环磷酰胺对大鼠脑缺血再灌注后脑组织中ICAM-1、白细胞影响的研究

目的观察环磷酰胺对大鼠脑缺血再灌注后脑组织中ICAM-1表达、白细胞浸润的影响。方法36只雄性SD大鼠分为假手术组和脑缺血再灌注组。脑缺血再灌注组中包括环磷酰胺治疗组和生理盐水对照组,每组按脑缺血2h后再灌注2h、4h、6h、24h又分为4个亚组。脑缺血再灌注组于脑缺血2h再灌注后2h、4h、6h、24h处死动物,用免疫组织化学法观察再灌注后不同时间点脑组织中ICAM-1的表达,HE染色法观察白细胞的浸润程度。结果(1)脑缺血再灌注后,脑缺血坏死区周边ICAM-1的表达及白细胞的浸润增多,并于24h达高峰;环磷酰胺组在相同再灌注时间点的ICAM-1表达及白细胞的浸润均明显低于对照组。(2)ICAM-1的表达与白细胞的浸润呈现正相关性。结论脑缺血再灌注后,ICAM-1的表达上调,介导了白细胞和内皮细胞的粘附,参与了脑缺血再灌注损伤的过程。环磷酰胺治疗组ICAM-1的表达、白细胞的浸润均少于对照组,说明环磷酰胺有抑制粘附分子表达上调、抑制白细胞浸润的作用,因而具有神经保护的作用,为该治疗应用于临床提供了理论基础。     

大鼠脑缺血再灌注模型ICAM-1表达与白细胞浸润关系及亚低温干预治疗的研究

目的　研究大鼠局灶性脑缺血再灌注后不同时程脑组织中的细胞间粘附分子 1 (ICAM 1 )表达规律 ,及其与白细胞浸润的关系 ,并探讨亚低温的治疗作用。方法　采用大鼠大脑中动脉 (MCA)线拴闭塞 /再通法建立大鼠局灶性脑缺血 再灌注模型 ,常温组分别于脑缺血 3小时再灌注 2小时、8小时、2 4小时、48小时、72小时后断头取脑 ;假手术组及亚低温组于脑缺血 3小时再灌注 2 4小时断头取脑 ,行ICAM 1免疫组化及组织HE染色 ,测定ICAM 1表达阳性微血管数及白细胞计数。结果　⑴脑缺血再灌注后 2小时 ,脑缺血的坏死周边区的微血管内皮细胞表达ICAM 1出现增高趋势 ,并于 2 4小时达到高峰 ,各组之间及各组与假手术组间均有显著差异 (均P <0 0 5) ;⑵脑缺血再灌注 8小时出现白细胞浸润 ,且浸润高峰也在 2 4小时 (P <0 0 1 ) ;⑶ICAM 1的表达与白细胞浸润呈正相关 (r =0 .82 7,P <0 0 1 ) ;⑷缺血早期进行亚低温治疗能明显减轻缺血再灌注后ICAM 1的表达及减少白细胞浸润 (P <0 0 1 )。结论　脑缺血再灌注后ICAM 1可介导白细胞和内皮细胞的粘附 ,加速白细胞的浸润 ,提示ICAM 1是造成脑缺血损伤的重要因素之一 ;亚低温的干预治疗能明显减轻缺血再灌注后脑组织病理形态学的损害程度。     

非甾体类抗炎药对局灶性脑缺血再灌注后炎症反应及黏附分子表达的影响

目的 探讨消炎痛和美洛昔康对再灌注后脑损伤的影响及作用机制.方法 线栓法制作大鼠大脑中动脉缺血模型（MCAO）.用免疫组化方法观察脑组织ICAM-1、E-选择素的表达.白细胞髓过氧化物酶（MPO）检测试剂盒检测MPO活性.用放射免疫方法检测PGE2的浓度.TTC染色观察梗死范围.结果 消炎痛和美洛昔康早期给药,都能减小皮层梗死体积.再灌注后4h给药,对梗死体积都无明显影响.再灌注后缺血侧皮层和纹体PGE2浓度明显升高,MPO活性增高,消炎痛和美洛昔康均能降低皮层和纹体区PGE2的水平,明显减少ICAM-1和E-选择素阳性血管数,降低皮层MPO活性.结论 消炎痛和美洛昔康早期给药能减小皮层的梗死体积,这种脑保护作用与抑制内皮黏附分子的表达,减轻再灌注后炎症反应.随着再灌注时间的延长,这种保护作用减弱.     

缺血预处理对大鼠脑缺血再灌注后ICAM-1表达的影响

目的 通过研究脑缺血预处理对大鼠脑缺血再灌注后细胞间粘附分子-1(Intowellular adhesion molecule-1，ICAM-1)表达及多形核白细胞(Ploymorphonuclear leukocytes，PMNLs)浸润变化的影响．探讨预处理的延迟保护作用机制。方法采用四血管阻断法建立大鼠全脑-全脑缺血预处理模型。应用HE染色和免疫组化染色技术，观察脑缺血预处理后ICAM-1表达及多形核白细胞浸润的变化。结果(1)脑缺血再灌注后ICAM-1表达的阳性血管数量、PMNLs浸润数量均明显增加；(2)脑缺血预处理可明显减少缺血再灌注后ICAM-1的表达阳性血管数及PMNLs的浸润数量。结论一次性缺血预处理3min后48h可以少再次长时间缺血(30min)／再灌注(24h)的ICAM-1的表达和多形核白细胞浸润。ICAM-表达的下调和PMNLs浸润减少参与缺血预处理的保护作用。     

米诺环素对大鼠局灶性脑缺血再灌注后ICAM-1表达的影响

目的观察米诺环素对大鼠短暂性脑缺血再灌注后细胞间黏附分子1表达的影响。方法采用改良线栓法制备大鼠大脑中动脉缺血2h再灌注24h模型,随机分为假手术组、缺血再灌注模型组、米诺环素处理组和米诺环素预处理组,每组16只。模型成功后观测各组大鼠的神经行为变化、脑梗死体积以及HE染色计数缺血区中性粒细胞的浸润数目,应用免疫组化方法检测细胞间黏附分子-1的表达情况。结果米诺环素能明显改善大鼠局灶性脑缺血再灌注引起的神经行为障碍,减少脑梗死体积,减少脑缺血引起细胞间黏附分子1的表达,抑制中性粒细胞的浸润。结论米诺环素对大鼠局灶脑缺血再灌足损伤有保护作用,抑制黏附分子可能是一种机制。     

The influence of antiadhesion therapies on leukocyte subset accumulation in central nervous system ischemia in rats

Although treatment with agents that block leukocyte function, including anti-ICAM-1 and doxycycline, reduces experimental central nervous system (CNS) ischemic injury, it is not known how leukocyte subset accumulation is affected by these agents. Using the rat two-vessel occlusion model and immunohistochemistry, we investigated granulocyte (PMN) and monocyte/macrophage (Mφ) accumulation at 1 and 4 d postischemia. A total of 24 animals were randomized to sham surgery, or to ischemia with saline, anti-ICAM-1, or doxycycline treatments. No leukocytes were observed in sham animals. At 24 h postischemia, there was a moderate infiltration of PMN and Mφ in untreated animals that was significantly decreased with either treatment. At 4 d after ischemia no PMN were identified, with extensive Mφ accumulation occurring in untreated animals that was only partially reduced with doxycycline treatment. These results confirm that both anti-ICAM-1 and doxycycline treatments reduce PMN and Mφ infiltration at 24 h. Delayed Mφ accumulation occurs despite treatment, suggesting that some of these cells represent transformed resident microglia.     

大鼠脑缺血再灌注区ICAM—1的表达与粘附性变化的实验研究

目的:观察大鼠脑缺血再灌注后缺血区ＩＣＡＭ－１ｍＲＮＡ和蛋白的表达及白细胞和血管内皮细胞间粘附性的变化。方法:４０只Ｗｉｓｔａｒ大鼠分为正常组、假手术组和缺血２ｈ再灌注２、４、１２、２４、４８、９６ｈ组,原位杂交和兔疫组化法检测ＩＣＡＭ、１ ｍＲＮＡ和蛋白表达,超高速摄录像系统观察缺血区微血管内白细胞与内皮细胞间的粘附性变化。结果:缺血再灌注后局部脑组织ＩＣＡＭ－１ｍＲＮＡ和蛋白的表达以及微动脉内白细胞与内皮细胞间粘附性均明显增高。结论:脑缺血再灌注后ＩＣＡＭ－１表达增高,介导了白细胞与血管内皮细胞间的粘附增强,参与了缺血再灌注损伤。     

The neurovascular unit as a selective barrier to polymorphonuclear granulocyte (PMN) infiltration into the brain after ischemic injury

The migration of polymorphonuclear granulocytes (PMN) into the brain parenchyma and release of their abundant proteases are considered the main causes of neuronal cell death and reperfusion injury following ischemia. Yet, therapies targeting PMN egress have been largely ineffective. To address this discrepancy we investigated the temporo-spatial localization of PMNs early after transient ischemia in a murine transient middle cerebral artery occlusion (tMCAO) model and human stroke specimens. Using specific markers that distinguish PMN (Ly6G) from monocytes/macrophages (Ly6C) and that define the cellular and basement membrane boundaries of the neurovascular unit (NVU), histology and confocal microscopy revealed that virtually no PMNs entered the infarcted CNS parenchyma. Regardless of tMCAO duration, PMNs were mainly restricted to luminal surfaces or perivascular spaces of cerebral vessels. Vascular PMN accumulation showed no spatial correlation with increased vessel permeability, enhanced expression of endothelial cell adhesion molecules, platelet aggregation or release of neutrophil extracellular traps. Live cell imaging studies confirmed that oxygen and glucose deprivation followed by reoxygenation fail to induce PMN migration across a brain endothelial monolayer under flow conditions in vitro. The absence of PMN infiltration in infarcted brain tissues was corroborated in 25 human stroke specimens collected at early time points after infarction. Our observations identify the NVU rather than the brain parenchyma as the site of PMN action after CNS ischemia and suggest reappraisal of targets for therapies to reduce reperfusion injury after stroke.     

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。     

尤瑞克林对大鼠局灶性脑缺血再灌注损伤后炎性反应的影响

目的研究尤瑞克林对大鼠局灶性脑缺血再灌注损伤后炎性反应的影响。方法将90只SD大鼠随机分为3组:假手术组,对照组,治疗组。采用线栓法建立大鼠大脑中动脉闭塞再灌注模型,缺血2 h后,拔出线栓,恢复灌注24 h,观察大鼠神经功能缺损症状、脑梗死体积、脑组织中白细胞浸润、MPO活性、IL-1和ICAM-1的表达。结果 (1)假手术组大鼠在神经功能缺损评分、脑梗死体积均低于对照组,有显著的统计学差异(P<0.01);脑组织中白细胞浸润程度、髓过氧化物酶(MPO)活性、ICAM-1和IL-1的表达均较对照组低,统计学差异明显(P<0.01);(2)治疗组与对照组相比,大鼠的神经功能缺损评分低、脑梗死体积小,有显著统计学差异(P<0.01);白细胞浸润程度、MPO活性、ICAM-1和IL-1的表达均较对照组减少(P<0.01)。结论尤瑞克林可通过抑制大鼠脑缺血再灌注损伤后的炎性反应来实现其神经保护作用。     

ICAM-1 Induction in the Mouse CNS Following Irradiation

Injury to the central nervous system (CNS) results in inflammation, increased trafficking of leukocytes into the CNS, induction of cytokines, and exacerbation of the primary injury. The increased trafficking of neutrophils into the CNS has been described following a number of injury models including stab, stroke, and excitotoxin-induced injury. This enhanced trafficking has largely been ascribed to the adhesion molecule intercellular adhesion molecule-1 (ICAM-1, CD54). In the current study, we wished to determine if the inflammation caused by irradiation of the CNS resulted in a similar induction of ICAM-1. C3H/HeJ mice were irradiated using gamma irradiation aimed over the right cerebral hemisphere. The relative induction of ICAM-1 mRNA levels was determined using quantitative RT-PCR 6 hours following irradiation with either 0, 5, 15, 25 or 35 Gy. ICAM-1 message was seen to exhibit a normal dose response curve with increasing mRNA levels seen at 15 Gy and higher. To determine the cellular distribution of the ICAM-1 protein following irradiation, mice were sacrificed at 4 hrs, 24 hrs, 48 hrs and 7 days following 25 Gy irradiation and the tissue was processed for ICAM-1 immunocytochemistry. ICAM-1 staining was seen to increase in both endothelial cells and astrocytes beginning as early as 4 hrs. The staining intensity continued to increase throughout the 7 day period observed. Together, these results suggest that irradiation of the CNS causes a rapid induction of both ICAM-1 mRNA and protein. This suggests that increased leukocyte trafficking into the CNS may exacerbate the inflammation induced by radiation injury.     

脑缺血-再灌注损伤白细胞渗出和L-选择素表达及人工合成E-选择素的干预作用

目的：研究人工合成E-选择素对脑缺血再灌注后白细胞渗出和L-选择素表达的影响。方法：应用线栓法建立局灶性脑缺血再灌注大鼠模型，采用流式细胞术检测脑缺血再灌注后白细胞渗出和L-选择素表达。结果：L-选择素在脑缺血再灌注2h后已经表达，再灌注6h达高峰；在再灌注12h后CD45表达逐渐增加。应用人工合成E-选择素后，L-选择素表达在各时间点均下降（P〈0．05）；在再灌注12.24%1148h时间点CD45的表达明显低于对照组（P〈0．05）。结论：人工合成E-选择素能够明显抑制L-选择素的表达，使白细胞的渗出明显减少，对大鼠脑缺血再灌注损伤起到保护作用。     

阿司匹林对沙土鼠脑缺血-再灌注损伤后细胞间黏附分子及降钙素基因相关肽表达的影响

目的 探讨阿司匹林对沙土鼠全前脑缺血-再灌注损伤后的脑保护作用及其对细胞间黏附分子及降钙素基因相关肽表达的影响。方法 采用夹闭双侧颈总动脉的方法，制备沙土鼠短暂性全前脑缺血-再灌注模型。63只沙土鼠随机分为假手术组、脑缺血-再灌注组（脑缺血组）和阿司匹林干预组（阿司匹林组）。应用免疫组化SABC法检测脑缺血-再灌注后细胞间黏附分子及降钙素基因相关肽表达水平的变化，以及阿司匹林干预对二者表达的影响。结果 （1）细胞间黏附分子表达的变化：脑缺血-再灌注24h，脑缺血组动物脑组织细胞间黏附分子的表达水平开始增加，3d后明显增强，至7d后仍维持在较高水平，与假手术组相比差异有显著性意义（P〈0．05）。而阿司匹林组动物细胞间黏附分子的表达水平在所有观察时限均明显低于脑缺血组，差异有显著性意义（P〈0．05）。（2）降钙素基因相关肽表达的变化：在脑缺血-再灌注后各时限，脑缺血组动物降钙素基因相关肽的表达均呈弱阳性；而阿司匹林组表达则呈强阳性。结论 脑缺血-再灌注可诱导细胞间黏附分子的表达上调，并抑制降钙素基因相关肽的表达。阿司匹林通过抑制细胞间黏附分子的表达水平和增强降钙素基因相关肽表达而获得较好的脑保护作用。     

Intercellular adhesion molecule-1-deficient mice are less susceptible to cerebral ischemia-reperfusion lnjury

Neutrophil emigration is mediated by adhesion proteins that are highly expressed on the endothelial surface during inflammatory processes in the brain. Intercellular adhesion molecule-1 (ICAM-1) is an inducible adhesion molecule that binds to leukocyte integrins and facilitates neutrophil adhesion and transendothelial migration. To study the role of ICAM-1 during ischemia and reperfusion in the brain, we analyzed the effect of transient focal cerebral ischemia in ICAM-1-deficient mice generated by gene targeting in embryonic stem cells. Transient focal ischemia was induced by occluding the left middle cerebral artery for 3 hours followed by a 21- or 45-hour reperfusion period. When compared with their wild-type littermates, ICAM-1-deficient mice were less susceptible to cerebral injury as demonstrated by a 5.6- or 7.8-fold reduction in infarction volume, respectively. These data support the premise that neutrophil adhesion in ischemic areas may be deleterious and that ICAM-1 deficiency reduces neurological damage after transient focal cerebral ischemia.     

Regional and temporal expression patterns of interleukin-10, interleukin-10 receptor and adhesion molecules in the rat spinal cord during chronic relapsing EAE

Adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) mediate leukocyte infiltration into the CNS, in experimental autoimmune encephalomyelitis (EAE) and multiple sclerosis (MS). Because exogenous interleukin-10 (IL-10) inhibits ICAM-1 and VCAM-1 expression and clinical EAE, we hypothesize that endogenous IL-10 signaling may suppress expression of adhesion molecules. In a rat model of chronic relapsing EAE, expression levels of IL-10 and its receptor (IL-10R1), ICAM-1 and VCAM-1 mRNA in the spinal cord are markedly increased, whereas levels of IL-10 mRNA remain relatively low. The temporal pattern of mRNA and protein expression showed marked differences between spinal cord levels. During relapse, IL-10, IL-10R1, ICAM-1, VCAM-1 mRNA levels and neurological scores show positive correlations. We conclude that endogenous IL-10 is not a crucial factor inhibiting adhesion molecule expression in this model.     

Doxycycline treatment reduces ischemic brain damage in transient middle cerebral artery occlusion in the rat

Agents that inhibit leukocyte adhesion including intercellular adhesion molecule-1 antibodies (anti-ICAM-1) have shown beneficial effects in experimental central nervous system (CNS) ischemia. Doxycycline inhibits leukocyte function in vitro by binding divalent cations and reduces spinal cord reperfusion injury. The authors used a clinically relevant model of focal CNS reperfusion injury to test whether treatment with doxycycline would reduce cerebral ischemic damage and improve functional outcome. Reversible middle cerebral artery occlusion was produced in adult Sprague-Dawley rats by advancing a filament into the internal carotid artery for 2 h. Animals received either IP doxycycline (10 mg/kg) (N=13) or saline (N=11) 30 min before ischemia, followed by 10mg/kg every 8 h×6. Both functional assessment (5 point neurologic scale) and infarct volume was evaluated at 48 h. Functional efficacy: doxycycline 0.5±0.2 (mean±SE) vs control 1.3±0.3 (p=0.03). Infarct volume: doxycycline 56±18 mm³ vs control 158±44 mm³ (p=0.03); This protective effect supports the role of doxycycline in reducing CNS reperfusion injury.     

Acupuncture for cerebral ischemia/reperfusion injury Does it reduce the inflammatory reaction?

BACKGROUND:Nuclear factor-κB (NF-κB), intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) in brain tissue can participate in inflammatory reactions after cerebral ischemia.Acupuncture treatment for acute cerebral ischemia produces abnormal protein expression.OBJECTIVE:To investigate the effects of acupuncture on NF-κB, ICAM-1, and VCAM-1 mRNA and protein expression in the brain tissue of rats with cerebral ischemia/reperfusion injury.DESIGN, TIME AND SETTING:Randomized...     

Expression of ICAM-1, VCAM-1, L-selectin, and leukosialin in the mouse central nervous system during the induction and remission stages of experimental allergic encephalomyelitis

Adhesion molecules facilitate infiltration of leukocytes into the central nervous system (CNS) of mice with experimental allergic encephalomyelitis (EAE). Expression of the adhesion molecules ICAM-1 (CD54), VCAM-1 (CD106), L-selectin (CD62L), and leukosialin (CD43) was analyzed via immunocytochemistry 4–28 days after the injection of encephalitogen into EAE-susceptible SWXJ mice. Constitutive ICAM-1 expression on large-diameter CNS vessels was upregulated on post-injection days 8, 11, 14 and 18 (concurrent with de novo expression on smaller capillaries and glial cells), partially downregulated by day 23, and back to control levels by day 28, Constitutive VCAM-1 expression was upregulated by day 14 and back to control levels by day 28. Upregulation of ICAM-1 temporally coincided with the immigration of CD4⁺ lymphocytes and L-selectin⁺ leukocytes into the CNS, while downregulation coincided with their emigration. The infiltration of CDA3⁺ leukocytes also coincided with the upregulation of vascular adhesion molecules, but CDA3⁺ cells remained in the CNS after ICAM-1 and VCAM-1 had returned to control levels. Cellular infiltration and adhesion-molecule expression preceded EAE clinical symptoms by a minimum of 3 days, suggesting a causal role of adhesion molecules in the initiation of CNS inflammation. However, prophylactic injections of monoclonal antibodies against either ICAM-1, L-selectin, or CD43, did not ameliorate the clinical severity of EAE in this model.     

Celecoxib对糖尿病大鼠缺血再灌注脑组织ICAM-1和MMP9表达及神经功能的影响

目的探讨celecoxib(塞来昔布)对糖尿病大鼠脑缺血再灌注后脑组织中ICAM-1和MMP9表达及神经功能的影响。方法将SD大鼠分为假手术组(S组)、脑缺血再灌注生理盐水组(NS组)、celecoxib低剂量组(LCIR组)和高剂量组(HCIR组)。采用腹腔注射链脲佐菌素(STZ)和线栓法制作糖尿病大鼠大脑中动脉缺血再灌注模型。分别于缺血后30min给予生理盐水或celecoxib溶液灌胃,再灌注后6、12、24、48h将大鼠断头取脑,免疫组化法检测脑组织中ICAM-1和MMP9的表达,并对大鼠进行神经功能缺损评分和死亡率的比较。结果 NS组、LCIR组、HCIR组大鼠神经功能缺损评分有显著差异(P<0.05);NS组、LCIR组、HCIR组MMP9及ICAM-1阳性血管主要表达于缺血周边区,较S组表达明显增强(P<0.05),LCIR组、HCIR组MMP9及ICAM-1阳性血管数较NS组减少(P<0.05),LCIR组、HCIR组之间差异不明显(P>0.05),各组不同时间点之间MMP9及ICAM-1阳性血管数均有明显差异(P<0.05)。经Celecoxib干预后大鼠死亡率明显下降。结论 celecoxib可能通过抑制脑组织中MMP9和ICAM-1的表达而减轻糖尿病大鼠脑缺血-再灌注后神经功能的损伤而降低实验大鼠的死亡。