Volatile Anesthetics Influence Blood-Brain Barrier Integrity by Modulation of Tight Junction Protein Expression in Traumatic Brain Injury

Disruption of the blood-brain barrier (BBB) results in cerebral edema formation, which is a major cause for high mortality after traumatic brain injury (TBI). As anesthetic care is mandatory in patients suffering from severe TBI it may be important to elucidate the effect of different anesthetics on cerebral edema formation. Tight junction proteins (TJ) such as zonula occludens-1 (ZO-1) and claudin-5 (cl5) play a central role for BBB stability. First, the influence of the volatile anesthetics sevoflurane and isoflurane on in-vitro BBB integrity was investigated by quantification of the electrical resistance (TEER) in murine brain endothelial monolayers and neurovascular co-cultures of the BBB. Secondly brain edema and TJ expression of ZO-1 and cl5 were measured in-vivo after exposure towards volatile anesthetics in native mice and after controlled cortical impact (CCI). In in-vitro endothelial monocultures, both anesthetics significantly reduced TEER within 24 hours after exposure. In BBB co-cultures mimicking the neurovascular unit (NVU) volatile anesthetics had no impact on TEER. In healthy mice, anesthesia did not influence brain water content and TJ expression, while 24 hours after CCI brain water content increased significantly stronger with isoflurane compared to sevoflurane. In line with the brain edema data, ZO-1 expression was significantly higher in sevoflurane compared to isoflurane exposed CCI animals. Immunohistochemical analyses revealed disruption of ZO-1 at the cerebrovascular level, while cl5 was less affected in the pericontusional area. The study demonstrates that anesthetics influence brain edema formation after experimental TBI. This effect may be attributed to modulation of BBB permeability by differential TJ protein expression. Therefore, selection of anesthetics may influence the barrier function and introduce a strong bias in experimental research on pathophysiology of BBB dysfunction. Future research is required to investigate adverse or beneficial effects of volatile anesthetics on patients at risk for cerebral edema.     

Involvement of ROS in BBB dysfunction

The blood–brain barrier (BBB) forms a protective barrier around the brain, with the important function of maintaining brain homeostasis. Pathways thought to initiate BBB dysfunction include the kinin system, excitotoxicity, neutrophil recruitment, mitochondrial alterations and macrophage/microglial activation, all of which converge on the same point—reactive oxygen species (ROS). Interestingly, ROS also provide a common trigger for many downstream pathways that directly mediate BBB compromise such as oxidative damage, tight junction (TJ) modification and matrix metalloproteinases (MMP) activation. These observations suggest that ROS are key mediators of BBB breakdown and implicate antioxidants as potential neuroprotectants in conditions like stroke and traumatic brain injury (TBI). This review explores some of the pathways both upstream and downstream of ROS that have been implicated in increased BBB permeability and discusses the role of ROS and antioxidants in neuropathology.     

Time course of Keap1-Nrf2 pathway expression after experimental intracerebral haemorrhage: correlation with brain oedema and neurological deficit

Abstract

Oxidative stress (OS) is involved in the progression of intracerebral haemorrhage (ICH)-induced secondary brain injury. The pathway involving Kelch-like ECH-associated protein 1 (Keap1) and nuclear factor erythroid 2-related factor 2 (Nrf2) is currently recognised as the major endogenous regulatory system against oxidative injury. Although its beneficial role has been described for ICH, the time course of Keap1-Nrf2 pathway expression, the activity of downstream antioxidative enzymes, and the association with brain oedema and neurological deficits have not been fully investigated. In this study, we investigated the temporal changes in expression of Keap1, Nrf2, and their downstream antioxidative proteins in the ICH rat brain. We additionally quantified the relationship between these gene and protein changes with brain water content and neurological behaviour scores. After blood infusion, Keap1 showed decreased expression starting at 8 h, whereas Nrf2 began to show a significant increase at 2 h with a peak at 24 h. Keap1 and Nrf2 are chiefly expressed in neuronal cells but not in glial cells. The downstream antioxidative enzymes such as haemeoxygenase-1 (HO-1), glutathione (GSH), thioredoxin (TRX), and glutathione-S-transferase (GST-α1) increased to different degrees during the early stages of ICH. Among these enzymes, HO-1 showed a significant time-dependent increase starting 8 h after ICH. In addition, there was a positive correlation between the HO-1 level and brain water content. In combination, these results suggest that activation of the Keap1-Nrf2 pathway may play an important endogenous neuroprotective role during OS after ICH. Because HO-1 expression is temporally associated with brain oedema – reflective of the severity of brain injury – it may be used as a biomarker of haeme-mediated oxidative damage after ICH.     

Oxidative stress activates protein tyrosine kinase and matrix metalloproteinases leading to blood-brain barrier dysfunction

The blood-brain barrier (BBB) formed by brain microvascular endothelial cells (BMVEC) regulates the passage of molecules and leukocytes in and out of the brain. Oxidative stress is a major underlying cause of neurodegenerative and neuroinflammatory disorders and BBB injury associated with them. Using human BMVEC grown on porous membranes covered with basement membrane (BM) matrix (BBB models), we demonstrated that reactive oxygen species (ROS) augmented permeability and monocyte migration across BBB. ROS activated matrix metalloproteinases (MMP-1, -2, and -9) and decreased tissue inhibitors of MMPs (TIMP-1 and -2) in a protein tyrosine kinase (PTK)-dependent manner. Increase in MMPs and PTK activities paralleled degradation of BM protein and enhanced tyrosine phosphorylation of tight junction (TJ) protein. These effects and enhanced permeability/monocyte migration were prevented by inhibitors of MMPs, PTKs, or antioxidant suggesting that oxidative stress caused BBB injury via degradation of BM protein by activated MMPs and by PTK-mediated TJ protein phosphorylation. These findings point to new therapeutic interventions ameliorating BBB dysfunction in neurological disorders such as stroke or neuroinflammation.     

Luteolin provides neuroprotection in models of traumatic brain injury via the Nrf2–ARE pathway

Luteolin has recently been proven to exert neuroprotection in a variety of neurological diseases; however, its roles and the underlying mechanisms in traumatic brain injury are not fully understood. The present study was aimed to investigate the neuroprotective effects of luteolin in models of traumatic brain injury (TBI) and the possible role of the Nrf2–ARE pathway in the putative neuroprotection. A modified Marmarou׳s weight-drop model in mice and the scratch model in mice primary cultured neurons were used to induce TBI. We determined that luteolin significantly ameliorated secondary brain injury induced by TBI, including neurological deficits, brain water content, and neuronal apoptosis. Furthermore, the level of malondialdehyde (MDA) and the activity of glutathione peroxidase (GPx) were restored in the group with luteolin treatment. in vitro studies showed that luteolin administration lowered the intracellular reactive oxygen species (ROS) level and increased the neuron survival. Moreover, luteolin enhanced the translocation of Nrf2 to the nucleus both in vivo and in vitro, which was proved by the results of Western blot, immunohistochemistry, and electrophoretic mobility shift assay (EMSA). Subsequently upregulation of the expression of the downstream factors such as heme oxygenase 1 (HO1) and NAD(P)H:quinone oxidoreductase 1 (NQO1) was also examined. However, luteolin treatment failed to provide neuroprotection after TBI in Nrf2^-/- mice. Taken together, these in vivo and in vitro data demonstrated that luteolin provided neuroprotective effects in the models of TBI, possibly through the activation of the Nrf2–ARE pathway.     

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic cerebrovascular accidents account for 80% of all strokes. A common cause of IR injury is the rapid inflow of fluids following an acute/chronic occlusion of blood, nutrients, oxygen to the tissue triggering the formation of free radicals.Ischemic stroke is followed by blood-brain barrier (BBB) dysfunction and vasogenic brain edema. Structurally, tight junctions (TJs) between the endothelial cells play an important role in maintaining the integrity of the blood-brain barrier (BBB). IR injury is an early secondary injury leading to a non-specific, inflammatory response. Oxidative and metabolic stress following inflammation triggers secondary brain damage including BBB permeability and disruption of tight junction (TJ) integrity.Our protocol presents an in vitro example of oxygen-glucose deprivation and reoxygenation (OGD-R) on rat brain endothelial cell TJ integrity and stress fiber formation. Currently, several experimental in vivo models are used to study the effects of IR injury; however they have several limitations, such as the technical challenges in performing surgeries, gene dependent molecular influences and difficulty in studying mechanistic relationships. However, in vitro models may aid in overcoming many of those limitations. The presented protocol can be used to study the various molecular mechanisms and mechanistic relationships to provide potential therapeutic strategies. However, the results of in vitro studies may differ from standard in vivo studies and should be interpreted with caution.     

How can microbial interactions with the blood–brain barrier modulate astroglial and neuronal function?

The vascular endothelium of the blood-brain barrier (BBB) is regarded as a part of the neurovascular unit (NVU). This emerging NVU concept emphasizes the need for homeostatic signalling among the neuronal, glial and vascular endothelial cellular compartments in maintaining normal brain function. Conversely, dysfunction in any component of the NVU affects another, thus contributing to disease. Brain endothelial activation and dysfunction is observed in various neurological diseases, such as (ischemic) stroke, seizure, brain inflammation and infectious diseases and likely contributes to or exacerbates neurological conditions. The role and impact of brain endothelial factors on astroglial and neuronal activation is unclear. Similarly, it is not clear which stages of BBB endothelial activation can be considered beneficial versus detrimental. Although the BBB plays an important role in context of encephalopathies caused by neurotropic microbes that must first penetrate into the brain, a crucial role of the BBB in contributing to neurological dysfunction may be seen in cerebral malaria (CM), where the Plasmodium parasite remains sequestered in the brain vasculature, does not enter the brain parenchyma, and yet causes coma and seizures. In this minireview some of the scenarios and factors that may play a role in BBB as a relay station to modulate astroneuronal functioning are discussed.     

Cerebral microvascular changes in permeability and tight junctions induced by hypoxia-reoxygenation

Cerebral microvessel endothelial cells that form the blood-brain barrier (BBB) have tight junctions (TJ) that are critical for maintaining brain homeostasis and low permeability. Both integral (claudin-1 and occludin) and membrane-associated zonula occluden-1 and -2 (ZO-1 and ZO-2) proteins combine to form these TJ complexes that are anchored to the cytoskeletal architecture (actin). Disruptions of the BBB have been attributed to hypoxic conditions that occur with ischemic stroke, pathologies of decreased perfusion, and high-altitude exposure. The effects of hypoxia and posthypoxic reoxygenation in cerebral microvasculature and corresponding cellular mechanisms involved in disrupting the BBB remain unclear. This study examined hypoxia and posthypoxic reoxygenation effects on paracellular permeability and changes in actin and TJ proteins using primary bovine brain microvessel endothelial cells (BBMEC). Hypoxia induced a 2.6-fold increase in [(14)C]sucrose, a marker of paracellular permeability. This effect was significantly reduced (~58%) with posthypoxic reoxygenation. After hypoxia and posthypoxic reoxygenation, actin expression was increased (1.4- and 2.3-fold, respectively). Whereas little change was observed in TJ protein expression immediately after hypoxia, a twofold increase in expression was seen with posthypoxic reoxygenation. Furthermore, immunofluorescence studies showed alterations in occludin, ZO-1, and ZO-2 protein localization during hypoxia and posthypoxic reoxygenation that correlate with the observed changes in BBMEC permeability. The results of this study show hypoxia-induced changes in paracellular permeability may be due to perturbation of TJ complexes and that posthypoxic reoxygenation reverses these effects.     

TNF-alpha induced NFκB signaling and p65 (RelA) overexpression repress Cldn5 promoter in mouse brain endothelial cells

Inflammatory cytokine TNFα enhances permeability of brain capillaries constituting blood brain barrier (BBB). In the monoculture endothelial models of BBB TNFα alters tight junction (TJ) structure and protein content. Claudin-5 (Cldn5) is a key TJ protein whose expression in the brain endothelial cells is critical to the function of BBB. TNFα reduces Cldn5 promoter activity and mRNA expression in mouse brain derived endothelial cells but the regulatory elements and signaling mechanism involved are not defined. Here we report that TNFα acts through NFκB signaling and requires a conserved promoter region for the down-regulation of Cldn5 expression. Overexpression of the NFκB subunit p65 (RelA) alone repressed Cldn5 promoter activity in mouse brain endothelial cells. We observed partial loss of Cldn5 protein expression after prolonged TNFα treatment in primary endothelial culture isolated from C56BL/6 mice brain. Taken together, our results confirm and extend previous observations of TNFα induced down-regulation of Cldn5 expression in mouse brain endothelial cells.     

Mechanisms of Blood Brain Barrier Disruption by Different Types of Bacteria,and Bacterial–Host Interactions Facilitate the Bacterial Pathogen Invading the Brain

This review aims to elucidate the different mechanisms of blood brain barrier (BBB) disruption that may occur due to invasion by different types of bacteria, as well as to show the bacteria–host interactions that assist the bacterial pathogen in invading the brain. For example, platelet-activating factor receptor (PAFR) is responsible for brain invasion during the adhesion of pneumococci to brain endothelial cells, which might lead to brain invasion. Additionally, the major adhesin of the pneumococcal pilus-1, RrgA is able to bind the BBB endothelial receptors: polymeric immunoglobulin receptor (pIgR) and platelet endothelial cell adhesion molecule (PECAM-1), thus leading to invasion of the brain. Moreover, Streptococcus pneumoniae choline binding protein A (CbpA) targets the common carboxy-terminal domain of the laminin receptor (LR) establishing initial contact with brain endothelium that might result in BBB invasion. Furthermore, BBB disruption may occur by S. pneumoniae penetration through increasing in pro-inflammatory markers and endothelial permeability. In contrast, adhesion, invasion, and translocation through or between endothelial cells can be done by S. pneumoniae without any disruption to the vascular endothelium, upon BBB penetration. Internalins (InlA and InlB) of Listeria monocytogenes interact with its cellular receptors E-cadherin and mesenchymal-epithelial transition (MET) to facilitate invading the brain. L. monocytogenes species activate NF-κB in endothelial cells, encouraging the expression of P- and E-selectin, intercellular adhesion molecule 1 (ICAM-1), and Vascular cell adhesion protein 1 (VCAM-1), as well as IL-6 and IL-8 and monocyte chemoattractant protein-1 (MCP-1), all these markers assist in BBB disruption. Bacillus anthracis species interrupt both adherens junctions (AJs) and tight junctions (TJs), leading to BBB disruption. Brain microvascular endothelial cells (BMECs) permeability and BBB disruption are induced via interendothelial junction proteins reduction as well as up-regulation of IL-1α, IL-1β, IL-6, TNF-α, MCP-1, macrophage inflammatory proteins-1 alpha (MIP1α) markers in Staphylococcus aureus species. Streptococcus agalactiae or Group B Streptococcus toxins (GBS) enhance IL-8 and ICAM-1 as well as nitric oxide (NO) production from endothelial cells via the expression of inducible nitric oxide synthase (iNOS) enhancement, resulting in BBB disruption. While Gram-negative bacteria, Haemophilus influenza OmpP2 is able to target the common carboxy-terminal domain of LR to start initial interaction with brain endothelium, then invade the brain. H. influenza type b (HiB), can induce BBB permeability through TJ disruption. LR and PAFR binding sites have been recognized as common routes of CNS entrance by Neisseria meningitidis. N. meningitidis species also initiate binding to BMECs and induces AJs deformation, as well as inducing specific cleavage of the TJ component occludin through the release of host MMP-8. Escherichia coli bind to BMECs through LR, resulting in IL-6 and IL-8 release and iNOS production, as well as resulting in disassembly of TJs between endothelial cells, facilitating BBB disruption. Therefore, obtaining knowledge of BBB disruption by different types of bacterial species will provide a picture of how the bacteria enter the central nervous system (CNS) which might support the discovery of therapeutic strategies for each bacteria to control and manage infection.     

Development of the blood-brain barrier

The endothelial cells forming the blood-brain barrier (BBB) are highly specialized to allow precise control over the substances that leave or enter the brain. An elaborate network of complex tight junctions (TJ) between the endothelial cells forms the structural basis of the BBB and restricts the paracellular diffusion of hydrophilic molecules. Additonally, the lack of fenestrae and the extremely low pinocytotic activity of endothelial cells of the BBB inhibit the transcellular passage of molecules across the barrier. On the other hand, in order to meet the high metabolic needs of the tissue of the central nervous system (CNS), specific transport systems selectively expressed in the membranes of brain endothelial cells in capillaries mediate the directed transport of nutrients into the CNS or of toxic metabolites out of the CNS. Whereas the characteristics of the mature BBB endothelium are well described, the cellular and molecular mechanisms that control the development, differentiation and maintenance of the highly specialized endothelial cells of the BBB remain unknown to date, despite the recent explosion in our knowledge of the growth factors and their receptors specifically acting on vascular endothelium during development. This review summarizes our current knowledge of the cellular and molecular mechanisms involved in the development and maintenance of the BBB.     

Melatonin stimulates antioxidant enzymes and reduces oxidative stress in experimental traumatic brain injury: the Nrf2–ARE signaling pathway as a potential mechanism

The goal of this study was to evaluate the potential involvement of melatonin in the activation of the nuclear factor erythroid 2-related factor 2 and antioxidant-responsive element (Nrf2–ARE) signaling pathway and the modulation of antioxidant enzyme activity in an experimental model of traumatic brain injury (TBI). In experiment 1, ICR mice were divided into four groups: sham group, TBI group, TBI + vehicle group, and TBI + melatonin group (n = 38 per group). Melatonin (10 mg/kg) was administered via an intraperitoneal (ip) injection at 0, 1, 2, 3, and 4 h post-TBI. In experiment 2, Nrf2 wild-type (Nrf2^+/+ group) and Nrf2-knockout (Nrf2^−/− group) mice received a TBI insult followed by melatonin administration (10 mg/kg, ip) at the corresponding time points (n = 35 per group). The administration of melatonin after TBI significantly ameliorated the effects of the brain injury, such as oxidative stress, brain edema, and cortical neuronal degeneration. Melatonin markedly promoted the translocation of Nrf2 protein from the cytoplasm to the nucleus; increased the expression of Nrf2–ARE pathway-related downstream factors, including heme oxygenase-1 and NAD(P)H:quinone oxidoreductase 1; and prevented the decline of antioxidant enzyme activities, including superoxide dismutase and glutathione peroxidase. Furthermore, knockout of Nrf2 partly reversed the neuroprotection of melatonin after TBI. In conclusion, melatonin administration may increase the activity of antioxidant enzymes and attenuate brain injury in a TBI model, potentially via mediation of the Nrf2–ARE pathway.     

The Involvement of CXCL11 in Bone Marrow-Derived Mesenchymal Stem Cell Migration Through Human Brain Microvascular Endothelial Cells

Bone marrow-derived mesenchymal stem cells (MSCs) transplant into the brain, where they play a potential therapeutic role in neurological diseases. However, the blood–brain barrier (BBB) is a native obstacle for MSCs entry into the brain. Little is known about the mechanism behind MSCs migration across the BBB. In the present study, we modeled the interactions between human MSCs (hMSCs) and human brain microvascular endothelial cells (HBMECs) to mimic the BBB microenvironment. Real-time PCR analysis indicated that the chemokine CXCL11 is produced by hMSCs and the chemokine receptor CXCR3 is expressed on HBMECs. Further results indicate that CXCL11 secreted by hMSCs may interact with CXCR3 on HBMECs to induce the disassembly of tight junctions through the activation of ERK1/2 signaling in the endothelium, which promotes MSCs transendothelial migration. These findings are relevant for understanding the biological responses of MSCs in BBB environments and helpful for the application of MSCs in neurological diseases.     

Hyperbaric oxygen preconditioning ameliorates hypoxia–ischemia brain damage by activating Nrf2 expression in vivo and in vitro

The present study aimed to investigate whether hyperbaric oxygen preconditioning (HBO-PC) could ameliorate hypoxia–ischemia brain damage (HIBD) by an increase of Nrf2 expression. P7 Sprague-Dawley rats (aged 7 d, n?=?195) were used in two in vivo experiments, including BO-PC exposure experiments in non-HIBD models and treatment experiments in HIBD models. 2,3,5-triphenyltetrazolium chloride (TTC) staining, Nissl Staining, and TUNEL staining were performed. And expressions of Nrf2, HO-1, and GSTs were measured. For in vitro studies, oxygen–glucose deprivation cells were established. Morphological and apoptotic staining and gene silencing of Nrf2 by siRNA transfection were investigated. For exposure experiments, HBO-PC for longer time increased the expression of Nrf2 significantly. And for treatment experiments, HBO-PC treatment significantly decreased infarction area, lessened neuronal injury, reduced apoptosis, and increased both the expression of Nrf2 and activities of its downstream proteins. Cytology tests confirmed effects of HBO-PC treatments. Besides, Nrf2 siRNA significantly reduced protective effects of HBO-PC. These observations demonstrated that an up-regulation of Nrf2 by HBO-PC might play an important role in the generation of tolerance against HIBD.     

Chronic inflammatory pain leads to increased blood-brain barrier permeability and tight junction protein alterations

The blood-brain barrier (BBB) maintains brain homeostasis by limiting entry of substances to the central nervous system through interaction of transmembrane and intracellular proteins that make up endothelial cell tight junctions (TJs). Recently it was shown that the BBB can be modulated by disease pathologies including inflammatory pain. This study examined the effects of chronic inflammatory pain on the functional and molecular integrity of the BBB. Inflammatory pain was induced by injection of complete Freund's adjuvant (CFA) into the right plantar hindpaw in female Sprague-Dawley rats under halothane anesthesia; control animals were injected with saline. Edema and hyperalgesia were assessed by plethysmography and infrared paw-withdrawal latency. At 72 h postinjection, significant edema formation and hyperalgesia were noted in the CFA-treated rats. Examination of permeability of the BBB by in situ perfusion of [14C]sucrose while rats were under pentobarbital anesthesia demonstrated that CFA treatment significantly increased brain sucrose uptake. Western blot analysis of BBB TJ proteins showed no change in expression of zonula occludens-1 (an accessory protein) or actin (a cytoskeletal protein) with CFA treatment. Expression of the transmembrane TJ proteins occludin and claudin-3 and -5 significantly changed with CFA treatment with a 60% decrease in occludin, a 450% increase in claudin-3, and a 615% increase in claudin-5 expression. This study demonstrates that during chronic inflammatory pain, alterations in BBB function are associated with changes in specific transmembrane TJ proteins.     

Novel insights into the development and maintenance of the blood–brain barrier

The blood–brain barrier (BBB) is essential for maintaining homeostasis within the central nervous system (CNS) and is a prerequisite for proper neuronal function. The BBB is localized to microvascular endothelial cells that strictly control the passage of metabolites into and out of the CNS. Complex and continuous tight junctions and lack of fenestrae combined with low pinocytotic activity make the BBB endothelium a tight barrier for water soluble moleucles. In combination with its expression of specific enzymes and transport molecules, the BBB endothelium is unique and distinguishable from all other endothelial cells in the body. During embryonic development, the CNS is vascularized by angiogenic sprouting from vascular networks originating outside of the CNS in a precise spatio-temporal manner. The particular barrier characteristics of BBB endothelial cells are induced during CNS angiogenesis by cross-talk with cellular and acellular elements within the developing CNS. In this review, we summarize the currently known cellular and molecular mechanisms mediating brain angiogenesis and introduce more recently discovered CNS-specific pathways (Wnt/β?catenin, Norrin/Frizzled4 and hedgehog) and molecules (GPR124) that are crucial in BBB differentiation and maturation. Finally, based on observations that BBB dysfunction is associated with many human diseases such as multiple sclerosis, stroke and brain tumors, we discuss recent insights into the molecular mechanisms involved in maintaining barrier characteristics in the mature BBB endothelium.     

Immunolocalization of tight junction proteins in blood vessels in human germinal matrix and cortex

Brain development occurs in a specialized environment maintained by a blood–brain barrier (BBB). An important structural element of the BBB is the endothelial tight junction (TJ). TJs are present during the embryonic period, but BBB impermeability accrues over an extended gestational interval. In studies of human premature infants, we used immunomicroscopy to determine if amounts of the TJ proteins ZO-1, claudin and occludin increase with gestational age in vessels of germinal matrix (GM) and cortex. By 24 weeks postconception (PC), TJ proteins were present in both GM and cortical vessels, but immunoreactivity in the GM of the youngest subjects was less than in older subjects. At 24 weeks PC, TJ protein immunoreactivity in GM vessels was less than in cortical vessels suggesting that TJ maturation progresses along a superficial to deep brain axis. This concept correlates with conclusions from previous analyses of the expression of brain endothelial cell alkaline phosphatase (AP) activity. AP appears in cortical vessels before appearing in deep white matter and GM vessels. Together, these data indicate that differentiation of some functional specializations is still in progress in GM vessels during the third trimester. This maturation could relate to the pathogenesis of germinal matrix hemorrhage–intraventricular hemorrhage.     

Alcohol-induced blood-brain barrier dysfunction is mediated via inositol 1,4,5-triphosphate receptor (IP3R)-gated intracellular calcium release

The blood-brain barrier (BBB) formed by brain microvascular endothelial cells (BMVEC), pericytes and astrocytes controls the transport of ions, peptides and leukocytes in and out of the brain. Tight junctions (TJ) composed of TJ proteins (occludin, claudins and zonula occludens) ensure the structural integrity of the BMVEC monolayer. Neuropathologic studies indicated that the BBB was impaired in alcohol abusers; however, the underlying mechanism of BBB dysfunction remains elusive. Using primary human BMVEC, we previously demonstrated that oxidative stress induced by ethanol (EtOH) metabolism in BMVEC activated myosin light chain kinase (MLCK), resulting in the enhanced phosphorylation of either cytoskeletal or TJ proteins, and in BBB impairment. We proposed that EtOH metabolites stimulated inositol 1,4,5-triphosphate receptor (IP(3)R)-operated intracellular calcium (Ca(2+)) release, thereby causing the activation of MLCK in BMVEC. Indeed, treatment of primary human BMVEC with EtOH or its metabolites resulted in the increased expression of IP(3)R protein and IP(3)R-gated intracellular Ca(2+) release. These functional changes paralleled MLCK activation, phosphorylation of cytoskeletal/TJ proteins, loss of BBB integrity, and enhanced leukocyte migration across BMVEC monolayers. Inhibition of either EtOH metabolism or IP(3)R activation prevented BBB impairment. These findings suggest that EtOH metabolites act as signaling molecules for the activation of MLCK via the stimulation of IP(3)R-gated intracellular Ca(2+) release in BMVEC. These putative events can lead to BBB dysfunction in the setting of alcoholism, and to neuro-inflammatory disorders promoting leukocyte migration across the BBB.     

Role of Nrf2 in host defense against influenza virus in cigarette smoke-exposed mice

Influenza virus is a common respiratory tract viral infection. Although influenza can be fatal in patients with chronic pulmonary diseases such as chronic obstructive pulmonary disease, its pathogenesis is not fully understood. The Nrf2-mediated antioxidant system is essential to protect the lungs from oxidative injury and inflammation. In the present study, we investigated the role of Nrf2 in protection against influenza virus-induced pulmonary inflammation after cigarette smoke exposure with both in vitro and in vivo approaches. For in vitro analyses, peritoneal macrophages isolated from wild-type and Nrf2-deficient mice were treated with poly(I:C) and/or cigarette smoke extract. For in vivo analysis, these mice were infected with influenza A virus with or without exposure to cigarette smoke. In Nrf2-deficient macrophages, NF-κB activation and the induction of its target inflammatory genes were enhanced after costimulation with cigarette smoke extract and poly(I:C) compared with wild-type macrophages. The induction of antioxidant genes was observed for the lungs of wild-type mice but not those of Nrf2-deficient mice after cigarette smoke exposure. Cigarette smoke-exposed Nrf2-deficient mice showed higher rates of mortality than did wild-type mice after influenza virus infection, with enhanced peribronchial inflammation, lung permeability damage, and mucus hypersecretion. Lung oxidant levels and NF-κB-mediated inflammatory gene expression in the lungs were also enhanced in Nrf2-deficient mice. Our data indicate that the antioxidant pathway controlled by Nrf2 is pivotal for protection against the development of influenza virus-induced pulmonary inflammation and injury under oxidative conditions.