We tested whether significant leukocyte infiltration occurs in a mouse model of permanent cerebral ischemia. C57BL6/J male mice underwent either permanent (3 or 24 hours) or transient (1 or 2 hours þ 22-to 23-hour reperfusion) middle cerebral artery occlusion (MCAO). Using flow cytometry, we observed B15,000 leukocytes (CD45 þ high cells) in the ischemic hemisphere as early as 3 hours after permanent MCAO (pMCAO), comprising B40% lymphoid cells and B60% myeloid cells. Neutrophils were the predominant cell type entering the brain, and were increased to B5,000 as early as 3 hours after pMCAO. Several cell types (monocytes, macrophages, B lymphocytes, CD8þ T lymphocytes, and natural killer cells) were also increased at 3 hours to levels sustained for 24 hours, whereas others (CD4 þ T cells, natural killer T cells, and dendritic cells) were unchanged at 3 hours, but were increased by 24 hours after pMCAO. Immunohistochemical analysis revealed that leukocytes typically had entered and widely dispersed throughout the parenchyma of the infarct within 3 hours. Moreover, compared with pMCAO, there were B50% fewer infiltrating leukocytes at 24 hours after transient MCAO (tMCAO), independent of infarct size. Microglial cell numbers were bilaterally increased in both models. These findings indicate that a profound infiltration of inflammatory cells occurs in the brain early after focal ischemia, especially without reperfusion.

Background and Purpose-Ly6Chi monocytes are generally thought to exert a proinflammatory role in acute tissue injury, although their impact after injuries to the central nervous system is poorly defined. CC chemokine receptor 2 is expressed on Ly6C hi monocytes and plays an essential role in their extravasation and transmigration into the brain after cerebral ischemia. We used a selective CC chemokine receptor 2 antagonist, INCB3344, to assess the effect of Ly6C hi monocytes recruited into the brain early after ischemic stroke. Methods-Male C57Bl/6J mice underwent occlusion of the middle cerebral artery for 1 hour followed by 23 hours of reperfusion. Mice were administered either vehicle (dimethyl sulfoxide/carboxymethylcellulose) or INCB3344 (10, 30 or 100 mg/kg IP) 1 hour before ischemia and at 2 and 6 hours after ischemia. At 24 hours, we assessed functional outcomes, infarct volume, and quantified the immune cells in blood and brain by flow cytometry or immunofluorescence. Gene expression of selected inflammatory markers was assessed by quantitative polymerase chain reaction. Results-Ly6Chi monocytes were increased 3-fold in the blood and 10-fold in the brain after stroke, and these increases were selectively prevented by INCB3344 in a dose-dependent manner. Mice treated with INCB3344 exhibited markedly worse functional outcomes and larger infarct volumes, in association with reduced M2 polarization and increased peroxynitrite production in macrophages, compared with vehicle-treated mice. Conclusions-Our data suggest that Ly6Chi monocytes exert an acute protective effect after ischemic stroke to limit brain injury and functional deficit that involves promotion of M2 macrophage polarization. Furthermore, there is evidence that CCR2/CCL2 mechanisms are important for the migration of neuroblasts from neurogenic regions to damaged regions of brain after cerebral ischemia in mice, suggesting a role in tissue recovery after stroke. 17 Thus, for the development of future therapies to target detrimental elements of the poststroke acute inflammatory response, it is vital to clarify the role of CCR2 + /Ly6C hi monocytes. To our knowledge, no study has examined the effect of a selective CCR2 antagonist in ischemic stroke. INCB3344 is a novel CCR2 antagonist that inhibits the binding of CCL2 to monocytes with high potency and it is highly selective for the CCR2 receptor.18 It effectively reduces monocyte/macrophage accumulation in autoimmune encephalomyelitis in mice and in inflammatory arthritis in rats 18 and in deoxycorticosterone acetate/salt-induced hypertension in mice.19 Its potency and selectivity for CCR2 inhibition have thus been used in this study to evaluate the effect of inhibiting Ly6C hi monocyte migration to the brain early during poststroke inflammation. Materials and Methods AnimalsThis study was approved by the Monash University Animal Ethics Committee (Project MARP/2011/112) and performed in accordance with the National Health and Medical Research Council of Australia guidelines for the care and use ...

CC chemokine receptor 2 (CCR2) plays important roles in extravasation and transmigration of monocytes under inflammatory conditions. CCR2 and its ligands have been extensively studied in a range of inflammatory diseases in the central nervous system (CNS), including multiple sclerosis, Alzheimer's disease and ischemic stroke. This brief review summarizes our current understanding of the physiologic and pathologic roles of CCR2, focusing on its involvement in CNS inflammatory diseases. There appears to be a rationale for exploring therapies involving CCR2 inhibition in multiple sclerosis and ischemic stroke, but there is also evidence for immunomodulatory and protective effects of CCR2 activity during CNS inflammation. The critical balance between protective and detrimental roles of CCR2-dependent recruitment of leukocytes must therefore be carefully examined to guide safe and effective development of any therapies involving CCR2 modulation.

O ur knowledge of estrogen signaling pathways during cerebral ischemia, including the relative importance of different estrogen receptors and identities of estrogen-regulated gene networks affecting neuronal death, remains incomplete. Until recently, estrogen was thought to elicit its neuroprotective actions solely via activation of the classical nuclear receptors, estrogen receptor(ER)α and ERβ.1,2 However, the discovery of a novel estrogen receptor called G protein-coupled estrogen receptor (GPER, formerly known as GPR30), with high expression in the brain 3,4 and cerebral circulation, 5 presents a third receptor that may influence estrogenmediated outcomes after stroke. Important differences exist between GPER and classical estrogen receptors in that GPER responds to estrogen with rapid cellular signaling, and it is a G protein-coupled receptor localized on the plasma membrane 6 and intracellular membranes such as the endoplasmic reticulum and Golgi apparatus. Similar to classical estrogen receptor signaling, there is evidence for neuroprotection after GPER activation in ovariectomized (OVX) animals subjected to stroke. Chronic pretreatment of OVX rodents with the selective GPER agonist, G-1, reduces brain injury after focal or global ischemia. 8,9Interestingly, we have found that GPER distribution and expression is increased in the brain of male mice, but not of intact female or OVX mice, after transient focal ischemia. 4This sex-dependent regulation of GPER expression after cerebral ischemia could help explain some of the complex effects of estrogen in the brain after stroke and also guide the Background and Purpose-Experimental studies indicate that estrogen typically, but not universally, has a neuroprotective effect in stroke. Ischemic stroke increases membrane-bound G protein-coupled estrogen receptor (GPER) distribution and expression in the brain of male but not female mice. We hypothesized that GPER activation may have a greater neuroprotective effect in males than in females after stroke. Methods-Vehicle (dimethyl sulfoxide), a GPER agonist (G-1, 30 μg/kg), or a GPER antagonist (G-15, 300 μg/kg) were administered alone or in combination to young or aged male mice, or young intact or ovariectomized female mice, 1 hour before or 3 hours after cerebral ischemia-reperfusion. Some mice were treated with a combination of G-1 and the pancaspase inhibitor, quinoline-Val-Asp(Ome)-CH2-O-phenoxy (Q-V D -OPh), 1 hour before stroke. We evaluated functional and histological end points of stroke outcome up to 72 hours after ischemia-reperfusion. In addition, apoptosis was examined using cleaved caspase-3 immunohistochemistry. Results-Surprisingly, G-1 worsened functional outcomes and increased infarct volume in males poststroke, in association with an increased expression of cleaved caspase-3 in peri-infarct neurons. These effects were blocked by G-15 or Q-VDOPh. Conversely, G-15 improved functional outcomes and reduced infarct volume after stroke in males, whether given before or after stroke. In contrast to find...

Systemic poststroke administration of hAECs elicits marked neuroprotection and facilitates mechanisms of repair and recovery.

Inflammatory cells may contribute to secondary brain injury following cerebral ischemia. The C57Bl/6 mouse strain is known to exhibit a T helper 1-prone, pro-inflammatory type response to injury, whereas the FVB strain is relatively T helper 2-prone, or anti-inflammatory, in its immune response. We tested whether stroke outcome is more severe in C57Bl/6 than FVB mice. Male mice of each strain underwent sham surgery or 1 h occlusion of the middle cerebral artery followed by 23 h of reperfusion. Despite no difference in infarct size, C57Bl/6 mice displayed markedly greater functional deficits than FVB mice after stroke, as assessed by neurological scoring and hanging wire test. Total numbers of CD45+ leukocytes tended to be larger in the brains of C57Bl/6 than FVB mice after stroke, but there were marked differences in leukocyte composition between the two mouse strains. The inflammatory response in C57Bl/6 mice primarily involved T and B lymphocytes, whereas neutrophils, monocytes and macrophages were more prominent in FVB mice. Our data are consistent with the concept that functional outcome after stroke is dependent on the immune cell composition which develops following ischemic brain injury.

Background and Purpose-Expression of numerous chemokine-related genes is increased in the brain after ischemicstroke. Here, we tested whether post-stroke administration of a chemokine-binding protein (CBP), derived from the parapoxvirus bovine papular stomatitis virus, might reduce infiltration of leukocytes into the brain and consequently limit infarct development. Methods-The binding spectrum of the CBP was evaluated in chemokine ELISAs, and binding affinity was determined using surface plasmon resonance. Focal stroke was induced in C57Bl/6 mice by middle cerebral artery occlusion for 1 hour followed by reperfusion for 23 or 47 hours. Mice were treated intravenously with either bovine serum albumin (10 μg) or CBP (10 μg) at the commencement of reperfusion. At 24 or 48 hours, we assessed plasma levels of the chemokines CCL2/MCP-1 and CXCL2/MIP-2, as well as neurological deficit, brain leukocyte infiltration, and infarct volume. Results-The CBP interacted with a broad spectrum of CC, CXC, and XC chemokines and bound CCL2/MCP-1 and CXCL2/MIP-2 with high affinity (pM range). Stroke markedly increased plasma levels of CCL2/MCP-1 and CXCL2/ MIP-2, as well as numbers of microglia and infiltrating leukocytes in the brain. Increases in plasma chemokines were blocked in mice treated with CBP, in which there was reduced neurological deficit, fewer brain-infiltrating leukocytes, and ≈50% smaller infarcts at 24 hours compared with bovine serum albumin-treated mice. However, CBP treatment was no longer protective at 48 hours. Conclusions-Post-stroke administration of CBP can reduce plasma chemokine levels in association with temporary attenuation of brain inflammation and infarct volume development.

Stroke triggers a complex inflammatory process in which the balance between pro- and antiinflammatory mediators is critical for the development of the brain infarct. However, systemic changes may also occur in parallel with brain inflammation. Here we demonstrate that administration of recombinant IL-33, a recently described member of the IL-1 superfamily of cytokines, promotes Th2-type effects following focal ischemic stroke, resulting in increased plasma levels of Th2-type cytokines and fewer proinflammatory (3-nitrotyrosine+F4/80+) microglia/macrophages in the brain. These effects of IL-33 were associated with reduced infarct size, fewer activated microglia and infiltrating cytotoxic (natural killer-like) T cells, and more IL-10-expressing regulatory T cells. Despite these neuroprotective effects, mice treated with IL-33 displayed exacerbated post-stroke lung bacterial infection in association with greater functional deficits and mortality at 24 hours. Supplementary antibiotics (gentamicin and ampicillin) mitigated these systemic effects of IL-33 after stroke. Our findings highlight the complex nature of the inflammatory mechanisms differentially activated in the brain and periphery during the acute phase after ischemic stroke. The data indicate that a Th2-promoting agent can provide neuroprotection without adverse systemic effects when given in combination with antibiotics.