摘要
目的:缺血性脑血管病是危害中老年人的常见病、多发病,具有高发病率、高致残率、高复发率和高死亡率的特点,其导致的肢体瘫痪,认知功能下降成为影响人类生存质量的重要因素。目前缺血性脑血管病尚缺乏理想的方法,迫切要求寻找其他的治疗策略。大脑中动脉阻断(middle cerebral artery obstructaion,MCAO)脑缺血模型是动物实验中最常用的模拟临床常见的大脑中动脉缺血所致的脑梗死的病理模型。近年来骨髓干细胞移植治疗缺血性脑血管病成为研究热点。目前报道的用于骨髓基质细胞(Bone Marrow Stromal Cells, BMSCs)移植的途径主要有三种方式:①经局部病变部位注射途径[1,2,3]。②经血液循环的动[4,5,6]、静[4,7,8,9,10]脉注射途径。③经脑脊液循环的途径[4,11,12,13,14,15]。即经脑室系统以及腰椎穿刺的蛛网膜下腔注射的方式。其中经脑脊液循环移植细胞是一种简便、易行、损伤轻微、费用少的治疗方式,尤其是蛛网膜下腔注射的方式便于临床推广应用。目前,较少文献报道经侧脑室移植BMSCs治疗MCAO大鼠的研究[11,12,13,14,15],发现侧脑室移植的BMSCs能够在MCAO大鼠脑内存活,分化,并迁移至脑缺血周边区,且可以改善其运动功能。但尚未有侧脑室移植BMSCs对MCAO大鼠认知功能的报道。我们采用线栓法制作MCAO缺血再灌注大鼠模型,在脑梗死的急性期经梗死侧侧脑室移植BMSCs,观察MCAO大鼠运动,认知及脑梗死灶大小的变化和植入的BMSCs的迁移路径。
方法:(1)动物:封闭群清洁级健康雄性(Sprague Dawley, SD)大鼠由河北医科大学实验动物中心提供,实验过程中对动物的处置符合动物伦理学标准。(2)实验方法:SD大鼠100只,淘汰有游泳障碍的大鼠,随机取10只归入正常组,其余大鼠制作MCAO缺血2h再灌注模型。麻醉苏醒后,观察评估并记录神经行为学评分,方法参考Longa等[16]的5级4分法标准,评分1~3分视为造模成功。将成功的MCAO模型大鼠随机分为BMSCs组25只,磷酸缓冲液(phosphate buffered solution, PBS)组20只,MCAO组20只。选取BMSCs组大鼠梗死侧侧脑室(坐标:前囟后1.0mm,正中线右侧1.5mm,硬膜下3.8mm)为注射点,注入经5-溴脱氧尿核苷(5-bromodeoxyuridine, BrdU)标记的BMSCs悬液5μl(即每只鼠移植约1.0x106个细胞),PBS组同法于大鼠梗死侧侧脑室注入等量的PBS,MCAO组和正常组不作任何处理。(3)实验评估:MCAO术后(移植前),移植后3、7、14天平衡木实验观察大鼠运动协调能力;移植后1、3、7、14天免疫组化观察BrdU阳性细胞的迁移路径,HE染色观察梗死灶大小的变化;移植后7~11天水迷宫实验观察其游泳速度及空间学习记忆能力。
结果:(1)MCAO大鼠运动功能的改变①平衡木评分:在BMSCs移植前、移植后3天时,3组比较无统计学意义(P>0.05);移植后7天、14天时,BMSCs组平衡木评分较MCAO组和PBS组均有显著改善,差异有统计学意义(P<0.01);在各时间点MCAO组和PBS组比较差异均无统计学意义(P>0.05)。BMSCs组在移植后3天时平衡木评分为(4.92±0.64),运动功能与移植前比就有明显好转(P<0.05),7天时平衡木评分为(3.6±0.52),运动功能有显著改善(P<0.01);而MCAO组和PBS组大鼠运动功能在7天时与移植前相比才有好转。②游泳速度:移植后第7,8天:正常组、MCAO组、BMSCs组大鼠的游泳速度均快于PBS组(P<0.01),正常组、MCAO组、BMSCs组,三组速度比较无统计学意义。移植后第9天:正常组、BMSC组的速度大于PBS组(P<0.01),BMSCs组的速度快于MCAO组(P<0.05),MCAO组的速度快于PBS组(P<0.05)。移植后第10天:正常组、BMSCs组的速度大于PBS组(P<0.01)和MCAO组(P<0.05),比较有统计学意义。PBS组的速度较前有所加快,与MCAO组接近,比较无统计学意义。(2)MCAO大鼠认知功能的改变①寻找平台潜伏期:MCAO组和PBS组与正常组比较大鼠寻找平台潜伏期的时间显著延长,差异有统计学意义(P<0.01);BMSCs组与MCAO组、PBS组相比大鼠寻找平台潜伏期的时间明显缩短,差异有统计学意义(P<0.05);MCAO组和PBS组比较,差异无统计学意义(P>0.05)②空间探索实验:大鼠在原平台象限所占时间百分比(T1%)和路程百分比(D1%)及穿越平台次数MCAO组和PBS组与正常组比较明显减少,差异有统计学意义(P<0.05);BMSCs组与MCAO组、PBS组相比大鼠在原平台象限所占时间百分比和路程百分比及穿越平台次数明显增加,差异有统计学意义(P<0.05);MCAO组和PBS组比较,差异无统计学意义(P>0.05)。空间探索轨迹图:MCAO组和PBS组大鼠为边缘式或随机式寻找,而BMSCs组和正常组大鼠为探索式寻找。(3)HE染色:移植后7、14天,MCAO组、PBS组、BMSCs组大鼠HE染色均可见典型的脑缺血梗死病灶。各组在移植后7、14天比较梗死面积百分比无差异。(4)BrdU染色:移植后1天时,阳性细胞都聚集在移植侧侧脑室,以紧密排列的细胞团形式存在于脑室壁;3天时大部分细胞穿越脑室壁以单个细胞形式向周围缺血区迁移,迁移具有明显的定向性;7天时大部分移植细胞迁移到缺血侧纹状体。14天时移植细胞在梗死的纹状体、皮层可见。
结论:(1)MCAO术后24h侧脑室移植BMSCs,移植后7天时可明显改善MCAO大鼠的运动协调能力。(2)MCAO术后24h侧脑室移植BMSCs可显著改善MCAO大鼠的空间学习记忆能力。(3)MCAO术后24h侧脑室移植BMSCs未能减小梗死灶大小,但侧脑室移植的BMSCs可存活并定向性地迁移至缺血的纹状体和皮层。
Objective: Ischemic cerebrovascular disease is a common and frequently encountered disease, which is hazardous to the aged people, and is characterized by the high rate of incidence, mutilation, recurrence and mortality. The palsy and decreased cognitive function are important impact factors of life quality of patients. At present, there is no ideal therapy for the ischemic cerebrovascular disease. It is urgent to find the other therapy strategy. Middle cerebral artery obstructaion (MCAO)model is the most commonly used as cerebral infarction model, which mimesis cerebral infarction due to middle cerebral artery obstructaion in clinic. In recent years, bone marrow stromal cells (BMSCs) in treatment ischemic cerebrovascular disease has been a hot research. At present, there are three pathways reported of transplanting BMSCs:①Injecting BMSCs into local ischemic place.②Injecting BMSCs into artery or vein.③Transplanting BMSCs into cerebrospinal fluid circulation. That is injecting BMSCs into ventricular system or subarachnoid cavity. Among the ways of transplantation BMSCs, injecting BMSCs into cerebrospinal fluid circulation is a convenient, easy operation, light injury, little expence therapy. Especially, the way of transplantation via subarachnoid space is available in clinic. Up to now, there are few research about transplantation BMSCs to treat MCAO rats via lateral cerebral ventricle. BMSCs transplanted into the lateral cerebral ventricle of MCAO rats could survive and migrate to the cerebral ischemia perimiter zone. In addition, the motor function of the MCAO rats was improve.Up to now, there is no report if BMSCs intracerebroventrcular transplantation could improve the cognitive function. Middle cerebral artery occlusion ischemia reperfusion models were made by inserting a line into the middle cerebral artery, and BMSCs were transplanted into the infarction side lateral cerebral ventricle on the acute stage. The movement, cognitive function, infarction size of the MCAO rats, and the migration way of the transplanted BMSCs were observed.
Methods: (1) Animals: Adult healthy male SD rats of closed group and clean grade were purchased from Laboratory Animal Center of Hebei Medical University. The treatment of animals in the experimental process was in accordance with the criteria of Animal Ethics. (2)Methods: after selecting 100 adult rats by using Morris water maze test, 10 rats were randomly allocated into the normal group. The other rats were made into MCAO 2h reperfusion cerebral ischemia model. After palinesthesia, evaluated the behavior of the MCAO rats and record the score according to Longa’s standard, which are the five grade four score. MCAO rats which evaluated one to three scors were regarded to the successful model rats. The successful MCAO model rats were randomly divided into BMSCs group (n=25), PBS group (n=20), MCAO group (n=20). The suspension of BMSCs(5μl, 1.0x106/μl)labled with BrdU and the same volume of phosphate buffer solution were injected seotaxically into the the infarction side lateral cerebral ventricle (the coordinate: A+1.0mm; R+1.5mm; V-3.8mm) of BMSCs group and PBS group respectively,while no treatment was did in the model group and normal group. (3)Experiment evaluation: the ability of motor coordination of the MCAO rats was observed in pre-transplantation and on the 3rd,7th,14th day after transplantation by the balance beam test. The swimming speed and the ability of space learning and memory were detected from the 7th day to the 11th day after transplantation by morris water maze.The migration way of the BrdU positive cells was detected by immunohisto- chemistry staining and the infarction size was detected by hematoxlinn eosine staining on the 1st,3rd,7th,14th day after transplantation.
Results: (1)The motor function of MCAO model rats:①Balance beam test: There were no difference among BMSC group, PBS group and MCAO group on the 3rd day after transplantation(P>0.05).Compared with PBS group and MCAO group, the score of the balance beam test of BMSCs group,was significantly lower on the 7th, 14th day after transplantation(P<0.01). The score of the balance beam test was no difference between PBS group and MCAO group in every point(P>0.05) .The score of the balance beam test in BMSCs group was(4.92±0.64)on the 3rd day after transplantation, the motor function was obviously improved compared with the pre- transplantation(P<0.05).The score of the balance beam test in BMSCs group was (3.6±0.52) on the 7th day after transplantation, the motor function was significantly improved improved compared with the pre-transplantation (P < 0.01). The motor function of MCAO group and PBS group rats had improved on the 7th day after transplantation compared with the pre- transplantation.②The swimming speed :On the 7th, 8th days after transplantation, the swimming speed in normal group, MCAO group and PBS group were faster than that of PBS group(P<0.01), and there was no difference among normal group, MCAO group and PBS group. On the 9th day after transplantation, the swimming speed in the normal group and BMSCs group were significantly faster than that of PBS group(P<0.01). The swimming speed in the BMSCs group were faster than that of MCAO group(P<0.05). The swimming speed in the MCAO group were faster than that of PBS group(P < 0.05).On the 10th day after transplantation, the swimming speed in normal group and BMSCs group were faster than that of MCAO group and PBS group. there was no difference between MCAO group and PBS group. (2)The cognitive function of MCAO model rats①The escape latency: Compared with normal group, the escape lantency in MCAO group and PBS group were significantly increased (P<0.05). Compared with MCAO group and PBS group, the escape lantency in BMSCs group was significantly decreased (P<0.05).There was no difference between MCAO group and PBS group.②Spatial probe test: The percentage of swimming time(T1%), swimming distance (D1%)in the plantform quadrant and the crossing times in BMSCs group were significantly increased compared with the MCAO group and PBS group(P<0.05).There were no difference of all indexes between MCAO group and PBS group(P>0.05).The spatial probe locus of each group: the way of looking for platform in MCAO group rats and PBS group rats were random,while in BMSCs group and normal group were telic. (3)The results of Hematoxylin eosine staining: Typical pathomorph of cerebral infarction was found on 7th and 14th day after transplantation in MCAO, PBS and BMSCs group rats. The infarction size was no differcnce among the groups on 7th and 14th day after transplantation. (4)The results of BrdU staining: On the 1st day after transplantation, the positive cells were gathered in the transplanted lateral cerebral ventricle, resided in the wall of the cerebral ventricle in the style of tigh disposed cell clump. On the 3rd day after transplantation, the most positive cells passed through the cerebral ventricle parietes directily migrated to the ischemic zone in the form of single cell. On the 7th day after transplantation, the most positive cells migrated to the striatum of the ischemia hemisphere. On the 14th day after transplantation, the most positive cells were found in the striatum and cortex of the ischemia hemisphere.
Conclusion: (1) On the 7th day, the motor coordination ability of MCAO rats was obviously improved via BMSCs intracerebroventrcular transplant- tation 24h after MCAO operation. (2)The function of space learning and memory of MCAO rats were obviously improved via BMSCs intracerebro- ventrcular transplantation 24h after MCAO operation. (3)The infarction size of MCAO rats was not diminished via BMSCs intracerebroventrcular transplantation 24h after MCAO operation. BMSCs were transplatation into later cerebal ventricle could survive and migrate to the striatum and cortical area of cerebral ischemia zone directily.
引文
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