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Objective: CD36 regulates biological and pathological processes including inflammation, resolution, phagocytosis, and angiogenesis. Highly expressed in monocytes/macrophages (MM), MM CD36 has been implicated in both detrimental and beneficial effects in acute stroke outcome and recovery. Using myeloid specific deficiency of CD36 (CD36KO MM ) mice, the present study addresses the role of MM CD36 on subset composition of MM in the acute and recovery phases of stroke. Methods: WT (CD36 fl/fl ) and CD36KO MM (M/F, 12w old) mice were subjected to transient focal ischemia. Brain immune cells were collected at 3d, 7d and 2m (n=8-12/timepoint). Infarct volume and hemispheric swelling were measured at 3d (n=13/genotype). In the isolated brain immune cells, CD11b+ cells were further distinguished by CD45 Hi and CD45 Low subsets. The extent of MM trafficking was determined by an intravenous infusion of control GFP+ splenocytes into the stroked WT and CD36KO MM mice 1d prior to sacrifice and MM subset analyses. Results: Compared to WT mice, CD36KO MM mice showed no difference in CD45 Low subset at 3d, 7d and 2m after stroke. However, CD36KO MM mice displayed a significant reduction of CD45 Hi subset, presumably infiltrating cells, at these time points (n=4-8 /group, P MM mice resulted in an increase of GFP+ cells in CD45 Low but a profound reduction in CD45 Hi subset. Despite the near absence of CD45 Hi and increased CD45 Low population in the post-ischemic brain, CD36KO MM mice displayed exacerbation of brain injury (15.1±13.7 vs 25.8±17.5 mm 3 , p Conclusion: The observed reduction of CD45 Hi subset and exacerbation of brain injury in CD36KO MM mice suggests a beneficial role of infiltrated CD45 Hi MMs in limiting stroke injury. The sustained trafficking at 2m indicates potential involvement of MM CD36 in the recovery process. Further studies to assess long-term outcomes of CD36KO MM mice will provide critical insights on the role of macrophage CD36 in stroke recovery.

Background and Purpose— Stroke-induced acute severe body weight (BW) loss is associated with a high rate of mortality during a critical poststroke period. Several interventions to reduce weight loss, however, have not been successful. Currently, the biological significance of this extraordinary catabolic process is not well understood. Spleen-derived monocytes/macrophages (MMs) are the major immune cells recruited to the injured brain. The trafficking of MMs has been shown to be important for tissue repair and recovery. The purpose of the study is to investigate whether the BW reduction is essential for MM-mediated immune response for mice to survive and whether a corticosterone-mediated catabolic event underlies the processes. Methods— C57BL/6 male mice (12-week-old) were subjected to transient middle cerebral artery occlusion. BW, total MMs, and their Ly-6C high and Ly-6C low subsets were determined in the spleen, blood, and the brain in poststroke mice. Poststroke survival rate and MM subsets were determined in mice with adrenalectomy, sham-adrenalectomy, and adrenalectomy mice supplemented with corticosterone. Results— Stroke reduced BW with a maximum reduction at day 3 poststroke (17.2±5.2%). The reduction at day 3 was positively linked to injury severity and selective depletion of MMs, but no other types of immune cells, in the spleen. Notably, the splenic MM depletion was significantly greater in mice with severe BW reduction (≥18% at day 3). In the blood, stroke depleted circulating MMs to a similar degree in animals with moderate and severe BW loss. Ly-6C+ monocyte infiltration in the poststroke brain was greater in mice with severe BW loss. Blocking the catabolic process by adrenalectomy significantly increased poststroke mortality, but the mortality was partially rescued by corticosterone supplement in adrenalectomy mice. Conclusions— Stroke-induced BW loss facilitates MM-mediated immune response, and the adrenal corticosterone-mediated catabolic process is necessary for poststroke survival. Visual Overview— An online visual overview is available for this article.

Objective: Post-stroke remote limb conditioning (PSLC) has been shown protective effect in preclinical and clinical studies. However, underlying protective mechanisms are multiple but not clearly defined. Although inflammation exacerbates stroke injury, studies showed inflammation also plays a key role in conditioning-mediated protection. This study investigated if PSLC induces changes of monocyte/macrophage (MM) subsets and the changes contribute to PSLC-induced functional benefits. Method: C57BL/6 male mice (12-week-old) were subjected to transient middle cerebral artery occlusion (MCAO) for 30 min. PSLC was induced 2h after MCAO by applying 5 cycles of 5 min inflation (200 mmHg) and 5 min deflation with a cuff on left hind limb. Mononuclear cells were isolated from the spleen, blood and brain at 3D and analyzed by flow cytometer. Acute injury size was measured at 3D. Motor and gait functions were assessed up to 4-month using Rotarod and Catwalk. Additionally, naïve spleen cells were treated sera obtained from sham-conditioned or PSLC animals, and MM subset shifts were determined. Results: PSLC significantly decreased anti-inflammatory (Ly-6C low ) monocytes without changing pro-inflammatory (Ly-6C high ) subset in blood, while there were no changes in the spleen (Fig A). Similarly, PSLC decreased Ly-6C low and increased Ly-6C high subset in the stroked brain (Fig B). The shift to a pro-inflammatory MM subset did not attenuate brain injury size but was associated with improved motor and gait function (Fig D). PSLC serum reduced Ly6-C low and increased Ly-6C high subset in splenocytes (Fig C). There was bigger MM shift and greater behavioral benefits of PSLC in animals with bigger injury (Fig E&F). Conclusion: The study demonstrates a contributing role of pro-inflammatory monocytes to PSLC-induced functional benefits in chronic stroke. This study provides a potential application of PSLC as a neuroimmune-based strategy for ischemic stroke patients.

Background and Purpose: Longitudinal imaging studies in stroke demonstrated that infarct development is a dynamic process and it matures over several days, even weeks depending on the severity of stroke. However, due to high cost and multiple exposure of anesthesia associated with repeated imaging, infarct volume has been typically measured at a fixed time point in preclinical studies, which prevent further assessment of behaviors during stroke recovery. In identifying a non-invasive physiological parameter that predicts the extent of stroke-induced brain injury, the study investigated whether acute body weight loss predicts the extent of brain injury and swelling. Methods: C57 male mice (10-12 wk old, 25-30 gr) were subjected to the proximal middle cerebral artery (MCA) occlusion for 30 min. Body weight was recorded daily. Infarct volume corrected with swelling (IVInd) and percent hemispheric swelling (%SW) were determined at 1, 3 and 7d after MCAO. In a retrospective study, we analyzed correlations between 3d %body weight reduction (%BWred) and stroke outcomes (IVInd & %SW) in C57 male animals that were subjected to MCAO by 3 different individuals over 8 years (n=91-96). Results: Stroke induced acute BW loss (%BWred; 1d, 88.9±3.3; 3d, 84.5±8.6; 5d, 86.8±10.6; 7d, 88.9±8.9). Correlation analyses in the post-ischemic time points showed IVind and %SW were significantly correlated with only at 3d BW reduction, but not 1d and 7d (Fig.1). A retrospective analysis in C57 mice also showed a significant correlation between 3d %BWred and IVInd & %SW (Fig.2). Conclusions: The study showed that acute post-stroke BWred is a simple and suitable index to predict the extent of stroke injury in C57 mice. Whether the prediction can be generalized across different strains and in comorbid conditions warrant further investigation.

Background and Purpose: Although postconditioning following stroke has indicated neuroprotective mechanisms, little is known about the mechanisms that lead to neuroprotection. Using flagellin, NF-kB activators and agonists of TLR5, we tested whether TLR5 plays an integral role in NF-kB activation and postconditioning-induced neuroprotection. Method: For the postconditioning group, 30 min of MCAO was induced, then 5 min of reperfusion was performed, after which the MCA was again occluded for 5 min. Using an anaerobic chamber, bEnd.3 and BV-2 cells in the postconditioning group were subjected to 180 min OGD (oxygen and glucose deprivation) and were allowed to recover for 10 min before being subjected to 10 more min of OGD. The control group was subjected to 180 min of OGD. Flagellin, an agonist of TLR5, was used to clarify the association between TLR5 and postconditioning in vivo (ICV; 50, 100 ng/mouse) and in vitro (0.1, 0.25, 0.5, 1 ng/ ml) experiments. Immunoprecipitation and immunoblotting were used to determine the interactions of TLR5, MyD88, p-Akt, and NF-κB. Results: Animals treated with postconditioning showed significant differences in infarct volumes compared with the MCAO group up to 60.51%. Postconditioning elevated NF-κB translocation to the nuclei (pin vivo and in vitro postconditioning. Additionally, inhibition of Akt phosphorylation by Akt inhibitor IV decreased NF-κB translocation after postconditioning. To clarify the role of NF-κB in postconditioning, flagellin was used as a postconditioning mimicking agent. The treatment of flagellin reduced hypoxic-ischemic injury and increased nuclear accumulation of NF-κB and activation of Akt up to 4 hr. Conclusions: Postconditioning has neuroprotective effects by activating NF-κB and Akt survival pathway under TLR5 after stroke. Agonist of TLR5, flagellin simulated NF-κB and Akt survival pathways in cerebral ischemia. Our results suggested new mechanisms of postconditioning through TLR-5 and NF-κB, which leads to the possibility of development of a postconditioning mimicking agent using NF-κB activators such as flagellin.

Background and Purpose: Hyperglycemia is frequently observed in patients with acute ischemic stroke, and conversely, diabetic patients showed a high frequency of stroke. This correlation indicates shared mechanisms in pathology. Glucagon like peptide-1 (GLP-1) stimulates glucose-dependent insulin secretion and is used to treat type 2 diabetes mellitus. Recently, beyond the glucose-lowering effect, GLP-1 has been reported to possess neuroprotective effects. We therefore investigated the neuroprotective mechanism of GLP-1 receptor (GLP-1R) agonist exendin-4 (ex-4) after cerebral ischemia-reperfusion injury. Methods: Male Sprague-Dawley rats were subjected to 60 min of middle cerebral artery occlusion (MCAO) with intracerebroventricular (i.c.v) pretreatment of ex-4. Oxygen glucose deprivation was induced to primary neuron cultures in an anaerobic chamber. Changes of genes were further confirmed by QPCR and Western blot, and siRNA was used to ascertain the mechanism. Results: Ischemia-reperfusion injury reduced the expressions of GLP-1R by 46.3% (p; higher oxidative stress in SOD2 KO mice induced lower expression of GLP-1R, but higher in SOD1 Tg mice. Down regulated GLP-1R by ischemic injury was 70% restored by GLP-1R agonist, ex-4 (p2 terminal kinase (JNK) signaling that stimulates activation of Cox-2 was 36% inhibited by the i.c.v injection of ex-4 at 24 h (p Conclusions: GLP-1R activation by ex-4 resulted in reduction of Cox-2 through increasing IB1, which led to anti-inflammatory neuroprotection in stroke. Our study suggests that anti-inflammatory action of GLP-1 could be a new strategy for the treatment of stroke accompanied by hyperglycemia.