Evaluation of infarct volumes and infiltrating immune cell populations in mice after middle cerebral artery occlusion (MCAO) strongly implicates a mixture of both pathogenic and regulatory immune cell subsets in stroke pathogenesis and recovery. Our goal was to evaluate the contribution of B-cells to the development of MCAO by comparing infarct volumes and functional outcomes in WT versus B-cell deficient μMT−/− mice. The results clearly demonstrate larger infarct volumes, higher mortality, more severe functional deficits and increased numbers of activated T-cells, macrophages, microglial cells and neutrophils in the affected brain hemisphere of MCAO-treated μMT−/− vs. WT mice. These MCAO-induced changes were completely prevented in B-cell restored μMT−/− mice after transfer of highly purified WT GFP+ B-cells that were detected in the periphery, but not the CNS. In contrast, transfer of B-cells from IL-10−/− mice had no effect on infarct volume when transferred into μMT−/− mice. These findings strongly support a previously unrecognized activity of IL-10-secreting WT B-cells to limit infarct volume, mortality rate, recruitment of inflammatory cells and functional neurological deficits 48h after MCAO. Our novel observations are the first to implicate IL-10-secreting B-cells as a major regulatory cell type in stroke and suggest that enhancement of regulatory B-cells might have application as a novel therapy for this devastating neurologic condition.

Background and Purpose The blood-brain barrier (BBB) is a selectively permeable cerebrovascular endothelial barrier that maintains homeostasis between the periphery and central nervous system (CNS). BBB disruption is a consequence of ischemic stroke and BBB permeability can be altered by infection/inflammation, but the complex cellular and molecular changes that result in this BBB alteration need to be elucidated to determine mechanisms. Methods Infection mimic (LPS) challenge on infarct volume, BBB permeability, infiltrated neutrophils and functional outcomes following murine transient middle cerebral artery occlusion (tMCAO) in vivo; mitochondrial evaluation of cerebrovascular endothelial cells (CVECs) challenged by LPS in vitro; pharmacological inhibition of mitochondria on BBB permeability in vitro and in vivo; the effects of mitochondrial inhibitor on BBB permeability, infarct volume and functional outcomes following tMCAO. Results We report here that LPS worsens ischemic stroke outcome and increases BBB permeability following tMCAO in mice. Further, we elucidate a novel mechanism that compromised mitochondrial function accounts for increased BBB permeability as evidenced by: LPS-induced reductions in oxidative phosphorylation and subunit expression of respiratory chain complexes in CVECs, a compromised BBB permeability induced by pharmacological inhibition of mitochondrial function in CVECs in vitro and in an in vivo animal model, and worsened stroke outcomes in tMCAO mice following inhibition of mitochondrial function. Conclusions We concluded that mitochondria are key players in BBB permeability. These novel findings suggest a potential new therapeutic strategy for ischemic stroke by endothelial cell mitochondrial regulation.

Tumor necrosis factor alpha (TNF-α) is known to exacerbate ischemic brain injury; however, the mechanism is unknown. Previous studies have evaluated the effects of TNF-α on neurons with long exposures to high doses of TNF-α, which is not pathophysiologically relevant. We characterized the rapid effects of TNF-α on basal respiration, ATP production, and maximal respiration using pathophysiologically relevant, post-stroke concentrations of TNF-α. We observed a reduction in mitochondrial function as early as 1.5 h after exposure to low doses of TNF-α, followed by a decrease in cell viability in HT-22 cells and primary neurons. Subsequently, we used the HT-22 cell line to determine the mechanism by which TNF-α causes a rapid and profound reduction in mitochondrial function. Pre-treating with TNF-R1 antibody, but not TNF-R2 antibody, ameliorated the neurotoxic effects of TNF-α, indicating that TNF-α exerts its neurotoxic effects through TNF-R1. We observed an increase in caspase 8 activity and a decrease in mitochondrial membrane potential after exposure to TNF-α which resulted in a release of cytochrome c from the mitochondria into the cytosol. These novel findings indicate for the first time that an acute exposure to pathophysiologically relevant concentrations of TNF-α has neurotoxic effects mediated by a rapid impairment of mitochondrial function.

Experimental cerebral ischemic stroke is exacerbated by inflammatory T-cells and is accompanied by systemic increases in CD4 + CD25 + Foxp3 + regulatory T-cells (Treg). To determine their effect on ischemic brain injury, Treg were depleted in Foxp3 DTR mice prior to stroke induction. In contrast to a recent Nature Medicine report, our results demonstrate unequivocally that Treg depletion did not affect stroke infarct volume, thus failing to implicate this regulatory pathway in limiting stroke damage.

Background and Purpose Evaluation of infarct volumes and infiltrating immune cell populations in mice after middle cerebral artery occlusion (MCAO) strongly implicates a mixture of both pathogenic and regulatory immune cell subsets that affect stroke outcome. Our goal was to evaluate the contribution of the well-described co-inhibitory pathway, Programmed Death (PD)-1, to the development of MCAO. Methods Infarct volumes, functional outcomes and effects on infiltrating immune cell populations were compared in wild type C57BL/6 versus PD-1 deficient mice after 60min MCAO and 96h reperfusion. Results The results clearly demonstrate a previously unrecognized activity of the PD-1 pathway to limit infarct volume, recruitment of inflammatory cells from the periphery, activation of macrophages and CNS microglia and functional neurological deficits. These regulatory functions were associated with increased percentages of circulating PD-Ligand (L)-1 and PD-L2 expressing CD19+ B-cells in blood, spleen and CNS with the capacity to inhibit activation of inflammatory T-cells and CNS macrophages and microglial cells through upregulated PD-1. Conclusions Our novel observations are the first to implicate PD-1 signaling as a major protective pathway for limiting CNS inflammation in MCAO. This inhibitory circuit would likely be pivotal in reducing stroke-associated TLR2- and TLR4-mediated release of neurotoxic factors by activated CNS microglia.

The blood-brain barrier is composed of cerebrovascular endothelial cells and tight junctions, and maintaining its integrity is crucial for the homeostasis of the neuronal environment. Recently, we discovered that mitochondria play a critical role in maintaining blood-brain barrier integrity. We report for the first time a novel mechanism underlying blood-brain barrier integrity: miR-34a mediated regulation of blood-brain barrier through a mitochondrial mechanism. Bioinformatics analysis suggests miR-34a targets several mitochondria-associated gene candidates. We demonstrated that miR-34a triggers the breakdown of blood-brain barrier in cerebrovascular endothelial cell monolayer in vitro, paralleled by reduction of mitochondrial oxidative phosphorylation and adenosine triphosphate production, and decreased cytochrome c levels.

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