小鼠中度挫伤性胸脊髓损伤模型的构建和评价

Construction and evaluation of a moderate contusion model of thoracic spinal cord injury in mice

中文关键词:  脊髓损伤  动物模型  小鼠  三维步态

英文关键词:Spinal cord injury  Animal model  Mouse  3D gait analysis

中文摘要:

  【摘要】 目的：建立一种双侧中度挫伤性胸脊髓损伤小鼠模型，并动态评估其病理生理功能。方法：采用28只SPF级8~10周龄雌性C57BL/6小鼠，随机分为Ⅰ~Ⅳ组，其中Ⅰ组作为对照组不做击打处理，Ⅱ~Ⅳ组分别用Infinite Horizon（IH）装置对小鼠脊髓T10水平施加60、80、100kdyn的击打力度建立模型，建模后即刻、3、7、14、21及28d进行体重测量和BMS量表（Basso Mouse Scale）评分，评估小鼠营养状态和运动功能。在建模后28d使用步态分析系统和运动诱发电位定量评测运动模式及电生理变化，随后进行脊髓组织取样，采用苏木精伊红（hematoxylin eosin，HE）和免疫荧光染色观察组织改变。结果：除Ⅳ组外，其他组小鼠在28d内体重均有所上升，Ⅲ组的体重和BMS评分恢复情况显著优于Ⅳ组。步态分析表明，Ⅱ组在运动持续时间和速度上优于Ⅲ组，表现出更好的运动协调性，但与对照组差异不显著。HE染色观察结果，Ⅰ~Ⅳ组损伤面积分别为0mm2、0.27±0.05mm2、0.44±0.10mm2、0.67±012mm2，Ⅲ组的脊髓结构显示出与正常状态相似的形态特征。对NeuN、GFAP、MBP的免疫荧光染色结果显示了不同损伤程度的梯度变化，Ⅰ组的NeuN数、胶质瘢痕空洞面积、MBP荧光强度分别为115.25±13.04个、0μm2、1，Ⅱ组分别为71.00±7.79个、147.02±11.6μm2、0.86±0.05，Ⅲ组分别为51.50±8.89个、175.01±16.28μm2、0.76±0.04，Ⅳ组分别为16.00±3.37个、212.65±18.64μm2、0.55±0.07，Ⅲ组脊髓结构保留良好，胶质瘢痕空洞、神经元损失及脱髓鞘程度均呈可控梯度。电生理评估显示，Ⅰ~Ⅳ组运动诱发电位潜伏期分别为1.03±0.21ms、1.31±0.43ms、1.58±0.80ms、1.99±0.74ms，波峰分别为0.83±0.06mV、0.58±0.19mV、0.46±0.17mV、0.37±0.19mV，潜伏期在挫伤后有增加的趋势，提示神经冲动传导速度明显减慢，可能与髓鞘损伤或轴突功能障碍相关，波幅随着挫伤力度增加而下降，提示神经纤维的兴奋传导能力随损伤程度加重而减弱。结论：使用80kdyn的挫伤参数建立的小鼠双侧中度挫伤性胸脊髓损伤模型有效且可靠，能够更好地反映脊髓损伤后的病理生理变化。

英文摘要:

  【Abstract】 Objectives: To establish a bilateral moderate contusion thoracic spinal cord injury mouse model and dynamically assess the pathophysiological functions. Methods: 28 specific pathogen-free(SPF) female C57BL/6 mice aged 8-10 weeks were randomly divided intoⅠ-Ⅳ groups, among which, groupⅠreceived no impact and served as the control, groups Ⅱ-Ⅳ received spinal cord impacts at the T10 level using a trauma impactor-Infinite Horizon(IH) with forces of 60, 80, and 100kdyn, respectively. Body weight measurements and Basso Mouse Scale(BMS) scores were recorded immediately following spinal cord injury and on 3, 7, 14, 21, and 28d post-injury to assess the nutritional status and motor function of the mice. Additionally, the gait analysis system and motor evoked potential(MEP) were used on 28d post-injury to quantitatively evaluate motor patterns and electrophysiological changes, after that, spinal cord tissue sampling was performed for hematoxylin eosin(HE) staining and immunofluorescence tests to observe tissue changes. Results: Except for group Ⅳ, the body weight of mice in other groups increased within 28 days, with group Ⅲ showing significantly better recovery in body weight and BMS scores compared to group Ⅳ. Gait analysis indicated that group Ⅱ had superior motor endurance and speed compared to group Ⅲ, demonstrating better motor coordination, but with no significant difference from the control group. HE staining showed the lesion areas in groups Ⅰ-Ⅳ were 0mm2, 0.27±0.05mm2, 0.44±0.10mm2, and 0.67±0.12mm2, respectively, group Ⅲ′s spinal cord structure displayed morphological characteristics similar to the normal state. Immunofluorescence staining for NeuN, GFAP, and MBP revealed gradient changes reflecting varying degrees of injury. In group I, the NeuN-positive neuron count, glial scar/cavity area, and MBP fluorescence intensity were 115.25±13.04, 0μm2, and 1(normalized); In group Ⅱ, the values were 71.00±7.79, 147.02±11.60μm2, and 0.86±0.05; In group III, the values were 51.50±8.89, 175.01±16.28μm2, and 0.76±0.04; In group IV, the values were 16.00±3.37, 212.65±18.64μm2, and 0.55±0.07. Group Ⅲ exhibited well-preserved spinal cord structure, with the extent of glial scarring cavities, neuronal loss, and demyelination showing a controlled gradient. The motor evoked potential(MEP) latencies in groups Ⅰ-Ⅳ were 1.03±0.21ms, 1.31±0.43ms, 1.58±0.80ms, and 1.99±0.74ms, respectively, while the corresponding amplitudes were 0.83±0.06mV, 0.58±0.19mV, 0.46±0.17mV, and 0.37±0.19mV. The latency exhibited a tendency to prolong post-injury, suggesting a significant reduction in nerve impulse conduction velocity, which maybe associated with myelin sheath damage or axonal dysfunction. The amplitude decreased with increasing contusion force, indicating that the excitatory conduction capacity of nerve fibers was impaired progressively as injury severity increased. Conclusions: The bilateral thoracic moderate contusion model of spinal cord injury in mice established with an 80kdyn contusion parameter is effective and reliable, which better reflects the pathophysiological changes following spinal cord injury.