamics were studied in pulmonary arterioles, capillaries and venules 4 h after CLP.

Results

Immunoneutralization of PSGL-1 decreased CLP-induced leukocyte rolling in pulmonary arterioles and venules significantly. Inhibition of PSGL-1 had no effect on leukocyte adhesion in venules, whereas the number of adherent leukocytes in lung arterioles and the number of trapped leukocytes in capillaries were markedly decreased. Moreover, immunoneutralization of PSGL-1 improved microvascular perfusion in the lung of septic animals.

Conclusions

Taken together, these results document that PSGL-1 mediates leukocyte rolling in arterioles and venules. However, inhibition of PSGL-1 only decreases leukocyte adhesion in arterioles, suggesting that leukocyte rolling is not a prerequisite for pulmonary venular adhesion of leukocytes in sepsis. In addition, our data show that capillary trapping of leukocytes is dependent on PSGL-1 function.     

Direct leukocyte migration across pulmonary arterioles and venules into the perivascular interstitium of murine lungs during bleomycin injury and repair

During acute lung injury and repair, leukocytes are thought to enter the lung primarily across alveolar capillaries and postcapillary venules. We hypothesized that leukocytes also migrate across pulmonary arterioles and venules, which serve as alternative sites for leukocyte influx into the lung during acute lung injury and repair. Lung sections from C57BL/6J mice up to 14 days after intratracheal bleomycin (3.33 U/kg) or saline instillation were assessed by light, fluorescence, confocal, and transmission electron microscopy for evidence of inflammatory cell sequestration and transmigration at these sites. After bleomycin treatment, large numbers of leukocytes (including neutrophils, eosinophils, and monocytes) were present in the vascular lumina and in perivascular interstitia of pulmonary arterioles and venules, as well as within the vascular walls. Leukocytes were observed within well-defined pathways in arteriolar walls and much less structured pathways in venular walls, apparently in the process of transmigration. Intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were expressed at sites of leukocyte interaction with the luminal surface, especially in arterioles. Leukocytes appeared to exit from the vessels near collagen fibers into the perivascular interstitium. Results indicate that leukocytes can directly migrate across arteriolar and venular walls into the perivascular interstitium, which may represent an important but under-recognized pathway for leukocyte influx into the lung during injury and repair.     

Mesenterial microcirculation and postischemic reperfusion of rat brain

We have conducted experiments on mesenterial microvessels (vessels of a remote organ) after postischemic reperfusion of brain in rats. The thrombogenic properties, leukocyte adhesion and vascular endothelium desquamation were studied. Brain ischemia was produced by bilateral clipping of both common carotid arteries. In venules the decrease of thromboresistant properties was significant. The quantity of circulating (desquamated) endothelial cells increased two-fold during the first 15 min after the beginning of reperfusion in comparison with controls. We found also increased leukocyte adhesion in mesenterial venules. Postischemic reperfusion of the brain influenced thus systemically microcirculation. The results suggest that the main mechanism of these changes was the inadequate release of nitric oxide. Leukocyte may play an active role.     

A murine model to study leukocyte rolling and intravascular trafficking in lung microvessels

The cascade of leukocyte interactions under conditions of blood flow is well established in the systemic microcirculation, but not in lung microcirculation. We have developed a murine model to study lung microcirculation by transplanting lung tissue into dorsal skin-fold window chambers in nude mice and examining the ability of leukocytes to traffic within revascularized lung microvessels by intravital microscopy. The revascularized lung allograft demonstrated a network of arterioles, capillaries, and postcapillary venules with continuous blood flow. Stimulation of the lung allograft with TNF-alpha induced leukocyte rolling and adhesion in both arterioles and venules. Treatment with function-blocking anti-selectin mAb revealed that P- and L-selectin are the predominant rolling receptors in the lung microvessels, with E-selectin strengthening P-selectin-dependent interactions. Intravital microscopic studies also demonstrated that during their transit in capillaries, some leukocytes undergo shape change and continue to roll as elongated cells in postcapillary venules. Furthermore, the revascularized microvessels demonstrated the ability to undergo vasoconstriction in response to superfusion with endothelin-1. Overall, these studies demonstrate that the revascularized lung allograft is responsive to various external stimuli such as cytokines and vaso-active mediators and serves as a model to evaluate the interaction of leukocytes with the vascular endothelium in the lung microcirculation under acute as well as chronic experimental conditions.     

Thy-1 antigen: selective association in lymphoid organs with the vascular basement membrane involved in lymphocyte recirculation.

The cell surface differentiation antigen, Thy-1, was demonstrated by immunofluorescence to be associated with collagen-based connective tissue (mainly basement membrane) around some blood vessels in rat lymphoid organs. This association is highly selective: only certain types of blood vessel within a given lymphoid organ were found to be Thy-1+; and different lymphoid organs (thymus, bone marrow, lymph node and spleen) had characteristic differences in the types of blood vessel that bear Thy-1. In lymph node and spleen the vessels that were Thy-1+ were those involved in lymphocyte recirculation and homing (post-capillary venules and arterioles of white pulp); the possibility that Thy-1 may function in mediating selective adhesion of small lymphocytes to extracellular substrates during recirculation is discussed.     

Receptor cleavage and P-selectin-dependent reduction of leukocyte adhesion in the spontaneously hypertensive rat

The SHR, a genetic model for hypertension and the metabolic syndrome, has attenuated leukocyte adhesion to the postcapillary endothelium by an unknown mechanism. Based on recent evidence of elevated levels of MMPs in plasma and on microvascular endothelium of the SHR with cleavage of several receptor types, we hypothesize that the reduced leukocyte-endothelial interaction is a result of enhanced proteolytic cleavage of P-selectin on the postcapillary endothelium and PSGL-1 on leukocytes. The attenuated rolling interactions of SHR leukocytes with the endothelium were restored by chronic treatment with a broad-spectrum MMP inhibitor (CGS) for 24 weeks. The SHR MMP levels, in plasma and mesentery, as well as the systolic blood pressure, decreased significantly with treatment. In the SHR mesentery, labeling of P-selectin in the postcapillary venules by immunohistochemistry demonstrated, on average, a 31% lower extracellular P-selectin density compared with the normotensive WKY. A significantly lower extracellular PSGL-1 density on the membranes of SHR neutrophils compared with the WKY also supported our hypothesis. In vivo stimulation of the mesenteric postcapillary venules with histamine demonstrated that the SHR had an attenuated response, as measured by leukocyte rolling velocity on the endothelium. The reduced P-selectin and PSGL-1 density, on SHR postcapillary endothelium and on SHR leukocytes, respectively, was restored significantly by chronic MMP inhibition. The impaired ability of SHR leukocytes to reduce rolling velocity upon inflammatory stimulation led to fewer firmly adhered leukocytes to the endothelium as a contributor to immune suppression.