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中华神经创伤外科电子杂志

中华神经创伤外科电子杂志 ›› 2023, Vol. 09 ›› Issue (06) : 325 -332. doi: 10.3877/cma.j.issn.2095-9141.2023.06.002

基础研究

改良大鼠挫伤型脊髓损伤模型的制备与评估
陈业煌, 陈恺钦, 薛亮, 吴箭午, 黄预备, 魏梁锋†(), 曾炳香, 王守森   
  1. 350025　福州，福建医科大学福总临床医学院（联勤保障部队第九〇〇医院）神经外科
  • 收稿日期:2022-11-09 出版日期:2023-12-15
  • 通信作者: 魏梁锋

Preparation and evaluation of an improved model of contusion spinal cord injury in rats

Yehuang Chen, Kaiqin Chen, Liang Xue, Jianwu Wu, Yubei Huang, Liangfeng Wei†(), Bingxiang Zeng, Shousen Wang   

  1. Department of Neurosurgery, Fuzong Clinical Medical College of Fujian Medical University (The 900th Hospital of Joint Logistic Team of PLA), Fuzhou 350025, China
  • Received:2022-11-09 Published:2023-12-15
  • Corresponding author: Liangfeng Wei
  • Supported by:
    Natural Science Foundation of Fujian Province(2021J011276); The Project of The 900th Hospital of Joint Logistic Team(2020L18); The Project of Joint Logistics Key Specialized Medicine(LQZD-SW)
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陈业煌, 陈恺钦, 薛亮, 吴箭午, 黄预备, 魏梁锋, 曾炳香, 王守森. 改良大鼠挫伤型脊髓损伤模型的制备与评估[J]. 中华神经创伤外科电子杂志, 2023, 09(06): 325-332.

Yehuang Chen, Kaiqin Chen, Liang Xue, Jianwu Wu, Yubei Huang, Liangfeng Wei, Bingxiang Zeng, Shousen Wang. Preparation and evaluation of an improved model of contusion spinal cord injury in rats[J]. Chinese Journal of Neurotraumatic Surgery(Electronic Edition), 2023, 09(06): 325-332.

目的

利用新型脊髓损伤（SCI）打击器制备大鼠挫伤型SCI模型，并对模型进行评估，为探究SCI后神经病理性疼痛（NP）发生机制及早期干预治疗奠定基础。

方法

100只健康成年雄性SD大鼠按随机数字表法分为Control组（对照组，20只）、Sham组（假手术组，40只）及SCI组（40只）。SCI组大鼠暴露T10节段并采用改进型Allen's法制备SCI模型，假手术组只暴露T10节段但不实施脊髓撞击，Control组不做任何干预措施。使用开放场地实验（BBB评分）评估伤前1 d及伤后1、3、7、14、21、28 d大鼠的运动功能。于伤前1 d及伤后14、21、28 d测定后肢机械缩足阈值（MWT），并采用苏木精-伊红（HE）染色检测脊髓组织病理学变化，评估SCI模型是否构建成功。

结果

（1）3组大鼠伤后1、3、7、14、21、28 d的BBB评分比较，差异均有统计学意义（P<0.05），其中SCI组的BBB评分均低于Control组和Sham组，差异有统计学意义（P<0.05）。SCI组伤后1 d的BBB评分均为0分，伤后3、7、14、21、28 d呈上升趋势，且任意两个时间点比较差异均有统计学意义（P<0.05）。（2）3组大鼠伤前1 d后肢MWT比较差异无统计学意义（P>0.05），伤后14、21、28 d后肢MWT比较，差异有统计学意义（P<0.05），且SCI组大鼠后肢MWT均明显低于Control组和Sham组，差异有统计学意义（P<0.05）。SCI组大鼠伤前1 d及伤后14、21、28 d时的后肢MWT呈现先下降后回升趋势，伤后21和28 d比较差异无统计学意义（P>0.05），其余各时间点后肢MWT比较差异均有统计学意义（P<0.05）。（3）伤后14 d，HE染色可见脊髓胶质细胞大量浸润；伤后21 d胶质细胞大量分化增生；伤后28 d大量胶质纤维生成。

结论

利用新型打击器建立的挫伤型SCI模型，具有数字化、稳定、标准化、操作性强、重复性好的特点，可为SCI后NP形成的病理学机制及靶向药物研究提供标准化的模型。

关键词: 挫伤型脊髓损伤, 改良动物模型, 神经病理性疼痛, 机械缩足阈值
Objective

To prepare a rat contusion SCI model using a novel spinal cord injury (SCI) impactor and evaluate the model, in order to lay the foundation for exploring the mechanism of neuropathic pain after SCI and early intervention and treatment.

Methods

One hundred adult male SD rats were randomly divided into Control group (n=20), Sham group (n=40) and SCI group (n=40). Rats in SCI group were exposed to T10 segments and the SCI model was established by modified Allen's method. Rats in Sham group were exposed to T10 segments without SCI, while rats in Control group were not intervened. Open field test (BBB score) was used to evaluate the motor function of rats 1 d before injury and 1, 3, 7, 14, 21 and 28 d after injury. The mechanical foot retraction threshold (MWT) of the hind limb was measured 1 d before injury and 14, 21 and 28 d after injury, and the histopathological changes of the spinal cord were detected by hematoxylin eosin (HE) staining to evaluate whether the SCI model was successfully constructed.

Results

(1) The BBB scores of three groups of rats at 1, 3, 7, 14, 21, and 28 d after injury were compared, and the differences were statistically significant (P<0.05). Among them, the BBB scores of the SCI group were lower than those of the Control group and the Sham group, and the differences were statistically significant (P<0.05). The BBB scores of the SCI group were all 0 points on the 1st day after injury, and showed an upward trend at 3, 7, 14, 21, and 28 d after injury, and the difference between any two time points in pairwise comparison was statistically significant (P<0.05). (2) There was no statistically significant difference in rat hindlimb MWT among the three groups 1 d before injury (P>0.05), but there was a statistically significant difference at 14, 21, and 28 d after injury (P<0.05). Moreover, the rat hindlimb MWT of SCI group was significantly lower than that of the Control group and Sham group, and the difference was statistically significant (P<0.05). The rat hindlimb MWT of SCI group rats showed a trend of first decreasing and then rebounding at 1 d before injury and 14, 21, and 28 d after injury. Except for no statistically significant difference between 21 d after injury and 28 d after injury (P>0.05), there were statistically significant differences in hindlimb MWT at all other time points (P<0.05). (3) On the 14 d after injury, HE staining showed extensive infiltration of spinal cord glial cells. A large number of glial cells differentiated and proliferated 21 d after injury, and a large amount of glial fibers were generated 28 d after injury.

Conclusion

The contusion SCI model established by using the new percussion device has the advantages of digitalization, stability, standardization, strong operability and good repeatability, can provide a standardized model for the pathological mechanism of neuropathic pain formation after SCI and the study of targeted drugs.

Key words: Contusion spinal cord injury, Improved animal model, Neuropathic pain, Mechanical withdrawal threshold
图1 大鼠T10棘突、脊髓暴露及SCI前后大体形态变化A：大鼠T10棘突（针头指示处）；B：暴露T10节段脊髓，硬脊膜及脊髓完整；C：打击器精准定位脊髓；D：打击器校正定位；E：脊髓后静脉形态清晰完整；F：打击后，脊髓后静脉形成淤血灶（箭头所示）
Fig.1 Exposure of T10 spinous process and spinal cord in rats, gross morphological changes before and after SCI
表1 3组大鼠SCI后BBB评分比较（分，±s）
Tab.1 Comparison of BBB scores after SCI in 3 groups (score, Mean±SD)
组别 只数 伤后1 d 伤后3 d 伤后7 d 伤后14 d 伤后21 d 伤后28 d F值 P值
Control组 5 20.60±0.89 20.60±0.89 20.60±0.89 20.60±0.89 20.60±0.55 20.60±0.55 <0.001 1.000
Sham组 10 20.20±0.92 20.20±1.40 20.00±1.49 20.50±0.85 20.70±0.67 20.80±0.63 2.978 0.128
SCI组 10 0.00±0.00ab 0.40±0.10ab 3.60±1.07ab 10.50±1.08ab 15.90±2.33ab 18.70±1.57ab 464.306 <0.001
F值   2527.148 1040.879 541.671 328.933 27.675 10.199    
P值   <0.001 <0.001 <0.001 <0.001 <0.001 0.001    
表2 3组大鼠SCI前后MWT比较（g，±s）
Tab.2 Comparison of MWT before and after SCI in 3 groups (g, Mean±SD)
组别 只数 伤前1 d 伤后14 d 伤后21 d 伤后28 d F值 P值
对照组 5 17.00±2.74 17.00±2.74 18.00±2.74 17.00±2.74 0.146 0.930
假手术组 10 17.50±2.64 16.50±2.42 16.00±2.11 17.50±2.64 1.976 0.167
SCI组 10 16.50±2.42 7.60±0.97abc 9.60±0.70abcd 9.90±0.32abcd 82.375 <0.001
F值   0.379 59.787 46.271 39.143    
P值   0.689 <0.001 <0.001 <0.001    
图2　大鼠SCI后脊髓背角灰白交界处神经元及胶质细胞形态结构变化（HE染色，×400）；图3　大鼠SCI后脊髓背角白质内组织结构及细胞形态学变化（HE染色，×400）；图4　大鼠SCI后脊髓背角灰质内神经细胞形态及组织结构变化（HE染色，×400）2A：假手术组神经元形态规则，灰白质界限明显；2B：伤后14 d白质结构逐渐稀疏，小胶质细胞浸润；2C：伤后21 d神经元肿胀变性，灰白质界限模糊；2D：伤后28 d灰白质结构疏松，大量胶质细胞浸润；3A：假手术组白质结构清晰；3B：伤后14 d白质结构疏松，小角质细胞大量浸润；3C：伤后21 d白质部髓鞘组织肿胀明显；3D：伤后28 d白质部大量胶质纤维生成；4A：假手术组灰质结构清晰；4B：伤后14 d灰质内部分神经元肿胀、变性；4C：伤后21 d灰质部神经元明显凋亡减少；4D：伤后28 d灰质部大量胶质纤维生成
Fig.2 Morphological and structural changes of neurons and glial cells in the dorsal horn of spinal cord after SCI (HE, ×400); Fig.3　Changes of tissue structure and cell morphology in white matter of dorsal horn of spinal cord after SCI (HE, ×400); Fig.4　Morphological and structural changes of neurons in the gray matter of dorsal horn of spinal cord after SCI (HE, ×400)
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