Abstract Body: Introduction: The cranial bone marrow has been recognized as a potential reservoir for myeloid cells, capable of entering the cerebral cortex through direct channels connecting the skull and cortical surface of the brain. The significance of cranial bone marrow-derived leukocytes following ischemic stroke in the setting of ischemia/reperfusion remains uncertain. We sought to characterize the fraction of cranial bone marrow-derived leukocytes entering the ischemic cortex following ischemia/reperfusion injury using a murine transient middle cerebral artery occlusion (tMCAO) model.
Methods: C57BL/6 LysM-GFP (green neutrophils and monocytes) mice underwent gamma irradiation with and without head shielding prior to adoptive transfer of bone marrow harvested from heterozygous CatchUp mice (Ly6G-tdTomato, red neutrophils). As a result, the exact percentages of cranial- and peripheral-derived neutrophils could easily be quantified and tracked, both in circulation and in the post-mortem brain. Mice underwent 90 min tMCAO with peripheral blood collection prior to and at 24 hours following ischemia/reperfusion. The relative fractions of green and red fluorescent Lys6G+ neutrophils post tMCAO in the brain were compared to the blood using flow cytometry and wide field fluorescence microscopy.
Results: The ratio of red and green Ly6G+ neutrophils in the brain most closely matched the ratio of circulating red and green neutrophils and was not particularly enriched in GFP+ neutrophils. The distribution of red and green cells in brain tissue was nearly identical and did not show an enrichment of either blood-derived or cranial bone marrow-derived cells in any particular region following tMCAO at 24 hours.
Conclusions: Together these data suggest that the majority of neutrophils entering the brain at 24 hours following stroke come from circulation with only a small contribution of neutrophils arriving directly from the cranial bone marrow. Furthermore, the distribution of red and green cells in tissue suggests that the neutrophils intermix regardless of origin. Further studies evaluating the subset of cranial bone marrow-derived neutrophils are needed to understand their significance specific to ischemia/reperfusion injury.