Abstract
BACKGROUND: Stem cell factors are hypoxia-induced neural regeneration factors. They stimulate animals' neural regeneration.
OBJECTIVE: To observe Nestin and stem cell factor mRNA expressions after ischemia/reperfusion injury in rat brain, and to analyze the time rule of the two.
DESIGN: A randomized controlled animal experiment.
SETTING: Department of Ultrasound Diagnosis, Affiliated Hospital of Qingdao University Medical College.
MATERIALS: Thirty-six healthy female adult Sprague-Dawley (SD) rats were provided by the Shanghai Laboratory Animal Center, Chinese Academy of Science. Nestin and stem cell factor mRNA in situ hybridization kits and 3,3'-diaminobenzidine (DAB) kit were provided by Boster Bioengineering Co.,Ltd (Wuhan, China).
METHODS: This study was performed at the Shangdong Key Laboratory for Prevention and Treatment of Encephalopathy from January to June 2005. Thirty-two rats were created into models of ischemia/reperfusion models by occluding left middle cerebral artery with suture. At ischemia 1.5 hours and reperfusion 2, 6, 12, 24 hours, 2, 3, 7, 14 days, 4 rats were separately used in order to observe the expressions of Nestin and stem cell factor mRNA. The other 4 rats were used for sham-operation, in which, suture insertion was omitted, and the other procedures were identical to experimental groups. The expressions of Nestin and stem cell factor mRNA were detected in the cortex, corpora striatum and paraventricular nucleus region in rat brain by in situ hybridization.
MAIN OUTCOME MEASURES: Nestin and stem cell factor mRNA expressions in the cortex, corpora striatum and paraventricular nucleus region in rat brain.
RESULTS: Thirty-six rats were included in the final analysis. Nestin mRNA and stem cell factor were weakly expressed in the cortex, corpora striatum and paraventricular nucleus region in rats of sham-operation group. After ischemia/reperfusion, Nestin mRNA expression at each time point was significantly higher in the experimental groups (except in the cortex at ischemia 1.5 hours and reperfusion 2 hours, in the corpora striatum at ischemia 1.5 hours and reperfusion 2 and 6 hours and in the paraventricular nucleus region at ischemia 1.5 hours and reperfusion 2, 6 hours and 14 days) than in the sham-operation group (P < 0.05). While stem cell factor mRNA expression at each time point was significantly higher in the experimental groups (except in the cortex at ischemia 1.5 hours and reperfusion 2,6 and 12 hours, in the corpora striatum at ischemia 1.5 hours and reperfusion 2 and 6 hours and in the paraventricular nucleus region at ischemia 1.5 hours and reperfusion 2 hours and 14 days) than in the sham-operation group (P < 0.05).
CONCLUSION: The time rule of stem cell factor mRNA expression is basically the same as that of neural stem cell proliferation. It indicates that following cerebral ischemia/reperfusion, stem cell factor mRNA expression may promote the proliferation of neural stem cells.

INTRODUCTION

Neural stem cells are a kind of cells possessing the capacity of proliferation and differentiation in the central nervous system. They are specified cell colony at the early stage of development of nervous system and can keep the capacity of proliferation and differentiation for long time [1]. Nestin is a molecule marker often used in the in vitro culture of neural stem cells. It is an intermediate filament protein only existing in the neuroepithelial stem cells. Its expression has a specified time sequence. Studies have demonstrated hypoxia induces neural regeneration in mouse, stem cell factor and fibroblast growth factor-2 are hypoxia-induced neural regeneration factors, and stem cell factor can promote neural regeneration in the whole animal body [2].
In this study, we measured Nestin and stem cell factor mRNA expressions by in situ hybridization in order to investigate the change in proliferation of neural stem cells after cerebral ischemia/reperfusion.
MATERIALS AND METHODS

Materials
This study was performed at the Shangdong Key Laboratory for Prevention and Treatment of Encephalopathy from January to June 2005.
Thirty-six healthy female adult Sprague-Dawley (SD) rats, weighing 230-280 g, were provided by the Shanghai Laboratory Animal Center, Chinese Academy of Science [Permission No. SCXK (hu) 2002-0010]. Nestin and stem cell factor mRNA in situ hybridization kits and 3,3'-diaminobenzidine (DAB) kit were provided by Boster Bioengineering Co.,Ltd (Wuhan, China).

Methods
Grouping and intervention
Thirty-two rats were created into models of ischemia/ reperfusion models by occluding left middle cerebral artery with suture. At ischemia 1.5 hours and reperfusion 2, 6, 12, 24 hours, 2, 3, 7, 14 days, 4 rats were separately used in order to observe Nestin and stem cell factor mRNA expressions. The other 4 rats were used for sham-operation, in which, suture insertion was omitted,

and the other procedures were identical to experimental groups.

Sampling and preparation of tissue sections
Rats were taken for the experiment at the set time in each group except sham-operation group (at 24 hours). After anesthetized with 100 g/L chloral hydrate (300 mg/kg), rats were intubated via left ventricle till ascending aorta, and then they were perfused 200 mL normal saline and then 300 mL paraformaldehyde(40 g/L). Thereafter, brain tissue with thickness of 5 mm between 2 mm anterior to bregma and 3 mm posterior to bregma was harvested, fixed in the 4% paraformaldehyde supplemented with diethyl pyrocarbonate (1/5 000) for 2 hours and soaked in distilled water for 4 hours. Subsequently, the brain tissue was routinely dehydrated with ethanol, cleared with dimethyl benzene, soaked and embedded with wax. Brain tissue 1 mm anterior to bregma and 2 mm posterior to bregma was sliced into successive coronal sections with the thickness of 7 μm. The coronal sections were placed on the polylysine-pretreated slide and preserved in the 37 ℃ baker, overnight, for Nestin and stem cell factor in situ hybridization tests and control test.

In situ hybridization test and cell counting
In situ hybridization tests of Nestin and stem cell factor mRNA were conducted according to the instructions of kits. The sections were developed with DAB. Under a light microscope, cells with brown granules were considered positive. Some sections were treated with 0.1 mol/L phosphate buffer solution without use of probe. Results showed that no positive cells were found, indicating that probe has a strong specificity. Under a high-fold microscope (×400), four visual fields were randomly chosen from the cortex, corpora striatum and paraventricular nucleus region in rat brain, separately, for counting positive cells.

Statistical analysis
Statistical analysis was performed by the first author with SPSS 10.0 software. Number of positive cells at each time point was expressed as Mean±SD. t test was used for intergroup comparison.

RESULTS

Quantitative analysis of experimental animals
Thirty-six rats were kept during the whole experiment.

Change in Nestin mRNA expression after cerebral ischemia/reperfusion
Nestin mRNA was weakly expressed in the cortex, corpora striatum and paraventricular nucleus region in rats of sham-operation group. In the experimental group, Nestin mRNA expression in the cortex of the affected side began to increase at reperfusion 2 hours, but it was not significantly different from that of sham-operation group. It increased significantly from 6 hours, reached its peak level at 24 hours, turned back on 2 days, reached the peak level again on 7 days, and then decreased gradually, but it was still higher than that of sham-operation group on 14 days. Nestin mRNA expression in the corpora striatum and paraventricular nucleus region was basically the same as that in the cortex, but they began to present significant difference from 12 hours, and Nestin mRNA expression in the paraventricular nucleus region had recovered to the level of sham-operation group by day 14. The concrete data are shown in Table 1.

Stem cell factor mRNA expression after cerebral ischemia/reperfusion
Stem cell factor mRNA was weakly expressed in the cortex, corpora striatum and paraventricular nucleus region in rats of sham-operation group. In the experimental group, stem cell factor mRNA expression in the cortex of the affected side began to increase at reperfusion 2 hours, but it was not significantly different from that of sham-operation group. It increased significantly from 24 hours, turned back on 2 days, increased on 3 days, reached the peak level again on 7 days, and then gradually decreased, but it was still higher than that of sham-operation group on 14 days. Nestin mRNA expression in the corpora striatum and paraventricular nucleus region was basically the same as that in the cortex. But stem cell factor mRNA expression in the corpora striatum began to increase from 12 hours and that in the paraventricular nucleus region began to increase from 6 hours and had recovered to the level of sham-operation group by day 14. The concrete data are shown in Table 2.

DISCUSSION

Cerebral ischemia injury and neural stem cell proliferation
Neural stem cells lack of the antigen possessed by adult nerve cells but have Nestin (i.e. neuroepithelial stem cell protein), as the marker of neuroepidermal layer and also the marker of original nerve cell. Therefore, immune reaction of Nestin can be used to confirm the existence of neural stem cells [3-6].
In the adult central nervous system, some neural stem cells survive in the brain in the status of quiescence and non-proliferation, and are regulated by central nervous system. When central nervous system is injured or degenerated, because of the alteration of cell microenvironment (for example, some cytokines appear), neural stem cells are activated (or inhibitory factors devitalized) and then appear in the injured region or present heteroplasia, and then they migrate to injured region under the effect of chemotatic factors [7-8]. Some investigators also reported that the reverse differentiation of adult nerve cells is one of sources of endogenous neural stem cells. They believed that under the cerebral ischemia or other pathological status of nervous system, adult cells could present embryo reply of cytoskeleton and re-expression of embryonic neuroepithelial cells. Such cells may be the precursor cells of astrocytes or oligodendrocytes[9-10]. Li et al [11] developed rat models of local cerebral ischemia by middle cerebral artery occlusion. They harvested brain tissue at reperfusion 2, 3, 6, 12 hours and 1, 2, 3, 7 and 8 days and made a quantitative analysis of Nestin by Western blot. Results showed that Nestin mRNA expression reached the peak level on 7 days. These findings have demonstrated after cerebral ischemia, body produces the compensative adaptive response to ischemia by inducing the proliferation of endogenous neural stem cells. The current experimental results are in accordance with this.

Whether stem cell factors promote the proliferation of neural stem cells
Stem cell factors are a kind of cytokines. They influence the function, growth and apoptosis of heart and other organs. They promote myocardial healing. Under the effect of nerve growth factor, brain-derived growth factor and neurenergen 3, stem cell factors, as the survival factors of neural stem cells, stimulate the proliferation of neural stem cells [12-15].
Cerebral ischemia can stimulate the neural regeneration in the hyperplasy region of rodents. Hypoxia promotes bromodeoxyuridine (BrdU) into cells, expressing the markers of proliferation and young neuron, while there is no evidence confirming the destruction of DNA and the activation of Caspase 3. Under the condition of hypoxia, stem cell factors stimulate BrdU to enter the culture medium. Stem cell factor receptors are expressed in the hyperplasy region of adult rats and the cultured cells. Perfusing stem cell factors into the whole body can increase the amount of BrdU-labeled young neurons. There exists study demonstrating that hypoxia promotes neural regeneration in cultured cells of mouse cortex. Such an effect is accomplished by secreting stem cell factors. Stem cell factors stimulate cultured cells and neural regeneration in the subventricular zone of cortex and the subgranular zone of hippocampal dentate area [2]. Many other factors also stimulate neural regeneration, including epithelial growth factor, basic fibroblast growth factor-2 and brain-derived nerve growth factor [16]. It is reported that adding neural growth factors or over-expressing such factors can promote the neural regeneration of general cerebrum in adults [17]. It is also reported that hemispheric ischemia triggers neural regeneration in the subgranular zone [18], local ischemia induces cortical regeneration in the peripheral ischemic region [19], while local cerebral ischemia is in correlation to the proliferation of basic fibroblast growth factor-2 in the precursor cells in the hippocampal dentate nucleus [20].
Previous studies have demonstrated after mouse transient prosencephalic ischemia 15 minutes, neural stem cells in the dentate nucleus proliferate fast, and the number of neural stem cells in the dentate nucleus and subventricular zone increases at reperfusion 3,7 and 10 days, especially at reperfusion 7 days [11]. Zhang et al [21] reported that neural stem cells existed in the subventricular zone and cerebral cortex of injured side of rats undergoing middle cerebral artery occlusion. They also reported that neural stem cells in the cerebral cortex increased 2-14 days after cerebral ischemia, reached the peak level on 7 days and greatly reduced 28 days later. The current experimental results showed that in the experimental groups, the expression of neural stem cells in the cortex of ischemic side began to significantly increase from 24 hours and reached the peak level on 7 days, and that in the corpora striatum and paraventricular nucleus region was basically the same. The time rule of stem cell factor mRNA expression in this study is basically the same as that of proliferation of neural stem cells in the above-mentioned studies. It indicates that stem cell factors may promote the proliferation of neural stem cells.

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脑缺血再灌注损伤后神经细胞巢蛋白和干细胞因子基因的表达★

摘要
背景：干细胞因子是缺氧诱导的神经再生因子，可能刺激动物的神经再生。
目的：观察大鼠脑缺血再灌注损伤后神经细胞巢蛋白和干细胞因子基因表达的变化，分析两者变化的时间规律。
设计：随机对照动物实验。
单位：青岛大学医学院附属医院超声诊断科。
材料：实验选用成年健康雌性SD大鼠36只，由中国科学院上海实验动物中心提供。实验所用巢蛋白和干细胞因子 mRNA原位杂交试剂盒、DAB试剂盒均由武汉博士德生物工程有限公司提供。
方法：实验于2005-01/06在山东省脑病防治重点实验室完成。选取32只大鼠，应用线栓法经左侧颈外一颈内动脉插线建立左侧大脑中动脉阻塞再灌注模型，在缺血1.5 h再灌注后2，6 ，1 2 ，24 h，2，3，7，14 d进行观察，每个时间点4只。其余4只为假手术组：除不插线外，其余步骤同实验组。应用原位杂交技术检测脑缺血再灌注后皮质、纹状体和室旁区巢蛋白和干细胞因子mRNA的表达。
主要观察指标：大鼠皮质、纹状体和室旁区神经细胞巢蛋白和干细胞因子基因表达。
结果：进入结果分析数量保持为36只。①巢蛋白 mRNA表达：假手术组皮质、纹状体和室旁区巢蛋白 mRNA表达很弱。缺血再灌注后，在皮质中的表达除再灌注后2 h、纹状体除再灌注后2,6 h以外，室旁区除2,6 h,14 d以外各时间点均明显高于假手术组，差异有显著性意义（P < 0.05）。②干细胞因子表达:假手术组皮质、纹状体和室旁区干细胞因子表达很弱。缺血再灌注后，在皮质中的表达除2,6,12 h以外，纹状体除2,6 h以外，室旁区除2 h,14 d以外各时间点均明显高于假手术组，差异有显著性意义（P < 0.05）。
结论: 干细胞因子表达的时间规律与神经干细胞增殖的时间规律基本一致，可以提示脑缺血再灌注后干细胞因子 mRNA表达可能具有促进神经干细胞增殖作用。
关键词：脑缺血;巢蛋白;干细胞因子;大鼠


李 翔1，王正滨1，房世保1，郑青立2
1 青岛大学医学院附属医院超声科，山东省青岛市 266003；2山东省青岛疗养院, 山东省青岛市 266003
李 翔★，女，1966年生，山东省青岛市人，汉族，2001年青岛大学医学院毕业，硕士，副主任医师，主要从事超声诊断工作。


中图分类号: R394.2 文献标识码: A 文章编号: 1673-8225(2008)12-02379-04
李翔，王正滨，房世保，郑青立.脑缺血再灌注损伤后神经细胞巢蛋白和干细胞因子基因的表达[J].中国组织工程研究与临床康复，2008，12(12):2379-2382
[www.zglckf.com/zglckf/ejournal/upfiles/08-12/12k-2379(ps).pdf]
(Edited by Rameshwar P/Song LP/Wang L)