摘要目的:构建反复性轻型颅脑损伤(rmTBI)后认知功能障碍小鼠模型，探讨其颅内早期病理改变。方法:将45只C57BL/6小鼠随机分为健康对照组( n＝15，简称对照组)与rmTBI建模组( n＝30，简称rmTBI组)。rmTBI组使用悬重式闭合性颅脑打击装置每日打击1次，连续7 d。建模后3、30 d进行头颅磁共振波谱扫描( n＝6)。建模后30 d采用转棒和Morris水迷宫实验评价两组小鼠的运动协调能力及认知功能。采用透射电子显微镜分析建模3 d rmTBI组( n＝3)、建模30 d rmTBI组( n＝3)和对照组( n＝3)小鼠内侧前额叶及海马CA2区域轴索外径、髓鞘厚度，并计算G-ratio[轴索外径/(轴索外径－2×髓鞘厚度)]。应用Spearman相关性检验分析轴索外径与髓鞘厚度的相关性。 结果:磁共振波谱结果显示，与对照组比较，rmTBI组建模3、30 d的皮质N-乙酰天冬氨酸/肌酸(NAA/Cr)值和海马NAA/Cr值的差异均无统计学意义(均 P>0.05)。转棒实验和Morris水迷宫实验结果显示，与对照组比较，rmTBI组小鼠的运动协调和认知功能均下降(均 P<0.05)。电子显微镜检查结果显示，在内侧前额叶区域，建模3 d rmTBI组、建模30 d rmTBI组与对照组神经轴索外径的差异无统计学意义( H＝2.11， P＝0.347)；三组间髓鞘厚度的差异具有统计学意义( H＝－2.52， P＝0.035)，其中建模30 d rmTBI组的髓鞘厚度小于对照组[ M( Q1， Q3)分别为0.11(0.09，0.13)μm、0.12(0.11，0.14)μm]。在海马CA2区域，建模3 d和30 d rmTBI组均可观察到髓鞘板层结构中存在空泡样改变，同时存在包绕轴索和髓鞘剥离现象；三组间轴索外径及髓鞘厚度的差异均具有统计学意义(均 P<0.05)，其中建模30 d rmTBI组的轴索外径小于建模3 d rmTBI组[0.67(0.59，0.84)μm对比0.82(0.72，0.99)μm]，建模3 d rmTBI组的髓鞘厚度[0.12(0.11，0.14)μm]大于对照组[0.11(0.09，0.12)μm]和建模30 d rmTBI组[0.08(0.06，0.11)μm]。与对照组比较，建模3 d和30 d rmTBI组小鼠海马CA2区域G-ratio的差异具有统计学意义( Z＝4.25， P<0.001)，其中建模30 d rmTBI组最高；而三组间内侧前额叶区域的差异无统计学意义( Z＝1.55， P＝0.267)。相关性分析结果显示，对照组、建模3 d和30 d rmTBI组小鼠内侧前额叶和海马CA2区域的轴索外径与髓鞘厚度均呈正相关(均 P<0.05)，其中对照组内侧前额叶区域的相关性最强( R＝0.66)，建模3 d rmTBI组小鼠海马CA2区域的相关性最小( R＝0.33)。 结论:rmTBI小鼠伤后30 d运动协调能力和认知功能均明显下降，动物模型病理结果提示其内侧前额叶存在少量髓鞘的损伤，但轴索结构相对完好，而海马CA2区域轴索－髓鞘结构不稳定，有脱髓鞘和髓鞘新生表现，可能与上述功能障碍存在因果关系。

abstractsObjective:To establish a mouse model of cognitive impairment following repetitive mild traumatic brain injury (rmTBI) and to investigate its intracranial early pathological changes.Methods:Forty-five C57BL/6 mice were randomly divided into a healthy control group ( n=15, referred to as the control group) and an rmTBI modeling group ( n=30, referred to as the rmTBI group). The rmTBI group underwent repetitive closed head impacts using a weight-drop device once a day for seven consecutive days. At 3 and 30 days post injury, head MRS scans were performed ( n=6). At 30 days post injury, motor coordination and cognitive function of the two groups of mice were evaluated using the rotarod and Morris water maze experiments. Transmission electron microscopy was used to analyze the axonal outer diameter and myelin sheath thickness in the medial prefrontal cortex and hippocampal CA2 region of mice in the rmTBI group at 3 days post injury ( n=3), the rmTBI group at 30 days post injury ( n=3) and the control group ( n=3), with the calculation of the G-ratio [axonal outer diameter / (axonal outer diameter-2 × myelin sheath thickness)]. Spearman′s correlation analysis was conducted for axonal outer diameter and myelin sheath thickness. Results:MRS (magnetic resonance spectroscopy) results showed that there were no statistically significant differences in cortical NAA/Cr values or hippocampal NAA/Cr values between the control group and the rmTBI group at 3 and 30 days post-injury (all P>0.05). The results of the rotarod and Morris water maze experiments indicated that the motor coordination and cognitive function of mice in the rmTBI group were impaired compared to the control group (all P<0.05). Electron microscopic examination revealed that in the medial prefrontal cortex, there were no statistically significant differences in the neuronal axonal outer diameter between the rmTBI group at 3 days post-injury, the rmTBI group at 30 days post-injury, and the control group ( H=2.11, P=0.347). However, there was a significant difference in myelin sheath thickness among the three groups ( H=-2.52, P=0.035), with the 30 days post-injury rmTBI group exhibiting smaller myelin sheath thickness compared to the control group [median( Q1, Q3): 0.11 (0.09, 0.13) μm and 0.12 (0.11, 0.14)μm, respectively]. In the hippocampal CA2 region, both the 3 days post-injury and 30 days post-injury rmTBI groups exhibited vacuolar changes in the myelin sheath layers, along with axonal wrapping and myelin sheath detachment. There were statistically significant differences in axonal outer diameter and myelin sheath thickness among the three groups (all P<0.05), with the 30 days post-injury rmTBI group showing the smallest axonal outer diameter compared to the 3 days post-injury rmTBI group [0.67 (0.59, 0.84)μm vs. 0.82 (0.72, 0.99)μm], and the myelin sheath thickness of the 3 days post-injury rmTBI group [0.12 (0.11, 0.14)μm] was greater than that of the control group [0.11 (0.09, 0.12)μm] and the 30 days post-injury rmTBI group [0.08 (0.06, 0.11)μm]. The G-ratio in the hippocampal CA2 region was significantly different between the control group and both the 3 days post-injury and 30 days post-injury rmTBI groups ( Z=4.25, P<0.001), with the highest value observed in the 30 days post-injury rmTBI group; whereas, there was no statistically significant difference in the medial prefrontal cortex region among the three groups ( Z=1.55, P=0.267). The correlation analysis results indicated a positive correlation between axonal outer diameter and myelin sheath thickness in the medial prefrontal cortex and hippocampal CA2 region of mice in the control group, and the 3 days and 30 days post-injury rmTBI groups (all P<0.05), with the strongest correlation observed in the medial prefrontal cortex region of the control group ( R=0.66) and the weakest correlation seen in the hippocampal CA2 region of the 3 days post-injury rmTBI group ( R=0.33). Conclusions:At 30 days following rmTBI, mice exhibit significantly decreased motor coordination and cognitive function. The pathological findings in the animal model suggest mild damage to the myelin sheath in the medial prefrontal cortex, with relatively intact axonal structures, while the hippocampal CA2 region exhibits unstable axon-myelin sheath structures, demyelination, and myelin regeneration, indicating a causal relationship with the aforementioned functional impairments.