Heng Hu

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.

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.

Evaluation of Bioenergetic Function in Cerebral Vascular Endothelial Cells.

The integrity of the blood-brain-barrier (BBB) is critical to prevent brain injury. Cerebral vascular endothelial (CVE) cells are one of the cell types that comprise the BBB; these cells have a very high-energy demand, which requires optimal mitochondrial function. In the case of disease or injury, the mitochondrial function in these cells can be altered, resulting in disease or the opening of the BBB. In this manuscript, we introduce a method to measure mitochondrial function in CVE cells by using whole, intact cells and a bioanalyzer. A mito-stress assay is used to challenge the cells that have been perturbed, either physically or chemically, and evaluate their bioenergetic function. Additionally, this method also provides a useful way to screen new therapeutics that have direct effects on mitochondrial function. We have optimized the cell density necessary to yield oxygen consumption rates that allow for the calculation of a variety of mitochondrial parameters, including ATP production, maximal respiration, and spare capacity. We also show the sensitivity of the assay by demonstrating that the introduction of the microRNA, miR-34a, leads to a pronounced and detectable decrease in mitochondrial activity. While the data shown in this paper is optimized for the bEnd.3 cell line, we have also optimized the protocol for primary CVE cells, further suggesting the utility in preclinical and clinical models.

Mitochondrial Impairment in Cerebrovascular Endothelial Cells is Involved in the Correlation between Body Temperature and Stroke Severity.

Stroke is the second leading cause of death worldwide. The prognostic influence of body temperature on acute stroke in patients has been recently reported; however, hypothermia has confounded experimental results in animal stroke models. This work aimed to investigate how body temperature could prognose stroke severity as well as reveal a possible mitochondrial mechanism in the association of body temperature and stroke severity. Lipopolysaccharide (LPS) compromises mitochondrial oxidative phosphorylation in cerebrovascular endothelial cells (CVECs) and worsens murine experimental stroke. In this study, we report that LPS (0.1 mg/kg) exacerbates stroke infarction and neurological deficits, in the mean time LPS causes temporary hypothermia in the hyperacute stage during 6 hours post-stroke. Lower body temperature is associated with worse infarction and higher neurological deficit score in the LPS-stroke study. However, warming of the LPS-stroke mice compromises animal survival. Furthermore, a high dose of LPS (2 mg/kg) worsens neurological deficits, but causes persistent severe hypothermia that conceals the LPS exacerbation of stroke infarction. Mitochondrial respiratory chain complex I inhibitor, rotenone, replicates the data profile of the LPS-stroke study. Moreover, we have confirmed that rotenone compromises mitochondrial oxidative phosphorylation in CVECs. Lastly, the pooled data analyses of a large sample size (n=353) demonstrate that stroke mice have lower body temperature compared to sham mice within 6 hours post-surgery; the body temperature is significantly correlated with stroke outcomes; linear regression shows that lower body temperature is significantly associated with higher neurological scores and larger infarct volume. We conclude that post-stroke body temperature predicts stroke severity and mitochondrial impairment in CVECs plays a pivotal role in this hypothermic response. These novel findings suggest that body temperature is prognostic for stroke severity in experimental stroke animal models and may have translational significance for clinical stroke patients - targeting endothelial mitochondria may be a clinically useful approach for stroke therapy.

Tert-butylhydroquinone compromises survival in murine experimental stroke

Tert-butylhydroquinone (tBHQ), an Nrf2 signaling pathway inducer that is widely used as a food additive in the U.S., prevents oxidative stress-induced cytotoxicity in neurons. This study assesses the effects of tBHQ on ischemic stroke outcomes in mice. We measured infarct size, neurological deficits, and brain volume after tBHQ treatments in murine permanent middle cerebral artery occlusion (pMCAO) model in vivo. Further, we evaluated the regulation of tBHQ on mitochondrial function in cerebrovascular endothelial cells in vitro, which is critical to the blood-brain barrier (BBB) permeability. Our results demonstrated that tBHQ increased post-stroke mortality and worsened stroke outcomes. Mitochondrial function was suppressed by tBHQ treatment of cerebrovascular endothelial cells, and this suppression was potentiated by co-treatment with lipopolysaccharide (LPS), the bacterial mimic. These data indicate that tBHQ-exacerbated stroke damage might due to the compromised BBB permeability in permanent stroke.