短暂性脑缺血发作的实验和临床研究
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摘要
第一部分短暂性脑缺血发作的实验研究
     第一节血小板激活在氧化应激诱发小鼠短暂性脑缺血样发作中的作用和替罗非班的干预作用
     目的:探讨氧化应激在诱发小鼠短暂性脑缺血样发作中的作用机制。
     方法:通过尾静脉注射叔丁基氢过氧化物(t-BHP, 0.11mol/L, 7.5ml/kg)加缺氧(15min后将小鼠置于0.3L密闭容器中10min)诱发昆明小鼠TIA样发作,替罗非班(0.25mg/kg)于造模前20min通过尾静脉注射给药。95只小鼠分为5组,分别为模型组、替罗非班组及对应的TIA样发作前血清可溶性P-选择素测定组各20只,对照组15只。比较小鼠症状出现时间、症状评分和血清可溶性P-选择素的变化。
     结果:小鼠TIA样发作前模型组血清可溶性P-选择素水平显著高于对照组,[(4.17±0.18)与(0.82±0.07 )ng/ml, P<0.05],替罗非班组与模型组比较无显著差异(P>0.05);替罗非班可延迟小鼠症状出现时间([4.70±0.92)与(3.75±1.12)d,P<0.05],降低第四天和第五天症状评分(P<0.05)。
     结论:氧化应激通过激活血小板,进而诱发微血栓的形成导致小鼠TIA样发作;血小板活化后伴随的炎症反应可能也起了一定的作用。
     第二节氧化应激诱发小鼠短暂性脑缺血样发作对血管内皮功能影响及银杏叶提取物的干预作用
     目的:探讨氧化应激诱发小鼠短暂性脑缺血样发作中对血管内皮功能的影响。
     方法:通过尾静脉注射t-BHP(0.11mol/L, 7.5ml/kg)加缺氧(15min后将小鼠置于0.3L密闭容器中10min)诱发昆明小鼠TIA样发作,银杏叶提取物(EGb761,剂量:10 mg/kg、20 mg/kg)于造模前30min腹腔注射。93只昆明小鼠分为7组,分别为模型组、EGb761低、高剂量组各15只,对应的TIA样发作前一氧化氮合酶(NOS)、一氧化氮(NO)、内皮素-1(ET-1)测定组和对照组各12只。比较小鼠症状出现时间、症状评分、血NOS活力和NO、ET-1水平。
     结果:与对照组相比,模型组小鼠TIA样发作前血NO水平[(29.61±3.08)与(43.43±4.24)umol/L, P<0.05]、总NOS活力[(22.27±2.31)与(27.17±3.27)U/ml, P<0.05]降低,ET-1含量升高([85.88±2.58)与(50.07±4.40)ng/ml , P<0.05],而诱导型NOS活力无明显变化(P>0.05);EGb761可升高小鼠血清NO水平和tNOS活力,降低ET-1含量,并降低症状出现率,延迟症状出现时间,降低症状评分(P<0.05),作用呈剂量依从性。
     结论:氧化应激损伤血管内皮功能,造成血管痉挛而诱发小鼠TIA样发作。
     第二部分短暂性脑缺血发作的临床研究
     第一节短暂性脑缺血发作的临床和磁共振弥散加权成像的研究
     目的:研究TIA患者的临床特征及其与磁共振弥散加权成像(DWI)异常改变的关系,探讨TIA新定义的临床应用价值。
     方法:以2006-04~2007-09住院的TIA患者为研究对象,入选患者完成常规MRI、DWI和磁共振成像血管造影(MRA)检查,前瞻性收集其临床资料和检查结果,并分析临床特征与DWI异常的关系。
     结果:84例TIA患者入组,其中40例(47.6%)DWI上有异常改变;22例持续时间≥1h的患者中19例DWI异常(86.4%);DWI异常更多见于持续时间≥1h、有失语、运动缺陷、颅内动脉病变、动脉易损斑块的患者(P<0.05);40例DWI异常的患者中31例常规MRI也发现相关病灶,但7例是经回顾分析后发现。
     结论:持续时间≥1h的TIA患者更易出现脑实质损伤;动脉易损斑块、颅内动脉病变是DWI异常的主要原因;新定义不仅可以明确界定TIA和脑梗死,也有助于TIA的早期评估和治疗;但是对影像学检查的依赖限制了其推广应用。
     第二节磁共振弥散加权成像异常对短暂性脑缺血发作短期预后的影响
     目的:研究TIA患者磁共振弥散加权成像(DWI)异常与短期预后的关系。
     方法:收集2006-04~2007-09间住院的TIA患者临床资料、DWI、MRA检查结果,所有患者完成ABCD评分,并随访90d;分析DWI异常与TIA患者7d和90d内出现卒中的关系。
     结果:TIA后90d内共35例(30.7%)患者出现脑梗死,其中24例(21.1%)发生于7d内,无脑出血发生。多因素Logistic回归分析发现:ABCD评分(OR, 2.75; 95% CI,1.35~5.59; P<0.05)是TIA患者7d内出现卒中的独立预测指标;ABCD评分(OR, 3.19; 95% CI,1.47~6.92; P<0.05)、DWI异常(OR, 9.37; 95% CI,1.42~61.66; P<0.05)、颈部动脉病变(OR, 7.92; 95% CI, 1.22~51.44; P<0.05)是TIA患者90d内出现卒中的独立危险因素。
     结论:有DWI异常的TIA患者短期内卒中发生的风险明显增高;ABCD评分和颈部动脉病变也有助于发现有卒中风险的高危患者。
     第三节伴磁共振弥散加权成像异常的短暂性脑缺血发作与急性脑梗死的临床对照研究
     目的:通过研究伴DWI异常TIA(transient symptoms associated with infarction,TSI)患者的临床和影像学特征,探讨TIA新定义对脑梗死的诊断价值和缺血性脑血管病分类的影响。
     方法:前瞻性收集84例TIA和同期住院45例急性脑梗死患者的一般资料、MRI(包括DWI、MRA)检查结果、住院期间脑血管病复发情况。根据DWI结果把TIA分为TSI和TIA无脑梗死(DWI阴性)患者,然后比较三组患者的临床、影像学特征。
     结果:TSI患者DWI上的病灶体积显著小于脑梗死患者(1.63cm3与10.34 cm3;P<0.05),但是二者的病灶体积之间有相互重叠的区域;脑梗死患者颅内动脉闭塞的发病率最高;住院期间TSI患者脑梗死的发生率显著高于TIA无脑梗死和急性脑梗死患者(27.5%与2.3%、2.2%,P<0.05)。
     结论:TSI可能是独立于TIA无脑梗死和脑梗死之外的脑缺血综合症。
PartⅠExperimental study on transient ischemic attack
     Section 1 Effects of platelet activation and tirofiban on oxidative stress-induced transient cerebral ischemia-like attack in mice
     Objective To explore the mechanism of transient cerebral ischemia-like attack(TIA-like attack) induced by oxidative stress in Kunming mice.
     Methods Mice model of TIA-like attack were established by intravenous injection of tert-butyl hydroperoxide (t-BHP, 0.11mol/L,0.75ml/kg) and hypoxia (the animal was put into a closed container 0.3L for 10 min). Tirofiban (0.25mg/kg) was given through vena caudalis before injection of t-BHP and hypoxia. Ninety-five Kunming mice were divided into 5 groups , including model group, tirofiban group and their corresponding groups for measurement of soluble P-selectin in serum before TIA-like attack (n = 20), control group (n = 15). The onset time of symptom,symptom score and serum P-selectin levels were determined in each group.
     Results Serum soluble level of P-selectin in the model group was significant higher than that in the control group before TIA-like attack [(4.17±0.18) vs (0.82±0.07)ng/ml, P<0.05], but there was no significant difference between the model group and the tirofiban group. The average time of symtom onset in the tirofiban group was later than that in the model group [(4.70±0.92) vs(3.75±1.12)days, P<0.05]. Symptom score was also lower on the fourth and fifth day in the tirofiban group than that in the model group(P<0.05).
     Conclusions Platelet activation and microthrombus were induced by oxidative stress in the mice model of TIA-like attack. Inflammatory response after platelet activation might also contribute to this model.
     Section 2 Effects Of endothelial dysfunction and ginkgo biloba extractin on xidative stress-induced transient cerebral ischemia-like attack in mice
     Objective To investigate the effects of vascular endothelial dysfunction on TIA-like attack induced by oxidative stress in Kunming mice.
     Methods: Mice model of TIA-like attack was established by intravenous injection of t-BHP(0.11mol/L,0.75ml/kg) and hypoxia (the animal was put into a closed container 0.3L for 10 min). Ginkgo biloba extract (EGb761) was given by intraperitoneal injection before injection of t-BHP and hypoxia. Ninety-three Kunming mice were divided into 7 groups, including model group, 10mg/kg and 20mg/kg EGb761 groups (n = 15), their corresponding groups for measurement of NOS (nitricoxide synthase), NO (nitric oxide), ET-1 (endothelin-1) in blood before TIA-like attack and control group (n = 12). The onset time of symptom,symptom score and blood levels of NO, ET-1 and NOS activity (before TIA-like attack) were determined in each group.
     Results Before TIA-like attack, the serum total NOS activity[(22.27±2.31)vs(27.17±3.27)U/ml, P<0.05] and NO level [(29.61±3.08)vs(43.43±4.24)umol/L, P<0.05]were significant lower in the model group than that in the control group, whereas the plasma level of ET-1 [(85.88±2.58)vs(50.07±4.40)ng/ml , P<0.05] was significant higher in the model group than that in the control group. There was no significant difference in serum iNOS (inducible nitric oxide synthase) activity between the model and the control group. EGb761 caused dose-dependent effects on mice model of TIA-like attack, such as increasing the serum level of NO and total NOS activity, decreasing the plasma level of ET-1, reducing the incidence of mice model, lowering the symptom score, delaying the time of symptom onset (P<0.05).
     Conclusions Vascular endothelial dysfunction and cerebral vasospasm induced by oxidative stress played an important role in the mice model of TIA-like attack.
     PartⅡClinical study on transient ischemic attack
     Section 1 Diffusion-Weighted MRI abnormolities and Clinical characteristics in patients with transient ischemic attack
     Objective We assessed the correlation between clinical characteristics and diffusion-weighted MRI (DWI) abnormalities in patients with transient ischemic attack (TIA) to further evaluate the usefulness of new definition of TIA.
     Methods A prospective analysis was performed on all TIA patients who had undergone a MRI scan after symptom onset and entered in the hospital during April 2006~September 2007. The relationship between patients’clinical presentation and DWI abnormalities was then analyzed. Results DWI-detected abnormalities were present in 40 of 84 cases (47.6%). Nineteen of the 22 patients with symptoms lasting equal or more than 1 hour had DWI lesions. Patients with positive DWI scans were more likely to have had symptom duration≥1 hour, aphasia and motor deficits than patients with negative DWI scans. Intracranial large-artery disease and vulnerable plaque were more common in patients with DWI abnormalities. In 31 of 40 cases, a DWI abnormality was present on both DWI and conventional imaging except that 7 patients were identified retrospectively.
     Conclusions TIA patients with symptom duration≥1 hour are more likely to have TIA-related infarcts. Intracranial large-artery disease and vulnerable plaque are the main cause of DWI abnormalities in patients with TIA. The new definition of TIA is not only useful for rapid evaluation and treatment of patients with TIA, but also helpful in diagnosising TIA accurately. On the other hand, its generalization is limited by the reliance on imaging techniques.
     Section 2 Short-term prognosis of TIA patients with diffusion-weighted MRI abnormalities
     Objective To investigate the relationship between the results of diffusion-weighted imaging and short-term prognosis of patients with TIA.
     Methods Clinical data, diffusion-weighted MRI (DWI) and magnetic resonance angiography (MRA) findings were collected in a cohort of hospitalized TIA patients from April 2006 to September 2007. The ABCD score was applied to all patients. Then the relationship between the results of DWI and stroke occurred during the 90-day follow-up was analyzed.
     Results A total of 35 (30.7%) patients had cerebral infarction within 90 days after symptoms onset, while 24(21.1%)patients occurred within 7 days. By multiple logistic regression analysis, ABCD score (odds ratio[OR],2.75; 95% confidence interval [CI],1.35~5.59; P<0.05) was an independent predictor of 7-day risk of stroke. At the same time , ABCD score (OR,3.19; 95%CI,1.47~6.92; P<0.05), DWI abnormalities (OR,9.37;95%CI,1.42~61.66;P < 0.05), and extracranial large-artery disease (OR,7.92;95%CI,1.22~51.44; P<0.05) were each independently associated with 90-day risk of stroke.
     Conclusions TIA patients with DWI abnormalities indicate an increased risk for future stroke. The ABCD score and extracranial large-artery disease were also helpful in detecting TIA patients at higher risk of stroke during the 90-day follow-up.
     Section 3 A comparative study on clinical characteristics of transient ischemic attack with diffusion-weighted MRI abnormalities and ischemic stroke
     Objective To establish similarities and differences among TIA with DWI abnormalities(transient symptoms associated with infarction,TSI),TIA without DWI abnormalities, and ischemic stroke (IS).
     Methods Demographic, Clinical, and in-hospital outcome data were collected in 84 consecutive TIA patients and a contemporaneous group of 45 completed stroke patients. All underwent diffusion-weighted imaging (DWI) and magnetic resonance angiography (MRA) on admission. Intracranial artery disease and infarct volume were determined in each group. Then the clinical and imaging features among them were analyzed.
     Results TIA-related infarcts were smaller than those associated with IS (mean, 1.63 vs 10.34 cm3; P<0.05), but a substantial overlap in infarct size was exist between the two groups. Intracranial large-arterial occlusive disease was more common in IS patients. There were 2 recurrent TIAs and 11 strokes (27.5%) among patients with TSI; 4 recurrent TIAs and 1 strokes (2.3%) among TIA patients without DWI abnormalities; and 1 recurrent stroke (2.2%) and no TIAs among IS patients. Patients with TSI were at significantly higher risk of in-hospital recurrent stroke (P<0.05).
     Conclusions TSI may be a separate clinical entity with unique features separate from TIA without DWI abnormalities and IS.
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    1. Zalba G, Fortu?o A, San JoséG,et al. Oxidative stress, endothelial dysfunction and cerebrovascular disease. Cerebrovasc Dis, 2007; 24(S1):24-29.
    2. Suzuki K, Nakazato K, Kusakabe T, et al. Role of oxidative stress on pathogenesis of hypertensive cerebrovascular lesions. Pathol Int, 2007; 57:133-139.
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