Anaemia worsens early functional outcome after traumatic brain injury: a preliminary study.

OBJECTIVE: To determine early effects on outcome from traumatic brain injury (TBI) induced by controlled cortical impact (CCI) associated with anaemia in mice. HYPOTHESIS: Outcome from TBI with concomitant anaemia would be worse than TBI without anaemia. METHODS: CCI was induced with electromagnetic impaction in four groups of C57BL/6J mice: sham, sham+anaemia; TBI; and TBI+anaemia. Anaemia was created by withdrawal of 30% of calculated intravascular blood volume and saline replacement of equal volume. Functional outcome was assessed by beam-walking test and open field test (after pre-injury training) on post-injury days 3 and 7. After functional assessment, brains removed from sacrificed animals were pathological reviewed with haematoxylin and eosin, cresyl violet, Luxol Fast Blue, and IBA-1 immunostains. RESULTS: Beam-walking was similar between animals with TBI and TBI+anaemia (p = 0.9). In open field test, animals with TBI+anaemia walked less distance than TBI alone or sham animals on days 3 (p < 0.001) and 7 (p < 0.05), indicating less exploratory and locomotion behaviours. No specific pathologic differences could be identified. CONCLUSIONS: Anaemia associated with TBI from CCI is associated with worse outcome as measured by less distance travelled in the open field test at three days than if anaemia is not present.

Cytosolic phospholipase A2 plays a crucial role in ROS/NO signaling during microglial activation through the lipoxygenase pathway.

BACKGROUND: Oxidative stress and inflammation are important factors contributing to the pathophysiology of numerous neurological disorders, including Alzheimer's disease, Parkinson's disease, acute stroke, and infections of the brain. There is well-established evidence that proinflammatory cytokines and glutamate, as well as reactive oxygen species (ROS) and nitric oxide (NO), are produced upon microglia activation, and these are important factors contributing to inflammatory responses and cytotoxic damage to surrounding neurons and neighboring cells. Microglial cells express relatively high levels of cytosolic phospholipase A2 (cPLA2), an enzyme known to regulate membrane phospholipid homeostasis and release of arachidonic acid (AA) for synthesis of eicosanoids. The goal for this study is to elucidate the role of cPLA2IV in mediating the oxidative and inflammatory responses in microglial cells. METHODS: Experiments involved primary microglia cells isolated from transgenic mice deficient in cPLA2α or iPLA2ß, as well as murine immortalized BV-2 microglial cells. Inhibitors of cPLA2/iPLA2/cyclooxygenase (COX)/lipoxygenase (LOX) were used in BV-2 microglial cell line. siRNA transfection was employed to knockdown cPLA2 expression in BV-2 cells. Griess reaction protocol was used to determine NO concentration, and CM-H2DCF-DA was used to detect ROS production in primary microglia and BV-2 cells. WST-1 assay was used to assess cell viability. Western blotting was used to assess protein expression levels. Immunocytochemical staining for phalloidin against F-actin was used to demonstrate cell morphology. RESULTS: In both primary and BV-2 microglial cells, stimulation with lipopolysaccharide (LPS) or interferon gamma (IFNγ) resulted in a time-dependent increase in phosphorylation of cPLA2 together with ERK1/2. In BV-2 cells, LPS- and IFNγ-induced ROS and NO production was inhibited by arachidonyl trifluoromethyl ketone (AACOCF3) and pyrrophenone as well as RNA interference, but not BEL, suggesting a link between cPLA2, and not iPLA2, on LPS/IFNγ-induced nitrosative and oxidative stress in microglial cells. Primary microglial cells isolated from cPLA2α-deficient mice generated significantly less NO and ROS as compared with the wild-type mice. Microglia isolated from iPLA2ß-deficient mice did not show a decrease in LPS-induced NO and ROS production. LPS/IFNγ induced morphological changes in primary microglia, and these changes were mitigated by AACOCF3. Interestingly, despite that LPS and IFNγ induced an increase in phospho-cPLA2 and prostaglandin E2 (PGE2) release, LPS- and IFNγ-induced NO and ROS production were not altered by the COX-1/2 inhibitor but were suppressed by the LOX-12 and LOX-15 inhibitors instead. CONCLUSIONS: In summary, the results in this study demonstrated the role of cPLA2 in microglial activation with metabolic links to oxidative and inflammatory responses, and this was in part regulated by the AA metabolic pathways, namely the LOXs. Further studies with targeted inhibition of cPLA2/LOX in microglia during neuroinflammatory conditions can be valuable to investigate the therapeutic potential in ameliorating neurological disease pathology.

Magnolia polyphenols attenuate oxidative and inflammatory responses in neurons and microglial cells.

BACKGROUND: The bark of magnolia has been used in Oriental medicine to treat a variety of remedies, including some neurological disorders. Magnolol (Mag) and honokiol (Hon) are isomers of polyphenolic compounds from the bark of Magnolia officinalis, and have been identified as major active components exhibiting anti-oxidative, anti-inflammatory, and neuroprotective effects. In this study, we investigate the ability of these isomers to suppress oxidative stress in neurons stimulated by the ionotropic glutamate receptor agonist N-methyl-D-aspartate (NMDA) and oxidative and inflammatory responses in microglial cells activated by interferon-γ (IFNγ) and lipopolysaccharide (LPS). We also attempt to elucidate the mechanism and signaling pathways involved in cytokine-induced production of reactive oxygen species (ROS) in microglial cells. METHODS: Dihydroethidium (DHE) was used to assay superoxide production in neurons, while CM-H2DCF-DA was used to test for ROS production in murine (BV-2) and rat (HAPI) immortalized microglial cells. NADPH oxidase inhibitors (for example, diphenyleneiodonium (DPI), AEBSF, and apocynin) and immunocytochemistry targeting p47phox and gp91phox were used to assess the involvement of NADPH oxidase. Western blotting was used to assess iNOS and ERK1/2 expression, and the Griess reaction protocol was employed to determine nitric oxide (NO) concentration. RESULTS: Exposure of Hon and Mag (1-10 µM) to neurons for 24 h did not alter neuronal viability, but both compounds (10 µM) inhibited NMDA-stimulated superoxide production, a pathway known to involve NADPH oxidase. In microglial cells, Hon and Mag inhibited IFNγ±LPS-induced iNOS expression, NO, and ROS production. Studies with inhibitors and immunocytochemical assay further demonstrated the important role of IFNγ activating the NADPH oxidase through the p-ERK-dependent pathway. Hon and, to a lesser extent, Mag inhibited IFNγ-induced p-ERK1/2 and its downstream pathway for ROS and NO production. CONCLUSION: This study highlights the important role of NADPH oxidase in mediating oxidative stress in neurons and microglial cells and has unveiled the role of IFNγ in stimulating the MAPK/ERK1/2 signaling pathway for activation of NADPH oxidase in microglial cells. Hon and Mag offer anti-oxidative or anti-inflammatory effects, at least in part, through suppressing IFNγ-induced p-ERK1/2 and its downstream pathway.

Phospholipases A2 and neural membrane dynamics: implications for Alzheimer's disease.

Phospholipases A(2) (PLA(2)s) are essential enzymes in cells. They are not only responsible for maintaining the structural organization of cell membranes, but also play a pivotal role in the regulation of cell functions. Activation of PLA(2) s results in the release of fatty acids and lysophospholipids, products that are lipid mediators and compounds capable of altering membrane microdomains and physical properties. Although not fully understood, recent studies have linked aberrant PLA(2) activity to oxidative signaling pathways involving NADPH oxidase that underlie the pathophysiology of a number of neurodegenerative diseases. In this paper, we review studies describing the involvement of cytosolic PLA(2) in oxidative signaling pathways leading to neuronal impairment and activation of glial cell inflammatory responses. In addition, this review also includes information on the role of cytosolic PLA(2) and exogenous secretory PLA(2) on membrane physical properties, dynamics, and membrane proteins. Unraveling the mechanisms that regulate specific types of PLA(2)s and their effects on membrane dynamics are important prerequisites towards understanding their roles in the pathophysiology of Alzheimer's disease, and in the development of novel therapeutics to retard progression of the disease.

Pro-inflammatory cytokines and lipopolysaccharide induce changes in cell morphology, and upregulation of ERK1/2, iNOS and sPLA2-IIA expression in astrocytes and microglia.

BACKGROUND: Activation of glial cells, including astrocytes and microglia, has been implicated in the inflammatory responses underlying brain injury and neurodegenerative diseases including Alzheimer's and Parkinson's diseases. Although cultured astrocytes and microglia are capable of responding to pro-inflammatory cytokines and lipopolysaccharide (LPS) in the induction and release of inflammatory factors, no detailed analysis has been carried out to compare the induction of iNOS and sPLA2-IIA. In this study, we investigated the effects of cytokines (TNF-alpha, IL-1beta, and IFN-gamma) and LPS + IFN-gamma to induce temporal changes in cell morphology and induction of p-ERK1/2, iNOS and sPLA2-IIA expression in immortalized rat (HAPI) and mouse (BV-2) microglial cells, immortalized rat astrocytes (DITNC), and primary microglia and astrocytes. METHODS/RESULTS: Cytokines (TNF-alpha, IL-1beta, and IFN-gamma) and LPS + IFN-gamma induced a time-dependent increase in fine processes (filopodia) in microglial cells but not in astrocytes. Filopodia production was attributed to IFN-gamma and was dependent on ERK1/2 activation. Cytokines induced an early (15 min) and a delayed phase (1 ~ 4 h) increase in p-ERK1/2 expression in microglial cells, and the delayed phase increase corresponded to the increase in filopodia production. In general, microglial cells are more active in responding to cytokines and LPS than astrocytes in the induction of NO. Although IFN-gamma and LPS could individually induce NO, additive production was observed when IFN-gamma was added together with LPS. On the other hand, while TNF-alpha, IL-1beta, and LPS could individually induce sPLA2-IIA mRNA and protein expression, this induction process does not require IFN-gamma. Interestingly, neither rat immortalized nor primary microglial cells were capable of responding to cytokines and LPS in the induction of sPLA2-IIA expression. CONCLUSION: These results demonstrated the utility of BV-2 and HAPI cells as models for investigation on cytokine and LPS induction of iNOS, and DITNC astrocytes for induction of sPLA2-IIA. In addition, results further demonstrated that cytokine-induced sPLA2-IIA is attributed mainly to astrocytes and not microglial cells.

mGluR7 genetics and alcohol: intersection yields clues for addiction.

Development of addiction to alcohol or other substances can be attributed in part to exposure-dependent modifications at synaptic efficacy leading to an organism which functions at an altered homeostatic setpoint. Genetic factors may also influence setpoints and the stability of the homeostatic system of an organism. Quantitative genetic analysis of voluntary alcohol drinking, and mapping of the involved genes in the quasi-congenic Recombinant QTL Introgression strain system, identified Eac2 as a Quantitative Trait Locus (QTL) on mouse chromosome 6 which explained 18% of the variance with an effect size of 2.09 g/kg/day alcohol consumption, and Grm7 as a quantitative trait gene underlying Eac2 [Vadasz et al. in Neurochem Res 32:1099-1112, 100, Genomics 90:690-702, 102]. In earlier studies, the product of Grm7 mGluR7, a G protein-coupled receptor, has been implicated in stress systems [Mitsukawa et al. in Proc Natl Acad Sci USA 102:18712-18717, 63], anxiety-like behaviors [Cryan et al. in Eur J Neurosci 17:2409-2417, 14], memory [Holscher et al. in Learn Mem 12:450-455, 26], and psychiatric disorders (e.g., [Mick et al. in Am J Med Genet B Neuropsychiatr Genet 147B:1412-1418, 61; Ohtsuki et al. in Schizophr Res 101:9-16, 72; Pergadia et al. in Paper presented at the 38th Annual Meeting of the Behavior Genetics Association, Louisville, Kentucky, USA, 76]. Here, in experiments with mice, we show that (1) Grm7 knockout mice express increased alcohol consumption, (2) sub-congenic, and congenic mice carrying a Grm7 variant characterized by higher Grm7 mRNA drink less alcohol, and show a tendency for higher circadian dark phase motor activity in a wheel running paradigm, respectively, and (3) there are significant genetic differences in Grm7 mRNA abundance in the mouse brain between congenic and background mice identifying brain areas whose function is implicated in addiction related processes. We hypothesize that metabotropic glutamate receptors may function as regulators of homeostasis, and Grm7 (mGluR7) is involved in multiple processes (including stress, circadian activity, reward control, memory, etc.) which interact with substance use and the development of addiction. In conclusion, we suggest that mGluR7 is a significant new therapeutic target in addiction and related neurobehavioral disorders.

Interleukin-1beta enhances nucleotide-induced and alpha-secretase-dependent amyloid precursor protein processing in rat primary cortical neurons via up-regulation of the P2Y(2) receptor.

The heterologous expression and activation of the human P2Y(2) nucleotide receptor (P2Y(2)R) in human 1321N1 astrocytoma cells stimulates alpha-secretase-dependent cleavage of the amyloid precursor protein (APP), causing extracellular release of the non-amyloidogenic protein secreted amyloid precursor protein (sAPPalpha). To determine whether a similar response occurs in a neuronal cell, we analyzed whether P2Y(2)R-mediated production of sAPPalpha occurs in rat primary cortical neurons (rPCNs). In rPCNs, P2Y(2)R mRNA and receptor activity were virtually absent in quiescent cells, whereas overnight treatment with the pro-inflammatory cytokine interleukin-1beta (IL-1beta) up-regulated both P2Y(2)R mRNA expression and receptor activity by four-fold. The up-regulation of the P2Y(2)R was abrogated by pre-incubation with Bay 11-7085, an IkappaB-alpha phosphorylation inhibitor, which suggests that P2Y(2)R mRNA transcript levels are regulated through nuclear factor-kappa-B (NFkappaB) signaling. Furthermore, the P2Y(2)R agonist Uridine-5'-triphosphate (UTP) enhanced the release of sAPPalpha in rPCNs treated with IL-1beta or transfected with P2Y(2)R cDNA. UTP-induced release of sAPPalpha from rPCNs was completely inhibited by pre-treatment of the cells with the metalloproteinase inhibitor TACE inhibitor (TAPI-2) or the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002, and was partially inhibited by the MAPK/extracellular signal-regulated kinase inhibitor U0126 and the protein kinase C inhibitor GF109203. These data suggest that P2Y(2)R-mediated release of sAPPalpha from cortical neurons is directly dependent on a disintegrin and metalloproteinase (ADAM) 10/17 and PI3K activity, whereas extracellular signal-regulated kinase 1/2 and PI3K activity may indirectly regulate APP processing. These results demonstrate that elevated levels of pro-inflammatory cytokines associated with neurodegenerative diseases, such as IL-1beta, can enhance non-amyloidogenic APP processing through up-regulation of the P2Y(2)R in neurons.

Cyclooxygenase-2 inhibition improves amyloid-beta-mediated suppression of memory and synaptic plasticity.

Non-steroidal anti-inflammatory agents (NSAIDs) are associated with a marked reduction in the risk of developing Alzheimer's disease, a form of dementia characterized by the accumulation of amyloid plaques containing the amyloid-beta protein (Abeta). Studies of the effects of NSAIDs upon the inflammatory response surrounding amyloid plaques and upon the generation of Abeta from the amyloid precursor protein (APP) have led to two proposed mechanisms by which NSAIDs may protect against Alzheimer's disease: one, the selective lowering of Abeta42 by a subset of NSAIDs; and two, the reduction of inflammation. Although Alzheimer's disease is a disorder of brain and synaptic function, the effects of NSAIDs on Abeta-mediated suppression of synaptic plasticity and memory function have never been reported. We therefore investigated how three different NSAIDs, chosen for their distinct effects on Abeta42 production and the inhibition of the cyclooxygenase (COX) isoenzymes, COX-1 and COX-2, affect memory function and synaptic plasticity. By focusing upon brain and synapse function, we made novel observations about the effects of NSAIDs on Abeta-mediated neural processes. Here we report that the selective inhibition of COX-2, but not COX-1, acutely prevented the suppression of hippocampal long-term plasticity (LTP) by Abeta. The non-selective NSAIDs, ibuprofen and naproxen, and a selective COX-2 inhibitor, MF-tricyclic, each restored memory function in Tg2576 mice over-expressing APP, and also blocked Abeta-mediated inhibition of LTP. There was no advantage of ibuprofen, a selective Abeta42-lowering agent (SALA), over the non-SALAs, naproxen and MF-tricyclic. The beneficial effects on memory did not depend upon lowered levels of Abeta42 or the inflammatory cytokines, tumour necrosis factor alpha (TNF-alpha) and interleukin 1beta (IL-1beta). Intriguingly, improved memory function was inversely related to prostaglandin E2 (PGE2) levels. Conversely, exogenous PGE2 prevented the restorative effects of COX-2 inhibitors on LTP. The data indicate that the inhibition of COX-2 blocks Abeta-mediated suppression of LTP and memory function, and that this block occurs independently of reductions in Abeta42 or decreases in inflammation. The results lead us to propose a third possible mechanism by which NSAIDs may protect against Alzheimer's disease, involving the blockade of a COX-2-mediated PGE2 response at synapses.

From Analysis of Ischemic Mouse Brain Proteome to Identification of Human Serum Clusterin as a Potential Biomarker for Severity of Acute Ischemic Stroke.

Ischemic stroke is a devastating neurological disease that can cause permanent brain damage, but to date, few biomarkers are available to reliably assess the severity of injury during acute onset. In this study, quantitative proteomic analysis of ischemic mouse brain detected the increase in expression levels of clusterin (CLU) and cystatin C (CST3). Since CLU is a secretary protein, serum samples (n = 70) were obtained from acute ischemic stroke (AIS) patients within 24 h of stroke onset and together with 70 matched health controls. Analysis of CLU levels indicated significantly higher levels in AIS patients than healthy controls (14.91 ± 4.03 vs. 12.79 ± 2.22 ng/L; P = 0.0004). Analysis of serum CST3 also showed significant increase in AIS patients as compared with healthy controls (0.90 ± 0.19 vs. 0.84 ± 0.12 ng/L; P = 0.0064). The serum values of CLU were also positively correlated with the NIH Stroke Scale (NIHSS) scores, the time interval after stroke onset, as well as major stroke risk factors associated with lipid profile. These data demonstrate that elevated levels of serum CLU and CST3 are independently associated with AIS and may serve as peripheral biomarkers to aid clinical assessment of AIS and its severity. This pilot study thus contributes to progress toward preclinical proteomic screening by using animal models and allows translation of results from bench to bedside.

Amyloid beta peptide and NMDA induce ROS from NADPH oxidase and AA release from cytosolic phospholipase A2 in cortical neurons.

Increase in oxidative stress has been postulated to play an important role in the pathogenesis of a number of neurodegenerative diseases including Alzheimer's disease. There is evidence for involvement of amyloid-beta peptide (Abeta) in mediating the oxidative damage to neurons. Despite yet unknown mechanism, Abeta appears to exert action on the ionotropic glutamate receptors, especially the N-methyl-D-aspartic acid (NMDA) receptor subtypes. In this study, we showed that NMDA and oligomeric Abeta(1-42) could induce reactive oxygen species (ROS) production from cortical neurons through activation of NADPH oxidase. ROS derived from NADPH oxidase led to activation of extracellular signal-regulated kinase 1/2, phosphorylation of cytosolic phospholipase A(2)alpha (cPLA(2)alpha), and arachidonic acid (AA) release. In addition, Abeta(1-42)-induced AA release was inhibited by d(-)-2-amino-5-phosphonopentanoic acid and memantine, two different NMDA receptor antagonists, suggesting action of Abeta through the NMDA receptor. Besides serving as a precursor for eicosanoids, AA is also regarded as a retrograde messenger and plays a role in modulating synaptic plasticity. Other phospholipase A(2) products such as lysophospholipids can perturb membrane phospholipids. These results suggest an oxidative-degradative mechanism for oligomeric Abeta(1-42) to induce ROS production and stimulate AA release through the NMDA receptors. This novel mechanism may contribute to the oxidative stress hypothesis and synaptic failure that underline the pathogenesis of Alzheimer's disease.

MGlu5 antagonism impairs exploration and memory of spatial and non-spatial stimuli in rats.

Metabotropic glutamate receptor subtype 5 (mGlu5) has been implicated in memory processing in some but not all learning tasks. The reason why this receptor is involved in some tasks but not in others remains to be determined. The present experiments using rats examined effects of the mGlu5-antagonist 2-methyl-6-(phenylethynyl)-pyridine (MPEP)--applied systemically i.p. (1-10mg/kg) or bilaterally into the prelimbic cortex (1-10 microg)---on the ability of rats to explore and remember new stimuli. A cross-maze, open field, and object recognition task were used to evaluate exploration and memory and it was found that: (1) locomotion during exploration of spatial environments and exploration time at novel objects were reduced by i.p. but not by prelimbic administration of MPEP, (2) spatial short-term memory was impaired in cross-maze and object discrimination was reduced after both types of administration, (3) long-term retention of spatial conditioning in the cross-maze was inhibited after i.p. applications which (4) also inhibited spontaneous alternation performance during maze-exploration. Reduced exploratory locomotion and exploration time after i.p. injections may have contributed to the observed retention impairments. However, the fact that prelimbic administration of MPEP inhibited retention without reducing exploration shows that memory formation was also impacted directly by prelimbic mGlu5 in both spatial and non-spatial learning.

Linking blast physics to biological outcomes in mild traumatic brain injury: Narrative review and preliminary report of an open-field blast model.

Blast exposures are associated with traumatic brain injury (TBI) and blast-induced TBIs are common injuries affecting military personnel. Department of Defense and Veterans Administration (DoD/VA) reports for TBI indicated that the vast majority (82.3%) has been mild TBI (mTBI)/concussion. mTBI and associated posttraumatic stress disorders (PTSD) have been called "the invisible injury" of the current conflicts in Iraq and Afghanistan. These injuries induce varying degrees of neuropathological alterations and, in some cases, chronic cognitive, behavioral and neurological disorders. Appropriate animal models of blast-induced TBI will not only assist the understanding of physical characteristics of the blast, but also help to address the potential mechanisms. This report provides a brief overview of physical principles of blast, injury mechanisms related to blast exposure, current blast animal models, and the neurological behavioral and neuropathological findings related to blast injury in experimental settings. We describe relationships between blast peak pressures and the observed injuries. We also report preliminary use of a highly reproducible and intensity-graded blast murine model carried out in open-field with explosives, and describe physical and pathological findings in this experimental model. Our results indicate close relationships between blast intensities and neuropathology and behavioral deficits, particularly at low level blast intensities relevant to mTBI.

Docosahexaenoic acid (DHA): An essential nutrient and a nutraceutical for brain health and diseases.

Docosahexaenoic acid (DHA), a polyunsaturated fatty acid (PUFA) enriched in phospholipids in the brain and retina, is known to play multi-functional roles in brain health and diseases. While arachidonic acid (AA) is released from membrane phospholipids by cytosolic phospholipase A2 (cPLA2), DHA is linked to action of the Ca2+-independent iPLA2. DHA undergoes enzymatic conversion by 15-lipoxygenase (Alox 15) to form oxylipins including resolvins and neuroprotectins, which are powerful lipid mediators. DHA can also undergo non-enzymatic conversion by reacting with oxygen free radicals (ROS), which cause the production of 4-hydoxyhexenal (4-HHE), an aldehyde derivative which can form adducts with DNA, proteins and lipids. In studies with both animal models and humans, there is evidence that inadequate intake of maternal n-3 PUFA may lead to aberrant development and function of the central nervous system (CNS). What is less certain is whether consumption of n-3 PUFA is important in maintaining brain health throughout one's life span. Evidence mostly from non-human studies suggests that DHA intake above normal nutritional requirements might modify the risk/course of a number of diseases of the brain. This concept has fueled much of the present interest in DHA research, in particular, in attempts to delineate mechanisms whereby DHA may serve as a nutraceutical and confer neuroprotective effects. Current studies have revealed ability for the oxylipins to regulation of cell redox homeostasis through the Nuclear factor (erythroid-derived 2)-like 2/Antioxidant response element (Nrf2/ARE) anti-oxidant pathway, and impact signaling pathways associated with neurotransmitters, and modulation of neuronal functions involving brain-derived neurotropic factor (BDNF). This review is aimed at describing recent studies elaborating these mechanisms with special regard to aging and Alzheimer's disease, autism spectrum disorder, schizophrenia, traumatic brain injury, and stroke.

Ultrastructural brain abnormalities and associated behavioral changes in mice after low-intensity blast exposure.

Explosive blast-induced mild traumatic brain injury (mTBI), a "signature wound" of recent military conflicts, commonly affects service members. While past blast injury studies have provided insights into TBI with moderate- to high-intensity explosions, the impact of primary low-intensity blast (LIB)-mediated pathobiology on neurological deficits requires further investigation. Our prior considerations of blast physics predicted ultrastructural injuries at nanoscale levels. Here, we provide quantitative data using a primary LIB injury murine model exposed to open field detonation of 350 g of high-energy explosive C4. We quantified ultrastructural and behavioral changes up to 30 days post blast injury (DPI). The use of an open-field experimental blast generated a primary blast wave with a peak overpressure of 6.76 PSI (46.6 kPa) at a 3-m distance from the center of the explosion, a positive phase duration of approximate 3.0 milliseconds (ms), a maximal impulse of 8.7 PSI × ms and a sharp rising time of 9 × 10-3 ms, with no apparent impact/acceleration in exposed animals. Neuropathologically, myelinated axonal damage was observed in blast-exposed groups at 7 DPI. Using transmission electron microscopy, we observed and quantified myelin sheath defects and mitochondrial abnormalities at 7 and 30 DPI. Inverse correlations between blast intensities and neurobehavioral outcomes including motor activities, anxiety levels, nesting behavior, spatial learning and memory occurred. These observations uncover unique ultrastructural brain abnormalities and associated behavioral changes due to primary blast injury and provide key insights into its pathogenesis and potential treatment.

Ethanol preconditioning protects against ischemia/reperfusion-induced brain damage: role of NADPH oxidase-derived ROS.

Ethanol preconditioning (EtOH-PC) refers to a phenomenon in which tissues are protected from the deleterious effects of ischemia/reperfusion (I/R) by prior ingestion of ethanol at low to moderate levels. In this study, we tested whether prior (24 h) administration of ethanol as a single bolus that produced a peak plasma concentration of 42-46 mg/dl in gerbils would offer protective effects against neuronal damage due to cerebral I/R. In addition, we also tested whether reactive oxygen species (ROS) derived from NADPH oxidase played a role as initiators of these putative protective effects. Groups of gerbils were administered either ethanol or the same volume of water by gavage 24 h before transient global cerebral ischemia induced by occlusion of both common carotid arteries for 5 min. In some experiments, apocynin, a specific inhibitor of NADPH oxidase, was administered (5 mg/kg body wt, i.p.) 10 min before ethanol administration. EtOH-PC ameliorated behavioral deficit induced by cerebral I/R and protected the brain against I/R-induced delayed neuronal death, neuronal and dendritic degeneration, oxidative DNA damage, and glial cell activation. These beneficial effects were attenuated by apocynin treatment coincident with ethanol administration. Ethanol ingestion was associated with translocation of the NADPH oxidase subunit p67(phox) from hippocampal cytosol fraction to membrane, increased NADPH oxidase activity in hippocampus within the first hour after gavage, and increased lipid peroxidation (4-hydroxy-2-nonenal) in plasma and hippocampus within the first 2 h after gavage. These effects were also inhibited by concomitant apocynin treatment. Our data are consistent with the hypothesis that antecedent ethanol ingestion at socially relevant levels induces neuroprotective effects in I/R by a mechanism that is triggered by ROS produced through NADPH oxidase. Our results further suggest the possibility that preconditioning with other pharmacological agents that induce a mild oxidative stress may have similar therapeutic value for suppressing stroke-mediated damage in brain.

TNFα alters occludin and cerebral endothelial permeability: Role of p38MAPK.

Occludin is a key tight junction (TJ) protein in cerebral endothelial cells (CECs) playing an important role in modulating blood-brain barrier (BBB) functions. This protein (65kDa) has been shown to engage in many signaling pathways and phosphorylation by both tyrosine and threonine kinases. Despite yet unknown mechanisms, pro-inflammatory cytokines and endotoxin (lipopolysaccharides, LPS) may alter TJ proteins in CECs and BBB functions. Here we demonstrate the responses of occludin in an immortalized human cerebral endothelial cell line (hCMEC/D3) to stimulation by TNFα (10 ng/mL), IL-1ß (10 ng/mL) and LPS (100 ng/mL). Exposing cells to TNFα resulted in a rapid and transient upward band-shift of occludin, suggesting of an increase in phosphorylation. Exposure to IL-1ß produced significantly smaller effects and LPS produced almost no effects on occludin band-shift. TNFα also caused transient stimulation of p38MAPK and ERK1/2 in hCMEC/D3 cells, and the occludin band-shift induced by TNFα was suppressed by SB202190, an inhibitor for p38MAPK, and partly by U0126, the MEK1/2-ERK1/2 inhibitor. Cells treated with TNFα and IL-1ß but not LPS for 24 h resulted in a significant (p < 0.001) decrease in the expression of occludin, and the decrease could be partially blocked by SB202190, the inhibitor for p38MAPK. Treatment with TNFα also altered cell morphology and enhanced permeability of the CEC layer as measured by the FITC-dextran assay and the trans-endothelial electrical resistances (TEER). However, treatment with SB202190 alone could not effectively reverse the TNFα -induced morphology changes or the enhanced permeability changes. These results suggest that despite effects of TNFα on p38MAPK-mediated occludin phosphorylation and expression, these changes are not sufficient to avert the TNFα-induced alterations on cell morphology and permeability.

Secretory PLA2-IIA: a new inflammatory factor for Alzheimer's disease.

Secretory phospholipase A2-IIA (sPLA2-IIA) is an inflammatory protein known to play a role in the pathogenesis of many inflammatory diseases. Although this enzyme has also been implicated in the pathogenesis of neurodegenerative diseases, there has not been a direct demonstration of its expression in diseased human brain. In this study, we show that sPLA2-IIA mRNA is up-regulated in Alzheimer's disease (AD) brains as compared to non-demented elderly brains (ND). We also report a higher percentage of sPLA2-IIA-immunoreactive astrocytes present in AD hippocampus and inferior temporal gyrus (ITG). In ITG, the majority of sPLA2-IIA-positive astrocytes were associated with amyloid beta (Abeta)-containing plaques. Studies with human astrocytes in culture demonstrated the ability of oligomeric Abeta1-42 and interleukin-1beta (IL-1beta) to induce sPLA2-IIA mRNA expression, indicating that this gene is among those induced by inflammatory cytokines. Since exogenous sPLA2-IIA has been shown to cause neuronal injury, understanding the mechanism(s) and physiological consequences of sPLA2-IIA upregulation in AD brain may facilitate the development of novel therapeutic strategies to inhibit the inflammatory responses and to retard the progression of the disease.

Apocynin protects against global cerebral ischemia-reperfusion-induced oxidative stress and injury in the gerbil hippocampus.

Increased production of reactive oxygen species (ROS) following cerebral ischemia-reperfusion (I/R) is an important underlying cause for neuronal injury leading to delayed neuronal death (DND). In this study, apocynin, a specific inhibitor for NADPH oxidase, was used to test whether suppression of ROS by the NADPH oxidase inhibitor can protect against ischemia-induced ROS generation and decrease DND. Global cerebral ischemia was induced in gerbils by a 5-min occlusion of bilateral common carotid arteries (CCA). Using measurement of 4-hydroxy-2-nonenal (HNE) as a marker for lipid peroxidation, apocynin (5 mg/kg body weight) injected i.p. 30 min prior to ischemia significantly attenuated the early increase in HNE in hippocampus measured at 3 h after I/R. Apocynin also protected against I/R-induced neuronal degeneration and DND, oxidative DNA damage, and glial cell activation. Taken together, the neuroprotective effects of apocynin against ROS production during early phase of I/R and subsequent I/R-induced neuronal damage provide strong evidence that inhibition of NADPH oxidase could be a promising therapeutic mechanism to protect against stroke damage in the brain.

Ischemia-induced increase in RGS7 mRNA expression in gerbil hippocampus.

The present study investigated the changes in the expression of regulators of G-protein-coupled signaling proteins RGS2, 7 and 8 in gerbil hippocampus to better understand alterations of G-protein-coupled receptors signaling after cerebral ischemia. In situ hybridization revealed a transient, robust early increase in RGS7 mRNA levels in the dentate gyrus after ischemia. RGS8 mRNA expression started to increase at a later time point in the CA3 region but no changes were found for RGS2. Our results show a subtype-, time-, and subregion-specific regulation in mRNA expression of RGS proteins after cerebral ischemia in gerbil hippocampus.

Botanical Polyphenols Mitigate Microglial Activation and Microglia-Induced Neurotoxicity: Role of Cytosolic Phospholipase A2.

Microglia play a significant role in the generation and propagation of oxidative/nitrosative stress, and are the basis of neuroinflammatory responses in the central nervous system. Upon stimulation by endotoxins such as lipopolysaccharides (LPS), these cells release pro-inflammatory factors which can exert harmful effects on surrounding neurons, leading to secondary neuronal damage and cell death. Our previous studies demonstrated the effects of botanical polyphenols to mitigate inflammatory responses induced by LPS, and highlighted an important role for cytosolic phospholipase A2 (cPLA2) upstream of the pro-inflammatory pathways (Chuang et al. in J Neuroinflammation 12(1):199, 2015. doi: 10.1186/s12974-015-0419-0 ). In this study, we investigate the action of botanical compounds and assess whether suppression of cPLA2 in microglia is involved in the neurotoxic effects on neurons. Differentiated SH-SY5Y neuroblastoma cells were used to test the neurotoxicity of conditioned medium from stimulated microglial cells, and WST-1 assay was used to assess for the cell viability of SH-SY5Y cells. Botanicals such as quercetin and honokiol (but not cyanidin-3-O-glucoside, 3CG) were effective in inhibiting LPS-induced nitric oxide (NO) production and phosphorylation of cPLA2. Conditioned medium from BV-2 cells stimulated with LPS or IFNγ caused neurotoxicity to SH-SY5Y cells. Decrease in cell viability could be ameliorated by pharmacological inhibitors for cPLA2 as well as by down-regulating cPLA2 with siRNA. Botanicals effective in inhibition of LPS-induced NO and cPLA2 phosphorylation were also effective in ameliorating microglial-induced neurotoxicity. Results demonstrated cytotoxic factors from activated microglial cells to cause damaging effects to neurons and potential use of botanical polyphenols to ameliorate the neurotoxic effects.