Abstract
BACKGROUND: Exogenous neural stem cells (NSCs) can repair nerve and promote recovery of neurofunction following cerebral hemorrhage.
OBJECTIVE: To observe the growth and development of NSCs in vitro, to evaluate the survival, migration and differentiation of transplanted NSCs surrounding hematoma and the possible recovery function of NSCs, and to investigate the repairing effect of NSCs on damaged neurofunction in cerebral hemorrhage model rats.
DESIGN: Completely randomized grouping design and controlled animal study.
SETTING: Department of Neurosurgery, Huashan Hospital, Fudan University.
MATERIALS: Eighteen adult healthy male SD rats weighing 280-320 g were provided by Shanghai Animal Center of Chinese Science Academy. BrdU was provided by Neomarkers Company; rat-anti-glial fibrillary acidic protein (GFAP) and rabbit-anti-microtubule-associated protein-2 (MAP-2) by Chemicon Company.
METHODS: This study was performed at the Laboratory of Anatomy and Histology & Embryology, Shanghai Medical College, Fudan University from February to December 2006. The NSCs was isolated, cultured, and evaluated from hippocampus of day E14 fetal SD rats. Eighteen rats were randomly divided into control group, PBS group and NSC transplantation group. Cerebral hemorrhage rat models were established via injection of autologous arterial blood in caudate nucleus. Thirty minutes after model establishment, 5 μL NSC suspension with the concentration of 2×1011 L-1 was transplanted at four points surrounding hematoma cavity in the NSC transplantation group. Transplantation of PBS and NSCs was the same as autoblood transplantation. Thirty minutes after model establishment, injuries at the four points were performed, and nothing was injected in the control group.
MAIN OUTCOME MEASURES: Neurofunction was evaluated with forward limb scale and turning scale just soon after transplantation and at 1, 3, 5, 14, and 28 days after transplantation. Brain was colleted by anesthesia 28 days after model establishment. Differentiation of transplanted NSCs was detected through testing GFAP, MAP-2, and BrdU by using immunohistochemistry.
RESULTS: ① Neurofunction scores: There was no significant difference 5 days after model establishment (P > 0.05). However, the scores were significantly improved in the NSC transplantation group 14-28 days after model establishment (P < 0.05). ② Immunofluorescent double labeling: Apoptosis cells around hemotoma in the NSC transplantation group were less than those in the PBS group. BrdU and MAP-2 or GFAP-positive cells were observed in cerebral tissue sections, and this suggested that NSCs could survive, migrate and differentiate in host brain and differentiate into neurons or astrocytes.
CONCLUSION: NSC Transplantation contributes to the recovery of neurofunction in cerebral hemorrhage rats through differentiation into neurons or astrocytes.

INTRODUCTION

Spontaneous intracerebral hemorrhage (SICH) is a blood clot that arises in the brain parenchyma in the absence of trauma or surgery. This entity accounts for 10% to 20% of all strokes in USA, 21% to 48% in China. It is associated with a mortality rate of 30%-50% and a morbidity rate over 30% one month later after ictus[1]. SICH injury mechanisms include: physical trauma and mass effect[2], cerebral blood flow[3], red blood cell lysis productions[4], thrombin[5-7], inflammation and complement activation[8-10], etc. The aim of surgically evacuating the clot is to decrease intracranial pressure and to get rid of red blood cell lysis production, but definitive evidence favoring surgical intervention is lacking. There are limited methods to cure SICH patients. It is urgently required to find other possible therapeutic method since SICH cause high mortality and morbidity rates. Reynolds et al isolated and cultured neural stem cells (NSCs) for the first time from adult mice straium in 1992, which had the ability of self-renewing and differentiation to neurons, astrocytes and oligodendrocytes. A lot of experiments have proved that the proliferation, migration and differentiation of endogenous NSCs could be induced in some neurological diseases[11]. NSCs transplantation can be an alternative to the traditional medication for treatment of a wide range of pathologies that occurs within the CNS.

MATERIALS AND METHODS

Materials
This study was performed from February 2006 to December 2006. A total of 18 adult male Sprague-Dawley rats weighing about 300 g and of clean grade were provided by Shanghai Animal Center of Chinese Science Academy [certification: SCXK (hu) 2002-0002]. Dulbecco’s modified eagle medium-F12 (DMEM/F12), N2, B27, epidermal growth factor (EGF), basic fibroblast growth factor (b-FGF), and PBS were provided by Gibco Company; Kopf stereotactic frame by David Kopf instruments, Tujunga; CA was used in the study.

Methods
NSCs culture
Procedures for the use of laboratory animals were approved by the Institutional Animal Care and Use Committee of the Shanghai Medical School, Fudan University, Shanghai, China. The hippocampus was isolated from day E14 fetal Sprague-Dawley rats [Shanghai Animal Center of Chinese Science Academy; certification: SCXK (hu) 2002-0002].


The tissues were transferred to cold phosphate buffered saline (PBS), minced, and dissociated. The suspension was transferred to a plastic conical 15-mL tube and centrifuged at 75 ×g for 10 minutes. The pellets were resuspended in Dulbecco’s modified Eagle’s medium-F12 medium supplemented with 1% N2, 2% B27 supplement, 2 mmol/L of glutamine, 20 ng/mL of epidermal growth factor, 20 ng/mL of basic fibroblast growth factor, 100 U/mL of penicillin, and 100 μg/mL of streptomycin. Cultured cells were seeded at a density of 5×105 cells per milliliter in growth medium and cultured at a humidified atmosphere of 5% CO2/95% air for 3 to 7 days. After passaging four generations, neurospheres were dissociated into single cells by incubation in 0.1% trypsin-ethylenediamine tetra-acetic acid at 37 °C for 2 minutes and then centrifugation in 10 mL of Dulbecco’s modified Eagle’s medium-F12 medium containing 4% bovine serum albumin at 110 ×g for 5 minutes. Neurospheres from single cell were transferred to a poly-l-ornithine-coated 96-well dish and cultured in the same medium. After 2 days of culture, neurospheres in NSC culture were differentiated using Dulbecco’s modified Eagle’s medium with 20% fetal bovine serum medium.

Labeling NSCs
NSCs were labeled with β-tublin III and glial fibrillary acidic protein (GFAP). Briefly, the primary antibody is rat anti-β-tublinIII (Chemicon, 1∶400), rat anti-GFAP (Chemicon, 1∶400). The secondary antibodies were goat anti-rat TRITC (Zhongshan Beijing, 1∶50), goat anti-rabbit FITC (Zhongshan Beijing, 1∶50).

Establishment of animal models
Experimental intracerebral hemorrhage (EICH) rats model were performed as described[4]. The rats were anesthetized with pentobarbital (40 mg/kg i.p.). The right femoral artery was catheterized for continuous blood pressure monitoring and blood sampling. The rats were placed in a stereotactic head frame (Kopf Instruments), and a 1-mm cranial burr hole was drilled on the right coronal suture 4 mm lateral to midline. Then, 100 mL autologous whole blood was infused into the right caudate at a rate of 10 mL/min through a 26-gauge needle (coordinates: 0.2 mm anterior, 5.5 mm ventral, and 3.5 mm lateral to the bregma). Body temperature was maintained at 37.5 °C with a feedback-controlled heating pad.
The transplanting point was A, B, C and D. Point A: 0.2 mm anterior, 5.5 mm ventral, and 3.5 mm lateral to the bregma. Point B: 0.2 mm anterior, 2.5 mm ventral, and 3.5 mm lateral to the bregma. Point C: 0.2 mm anterior, 3 mm ventral, and 5.5 mm lateral to the bregma. Point D: 0.2 mm anterior, 8 mm ventral, and 5.5 mm lateral to the bregma.

Grouping and transplantation
Eighteen rats were randomly divided into three groups: group A, group B and group C, with 6 rats in each group. In group A, the rats received a needle insertion without donor transplantation, the rats in group B received 5 μL PBS and group C received 5 μL NSCs (2×108 mL-1, cocultured with BrdU for 24 hours before transplantation) at every point as described before.

Behavioral test
In a blind test of all experimental groups, behavioral tests were performed in all animals just soon after transplantation and at 1, 3, 5, 14, and 28 days after transplantation.
The forelimb placing test and corner turn test scores were used to evaluate the neurological function of each animal according to the method used by Hua et al[12]. The first behavioral test was a vibrissae-elicited forelimb placing test. Animals were held by their torsos, which allowed the forelimb to hang free. The animal was gently moved up and down before the placement of testing to facilitate muscle relaxation and eliminate any struggling movement. Trials during which extreme muscle tension, struggling, or placing of any of the limbs onto the experimenter’s hand occurred were not counted. Independent testing of each forelimb was induced by brushing the respective vibrissae on the corner edge of a countertop. Intact animals place the forelimb ipsilateral to the stimulated vibrissae quickly onto the countertop. Depending on the extent of injury, placing of the forelimb contralateral to the injury in response to contralateral vibrissae contact with countertop may be impaired. In the experiments each rat was tested 10 times for each forelimb, and the percentage of trials in which the rat was placed the appropriate forelimb on the edge of the countertop in response to the vibrissae stimulation was determined. Testers were highly experienced and blind to the condition of the animal.
Corner Turn Test is done as following: The rat was allowed to proceed into a corner, the angle of which was 30 degrees. To exit the corner, the rat could turn either to the left or the right, and this was recorded. This was repeated 10 to 15 times, with at least 30 seconds between trials, and the percentage of right turns was calculated. Only turns involving full rearing along either wall were included (i.e., ventral tucks or horizontal turns were excluded). The rats were not picked up immediately after each turn so that they did not develop an aversion for their prepotent turning response.

Immunohistochemistry
Immunohistochemistry was performed as follows: Briefly, rats were anesthetized with pentobarbital (60 mg/kg, i.p.) 28 days after EICH, followed by intracardiac perfusion with 4% paraformaldehyde in 0.1 mol/L hosphate-buffered saline, pH 7.4. After perfusion, the brains were removed, kept in 4% paraformaldehyde for 6 hours, and then immersed in 25% sucrose for 3 to 4 days at 4 °C. They were then placed in embedding compound and sectioned (20 μm slices) on a cryostat. Sections were incubated with avidinbiotin complex technique. Primary antibodies were rat anti-BrdU (neomarkers, 1∶100), rabbit anti-MAP-2 and rabbit anti-GFAP. Secondary antibodies were goat anti-rabbit TRITC, goat anti-rabbit FITC and goat anti-rat TRITC, goat anti-rat FITC.

Statistical analysis
Statistical analysis was done by Dr. Zhu, who was blind to the condition of the animal. All data in this study were expressed as Mean ± SD. Data were analyzed by ANOVA, using SPSS (11.0). Differences were considered significant at P < 0.05.

RESULTS

NSCs culture
Cultured cells survived and small aggregates of cells appeared approximately 3 days after isolation from the E14 fetal rat hippocampus. These aggregates demonstrated typical neurosphere morphology and continued to expand for several weeks. When the dissociated cells were plated into a 96-well dish, a small number of new neurospheres were generated, suggesting that the neurosphere-forming cells have the capacity of self-renewing. Immunofluorescence staining demonstrated that these cells were nestin, β-tublin III, and GFAP-positive, indicating that these cells were NSCs, which differentiated into neurons or astrocytes (Figure 1).

Magnetic resonance image (MRI) of EICH models
MRI shows the site of hematoma in rat (Figure 2). The dark spot site demonstrated as arrow, and showed the hematoma located at the caudate nucleus of rat cerebrum.

Behavioral tests
There was no difference in physiological parameters, arterial blood pressure, or body temperature during EICH among the different experimental groups (data not presented). Also, no significant differences in scores were detected among the groups soon after transplantation or after 5 days of EICH. Behavioral deficits evaluated by both methods demonstrated progressive recovery during the period from 1 to 28 days after EICH in each group. Interestingly, rats transplanted with NSCs demonstrated a significant improvement in both score from 14 to 28 days compared with the other groups (P < 0.05). There were no differences in both scores between the PBS transplanted group and sham group (Figure 3).

Immunofluorescent double labeling
No tumor was found in any group. TUNEL labeling showed that apoptosis cells around hemotoma in NSC transplantation group were less than those in PBS transplantation group (Figure 4).

BrdU and microtubule-associated protein-2 (MAP-2) or GFAP-positive showed that the transplanted NSCs differentiated to neurons or astrocytes (Figures 5, 6).

Owing to a lack of electrophysiology test data, whether the euron differentiated form transplanted NSCs was functionally useful need to be investigated further.

DISCUSSION

SICH injury mechanisms include: physical trauma and mass effect[2], cerebral blood flow, red blood cell lysis productions, thrombin, inflammation and complement activation etc[2-10]. Although large hematoma can be evacuated, the little one remain a big problem, and some old patients give up surgery due to systematic problems. It is necessary to find new methods to cure SICH.
NSCs were first defined by Anderson in 1989. Mckay[13] defined NSC as a kind of cell that can self-renew and differentiate to astrocyte, neuron and oligodendrocyte. Its characteristic is self-renewal and multi-differentiation. NSCs mainly locate at dentate gyrus (DG) and subventricular zone (SVZ)[14]. A lot of experiments have proved that the endogenous NSCs in adult CNS can proliferate, migrate, differentiate and they can replace lost neurons[11]. Many investigators tried to implant NSCs into the brain of EICH rats to promote function recovery. In this study we found that transplanting NSCs to the surrounding area of intracerebral hematoma can improve function recovery 14 days after ictus. We also found that NSCs in vivo could differentiate to neurons and astrocytes. An et al[15] transplanted NSCs to ICH caudate nucleus and found that the motor function of rats that received ipsilateral transplantation recovered much better than the rats that received contralateral transplantation or those without transplantation. immunohistological results confirmed that the transplanted NSCs differentiated into neurons or glials and migrated to the injured area. They implanted NSCs half an hour after EICH. Xue et al[16] found that the NSCs isolated and cultured from embryonic rats were induced to differentiate into neurons , oligodendrocyte and glial cells. The motor function of rats with NSCs transplantaion 7 days after cerebral hemorrhage recovered much better than those with NSCs transplantaion 3 days after cerebral hemorrhage and the control group. Further study is needed to reveal the appropriate time for NSCs transplantation.
Transplanting NSCs can promote neural function recovery of EICH rats. We consider that it may lies in four reasons. ① NSCs can differentiated to neurons and astrocytes in vivo, and these cells can replace lost neurons and astrocytes[15-16]. ② NSCs, and the cells they differentiate to, can secrete neurotrophin. Until now many neurotrophic factors have been identified, including neurotrophin factor (NTF), brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), neurotrophins (NT3-6), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), etc. Xue et al[17] transplanted BFGF to EICH rats and found that the motor function of bFGF group rats recovered much better than the control group. When compared to the control group, the number of nestin positive cells in bFGF group significantly increased. This experiment indicates that bFGF is important for NSCs survival and differentiation in vivo. ③ NSCs can repair damaged neurons. In our experiment we found less apoptosis cells arounding hematoma in NSCs transplantation group than in PBS transplantation group. The result may relate to NGF protection function. ④ NSCs may alleviate edema around hematoma of EICH rats. This needs to be investigated further.
We transplanted NSCs into the caudate nucleus of EICH rats, in case caudate nucleus is near the hematoma and NSCs could easily move to damage part. We found NSCs in caudate nucleus survived and differentiated, although the environment near hematoma was unfit for NSCs survival because of edema due to hematoma.
Some investigators transplanted NSCs to EICH rats by lateral ventricle or peripheral vessels. Nonaka et al[18] transplanted embryonic stem (ES) cells into the lateral ventricle in the hemisphere contralateral to the hemorrhage 7 days after collagenase infusion. Twenty-eight days after transplantation ES-derived neurons and astrocytes were detected around the hematoma cavities of the brain in all of ten rats receiving grafts. Mahmood et al[19] transplanted mesenchymal stem cells (MSC) isolated form male rats to female rats by tailor vein and found MSCs could migrate to brain through blood brain barrier. Jeong et al[20] transplanted NSCs to EICH rats by tail vein and found NSCs could migrate to the brain near hematoma. The NSCs could survive and promote motor function. What is the best approach for NSCs transplantation into the EICH rats needs further investigation.
In conclusion, NSCs show high proliferating potential, multipotential in vitro and in vivo. It also showed good adaptation in the host EICH rat brain. NSCs can accumulate in and around the hematoma and differentiate in response to the host microenvironment. NSCs transplantation to the caudate nucleus provides an efficient tool for ICH treatment.

REFERENCES

1 Butcher K, Laidlaw J.Current intracerebral haemorrhage management.J Clin Neurosci 2003;10(2):158-167
2 Nath PF, Jenkins A, Mendelow DA, et al. Early hemodynamic changes in experimental intracerebral hemorrhage. J Neurosurg 1986;65:697-703
3 Yang GY, Berz AL, Chencvert TL, et al. Experimental intracerebral hemorrhage: relationship between brain edema, blood flow, and blood-brain barrier permeability in rats. J Neurosurg 1994;81:93-102
4 Huang FP, Xi GH, Keep FR. Brain edema after experimental intracerebral hemorrhage: role of hemoglobin degradation products. J Neurosurg 2002;96:287-293
5 Rohde V, Rohde I, Thiex R, et al. Fibrinolysis therapy achieved with tissue plasminogen activator and aspiration of the liquefied clot after experimental intracerebral hemorrhage: rapid reduction in hematoma volume but intensification of delayed edema formation. J Neurosurg 2002;97:954-962
6 Kitaoka T, Hua Y, Xi GH, et al. Delayed argatroban treatment reduces edema in a rat model of intracerebral hemorrhage. Stroke 2002;33:3012-3018
7 Hua Y, Keep RF, Hoff JT, et al.Thrombin preconditioning attenuates brain edema induced by erythrocytes and iron.J Cereb Blood Flow Metab 2003;23(12):1448-1454
8 Gong C, Hoff JT, Keep RF.Acute inflammatory reaction following experimental intracerebral hemorrhage in rat.Brain Res 2000;871(1): 57-65
9 Hua Y, Xi GY, Keep FR, et al. Complement activation in the brain after experimental intracerebral hemorrhage. J Neurosurg 2000;92:1016-1022
10 Xi GH, Hua Y, Keep FR, et al. Systemic complement depletion diminishes perihematomal brain edema in rats. Stroke 2001;32:162-167
11 Bjorklund A, Lindvall O. Self-repair in the brain. Nature 2000;405: 892-895
12 Hua Y, Schallert T, Keep RF, et al Behavioral tests after intracerebral hemorrhage in the rat.Sorke 2002;33:2478-2484
13 Mckay R. Stem cells in the central nerve system. Science 1997;276:66-71
14 Rietze R, Poulin P, Weiss S. Mitotically active cells that generate neurons and astrocytes are present in multiple regions of the adult mouse hippocampus. J Comp Neurobiol 1999;135:135-141
15 An YH, Wang HY, Zhang XT, et al. Rat embryonic neural stem cells transplantation to treat intracerebral hemorrhage.Zhonghua Shenjing Waike Zazhi 2002;18:50-53
16 Xue CS, Li GL, He JF, et al.Experimental study on neural stem cells transplantation to cerebral hemorrhage rat. Zhongxiyi Jiehe Xinnao Xueguanbing Zazhi 2004;2(5):283-285
17 Xue CS, Li GL, Liu YF. Influence of basic fibroblast growth factor on proliferation of neural stem cells from cerebral hemorrhage rat. Zhongxiyi Jiehe Xinnao Xueguanbing Zazhi 2004;2(7):405-406
18 Nonaka M, Yoshikawa M, Nishimura F, et al.Intraventricular transplantation of embryonic stem cell-derived neural stem cells in intracerebral hemorrhage rats.Neurol Res 2004;26(3):265-272
19 Mahmood A, Lu D, Wang L. et al. Treatment of traumatic brain injury in female rats with intravenous administration of bone marrow stromal cells. Neurosurgery 2001;49:1196-1204
20 Jeong SW, Chu K, Jung KH, et al. Human neural stem cell transplantation promotes functional recovery in rats with experimental intracerebral hemorrhage. Stroke 2003;34:2258-2263

神经干细胞移植对脑出血模型大鼠神经功能的影响☆

安庆祝1，朱 巍1，汪 洋2，毛 颖1，张 荣1，周良辅1
1 复旦大学附属华山医院神经外科，上海市 200040；2复旦大学上海医学院解剖组胚教研室，上海市 200032

安庆祝☆，男，1978年生，山东省寿光市人，汉族，复旦大学在读博士，主要从事神经干细胞治疗自发性脑出血的研究。
摘要
背景：外源性神经干细胞具有神经修复作用，可能对脑出血后的神经功能恢复起到一定的作用。
目的：观察胎鼠神经干细胞的体外生长、分化及移植到脑出血大鼠后的存活、迁徙、分化情况，探讨神经干细胞对脑出血模型大鼠受损神经功能的修复作用。
设计：完全随机分组设计，对照动物实验。
单位：复旦大学附属华山医院神经外科
材料：选用健康雄性成年SD大鼠18只为受体，体质量280~320 g，由中国科学院上海实验动物中心提供。实验用鼠抗BrdU为Neomarkers产品, 鼠抗胶质纤维酸性蛋白和兔抗微管相关蛋白2 为Chemicon产品。
方法：实验于2006-02/12在复旦大学附属上海医学院解剖组胚实验室完成。从胎龄14 d的胎鼠海马中分离、培养、鉴定神经干细胞。16只受体SD大鼠被随机分为3组：对照组，PBS组和移植神经干细胞组。均通过尾状核内注射自体动脉血制作大鼠脑出血模型。移植NSC组在造模后30 min在血肿腔周围四点分别移植浓度为2×1011 L-1神经干细胞悬液5μL；PBS组于相同时间点在脑内相同部位注射PBS；PBS和神经干细胞的移植方法同自体血的移植方法。对照组大鼠在造模后30 min只造成四点损伤，不注射任何物质。
主要观察指标：在造模后立即，1，3，5，14，21，28 d采用前肢评分和转身评分对大鼠神经功能进行评估。大鼠于造模后28 d麻醉后取脑，并通过双标胶质纤维酸性蛋白、微管相关蛋白2、BrdU免疫组化来检测移植入脑的神经干细胞在体内的分化情况。
结果：①神经功能评分：造模后5 d，各组差异无显著性意义（P > 0.05）。造模后14~28 d，干细胞移植组较其他3组明显改善（P < 0.05）。②脑组织切片双免疫组织学双标染色结果：干细胞移植组血肿周围凋亡细胞少于PBS组。受体大鼠脑组织切片显示有BrdU, 微管相关蛋白2,胶质纤维酸性蛋白阳性细胞，说明神经干细胞可以在宿主脑内存活、迁徙和分化，可以分化为神经元样细胞和神经胶质样细胞。
结论：神经干细胞移植可能通过分化为神经元样细胞和神经胶质细胞促进大鼠脑出血的神经功能恢复。
关键词：神经干细胞；脑出血；大鼠；移植
中图分类号: R394.2 文献标识码: A 文章编号: 1673-8225(2008)12-02364-05
安庆祝，朱巍，汪洋，毛颖，张荣，周良辅.神经干细胞移植对脑出血模型大鼠神经功能的影响[J].中国组织工程研究与临床康复，2008，12(12):2364-2368
[www.zglckf.com/zglckf/ejournal/upfiles/08-12/12k-2364(ps).pdf]
(Edited by SSW Tay/Ji H/Wang L)