Fig. 7

The neuroprotective effect of B cells in TBI requires infiltrating peripheral monocytes/ macrophages at the site of injury. A Overview of the CCI injury site at 18 h, showing abundant infiltration of CD11b⁺ monocytes (red) throughout the region. The dashed line indicates the pial surface of the injured brain hemisphere. The asterisk indicates the hippocampus. Under the selected imaging conditions, CD11b immunolabeling preferentially identified large infiltrating myeloid cells, rather than resident microglia, which are not apparent, likely due to lower expression of this marker. B Intravenous administration of clodronate liposomes 18 h prior to CCI resulted in the specific depletion of CD45^hiCD11b⁺ infiltrating immune cells in the injured brain hemisphere. No reduction in cell numbers was observed in the infiltrating CD11b^low/− lymphocyte population. C, D. Quantitative analysis confirmed a significant (~ 90%) depletion of Ly6C⁺CD11b⁺ classical monocytes in the brain as well as in the spleen of animals treated with a single dose of clodronate 18 h prior to CCI (p < 0.01, two-tailed unpaired t-test; N = 4 animals/ treatment). E Clodronate administration did not deplete the resident microglia in the injured hemisphere. F, G Quantitative analysis confirmed no significant depletion of CD45^midCD11b⁺ microglia, either as a proportion of total CD45⁺ immune cells F, or in absolute cell counts G (p < 0.01, two-tailed unpaired t-test; N = 4 animals/ treatment). H Experimental paradigm for assessing the impact of clodronate-mediated depletion of peripheral myeloid cells on the functional neuroprotective effect of B cell administration. I, J Rotarod assessment of motor learning and coordination. In injured animals (N = 10/group) that received injections with control liposomes, B cell administration was associated with strong functional neuroprotection, with treated animals performing significantly better than saline-treated controls, and indistinguishable from sham-injured controls (p < 0.001 interaction, two-way repeated-measures ANOVA followed by Tukey's multiple comparisons test). This effect was maintained up to 72 days post-injury I. By contrast, in animals treated with clodronate liposomes 18 h before and during the first week post-CCI, the neuroprotective effect of B cell treatment observed in this test was mostly abolished J. K, L Y-maze assessment of short-term spatial learning. K The neuroprotective effect of B cell treatment was lost in injured animals treated with clodronate, which performed similar to saline-treated injured controls and significantly worse than sham-injured controls. In the control condition, when myeloid cells were not depleted, injured B cell-treated animals performed significantly better than saline-treated injured controls and similarly to sham-injured animals (p < 0.0001, three-way ANOVA, followed by Tukey's multiple comparisons test). L No difference was observed in the number of total arm entries between any of the injured and sham groups, indicating no bias in total motor and exploratory activity between treatment groups. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001