Design

A randomized, controlled animal experiment.

Time and setting

Experiments were performed in the First Affiliated Hospital of General Hospital of Chinese PLA from March 2008 to September 2011.

Materials

Embryonic (14 ± 0.5 days old) transgenic donor Sprague-Dawley rats (SOD G93A, RRRC, USA) and 136 adult female Sprague-Dawley rats, weighing approximately 250–300 g (Vital River Laboratory Animal Technology Co., Ltd., Beijing, China; license No. SCXK (Jing) 2007-0001) were used in this study. All experimental protocols were in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals, formulated by the Ministry of Science and Technology of China^[31].

Methods

Neural progenitor cell culture

Neural progenitor cells were isolated from embryonic cerebral vesicles of transgenic donor rats (outbred Sprague-Dawley) expressing green fluorescence protein (RRRC #65). Briefly, embryos were isolated in a dish containing Dulbecco’s modified Eagle’s medium/F12 (Sigma, St. Louis, MO, USA). After peeling away the meninges, the cerebral vesicles were incubated in collagenase type I (10 mg/mL) / dispase II (20 ng/mL)/ Hank’s Buffered Salt for 30 minutes at 37°C . Cells were then plated in neural progenitor cell complete media (Dulbecco’s modified Eagle’s medium/F12) containing 10% bovine serum albumin (1 mg/mL, Sigma) B27      (5 ng/mL; Invitrogen, Carlsbad, CA, USA), basic fibroblast growth factor (20 ng/mL; PeproTech, Rocky Hill, NJ, USA), N2 (10 ng/mL; Invitrogen), epidermal growth factor     (20 ng/mL; PeproTech) and chick embryo extract (10%, SLI Ltd., West Sussex, UK) on laminin- coated (20 ng/mL; Invitrogen) plastic plates. Cells were passed every      3–5 days for a total of 10 passages.

The neural progenitor cell population was dissociated from culture plates using 0.05% trypsin/ ethylenediaminetetraacetic acid, washed and re-suspended at a concentration of 4 × 10⁶ cells (in Dulbecco’s modified Eagle’s medium/F12) for transplantation. Cells were placed on ice throughout the grafting process. Before completion of the grafting procedure, cell viability was assessed using the trypan blue assay^[29]. The phenotype of neural progenitor cells was verified before grafting by staining for nestin and stem cell marker A2B5 (A2B5 for neural progenitor cells), neuronal nuclei (for mature neurons), glial fibrillary acidic protein (for astrocytes) and O4 (for oligodendrocytes)^[29].

Establishment of thoracic spinal cord injury model

A laminectomy was performed at the T_9-10 region under pentobarbital sodium (40 mg/kg) anesthesia. The rod of the impactor (10 g) was centered above T₁₀ and dropped from a height of 25 mm to induce a consistent partial and incomplete spinal cord injury^[32]. Muscle and skin were then sutured in layers after injury. Cefazolin (25 mg/kg) was administered for 5 days to prevent urinary tract infections. Urinary bladders were emptied manually twice daily until urinary function was recovered. All surgeries were performed by one blinded operator.

Cell transplantation

Neural progenitor cells were injected into the lumbar subarachnoid space at the corresponding time point. Under sodium pentobarbital (40 mg/kg) anesthesia, a 1 cm midline incision was cut into the skin over the L_3–5 spinous process region, and the skin was retracted. A 29-gauge needle (BD Neonatal, 29G1; BD Medical Systems, San Jose, CA, USA) was inserted into the spinal canal at L_4–5. Proper needle placement was confirmed by a sudden loss of resistance at the time of entry, a tail flick and cerebral spinal fluid flow into the needle hub. Cells were then administered over a period of 1 minute. 80 µL of F12 media solution containing neural progenitor cells (4 × 10⁶ cells), or the same volume of F12 media, was injected as a single dose into the subarachnoid space. The muscle and skin were sutured tightly. The control group was considered as an untreated control and received no cell transplantation or media injection. Cell transplantations were performed by one blinded operator.

Immunohistochemical and immunofluorescent staining  

Animals were deeply anesthetized with pentobarbital sodium. Rats were sacrificed by transaortic perfusion with 100 mL of 4°C PBS followed by 150 mL of 4% paraformaldehyde. The whole spinal cord and brain were removed from the spinal column and skull after perfusion. One spinal cord specimen from each group was cut sagittally, while the others were cut coronally at 1, 2, 4 and 8 weeks. All brain specimens were cut coronally. All brain and spinal cord tissues were post-fixed in 4% paraformaldehyde overnight and transferred into a 30% sucrose solution, frozen, and cut into 6 µm-thick slices using a cryostat (Leica, Heidelberg, Germany). Sections were mounted on glass slides and stored at –70°C. Tissue sections and cultured cells were washed in PBS, blocked in 10% goat serum (Invitrogen) for 1 hour at room temperature and incubated in primary antibody solution at 4°C overnight. Both monoclonal and polyclonal antibodies were used to identify the grafted cells. A number of primary antibodies were used to assess the phenotype of the cells. Mouse anti-rat nestin (1:300; Chemicon, Santa Cruz, CA, USA) and A2B5 (1:500; Chemicon) monoclonal antibody were used to identify undifferentiated neural progenitor cells. Neurons were identified using mouse anti-rat neuronal nuclei monoclonal antibody (1:100; Chemicon). Astrocytes were identified using mouse anti-rat glial fibrillary acidic protein monoclonal (1:100; Chemicon) and rabbit anti-glial fibrillary acidic protein polyclonal (1:200; Dako, Glostrup, Denmark) antibodies. Oligodendrocytes were identified using rabbit anti-O4 polyclonal antibody (1:250; Chemicon). Rabbit anti-neurofilament polyclonal antibody (1:100; Chemicon) and mouse anti-rat smooth muscle actin monoclonal antibody (1:50; Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used to label axons and blood vessels respectively. Samples were incubated for 2 hours at room temperature with secondary antibodies: Alexa 488- or 546-labeled goat-anti-mouse/ rabbit IgG (1:400; Molecular Probes, Eugene, OR, USA) or Cy5-labeled goat-anti-mouse IgG (1:400, Jackson Immuno Research Labs, Inc., West Grove, PA, USA). Samples were counterstained with 4',6-diamidino-2- phenylindole (1:1 000; Sigma) to identify nuclei and mounted under coverslips with anti-fade mounting media (Fluorosave, CN Biosciences, La Jolla, CA, USA). Images were acquired using confocal microscopy (Zeiss LSM 510 Meta, Oberkochen, Germany).

Basso, Beattie and Bresnahan scale score

Hind limb locomotor activity of the animals in each group (10 rats/group) was assessed by two blinded examiners using the Basso, Beattie and Bresnahan scoring scale^[8] once a week for 12 weeks after injury. Assessments were conducted 1 hour after bladder evacuation every Monday morning.

Statistical analysis

SPSS 13.0 software(SPSS, Chicago, IL, USA) was used for statistical analyses. All data are represented as  mean ± SD. Statistical analysis was performed using analysis of variance and Dunnett’s test. P values < 0.05 were regarded as statistically significant.

1 Neural progenitor cells transplanted via the subarachnoid space at the acute, subacute and chronic stages following spinal cord injury could survive and improve neurological function in injured rats.
2 Grafted neural progenitor cells migrated and aggregated around blood vessels of the injured region, and infiltrated the spinal cord parenchyma along the tissue spaces after acute stage transplantation.
3 Grafted neural progenitor cells survived long term (8 weeks) and differentiated into astrocytes or oligodendrocytes after chronic stage transplantation.
4 Grafted neural progenitor cells localized to the parenchyma of injured spinal cords and improved neurological function at the acute stage following injury in rats. Efficacy was enhanced in comparison with transplantation at subacute and chronic stages.
1 脊髓损伤急性、亚急性及慢性期经蛛网膜下腔移植的神经祖细胞均可存活，并可改善脊髓损伤大鼠的神经功能。
2 脊髓损伤急性期经蛛网膜下腔移植的神经祖细胞多聚集在损伤区血管周围，并沿神经纤维间隙向损伤区脊髓实质内迁移。
3 脊髓损伤慢性期经蛛网膜下腔移植的神经祖细胞存活时间长，且可向胶质细胞或少突胶质细胞分化。
4 脊髓损伤急性期移植的祖细胞细胞可到达脊髓损伤区实质，在改善脊髓损伤大鼠的神经功能方面优于亚急性期和慢性期。

1 实验设计构思介绍

近几十年，脊髓损伤后促进中枢神经再生修复及功能康复的研究较多，多是以细胞移植为背景，尤其是干细胞移植治疗中枢神经系统疾病及损伤已成为热点，其中神经祖细胞移植有利于损伤的神经组织修复。神经祖细胞具有较高的神经细胞分化潜能，但多数研究是将细胞移植入损伤区，该途径是比较有效的方法。但是在临床应用中，这种方法具有侵袭性，常需要开放性手术治疗，费用也较高，存在进一步损伤脊髓的风险且不易被患者接受。蛛网膜下腔神经干细胞移植治疗脊髓损伤实验研究报道具有一定疗效，临床应用也有报道，但疗效不确定，是否移植时机对治疗效果有影响，因此进行该途径移植神经祖细胞的时间窗研究。

2 国内外同类研究水平的介绍

国内外研究多为实验研究，发现移植的神经祖细胞可以向脊髓损伤区迁移，且可以分化为成熟的神经元或神经胶质细胞，可以促进实验动物脊髓神经功能恢复，也对移植细胞种类进行了研究，临床研究多为亚急性期细胞移植的临床观察，只是得出安全的结论，有效性尚存争议。但是针对最佳移植时间窗的研究尚未见报道。

3 与其他同类研究的比较

课题组在脊髓损伤后不同时期（急性期、亚急性期及慢性期）进行细胞移植治疗脊髓损伤，首先观察移植细胞是否可以进入损伤区脊髓实质，因为只有移植细胞进入脊髓实质内才能发挥作用，其次观察移植细胞的分布及分化特点，实验在其他同类研究的基础上进一步探讨该方法的时限性，精细探讨经蛛网膜下腔移植神经祖细胞治疗脊髓损伤的适用范围，提高临床疗效，从而为临床应用提供实验数据。

4 文章先进性

PubMed（Medline）检索时间1977—2012年3月、EMBASE数据库（1977—2012年3月）、中国生物医学文献数据库(1984年2012～3月)，手工检索相关杂志。检索词：spinal cord injury；subarachnoid space；cell transplantation；neural progenitor cells；neural stem cell; lumbar puncture;脊髓损伤；蛛网膜下腔；细胞移植；神经干细胞；神经祖细胞；腰椎穿次。共检索相关文献123篇，最终选定62篇，综述成文《经蛛网膜下腔细胞移植治疗脊髓损伤的研究进展》，在《中国脊柱脊髓杂志》发表。

5 专家意见与答疑

专家意见1：材料与方法部分出现了NF的说明，但结果中没有，建议补充说明。

作者答疑：已经补充。NF主要标记宿主神经轴突（神经纤维）目的是观察移植细胞与宿主脊髓白质相对位置关系，本研究未作轴突再生方面的观察，只是探寻蛛网膜下腔细胞移植，神经祖细胞迁入脊髓损伤区实质内的最佳时间窗，因为只有移植细胞进入脊髓实质内才具有促进再生及神经功能修复的物质基础，具体促进脊髓神经功能修复机制是细胞替代、促进轴突再生、促进再髓鞘化还是作为中转重建神经环路等问题是我们的下一步研究计划，故NF在本文中未做重点描述。

专家意见2：本文最主要的结果是急性期移植细胞可迁移至脊髓损伤部位实质中，促进模型动物BBB评分改善，但移植细胞缺乏分化为胶质细胞或神经元的证据，那么，其促进BBB评分提高的依据可能是什么呢？建议结合文献适当补充。

作者答疑：已经补充下述讨论内容：没有观察到神经祖细胞的分化证据，可能的原因（1）细胞移植入蛛网膜下腔的时间较短；（2）急性期局部炎症反应显著，可能炎性因子对神经祖细胞的分化具有抑制作用；（3）炎症反应旺盛期巨噬细胞大量迁入，导致移植的异种神经祖细胞被吞噬，使得短时间内神经祖细胞细胞数量急剧减少，从而分化表达较弱，不易观察。BBB评分提高可能的机制：（1）移植的神经祖细胞进入脊髓实质内可提供轴突附着基质，有助于轴突生长；（2）分泌神经营养因子改善局部微环境，减轻继发性损伤，促进轴突发芽再生从而保留更多的脊髓神经功能；（3）少量神经祖细胞可分化为胶质细胞或神经元，使得脱髓鞘轴突再髓鞘化或作为中转神经元重建神经环路，从而促进神经功能修复。