Neutrophils are rapidly recruited in response to local tissue infection or inflammation. Stroke triggers a strong inflammatory reaction but the relevance of neutrophils in the ischemic brain is not fully understood, particularly in the absence of reperfusion. We investigated brain neutrophil recruitment in two murine models of permanent ischemia induced by either cauterization of the distal portion of the middle cerebral artery (c-MCAo) or intraluminal MCA occlusion (il-MCAo), and three fatal cases of human ischemic stroke. Flow cytometry analyses revealed progressive neutrophil recruitment after c-MCAo, lesser neutrophil recruitment following il-MCAo, and absence of neutrophils after sham operation. Confocal microscopy identified neutrophils in the leptomeninges from 6 h after the occlusion, in the cortical basal lamina and cortical Virchow-Robin spaces from 15 h, and also in the cortical brain parenchyma at 24 h. Neutrophils showed signs of activation including histone-3 citrullination, chromatin decondensation, and extracellular projection of DNA and histones suggestive of extracellular trap formation. Perivascular neutrophils were identified within the entire cortical infarction following c-MCAo. After il-MCAo, neutrophils prevailed in the margins but not the center of the cortical infarct, and were intraluminal and less abundant in the striatum. The lack of collaterals to the striatum and a collapsed pial anastomotic network due to brain edema in large hemispheric infarctions could impair neutrophil trafficking in this model. Neutrophil extravasation at the leptomeninges was also detected in the human tissue. We concluded that neutrophils extravasate from the leptomeningeal vessels and can eventually reach the brain in experimental animal models and humans with prolonged arterial occlusion.

Stroke attracts neutrophils to the injured brain tissue where they can damage the integrity of the blood–brain barrier and exacerbate the lesion. However, the mechanisms involved in neutrophil transmigration, location and accumulation in the ischemic brain are not fully elucidated. Neutrophils can reach the perivascular spaces of brain vessels after crossing the endothelial cell layer and endothelial basal lamina of post-capillary venules, or migrating from the leptomeninges following pial vessel extravasation and/or a suggested translocation from the skull bone marrow. Based on previous observations of microglia phagocytosing neutrophils recruited to the ischemic brain lesion, we hypothesized that microglial cells might control neutrophil accumulation in the injured brain. We studied a model of permanent occlusion of the middle cerebral artery in mice, including microglia- and neutrophil-reporter mice. Using various in vitro and in vivo strategies to impair microglial function or to eliminate microglia by targeting colony stimulating factor 1 receptor (CSF1R), this study demonstrates that microglial phagocytosis of neutrophils has fundamental consequences for the ischemic tissue. We found that reactive microglia engulf neutrophils at the periphery of the ischemic lesion, whereas local microglial cell loss and dystrophy occurring in the ischemic core are associated with the accumulation of neutrophils first in perivascular spaces and later in the parenchyma. Accordingly, microglia depletion by long-term treatment with a CSF1R inhibitor increased the numbers of neutrophils and enlarged the ischemic lesion. Hence, microglial phagocytic function sets a critical line of defense against the vascular and tissue damaging capacity of neutrophils in brain ischemia. Electronic supplementary material The online version of this article (10.1007/s00401-018-1954-4) contains supplementary material, which is available to authorized users.