The Chronic Effects of a Single Low-Intensity Blast Exposure on Phosphoproteome Networks and Cognitive Function Influenced by Mutant Tau Overexpression.

Blast-induced neurotrauma (BINT) is a pressing concern for veterans and civilians exposed to explosive devices. Affected personnel may have increased risk for long-term cognitive decline and developing tauopathies including Alzheimer's disease-related disorders (ADRD) or frontal-temporal dementia (FTD). The goal of this study was to identify the effect of BINT on molecular networks and their modulation by mutant tau in transgenic (Tg) mice overexpressing the human tau P301L mutation (rTg4510) linked to FTD or non-carriers. The primary focus was on the phosphoproteome because of the prominent role of hyperphosphorylation in neurological disorders. Discrimination learning was assessed following injury in the subsequent 6 weeks, using the automated home-cage monitoring CognitionWall platform. At 40 days post injury, label-free phosphoproteomics was used to evaluate molecular networks in the frontal cortex of mice. Utilizing a weighted peptide co-expression network analysis (WpCNA) approach, we identified phosphopeptide networks tied to associative learning and mossy-fiber pathways and those which predicted learning outcomes. Phosphorylation levels in these networks were inversely related to learning and linked to synaptic dysfunction, cognitive decline, and dementia including Atp6v1a and Itsn1. Low-intensity blast (LIB) selectively increased pSer262tau in rTg4510, a site implicated in initiating tauopathy. Additionally, individual and group level analyses identified the Arhgap33 phosphopeptide as an indicator of BINT-induced cognitive impairment predominantly in rTg4510 mice. This study unveils novel interactions between ADRD genetic susceptibility, BINT, and cognitive decline, thus identifying dysregulated pathways as targets in potential precision-medicine focused therapeutics to alleviate the disease burden among those affected by BINT.

Low-intensity open-field blast exposure effects on neurovascular unit ultrastructure in mice.

Mild traumatic brain injury (mTBI) induced by low-intensity blast (LIB) is a serious health problem affecting military service members and veterans. Our previous reports using a single open-field LIB mouse model showed the absence of gross microscopic damage or necrosis in the brain, while transmission electron microscopy (TEM) identified ultrastructural abnormalities of myelin sheaths, mitochondria, and synapses. The neurovascular unit (NVU), an anatomical and functional system with multiple components, is vital for the regulation of cerebral blood flow and cellular interactions. In this study, we delineated ultrastructural abnormalities affecting the NVU in mice with LIB exposure quantitatively and qualitatively. Luminal constrictive irregularities were identified at 7 days post-injury (DPI) followed by dilation at 30 DPI along with degeneration of pericytes. Quantitative proteomic analysis identified significantly altered vasomotor-related proteins at 24 h post-injury. Endothelial cell, basement membrane and astrocyte end-foot swellings, as well as vacuole formations, occurred in LIB-exposed mice, indicating cellular edema. Structural abnormalities of tight junctions and astrocyte end-foot detachment from basement membranes were also noted. These ultrastructural findings demonstrate that LIB induces multiple-component NVU damage. Prevention of NVU damage may aid in identifying therapeutic targets to mitigate the effects of primary brain blast injury.

Gelatin Zymography Can Be Performed on Fixed Brain Tissue.

Gelatin zymography is widely used to detect gelatinase activity, which is performed on unfixed tissue because it is assumed that fixation inactivates enzymes. However, using fixed tissues has several advantages over using fresh tissues for such prevention of tissue decay, thereby preserving the proteins as well as the morphology and structure of the specimens. In this study, we investigated the effects of the four commonly used fixatives (ethanol, acetone, zinc-based fixative (ZBF), and paraformaldehyde (PFA)) on the gelatinolytic activity in mouse brain tissue. Multiple protocols were employed to extract proteins from the fixed brain tissue. Western blotting and in-gel zymography (IGZ) were used to detect the gelatinase proteins and gelatinolytic activity of the extractions, respectively. In situ zymography (ISZ) revealed that ethanol, acetone, ZBF, and short-time PFA fixation did not inhibit gelatinolytic activity. Neither 1% Triton + 1 M NaCl nor 10% DMSO + 1 M NaCl was effective in extracting proteins from ethanol-, acetone-, ZBF-, or PFA-fixed brain tissues. However, 8 M urea + 4% CHAPS effectively extracted gelatinase proteins from ethanol- and acetone-fixed tissues while retaining the gelatinolytic activity. 2% SDS effectively extracted gelatinase proteins from ethanol-, acetone-, and ZBF-fixed tissues while retaining the gelatinolytic activity. Although 2% SDS + heating extracted gelatinase proteins from ethanol-, acetone-, ZBF-, and even long-term PFA-fixed tissues, the gelatinolytic activity was not retained. Our findings suggest that both ISZ and IGZ can be performed on fixed brain tissue, which is anticipated to be an improvement over the conventionally used gelatin zymography methods. (J Histochem Cytochem 71: 481-493, 2023).

Microglia Signaling Pathway Reporters Unveiled Manganese Activation of the Interferon/STAT1 Pathway and Its Mitigation by Flavonoids.

Neuroinflammatory responses to neurotoxic manganese (Mn) in CNS have been associated with the Mn-induced Parkinson-like syndromes. However, the framework of molecular mechanisms contributing to manganism is still unclear. Using an in vitro neuroinflammation model based on the insulated signaling pathway reporter transposon constructs stably transfected into a murine BV-2 microglia line, we tested effects of manganese (II) together with a set of 12 metal salts on the transcriptional activities of the NF-κB, activator protein-1 (AP-1), signal transducer and activator of transcription 1 (STAT1), STAT1/STAT2, STAT3, Nrf2, and metal-responsive transcription factor-1 (MTF-1) via luciferase assay, while concatenated destabilized green fluorescent protein expression provided for simultaneous evaluation of cellular viability. This experiment revealed specific and strong responses to manganese (II) in reporters of the type I and type II interferon-induced signaling pathways, while weaker activation of the NF-κB in the microglia was detected upon treatment of cells with Mn(II) and Ba(II). There was a similarity between Mn(II) and interferon-γ in the temporal STAT1 activation profile and in their antagonism to bacterial LPS. Sixty-four natural and synthetic flavonoids differentially affected both cytotoxicity and the pro-inflammatory activity of Mn (II) in the microglia. Whereas flavan-3-ols, flavanones, flavones, and flavonols were cytoprotective, isoflavones enhanced the cytotoxicity of Mn(II). Furthermore, about half of the tested flavonoids at 10-50 µM could attenuate both basal and 100-200 µM Mn(II)-induced activity at the gamma-interferon activated DNA sequence (GAS) in the cells, suggesting no critical roles for the metal chelation or antioxidant activity in the protective potential of flavonoids against manganese in microglia. In summary, results of the study identified Mn as a specific elicitor of the interferon-dependent pathways that can be mitigated by dietary polyphenols.

Inulin Supplementation Mitigates Gut Dysbiosis and Brain Impairment Induced by Mild Traumatic Brain Injury during Chronic Phase.

Mild traumatic brain injury (mTBI) has been shown to acutely alter the gut microbiome diversity and composition, known as dysbiosis, which can further exacerbate metabolic and vascular changes in the brain in both humans and rodents. However, it remains unknown how mTBI affects the gut microbiome in the chronic phase recovery (past one week post injury). It is also unknown if injury recovery can be improved by mitigating dysbiosis. The goal of the study is to fill the knowledge gap. First, we aim to understand how mTBI alters the gut microbiome through the chronic period of recovery (3 months post injury). In addition, as the gut microbiome can be modulated by diet, we also investigated if prebiotic inulin, a fermentable fiber that promotes growth of beneficial bacteria and metabolites, would mitigate dysbiosis, improve systemic metabolism, and protect brain structural and vascular integrity when administered after 3 months post closed head injury (CHI). We found that CHI given to male mice at 4 months of age induced gut dysbiosis which peaked at 1.5 months post injury, reduced cerebral blood flow (CBF) and altered brain white matter integrity. Interestingly, we also found that Sham mice had transient dysbiosis, which peaked 24 hours after injury and then normalized. After 8 weeks of inulin feeding, CHI mice had increased abundance of beneficial/anti-inflammatory bacteria, reduced abundance of pathogenic bacteria, enriched levels of short-chain fatty acids, and restored CBF in both hippocampi and left thalamus, compared to the CHI-control fed and Sham groups. Using machine learning, we further identified top bacterial species that separate Sham and CHI mice with and without the diet. Our results indicate that there is an injury- and time-dependent dysbiosis between CHI and Sham mice; inulin is effective to mitigate dysbiosis and improve brain injury recovery in the CHI mice. As there are currently no effective treatments for mTBI, the study may have profound implications for developing therapeutics or preventive interventions in the future.

Long-Term Effects of Low-Intensity Blast Non-Inertial Brain Injury on Anxiety-Like Behaviors in Mice: Home-Cage Monitoring Assessments.

Mild traumatic brain injury induced by low-intensity blast (LIB) exposure poses concerns in military personnel. Using an open-field, non-inertial blast model and assessments by conventional behavioral tests, our previous studies revealed early-phase anxiety-like behaviors in LIB-exposed mice. However, the impact of LIB upon long-term anxiety-like behaviors requires clarification. This study applied a highly sensitive automated home-cage monitoring (HCM) system, which minimized human intervention and environmental changes, to assess anxiety-like responses in mice 3 months after LIB exposure. Initial assessment of 72-h spontaneous activities in a natural cage condition over multiple light and dark phases showed altered sheltering behaviors. LIB-exposed mice exhibited a subtle, but significantly decreased, duration of short shelter visits as compared to sham controls. Other measured responses between LIB-exposed mice and sham controls were insignificant. When behavioral assessments were performed in a challenged condition using an aversive spotlight, LIB-exposed mice demonstrated a significantly higher frequency of movements of shorter distance and duration per movement. Taken together, these findings demonstrated the presence of chronic anxiety-like behaviors assessed by the HCM system under both natural and challenged conditions in mice occurring post-LIB exposure. This model thus provides a platform to test for screening and interventions on anxiety disorders occurring after LIB non-inertial brain injury.

Bidirectional Responses of Eight Neuroinflammation-Related Transcriptional Factors to 64 Flavonoids in Astrocytes with Transposable Insulated Signaling Pathway Reporters.

Neuroinflammation is implicated in a variety of pathologies and is mechanistically linked to hyperactivation of glial cells in the central nervous system (CNS), predominantly in response to external stimuli. Multiple dietary factors were reported to alter neuroinflammation, but their actions on the relevant transcription factors in glia are not sufficiently understood. Here, an in vitro protocol employing cultured astroglial cells, which carry reporters of multiple signaling pathways associated with inflammation, was developed for screening environmental factors and synthetic drugs. Immortalized rat astrocyte line DI TNC1 was stably transfected with piggyBac transposon vectors containing a series of insulated reporters for the transcriptional activity of NF-κB, AP-1, signal transducer and activator of transcription 1 (STAT1), signal transducer and activator of transcription 3 (STAT3), aromatic hydrocarbon receptor (AhR), Nrf2, peroxisome proliferator-activated receptor γ (PPARγ), and HIF-1α, which is quantified via luciferase assay. Concatenated green fluorescent protein (GFP) expression was employed for simultaneous evaluation of cellular viability. Responses to a set of 64 natural and synthetic monomeric flavonoids representing six main structural classes (flavan-3-ols, flavanones, flavones, flavonols, isoflavones, and anthocyan(id)ins) were obtained at 10 and 50 µM concentrations. Except for HIF-1α, the activity of NF-κB and other transcription factors (TFs) in astrocytes was predominantly inhibited by flavan-3-ols and anthocyan(id)ins, while flavones and isoflavones generally activated these TFs. In addition, we obtained dose-response profiles for 11 flavonoids (apigenin, baicalein, catechin, cyanidin, epigallocatechin gallate, genistein, hesperetin, kaempferol, luteolin, naringenin, and quercetin) within the 1-100 µM range and in the presence of immune-stimulants and immune-suppressors. The flavonoid concentration profiles for TF-activation reveal biphasic response curves from the astrocytes. Apart from epigallocatechin gallate (EGCG), flavonoids failed to inhibit the NF-κB activation by proinflammatory agents [lipopolysaccharide (LPS), cytokines], but most of the tested polyphenols synergized with STAT3 inhibitors (stattic, ruxolitinib) against the activation of this TF in the astrocytes. We conclude that transposable insulated reporters of transcriptional activation represent a convenient neurochemistry tool in screening for activators/inhibitors of signaling pathways.

Low-intensity blast induces acute glutamatergic hyperexcitability in mouse hippocampus leading to long-term learning deficits and altered expression of proteins involved in synaptic plasticity and serine protease inhibitors.

Neurocognitive consequences of blast-induced traumatic brain injury (bTBI) pose significant concerns for military service members and veterans with the majority of "invisible injury." However, the underlying mechanism of such mild bTBI by low-intensity blast (LIB) exposure for long-term cognitive and mental deficits remains elusive. Our previous studies have shown that mice exposed to LIB result in nanoscale ultrastructural abnormalities in the absence of gross or apparent cellular damage in the brain. Here we tested the hypothesis that glutamatergic hyperexcitability may contribute to long-term learning deficits. Using brain slice electrophysiological recordings, we found an increase in averaged frequencies with a burst pattern of miniature excitatory postsynaptic currents (mEPSCs) in hippocampal CA3 neurons in LIB-exposed mice at 1- and 7-days post injury, which was blocked by a specific NMDA receptor antagonist AP5. In addition, cognitive function assessed at 3-months post LIB exposure by automated home-cage monitoring showed deficits in dynamic patterns of discrimination learning and cognitive flexibility in LIB-exposed mice. Collected hippocampal tissue was further processed for quantitative global-proteomic analysis. Advanced data-independent acquisition for quantitative tandem mass spectrometry analysis identified altered expression of proteins involved in synaptic plasticity and serine protease inhibitors in LIB-exposed mice. Some were correlated with the ability of discrimination learning and cognitive flexibility. These findings show that acute glutamatergic hyperexcitability in the hippocampus induced by LIB may contribute to long-term cognitive dysfunction and protein alterations. Studies using this military-relevant mouse model of mild bTBI provide valuable insights into developing a potential therapeutic strategy to ameliorate hyperexcitability-modulated LIB injuries.

Impaired Kv7 channel activity in the central amygdala contributes to elevated sympathetic outflow in hypertension.

AIMS: Elevated sympathetic outflow is associated with primary hypertension. However, the mechanisms involved in heightened sympathetic outflow in hypertension are unclear. The central amygdala (CeA) regulates autonomic components of emotions through projections to the brainstem. The neuronal Kv7 channel is a non-inactivating voltage-dependent K+ channel encoded by KCNQ2/3 genes involved in stabilizing the neuronal membrane potential and regulating neuronal excitability. In this study, we investigated if altered Kv7 channel activity in the CeA contributes to heightened sympathetic outflow in hypertension. METHODS AND RESULTS: The mRNA and protein expression levels of Kv7.2/Kv7.3 in the CeA were significantly reduced in spontaneously hypertensive rats (SHRs) compared with Wistar-Kyoto (WKY) rats. Lowering blood pressure with coeliac ganglionectomy in SHRs did not alter Kv7.2 and Kv7.3 channel expression levels in the CeA. Fluospheres were injected into the rostral ventrolateral medulla (RVLM) to retrogradely label CeA neurons projecting to the RVLM (CeA-RVLM neurons). Kv7 channel currents recorded from CeA-RVLM neurons in brain slices were much smaller in SHRs than in WKY rats. Furthermore, the basal firing activity of CeA-RVLM neurons was significantly greater in SHRs than in WKY rats. Bath application of specific Kv7 channel blocker 10, 10-bis (4-pyridinylmethyl)-9(10H)-anthracnose (XE-991) increased the excitability of CeA-RVLM neurons in WKY rats, but not in SHRs. Microinjection of XE-991 into the CeA increased arterial blood pressure (ABP) and renal sympathetic nerve activity (RSNA), while microinjection of Kv7 channel opener QO-58 decreased ABP and RSNA, in anaesthetized WKY rats but not SHRs. CONCLUSIONS: Our findings suggest that diminished Kv7 channel activity in the CeA contributes to elevated sympathetic outflow in primary hypertension. This novel information provides new mechanistic insight into the pathogenesis of neurogenic hypertension.

Shock Wave Physics as Related to Primary Non-Impact Blast-Induced Traumatic Brain Injury.

INTRODUCTION: Blast overpressure exposure, an important cause of traumatic brain injury (TBI), may occur during combat or military training. TBI, most commonly mild TBI, is considered a signature injury of recent combat in Iraq and Afghanistan. Low intensity primary blast-induced TBI (bTBI), caused by exposure to an explosive shock wave, commonly leaves no obvious physical external signs. Numerous studies have been conducted to understand its biological effects; however, the role of shock wave energy as related to bTBI remains poorly understood. This report combines shock wave analysis with established biological effects on the mouse brain to provide insights into the effects of shock wave physics as related to low intensity bTBI outcomes from both open-air and shock tube environments. METHODS: Shock wave peak pressure, rise time, positive phase duration, impulse, shock velocity, and particle velocity were measured using the Missouri open-air blast model from 16 blast experiments totaling 122 mice to quantify physical shock wave properties. Open-air shock waves were generated by detonating 350-g 1-m suspended Composition C-4 charges with targets on 1-m elevated stands at 2.15, 3, 4, and 7 m from the source. RESULTS: All mice sustained brain injury with no observable head movement, because of mice experiencing lower dynamic pressures than calculated in shock tubes. Impulse, pressure loading over time, was found to be directly related to bTBI severity and is a primary shock physics variable that relates to bTBI. DISCUSSION: The physical blast properties including shock wave peak pressure, rise time, positive phase duration, impulse, shock velocity, and particle velocity were examined using the Missouri open-air blast model in mice with associated neurobehavioral deficits. The blast-exposed mice sustained ultrastructural abnormalities in mitochondria, myelinated axons, and synapses, implicating that primary low intensity blast leads to nanoscale brain damage by providing the link to its pathogenesis. The velocity of the shock wave reflected back from the target stand was calculated from high-speed video and compared with that of the incident shock wave velocity. Peak incident pressure measured from high sample rate sensors was found to be within 1% of the velocity recorded by the high-speed camera, concluding that using sensors in or close to an animal brain can provide useful information regarding shock velocity within the brain, leading to more advanced knowledge between shock wave physics and tissue damage that leads to bTBIs.

Docosahexaenoic Acid (DHA) Supplementation Alters Phospholipid Species and Lipid Peroxidation Products in Adult Mouse Brain, Heart, and Plasma.

The abundance of docosahexaenoic acid (DHA) in phospholipids in the brain and retina has generated interest to search for its role in mediating neurological functions. Besides the source of many oxylipins with pro-resolving properties, DHA also undergoes peroxidation, producing 4-hydroxyhexenal (4-HHE), although its function remains elusive. Despite wide dietary consumption, whether supplementation of DHA may alter the peroxidation products and their relationship to phospholipid species in brain and other body organs have not been explored sufficiently. In this study, adult mice were administered a control or DHA-enriched diet for 3 weeks, and phospholipid species and peroxidation products were examined in brain, heart, and plasma. Results demonstrated that this dietary regimen increased (n-3) and decreased (n-6) species to different extent in all major phospholipid classes (PC, dPE, PE-pl, PI and PS) examined. Besides changes in phospholipid species, DHA-enriched diet also showed substantial increases in 4-HHE in brain, heart, and plasma. Among different brain regions, the hippocampus responded to the DHA-enriched diet showing significant increase in 4-HHE. Considering the pro- and anti-inflammatory pathways mediated by the (n-6) and (n-3) polyunsaturated fatty acids, unveiling the ability for DHA-enriched diet to alter phospholipid species and lipid peroxidation products in the brain and in different body organs may be an important step forward towards understanding the mechanism(s) for this (n-3) fatty acid on health and diseases.

Perspectives on Primary Blast Injury of the Brain: Translational Insights Into Non-inertial Low-Intensity Blast Injury.

Most traumatic brain injuries (TBIs) during military deployment or training are clinically "mild" and frequently caused by non-impact blast exposures. Experimental models were developed to reproduce the biological consequences of high-intensity blasts causing moderate to severe brain injuries. However, the pathophysiological mechanisms of low-intensity blast (LIB)-induced neurological deficits have been understudied. This review provides perspectives on primary blast-induced mild TBI models and discusses translational aspects of LIB exposures as defined by standardized physical parameters including overpressure, impulse, and shock wave velocity. Our mouse LIB-exposure model, which reproduces deployment-related scenarios of open-field blast (OFB), caused neurobehavioral changes, including reduced exploratory activities, elevated anxiety-like levels, impaired nesting behavior, and compromised spatial reference learning and memory. These functional impairments associate with subcellular and ultrastructural neuropathological changes, such as myelinated axonal damage, synaptic alterations, and mitochondrial abnormalities occurring in the absence of gross- or cellular damage. Biochemically, we observed dysfunctional mitochondrial pathways that led to elevated oxidative stress, impaired fission-fusion dynamics, diminished mitophagy, decreased oxidative phosphorylation, and compensated cell respiration-relevant enzyme activity. LIB also induced increased levels of total tau, phosphorylated tau, and amyloid ß peptide, suggesting initiation of signaling cascades leading to neurodegeneration. We also compare translational aspects of OFB findings to alternative blast injury models. By scoping relevant recent research findings, we provide recommendations for future preclinical studies to better reflect military-operational and clinical realities. Overall, better alignment of preclinical models with clinical observations and experience related to military injuries will facilitate development of more precise diagnosis, clinical evaluation, treatment, and rehabilitation.

Bioactive components from garlic on brain resiliency against neuroinflammation and neurodegeneration.

Garlic (Allium sativum) has been widely used for culinary and medicinal purposes. Aged garlic extract (AGE) and sulfur-containing compounds, including S-allylcysteine (SAC) are well documented botanical active components of garlic. AGE is prepared by the prolonged extraction of fresh garlic with aqueous ethanol and is considered a nutritional supplement with potential to promote human health. SAC is a water-soluble organosulfur compound and the most abundant component of AGE. Studies have demonstrated that both AGE and SAC can exert neuroprotective effects against neuroinflammation and neurodegeneration. Another bioactive component in AGE is N-α-(1-deoxy-D-fructos-1-yl)-L-arginine (FruArg) although less is known about the metabolic activity of this compound. The main aim of this review was to provide an undated overview of the neuroprotective perspectives of these active garlic components (AGE, SAC and FruArg). Of interest, our studies and those of others indicate that both AGE and FruArg are involved in the regulation of gene transcription and protein expression. AGE has been shown to reverse 67% of the transcriptome alteration induced by endotoxins-lipopolysaccharide (LPS), and FruArg has been shown to account for the protective effects by reversing 55% of genes altered in a cell-based neuroinflammation paradigm stimulated by LPS in murine BV-2 microglial cells. AGE and FruArg can alleviate neuroinflammatory responses through a variety of signaling pathways, such as Toll-like receptor and interleukin (IL)-6 signaling, as well as by upregulating the nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated oxidative stress pathways known to promote microglial resiliency against neuroinflammation and neurodegeneration. The capability of FruArg to pass through the blood-brain barrier further supports its potential as a therapeutic compound. In summary, these experimental results provide new insight into the understanding of the neuroprotective effects of garlic components in promoting brain resiliency for health benefits.

Harpagophytum procumbens Extract Ameliorates Allodynia and Modulates Oxidative and Antioxidant Stress Pathways in a Rat Model of Spinal Cord Injury.

Spinal cord injury (SCI) is a deliberating disorder with impairments in locomotor deficits and incapacitating sensory abnormalities. Harpagophytum procumbens (Hp) is a botanical widely used for treating inflammation and pain related to various inflammatory and musculoskeletal conditions. Using a modified rodent contusion model of SCI, we explored the effects of this botanical on locomotor function and responses to mechanical stimuli, and examined possible neurochemical changes associated with SCI-induced allodynia. Following spinal cord contusion at T10 level, Hp (300 mg/kg, p.o.) or vehicle (water) was administered daily starting 24 h post-surgery, and behavioral measurements made every-other day until sacrifice (Day 21). Hp treatment markedly ameliorated the contusion-induced decrease in locomotor function and increased sensitivity to mechanical stimuli. Determination of Iba1 expression in spinal cord tissues indicated microglial infiltration starting 3 days post-injury. SCI results in increased levels of 4-hydroxynonenal, an oxidative stress product and proalgesic, which was diminished at 7 days by treatment with Hp. SCI also enhanced antioxidant heme oxygenase-1 (HO-1) expression. Concurrent studies of cultured murine BV-2 microglial cells revealed that Hp suppressed oxidative/nitrosative stress and inflammatory responses, including production of nitric oxide and reactive oxygen species, phosphorylation of cytosolic phospholipases A2, and upregulation of the antioxidative stress pathway involving the nuclear factor erythroid 2-related factor 2 and HO-1. These results support the use of Hp for management of allodynia by providing resilience against the neuroinflammation and pain associated with SCI and other neuropathological conditions.

Yin-Yang Mechanisms Regulating Lipid Peroxidation of Docosahexaenoic Acid and Arachidonic Acid in the Central Nervous System.

Phospholipids in the central nervous system (CNS) are rich in polyunsaturated fatty acids (PUFAs), particularly arachidonic acid (ARA) and docosahexaenoic acid (DHA). Besides providing physical properties to cell membranes, these PUFAs are metabolically active and undergo turnover through the "deacylation-reacylation (Land's) cycle". Recent studies suggest a Yin-Yang mechanism for metabolism of ARA and DHA, largely due to different phospholipases A2 (PLA2s) mediating their release. ARA and DHA are substrates of cyclooxygenases and lipoxygenases resulting in an array of lipid mediators, which are pro-inflammatory and pro-resolving. The PUFAs are susceptible to peroxidation by oxygen free radicals, resulting in the production of 4-hydroxynonenal (4-HNE) from ARA and 4-hydroxyhexenal (4-HHE) from DHA. These alkenal electrophiles are reactive and capable of forming adducts with proteins, phospholipids and nucleic acids. The perceived cytotoxic and hormetic effects of these hydroxyl-alkenals have impacted cell signaling pathways, glucose metabolism and mitochondrial functions in chronic and inflammatory diseases. Due to the high levels of DHA and ARA in brain phospholipids, this review is aimed at providing information on the Yin-Yang mechanisms for regulating these PUFAs and their lipid peroxidation products in the CNS, and implications of their roles in neurological disorders.

Maternal Dietary Docosahexaenoic Acid Alters Lipid Peroxidation Products and (n-3)/(n-6) Fatty Acid Balance in Offspring Mice.

The abundance of docosahexaenoic acid (DHA) in the mammalian brain has generated substantial interest in the search for its roles in regulating brain functions. Our recent study with a gene/stress mouse model provided evidence to support the ability for the maternal supplement of DHA to alleviate autism-associated behavior in the offspring. DHA and arachidonic acid (ARA) are substrates of enzymatic and non-enzymatic reactions, and lipid peroxidation results in the production of 4-hydroxyhexenal (4-HHE) and 4-hydroxynonenal (4-HNE), respectively. In this study, we examine whether a maternal DHA-supplemented diet alters fatty acids (FAs), as well as lipid peroxidation products in the pup brain, heart and plasma by a targeted metabolite approach. Pups in the maternal DHA-supplemented diet group showed an increase in DHA and a concomitant decrease in ARA in all brain regions examined. However, significant increases in 4-HHE, and not 4-HNE, were found mainly in the cerebral cortex and hippocampus. Analysis of heart and plasma showed large increases in DHA and 4-HHE, but a significant decrease in 4-HNE levels only in plasma. Taken together, the DHA-supplemented maternal diet alters the (n-3)/(n-6) FA ratio, and increases 4-HHE levels in pup brain, heart and plasma. These effects may contribute to the beneficial effects of DHA on neurodevelopment, as well as functional changes in other body organs.

Multi-Focal Neuronal Ultrastructural Abnormalities and Synaptic Alterations in Mice after Low-Intensity Blast Exposure.

Service members during military actions or combat training are exposed frequently to primary blast generated by explosive weaponry. The majority of military-related neurotrauma are classified as mild and designated as "invisible injuries" that are prevalent during current conflicts. While the previous experimental blast injury studies using moderate- to high-intensity exposures focused mainly on gross and microscopic neuropathology, our previous studies have shown that low-intensity blast (LIB) exposures resulted in nanoscale subcellular myelin and mitochondrial damages and subsequent behavioral disorders in the absence of gross or detectable cellular damage. In this study, we used transmission electron microscopy to delineate the LIB effects at the ultrastructural level specifically focusing on the neuron perikaryon, axons, and synapses in the cortex and hippocampus of mice at seven and 30 days post-injury (DPI). We found dysmorphic dark neuronal perikaryon and "cytoplasmic aeration" of dendritic processes, as well as increased microtubular fragmentation of the myelinated axons along with biochemically measured elevated tau/phosphorylated tau/Aß levels. The number of cortical excitatory synapses decreased along with a compensatory increase of the post-synaptic density (PSD) thickness both at seven and 30 DPI, while the amount of hippocampal CA1 synapses increased with the reduced PSD thickness. In addition, we observed a significant increase in protein levels of PSD95 and synaptophysin mainly at seven DPI indicating potential synaptic reorganization. These results demonstrated that a single LIB exposure can lead to ultrastructural brain injury with accompanying multi-focal neuronal organelle alterations. This pre-clinical study provides key insights into disease pathogenesis related to primary blast exposure.

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.

Proteomic Analysis and Biochemical Correlates of Mitochondrial Dysfunction after Low-Intensity Primary Blast Exposure.

Service members during military actions or combat training are frequently exposed to primary blasts by weaponry. Most studies have investigated moderate or severe brain injuries from blasts generating overpressures >100 kPa, whereas understanding the pathophysiology of low-intensity blast (LIB)-induced mild traumatic brain injury (mTBI) leading to neurological deficits remains elusive. Our recent studies, using an open-field LIB-induced mTBI mouse model with a peak overpressure at 46.6 kPa, demonstrated behavioral impairments and brain nanoscale damages, notably mitochondrial and axonal ultrastructural changes. In this study, we used tandem mass tagged (TMT) quantitative proteomics and bioinformatics analysis to seek insights into the molecular mechanisms underlying ultrastructural pathology. Changes in global- and phospho-proteomes were determined at 3 and 24 h and at 7 and 30 days post injury (DPI), in order to investigate the biochemical and molecular correlates of mitochondrial dysfunction. Results showed striking dynamic changes in a total of 2216 proteins and 459 phosphorylated proteins at vary time points after blast. Disruption of key canonical pathways included evidence of mitochondrial dysfunction, oxidative stress, axonal/cytoskeletal/synaptic dysregulation, and neurodegeneration. Bioinformatic analysis identified blast-induced trends in networks related to cellular growth/development/movement/assembly and cell-to-cell signaling interactions. With observations of proteomic changes, we found LIB-induced oxidative stress associated with mitochondrial dysfunction mainly at 7 and 30 DPI. These dysfunctions included impaired fission-fusion dynamics, diminished mitophagy, decreased oxidative phosphorylation, and compensated respiration-relevant enzyme activities. Insights on the early pathogenesis of primary LIB-induced brain damage provide a template for further characterization of its chronic effects, identification of potential biomarkers, and targets for intervention.

Proteomic Profiling of Mouse Brains Exposed to Blast-Induced Mild Traumatic Brain Injury Reveals Changes in Axonal Proteins and Phosphorylated Tau.

Alzheimer's disease (AD), the most prevalent form of dementia, is characterized by two pathological hallmarks: Tau-containing neurofibrillary tangles and amyloid-ß protein (Aß)-containing neuritic plaques. The goal of this study is to understand mild traumatic brain injury (mTBI)-related brain proteomic changes and tau-related biochemical adaptations that may contribute to AD-like neurodegeneration. We found that both phosphorylated tau (p-tau) and the ratio of p-tau/tau were significantly increased in brains of mice collected at 3 and 24 h after exposure to 82-kPa low-intensity open-field blast. Neurological deficits were observed in animals at 24 h and 7 days after the blast using Simple Neuroassessment of Asymmetric imPairment (SNAP) test, and axon/dendrite degeneration was revealed at 7 days by silver staining. Liquid chromatography-mass spectrometry (LC-MS/MS) was used to analyze brain tissue labeled with isobaric mass tags for relative protein quantification. The results from the proteomics and bioinformatic analysis illustrated the alterations of axonal and synaptic proteins in related pathways, including but not being limited to substantia nigra development, cortical cytoskeleton organization, and synaptic vesicle exocytosis, suggesting a potential axonal damage caused by blast-induced mTBI. Among altered proteins found in brains suffering blast, microtubule-associated protein 1B, stathmin, neurofilaments, actin binding proteins, myelin basic protein, calcium/calmodulin-dependent protein kinase, and synaptotagmin I were representative ones involved in altered pathways elicited by mTBI. Therefore, TBI induces elevated phospho-tau, a pathological feature found in brains of AD, and altered a number of neurophysiological processes, supporting the notion that blast-induced mTBI as a risk factor contributes to AD pathogenesis. LC/MS-based profiling has presented candidate target/pathways that could be explored for future therapeutic development.