In vivo Neuroprotective Effect of a Self-assembled Peptide Hydrogel.

Traumatic brain injury (TBI) is associated with poor intrinsic healing responses and long-term cognitive decline. A major pathological outcome of TBI is acute glutamate-mediated excitotoxicity (GME) experienced by neurons. Short peptides based on the neuroprotective extracellular glycoprotein ependymin have shown the ability to slow down the effect of GME - however, such short peptides tend to diffuse away from target sites after in vivo delivery. We have designed a self-assembling peptide containing an ependymin mimic that can form nanofibrous matrices. The peptide was evaluated in situ to assess neuroprotective utility after an acute fluidpercussion injury. This biomimetic matrix can conform to the intracranial damaged site after delivery, due its shear-responsive rheological properties. We demonstrated the potential efficacy of the peptide for supporting neuronal survival in vitro and in vivo. Our study demonstrates the potential of these implantable acellular hydrogels for managing the acute (up to 7 days) pathophysiological sequelae after traumatic brain injury. Further work is needed to evaluate less invasive administrative routes and long-term functional and behavioral improvements after injury.

Activation of NLRP3 inflammasome by cholesterol crystals in alcohol consumption induces atherosclerotic lesions.

Epidemiological studies showed a strong association between alcoholism and incidence of stroke, for which the underlying causative mechanisms remain to be understood. Here we found that infiltration of immune cells and deposition of cholesterol at the site of brain artery/capillary injury induced atherosclerosis in chronic alcohol (ethanol) consumption in the presence or absence of high-fat diet. Conversion of cholesterol into sharp edges of cholesterol crystals (CCs) in alcohol intake was key to activation of NLRP3 inflammasome, induction of cerebral atherosclerosis, and development of neuropathy around the atherosclerotic lesions. The presence of alcohol was critical for the formation of CCs and development of the neuropathology. Thus, we observed that alcohol consumption elevated the level of plasma cholesterol, deposition and crystallization of cholesterol, as well as activation of NLRP3 inflammasome. This led to arteriole or capillary walls thickening and increase intracranial blood pressure. Distinct neuropathy around the atherosclerotic lesions indicated vascular inflammation as an initial cause of neuronal degeneration. We demonstrated the molecular mechanisms of NLRP3 activation and downstream signaling cascade event in primary culture of human brain arterial/capillary endothelial cells in the setting of dose-/time-dependent effects of alcohol/CCs using NLRP3 gene silencing technique. We also detected CCs in blood samples from alcohol users, which validated the clinical importance of the findings. Finally, combined therapy of acetyl-l-carnitine and Lipitor® prevented deposition of cholesterol, formation of CCs, activation of NLRP3, thickening of vessel walls, and elevation of intracranial blood pressure. We conclude that alcohol-induced accumulation and crystallization of cholesterol activates NLRP3/caspase-1 in the cerebral vessel that leads to early development of atherosclerosis.

The mechanisms of cerebral vascular dysfunction and neuroinflammation by MMP-mediated degradation of VEGFR-2 in alcohol ingestion.

OBJECTIVE: Blood-brain barrier (BBB) dysfunction caused by activation of matrix metalloproteinases (MMPs) is a pathological feature in vascular/neurological disease. We describe the mechanisms of BBB dysfunction and neuroinflammation as a result of MMP-3/9 activation and disruption of vascular endothelial growth factor (VEGF)-A/VEGFR-2 interaction, impairing effective angiogenesis. METHODS AND RESULTS: We investigate the hypothesis in human brain endothelial cells and animal model of chronic alcohol ingestion. Proteome array analysis, zymography, immunofluorescence, and Western blotting techniques detected the activation, expression, and levels of MMP-3 and MMP-9. We found that degradation of VEGFR-2 and BBB proteins, for example, occludin, claudin-5, and ZO-1 by MMP-3/9, causes rupture of capillary endothelium and BBB leakiness. Impairment of BBB integrity was demonstrated by increased permeability of dye tracers and Fluo-3/calcein-AM-labeled monocyte adhesion or infiltration and decrease in transendothelial electric resistance. Alcohol-induced degradation of endothelial VEGFR-2 by MMP-3/9 led to a subsequent elevation of cellular/serum VEGF-A level. The decrease in VEGFR-2 with subsequent increase in VEGF-A level led to apoptosis and neuroinflammation via the activation of caspase-1 and IL-1ß release. The use of MMPs, VEGFR-2, and caspase-1 inhibitors helped to dissect the underlying mechanisms. CONCLUSIONS: Alcohol-induced MMPs activation is a key mechanism for dysfunction of BBB via degradation of VEGFR-2 protein and activation of caspase-1 or IL-1ß release. Targeting VEGF-induced MMP-3/9 activation can be a novel preventive approach to vascular inflammatory disease in alcohol abuse.

Hemolytic iron regulation in traumatic brain injury and alcohol use.

Hemorrhage is a major component of traumatic brain injury (TBI). Red blood cells, accumulated at the hemorrhagic site, undergo hemolysis upon energy depletion and release free iron into the central nervous system. This iron must be managed to prevent iron neurotoxicity and ferroptosis. As prior alcohol consumption is often associated with TBI, we examined iron regulation in a rat model of chronic alcohol feeding subjected to fluid percussion-induced TBI. We found that alcohol consumption prior to TBI altered the expression profiles of the lipocalin 2/heme oxygenase 1/ferritin iron management system. Notably, unlike TBI alone, TBI following chronic alcohol consumption sustained the expression of all three regulatory proteins for 1, 3, and 7 days post-injury. In addition, alcohol significantly affected TBI-induced expression of ferritin light chain at 3 days post-injury. We also found that alcohol exacerbated TBI-induced activation of microglia at 7 days post-injury. Finally, we propose that microglia may also play a role in iron management through red blood cell clearance.

Proteasome activity and autophagosome content in liver are reciprocally regulated by ethanol treatment.

UNLABELLED: The proteasome and autophagy are two major intracellular protein degradation pathways and the regulation of each by ethanol metabolism affects cellular integrity. Using acute and chronic ethanol feeding to mice in vivo, and precision-cut rat liver slices (PCLS) ex vivo, we examined whether ethanol treatment altered these proteolytic pathways. In acute studies, we gave C57Bl/6 mice either ethanol or phosphate-buffered saline (PBS) by gastric intubation and sacrificed them 12h later. PCLS were exposed to 0 or 50mM ethanol for 12 and 24h with or without 4-methylpyrazole (4MP). In chronic studies we pair-fed control and ethanol liquid diets for 4-6 weeks to transgenic mice, expressing the green fluorescent protein (GFP) fused to the autophagic marker, microtubule associated protein-1 light chain 3 (LC3). Acute ethanol administration elevated autophagosomes (AVs), as judged by a 1.5-fold increase in LC3II content over PBS-gavaged control mice. Hepatic proteasome activity was unaffected by this treatment. Compared with controls, ethanol exposure for 12 and 24h to PCLS inhibited proteasome activity by 1.5- to 3-fold and simultaneously enhanced AVs by 2- to 5-fold. The decrease in proteasome activity and the rise in AVs both depended on ethanol oxidation as its inhibition by 4-methylpyrazole (4MP) blocked both proteasome inhibition and AV induction. Hepatocytes from mice chronically consuming ethanol exhibited a 1.6-fold decline in proteasome activity, and a 4-fold rise in GFP-LC3 puncta compared with pair-fed control mice. When we exposed hepatocytes from these animals to MG262, a proteasome inhibitor, LC3II puncta per cell further increased 2- to 5-fold over untreated cells. CONCLUSION: Our findings demonstrate that ethanol metabolism generates oxidants, the levels of which differentially influence the activities of the proteasome and autophagy.

Synergistic effects of alcohol and HIV TAT protein on macrophage migration and neurotoxicity.

The trans-activator of transcription (TAT) is a human immunodeficiency virus (HIV-1) regulatory protein that is actively sloughed by infected cells. Once released, TAT can injure bystander cells and bring about their dysfunction. In the presence of ethanol, TAT-induced toxicity potentiates and, in so doing, exacerbates inflammation. One key aspect of neuroinflammation involves the infiltration of peripheral macrophage to the central nervous system. Here, we use an interactive neuroimmune cell coculture of brain endothelial, astrocyte, neuron, and macrophage cells to model the blood-brain barrier and evaluate macrophage migration upon challenge with ethanol and TAT concentrations. We have limited this study to examine TAT concentrations found in people living with HIV-1 with (5 ng/mL) or without (25 ng/mL) viral suppression and ethanol doses below the legal driving limit (10 mM). In so doing, we study the effects of casual drinking on people living with HIV-1 but experiencing the best possible clinical outcome. We found that TAT alone increases macrophage migration between 0.5 and 4 h. while ethanol alone increases migration in a delayed manner (occurring at 48 h.). Ethanol-induced NO production by endothelial cells and TAT's chemoattractant properties may explain this dichotomy in migration pattern. Combined low dose ethanol significantly increased migration under both 5 ng/mL and 25 ng/mL TAT injuries across all timepoints. Our findings suggest that co-presence of ethanol and TAT may be the combination of an initial TAT effect followed by subsequent ethanol treatment. We also examined the structural and behavioral changes of neurons treated with TAT and ethanol to understand their contribution to neurotoxicity. The lowest concentration of TAT still induced neurotoxicity while alcohol potentiated neuronal death, even at low doses.

The inflammatory footprints of alcohol-induced oxidative damage in neurovascular components.

Microvessels, the main components of the blood-brain barrier (BBB) are vulnerable to oxidative damage during alcohol-induced stress. Alcohol produces oxidative damage within the vessels and in the brain. Using our animal model of catheter implant into the common carotid artery (CCA), we trace the footprints of alcohol-induced oxidative damage and inflammatory process at the BBB and into the brain. The uniqueness of the finding is that ethanol causes oxidative damage in all neurovascular components by activating NADPH oxidase and inducible nitric oxide synthase in the brain. It is not the oxidants but the ethanol that traverses through the BBB because we found that the highly reactive peroxynitrite does not cross the BBB. Thus, oxidative damage is caused at the site of oxidant production in the microvessels and in the brain. Our data indicate that acetaldehyde (the primary metabolite of ethanol) is the inducer/activator of these enzymes that generate oxidants in brain neurovascular cells. Evidence for alcohol-induced BBB damage is indicated by the alterations of the tight junction protein occludin in intact microvessels. Importantly, we demonstrate that the site of BBB oxidative damage is also the site of immune cells aggregation in the microvessels, which paves the path for inflammatory footprints. These findings reveal the underlying mechanisms that ethanol-elicited BBB oxidative damage initiates the brain vascular inflammatory process, which ultimately leads to neuroinflammation.

Biphasic Effects of Ethanol Exposure on Waste Metabolites Clearance in the CNS.

We have shown that the effects of low-dose ethanol promote the clearance of waste metabolites, such as amyloid-beta (Aß) proteins, from the brain through the perivascular space (PVS). We demonstrated that dilative reactivity of arterial smooth muscle and endothelial cells regulate this clearance. These findings indicate the importance of blood-brain barrier (BBB) transvascular clearance of large size metabolites from the central nervous system (CNS), where the lymphatic clearance system is absent. We next examined the contrasting effects of acute low-dose and chronic moderate ethanol exposure on BBB-associated perivascular clearance. We injected a high molecular weight fluorescent dye into the interstitial space or directly into the cerebrospinal fluid (CSF). Bio-distribution of this tracer was then examined in different brain regions by multiphoton imaging and whole brain tissue section scanning. Ethanol-induced molecular/cellular mechanisms that drive the increase or decrease in movement of the fluorescent tracer were correlated to BBB integrity and arterial vessel reactivity. We found that activation of endothelial nitric oxide synthase (eNOS) under low-dose ethanol conditions with a shift to activation of inducible NOS (iNOS) under chronic high ethanol exposure conditions, which appeared to regulate these contrasting effects. We validated these observations by qualitative and quantitative investigation of eNOS, iNOS, BBB integrity, and perivascular clearance of waste metabolites. We concluded that the effects of low-dose ethanol increased the diffusive movement of waste metabolites via eNOS-derived NO, which increased the arterial endothelial-smooth muscle cell dilative reactivity without affecting BBB integrity, whereas a prolonged induction of iNOS under chronic ethanol exposure conditions caused oxidative damage of the arterial endothelial-smooth muscle layers resulting in cerebral amyloid-like angiopathy. This led to dysfunction of the BBB, dilative reactivity, and impaired waste metabolites movement from the interstitial space or subarachnoid space (SAS) through perivascular clearance.

Possible mechanisms of HIV neuro-infection in alcohol use: Interplay of oxidative stress, inflammation, and energy interruption.

Alcohol use and HIV-1 infection have a pervasive impact on brain function, which extends to the requirement, distribution, and utilization of energy within the central nervous system. This effect on neuroenergetics may explain, in part, the exacerbation of HIV-1 disease under the influence of alcohol, particularly the persistence of HIV-associated neurological complications. The objective of this review article is to highlight the possible mechanisms of HIV/AIDS progression in alcohol users from the perspective of oxidative stress, neuroinflammation, and interruption of energy metabolism. These include the hallmark of sustained immune cell activation and high metabolic energy demand by HIV-1-infected cells in the central nervous system, with at-risk alcohol use. Here, we discussed the point that the increase in energy supply requirement by HIV-1-infected neuroimmune cells as well as the deterrence of nutrient uptake across the blood-brain barrier significantly depletes the energy source and neuro-environment homeostasis in the CNS. We also described the mechanistic idea that comorbidity of HIV-1 infection and alcohol use can cause a metabolic shift and redistribution of energy usage toward HIV-1-infected neuroimmune cells, as shown in neuropathological evidence. Under such an imbalanced neuro-environment, meaningless energy waste is expected in infected cells, along with unnecessary malnutrition in non-infected neuronal cells, which is likely to accelerate HIV neuro-infection progression in alcohol use. Thus, it will be important to consider the factor of nutrients/energy imbalance in formulating treatment strategies to help impede the progression of HIV-1 disease and associated neurological disorders in alcohol use.

Hemorrhage Associated Mechanisms of Neuroinflammation in Experimental Traumatic Brain Injury.

Traumatic brain injury (TBI) is a major health problem for over 3.17 million people in the US, attracting increasing public attentions. Understanding the underlying mechanism of TBI is urgent for better diagnosis and treatment. Here, we examined the hypothesis that cerebral hemorrhagic coagulation and subsequent immune cells infiltration causes the progressive mechanisms of brain injury in moderate fluid percussion injury model. This represents a subdural hematoma and hemorrhagic head injury. We found increased hemorrhagic lesions and infarct volume in the injured brain with increment of pressure. The extent of hemorrhage was also validated by the bio-distribution of fluorescent tracer in cerebrospinal fluid (CSF) pathway after the injury. Bio-distribution of tracer was specifically diminished at the site of hemorrhage resulting from coagulation, which blocked the interstitial and CSF movement of the tracer. Increased expression of coagulation factor XII and necrotic cell death in and around the impact site confirmed the reason for this blockade. Different biomarkers, including immune cells accumulation and neuronal death showed that blood-brain barrier disruption played an important role for induction of neuroinflammation and neurodegeneration around the impact site. Our results suggest that instant hemorrhagic injury resulting from rupturing the brain blood vessels intertwined with coagulation causes onsite perivascular inflammation and neurodegeneration. Understanding of this sequential event should be valuable for development of therapeutic treatment in TBI. Graphical Abstract Underlying mechanisms in moderate/severe blunt TBI: hemorrhage following cerebrovascular disruption results in coagulation, thrombotic necrosis, and acute immune cell infiltration.

Alcohol induces programmed death receptor-1 and programmed death-ligand-1 differentially in neuroimmune cells.

Engagement of programmed death-1 (PD-1) receptor by its ligands (PD-L1/PD-L2) in activated immune cells is known to be involved in inflammatory neurological disease via a co-inhibitory signal pathway. Interaction of PD-1/PD-L1 is believed to occur only in activated neuroimmune cells because there are undetectable levels of PD-1/PD-L1 in normal physiological conditions. Here, we evaluated whether activation of neuroimmune cells such as human macrophage, brain endothelial cells (hBECs), astrocytes, microglia, and neurons by non-toxic concentrations of ethanol (EtOH) exposure can alter PD-1/PD-L1 expression. Thus, the present study is limited to the screening of PD-1/PD-L1 alterations in neuroimmune cells following ethanol exposure. We found that exposure of human macrophage or microglia to EtOH in primary culture immediately increased the levels of PD-L1 and gradually up-regulated PD-1 levels (beginning at 1-2 h). Similarly, ethanol exposure was able to induce PD-1/PD-L1 levels in hBECs and neuronal culture in a delayed process (occurring at 24 h). Astrocyte culture was the only cell type that showed endogenous levels of PD-1/PD-L1 that was decreased by EtOH exposure time-dependently. We concluded that ethanol (alcohol) mediated the induction of PD-1/PD-L1 differentially in neuroimmune cells. Taken together, our findings suggest that up-regulation of PD-1/PD-L1 by chronic alcohol use may dampen the innate immune response of neuroimmune cells, thereby contributing to neuroinflammation and neurodegeneration.

Angiogenic peptide hydrogels for treatment of traumatic brain injury.

Traumatic brain injury (TBI) impacts over 3.17 million Americans. Management of hemorrhage and coagulation caused by vascular disruption after TBI is critical for the recovery of patients. Cerebrovascular pathologies play an important role in the underlying mechanisms of TBI. The objective of this study is to evaluate a novel regenerative medicine for the injured tissue after brain injury. We utilized a recently described synthetic growth factor with angiogenic potential to facilitate vascular growth in situ at the injury site. Previous work has shown how this injectable self-assembling peptide-based hydrogel (SAPH) creates a regenerative microenvironment for neovascularization at the injury site. Supramolecular assembly allows for thixotropy; the injectable drug delivery system provides sustained in vivo efficacy. In this study, a moderate blunt injury model was used to cause physical vascular damage and hemorrhage. The angiogenic SAPH was then applied directly on the injured rat brain. At day 7 post-TBI, significantly more blood vessels were observed than the sham and injury control group, as well as activation of VEGF-receptor 2, demonstrating the robust angiogenic response elicited by the angiogenic SAPH. Vascular markers von-Willebrand factor (vWF) and α-smooth muscle actin (α-SMA) showed a concomitant increase with blood vessel density in response to the angiogenic SAPH. Moreover, blood brain barrier integrity and blood coagulation were also examined as the parameters to indicate wound recovery post TBI. Neuronal rescue examination by NeuN and myelin basic protein staining showed that the angiogenic SAPH may provide and neuroprotective benefit in the long-term recovery.

How does the brain remove its waste metabolites from within?

The brain is the command center of the body that regulates the vital functions of circulation, respiration, motor function, metabolic activities, or autonomic nervous system outcomes. The brain coordinates these continuous activities at the expense of huge energy utilization. This energy demand is achieved by active transport of nutrients across the endothelial blood-brain barrier (BBB). This review discusses the barrier interfaces in the CNS that include the BBB, blood-spinal cord barrier, the epithelial choroid plexus, and the epithelial arachnoid. While transporting of nutrients across the BBB is a normal physiological function, the trafficking of xenobiotics and inflammatory cells/agents across these interfaces is harmful to brain cells. This leads to production of waste metabolites in the brain. Clearance of these waste metabolites maintains the normal brain homeostasis, while aggregation is detrimental to neurological complications. Since the CNS lacks lymphatic system, the CSF serves as the clearance path for water-soluble peptides/solutes, but not large size waste metabolites like Aß protein. In particular, this review will focus on the mechanisms of waste metabolites clearance paths in the CNS. This will include the recently discovered waste metabolites movement from interstitial space (IS) directly into perivascular clearance (PVC), or via IS-CSF-PVC, and its exchange from PVC to circulation. Concluding remarks will discuss the therapeutic approach to improve the clearance mechanisms for ameliorating neurological diseases.

Antiretroviral drug-S for a possible HIV elimination.

Although the combination of highly active antiretroviral therapy (cART) can remarkably control human immunodeficiency virus type-1 (HIV-1) replication, it fails to cure HIV/AIDS disease. It is attributed to the incapability of cART to eliminate persistent HIV-1 contained in latent reservoirs in the central nervous system (CNS) and other tissue organs. Thus, withdrawal of cART causes rebound viral replication and resurgent of HIV/AIDS. The lack of success on non-ART approaches for elimination of HIV-1 include the targeted molecules not reaching the CNS, not adjusting well with drug-resistant mutants, or unable to eliminate all components of viral life cycle. Here, we show that our newly discovered Drug-S can effectively inhibit HIV-1 infection and persistence at the low concentration without causing any toxicity to neuroimmune cells. Our results suggest that Drug-S may have a direct effect on viral structure, prevent rebounding of HIV-1 infection, and arrest progression into acquired immunodeficiency syndrome. We also observed that Drug-S is capable of crossing the blood-brain barrier, suggesting a potential antiretroviral drug for elimination of CNS viral reservoirs and self-renewal of residual HIV-1. These results outlined the possible mechanism(s) of action of Drug-S as a novel antiretroviral drug for elimination of HIV-1 replication by interfering the virion structure.

Alcohol promotes waste clearance in the CNS via brain vascular reactivity.

The efficient clearance of the interstitial waste metabolites is essential for the normal maintenance of brain homeostasis. The brain lacks the lymphatic clearance system. Thus, the drainage of waste metabolites in the brain is dependent on a slow flow of cerebrospinal fluid (CSF) system. Glymphatic system claims the direct bulk flow transport of small size water-soluble waste metabolites into to the perivenous space by aquaporin-4 water channels of the astrocyte end-feet, but it did not address the diffusive clearance of large size waste metabolites. Here, we addressed the clearance mechanisms of large size waste metabolites from interstitial fluid to perivascular space as well as from CSF subarachnoid into perivascular space via the paravascular drainage. A low dose ethanol acting as a potent vasodilator promotes the dynamic clearance of waste metabolites through this perivascular-perivenous drainage path. We observed that ethanol-induced increased in vascular endothelial and smooth muscle cell reactivity regulated the enhanced clearance of metabolites. Here, activation of endothelial specific nitric oxide synthase (eNOS) by ethanol and generation of vasodilator nitric oxide mediates the interactive reactivity of endothelial-smooth muscle cells and subsequent diffusion of the CNS waste metabolites towards perivascular space. Detection of tracer dye (waste metabolite) in the perivenous space and in the blood samples further confirmed the improved clearance of waste metabolites through this unraveled interstitial-perivascular-perivenous clearance path. Our results suggest that alcohol intake at low-dose levels may promote clearance of neurological disease associated entangled proteins.

Animal Models of Traumatic Brain Injury and Assessment of Injury Severity.

Traumatic brain injury (TBI) contributes a major cause of death, disability, and mental health disorders. Most TBI patients suffer long-term post-traumatic stress disorder, cognitive dysfunction, and disability. The underlying molecular and cellular mechanisms of such neuropathology progression in TBI remain elusive. In part, it is due to non-standardized classification of mild, moderate, and severe injury in various animal models of TBI. Thus, a better diagnosis and treatment requires a better understanding of the injury mechanisms in a well-defined severity of mild, moderate, and severe injury in different models that may potentially reflect the various types of human brain injuries. The purpose of this review article is to highlight the classification of mild, moderate, and severe injury in various animal models of TBI with special focus on mixed injury that represents a translational concussive head injury. We will classify animal models of TBI broadly into focal injury, diffuse injury, and mixed injury. Focal injury, a localized injury, is represented by animal models of controlled cortical impact, penetrating ballistic-like brain injury, and Feeney or Shohami weight drop injury. A global diffuse injury is best represented by shock tube model of primary blast injury, and Marmarou or Maryland weight drop model. A mixed injury consists of focal and diffuse injury which reproduces the concussive clinical syndrome, and it is best studied in animal model of lateral fluid percussion injury.

Mechanism of alcohol-induced oxidative stress and neuronal injury.

Neuro-cognitive deficits, neuronal injury, and neurodegeneration are well documented in alcoholics, yet the underlying mechanisms remain elusive. Oxidative damage of mitochondria and cellular proteins intertwines with the progression of neuroinflammation and neurological disorders initiated by alcohol abuse. Here, we present the evidence that metabolism of ethanol in primary human neurons by alcohol dehydrogenase (ADH) or cytochrome P450-2E1 (CYP2E1) generates reactive oxygen species (ROS) and nitric oxide (NO) via induction of NADPH/xanthine oxidase (NOX/XOX) and nitric oxide synthase (NOS) in human neurons. The acetaldehyde-mediated increase in NOX, XOX, or NOS activity is regulated as a transcriptional rather than a translational process. Marked increase in the lipid peroxidation product (4-hydroxynonenal) and enhanced ROS generation coincides with decreased neuronal viability and diminished expression of neuronal marker (neurofilaments). Novel quantitative methods of ROS and NO detection help dissect the mechanisms of alcohol-induced neurodegeneration. Uncovering the basic mechanisms of oxidative neuronal injury will serve as the basis for development of new therapies.

Impairment of Thiamine Transport at the GUT-BBB-AXIS Contributes to Wernicke's Encephalopathy.

Wernicke's encephalopathy, a common neurological disease, is caused by thiamine (vitamin B1) deficiency. Neuropathy resulting from thiamine deficiency is a hallmark of Wernicke-Korsakoff syndrome in chronic alcohol users. The underlying mechanisms of this deficiency and progression of neuropathy remain to be understood. To uncover the unknown mechanisms of thiamine deficiency in alcohol abuse, we used chronic alcohol consumption or thiamine deficiency diet ingestion in animal models. Observations from animal models were validated in primary human neuronal culture for neurodegenerative process. We employed radio-labeled bio-distribution of thiamine, qualitative and quantitative analyses of the various biomarkers and neurodegenerative process. In the present studies, we established that disruption of thiamine transport across the intestinal gut blood-brain barrier axis as the cause of thiamine deficiency in the brain for neurodegeneration. We found that reduction in thiamine transport across these interfaces was the cause of reduction in the synthesis of thiamine pyrophosphate (TPP), an active cofactor for pyruvate dehydrogenase E1α (PDHE1α). Our findings revealed that decrease in the levels of PDHE1α cofactors switched on the activation of PD kinase (PDK) in the brain, thereby triggering the neuronal phosphorylation of PDHE1α (p-PDHE1α). Dysfunctional phosphorylated PDHE1α causes the reduction of mitochondrial aerobic respiration that led to neurodegeneration. We concluded that impairment of thiamine transport across the gut-BBB-axis that led to insufficient TPP synthesis was critical to Wernicke-neuropathy, which could be effectively prevented by stabilizing the thiamine transporters.

Partial purification from hot dogs of N-nitroso compound precursors and their mutagenicity after nitrosation.

Hot dogs contain apparent N-nitroso compounds (ANC) and ANC precursors (ANCP). ANCP purification was followed by nitrosation, sulfamic acid treatment, and analysis for ANC. Aqueous hot dog extracts were adsorbed on silica gel, which was eluted with MeCN and MeOH. The MeOH eluate was adsorbed on cation exchange resin (H+ form) and eluted with NH4OH. Eluted ANCP traveled at moderate speeds in high-performance liquid chromatography (HPLC) on amino and Pb2+ columns. Gas chromatography-mass spectrometry (GC-MS) of trimethylsilyl (TMS) derivatives of crude water extract indicated the presence of glycerol, phosphate, lactic acid, and two monosaccharides. GC-MS of TMS derivatives of Pb2+ column HPLC eluates indicated that ANCP included 1-deoxy-N-1-glucosyl glycine. The nitrosated NH4OH eluate showed 4x background mutagenic activity for Salmonella typhimurium TA-100. Un-nitrosated fractions showed 2x background activity. Although tryptophan nitrosation gave 88% ANC yield, tryptophan is probably not a major ANCP in hot dogs. Hot dog patties prepared with or without sucrose or glucose showed similar ANC and ANCP levels. We discuss possible implications of these findings for the etiology of colon cancer.

Primary blast causes mild, moderate, severe and lethal TBI with increasing blast overpressures: Experimental rat injury model.

Injury severity in blast induced Traumatic Brain Injury (bTBI) increases with blast overpressure (BOP) and impulse in dose-dependent manner. Pure primary blast waves were simulated in compressed gas shock-tubes in discrete increments. Present work demonstrates 24 hour survival of rats in 0-450 kPa (0-800 Pa∙s impulse) range at 10 discrete levels (60, 100, 130, 160, 190, 230, 250, 290, 350 and 420 kPa) and determines the mortality rate as a non-linear function of BOP. Using logistic regression model, predicted mortality rate (PMR) function was calculated, and used to establish TBI severities. We determined a BOP of 145 kPa as upper mild TBI threshold (5% PMR). Also we determined 146-220 kPa and 221-290 kPa levels as moderate and severe TBI based on 35%, and 70% PMR, respectively, while BOP above 290 kPa is lethal. Since there are no standards for animal bTBI injury severity, these thresholds need further refinements using histopathology, immunohistochemistry and behavior. Further, we specifically investigated mild TBI range (0-145 kPa) using physiological (heart rate), pathological (lung injury), immuno-histochemical (oxidative/nitrosative and blood-brain barrier markers) as well as blood borne biomarkers. With these additional data, we conclude that mild bTBI occurs in rats when the BOP is in the range of 85-145 kPa.