Delayed adoptive transfer of bone marrow-derived macrophages modulates post-ischemic inflammation in a model of neonatal hypoxia-ischemia

Neonatal hypoxic-ischemia (HI) represents a major cause of brain injury in the perinatal period. Post-ischemic neuroinflammation plays a key role in HI pathophysiology and is characterized by the infiltration of peripheral immune cells in support of resident microglia. To investigate the effects of peripheral macrophages as a possible treatment for HI, 10-day old CX3CXR1^GFP/+/CCR2^RFP/+ mice were subjected to electrocoagulation of the right carotid artery and 1 hour hypoxia with 10 % oxygen. CCR2^RFP/+ cell migration to the brain was assessed by flow cytometry 1, 3, 5, 7, 10 and 14 days after HI, and a biphasic infiltration pattern was identified. Five days post-surgery injured and sham pups received an intraperitoneal administration of bone marrow-derived macrophages (BMDMs) previously polarized into M0 or M2 states in vitro. Open field, beam walk and rotarod behavioral tests were performed 3 weeks after HI, and brains were then collected to assess injury extent. Injured mice treated with M2 cells showed significant functional recovery and reduced brain atrophy specifically in males. In contrast, treatment with M0 cells led to a significant worsening of behavioral performances and a greater brain injury specifically in females if compared to HI mice receiving PBS. To further define BMDM plasticity in a post-ischemic environment, M0 and M2 cells were co-cultured in vitro with hippocampal organotypic slices previously subjected to oxygen-glucose deprivation. After 48 h, M2 cells showed significant downregulation of Il1b, Cd68 and Tnfa, while significant upregulation of Il1b, Il6 and Ccl2 was observed in M0 cells, suggesting their switch into a M1 pro-inflammatory polarization state. Overall, these results suggest the therapeutic potential of M2 BMDMs for neonatal HI.