Fig 2: TLR4 deficiency attenuated cognitive impairment and white matter lesions induced by BCAS. (A) The working memory and reference memory of mice were assessed by the 8-arm maze test at 1-month post injury. WT mice suffered from BCAS made much more revisiting errors (P<0.001) and less different arm choices (P<0.001) comparing to the sham-operated mice. TLR4 knockout mice with BCAS made much less revisiting errors (P<0.001) and more different arm choices (P=0.007) comparing to the WT group. No impairment in spatial reference memory was revealed between different groups (P>0.05). Two-way analysis of variance (ANOVA) with repeated analysis, n=9 in each group. (B) White matter lesions were detected by LFB staining in different groups. Scale bar, 200 µm. Dot-plot with median and interquartile range of the severity of white matter lesions in in CCm, CPu, AC, IC, F and OT in histogram. Two-way ANOVA with Dunnett's post-hoc test, **P<0.01 versus WT Sham, #P<0.05, ##P<0.01 versus WT BCAS. n=6 per group. (C) Representative images depicting immunofluorescent labeling of myelin-associated glycoprotein (MAG), myelin basic protein (MBP), and nerve fiber (NF) in the corpus callosum of coronal slices from different groups. Scale bar, 100µm. The expression of MAG, MBP and NF (three major NF subunits based upon their molecular mass: the lowest (NF-L), the middle (NF-M) and the highest (NF-H)) was determined by Western blot in mice from different groups. Quantitative analysis of Western blot results was performed. Two-way ANOVA with Dunnett's post-hoc test, **P?0.01 versus WT Sham, ##P<0.01 versus WT BCAS. n=8 per group.

Fig 3: H&E staining, LFB staining, Masson staining and immunocytochemistry staining of spinal cord at 6-week after SCI. (A-C) The longitudinal sections of spinal cord were analyzed by H&E staining, Luxol fast blue staining, Masson staining. Scale bar: 500 μm; (D-E) Immunohistochemistry assay was performed to detect NF and GFAP positive cells in transections of spinal cord. Scale bar: 25 μm; (I) Schematic diagram of spinal cord. The areas of lesion cavity (F), myelin (G), collagen (H), NF-H (J) and GFAP (K) were analyzed by IPP software. Each data point was presented as mean± SEM (n=3, *P< 0.05, **P< 0.01).

Fig 4: CB2R ameliorated secondary neuronal apoptosis and demyelination post-SCI. (A) LFB staining of spinal cords on day 7 post-SCI. Scale bar = 200 μm. (B) Quantitative analysis of LFB-labeled area. (C) IF staining of NF200 or myelin basic protein (green) on day 7 post-SCI. Scale bar = 500 μm. (D) Quantitative analysis of NF200. (E) Quantitative analysis of myelin basic protein. (F) TUNEL staining of spinal cords on day 7 post-SCI. Scale bar = 100 μm. (G) Quantitative analysis of TUNEL-positive cells. (H) IF staining of Neun (green) and Annexin V (red) on day 7 post-SCI. Scale bar = 100 μm. (I) Quantitative analysis of Annexin V-positive neurons. (J) Nissl staining of spinal cords on day 7 post-SCI. Scale bar = 200 μm. (K) Quantitative analysis of the number of neurons. * p < 0.05. n.s means no significance.

Fig 5: Immunohistochemical and Glees-Marsland staining of the hippocampal CA1 region and brainstem. The axons became varicose, swollen and presented the formation of axonal retraction balls in the injured group (magnification, ×400). (A) The axonal pathological alterations following DAI, as conducted by Glees-Marsland silver staining (white arrowheads, axonal retraction balls; black arrowheads, axonal varicose; black triangles, axonal swelling). (B) The axonal pathological alterations following DAI as conducted by immunohistochemical staining on NF-H (white arrowheads, axonal retraction balls; black arrowheads, axonal varicose; black triangles, axonal swelling). (C) Bar chart presenting the number of axonal retractions balls in the field of view of the hippocampal CA1 region and brainstem in Sham, DAI+Vehicle and DAI+FK506 groups. (D) Bar chart presenting the amount of axonal swelling in the field of view of the hippocampal CA1 region and brainstem in Sham, DAI+Vehicle and DAI+FK506 groups. *P<0.05 vs. Sham group; #P<0.05 vs. DAI+Vehicle group. DAI, diffuse axonal injury; NF-H, neurofilament-H.