【作者】 尹勇；

【导师】 冯忠堂；

【作者基本信息】 昆明医科大学 ， 外科学， 2013， 博士

【摘要】 目的运动再学习疗法(Motor relearning programme for stroke, MRP)为现代先进康复医学治疗新技术,已经成为治疗缺血性脑血管病所导致各种神经功能障碍的一项重要功能性训练方法,具有较好的脑保护作用,但是其分子生物学机制尚不十分清楚,推测可能与脑缺血后受损脑组织的神经再生(neurogenesis)和/或血管新生(angiogenesis, AG)存在一定的关联性。如果运动再学习疗法促使脑缺血所致受损神经功能的恢复确与受损脑组织神经再生和/或血管新生相关,那么使用运动再学习疗法对脑缺血区周围神经再生和/或血管新生的影响,是以促进神经生长发育、损伤修复再生为主,还是以促进血管新生为主,或者是以共同促进神经再生和血管新生来促使受损神经功能恢复呢?为进一步证实这个假设,本实验在以往研究的基础上选择非人灵长类恒河猴为研究系统,通过成功制备建立恒河猴脑缺血动物模型,应用运动再学习疗法训练实验动物,观察运动再学习疗法这种功能训练技术对脑缺血两侧脑组织会产生什么影响?对于脑缺血受损脑组织中神经元和星形胶质细胞又会有何影响?能否引起受损神经元和胶质细胞分泌一些神经营养因子来实现受损神经组织生长发育、修复或者再生?对缺血损伤区或者周围缺血半暗带区的血管和血流量又有什么影响?能否促进受损脑区新生血管的形成和相应促血管生成的因子分泌和生长?我们选用在遗传和生理上较啮齿类或其他动物,更接近人类的非人灵长类恒河猴作为实验对象,通过电凝法阻断恒河猴右侧大脑中动脉M1段制备建立猴脑缺血损伤模型,使用运动再学习疗法(MRP)训练造模成功实验恒河猴60天,采用免疫组织化学的方法,通过定量形态学分析受损脑部相关脑组织区域能够较好地表达神经元功能状况、提示神经元细胞生长状态内在标记的神经丝蛋白(neurofilament protein, NF)与作为全脑所有成熟和反应性星形胶质细胞(astrocyte, Ast)的免疫组化特征性标记的胶质纤维酸性蛋白(glial fibrillary acidic protein, GFAP)(这两个因子的含量和表达水平可以反映受损脑组织神经损伤修复、再生状况)以及能够较好地促进血管形成的血管内皮细胞生长因子(vascular endothelial growth factor, VEGF)和启动血管新生的碱性成纤维细胞生长因子(basic fibroblast growth factor, bFGF)(这两个因子的含量和表达水平可以反映受损脑组织血管新生状况)的表达变化；通过单光子发射计算机体层摄影(single photon emission computed tomography, SPECT)技术半定量检测训练前后实验恒河猴相关脑区脑血流量的改变；使用对判定脑损伤猴行为学较为常用,信度和效度较高的“卒中临床等级评定量表(stroke clinical rating scale, SCRS)"评定训练前后实验动物自身神经功能的变化,探索运动再学习疗法这种康复治疗技术是否具有良好的促进神经再生、血管新生的趋势,尤其想了解运动再学习疗法对脑缺血受损脑组织脑可塑性和功能重建的影响以及与神经再生和血管新生的关系?试图阐明运动再学习疗法促进脑缺血损伤神经功能恢复的机制,为临床广泛运用该治疗技术提供理论依据。方法第一部分：实验设计将9只雄性成年恒河猴随机等分为3组,每组3只：训练组(大脑中动脉缺血模型+运动再学习疗法训练)、自然恢复组(大脑中动脉缺血模型+未进行运动再学习疗法训练)和假手术组(仅暴露大脑中动脉,没有阻断大脑中动脉,未进行运动再学习疗法训练)进行随机对照试验(randomized controlled trial, RCT)。采用电凝阻断恒河猴右侧大脑中动脉M1段方法(与Marahall等改良的闭塞单一大血管法一致)制备建立恒河猴脑缺血损伤模型,但在实施手术造模过程中,发现训练组、自然恢复组和假手术组三组均有实验猴死亡,整个实验动物建模过程完成后9只恒河猴中仅有3只猴造模成功,5只猴死亡,1只猴存活但造模未成功,由于实验恒河猴价格昂贵成本较高,无法进一步补充实验动物数量,所以不能实现原来实验设计的实验组、自然恢复组和假手术组三组分组,只能把仅有的3只造模成功实验动物随机设为训练组。3只造模成功恒河猴残疾程度高度一致,均表现为左侧中枢性面舌瘫、左侧肢体完全性瘫痪、偏身感觉障碍等症状和体征。最终3只恒河猴进入结果分析。并且,根据实验动物恒河猴的特性,造模成功后第3天起采用促进实验动物的觉醒意识水平、增强肌力、改善平衡协调能力等一系列改良运动再学习疗法的康复治疗综合技术训练实验组3只恒河猴,每天1小时,共60天；同时,于建模前1天,建模后3天,10天,60天分别应用“卒中临床等级量表”对实验动物的意识状态、防御反应、抓握反射、四肢运动、步态、平衡能力等神经功能进行评定,观察运动再学习疗法对脑缺血受损神经功能恢复程度的影响。第二部分：应用94mTC-双半胱乙酯(99mTc-ECD) SPECT灌注成像技术对9只实验恒河猴均进行半定量检测相关脑区脑血流量情况,扫描时间为建模前1天、建模后3天,10天,60天,但由于实验过程中存在实验动物不断死亡和动物建模未成功,无法完成所有9只实验动物的SPECT检测和收集所有实验动物完整的SPECT检测数据,所以只能选择把造模失败已经死亡和即使存活但未能成功建模的实验动物剔除,仅把电凝法阻断恒河猴右侧大脑中动脉M1段制备成功脑缺血损伤模型的的3只恒河猴实验动物所进行的所有SPECT检测数据纳入实验数据进行统计,应用SPECT灌注成像检测仪自带软件开展相关检测指标对照分析,了解运动再学习疗法对脑缺血损伤后各脑区局部脑血流量的影响。第三部分：将电凝法阻断右侧大脑中动脉M1段制备成功脑缺血损伤模型的3只恒河猴实验动物,使用运动再学习疗法(MRP)在动物饲养笼内外进行60天的综合性康复训练,每天1小时。60天时麻醉处死动物,4%多聚甲醛灌注固定后取实验动物脑,收集两侧大脑中动脉供血区中央前回运动区(Precentral Motor Area)脑组织标本,采用实验动物手术侧与非手术侧的自身对照方法,通过免疫组织化学的方法,定量检测分析实验恒河猴两侧脑组织中央前回运动区神经元和胶质细胞中NF、GFAP、VEGF、bFGF阳性细胞数以及这些因子的表达变化,以反映运动再学习疗法对于脑缺血受损脑组织神经神经生长发育、修复再生和／或血管新生的影响。结果第一部分：从实验动物神经功能评定曲线可知,建模前1天,实验猴神经功能未受损；建模后3天,神经功能严重受损,损伤程度达到高峰；建模后10天,受损神经功能逐渐改善；建模后60天,受损神经功能改善明显,但尚未恢复到正常。本实验结果进一步显示运动再学习疗法训练对3只脑缺血损伤恒河猴神经功能障碍改善显著(P<0.01),不仅促进其运动功能的恢复,而且对实验恒河猴的意识状态、自我照顾能力、平衡能力(肢体和面部肌肉协调性)等症状和体征改善明显,但是对于瘫痪侧肢体肌肉力量改善不显著,可能与恒河猴为四肢动物,善于调整自身的平衡协调性以便更好地适应生存需要有关。第二部分：SPECT分析结果显示通过手术造模引起的脑缺血可以导致恒河猴全脑平均局部脑血流(rCBF)的显著性改变(P=0.042),尤其是左侧小脑(P=0.042)、左侧枕叶(P=0.023)、右侧颞叶(P=0.039)、左侧颞叶(P=0.033)、右侧顶叶(P=0.042)、右侧额叶(P=0.018)以及左侧脑半球(P=0.042),局部脑血流发生明显降低。与建模前1天相比,建模后3天恒河猴各脑区rCBF均显著降低；建模后10天,恒河猴各脑区rCBF开始逐步恢复；建模后60天恒河猴各脑区rCBF基本恢复正常,有些脑区rCBF甚至高于建模前,包括右侧小脑、左侧小脑、右侧枕叶、左侧枕叶、左侧颞叶、左侧顶叶、左侧额叶、左侧脑半球以及全脑。分析其主要原因,可能是建模后通过运动再学习疗法训练可以明显地改善损伤恒河猴不同脑区的脑血流量,不仅梗死侧大脑各脑区血流恢复显著,而且双侧大脑中一些脑区脑血流量比建模前还增加,说明运动再学习疗法一方面通过提高双侧脑血流自身调节功能,实现脑血液循环的生理学重组；另一方面,运动再学习疗法也可能通过整体训练实验动物的肌肉力量、平衡、协调、坐位、站立、步行、上肢、头面口腔功能等促进脑血流量的增加。同时,还发现一个值得思索的现象,尽管MRP可以明显改善脑缺血损伤恒河猴两侧的局部脑血流量,但是仍然无法改变脑缺血损伤所导致的右/左脑区脑血流量的不对称性。第三部分：脑缺血恒河猴进行改良运动再学习疗法综合训练60天后,处死动物,收集脑组织标本,大体病理学显示脑缺血猴梗死侧脑组织发生液化坏死变软,脑组织萎缩,形成中风囊。免疫组化定量分析表明,与非手术侧相比,NF、GFAP,VEGF, bFGF阳性细胞主要集中在缺血损伤侧缺血周围区,缺血损伤侧NF、GFAP、VEGF和bFGF含量显著增加,缺血损伤侧大脑皮质中央前回运动区NF.GFAP、 VEGF和bFGF表达显著增强(P<0.001),说明运动再学习疗法能够促进和增加脑缺血受损脑组织神经元和胶质细胞分泌一些因子反映受损脑组织神经生长发育、损伤修复再生和/或者受损区域血管新生的状况,进而揭示该治疗方法具有一定的促进神经再生和血管新生能力的趋势。结论1.运动再学习疗法可以不同程度地改善脑缺血损伤所导致的各种神经功能障碍。2.运动再学习疗法可以明显改善脑缺血损伤周围及相关脑区局部脑血流量。3.运动再学习疗法可以显著增加脑缺血区脑组织中神经元和胶质细胞NF、GFAP、VEGF、bFGF含量,提高这些因子的表达水平。4.研究结果揭示运动再学习疗法具有脑缺血损伤后脑保护作用的趋势。5.从功能学和形态学角度,阐明运动再学习疗法促进脑缺血损伤功能恢复的治疗机制,可能与运动再学习疗法能够同时促进脑缺血损伤周围区脑组织神经生长发育、修复再生、血管新生和改善脑血流量相关。更多还原

【Abstract】 ObjectiveMotor relearning program (MRP), as an advanced modern technology, has become an important method of treatment for ischemic cerebrovascular disease caused by a variety of neurological dysfunction, with better brain protection. However, its molecular mechanism is not yet very clear. Presumably it involves nerve repair, nerve regeneration or angiogenesis. This study plans to assess the effects of MRP therapy on ischemic brain, neurons, astrocytes, blood vessels and blood flow. This study may help reveal the mechanism of MRP therapy, and determine whether MRP therapy promotes nerve repair, nerve regeneration or angiogenesis.In this study, we select the rhesus monkeys as model animals because they are genetically closer to humans than rodents and other animals. The monkeys had an occlusion of the M1segment of the right middle cerebral artery (MCA) by using electrocoagulation. They were then trained with MRP for60days. The expression of NF (neurofilament protein), GFAP (glial fibrillary acidic protein), VEGF (vascular endothelial growth factor), and bFGF (basic fibroblast growth factor) between injured and non-injured areas were detected and comparatively analyzed by immunohistochemical staining. SPECT (single photon emission computed tomography) was used before and after MCA occlusion to semi-quantify the changes of rCBF (regional cerebral blood flow). A stroke clinical rating scale was used before and after MCA occlusion to quantify the results of a clinical neurological examination. In the present study, we hope to explore whether the MRP therapy has promoted nerve regeneration and/or angiogenesis, especially want to learn more about the influences of MRP therapy on ischemic brain. This study attempts to clarify the mechanism of MRP on recovery of brain ischemia.MethodsPart Ⅰ:In this study,9adult male rhesus monkeys were randomly divided into three groups:the training group (middle cerebral artery occlusion model plus MRP therapy), the spontaneous recovery group (middle cerebral artery occlusion, not for MRP training), and the sham operation group (exposed only to the middle cerebral artery, without blocking the middle cerebral artery, not for training). Rhesus monkeys had an occlusion of the M1segment of the right middle cerebral artery (MCA) by using electrocoagulation. However, in the course of the surgery, it was found that there were monkeys died in the training group, the natural recovery group, and the sham group. Eventually, there were only3successful modeling monkeys.5monkeys died in the course of the surgery, and1monkey was not successfully modeled. Due to insufficient funds, the number of experimental animals cannot be further added. Therefore, the original design of experiments cannot be achieved. The remaining3monkeys were put in the training group. These three monkeys reached a considerable measure of agreement in degree of disability, with paralysis of the left facial tongue, complete paralysis of the left limb, and hemidysesthesia. The data from these three monkeys was used in the final analysis. Three days after the surgery, the monkeys were trained to promote the awakening of consciousness, enhance muscle strength, and improve the balance and coordination ability with a series of modified MRP therapy, according to their respective characteristics. Each monkey was trained for60days,1hour per day. On1day before the surgery,3days,10days and60days after the surgery, a stroke clinical rating scale was used to evaluate the state of consciousness, defensive reaction, grasping reflex, limb movement, gait, balance and other neurological functions, which might help to observe the effects of MRP therapy on cerebral ischemia.Part Ⅱ:99mTc-ECD SPECT was performed for the9monkeys to semi-quantify the rCBF. The scan time was set on1day before surgery,3days,10days, and60days after surgery. Because there were monkeys died in the course of surgery, only three successfully modeled monkeys completed the SPECT scans. The data from these three monkeys was used in the final analysis.Part III:Three successfully modeled monkeys were trained with MRP inside and outside the cage. The MRP therapy lasted for60days,1hour per day. In the end, these three monkeys were killed. The brain was fixed with40%paraformaldehyde and the brain tissue from both sides of the central gyrus was obtained. By using immunohistochemical staining, the expression of NF (neurofilament protein), GFAP (glial fibrillary acidic protein), VEGF (vascular endothelial growth factor), and bFGF (basic fibroblast growth factor) were detected and compared between injured areas and non-injured areas to reflect the effects of MRP therapy on nerve repair, regeneration and/or angiogenesis.ResultsPart Ⅰ:1day before surgery, the monkeys" neurological function was not impaired. On3days after surgery, the neurological function was severely damaged. On10days after surgery, the damaged nerve began slowly to recover. On60days after surgery, the neurological function had improved significantly, but had not yet returned to normal. These results showed that MRP therapy could significantly improve the neurological function caused by cerebral ischemia (P<0.01) This therapy not only promoted the recovery of motor function, but also improved the state of consciousness, self-care skills and balance (limb and facial muscle coordination). However, the improvement of paralyzed limb muscle strength was not significant, possibly because rhesus monkeys walked on all four limbs. They were skilled at adjusting their balance to better adapt to environments.Part II:SPECT analysis showed that the surgery can lead to significant decreases in regional cerebral blood flow (rCBF)(P=0.042), especially the left cerebellum (P=0.042), left occipital lobe (P=0.023), right temporal lobe (P=0.039), left temporal lobe (P=0.033), right parietal lobe (P=0.042), right frontal lobe (P=0.018) and the left hemisphere (P=0.042). Compared with preoperation, the rCBF was significantly reduced on3days after surgery. On10days after surgery, the rCBF gradually restored. On60days after surgery, the rCBF returned to normal, and the rCBF of some brain regions was even higher than before modeling, including the right cerebellum, the left cerebellum, right occipital lobe, left occipital lobe, left temporal lobe, left parietal lobe, left frontal lobe, left cerebral hemisphere and the whole brain. Although the MRP therapy can significantly improve the rCBF in rhesus monkeys, it cannot change the asymmetry of brain induced by cerebral ischemia. These results indicated that the MCA occlusion had significantly affected the rCBF, and the MRP therapy can significantly improve the rCBF. Not only the rCBF of injured brain areas was significantly improved, but also the rCBF of some brain areas had even reached a higher level than pre-surgery. This showed that the MRP therapy improved the rCBF to achieve physiological reorganization on the one hand and exercised whole muscles, balance, coordination, sitting, standing, walking, upper limbs, head, face and mouth to further promote an increase in rCBF on the other.The expression of NF, GFAP, VEGF and bFGF in injured areas, especially precentral motor area, was significantly higher than non-injured areas. MRP has greatly improved rCBF of cerebral ischemic stroke monkeys, but cannot change the functional asymmetry of cerebral hemispheres. Moreover, MRP has greatly improved neurological function of cerebral ischemic stroke monkeys, not only promoted the recovery of motor function, but also improved the monkeys’state of consciousness, abilities to care for themselves, and balance (coordination of facial and limb muscles). However, MRP cannot improve the strength of paralysed limb’s muscles, possibly because monkeys walk on all fours.Part III:After60days’MRP training, the monkeys were sacrificed and the brain tissue was obtained. The results of pathologic examination indicated that the brain tissue formed stroke capsule. Immunohistochemical analysis showed that the positive cells of NF, GFAP, VEGF and bFGF were mainly concentrated in the ischemic damage areas. Compared to non-operated side, the expression of NF, GFAP, VEGF and bFGF were significantly increased (P<0.001). It was confirmed that MRP therapy can increase the expression of neurotrophic factor, suggesting this therapy had the ability to promote nerve regeneration and/or angiogenesis.Conclusion1. MRP therapy can improve a variety of neurological dysfunction induced by cerebral ischemia.2. MRP therapy can significantly increase the cerebral blood flow of ischemic injuried areas and related brain areas.3. MRP therapy can significantly increase the expression levels of NF, GFAP, VEGF and bFGF.4. MRP therapy can effectively improve neurological dysfunction, possibly because it promotes neuronal regeneration or angiogenesis in injured areas to have a significant improvement on neurological deficits caused by cerebral ischemia.5. This will provide a theoretical basis for carrying out MRP therapy in patients withcerebral ischemia.更多还原