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. 2022 Oct 18;10:147. doi: 10.1186/s40478-022-01449-x

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© The Author(s) 2022

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 6 — Evaluation of the extent of white matter integrity at 6 months after injury in the corpus callosum. a–d Luxol fast blue/cresyl violet (LFB/CV) staining indicated changes in white matter integrity with a thinning of the corpus callosum (CC) by approximately 15% in the GW treated group, 40% in the r-mTBI group and 35% in the r-TBI/GW group (P < 0.0001) when to compared to Sham/V. aa Quantitative analysis of the thickness of the corpus callosum. (*P < 0.05, Two-Way ANOVA-followed by Tukey post hoc comparisons). A three-Way ANOVA analysis revealed a Sex and Injury effect: F_{(1, 56)} = 13.58 P=0.0005; F_{(1, 56)} = 87.58, P < 0.0001; but not Treatment effect F_{(1, 56)} = 0.68, P = 0.41. e, h Granular axonal profiles stained positively with the axonal injury marker (amyloid precursor protein [APP]) were predominantly seen in both r-mTBI groups within the CC when compared to their respective sham control. bb The density of APP-immunoreactive profiles per unit area in the body of the CC of the r-mTBI/GW group was greater than in the r-mTBI/V group (r-mTBI/V, 10.25 ± 1.0 vs r-mTBI/GW, 20.44 ± 2.3 axonal profiles/body of CC, P < 0.0001; Data are presented as mean ± standard error of the mean). Magnification of the boxed inset represents the area indicated by the arrow. A three-Way ANOVA analysis revealed no Sex effect F_{(1, 56)} = 2.6, P = 0.1, an Injury effect: F_{(1, 56)} = 290.3 P < 0.0001 and a Treatment effect; F_{(1, 56)} = 52.16 P < 0.0001. i, l The number of Oligodendrocytes cells was counted using the Oligo2 pan marker, a transcription factor that is expressed in both mature oligodendrocytes as well as oligodendrocyte precursor cells. bb At 6 months post injury an increase of the number of Oligo2⁺ cells were observed in the GW group with 35.7 cells per 200 µm², followed by the r-mTBI group with 41.7 cells per 200 µm² while the r-mTBI/GW group showed the highest number of positive cells 58.1 cells per 200 µm² (P < 0.0001, Two-Way ANOVA-followed by Tukey post hoc comparisons). A three-Way ANOVA analysis revealed no Sex effect F_{(1, 67)} = 0.13, P = 0.7, an Injury effect: F_{(1, 67)} = 208.0 P < 0.0001 and a Treatment effect; F_{(1, 56)} = 90.88, P < 0.0001. m–p Strong reactive astrogliosis was observed in the body of the CC in both r-mTBI groups when compared to the r-sham group while no difference was observed between the sham/V and the sham/GW. dd Quantitative analysis of glial fibrillary acidic protein (GFAP) staining in the body of the CC (Sham/V, 4.9 ± 0.3% vs Sham/GW, 8.4 ± 2.5%; P > 0.05; r-mTBI/V 13.3 ± 5.0% vs r-mTBI/GW, 24.6 ± 7.0%; P < 0.0001). A three-Way ANOVA analysis revealed a Sex effect F_{(1, 67)} = 0.13 P < 0.0001, an Injury effect: F_{(1, 67)} = 208.0 P < 0.0001 and a Treatment effect; F_{(1, 56)} = 90.88 P < 0.0001. q–t Iba-1 immunostaining showed moderate immunoreactivity in both TBI while no difference was observed between the Sham/V and the Sham/GW. ee Quantitative analysis of Iba-1 staining in the body of the CC (Sham/V, 1.54 ± 1.0% vs Sham/GWI, 1.79 ± 0.8%; P > 0.05; TBI/V 5.3 ± 1.5% vs r-mTBI/GW, 7.6 ± 1.4%; P=0.019). A three-Way ANOVA analysis revealed a Sex effect F_{(1, 79)} = 24.66 P < 0.0001, an Injury effect: F_{(1, 79)} = 98.5 P < 0.0001 and a Treatment effect; F_{(1, 79)} = 7.06 P=0.0095. u–y A second immunostaining with cluster of differentiation receptors 45 (CD45) probed for activated microglia and peripherical macrophage. ff Quantitative analysis of CD45 staining in the body of the CC (Sham/V, 0.97 ± 0.2 vs Sham/GW, 2.9 ± 0.49%; P > 0.05; r-mTBI/V 3.7 ± 0.07% vs r-mTBI/GW, 5.9 ± 0.12%; P = 0.042). A three-Way ANOVA analysis revealed no Sex effect F_{(1, 64)} = 0.32, P > 0.05, an Injury effect: F_{(1, 64)} = 23.97 P < 0.0001 and a Treatment effect; F_{(1, 79)} = 12.44, P = 0.008. Tissue sections were counterstained with hematoxylin. Data are presented as Whiskers: Min to Max. Show all points; symbol represents 1 mouse N = 8/12 per group per Sex)