Abstract
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.
Highlights
Neutrophil infiltration under conditions of sterile inflammation can contribute to tissue injury
After 2 months, we induced ischemia and 4 days later we studied the brain by immunofluorescence (n = 5) (a, b) and flow cytometry (n = 5) (c, e). a CD45hiCD11bhi cells infiltrating the ipsilateral hemisphere are mostly DsRed+ whereas CD45dimCD11bdim microglial cells are DsRed−. b Flow cytometry shows an increase in infiltrating DsRed leukocytes (CD11bhiCD45hi) (Mann–Whitney test, **p = 0.008). c Immunostaining of astrocytes (GFAP, green) showed the presence of DsRed cells at the border and core of the lesion separated from the peripheral area that shows a prominent astroglial reaction. d Microglial cells were stained with an antibody against P2RY12, which did not co-localize with D sRed+ leukocytes
This study supports the concept that microglia phagocytose and remove neutrophils after brain ischemia [14, 50,51,52] and demonstrates that neutrophil accumulation in the brain parenchyma is associated with reduced microglial phagocytic activity, attributable to ischemia-induced microglial cell dysfunction due to loss or dystrophy
Summary
Neutrophil infiltration under conditions of sterile inflammation can contribute to tissue injury. Neutrophils are transiently detected in the brain after stroke since they are. Several aspects of neutrophil infiltration after acute ischemic brain damage remain controversial. The presence of neutrophils in the brain parenchyma has been reported in rodent models of permanent ischemia [23, 51, 55], but it is more controversial in experimental models of transient ischemia [17, 64]. Several studies reported the presence of neutrophils in the brain parenchyma in postmortem samples of patients deceased between day 1 and 5 [55], or 3 days after stroke onset but not at other time points [56, 76]. Neutrophils were not detected in the brain parenchyma of stroke patients [17]. The molecular determinants underlying perivascular neutrophil accumulation and the conditions facilitating the potential access of neutrophils to the brain parenchyma need further clarification
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