Thrombolytic properties of leukocytes from peripheral blood in healthy subjects and in patients with acute cerebral ischemia

Polymorphonuclear leukocytes are activated in acute ischemic stroke. Activated polymorphonuclear leukocytes may contribute to thrombolysis by proteolytic degradation of fibrin and by modification of the plasminogen system. We used an in vitro thrombolysis model to investigate (1) thrombolytic properties of leukocytes in young and healthy subjects, (2) to test the hypothesis of increased polymorphonuclear leukocyte-associated thrombolysis in patients with acute cerebral ischemia, and (3) to assess plasminogen-dependent and -independent thrombolytic properties of polymorphonuclear leukocyte elastase. Coincubation of polymorphonuclear leukocytes with fibrin clots led to increased thrombolysis, a process reaching statistical significance after 8 hours [1x10(7) polymorphonuclear leukocytes/mL; 12.8+/-1.9% (mean+/-SEM), spontaneous clot lysis: 7.3+/-0.7%]. Polymorphonuclear leukocytes inside clots caused more efficient thrombolysis than polymorphonuclear leukocytes in the incubation medium. Spontaneous and polymorphonuclear leukocyte-associated lysis tended to be lower in patients with acute cerebral ischemia (n=9, 24 hours, 9.5+/-1.8% and 12.9+/-2.2%) than in age- and sex-matched control subjects (n=8; 12.2+/-2.0% and 17.4+/-1.9%). In the presence of alpha(2)-antiplasmin, thrombolysis tended to be faster with elastase-digested plasminogen (miniplasminogen) than with native plasminogen. Purified polymorphonuclear leukocyte elastase itself had no thrombolytic effect. We conclude that the thrombolytic capacity of polymorphonuclear leukocytes from peripheral blood is small and slow and may have been overestimated in previous reports. Polymorphonuclear leukocyte thrombolytic activity may not be increased in acute cerebral ischemia. Miniplasminogen may be an interesting adjunct to plasminogen activators in acute stroke models.     

Leukocyte-endothelium interactions during permanent focal cerebral ischemia in mice.

The contribution of leukocyte infiltration to brain damage after permanent focal cerebral ischemia and the underlying molecular mechanisms are still unclear. Therefore, the aim of this study was to establish a mouse model for the visualization of leukocytes in the cerebral microcirculation in vivo and to investigate leukocyte-endothelial interaction (LEI) after permanent middle cerebral artery occlusion (MCAO). Sham-operated 129/Sv mice showed physiologic LEI in pial venules as observed by intravital fluorescent microscopy. Permanent focal cerebral ischemia induced a significant increase of LEI predominantly in pial venules. The number of rolling and adherent leukocytes reached 36.5 +/- 13.2/100 microm x min and 22.5 +/- 7.9/100 microm x min, respectively at 120 minutes after MCAO (P = 0.016 vs. control). Of note, rolling and adherent leukocytes were also observed in arterioles of ischemic animals (7.3 +/- 3.0/100 microm x min rolling and 3.0 +/- 3.6/100 microm x min adherent). Capillary density was not different between groups. These results demonstrate that leukocytes accumulate in the brain not only after transient but also after permanent focal cerebral ischemia and may therefore contribute to brain damage after stroke without reperfusion.     

Protective effects of inhibiting both blood and vascular selectins after stroke and reperfusion

Early intervention after acute ischemic stroke is essential to minimize brain cell injury. Although reperfusion of the ischemic brain is the treatment of choice for acute stroke, reperfusion itself may cause additional injury. The inflammatory cascade, characterized in part by early leukocyte interaction with endothelium, may contribute to this additional injury to blood vessels and surrounding brain tissue, extending the area of infarction. The selectin family of adhesion molecules mediates the initial, rolling and tethering of leukocytes to endothelium. P-selectin is rapidly expressed on ischemic endothelium in the brain vasculature, and L-selectin is expressed on leukocytes. Blocking the selectin-mediated tethering step may limit the inflammatory component of reperfusion injury in the brain. Fucoidin (FCN), a competitive inhibitor of P- and L-selectin, has been reported to decrease leukocyte accumulation during reperfusion of other organs. The effect of both leukocyte and endothelial selectin inhibition after cerebral ischemia and reperfusion has not been previously examined. The purpose of this study was to determine the effects of selectin adhesion molecule blockade on cerebral infarction size and neurological function after middle cerebral artery occlusion and reperfusion (MCAO-R) in the rat. MCAO was induced using the filament method. All animals were subjected to 4 h of MCAO and 24 h of reperfusion. After 24 h, brains were analyzed for size of infarction. Neurological function was assessed during stroke and 24 h after reperfusion. Two groups were studied, an untreated control group (n = 9) and a group treated with the selectin inhibitor, fucoidin (25 mg kg(-1)) (n = 9). We found that selectin blockade significantly reduced cerebral infarction size by 50% (p < 0.05) and improved neurological function (p < 0.05). In addition, a trend toward decreased cerebral edema was demonstrated with selectin inhibition. These results indicate that treatment of the blood and the endothelium with a selectin anti-inflammatory agent is protective after focal stroke and reperfusion in the rat.     

Regional differences in the Purkinje cells settled pattern: a comparative autoradiographic study in control and homozygous weaver mice

Data accumulated over the last 10 years have led to the popular hypothesis that neutrophils and other inflammatory cells play a prominent role in the neuropathology of cerebral ischemia. This hypothesis was derived from a large number of studies involving three general observations: (1) leukocytes, particularly neutrophils, are present in ischemic tissue at the approximate time that substantial neuronal death occurs; (2) neutropenia is sometimes associated with reduced ischemic damage; and (3) treatments that prevent leukocyte vascular adhesion and extravasation into the brain parenchyma can be neuroprotective. This review reexamines the literature to ascertain its support for a pathogenic role for neutrophils in ischemia-induced neuronal loss. To accomplish this goal, we employed several logical theorems of "cause-effect" relationships, as they pertain to leukocytes and ischemic brain damage. Since the majority of studies focused on neutrophils as the most likely pathogenic inflammatory cell, this review necessarily does so here. We reasoned that if neutrophils play an important pathogenic (i.e., cause-effect) role in the neuronal damage that follows a stroke, then one should expect to find clear evidence that: (1) neutrophils invade the ischemic area prior to terminal stage infarction, (2) greater numbers of early appearing neutrophils are accompanied by evidence of greater neuronal loss, and (3) dose-related inhibition of neutrophil trafficking or activity produces a corresponding decrease in the degree of brain damage following ischemia. This review of the literature reveals that the existing evidence does not readily support any of these predictions and that, therefore, it consistently falls short of establishing a clear cause-effect relationship between leukocyte recruitment and the pathogenesis of ischemia. While the available evidence does not necessarily rule out a potential pathogenic role of neutrophils and other leukocytes, it nevertheless does expose serious weaknesses in the existing studies intended to support that hypothesis. For this reason we also offer suggestions for additional experiments and the inclusion of control groups that, in the future, might provide more effective or conclusive tests of the hypothesis.     

Leukocyte Recruitment and Ischemic Brain Injury

Leukocytes are recruited into the cerebral microcirculation following an ischemic insult. The leukocyte–endothelial cell adhesion manifested within a few hours after ischemia (followed by reperfusion, I/R) largely reflects an infiltration of neutrophils, while other leukocyte populations appear to dominate the adhesive interactions with the vessel wall at 24 h of reperfusion. The influx of rolling and adherent leukocytes is accompanied by the recruitment of adherent platelets, which likely enhances the cytotoxic potential of the leukocytes to which they are attached. The recruitment of leukocytes and platelets in the postischemic brain is mediated by specific adhesion glycoproteins expressed by the activated blood cells and on cerebral microvascular endothelial cells. This process is also modulated by different signaling pathways (e.g., CD40/CD40L, Notch) and cytokines (e.g., RANTES) that are activated/released following I/R. Some of the known risk factors for cardiovascular disease, including hypercholesterolemia and obesity appear to exacerbate the leukocyte and platelet recruitment elicited by brain I/R. Although lymphocyte–endothelial cell and –platelet interactions in the postischemic cerebral microcirculation have not been evaluated to date, recent evidence in experimental animals implicate both CD4+ and CD8+ T-lymphocytes in the cerebral microvascular dysfunction, inflammation, and tissue injury associated with brain I/R. Evidence implicating regulatory T-cells as cerebroprotective modulators of the inflammatory and tissue injury responses to brain I/R support a continued focus on leukocytes as a target for therapeutic intervention in ischemic stroke.     

白细胞在实验性脑缺血中作用及中药川芎嗪和丹皮酚对其影响的研究

目的　研究沙鼠脑缺血后再灌注期周围血白细胞 (WBC)、脑实质中小胶质细胞和WBC数的变化规律及中药川芎嗪、丹皮酚对其的影响。　　方法　建立沙鼠脑缺血及再灌注模型 ,取周围血行WBC计数 ,脑组织切片在光镜下行WBC及小胶质细胞计数。　　结果　周围血WBC数在再灌注 4h升高 ,至 1 2h达到高峰 ,以中性粒细胞 (NC)增多为主 ;脑实质中小胶质细胞于再灌注 4h达到高峰 ,WBC在再灌注 2 4h增多最明显 ;川芎嗪、丹皮酚均可降低脑缺血再灌注后增加的周围血WBC数 ,亦能使脑实质中小胶质细胞和WBC数明显减少 ,　　结论　白细胞、小胶质细胞在脑缺血时明显增多 ,丹皮酚、川芎嗪能减少增多的白细胞和小胶质细胞。     

Leukocyte response in patients suffering from acute stroke

The oxidation of adrenaline to adrenochrome has been shown to reflect the activation of leukocytes in vivo. We tested the in vivo activation of leukocytes by measuring plasma oxidation of adrenaline to adrenochrome in patients suffering from cerebral ischemia, cerebral hemorrhage, and transient ischemic attacks and in healthy subjects. Patients with cerebral ischemia and cerebral hemorrhage had significantly higher values than healthy subjects, while patients with transient ischemic attacks had values similar to those of healthy subjects. In some patients with cerebral ischemia, the test was repeated 4 and 15 days after the acute event, but the follow-up data did not differ from baseline values. Our study shows that leukocyte activation occurs in cerebral ischemia and cerebral hemorrhage.     

Leukocyte activation: relation to cardiovascular mortality after cerebrovascular ischemia

Activated leukocytes are believed to be involved in the pathogenesis and progression of atherosclerotic vascular disease and its consequences. In a 4-year observational follow-up study, we investigated whether markers for systemic leukocyte activation (leukocyte-derived inflammatory mediators) were related to cardiovascular mortality after cerebrovascular ischemia. Using enzyme-linked immunosorbent assays, we measured the plasma levels of soluble tumor necrosis factor receptor protein-1 (sTNFR-1), neutrophil gelatinase-associated lipocalin (NGAL) and neutrophil protease-4 (NP4) in 144 patients (90 stroke, 54 transient ischemic attack) 1-3 days after cerebral ischemia. During the 4 years of follow-up, 42 (29%) of the 144 patients died; 38 of cardiovascular causes and 4 of other causes. Patients with evidence of higher leukocyte activation (n = 47) had a higher 4-year cardiovascular mortality rate than those without evidence of leukocyte activation (n = 97; p < 0.005). Logistic regression analysis with age, sex and other significant predictors as covariates showed higher plasma levels of sTNFR1 and NGAL both to be significant independent predictors of cardiovascular mortality, the respective odds ratio, 95% confidence intervals, and p values being 2.0, 1.2-3.4, p < 0.01, and 3.6, 1.2-10.5, p = 0.02, respectively. We concluded that in patients with acute cerebral ischemia, plasma markers of leukocyte activation were significant predictors of long-term cardiovascular mortality. This may indicate an important role of activated leukocytes in the progression of these diseases.     

糖酐酯对脑栓塞大鼠溶栓治疗影响的研究

目的　观察糖酐酯对脑栓塞大鼠白细胞浸润的抑制作用及对溶栓治疗后梗死灶体积、细胞凋亡等的影响。方法　用自体血栓子栓塞大鼠大脑中动脉 0 .5h后 ,静脉给予糖酐酯或生理盐水 ,2h或 4h后应用尿激酶溶栓治疗 ,12h或 2 4h后 ,采用TTC染色测定梗死灶大小 ,免疫组化法检测白细胞浸润和细胞间黏附分子 1(ICAM 1)表达 ,TUNEL法检测细胞凋亡 ,透射电镜观察血脑屏障 (BBB)及细胞坏死。结果　联合溶栓组与单纯溶栓组比较 ,梗死灶减小 (P <0 .0 5 ) ,缺血周边区浸润白细胞数和凋亡细胞数明显减少 (均P <0 0 1) ,BBB损伤及细胞坏死程度减轻 ,颅内出血发生例数减少。但ICAM 1表达两组比较无显著性差异 (P >0 0 5 )。结论　糖酐酯能明显抑制白细胞浸润 ,其作用机制与ICAM 1表达无关 ;抑制白细胞浸润可以增强溶栓治疗疗效、保护BBB和减少溶栓治疗后神经细胞凋亡。     

Heparin and oral anticoagulants in the treatment of brain ischemia

The use of heparin, heparin analogues, and oral anticoagulation in the treatment of brain ischemia remains controversial. We review the mechanism of action of the heparins and warfarin as well as the data regarding their efficacy and complications in patients with ischemic stroke and venous sinus thrombosis. None of these drugs have proven effective for all forms of cerebral ischemia. Further studies examining the efficacy of anticoagulants in different stroke subgroups will be necessary in order to clarify optimal clinical practice.     

Inflammatory response after acute ischemic stroke

BACKGROUND AND PURPOSE: This study aimed to characterize the time course of inflammatory parameters after acute ischemic stroke. METHODS: We serially determined high sensitivity C-reactive protein (CRP), fibrinogen, and leukocyte counts at 10 time points between days 1 and 90 after ischemic stroke and in control subjects. RESULTS: CRP did not significantly change, whereas fibrinogen increased after stroke. At all time points, CRP and fibrinogen were higher than in healthy control subjects, but not risk factor control subjects. The leukocyte count declined after stroke and was significantly elevated as compared to both control groups only on day 1 but not later. NIHSS levels were positively correlated with CRP and fibrinogen at all time points. Larger infarcts were associated with a higher CRP and leukocyte counts on day 90. Treatment with aspirin was associated with lower values for all three inflammatory parameters in the subacute phase after ischemia. CONCLUSIONS: The course after stroke was different between the parameters of inflammation. Only the leukocytes followed the paradigm of an acute phase response.     

Blood cell rheology in acute cerebral infarction

Recently it has been hypothesized that leukocyte rheology could be a relevant variable of the microcirculation during cerebral ischemia. However, relatively few studies have been carried out on the rheologic behavior of leukocytes in vascular diseases. This study aimed at quantifying the filterability through Nuclepore filters (mean pore diameter 5 microns) of both leukocyte subpopulations and red blood cells in patients with acute stroke compared with age-matched healthy controls. Leukocytes were separated by density into polymorphonuclear and mononuclear cells. Filterability of the red blood cells and polymorphonuclear and mononuclear subpopulations in buffer was measured using a constant-flow and low-positive pressure system. We used one-way analysis of variance, signed rank sum, and simple and multiple regression tests for statistical analysis. Twenty consecutive male patients with acute ischemic infarction were compared with 20 age-matched healthy subjects. Mononuclear cell filterability was impaired in acute stroke (7.26 +/- 2.00) compared with the controls (5.55 +/- 1.23) (p less than 0.01). Polymorphonuclear cell filterability was less, but still significantly (p less than 0.05), impaired in acute infarction (5.75 +/- 0.87 vs. 4.19 +/- 0.43). The results show that leukocyte and, especially, mononuclear cell filterability is impaired in acute infarction, while no differences exist in red blood cell filterability.     

Persistent increase of leukocyte aggregation in a patient with relapsing ischemic stroke and ulcerative colitis

There is an increased incidence of central nervous system thromboembolic events in patients with inflammatory bowel disease. The possible pathogenetic link between the two diseases is not clear. Experimental and clinical observations suggest the possible role of leukocyte activation in the induction of ischemic and inflammatory events. In a patient with relapsing ischemic stroke and ulcerative colitis, serial determinations of leukocyte aggregation, by means of the modified leukergy test, were performed. A persistent increase of the percentage of aggregated leukocytes with values much higher than those of healthy subjects and patients with ischemic stroke was found during and between the episodes of ischemia and inflammation. The particular temporal pattern of clinical and hemorheological alterations suggests that the persistent activation of leukocytes may represent a risk factor for the development of both ischemic and inflammatory disease.     

Slit modulates cerebrovascular inflammation and mediates neuroprotection against global cerebral ischemia

Cerebrovascular inflammation contributes to secondary brain injury following ischemia. Recent in vitro studies of cell migration and molecular guidance mechanisms have indicated that the Slit family of secreted proteins can exert repellant effects on leukocyte recruitment in response to chemoattractants. Utilizing intravital microscopy, we addressed the role of Slit in modulating leukocyte dynamics in the mouse cortical venular microcirculation in vivo following TNFalpha application or global cerebral ischemia. We also studied whether Slit affected neuronal survival in the mouse global ischemia model as well as in mixed neuronal-glial cultures subjected to oxygen-glucose deprivation. We found that systemically administered Slit significantly attenuated cerebral microvessel leukocyte-endothelial adherence occurring 4 h after TNFalpha and 24 h after global cerebral ischemia. Administration of RoboN, the soluble receptor for Slit, exacerbated the acute chemotactic response to TNFalpha. These findings are indicative of a tonic repellant effect of endogenous Slit in brain under acute proinflammatory conditions. Three days of continuous systemic administration of Slit following global ischemia significantly attenuated the delayed neuronal death of hippocampal CA1 pyramidal cells. Moreover, Slit abrogated neuronal death in mixed neuronal-glial cultures exposed to oxygen-glucose deprivation. The ability of Slit to reduce the recruitment of immune cells to ischemic brain and to provide cytoprotective effects suggests that this protein may serve as a novel anti-inflammatory and neuroprotective target for stroke therapy.     

Glycerol attenuates the adherence of leukocytes in rat pial venules after transient middle cerebral artery occlusion

Abstract

Intravenous infusion of glycerol has been used in patients with a cerebral infarction, expecting improvement in brain edema and cerebral blood flow (CBF). However, the mechanism of the improvement of CBF has not been clearly demonstrated. The aim of this study in the rat pial microvasculature after transient middle cerebral artery occlusion (MCAO) is to examine the effects of glycerol on leukocyte- endothelium interaction, which plays a critical role in the pathogenesis of brain injury by ischemia/ reperfusion and concerns induction of secondary brain damage. Rhodamine 6G-labeled leukocytes at the brain surface were visualized with intra-vital fluorescence videomicroscopy through a closed cranial window and an analysis was made of the number of adherent leukocytes and the centerline leukocyte velocity in the venule before MCAO, after reperfusion of MCAO and after infusion of glycerol (Croup 1) or saline (Croup 2). The number of adherent leukocytes decreased and the centerline leukocyte velocity increased statistically significantly immediately after the infusion of glycerol in Croup I, but there was no significant change in Croup 2. The infusion of glycerol washes away the adherent leukocytes and prevents them from interfering with the blood cell and plasma flow. Furthermore, secondary brain damage may be relieved by decreasing the adherence of leukocytes. In conclusion, modulating the adherence of leukocytes is one of the important factors in the neuroprotective effect of glycerol. [Neurol Res 1999; 21: 785-790]     

小鼠局灶性脑缺血模型中细胞间粘附分子-1表达升高

目的　白细胞可以导致缺血细胞损伤,内皮细胞上表达的细胞间粘附分子－１（ＩＣＡＭ－１）有利于白细胞迁移至组织。本研究目的是对小鼠大脑中动脉栓塞（ＭＣＡＯ）后脑内ＩＣＡＭ－１ 蛋白在组织中表达和含量进行检测。方法　通过对成年雄性ＣＤ－１ 小鼠使用血管腔内尼龙线栓塞术,造成０、３、６、１２、２４、４８ 和７２ ｈ 的持续性大脑中动脉栓塞。缺血程度由激光多普勒流量仪确定,缺血脑组织ＩＣＡＭ－１ 的阳性表达由免疫组化技术检测,并用免疫沉淀和Ｗｅｓｔｅｒｎ 印迹来定量。结果　在大脑中动脉栓塞后,小鼠缺血脑半球的表面脑血流量减少到基准值的９％ ～１５％ 。各组间大脑中动脉栓塞过程中的脑血流量无显著差异。免疫组化技术显示,缺血中心区和末影区都见ＩＣＡＭ－１ 阳性的微血管内皮细胞,从缺血中心到缺血边缘区微血管内皮细胞表达ＩＣＡＭ－１ 出现增高的趋势。免疫沉淀和Ｗｅｓｔｅｒｎ 印迹分析结果表明,缺血区ＩＣＡＭ－１的表达在大脑中动脉栓塞后３ ｈ 增高,６～１２ ｈ 达到高峰,并持续到７２ ｈ。结论　研究表明,在持续性大脑中动脉栓塞的小鼠中检测到ＩＣＡＭ－１ 表达明显升高,因为在持续局灶性大脑中动脉缺血后ＩＣＡＭ－１ 可介导白细胞和内皮细胞粘附,加速     

Hyperbaric oxygen therapy of cerebral ischemia

BACKGROUND: Hyperbaric oxygen (HBO) therapy of cerebral ischemia has been evaluated in a number of human and animal studies; however, there is presently no consensus on its efficacy. METHODS: We present a review of animal and human studies on HBO therapy of cerebral ischemia as well as present potential mechanisms of action of HBO. RESULTS: Animal studies of HBO have shown promise by reducing infarct size and improving neurologic outcome. HBO has also been shown to inhibit inflammation and apoptosis after cerebral ischemia. Early reports in humans also suggested benefit in stroke patients treated with HBO. Recent randomized, controlled human studies, however, have not shown benefit, although all were limited by small sample size. Important differences between animal and human studies suggest HBO might be more effective in stroke within the first few hours and at a pressure of 2-3 ATA. CONCLUSIONS: The clinical usefulness of HBO in the treatment of cerebral ischemia is not yet certain. Attention to emerging pathophysiologic data should be taken into consideration in design of any future clinical trials of HBO in acute ischemic stroke.     

Nitric oxide synthase inhibitor augments post-ischemic leukocyte adhesion in the cerebral microcirculation in vivo.

The objective was to examine the effect of the nitric oxide synthase inhibitor, N omega-nitro-L-arginine methyl ester (L-NAME) on leukocyte adhesion in the cerebral microcirculation during reperfusion following partial forebrain ischemia in the rat. Intravital fluorescence video-microscopy through a closed cranial window was used to visualize leukocyte-endothelium interaction in small pial veins of 15-100 microns diameter. Forebrain ischemia was produced by the ligation of both common carotid arteries plus elevation of the intracranial pressure to 20 mmHg for 60 min. The number of leukocytes adhering to the endothelium for longer than 3 sec was determined during ischemia (5 min and 60 min) and during reperfusion (5 min and 60 min). Two experimental groups were treated with either L-NAME or its inactive enantiomer D-NAME (20 mg kg-1 i.v.) 30 min prior to reperfusion. In a third group, also treated with D-NAME, post-ischemic hyperemia was prevented by lowering the ICP without removing the occlusion of common carotid arteries (partial reperfusion). The velocity of flow adjacent to the endothelial surface of pial veins was measured by tracking the movement of fluorescently labeled red blood cells as flow markers before and after ischemia. During ischemia, the number of adhering leukocytes increased approximately two-fold at 5 min, and three-fold at 60 min. In the D-NAME-treated group with complete reperfusion, leukocyte adhesion returned to the baseline level by 60 min of reperfusion. However, in the L-NAME-treated group, leukocyte adhesion remained elevated at 60 min of reperfusion. Post-ischemic flow velocity was significantly decreased (-66%) from control after L-NAME treatment whereas it was increased (+53%) in the D-NAME-treated group. In the partial reperfusion group, leukocyte adhesion continued to increase after the first hour of ischemia and reached a level 2.7-fold over baseline at 60 min reperfusion. Flow velocity remained below control (-26%) at 60 min reperfusion. Leukocyte adhesion was absent in pial arteries and no plugging by leukocytes was observed in cortical capillaries. The results suggest that leukocyte adhesion in small pial veins increases during 1 h forebrain ischemia and continues to increase during reperfusion if the velocity of flow or shear rate is low. The increase in leukocyte adhesion is reversible if flow velocity is elevated during reperfusion. L-NAME prevents post-ischemic hyperemia and augments leukocyte adhesion principally via a decrease in velocity or shear rate.     

Glycerol attenuates the adherence of leukocytes in rat pial venules after transient middle cerebral artery occlusion

Intravenous infusion of glycerol has been used in patients with a cerebral infarction, expecting improvement in brain edema and cerebral blood flow (CBF). However, the mechanism of the improvement of CBF has not been clearly demonstrated. The aim of this study in the rat pial microvasculature after transient middle cerebral artery occlusion (MCAO) is to examine the effects of glycerol on leukocyte-endothelium interaction, which plays a critical role in the pathogenesis of brain injury by ischemia/reperfusion and concerns induction of secondary brain damage. Rhodamine 6G-labeled leukocytes at the brain surface were visualized with intra-vital fluorescence videomicroscopy through a closed cranial window and an analysis was made of the number of adherent leukocytes and the centerline leukocyte velocity in the venule before MCAO, after reperfusion of MCAO and after infusion of glycerol (Group 1) or saline (Group 2). The number of adherent leukocytes decreased and the centerline leukocyte velocity increased statistically significantly immediately after the infusion of glycerol in Group 1, but there was no significant change in Group 2. The infusion of glycerol washes away the adherent leukocytes and prevents them from interfering with the blood cell and plasma flow. Furthermore, secondary brain damage may be relieved by decreasing the adherence of leukocytes. In conclusion, modulating the adherence of leukocytes is one of the important factors in the neuroprotective effect of glycerol.     

小鼠大脑中动脉暂时性闭塞引起脑皮质局部炎性细胞浸润的变化

目的 研究局部脑缺血模型小胶质细胞的激活和白细胞的浸润情况，以进一步研究脑卒中后引起炎症反应的细胞及分子机制。方法 用免疫组化方法研究单克隆抗体CD11b，Ly-6G，CD3和CD45／B220在大脑中动脉闭塞(MCAO)60min进行不同时段灌注后在脑组织中的表达。结果 灌注早期(18～24h)在脑梗塞区就可见被激活的CD11b阳性的小胶质细胞，在18～24h灌注期，可见CD11b阳性的巨噬细胞和Ly—6G阳性的中性白细胞黏附在脑膜的血管壁。灌流48～72h，CD11b阳性的巨噬细胞和Ly-6G阳性的中性白细胞从血管壁迁移到脑梗塞区。在灌流24～48h，我们仅见到个别CD3阳性T淋巴细胞和CD45／B220阳性B淋巴细胞。然而，在灌流72h后，可容易地检测到CD3阳性T淋巴细胞。结论 研究结果表明单核吞噬细胞系统在MCAO脑卒中模型的炎症反应中起重要作用。在灌注早期CD11b阳性细胞主要为脑实质内被激活的小胶质细胞，灌注24～72h，外周血循环中的巨噬细胞和其他白细胞从血管迁移到缺血区以及被激活的小胶质细胞一同在脑卒中炎症反应中起作用。