图1 反复轻度脑损伤导致小鼠死亡和行为学改变

Fig.1 Repeated mild traumatic brain injury (rmTBI) results in death and behavioral changes of mice. A: The free-fall device for inducing rmTBI in mice. B: Impact site on the skull of mice shown by the dashed line circle. C: Wound in the skull after rmTBI. D: The mortality rate of mice increases with the strike number. E: Neurological severity scores of the mice surviving rmTBI. F: The mice surviving rmTBI show delayed recovery of righting reflex (RORR). G: The mice surviving rmTBI show increased immobility time in forced swimming test. *P<0.05, ***P<0.001 vs sham group (nsham=8, nrmTBI-S=10-14).

图2 反复轻度脑损伤死亡组小鼠顶叶皮层和延髓组织病理学改变

Fig.2 Histopathological changes in the parietal cortex and medulla oblongata of the mice that died after rmTBI (Original magnification: ×20). A: No abnormalities are observed in the parietal cortical neurons in the sham group. B: Tissue defects, bleeding, neuronal edema and deposition of hemosiderin particles are observed in the parietal cortex of a mouse that did not survive rmTBI. C: No abnormalities are observed in the neurons and nerve fibers in the medulla oblongata of a sham-operated mouse. D: Loose, twisted and broken nerve fibers and swollen neurons are observed in the medulla oblongata of a mouse that did not survive rmTBI.

图3 反复轻度脑损伤死亡组小鼠顶叶皮层和延髓神经元尼氏小体消失

Fig.3 Loss of Nissl bodies in the neurons in the parietal cortex and medulla oblongata of the mice that did not survive rmTBI. The figures in the lower panel (B1-B4; ×40, scale bar=100 μm) are enlarged views of the boxed areas in A1-A4 (×20, scale bar=100 μm). C, D: Statistical analysis of positive cells in the parietal cortex (C) and medulla oblongata (D) (n=6). **P<0.01 vs sham group.

图4 Western blotting分析NLG-1、PSD-95蛋白表达

Fig.4 Western blotting of NLG-1 and PSD-95 in the parietal cortex and medulla oblongata of the mice. A-C: rmTBI decreases the expression levels of NLG-1 and PSD-95 in the parietal cortex, and their expression levels are lower in the mice that died after rmTBI (rmTBI-D group) than in the survivors (rmTBI-S group). D-F: rmTBI decreases the expression levels of NLG-1 and PSD-95 in the medulla oblongata, and their expression levels are lower in rmTBI-D than in rmTBI-S group. *P<0.05, ***P<0.001 vs sham group, #P<0.05, ##P<0.01 vs rmTBI-S group (n=3).

图5 顶叶皮层NLG-1和神经元标记物NeuN免疫荧光染色的共表达

Fig.5 Immunofluorescent co-expression of NLG-1 and neuronal marker NeuN in the parietal cortex (×40, scale bar=100 μm). A1-A3: Sham group; B1-B3: rmTBI-S group; C1-C3: rmTBI-D group; NeuN,DAPI and NLG-1 present green,blue and red fluorescence rspectively.

图6 顶叶皮层PSD-95和神经元标记物NeuN免疫荧光染色的共表达

Fig.6 Immunofluorescent co-expression of PSD-95 and NeuN in the parietal cortex (×40, scale bar=100 μm).A1-A3: the sham group; B1-B3: rmTBI-S group; C1-C3: rmTBI-D group; PSD-95 presents red fluorescence.

图7 NLG-1、PSD-95荧光强度统计分析

Fig.7 Statistical analysis of immunofluorescence intensity of NLG-1 and PSD-95. A, B: rmTBI decreases the integrated densities of NLG-1 and PSD-95 in the parietal cortex, which are lowered in rmTBI-D group than in rmTBI-S group. C, D: rmTBI decreases the integrated densities of NLG-1 and PSD-95 in the medulla oblongata, which are lower in rmTBI-D group than in rmTBI-S group. ***P<0.001 vs sham group, ##P<0.01, ###P<0.001 vs rmTBI-S group (n=8).