Establishment and evaluation of the animal model of acute spinal cord injury

doi:10.3969/j.issn.2095-4344.2016.49.007

Abstract

Abstract:

BACKGROUND: There are a variety of methods for modeling spinal cord injury at home and abroad, showing their own advantages and disadvantages, but not yet a kind of objective and standardized model meeting the clinical research.
OBJECTIVE: To design a simple impactor used for establishing the spinal cord injury model, then to evaluate the parameters and stability of the Allen’s spinal cord injury model through comparing with the New York University (NYU) impactor established model.
METHODS: Female rats were equivalently allotted into five groups: sham-operation group (group A), self-made impactor 5 and 10 cm groups (group B1 and B2) and NYU 1.25 and 2.5 cm groups (group C1 and C2). All groups except group A were subjected to striking at different heights. Behavioral scores were detected at 1, 3, 5 and 7 days after modeling. Nissl staining was used to observe the morphological changes after freezing section and semiquantitative analysis.
RESULTS AND CONCLUSION: (1) At the same time point, the behavioral scores, damaged area ratio of spinal cord and semiquantitative analysis of dorsal horn neurons showed significant differences in the group A compared with the other groups, also between groups B1 and B2, and groups C1 and C2 (P < 0.05), but no significant differences were found between groups B1 and C1, as well as groups B2 and C2 (P > 0.05). (2) In the group A, there was a clear boundary between the grey and white matter in a butterfly shape, numerous neurons with large nucleus and obvious nucleolus, and plaques or tabby of Nissl body appeared in the cytoplasm. In contrast, in the other four groups, there were few neurons, different degrees of Gore focal, swelling, demyelinating, and vacuole of cellula could be found, and Nissl body was fuzzy or disappeared. These results administrate that the self-made impactor can establish different damage degrees of spinal cord inury models, which are close to the NYU-made models. Moreover, it can be helpful for study on spinal cord injury for reasons of good stability, low cost, and operated easily.

中国组织工程研究杂志出版内容重点：肾移植；肝移植；移植；心脏移植；组织移植；皮肤移植；皮瓣移植；血管移植；器官移植；组织工程

Key words: Spinal Cord Injury, Animal Models, Neurobehavioral Manifestations, Tissue Engineering

CLC Number:

R318

Wu Yang-peng, Fan Xiao, Zhang Li. Establishment and evaluation of the animal model of acute spinal cord injury[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(49): 7341-7348.

Figures/Tables 2

2.1 实验动物数量分析选用30只雌性SD大鼠，随机分为5组，在实验过程中，由于麻醉剂量过大和术后感染导致2只大鼠死亡，死亡后立即选2只体质量接近的大鼠按原来造模方法造模后补上。 2.2 模型稳定性脊髓损伤模型研究众多，包括脊髓撞击损伤模型、脊髓缺血损伤模型、脊髓压迫损伤模型等等，然而临床中由于车祸撞击或地震等重物撞击脊柱造成的脊髓损伤更为多见，故经典的Allen’s重物坠落法建立的脊髓损伤模型，其临床相似性高。而本实验基于经典Allen’s法的基本原理进行改良，模型更接近临床，并与稳定性高的NYU打击模型进行比较，来证实本实验模型的稳定性，结果可靠，可控制好，操作简便，易于复制。 2.3 两种打击器制备的脊髓损伤模型脊髓外观形态造模时，正常脊髓节段呈乳白色透亮条索状，后正中沟明显，且伴有血管走行，打击后，硬脊膜完整，硬脊膜下出现不同程度水肿、淤血(见图2a)；脊髓损伤术后1周，损伤局部组织间有不同程度的粘连，不同打击器及不同打击高度损伤后，受损脊髓段出现不同程度的淤血，但硬脊膜均保持完整，B2、C2组淤血明显(见图2b)。 2.4 急性脊髓损伤动物模型的评估 2.4.1 术后常见症状 ①死亡：A组1只，因麻醉剂量过大，麻醉后死亡，又随机抽取1只体质量相近的正常雌鼠补上；B2组1只，术后见双下肢浮肿、血尿明显，并有自噬足指、眼睛红色变淡、体质量明显减轻等表现，于术后5 d出现死亡，可能与术后泌尿系统感染、贫血有关；②无尿便：B2、C2组损伤较重，大部分术后3 d均无尿便，需行人工按摩促排便；③血尿：A组及大部分B1、C1轻度损伤的大鼠均无血尿，而B2、C2组损伤较重者，部分出现不同程度的血尿，经抗生素治疗及加强术后护理后，基本恢复；④伤口渗血：C2组出现一只伤口少量渗血，经碘伏消毒纱布包扎后，第2天未见渗血。 2.4.2 行为学评分 BBB评分：A组术后1 d，部分大鼠运动功能未完全恢复，术后3 d全部恢复到21分；余各组均出现不同程度的运动功能障碍，其中B2、C2组术后1 d，大鼠后肢几乎全瘫，术后3-7 d均出现不同程度的功能恢复(见图3a)，B1和C1、B2和C2同一时间点BBB评分比较，差异无显著性意义(P > 0.05)，余各组组间BBB评分比较，差异有显著性意义(P < 0.05)(见表1)，说明同一打击器，随着打击高度的增加，相应BBB评分则降低。感觉评分：A组术后1 d，全部感觉功能正常；余各组均出现不同程度的感觉功能障碍，其中B2、C2组术后1 d肌力、肌张力、牵张发射消失，部分大鼠可见背部感觉部分存在，疼痛回收迟钝，而B1、C1组术后1d肌力消失，肌张力、牵张反射、疼痛回缩反射、背部感觉存在，术后3-7 d均出现不同程度的功能恢复(见图3b)，B1和C1、B2和C2同一时间点感觉评分比较，差异无显著性意义(P > 0.05)，余各组组间感觉评分比较，差异有显著性意义(P < 0.05)，见表2。 2.4.3 组织学观察尼氏染色后，A组见脊髓灰、白质分界清晰，呈蝴蝶状，神经元细胞核大、核仁明显、数量较多，神经细胞质内有明显的斑块状或虎斑样尼氏体；余各组在灰质中央或两侧均有不同程度的淤血灶，组织破坏明显(见图4a)。损伤处可见灰质神经元肿胀，细胞间质水肿，轴突有脱髓鞘改变，部分细胞呈空泡样改变，神经元数目明显减少，尼氏体模糊或消失(见图4b)。 2.4.4 不同方法打击后脊髓受损面积比用Motic Med 6.0显微图像分析系统，选则手动分析法测得损伤处像素数与脊髓横截面总像素数，从而计算出受损面积比。A组未见明显受损区，比值为0；B1和B2、C1和C2组随打击的高度的增加而升高，组间差异有显著性意义(P < 0.05)，B1和C1、B2和C2损伤面积比差异无显著性意义(P > 0.05)，见表3。CV-B1=0.141，CV-B2=0.113， CV-C1=0.184，CV-C2=0.093，证明两种打击器每次打击的结果差异较小，稳定性较好。 2.4.5 不同方法打击后脊髓前、后角神经元计数用Motic Med 6.0显微图像分析系统，运用免疫组化图像分析法对前、后角神经元阳性目标进行半定量分析；A组前、后角神经元数目最多，尼氏体明显；B1和B2、C1和C2组随打击的高度的增加而减少，组间差异有显著性意义 (P < 0.05)，B1和C1、B2和C2损伤后前、后角神经元及尼氏体半定量分析差异无显著性意义(P > 0.05)，见表4。"

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