Effects Of BMSCs Intracerebroventrcular Transplantation On The Cerebral Ischemic Reperfusion Rats

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.