Abstract

BACKGROUND: Transplantation of bone marrow mesenchymal stem cells (MSCs) has been used in the field of repair of nerve injury. Brain stereotactic transplantation and transvascular transplantation are two transplantation methods.
OBJECTIVE: We infused MSCs into rat peripheral cerebral infarct focus, in order to investigate the improvement of rat neurological dysfunction by forelimb use asymmetry test and postural reflex test.
DESIGN: A randomized controlled animal experiment.
SETTING: Department of Neurology, First Hospital of Jilin University.
MATERIALS: This study was performed at the Key Laboratory of Zoonosis, Ministry of Education, Institute of Zoonosis, Jilin University between October 2006 and April 2007. Healthy male Wistar rats of clean grade, weighing 250-280 g, were provided by the Laboratory Animal Center of Jilin University. The protocol was performed in accordance with ethical guidelines for the use and care of animals.
METHODS: MSCs from healthy adult volunteers were in vitro cultured and proliferated by density gradient separation and adherence screening method. Their immunophenotypes were identified by a flow cytometer. The Wistar rats were randomized into 5 groups with 10 rats in each: normal control group, sham-operated group, model group, serum-free DMEM-treated group (DMEM group) and MSCs -treated group (MSCs group). Rat models of cerebral ischemia/reperfusion were developed by occluding rat right middle cerebral artery following suture occlusion method modified by Longa et al. Rats in the normal control group were untouched. In the sham-operated group, operation was not ended till cervical interior and exterior arteries were exposed and sutured, and the other disposals were the same as those in the model group. At ischemia 90 minutes reperfusion 1 hour, a stereotaxic apparatus was used to take rat right peripheral cerebral ischemic region as transplantation site: 3 mm lateral to, 1mm caudal to and 4 mm posterior to Bregma. Rats in the MSCs group were slowly injected 5 μL BrdU-labeled MSCs (4×1011 L-1) serum-free medium. Rats in the DMEM group were injected 5 μL serum-free medium. After perfusion, inserted needle was retained for 5 minutes and then slowly withdrawn in order to avoid the back flow of liquid from needle pole. The survival of MSCs in rats was detected by immunohistochemical technique, and rat behavioral changes of observed on days1, 3, 7 and 28 after transplantation by forelimb use asymmetry test and postural reflex test.
MAIN OUTCOME MEASURES: ① The immunophenotype of MSCs were identified by a flow cytometer. ② The survival of transplanted MSCs in the rat brain. ③ Rat behavioral changes.
RESULTS: All the 50 rats were included in the final analysis. ① High purity of MSCs were harvested in the experiment. Flow cytometer detection showed that both CD44 and CD29 were positive, while CD34, CD45 and CD31 were negative. ② MSCs transplanted into the brains of rats in the MSCs group gathered in the peripheral cerebral ischemic region and survived. ③ Behavioral scores of rats in the MSCs group were significantly lower than those in the other groups (P < 0.05). They were gradually decreased with time after transplantation, and reached the valley value on day 7 after transplantation (P < 0.01).
CONCLUSION: Neurological function of rats recovers in all the groups except normal control group. But the recovery differs in different groups, and neurological function of rats in the MSCs group recovers better than that in other groups.

INTRODUCTION

Bone marrow mesenchymal stem cells (MSCs) are non-hematopoietic stem cells from bone marrow. They possess a strong capacity of self-renewal and reproductive activity as well as multidirectional differentiation potential. Under certain culture condition, they can differentiate into adipocytes, osteoblast, chondrocyte and other cells [1-3] and transdifferentiate into nerve cells from epiblastic ectoderm. This indicates that MSCs may be promising in the treatment of ischemic cerebrovascular disease.

MATERIALS AND METHODS

Materials
This study was carried out in the Key Laboratory of Zoonosis, Ministry of Education, Institute of Zoonosis, Jilin University between October 2006 and April 2007. Fifty healthy male Wistar rats of clean grade, weighing 250-280 g, were provided by the Laboratory Animal Center of Jilin University. The protocol was carried out in accordance with ethical guidelines for the use and care of animals. L-DMEM medium, 10% fetal bovine serum (Hyclone Company, USA), percoll cell separation solution (Pharmacia Company, 1.073 g/mL), phosphate buffer solution (PBS, pH 7.2, 0.01 mol/L), 0.25% trypsinase (Promega Company), rat anti-human CD44, CD29, CD31, CD34 and CD45 antibody working fluid (PE labeled, 1:30,Boster Company, Wuhan, China), monoclonal antibody of mouse anti-BrdU (Sigma Company, USA), SP kit, diaminobenzidine (DAB) kit (Zhongshan BioTech Co., Ltd, Beijing, China), rat stereotaxis instrument (BUNKYO-KV113, Tokyo) and FACScan flow cytometer (Becton Dickinson, USA) were used in this study.
Methods
Isolation, culture, proliferation and purification of MSCs
About 5 mL fresh bone marrow was taken from the os illium of healthy adult volunteers who had no hematological system disease, and it was cultured by percoll density gradient centrifugation as follows. The same volume of L-DMEM medium supplemented with 10% fetal bovine serum was added into the fresh bone marrow for centrifugation. Supernatant fluid was discarded. Medium was added till the total volume was 5 mL. Cells were re-suspended and then slightly stacked on the same volume of percoll cell separation solution. After centrifugation, monocytes were sucked, and inoculated to 24-well plate at （3-4）×106/cm2. Medium was renewed firstly on day 4 and then once every 3 or 4 days. Cell morphology was observed under an inverted microscope. When primary cells reached 80% confluence, they were digested with 0.25% trypsinase, but terminated with culture fluid. Then, they were cultured at 1:3 (primary cell concentration: passage cell concentration). Such cells were the first generation. On day 2 after passage, medium was renewed, and cell debris and remaining trypsinase were cleaned away; The 3rd generation of cells were kept for later test of 5-bromo-2-deoxyuridine (BrdU)-labeled incubation.

Determination of immunological phenotype of bone marrow mesenchymal stem cell surface antigen by flow cytometry
After routinely digested with 0.25% trypsinase, the 3rd generation of MSCs with good growth state were harvested, rinsed twice with PBS (Ph 7.2, 0.01 mol/L) and incubated at 1×1010 L-1 with 5 μL CD44, CD29, CD31, CD34 and CD45 antibody working fluid for 50 minutes at 4 ℃.Subsequently, the cells were re-rinsed twice with PBS, and re-suspended in PBS for later analysis of flow cytometer. In each group, corresponding negative control was made, i.e. PBS replaced primary antibody.

BrdU incubation labeling
The 3rd generation of MSCs were taken and spread on the bottom of culture flask. When adhered to the wall, they were incubated for 48 hours after filtered and sterilized BrdU (final concentration 5 μmol/L) and L-DMEM supplemented with 10% fetal bovine serum being added. Cell density was regulated to 4×1011 L-1 for later directional transplantation.

Grouping, model preparation and processing
The 50 Wistar rats were randomized into 5 groups with 10 rats in each: normal control group, sham-operated group, model group, serum-free DMEM-treated group (DMEM group) and MSCs -treated group (MSCs group). Rat models of cerebral ischemia/reperfusion were developed by occluding rat right middle cerebral artery following suture occlusion method modified by Longa et al [4]. After operation, the residual part of suture was left outside. After ischemia 90 minutes, suture was slightly withdrawn for ischemia/reperfusion 1 hour. Intra- and post-operatively, rat temperature was kept at 37 ℃ until rat recovered. Rats in the normal control group were untouched. In the sham-operated group, operation was not ended till cervical interior and exterior arteries were exposed and sutured, and the other disposals were the same as those in the model group.

Assessment of cerebral infarction models
Behavioral score: When the operated rats are recovered, their neurological functions were scored according to the scoring standard of Longa et al [4].
Morphological assessment: Twenty-four hours after ischemia, normal saline was infused into the hearts of operated rats. Then, their brains were rapidly taken out, and brain tissue from frontal to occipital region was sliced and equally divided into sections A, B, C and D. Subsequently, the sections were incubated in 2% 2, 3, 5-triphenyltetrazolium chlorid (TTC) for 15 minutes at 37 ℃ away from light for determination of infarct focus.

Stereotaxic directional transplantation of MSCs
At ischemia 90 minutes reperfusion 1 hour, a stereotaxic apparatus was used to take rat right peripheral cerebral ischemic region as transplantation site: 3 mm lateral to, 1mm caudal to and 4 mm posterior to Bregma. Parietal bone was drilled with a small dental drill until dura mater. Rats in the bone marrow mesenchymal stem cells group were injected 5 μL BrdU-labeled MSCs (4×1011 L-1) serum-free medium at 1 μL/min. Rats in the DMEM group were injected 5 μL serum-free medium. After perfusion, inserted needle was retained for 5 minutes and then slowly withdrawn in order to avoid the back flow of liquid from needle pole. Postoperatively, immunological rejections were inhibited by intraperitoneal injection of 0.1 mL dexamethasone (5 g/L). Pre- and post-operatively, the rats were raised under the standard condition, and they were free to access food and water for behavioral observation.

Immunohistochemical BrdU staining
With SP staining method, rats in each group were fixed with 4% paraformaldehyde. Their brain tissues were taken, embedded with paraffin, routinely de-waxed, hydrated and incubated with 3% H2O2 for 10 minutes to block the activity of endogenous peroxydase. Afterwards, the brain tissues were enveloped for 15 minutes with 10% normal goat serum, cultured with monoclonal antibody of mouse anti-BrdU (1∶100) at 4 ℃, overnight, with biotin labeled second antibody (goat anti-rat IgG) for 30 minutes at 37 ℃, with horseradish peroxidase-labeled strepto-avidin working fluid for 30 minutes at 37 ℃ and developed with DAB.

Behavioral assessment
The behavioral changes of rats in each group were assessed on days 1, 3, 7 and 28 after bone marrow mesenchyal stem cells were directionally transplanted. Behavioral scores were blindly evaluated by a researcher who received the training but did not know the grouping condition.
Forelimb use asymmetry test [5]: Rats were placed in a transparent glass round bucket with bottom diameter of 15 cm and height of 25 cm. They wound stand to close to bucket wall and observe surrounding environment by alternately and simultaneously touching bucket wall with their two forelimbs. The bilateral forelimb use frequency was the same. While the use frequency of limb on the side of hemiplegia caused by middle cerebral artery occlusion was small. Bilateral forelimb touching wall condition of each rat was observed and recorded 20 times once. Following formula, behavioral score=(R-L)/(R+L+D), forelimb use condition was assessed (R: single use times of right forelimb; L: single use times of left forelimb; D: simultaneous use times of bilateral forelimbs).
Postual reflex test: Neurological impairment was assessed by postural reflex test modified by Bederson et al [6]. Firstly, each rat with ischemia/reperfusion slightly raised its tail 1 m high away from the ground, and normal rat stretched out its bilateral forelimbs to ground direction, while when its middle cerebral artery was occluded, forelimb opposite to injured hemisphere (left side) presented flection posture. Then, each was slightly placed on the plastic plate, and its anterior part of the body as well as forelimbs glided several centimeters. Power of resistance of rats with normal or slightly injured neurological impairment was equal on both sides. Neurological score standard was as follows: normal(0): double forelimbs fully extended, without neurological signs; slight (1 point): left forelimb presented flection posture, right forelimbs extended, without other abnormal signs; severe (2 points): when resisting to side force, left powder of resistance was decreased, forelimbs presented flection posture, without rotating to paralyzed side, and extremely severe (3 points): when resisting to side force, left powder of resistance was decreased, forelimbs presented flection posture and with rotating to paralyzed side. Higher scoring grade indicated severer neurological impairments.

Statistical analysis
Statistical processing was carried out by the fourth author with SPSS10.0 software. All data were expressed as Mean ±SD. One-way ANOVA was carried out. When variable was not belonged to normal distribution, nonparametric rank sum test and Kruskal-Wallis test were used for behavioral scoring. If significant meaning exists, comparison pairwise was conducted.

RESULTS

Quantitative analysis of experimental animals
Fifty rats were included in the final analysis.

Morphology observation of MSCs cultured primarily
After primarily cultured for 24 hours, MSCs adhered to wall with neurite stretching out. With time going, suspended hematopoietic cells gradually became necrotic and broken, and finally were discarded. Adhered cells presented long shuttle-shaped, being single or several cell clones.

Morphology observation of MSCs for serial subcultivation
One week later, adhered cells were enlarged in body volume, presenting spindle-shapedappearance, stretching out bulk prominence. Two weeks later, cells fused in monolayer, clones overlapped, without contact inhibition, spindle-shapedprominence become longer and arranged directionally in whirlpool and radiat. Adhered cells had relatively consistent cell morphology. They could propagate until the 3rd passage with uniform cell morphology, presenting spindle-shapedand clone-like growth (Figure 1).

Detection of immunological phenotype of MSCs
MSCs are cultured by adherent method. 83% of them expressed CD90, and 80% expressed CD71, indicating that most of them are in the undifferentiated status, and the purity of MSCs isolated by adherent method is over 80% [7]. The expression rate of CD45 was 3.53%. 3.51% of CD14 was expressed in the monocytes, macrophages and polymorphonuclear leukocytes. The expression rate of CD34 was 3.71%; 4.67% of HLA-DR was mainly expressed in macrophages. After identification, both CD44 (73.9%) and CD29 were negative, and the positive rate of CD44 was the highest. CD34, CD45 and CD31 (4.37%) were negative (Figure 2).

Behavioral scoring
Behavioral score of rats in each group turned better with time going. Statistical analysis results showed that there was no significant difference of behavioral score between MSCs group and model group and DMEM group (P < 0.05), indicating that after MSCs transplantation, behaviors of rats were markedly improved, and the movements of limbs on the affected side noticeably turned better. No significant difference of behavioral score existed between model group and DMEM group (P > 0.05); There was no significant meaning in behavioral meaning between sham-operation group and normal control group (P > 0.05). In addition, behavioral score of rats in the MSCs group obviously improved on day 7 after operation in comparison with other time points (P < 0.01) (Table 1).

DISCUSSION

Transplantation of MSCs has been used in the field of repair of nerve injury [8,9]. Brain stereotactic transplantation and transvascular transplantation are two transplantation methods [10-12]. In this study, we stereotactically infused MSCs into peripheral infarction focus to save ischemic penumbra. By BrdU immunohistological staining, we found that MSCs transplanted into peripheral infarct focus survived, and rat neurological impairment markedly recovered, indicating that MSCs promote neurological function improvement after cerebral infarction. In the experiment, we did not select infract center as perfusion region because infarct center lacks of blood supply, and necrotic tissue there will destroy the microenvironment, which is for transplanted cell survival and differentiation, and a small number of transplanted cells are needed for direct infusion to target site. In some other studies, scholars infused MSCs into rat internal carotid artery, and they found that MSCs were widely distributed in the ischemic center and penumbral region[13]. However, it is necessary to make sure the open of middle cerebral artery and its branches in intraarterial perfusion, and during which, the risk of embolism will increases and be bound to influence future clinical application. It is also reported that intravenous perfusion of MSCs promotes functional recovery after cerebral ischemia. Generally, rodents have a marked tendency of spontaneous neurological recovery in the 1st week after stroke. Therefore, in order to better reflect the long-time effect of interventional factors of MSCs on the recovery of neurological function, we selected forelimb use asymmetry test, which was suitable for long-time observation, and followed postural reflex test modified by Bederson et al to observe the improvement of neurological functional impairment of cerebral infarction rats undergoing MSCs transplantation. Our experimental results indicate that MSCs transplantation promotes rat neurological function recovery after cerebral ischemia, which is in accordance with previous result. It was also found that neurological function better recovered on day 7 postoperatively in MSCs group than in other groups. It is presumed that MSCs just widely migrate to peripheral ischemic region and take some way, for example, MSCs secrete neuroprotective factors, such as nerve growth factor, brain-derived neurotrophic factor, and so on, or differentiate into neurons in vivo, to establish association between MSCs and peripheral microenvironment and reduce the apoptosis and necrosis of neurons. At present, the fact that 80% of MSCs can be induced into neurons in vitro, but only 3%-10% of them can be transplanted into cerebral ischemic region by no matter what ways puzzle the investigators. Therefore, it is thought that it is extremely difficult for MSCs to completely replace injured nerve tissue and reconstruct after induction and differentiation. Its mechanism needs to be further confirmed.

REFERENCES

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骨髓间充质干细胞定向移植局灶性 脑缺血大鼠的行为学变化*☆○

马学玲1，王心蕊2，江新梅1，陈柏竹○3，李 霞1，刘亢丁1
1吉林大学第一医院神经内科，吉林省长春市 130021；2吉林大学人兽共患病研究所,人兽共患病教育部重点实验室，吉林省长春市 130062；3 Division of Business and Finance, Utah University, 8600 Old Main Hill, Logan Utah, USA
马学玲☆，女，1976年生，黑龙江省哈尔滨市人，汉族，主治医师，吉林大学第一医院在读博士，主要从事干细胞及脑血管病研究。
通讯作者：刘亢丁，教授，主任医师，博士（后），硕士生导师，吉林大学第一医院，吉林省长春市 130021
国家自然科学基金资助项目(30470588) *
摘要
背景：骨髓间充质干细胞移植已经应用到神经损伤修复领域，脑内立体定位移植和经血管移植均为其移植途径。
目的：经立体定位将骨髓间充质干细胞注入大鼠脑梗死灶周边区，通过前肢不对称应用试验及姿势反射试验观察大鼠神经功能障碍的改善情况。
设计：随机对照动物实验。
单位：吉林大学第一医院神经内科。
材料：实验于2006-10/2007-04在吉林大学人兽共患病研究所，人兽共患病教育部重点实验室进行。健康雄性Wistar大鼠由吉林大学实验动物中心提供，体质量250~280 g，清洁级，实验过程中对动物处置符合动物伦理学标准。
方法：采用密度梯度分离法、贴壁筛选法体外培养扩增健康成人志愿者骨髓间充质干细胞，采用流式细胞仪鉴定其免疫表型。将Wistar大鼠随机分为正常对照组、假手术组、模型组、模型+无血清DMEM组、模型+骨髓间充质干细胞组，每组10只。采用Longa等改良线栓法制备大鼠右侧大脑中动脉闭塞脑缺血再灌注模型，正常对照组无任何处理，假手术组手术操作至暴露颈内外动脉后即缝合，其余处理同模型组。骨髓间充质干细胞立体定向移植：缺血90 min再灌注后1 h，采用立体定位仪取大鼠右侧大脑缺血周边区为移植点：前囟旁开3 mm、尾侧1 mm、深4 mm。缓慢注入5 μL经BrdU标记的骨髓间充质干细胞(4×1011 L-1)无血清培养基悬液于模型+骨髓间充质干细胞组大鼠，模型+无血清DMEM组大鼠注射5 μL无血清培养基，注射后留针5 min，缓慢退针，防止液体随针道返流。采用免疫组化技术检测骨髓间充质干细胞在大鼠体内存活情况，并于移植后1，3，7，28 d采用前肢不对称应用试验及姿势反射试验观察大鼠行为学变化。
主要观察指标：①以流式细胞仪鉴定骨髓间充质干细胞免疫表型。②移植大鼠脑内骨髓间充质干细胞的存活情况。③大鼠行为学变化。
结果：50只大鼠全部进入结果分析。①实验获得了高纯度的骨髓间充质干细胞，流式细胞仪检测显示，CD44、CD29均呈阳性，CD34、CD45、CD31均呈阴性。②模型+骨髓间充质干细胞组移植的骨髓间充质干细胞在脑缺血周边区聚集并存活。③模型+骨髓间充质干细胞组大鼠行为学评分较其他各组降低明显 (P < 0.05)，并随着细胞移植后时间延长逐渐降低，并且移植后7 d行为学评分低于同组内其他时间点(P < 0.01)。
结论：除正常对照组外，其他组大鼠神经功能均有所恢复，但各组恢复情况明显不同，脑梗死周边区域注射骨髓间充质干细胞组大鼠功能恢复明显好于各对照组。
关键词：骨髓祖代细胞；干细胞移植；大鼠,Wistar；缺血,脑；行为学
中图分类号: R394.2 文献标识码: A 文章编号: 1673-8225(2008)03-00578-05
马学玲，王心蕊，江新梅，陈柏竹，李霞，刘亢丁.骨髓间充质干细胞定向移植局灶性脑缺血大鼠的行为学变化[J].中国组织工程研究与临床康复，2008，12(3):578-582
[www.zglckf.com/zglckf/ejournal/upfiles/08-3/3k-578 (ps).pdf]
（Edited by Yuan QL/Song LP/Wang L）