Repeated mild traumatic brain injury in the parietal cortex inhibits expressions of NLG-1 and PSD-95 in the medulla oblongata of mice

doi:10.12122/j.issn.1673-4254.2024.05.18

Abstract

Abstract:

Objective To assess the effects of repeated mild traumatic brain injury (rmTBI) in the parietal cortex on neuronal morphology and synaptic plasticity in the medulla oblongata of mice. Methods Thirty-two male ICR mice were randomly divided into sham operation group (n=8) and rmTBI group (n=24). The mice in the latter group were subjected to repeated mild impact injury of the parietal cortex by a free-falling object. The mice surviving the injuries were evaluated for neurological deficits using neurological severity scores (NSS), righting reflex test and forced swimming test, and pathological changes of the neuronal cells in the medulla oblongata were observed with HE and Nissl staining. Western blotting and immunofluorescence staining were used to detect the expressions of neuroligin 1 (NLG-1) and postsynaptic density protein 95 (PSD-95) in the medulla oblongata of the mice that either survived rmTBI or not. Results None of the mice in the sham-operated group died, while the mortality rate was 41.67% in rmTBI group. The mice surviving rmTBI showed significantly reduced NSS, delayed recovery of righting reflex, increased immobility time in forced swimming test (P<0.05), and loss of Nissl bodies;swelling and necrosis were observed in a large number of neurons in the medulla oblongata, where the expression levels of NLG-1 and PSD-95 were significantly downregulated (P<0.05). The mice that did not survive rmTBI showed distorted and swelling nerve fibers and decreased density of neurons in the medulla oblongina with lowered expression levels of NLG-1 and PSD-95 compared with the mice surviving the injuries (P<0.01). Conclusion The structural and functional anomalies of the synapses in the medulla oblongata may contribute to death and neurological impairment following rmTBI in mice.

Key words: repeated mild traumatic brain injury, medulla oblongata, neuroligin 1, postsynaptic density protein 95, synaptic plasticity

Mingming LI, Liangchao HE, Tianyu LI, Yan BAO, Xiang XU, Guang CHEN. Repeated mild traumatic brain injury in the parietal cortex inhibits expressions of NLG-1 and PSD-95 in the medulla oblongata of mice[J]. Journal of Southern Medical University, 2024, 44(5): 960-966.

Figures/Tables 7

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).

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.

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.

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).

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.

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.

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).

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