患侧与.双侧肢体训练促进脑梗死大鼠内源性神经干细胞增殖的比较

doi:10.3969/j.issn.1673-5374.2012.34.007

摘要/Abstract

摘要：

为探讨患侧、双侧肢体训练对大鼠脑梗死周围区和未受累半球相应脑组织内源性神经干细胞的增殖和分化的影响。在Wistar大鼠建立中脑中动脉阻塞模型后，用胶布缠住大鼠健侧前肢，强迫其使用患侧前肢；或不限制大鼠健肢运动，让其双侧肢体均参与运动。训练内容：转动、行走及抓握训练共40min，1次/d。同时，以不进行运动训练的大鼠为对照。发现功能训练后14d，双侧肢体功能训练大鼠的神经损害较患侧功能训练和未训练大鼠减轻，双侧功能训练大鼠梗死灶周围区和未受累半球相应脑组织区Nestin/GFAP、Nestin/MAP2免疫荧光双标阳性细胞在功能训练后3和14d较患侧功能训练明显增多。说明健肢与患肢共同进行功能训练可促进双侧半球内源性神经干细胞的增殖和分化，促进神经功能的恢复，且效果较单纯患肢功能训练更佳。

关键词: 神经再生, 脑损伤, 康复训练, 患侧肢体, 双侧肢体, 脑梗死灶周围区, 未受累半球, 神经干细胞, 增殖, 分化, 脑损伤, 脑, 可塑性

Abstract:

We investigated the effects of ipsilateral versus bilateral limb-training on promotion of endogenous neural stem cells in the peripheral infarct zone and the corresponding cerebral region in the unaffected hemisphere of rats with cerebral infarction. Middle cerebral artery occlusion was induced in Wistar rats. The rat forelimb on the unaffected side was either wrapped up with tape to force the use of the paretic forelimb in rats or not braked to allow bilateral forelimbs to participate in training. Daily training consisted of mesh drum training, balance beam training, and stick rolling training for a total of 40 minutes, once per day. Control rats received no training. At 14 days after functional training, rats receiving bilateral limb-training exhibited milder neurological impairment than that in the ipsilateral limb-training group or the control group. The number of nestin/glial fibrillary acidic protein-positive and nestin/microtubule-associated protein 2-positive cells in the peripheral infarct zone and in the corresponding cerebral region in the unaffected hemisphere was significantly higher in rats receiving bilateral limb-training than in rats receiving ipsilateral limb-training. These data suggest that bilateral limb-training can promote the proliferation and differentiation of endogenous neural stem cells in the bilateral hemispheres after cerebral infarction and accelerate the recovery of neurologic function. In addition, bilateral limb-training produces better therapeutic effects than ipsilateral limb-training.

Key words: bilateral rehabilitation training, affected limb, bilateral limbs, peripheral infarct zone, unaffected hemisphere, middle cerebral artery occlusion, brain, neural stem cells, proliferation, differentiation, plasticity, neural regeneration

. 患侧与.双侧肢体训练促进脑梗死大鼠内源性神经干细胞增殖的比较[J]. 中国神经再生研究(英文版), 2012, 7(34): 2698-2704.

Xiyao Yang, Feng Zhu, Xiaomei Zhang, Zhuo Gao, Yunpeng Cao. Ipsilateral versus bilateral limb-training in promoting the proliferation and differentiation of endogenous neural stem cells following cerebral infarction in rats[J]. Neural Regeneration Research, 2012, 7(34): 2698-2704.

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引言

材料方法

Design

A randomized, controlled animal experiment.

Time and setting

This study was performed at the Laboratory Animal Center, General Hospital of Shenyang Military Region, Research Institute of Shenyang Brain Hospital, China between April 2010 and April 2011.

Materials

Healthy male Wistar rats of clean grade, weighing 250–300 g, were provided by the Laboratory Animal Center, China Medical University (license No. SCXK (Liao) 2003-0009). All procedures were performed according to the Guidance Suggestions for the Care and Use of Laboratory Animals ^[28] issued by the Ministry of Science and Technology of China.

Methods

Preparation of MCAO rat models

Following anesthesia by intraperitoneal injection of 1% sodium pentobarbital (30–40 mg/kg), a 1.5 cm long longitudinal incision was made in the midline of the ventral cervical skin. The right common carotid artery, right internal carotid artery, and right external carotid artery were identified and separated from the vagus nerve. A silk suture was inserted into the stump of the external carotid artery and entered into the anterior cerebral artery via the bifurcation of the carotid artery until some resistance was felt. The depth of insertion was approximately 17–19 mm. The common carotid artery was ligated with sutures at the bifurcation, followed by skin suture. Thereafter, rats were returned to their cages and raised normally^[29]. After recovery of consciousness, MCAO induction was considered successful when rats exhibited the following signs: flexion of the left forelimb when lifting the tail, ipsilateral Hornor syndrome, left circling while climbing, and falling to the left while standing^[30]. At 24 hours after MCAO, rat neurologic function was evaluated with Zea Longa’s scale^[31]: a score of 0, no neurologic deficit; 1, mild focal neurologic deficit, failure to extend left forepaw fully; 2, moderate focal neurologic deficits, circling to the left; 3, severe focal neurological deficits, falling to the left; 4, did not walk spontaneously and had a depressed level of consciousness; 5, died. In each group, rats were fixed by mesh equipment. Rats that scored 2–3 were included for further experimentation.

Rehabilitation training

The daily training consisted of mesh drum training, balance beam training, and stick rolling training^[32], for a total of 40 minutes, once per day. The training commenced at 24 hours after surgery and was terminated when the rats died. Ipsilateral training group: the forelimb on the unaffected side was wrapped with the tape, forcing the use of the paretic forelimb. Bilateral limb-training group: the bilateral limbs were braked. Untreated group: no rehabilitation training was performed.

Assessment of neurologic function

At 1, 3, and 14 days after MCAO, neurological function in the rats was assessed including spontaneous activity, symmetry in the movement of four limbs, forepaw outstretching, climbing, body proprioception and response to vibrissae touch^[33]. The examiners had no knowledge of the procedure that each rat had received. The score given to each rat at the completion of the evaluation was the summation of all six individual test scores. The minimum neurological score was 3 and the maximum was 18.

TTC staining for cerebral infarct volume

Rats were anesthetized under 4% chloral hydrate (100 mg/kg) and decapitated at 3 and 14 days after MCAO. Brains were rapidly removed and cut into 2 mm-thick coronal sections. Slices were stained with 1% TTC (Sigma, St. Louis, MO, USA) in 0.1 M PBS (PH 7.4) for 30 minutes at room temperature, and fixed in 10% buffered formalin overnight. The staining was photographed with an image acquirement system HPIAS-1000 (Beijing Kong Hai, China). Infarct volumes were blindly quantified using the formula: V = t (A1+ . . . An) – (A1+ An) t/2, where t = slice thickness and A = infarct area.

Sampling and sections

At postoperative days 3 and 14, rats were rapidly perfused with heparinized saline, fixed with 4% paraformaldehyde (w/v), and rinsed with 0.1 M PBS (pH 7.4) for 10 minutes at 4°C. After rats were euthanized by cervical dislocation, continuous coronary blocks were cut from the frontal pole at 1.5 mm intervals, which were then fixed with 4% paraformaldehyde (w/v) and rinsed with 0.1 M PBS (pH 7.4) at 4°C for 8 hours, followed by conventional dehydration, paraffin embedding, and cutting into 4 µm thick sections.

Immunofluorescent double staining of nestin/MAP2 and nestin/GFAP

Brain slices were incubated with rabbit anti-rat nestin (1:200; Wuhan Boster, China) at 37°C for 2 hours and goat anti-rabbit IgG/Dylight 488 (1:100; Jackson ImmunoResearch Inc., West Grove, PA, USA) at 4°C overnignt, then with rabbit anti-rat GFAP or rabbit anti-rat MAP2 (1:200; Wuhan Boster) at 37°C for 2 hours followed by 4°C overnight. On the second day, slices were rinsed with PBS four times for 5 minutes each, and incubated with goat anti-rabbit IgG/DyLight 594 (1:100; Jackson ImmunoResearch Inc.) at 37°C in the dark for 1 hour, rinsed with PBS four times for 5 minutes each, and then mounted with glycerol and immediately photographed under laser confocal microscope (Leica, Wetzlar, Germany) for fluorescence observation. Negative controls for immunohistochemistry were performed by incubation with 1% bovine serum protein rather than antibodies.

Double immunofluorescent staining for nestin/GFAP double-labeled cells showed that nestin-positive cells exhibited a green cytoplasm, while the GFAP-positive cells were mainly red; in the cytoplasm, nestin/GFAP- positive cells were labeled yellow. For nestin/MAP2 double-labeled cells, the nestin-positive cells exhibited a green cytoplasm, with uniform size and irregular shape, while the MAP2-positive cells were red; in the cytoplasm, nestin/MAP2-positive cells were mainly labeled yellow. Three discontinuous sections randomly selected from each rat were used to calculate the number of positive cells in seven fields of view at high magnification (× 400).

Statistical analysis

Data were expressed as mean ± SD, and all values were tested by the homogeneity of variance and the normality test. Statistical processing was performed using SPSS 13.0 software (SPSS, Chicago, IL, USA). Intergroup comparisons were performed using one-way analysis of variance. P < 0.05 was considered statistically significant.

Author contributions:Xiyao Yang and Feng Zhu were responsible for providing and integrating experimental data. Xiaomei Zhang and Zhuo Gao contributed to conception and design of the study, analysis and interpretation of the data, and writing of the manuscript. Yunpeng Cao guided and supervised the study. All authors approved the final version of the paper.

Conflicts of interest:None declared.

Ethical approval: This study received ethical permission from the Animal Ethics Committee of China Medical University, China.

Author statements: The manuscript is original, has not been submitted to or is not under consideration by another publication, has not been previously published in any language or any form, including electronic, and contains no disclosure of confidential information or authorship/patent application disputations.