Matthew D. Hammond

CCR2+Ly6Chi Inflammatory Monocyte Recruitment Exacerbates Acute Disability Following Intracerebral Hemorrhage

Intracerebral hemorrhage (ICH) is a devastating type of stroke that lacks a specific treatment. An intense immune response develops after ICH, which contributes to neuronal injury, disability, and death. However, the specific mediators of inflammation-induced injury remain unclear. The objective of the present study was to determine whether blood-derived CCR2+Ly6Chi inflammatory monocytes contribute to disability. ICH was induced in mice and the resulting inflammatory response was quantified using flow cytometry, confocal microscopy, and neurobehavioral testing. Importantly, blood-derived monocytes were distinguished from resident microglia by differential CD45 staining and by using bone marrow chimeras with fluorescent leukocytes. After ICH, blood-derived CCR2+Ly6Chi inflammatory monocytes trafficked into the brain, outnumbered other leukocytes, and produced tumor necrosis factor. Ccr2−/− mice, which have few circulating inflammatory monocytes, exhibited better motor function following ICH than control mice. Chimeric mice with wild-type CNS cells and Ccr2−/− hematopoietic cells also exhibited early improvement in motor function, as did wild-type mice after inflammatory monocyte depletion. These findings suggest that blood-derived inflammatory monocytes contribute to acute neurological disability. To determine the translational relevance of our experimental findings, we examined CCL2, the principle ligand for the CCR2 receptor, in ICH patients. Serum samples from 85 patients were collected prospectively at two hospitals. In patients, higher CCL2 levels at 24 h were independently associated with poor functional outcome at day 7 after adjusting for potential confounding variables. Together, these findings suggest that inflammatory monocytes worsen early disability after murine ICH and may represent a therapeutic target for patients.

Differential effects of aging and sex on stroke induced inflammation across the lifespan

Aging and biological sex are critical determinants of stroke outcome. Post-ischemic inflammatory response strongly contributes to the extent of ischemic brain injury, but how this response changes with age and sex is unknown. We subjected young (5-6 months), middle aged (14-15 months) and aged (20-22 months), C57BL/6 male and female mice to transient middle cerebral artery occlusion (MCAO) and found that a significant age by sex interaction influenced histological stroke outcomes. Acute functional outcomes were worse with aging. Neutrophils, inflammatory macrophages, macrophages, dendritic cells (DCs) and microglia significantly increased in the brain post MCAO. Cycling females had higher Gr1(-) non-inflammatory macrophages and lower T cells in the brain after stroke and these correlated with serum estradiol levels. Estrogen loss in acyclic aged female mice exacerbated stroke induced splenic contraction. Advanced age increased T cells, DCs and microglia at the site of injury, which may be responsible for the exacerbated behavioral deficits in the aged. We conclude that aging and sex have differential effects on the post stroke inflammatory milieu. Putative immunomodulatory therapies need to account for this heterogeneity.

Chronic metformin treatment improves post‐stroke angiogenesis and recovery after experimental stroke

Metformin is currently the first‐line treatment drug for type 2 diabetes. Metformin is a well‐known activator of AMP‐activated protein kinase (AMPK). In experimental studies, metformin has been shown to exert direct vascular effects by increasing vascular endothelial growth factor expression and improving microvascular density. As stroke is the leading cause of long‐term disability and angiogenesis is implicated as an important mechanism in functional recovery, we hypothesized that chronic metformin treatment would improve post‐stroke functional recovery by enhancing functional microvascular density. For this study, C57BL/6N male mice were subjected to a 60‐min middle cerebral artery occlusion, and were given 50 mg/kg/day metformin beginning 24 h post‐stroke for 3 weeks. Behavioral recovery was assessed using adhesive‐tape removal and the apomorphine‐induced turning test. The role of angiogenesis was assessed by counting vessel branch points from fluorescein‐conjugated lectin‐perfused brain sections. Importantly even if metformin treatment was initiated 24 h after injury it enhanced recovery and significantly improved stroke‐induced behavioral deficits. This recovery occurred in parallel with enhanced angiogenesis and with restoration of endogenous cerebral dopaminergic tone and revascularization of ischemic tissue. We assessed if the effects on recovery and angiogenesis were mediated by AMPK. When tested in AMPK α‐2 knockout mice, we found that metformin treatment did not have the same beneficial effects on recovery and angiogenesis, suggesting that metformin‐induced angiogenic effects are mediated by AMPK. The results from this study suggest that metformin mediates post‐stroke recovery by enhancing angiogenesis, and these effects are mediated by AMPK signaling.

TGF-β1 modulates microglial phenotype and promotes recovery after intracerebral hemorrhage

Intracerebral hemorrhage (ICH) is a devastating form of stroke that results from the rupture of a blood vessel in the brain, leading to a mass of blood within the brain parenchyma. The injury causes a rapid inflammatory reaction that includes activation of the tissue-resident microglia and recruitment of blood-derived macrophages and other leukocytes. In this work, we investigated the specific responses of microglia following ICH with the aim of identifying pathways that may aid in recovery after brain injury. We used longitudinal transcriptional profiling of microglia in a murine model to determine the phenotype of microglia during the acute and resolution phases of ICH in vivo and found increases in TGF-&bgr;1 pathway activation during the resolution phase. We then confirmed that TGF-&bgr;1 treatment modulated inflammatory profiles of microglia in vitro. Moreover, TGF-&bgr;1 treatment following ICH decreased microglial Il6 gene expression in vivo and improved functional outcomes in the murine model. Finally, we observed that patients with early increases in plasma TGF-&bgr;1 concentrations had better outcomes 90 days after ICH, confirming the role of TGF-&bgr;1 in functional recovery from ICH. Taken together, our data show that TGF-&bgr;1 modulates microglia-mediated neuroinflammation after ICH and promotes functional recovery, suggesting that TGF-&bgr;1 may be a therapeutic target for acute brain injury.

α4 Integrin Is a Regulator of Leukocyte Recruitment After Experimental Intracerebral Hemorrhage

Background and Purpose— Intracerebral hemorrhage (ICH) is swiftly followed by an inflammatory response. A key component of this response is the recruitment of leukocytes into the brain, which promotes neurological injury in rodent models. However, the mechanisms by which leukocytes transmigrate across the endothelium into the injured brain are unclear. The present study examines leukocyte adhesion molecules (&agr;4 integrin, L-selectin, and &agr;L&bgr;2 integrin) on 4 leukocyte subtypes to determine which are important for leukocyte recruitment after ICH. Methods— We used the blood injection mouse model of ICH, whereby 25 &mgr;L of blood was injected into the striatum. Flow cytometry was used to quantify leukocyte populations and adhesion molecule expression in brain and blood. An &agr;4 integrin–blocking antibody was administered to evaluate the contribution of &agr;4 integrin in leukocyte migration and neurological injury. Results— &agr;4 integrin was elevated on all leukocyte populations in brain after ICH, whereas L-selectin was unchanged and &agr;L&bgr;2 was increased only on T cells. Antagonism of &agr;4 resulted in decreased leukocyte transmigration and lessened neurobehavioral disability. Conclusions— &agr;4 integrin is an important cell adhesion molecule involved in neuroinflammation after ICH.

Erythrocyte efferocytosis modulates macrophages towards recovery after intracerebral hemorrhage

Macrophages are a source of both proinflammatory and restorative functions in damaged tissue through complex dynamic phenotypic changes. Here, we sought to determine whether monocyte-derived macrophages (MDMs) contribute to recovery after acute sterile brain injury. By profiling the transcriptional dynamics of MDMs in the murine brain after experimental intracerebral hemorrhage (ICH), we found robust phenotypic changes in the infiltrating MDMs over time and demonstrated that MDMs are essential for optimal hematoma clearance and neurological recovery. Next, we identified the mechanism by which the engulfment of erythrocytes with exposed phosphatidylserine directly modulated the phenotype of both murine and human MDMs. In mice, loss of receptor tyrosine kinases AXL and MERTK reduced efferocytosis of eryptotic erythrocytes and hematoma clearance, worsened neurological recovery, exacerbated iron deposition, and decreased alternative activation of macrophages after ICH. Patients with higher circulating soluble AXL had poor 1-year outcomes after ICH onset, suggesting that therapeutically augmenting efferocytosis may improve functional outcomes by both reducing tissue injury and promoting the development of reparative macrophage responses. Thus, our results identify the efferocytosis of eryptotic erythrocytes through AXL/MERTK as a critical mechanism modulating macrophage phenotype and contributing to recovery from ICH.

CX3CR1 Signaling on Monocytes Is Dispensable after Intracerebral Hemorrhage

Intracerebral hemorrhage is a subset of stroke for which there is no specific treatment. The Ly6Chi CCR2+ monocytes have been shown to contribute to acute injury after intracerebral hemorrhage. The other murine monocyte subset expresses CX3CR1 and lower Ly6C levels, and contributes to repair in other disease models. We hypothesized that the Ly6Clo CX3CR1+ monocytes would contribute to recovery after intracerebral hemorrhage. Intracerebral hemorrhage was modeled by blood injection in WT and CX3CR1-null bone marrow chimeras. Neurological outcomes and leukocyte recruitment were quantified at various time points. Functional outcomes were equal at 1, 3, 7, and 14 days after intracerebral hemorrhage in both genotypes. No differences were observed in leukocyte recruitment between genotypes on either 3 or 7 days after intracerebral hemorrhage. A few hundred Ly6Clo monocytes were found in the ipsilateral hemisphere in each genotype and they did not change over time. Peripherally derived CX3CR1+ monocytes were observed in the perihematomal brain 7 and 14 days after intracerebral hemorrhage. Our data suggests CX3CR1 signaling on monocytes does not play an influential role in acute injury or functional recovery after intracerebral hemorrhage and therefore CX3CR1 is not a therapeutic target to improve outcome after intracerebral hemorrhage.