【作者】 邢宏义；

【导师】 孙圣刚； 梅元武；

【作者基本信息】 华中科技大学 ， 神经病学， 2006， 博士

【摘要】 目的脑缺血后神经可塑性与修复是近年来研究的热点，选择怎样的动物模型及脑保护剂是其研究的基础。本研究介绍一种标准的小鼠局灶性脑缺血／再灌注模型的制作，并观察脑梗死体积和脑水肿的变化。研究罗伐他汀(rosuvastatin)、氨甲酰促红细胞生成素(CEPO)、促红细胞生成素(EPO)对缺血性脑损伤的保护作用及机制。探讨多药耐药蛋白(P糖蛋白，P-glycoprotein,P-gp)对FK506通过血脑屏障(BBB)进入脑内浓度及其脑保护功能的影响。应用三级康复方案对急性脑卒中患者生理功能、生存质量的影响及卫生经济学评价，为脑血管病的康复制定最佳策略。方法用腔内线栓法制作脑缺血／再灌注动物模型(MCAO)；用TFC染色法进行脑大体观察；用甲酚紫染色法观察脑切片梗死灶；用脑血流激光多普勒监测脑缺血过程中脑血流的变化；用ImageJ软件计算脑梗死体积和脑水肿；用蛋白免疫印迹法分析脑缺血前后内皮型一氧化氮合酶(eNOS)和活化型caspase-3(activated caspase-3)的变化；用免疫组织化学方法观察脑缺血再灌注后诱导型一氧化氮合酶(iNOS)的改变。用免疫组织化学方法和western blot观察P-gp的表达；用ELISA法检测血和脑组织FK506浓度；用TUNEL法观察凋亡细胞。42例脑卒中偏瘫患者被随机分成康复组和对照组。两组患者急性期(21天内)均进行早期康复治疗。恢复期康复组于康复机构康复治疗2个月，再到社区或家庭康复治疗3个月，对照组自行在家练习。分别采用美国国立卫生院卒中量表(NIHSS)、Fugl-Meyer运动功能评定(Fugl-Meyer assessment，FMA)、改良巴氏指数(modified barthel index，MBI)、SF-36量表来评定疗效。采用成本—效果分析及增量分析进行卫生经济学评价。结果(一)小鼠局灶性脑缺血／再灌注模型的制作及可重复性论证：(1)当线栓封闭大脑中动脉时，脑血流就会急剧下降至最低水平，拔出线栓后脑血流迅速上升至缺血前水平。(2)脑缺血后，脑片上呈现明显的梗死灶，脑梗死体积和脑水肿的大小较恒定。(3)脑缺血30 min再灌注24 h组梗死体积、脑水肿体积和脑水肿百分数分别为81.3±5.6 mm~3、61.6±3.5 mm~3、38.5±2.3％，显著小于脑缺血90 min再灌注24 h组(分别为105.2±8.9 mm~3、79.3±6.4 mm~3、49.6±3.9％)(P＜0.001)。(4)脑缺血30 min再灌注24 h和72 h脑水肿非常明显，分别为61.6±3.5 mm~3、72.6±4.3 mm~3(P＜0.001)，再灌注7天时脑水肿开始减退，仅为50.9±4.1 mm~3，再灌注30天时脑容积出现萎缩，脑水肿呈负值(-20.1±1.8 mm~3)。(二)罗伐他汀对缺血性脑损伤的保护作用及机制：(1)rosuvastatin 0.5 mg／kg、rosuvastatin 5 mg／kg组与对照组的梗死体积、脑水肿的差异无显著性意义，但rosuvastatin 20 mg／kg可以明显减少梗死体积，减轻脑水肿。(2) western blots显示脑缺血前脑组织eNOS表达为100±43.3％，缺血后明显升高至1668.9±112.2％(P＜0.001)，rosuvastatin可以使非缺血区脑组织eNOS表达从100±43.3％上调至511.4±68.7％(P＜0.001)，但rosuvastatin组缺血区脑组织eNOS表达为1678.8±121.3％，与对照组缺血区脑组织eNOS表达(1668.9±112.2％)比较差异无显著性意义(P＞0.05)。(3)非缺血区皮层无activated caspase-3表达，脑缺血后activated caspase-3表达上升至88.3±15.6％，rosuvastatin可使之显著降至42.1±11.2％(P＜0.01)。(4)免疫组织化学显示非缺血脑组织无iNOS阳性细胞表达，脑缺血再灌注后iNOS表达增至2.6±1.8cells／sq．，rosuvastatin 20mg／kg可明显减少iNOS的表达(0.6±0.3 cells／sq．)(P＜0.05)。(三)抑制P-糖蛋白可加强FK506对缺血性脑损伤的保护作用：(1) MCAO 30min再灌注3小时大脑皮质和纹状体中P-gp的表达即开始显著升高，持续至24小时，分别升高55.3％和67.9％(P＜0.05)；但再灌注72小时P-gp表达已不再升高，MCAO90分钟组P-gp的表达升高幅度较MCAO 30分钟组大。提示脑缺血后P-gp的表达在早期一过性升高。(2)脑缺血前后，P-gp抑制剂tariquidar(TQD)对血中FK506浓度的影响无显著性差异(P＞0.05)；使用TQD后，非缺血侧大脑半球FK506浓度无显著性变化，但在缺血侧大脑半球FK506浓度显著升高，脑与血的浓度比率平均从6.3上升至42.2(P＜0.001)，提示脑缺血后P-gp表达过多，阻止了FK506进入脑组织，当TQD抑制P-gp的活性后，FK506进入脑组织的量迅猛上升。(3) FK506在较低剂量(1mg／kg和3 mg／kg)无抗细胞凋亡作用，只有较大剂量(5 mg／kg)才有抗细胞凋亡作用，凋亡的细胞数从对照组的81.2±12.3下降至46.5±9.2(TUNEL(+)细胞数／square)(P＜0.001)。单独使用TQD无抗细胞凋亡作用，当TQD与FK506合用时，FK506较低剂量(1 mg／kg)即可发挥抗细胞凋亡作用。(4)单独使用TQD或FK506 3mg／kg无显著减少脑梗死体积的作用，当FK506 3mg／kg与TQD合用时，脑梗死体积从113.5±11.1 mm~3显著降低至70.6±10.2 mm~3(P＜0.01)，而FK506 5mg／kg可使脑梗死体积降低至85.2±10.1 mm~3(P＜0.01)。提示TQD抑制P-gp活性，使FK506对缺血性脑保护作用的阈值从5 mg／kg降低至1 mg／kg。(四)氨甲酰促红细胞生成素对缺血性脑损伤的保护作用：(1) CEPO 50μg／kg与较高剂量EPO(50μg／kg)具有相同的减少神经功能缺损评分作用，而低剂量EPO(5μg／kg)时无此作用；(2) NS组、EPO 5μg／kg组、EPO 50μg／kg组、CEPO 50μg／kg组的脑梗死体积分别为103.2±10.1、98.7±11.2、66.9±8.3、67.1±8.5 mm~3，脑水肿体积分别为78.9±6.8、76.8±7.3、52.2±5.4、53.1±5.6 mm~3；(3) EPO 50μg／kg使大脑皮层细胞凋亡数从对照组的94.2±15.2／sq．下降至40.5±9.8／sq．(P＜0.001)，CEPO 50μg／kg的抗细胞凋亡作用与EPO 50μg／kg相似；(4) CEPO和EPO脑缺血后大脑皮层eNOS无明显影响，但CEPO 50μg／kg可使activated caspase-3表达从95.4±16.7％明显下降至43.5±13.1％(P＜0.001)，EPO 50μg／kg也有类似作用，而EPO 5μg／kg无此作用；(5)CEPO 50μg／kg使缺血皮层iNOS表达从NS组的3.1±1.9 cells／sq．下降至0.7±0.2cells／sq．，(P＜0.05)，EPO 50μg／kg组的表达与CEPO 50μg／kg组相似，为0.8±0.2／sq．。(五)急性脑卒中患者应用三级康复程序的效果和卫生经济学评价：(1)脑卒中急性期(21天内)，康复组与对照组患者NIHSS、FMA及MBI的改善程度，差异无显著性意义(P＞0.05)；而恢复期(21 d后～6个月)的各个阶段，康复组明显好于对照组(P＜0.01)。(2)在康复后6个月及2年随访时，康复组的生存质量各个维度明显改善，与对照组相比差异有显著性意义(P＜0.05)；(3)康复组患者神经功能缺损积分每减少1分、运动功能及日常生活活动能力每提高1分需分别花费人民币2412.5元、442元和332.1元，而对照组则分别需花费3285.4元、637.8元和447.5元。结论(1)该小鼠局灶性脑缺血／再灌注模型具有重复性好、容易操作。(2) rosuvastatin通过增加eNOS的表达、抑制iNOS和activated caspase-3的表达而起到神经保护作用。(3) TQD抑制P-gp活性，增加FK506进入脑内的浓度，降低FK506脑保护作用的阈值，从而加强FK506的神经保护作用。(4)低剂量的EPO(5μg／kg)无脑保护作用。CEPO 50μg／kg与较高剂量EPO(50μg／kg)具有相似的减少梗死体积、缩小脑水肿、抗细胞凋亡作用，它们通过减少activated caspase-3和iNOS的表达而发挥神经保护作用。(5)三级康复方案对脑卒中患者功能恢复及生存质量具有良好的促进作用，而且更为经济。更多还原

【Abstract】 ObjectiveNowadays neural plasticity and repair after cerebral ischemia is a hot issue in neuroscience. Nonetheless, what kind of animal model of stroke and neuroprotective agents are critical questions. This study would like to introduce a reproducible focal cerebral ischemia/ reperfusion animal model in mice. To study the protection and their mechanism of rosuvastatin, erythropoietin(EPO) ,carbamylated erythropoietin (CEPO) on ischemic brain injury. To observe the effect of multidrug resistance protein (P-glycoprotein,P-gp) on the concentration in brain and neuroprotection of FK506. To investigate the effects and health economic evaluation of rehabilitation procedure on patients with stroke.MethodsFocal cerebral ischemia/reperfusion was induced by occlusion of the middle cerebral artery(MCA) using the intraluminal filament technique. The whole mice brain were stained with TTC, and the slices of brain tissue stained with cresyl-violet. The cerebral blood flow were monitored with laser-Doppler flow metry(LDF). The cerebral volume of infarction and edema were quantified with ImageJ software. The expression of endothelial NO synthase(eNOS) and activated caspase-3 were detected with western blot. The inducible NO synthase(iNOS) positive cells were observed with immunohistochemistry. Theexpression of P-gp was detected with immunohistochemistry and western blot. The concentration of FK506 in blood and brain tissue were determinated with ELISA. The apoptotic cell was detected with TUNEL staining. 42 patients were randomly divided into two groups, rehabilitation group and control group. Both groups were given early rehabilitation during the first 21 days following the stroke. Then, the rehabilitation group went on the same rehabilitation in the rehabilitation center for 2 months ,and then go to community medical service or go home with rehabilitation for 3 months. Whereas the control group were given self-training at home. Outcome measures were National Institute of Health Stroke Scale ( NIHSS), Fugl-Mayer Assessment(FMA), Modified Barthel Index(MBI) and the Medical Outcomes Study 36-Item Short-Form Health Survey (SF-36). The health economic evaluation was made by using cost-effectiveness analysis and increment analysis.Results1. A reproducible focal cerebral ischemia/ reperfusion animal model in mice: (1) The cerebral blood flow droped sharply after MCAO, and jumped rapidly after reperfusion. (2) After cerebral ischemia, the infarct area was very clear, and the volume of infarction and edema were stable. (3) In group ischemia 30 min / reperfusion 24h, the volume of infarction and edema were significantly smaller than those in group ischemia 90 min / reperfusion 24 h (P<0.001) . (4)After cerebral ischemia 30 min / reperfusion 24 h and reperfusion 72 h, remarkable edema appeared (61.6±3.5 mm~3,72.6±4.3 mm~3 respectively) (P<0.001) , whereas the volume of edema was only 50.9±4.1 mm~3 after reperfusion 7 d. However, after reperfusion 30 d, the volume of whole brain shrinked, and the volume of edema came to negative value (-20.1±1.8 mm~3) 2. The protection and its mechanism of rosuvastatin on ischemic brain injury :(1) rosuvastatin 20 mg/kg could remarkably decrease infarct volume and cerebral edemaafter MCAO 90 min/reperfusion 24 h. (2) Western blots showed the expression of eNOS incerebral cortex before and after ischemia were 100±43.3%, 1668.9±112.2% respectively( P<0.001 ) , rosuvastatin significantly up-regulated the expression of eNOS innon-ischemic cortex from 100±43.3% to 511.4±68.7% (P<0.001), whereas in ischemic cortex rosuvastatin did not up-regulated the expression of eNOS (rosuvastatin vs NS: 1678.8±121.3% vs 1668.9±112.2%) (P>0.05). (3) There was no expression of activated caspase-3 in non-ischemic cortex, nonetheless the expression of activated caspase-3 gained 88.3±15.6% after ischemia, rosuvastatin significantly diminished it to 42.1±11.2% (P<0.01) (4) Immunohistochemistry revealed no iNOS positive cells in non-ischemic brain area, while in ischemic brain area the number of iNOS positive cells go up 2.6±1.8 cells/sq., rosuvastatin 20mg/kg reduced them to 0.6±0.3 cells/sq (P<0.05).3. Potentiating the neuroprotection of FK506 on ischemic brain injury by inhibiting P-glycoprotein: (1) The expression of P-gp in cortex and striatum started to rise after MCAO 30 min reperfusion 3 h. Until reperfusion 24 h, the expression of P-gp in cortex and striatum increased 55.3% and 67.9% respectively (P<0.05); However, after reperfusion 72 h, the expression of P-gp went down to control level. It revealed the expression of P-gp transiently grew at the early stage of cerebral ischemia. (2) The inhibitor of P-gp(tariquidar,TQD) had no significant effect on the concentration of FK506 in blood and brain tissue before or after MCAO (P>0.05); After application of TQD, the level of FK506 in non-ischemic hemispheres had no markedly change following MCAO. While the level of FK506 in ischemic hemispheres rose significantly, the average of brain-to-blood-concentration ratio jumped from 6.3 to 42.2 (P<0.001) . It indicated that the expression of P-gp upregulated following cerebral ischemia, which prevented FK506 from entering brain tissue. TQD, which suppressed P-gp, increased the level of FK506 in brain tissue following cerebral ischemia. (3) The lower doses of FK506 (1 mg/kg 和 3 mg/kg) had no anti-apoptosis after MCAO, whereas a higher dose of FK506 (5 mg/kg) had anti-apoptosis, from 81.2+12.3 TUNEL(+)cells /square to 46.5±9.2 TUNEL(+)cells /square (P<0.001) TQD had no anti-apoptosis after MCAO. Fortunately, FK506 1 mg/kg had anti-apoptosis when TQD and FK506 were applied together following MCAO. (4) TQD or FK506 3mg/kg could not reduce the infarct volume following MCAO 90 min reperfusion 24 h, while infarct volume significantly fell from 113.5±11.1 mm~3 to 70.6±10.2mm3 when TQD and FK506 were applied together (P<0.01). It noted that TQD (the inhibitor of P-gp) could lower the neuroprotective threshold of FK506 from 5 mg/kg to 1 mg/kg.4. The protection and its mechanism of carbamylated erythropoietin on ischemic brain injury : (1) In terms of decreasing of neurological deficit scores , CEPO 50μg/kg and EPO(50μg/kg)had equal function except for EPO(5μg/kg); (2) In group NS EPO 5μg/kg EPO 50μg/kg CEPO 50μg/kg, the infarct volume was 103.2±10.1 98.7±11.2, 66.9±8.3 67.1±8.5 mm~3, respectively. The edema volume was 78.9±6.8 76.8±7.3 52.2±5.4 53.1±5.6 mm~3 , respectively ; (3) The apoptotic cells in ischemic cortex fell from 94.2±15.2/sq. in group NS to 40.5±9.8/sq. in group CEPO 50μg/kg (P<0.001), the anti-apoptosis of EPO 50μg/kg was similar to CEPO 50μg/kg; (4) The influence of CEPO and EPO on eNOS in ischemic cortex were not significantly different. However, the expression of activated caspase-3 markedly dropped from 95.4±16.7% in group NS to 43.5±13.1% in group CEPO 50μg/kg (P<0.001) , and 45.1±11.2% in group EPO 50μg/kg, instead of group EPO 5μg/kg; (5) Immunohistochemistry revealed iNOS positive cells in ischemic cortex was 3.1±1.9 cells/sq., CEPO and EPO remarkably reduced them to 0.7±0.2/sq. and 0.8±0.2/sq.,respectively. (P<0.05).5. Effects and health economic evaluation of three-stage rehabilitation procedure on patients with acute stroke: (1) At acute stage of stroke (within 21 d), the scores of NIHSS, FMA and MBI in two groups were not significantly different (P>0.05). However, the patients in the rehabilitation group significantly got much better scores of NIHSS, FMA and MBI than the control group in recovery stage (21 d later -6 months) after the onset of stroke (P<0.01). (2) The patients in the rehabilitation group got much better scores in all 8 dimensions of SF-36 than the control group after 6 months and 2 years following the onset of stroke, the difference was statistically significant (P<0.05). (3) When the NIHSS scores were reduced one point, FMA and MBI scores were improved one point, the cost in the rehabilitation group were 2412.5, 442.0, 332.1 yuan RMB, respectively; Whereas in the control group those were 3285.4, 637.8, 447.5 yuan RMB, respectively.Conclusion(1) This kind of cerebral ischemia/ reperfusion animal model in mice is reproducible and easy to operate. (2) The mechanisms of rosuvastatin’s neural protection on ischemic brain injury are to enhance expression of eNOS, to inhibit expression of iNOS and activated caspase-3. (3) TQD, which is the inhibitor of P-gp, potentiates the neuroprotection of FK506 by means of increasing the level of FK506 and lowering the neuroprotective threshold of FK506. (4) Lower dose EPO (5μg/kg) has no brain protection. CEPO 50μg/kg and EPO 50μg/kg have equal role of decreasing infarct volume, diminishing brain edema, anti-apoptosis by means of inhibiting expression of activated caspase-3 and iNOS. (5) Three-stage rehabilitation scheme can obviously improve patients’ general functional abilities, decrease the disability and increase patients’ quality of life, and is more economical.更多还原