Xuefang Ren

Regulatory B Cells Limit CNS Inflammation and Neurologic Deficits in Murine Experimental Stroke

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 wild-type (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−/− versus 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 48 h 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.

CD4+FoxP3+ regulatory T-cells in cerebral ischemic stroke.

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 Foxp3DTR 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.

Rapid mitochondrial dysfunction mediates TNF-alpha-induced neurotoxicity

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.

Mitochondrial Crisis in Cerebrovascular Endothelial Cells Opens the Blood–Brain Barrier

Background and Purpose— The blood–brain barrier (BBB) is a selectively permeable cerebrovascular endothelial barrier that maintains homeostasis between the periphery and the central nervous system. 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 (lipopolysaccharide) challenge on infarct volume, BBB permeability, infiltrated neutrophils, and functional outcomes after murine transient middle cerebral artery occlusion in vivo; mitochondrial evaluation of cerebrovascular endothelial cells challenged by lipopolysaccharide 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 after transient middle cerebral artery occlusion. Results— We report here that lipopolysaccharide worsens ischemic stroke outcome and increases BBB permeability after transient middle cerebral artery occlusion in mice. Furthermore, we elucidate a novel mechanism that compromised mitochondrial function accounts for increased BBB permeability as evidenced by: lipopolysaccharide-induced reductions in oxidative phosphorylation and subunit expression of respiratory chain complexes in cerebrovascular endothelial cells, a compromised BBB permeability induced by pharmacological inhibition of mitochondrial function in cerebrovascular endothelial cells in vitro and in an in vivo animal model, and worsened stroke outcomes in transient middle cerebral artery occlusion mice after 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.

Programmed Death-1 Pathway Limits Central Nervous System Inflammation and Neurologic Deficits in Murine Experimental Stroke

Background and Purpose— Evaluation of infarct volumes and infiltrating immune cell populations in mice after middle cerebral artery occlusion 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 coinhibitory pathway, programmed death (PD)-1, to the development of middle cerebral artery occlusion. 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 60 minutes middle cerebral artery occlusion and 96 hours 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 central nervous system microglia, and functional neurological deficits. These regulatory functions were associated with increased percentages of circulating PD-ligand-1 and PD-ligand-2 expressing CD19+ B-cells in blood, the spleen, and central nervous system with the capacity to inhibit activation of inflammatory T-cells and central nervous system 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 central nervous system inflammation in middle cerebral artery occlusion. This inhibitory circuit would likely be pivotal in reducing stroke-associated Toll-like receptor-2- and Toll like receptor-4-mediated release of neurotoxic factors by activated central nervous system microglia.

Sequential Upregulation of Superoxide Dismutase 2 and Heme Oxygenase 1 by tert-Butylhydroquinone Protects Mitochondria during Oxidative Stress

Oxidative stress is linked to mitochondrial dysfunction in aging and neurodegenerative conditions. The transcription factor nuclear factor E2–related factor 2 (Nrf2)–antioxidant response element (ARE) regulates intracellular antioxidative capacity to combat oxidative stress. We examined the effect of tert-butylhydroquinone (tBHQ), an Nrf2-ARE signaling pathway inducer, on mitochondrial function during oxidative challenge in neurons. tBHQ prevented glutamate-induced cytotoxicity in an HT-22 neuronal cell line even with an 8-hour exposure delay. tBHQ blocked glutamate-induced intracellular reactive oxygen species (ROS) and mitochondrial superoxide accumulation. It also protected mitochondrial function under glutamate toxicity, including maintaining mitochondrial membrane potential, mitochondrial Ca2+ hemostasis, and mitochondrial respiration. Glutamate-activated, mitochondria-mediated apoptosis was inhibited by tBHQ as well. In rat primary cortical neurons, tBHQ protected cells from both glutamate and buthionine sulfoximine toxicity. We found that tBHQ scavenged ROS and induced a rapid upregulation of superoxide dismutase 2 (SOD2) expression and a delayed upregulation of heme oxygenase 1 (HO-1) expression. In HT-22 cells with a knockdown of SOD2 expression, delayed treatment with tBHQ failed to prevent glutamate-induced cell death. Briefly, tBHQ rescues mitochondrial function by sequentially increasing SOD2 and HO-1 expression during glutamate-mediated oxidative stress. This study is the first to demonstrate the role of tBHQ in preserving mitochondrial function during oxidative challenge and provides a clinically relevant argument for using tBHQ against acute neuron-compromising conditions.

NF-κB is involved in brain repair by stem cell factor and granulocyte-colony stimulating factor in chronic stroke

Chronic stroke is the phase of brain recovery and repair generally beginning 3 months after stroke onset. No pharmaceutical approach is currently available to enhance brain repair in chronic stroke. We have previously determined the therapeutic effects of stem cell factor (SCF) and granulocyte-colony stimulating factor (G-CSF) alone or in combination (SCF+G-CSF) in an animal model of chronic stroke and demonstrated that only SCF+G-CSF induces long-term functional recovery. However, the mechanism underlying the SCF+G-CSF-induced brain repair in chronic stroke remains largely elusive. In the present study, we determined the role of nuclear factor-kappa B (NF-κB) in neurovascular network remodeling and motor function improvement by SCF+G-CSF treatment in chronic stroke. SCF+G-CSF was subcutaneously administered for 7 days beginning 17 weeks after induction of experimental stroke. To inhibit NF-κB activation, NF-κB inhibitor was infused into the brain before SCF+G-CSF treatment. We observed that NF-κB inhibitor abolished the SCF+G-CSF-induced axonal sprouting, synaptogenesis and angiogenesis in the ipsilesional somatosensorimotor cortex. In addition, blockage of NF-κB activation resulted in elimination of the SCF+G-CSF-induced motor functional restoration in chronic stroke. These data suggest that NF-κB is required for the SCF+G-CSF-induced neuron-vascular network remodeling in the ipsilesional somatosensorimotor cortex and motor functional recovery in chronic stroke.

MiR-34a regulates blood–brain barrier permeability and mitochondrial function by targeting cytochrome c

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.

Impacts of prenatal nanomaterial exposure on male adult Sprague-Dawley rat behavior and cognition.

ABSTRACT It is generally accepted that gestational xenobiotic exposures result in systemic consequences in the adult F1 generation. However, data on detailed behavioral and cognitive consequences remain limited. Using our whole-body nanoparticle inhalation facility, pregnant Sprague-Dawley rats (gestational day [GD] 7) were exposed 4 d/wk to either filtered air (control) or nano-titanium dioxide aerosols (nano-TiO2; count median aerodynamic diameter of 170.9 ± 6.4 nm, 10.4 ± 0.4 mg/m3, 5 h/d) for 7.8 ± 0.5 d of the remaining gestational period. All rats received their final exposure on GD 20 prior to delivery. The calculated daily maternal deposition was 13.9 ± 0.5 µg. Subsequently, at 5 mo of age, behavior and cognitive functions of these pups were evaluated employing a standard battery of locomotion, learning, and anxiety tests. These assessments revealed significant working impairments, especially under maximal mnemonic challenge, and possible deficits in initial motivation in male F1 adults. Evidence indicates that maternal engineered nanomaterial exposure during gestation produces psychological deficits that persist into adulthood in male rats.

Deciphering the Blood-Brain Barrier Damage in Stroke: MitochondrialMechanism

Stroke is a complex vascular and neurological syndrome that can lead to death and disability. About 15 million people worldwide and 800,000 people in the United States suffer stroke each year . On average, in the U.S. every 40 seconds someone has a stroke and every 4 minutes someone dies from a stroke. This medical emergency has very limited treatments, and causes serious health and economic burdens in the United States and globally. Blood-brain barrier (BBB) is composed of highly specialized cerebrovascular endothelial cells, seals brain tissue from the circulating blood, and prevents blood, bacteria or toxins from reaching the central nervous system (CNS). In acute ischemic stroke, BBB is disrupted, and blood solutes penetrate into the CNS parenchymal extracellular space then cause cerebral edema . Although much has been observed in this stroke-induced brain edema, such as inflammatory infiltration, releasing of chemokines and cytokines, much less has the mechanism of BBB disruption been understood.