Modulation of Brain Injury After Intracerebral Hemorrhage via the Scalp-Skull-Dura Interface in Mice.

Abstract

Background: Neuroinflammation critically contributes to secondary brain injury after intracerebral hemorrhage (ICH). The skull-dura interface allows immune trafficking from skull bone marrow (SBM) to the brain, but its role in ICH remains unclear.

Methods: ICH was induced in male C57BL/6J mice by collagenase injection. To compare the immune landscape of SBM and femur bone marrow (FBM), single-cell RNA sequencing was performed on sham-operated and ICH mice. Flow cytometry at serial time points, combined with cell-tracking experiments, was used to assess time-dependent neutrophil accumulation in SBM, dura, and brain, as well as the relative contribution of SBM- versus FBM-derived neutrophils after ICH. To test whether SBM-derived neutrophil migration contributes to neuroinflammation, the CXCR2 (C-X-C motif chemokine receptor 2) antagonist SB225002 was delivered subscalp. Finally, dural delivery of anti-CCL3 (C-C motif chemokine ligand 3) neutralizing antibodies was performed to determine the role of dural CCL3 in SBM neutrophil priming. Outcomes included evaluations of behavioral deficits, brain edema, integrity of the blood-brain barrier, hematoma removal, and brain neutrophil infiltration.

Results: Single-cell RNA sequencing revealed that ICH shifted hematopoiesis toward the myeloid lineage in both SBM and FBM; however, SBM neutrophils exhibited significantly stronger proinflammatory and migratory gene signatures than FBM neutrophils. Skull-derived neutrophils preferentially migrated to the dura and brain parenchyma post-ICH. Subscalp delivery of SB225002 reduced neutrophil infiltration, attenuated brain edema and blood-brain barrier disruption, and improved short-term and long-term neurological deficits. In addition, CCL3 was specifically upregulated in dural neutrophils after ICH. Unexpectedly, dural delivery of an anti-CCL3 antibody markedly reduced neutrophil accumulation in the brain and protected against ICH-induced brain injury, accompanied by inhibition of neutrophil activation in SBM but not in FBM.

Conclusions: SBM may serve as a primary source of pathogenic neutrophils driving neuroinflammation after ICH. Targeting this immune compartment through the scalp-skull-dura interface represents an accessible strategy for mitigating ICH-induced brain injury.