BackgroundThe brain’s initial innate response to stroke is primarily mediated by microglia, the resident macrophage of the CNS. However, as early as 4 h after stroke, the blood–brain barrier is compromised and monocyte infiltration occurs. The lack of discriminating markers between these two myeloid populations has led many studies to generate conclusions based on the grouping of these two populations. A growing body of evidence now supports the distinct roles played by microglia and monocytes in many disease models.MethodsUsing a flow cytometry approach, combined with ex-vivo functional assays, we were able to distinguish microglia from monocytes using the relative expression of CD45 and assess the function of each cell type following stroke over the course of 7 days.ResultsWe found that at 72 h after a 90-min middle cerebral artery occlusion (MCAO), microglia populations decrease whereas monocytes significantly increase in the stroke brain compared to sham. After stroke, BRDU incorporation into monocytes in the bone marrow increased. After recruitment to the ischemic brain, these monocytes accounted for nearly all BRDU-positive macrophages. Inflammatory activity peaked at 72 h. Microglia produced relatively higher reactive oxygen species and TNF, whereas monocytes were the predominant IL-1β producer. Although microglia showed enhanced phagocytic activity after stroke, monocytes had significantly higher phagocytic capacity at 72 h. Interestingly, we found a positive correlation between TNF expression levels and phagocytic activity of microglia after stroke.ConclusionsIn summary, the resident microglia population is vulnerable to the effects of severe ischemia, show compromised cell cycle progression, and adopt a largely pro-inflammatory phenotype after stroke. Infiltrating monocytes are primarily involved with early debris clearance of dying cells. These findings suggest that the early wave of infiltrating monocytes may be beneficial to stroke repair and future therapies aimed at mitigating microglia cell death may prove more effective than attempting to elicit targeted anti-inflammatory responses from damaged cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-015-0329-1) contains supplementary material, which is available to authorized users.

The peripheral immune system plays a critical role in aging and in the response to brain injury. Emerging data suggest inflammatory responses are exacerbated in older animals following ischemic stroke; however, our understanding of these age-related changes is poor. In this work, we demonstrate marked differences in the composition of circulating and infiltrating leukocytes recruited to the ischemic brain of old male mice after stroke compared to young male mice. Blood neutrophilia and neutrophil invasion into the brain were increased in aged animals. Relative to infiltrating monocyte populations, brain-invading neutrophils had reduced phagocytic potential, and produced higher levels of reactive oxygen species and extracellular matrix-degrading enzymes (i.e., MMP-9), which were further exacerbated with age. Hemorrhagic transformation was more pronounced in aged versus young mice relative to infarct size. High numbers of myeloperoxidase-positive neutrophils were found in postmortem human brain samples of old (> 71 years) acute ischemic stroke subjects compared to non-ischemic controls. Many of these neutrophils were found in the brain parenchyma. A large proportion of these neutrophils expressed MMP-9 and positively correlated with hemorrhage and hyperemia. MMP-9 expression and hemorrhagic transformation after stroke increased with age. These changes in the myeloid response to stroke with age led us to hypothesize that the bone marrow response to stroke is altered with age, which could be important for the development of effective therapies targeting the immune response. We generated heterochronic bone marrow chimeras as a tool to determine the contribution of peripheral immune senescence to age- and stroke-induced inflammation. Old hosts that received young bone marrow (i.e., Young → Old) had attenuation of age-related reductions in bFGF and VEGF and showed improved locomotor activity and gait dynamics compared to isochronic (Old → Old) controls. Microglia in young heterochronic mice (Old → Young) developed a senescent-like phenotype. After stroke, aged animals reconstituted with young marrow had reduced behavioral deficits compared to isochronic controls, and had significantly fewer brain-infiltrating neutrophils. Increased rates of hemorrhagic transformation were seen in young mice reconstituted with aged bone marrow. This work suggests that age alters the immunological response to stroke, and that this can be reversed by manipulation of the peripheral immune cells in the bone marrow.Electronic supplementary materialThe online version of this article (10.1007/s00401-018-1859-2) contains supplementary material, which is available to authorized users.

BackgroundNeonatal hypoxic-ischemic encephalopathy (HIE) is an important cause of motor and cognitive impairment in children. Clinically, male infants are more vulnerable to ischemic insults and suffer more long-term deficits than females; however, the mechanisms underlying this sex difference remain elusive. Inflammatory processes initiated by microglial activation are fundamental in the pathophysiology of ischemia. Recent studies report a sexual dimorphism in microglia numbers and expression of activation markers in the neonatal brain under normal conditions. How these basal sex differences in microglia affect HIE remains largely unexplored. This study investigated sex differences in ischemic outcomes and inflammation triggered by HIE. We hypothesize that ischemia induces sex-specific brain injury in male and female neonates and that microglial activation and inflammatory responses play an important role in this sexual dimorphism.MethodsMale and female C57BL6 mice were subjected to 60-min Rice-Vanucci modeling (RVM) at post-natal day 10 (P10) to induce HIE. Stroke outcomes were measured 1, 3, 7, and 30 days after stroke. Microglial activation and inflammatory responses were evaluated by flow cytometry and cytokine analysis.ResultsOn day 1 of HIE, no difference in infarct volumes or seizure scores was seen between male and female neonates. However, female neonates exhibited significantly smaller infarct size and fewer seizures compared to males 3 days after HIE. Females also had less brain tissue loss and behavioral deficits compared to males at the chronic stage of HIE. Male animals demonstrated increased microglial activation and up-regulated inflammatory response compared to females at day 3.ConclusionsHIE leads to an equivalent primary brain injury in male and female neonates at the acute stage that develops into sexually dimorphic outcomes at later time points. An innate immune response secondary to the primary injury may contribute to sexual dimorphism in HIE.

Chronic neuroinflammation with sustained microglial activation occurs following severe traumatic brain injury (TBI) and is believed to contribute to subsequent neurodegeneration and neurological deficits. Microglia, the primary innate immune cells in brain, are dependent on colony stimulating factor 1 receptor (CSF1R) signaling for their survival. In this preclinical study, we examined the effects of delayed depletion of chronically activated microglia on functional recovery and neurodegeneration up to 3 months postinjury. A CSF1R inhibitor, Plexxikon (PLX) 5622, was administered to adult male C57BL/6J mice at 1 month after controlled cortical impact to remove chronically activated microglia, and the inhibitor was withdrawn 1-week later to allow for microglial repopulation. Following TBI, the repopulated microglia displayed a ramified morphology similar to that of Sham uninjured mice, whereas microglia in vehicle-treated TBI mice showed the typical chronic posttraumatic hypertrophic morphology. PLX5622 treatment limited TBI-associated neuropathological changes at 3 months postinjury; these included a smaller cortical lesion, reduced hippocampal neuron cell death, and decreased NOX2and NLRP3 inflammasome-associated neuroinflammation. Furthermore, delayed depletion of chronically activated microglia after TBI led to widespread changes in the cortical transcriptome and altered gene pathways involved in neuroinflammation, oxidative stress, and neuroplasticity. Using a variety of complementary neurobehavioral tests, PLX5622-treated TBI mice also had improved long-term motor and cognitive function recovery through 3 months postinjury. Together, these studies demonstrate that chronic phase removal of neurotoxic microglia after TBI using CSF1R inhibitors markedly reduce chronic neuroinflammation and associated neurodegeneration, as well as related motor and cognitive deficits.

Microglia are highly active and vigilant housekeepers of the central nervous system that function to promote neuronal growth and activity. With advanced age, however, dysregulated inflammatory signaling and defects in phagocytosis impede their ability to perform the most essential of homeostatic functions, including immune surveillance and debris clearance. Microglial activation is one of the hallmarks of the aging brain and coincides with age-related neurodegeneration and cognitive decline. Age-associated microglial dysfunction leads to cellular senescence and can profoundly alter the response to sterile injuries and immune diseases, often resulting in maladaptive responses, chronic inflammation, and worsened outcomes after injury. Our knowledge of microglia aging and the factors that regulate age-related microglial dysfunction remain limited, as the majority of pre-clinical studies are performed in young animals, and human brain samples are difficult to obtain quickly post-mortem or in large numbers. This review outlines the impact of normal aging on microglial function, highlights the potential mechanisms underlying age-related changes in microglia, and discusses how aging can shape the recovery process following injury.

Aging is an important risk factor for post-stroke infection, which accounts for a large proportion of stroke-associated mortality. Despite this, studies evaluating post-stroke infection rates in aged animal models are limited. In addition, few studies have assessed gut microbes as a potential source of infection following stroke. Therefore we investigated the effects of age and the role of bacterial translocation from the gut in post-stroke infection in young (8-12 weeks) and aged (18-20 months) C57Bl/6 male mice following transient middle cerebral artery occlusion (MCAO) or sham surgery. Gut permeability was examined and peripheral organs were assessed for the presence of gut-derived bacteria following stroke. Furthermore, sickness parameters and components of innate and adaptive immunity were examined. We found that while stroke induced gut permeability and bacterial translocation in both young and aged mice, only young mice were able to resolve infection. Bacterial species seeding peripheral organs also differed between young (Escherichia) and aged (Enterobacter) mice. Consequently, aged mice developed a septic response marked by persistent and exacerbated hypothermia, weight loss, and immune dysfunction compared to young mice following stroke.

Aging is associated with an increase in basal inflammation in the central nervous system (CNS) and an overall decline in cognitive function and poorer recovery following injury. Growing evidence suggests that leukocyte recruitment to the CNS is also increased with normal aging, but to date, no systematic evaluation of these “age-associated” leukocytes have been performed. In this work the effect of aging on CNS leukocyte recruitment was examined. Aging was associated with an increased number of CD45hi leukocytes, primarily composed of conventional CD8+ T cells. These results were strain-independent and seen in both sexes. Intravascular labeling and immunohistology revealed the presence of parenchymal CD8+ T cells in several regions of the brain including the choroid plexus and meninges. These cells had effector memory (CD44+CD62L−) and tissue-resident phenotypes and expressed markers associated with T cell receptor (TCR) activation. Analysis of TCRvβ repertoire usage suggested that entry into the CNS is likely stochastic rather than antigen-driven. Correlational analyses revealed a positive association between CD8 T cell numbers and decreased pro-inflammatory function of microglia. However, the effects of cerebral ischemia and ex-vivo stimulation of these cells dramatically increased production of tumor necrosis factor (TNF), interferon gamma (IFNγ), and monocyte-chemotactic protein-1 (MCP-1/CCL2). Taken together, we identified a novel population of resident memory, immunosurveillant CD8 T cells that represent a hallmark of CNS aging and appear to modify microglia homeostasis under normal conditions, but are primed to potentiate inflammation and leukocyte recruitment following ischemic injury.

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