Rationale: Peripheral blood monocytes are recruited into the ischemic brain and transform into macrophages after stroke. Nevertheless, the exact role of CCR2-dependent monocytes/macrophages in brain injury after stroke remains elusive.Methods: We used CCR2 knockout (KO) mice and the CCR2 pharmacological inhibitor, propagermanium (PG), to address the role of CCR2-dependent monocytes/macrophages in the acute stage and neurological functional recovery after middle cerebral artery (MCA) occlusion and reperfusion.Results: CCR2 KO resulted in smaller infarct size and lower mortality than in wild type (WT) mice, when measured 3 days after stroke. However, from 5 to 28 days after stroke, the KO mice had higher mortality and showed no obvious neurological functional recovery. In addition, WT mice treated with PG had similar stroke outcomes compared with CCR2 KO, as measured by T2 weighted MRI. Flow cytometry and real-time PCR analyses suggest that monocyte-derived macrophages (MoDMs) in the stroke brains mainly polarized to pro-inflammatory macrophages at the early stage, but gradually switched to anti-inflammatory macrophages at 7 days after stroke. In addition, adoptive transfer of anti-inflammatory macrophages into CCR2 KO mice at 4 and 6 days after stroke alleviated mortality and promoted neurological recovery.Conclusion: CCR2-dependent monocytes/macrophages are a double-edged sword; they worsen acute brain injury, but are essential for neurological recovery by promoting anti-inflammatory macrophage polarization.

Background and Purpose: Brain acidosis is prevalent in stroke and other neurological diseases. Acidosis can have paradoxical injurious and protective effects. The purpose of this study is to determine whether a proton receptor exists in neurons to counteract acidosis-induced injury. Methods: We analyzed the expression of proton-sensitive GPCRs (G protein-coupled receptors) in the brain, examined acidosis-induced signaling in vitro, and studied neuronal injury using in vitro and in vivo mouse models. Results: GPR68, a proton-sensitive GPCR, was present in both mouse and human brain, and elicited neuroprotection in acidotic and ischemic conditions. GPR68 exhibited wide expression in brain neurons and mediated acidosis-induced PKC (protein kinase C) activation. PKC inhibition exacerbated pH 6-induced neuronal injury in a GPR68-dependent manner. Consistent with its neuroprotective function, GPR68 overexpression alleviated middle cerebral artery occlusion–induced brain injury. Conclusions: These data expand our knowledge on neuronal acid signaling to include a neuroprotective metabotropic dimension and offer GPR68 as a novel therapeutic target to alleviate neuronal injuries in ischemia and multiple other neurological diseases.

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Brain acid signaling plays important roles in both physiological and disease conditions. One key neuronal metabotropic proton receptor in the brain is GPR68, which contributes to hippocampal long-term potentiation (LTP) and mediates neuroprotection in acidotic and ischemic conditions. Here, to gain greater understanding of GPR68 function in the brain, we performed mRNA-Seq analysis in mice. First, we studied sham-operated animals to determine baseline expression. Compared to wild type (WT), GPR68−/− (KO) brain downregulated genes that are enriched in Gene Ontology (GO) terms of misfolding protein binding, response to organic cyclic compounds, and endoplasmic reticulum chaperone complex. Next, we examined the expression profile following transient middle cerebral artery occlusion (tMCAO). tMCAO-upregulated genes cluster to cytokine/chemokine-related functions and immune responses, while tMCAO-downregulated genes cluster to channel activities and synaptic signaling. For proton-sensitive receptors, tMCAO downregulated ASIC1a and upregulated GPR4 and GPR65, but had no effect on ASIC2, PAC, or GPR68. GPR68 deletion did not alter the expression of these proton receptors, either at baseline or after ischemia. Lastly, we performed GeneVenn analysis of differential genes at baseline and post-tMCAO. Ischemia upregulated the expression of three hemoglobin genes, along with H2-Aa, Ppbp, Siglece, and Tagln, in WT but not in KO. Immunostaining showed that tMCAO-induced hemoglobin localized to neurons. Western blot analysis further showed that hemoglobin induction is GPR68dependent. Together, these data suggest that GPR68 deletion at baseline disrupts chaperone functions and cellular signaling responses and imply a contribution of hemoglobin-mediated antioxidant mechanism to GPR68-dependent neuroprotection in ischemia.

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