Ketamine Produces Antidepressant Effects by Inhibiting Histone Deacetylases and Upregulating Hippocampal Brain-Derived Neurotrophic Factor Levels in a Diisopropyl Fluorophosphate-Based Rat Model of Gulf War Illness.

Approximately one-third of Gulf War veterans suffer from Gulf War Illness (GWI), which encompasses mood disorders and depressive symptoms. Deployment-related exposure to organophosphate compounds has been associated with GWI development. Epigenetic modifications have been reported in GWI veterans. We previously showed that epigenetic histone dysregulations were associated with decreased brain-derived neurotrophic factor (BDNF) expression in a GWI rat model. GWI has no effective therapies. Ketamine (KET) has recently been approved by the Food and Drug Administration for therapy-resistant depression. Interestingly, BDNF upregulation underlies KET's antidepressant effect in GWI-related depression. Here, we investigated whether KET's effect on histone mechanisms signals BDNF upregulations in GWI. Male Sprague-Dawley rats were injected once daily with diisopropyl fluorophosphate (DFP; 0.5 mg/kg, s.c., 5 days). At 6 months following DFP exposure, KET (10 mg/kg, i.p.) was injected, and brains were dissected 24 hours later. Western blotting was used for protein expression, and epigenetic studies used chromatin immunoprecipitation methods. Dil staining was conducted for assessing dendritic spines. Our results indicated that an antidepressant dose of KET inhibited the upregulation of histone deacetylase (HDAC) enzymes in DFP rats. Furthermore, KET restored acetylated histone occupancy at the Bdnf promoter IV and induced BDNF protein expression in DFP rats. Finally, KET treatment also increased the spine density and altered the spine diversity with increased T-type and decreased S-type spines in DFP rats. Given these findings, we propose that KET's actions involve the inhibition of HDAC expression, upregulation of BDNF, and dendritic modifications that together ameliorates the pathologic synaptic plasticity and exerts an antidepressant effect in DFP rats. SIGNIFICANCE STATEMENT: This study offers evidence supporting the involvement of epigenetic histone pathways in the antidepressant effects of ketamine (KET) in a rat model of Gulf War Illness (GWI)-like depression. This effect is achieved through the modulation of histone acetylation at the Bdnf promoter, resulting in elevated brain-derived neurotrophic factor expression and subsequent dendritic remodeling in the hippocampus. These findings underscore the rationale for considering KET as a potential candidate for clinical trials aimed at managing GWI-related depression.

Fingolimod mitigates memory loss in a mouse model of Gulf War Illness amid decreasing the activation of microglia, protein kinase R, and NFκB.

Gulf War Illness (GWI) is an unrelenting multi-symptom illness with chronic central nervous system and peripheral pathology affecting veterans from the 1991 Gulf War and for which effective treatment is lacking. An increasing number of studies indicate that persistent neuroinflammation is likely the underlying cause of cognitive and mood dysfunction that affects veterans with GWI. We have previously reported that fingolimod, a drug approved for the treatment of relapsing-remitting multiple sclerosis, decreases neuroinflammation and improves cognition in a mouse model of Alzheimer's disease. In this study, we investigated the effect of fingolimod treatment on cognition and neuroinflammation in a mouse model of GWI. We exposed C57BL/6 J male mice to GWI-related chemicals pyridostigmine bromide, DEET, and permethrin, and to mild restraint stress for 28 days (GWI mice). Control mice were exposed to the chemicals' vehicle only. Starting 3 months post-exposure, half of the GWI mice and control mice were orally treated with fingolimod (1 mg/kg/day) for 1 month, and the other half were left untreated. Decreased memory on the Morris water maze test was detected in GWI mice compared to control mice and was reversed by fingolimod treatment. Immunohistochemical analysis of brain sections with antibodies to Iba1 and GFAP revealed that GWI mice had increased microglia activation in the hippocampal dentate gyrus, but no difference in reactive astrocytes was detected. The increased activation of microglia in GWI mice was decreased to the level in control mice by treatment with fingolimod. No effect of fingolimod treatment on gliosis in control mice was detected. To explore the signaling pathways by which decreased memory and increased neuroinflammation in GWI may be protected by fingolimod, we investigated the involvement of the inflammatory signaling pathways of protein kinase R (PKR) in the cerebral cortex of these mice. We found increased phosphorylation of PKR in the brain of GWI mice compared to controls, as well as increased phosphorylation of its most recognized downstream effectors: the α subunit of eukaryotic initiation factor 2 (eIF2α), IκB kinase (IKK), and the p65 subunit of nuclear factor-κB (NFκB-p65). Furthermore, we found that the increased phosphorylation level of these three proteins were suppressed in GWI mice treated with fingolimod. These results suggest that activation of PKR and NFκB signaling may be important for the regulation of cognition and neuroinflammation in the GWI condition and that fingolimod, a drug already approved for human use, may be a potential candidate for the treatment of GWI.

Brain-Specific Increase in Leukotriene Signaling Accompanies Chronic Neuroinflammation and Cognitive Impairment in a Model of Gulf War Illness.

Persistent cognitive impairment is a primary central nervous system-related symptom in veterans afflicted with chronic Gulf War Illness (GWI). Previous studies in a rat model have revealed that cognitive dysfunction in chronic GWI is associated with neuroinflammation, typified by astrocyte hypertrophy, activated microglia, and enhanced proinflammatory cytokine levels. Studies in a mouse model of GWI have also shown upregulation of several phospholipids that serve as reservoirs of arachidonic acid, a precursor of leukotrienes (LTs). However, it is unknown whether altered LT signaling is a component of chronic neuroinflammatory conditions in GWI. Therefore, this study investigated changes in LT signaling in the brain of rats displaying significant cognitive impairments six months after exposure to GWI-related chemicals and moderate stress. The concentration of cysteinyl LTs (CysLTs), LTB4, and 5-Lipoxygenase (5-LOX), the synthesizing enzyme of LTs, were evaluated. CysLT and LTB4 concentrations were elevated in the hippocampus and the cerebral cortex, along with enhanced 5-LOX expression in neurons and microglia. Such changes were also associated with increased proinflammatory cytokine levels in the hippocampus and the cerebral cortex. Enhanced CysLT and LTB4 levels in the brain could also be gleaned from their concentrations in brain-derived extracellular vesicles in the circulating blood. The circulating blood in GWI rats displayed elevated proinflammatory cytokines with no alterations in CysLT and LTB4 concentrations. The results provide new evidence that a brain-specific increase in LT signaling is another adverse alteration that potentially contributes to the maintenance of chronic neuroinflammation in GWI. Therefore, drugs capable of modulating LT signaling may reduce neuroinflammation and improve cognitive function in GWI. Additional findings demonstrate that altered LT levels in the brain could be tracked efficiently by analyzing brain-derived EVs in the circulating blood.

Sex-specific differences in plasma lipid profiles are associated with Gulf War Illness.

BACKGROUND: Nearly 250,000 veterans from the 1990-1991 Gulf War have Gulf War Illness (GWI), a condition with heterogeneous pathobiology that remains difficult to diagnose. As such, availability of blood biomarkers that reflect the underlying biology of GWI would help clinicians provide appropriate care to ill veterans. In this study, we measured blood lipids to examine the influence of sex on the association between blood lipids and GWI diagnosis. METHODS: Plasma lipid extracts from GWI (n = 100) and control (n = 45) participants were subjected to reversed-phase nano-flow liquid chromatography-mass spectrometry analysis. RESULTS: An influence of sex and GWI case status on plasma neutral lipid and phospholipid species was observed. Among male participants, triglycerides, diglycerides, and phosphatidylcholines were increased while cholesterol esters were decreased in GWI cases compared to controls. In female participants, ceramides were increased in GWI cases compared to controls. Among male participants, unsaturated triglycerides, phosphatidylcholine and diglycerides were increased while unsaturated cholesterol esters were lower in GWI cases compared to controls. The ratio of arachidonic acid- to docosahexaenoic acid-containing triglyceride species was increased in female and male GWI cases as compared to their sex-matched controls. CONCLUSION: Differential modulation of neutral lipids and ratios of arachidonic acid to docosahexaenoic acid in male veterans with GWI suggest metabolic dysfunction and inflammation. Increases in ceramides among female veterans with GWI also suggest activation of inflammatory pathways. Future research should characterize how these lipids and their associated pathways relate to GWI pathology to identify biomarkers of the disorder.

Persistent exercise fatigue and associative learning deficits in combination with transient glucose dyshomeostasis in a mouse model of Gulf War Illness.

AIMS: To characterize exercise fatigue, metabolic phenotype and cognitive and mood deficits correlated with brain neuroinflammatory and gut microbiome changes in a chronic Gulf War Illness (GWI) mouse model. The latter have been described in an accompanying paper [1]. MAIN METHODS: Adult male C57Bl/6N mice were exposed for 28 days (5 days/week) to pyridostigmine bromide: 6.5 mg/kg, b.i.d., P.O. (GW1) or 8.7 mg/kg, q.d., P.O. (GW2); topical permethrin (1.3 mg/kg in 100% DMSO) and N,N-diethyl-meta-toluamide (DEET 33% in 70% EtOH) and restraint stress (5 min). Exercise, metabolic and behavioral endpoints were compared to sham stress control (CON/S). KEY FINDINGS: Relative to CON/S, GW2 presented persistent exercise intolerance (through post-treatment (PT) day 161), deficient associative learning/memory, and transient insulin insensitivity. In contrast to GW2, GW1 showed deficient long-term object recognition memory, milder associative learning/memory deficit, and behavioral despair. SIGNIFICANCE: Our findings demonstrate that GW chemicals dose-dependently determine the presentation of exercise fatigue and severity/type of cognitive/mood-deficient phenotypes that show persistence. Our comprehensive mouse model of GWI recapitulates the major multiple symptom domains characterizing GWI, including fatigue and cognitive impairment that can be used to more efficiently develop diagnostic tests and curative treatments for ill Gulf War veterans.

Diagnosis of Gulf War Illness Using Laser-Induced Spectra Acquired from Blood Samples.

Gulf War illness (GWI) is a chronic illness with no known validated biomarkers that affects the lives of hundreds of thousands of people. As a result, there is an urgent need for the development of an untargeted and unbiased method to distinguish GWI patients from non-GWI patients. We report on the application of laser-induced breakdown spectroscopy (LIBS) to distinguish blood plasma samples from a group of subjects with GWI and from subjects with chronic low back pain as controls. We initially obtained LIBS data from blood plasma samples of four GWI patients and four non-GWI patients. We used an analytical method based on taking the difference between a mean LIBS spectrum obtained with those of GWI patients from the mean LIBS spectrum of those of the control group, to generate a "difference" spectrum for our classification model. This model was cross-validated using different numbers of differential LIBS emission peaks. A subset of 17 of the 82 atomic and ionic transitions that provided 70% of correct diagnosis was selected test in a blinded fashion using 10 additional samples and was found to yield 90% classification accuracy, 100% sensitivity, and 83.3% specificity. Of the 17 atomic and ionic transitions, eight could be assigned unambiguously to species of Na, K, and Fe.

Exposure to Gulf War Illness-related agents leads to the development of chronic pain and fatigue.

AIMS: A substantial contingent of veterans from the first Gulf War continues to suffer from a number of Gulf War-related illnesses (GWI) affecting the neurological and musculoskeletal systems; the most common symptoms include chronic pain and fatigue. Although animal models have recapitulated several aspects of cognitive impairments in GWI, the pain and fatigue symptoms have not been well documented to allow examination of potential pathogenic mechanisms. MAIN METHODS: We used a mouse model of GWI by exposing mice repeatedly to a combination of Gulf War chemicals (pyridostigmine bromide, permethrin, DEET, and chlorpyrifos) and mild immobilization stress, followed by investigating their pain susceptibilities and fatigue symptoms. To assess whether enhanced antioxidant capacity can counter the effects of GW agents, transgenic mice overexpressing extracellular superoxide dismutase (SOD3OE) were also examined. KEY FINDINGS: The mouse model recapitulated several aspects of the human illness, including hyperalgesia, impaired descending inhibition of pain, and increased tonic pain. There is a close association between chronic pain and fatigue in GWI patients. Consistent with this observation, the mouse model showed a significant reduction in physical endurance on the treadmill. Examination of skeletal muscles suggested reduction in mitochondrial functions may have contributed to the fatigue symptoms. Furthermore, the negative impacts of GW agents in pain susceptibilities were largely diminished in SOD3OE mice, suggesting that increased oxidative stress was associated with the emergence of these Gulf War symptoms. SIGNIFICANCE: the mouse model will be suitable for delineating specific defects in the pain pathways and mechanisms of fatigue in GWI.

Emerging role of glutamate in the pathophysiology and therapeutics of Gulf War illness.

Gulf War illness (GWI) is a chronic and multi-symptomatic disorder affecting veterans who served in the Gulf War. The commonly reported symptoms in GWI veterans include mood problems, cognitive impairment, muscle and joint pain, migraine/headache, chronic fatigue, gastrointestinal complaints, skin rashes, and respiratory problems. Neuroimaging studies have revealed significant brain structure alterations in GWI veterans, including subcortical atrophy, decreased volume of the hippocampus, reduced total grey and white matter, and increased brain white matter axial diffusivity. These brain changes may contribute to or increase the severities of the GWI-related symptoms. Epidemiological studies have revealed that neurotoxic exposures and stress may be significant contributors to the development of GWI. However, the mechanism underlying how the exposure and stress could contribute to the multi-symptomatic disorder of GWI remains unclear. We and others have demonstrated that rodent models exposed to GW-related agents and stress exhibited higher extracellular glutamate levels, as well as impaired structure and function of glutamatergic synapses. Restoration of the glutamatergic synapses ameliorated the GWI-related pathological and behavioral deficits. Moreover, recent studies showed that a low-glutamate diet reduced multiple symptoms in GWI veterans, suggesting an important role of the glutamatergic system in GWI. Currently, growing evidence has indicated that abnormal glutamate neurotransmission may contribute to the GWI symptoms. This review summarizes the potential roles of glutamate dyshomeostasis and dysfunction of the glutamatergic system in linking the initial cause to the multi-symptomatic outcomes in GWI and suggests the glutamatergic system as a therapeutic target for GWI.

Heart rate and heart rate variability as outcomes and longitudinal moderators of treatment for pain across follow-up in Veterans with Gulf War illness.

AIMS: Accumulating evidence suggests Gulf War illness (GWI) is characterised by autonomic nervous system dysfunction (higher heart rate [HR], lower heart rate variability [HRV]). Yoga - an ancient mind-body practice combining mindfulness, breathwork, and physical postures - is proposed to improve autonomic dysfunction yet this remains untested in GWI. We aimed to determine (i) whether HR and HRV improve among Veterans with GWI receiving either yoga or cognitive behavioural therapy (CBT) for pain; and (ii) whether baseline autonomic functioning predicts treatment-related pain outcomes across follow-up. MAIN METHODS: We present secondary analyses of 24-hour ambulatory cardiac data (mean HR, square root of the mean squared differences between successive R-R intervals [RMSSD], high frequency power [HF-HFV], and low-to-high frequency ratio [LF/HF] extracted from a 5-min window during the first hour of sleep) from our randomised controlled trial of yoga versus CBT for pain among Veterans with GWI (ClinicalTrials.govNCT02378025; N = 75). KEY FINDINGS: Veterans who received CBT tended towards higher mean HR at end-of-treatment. Better autonomic function (lower mean HR, higher RMSSD/HF-HRV) at baseline predicted greater reductions in pain across follow-up, regardless of treatment group. Better baseline autonomic function (mid-range-to-high RMSSD/HF-HRV) also predicted greater pain reductions with yoga, while worse baseline autonomic function (higher mean HR, lower RMSSD/HF-HRV) predicted greater pain reductions with CBT. SIGNIFICANCE: To our knowledge, this is the first study to suggest that among Veterans with GWI, HR may increase with CBT yet remain stable with yoga. Furthermore, HR and HRV moderated pain outcome across follow-up for yoga and CBT.

Effect of the low glutamate diet on inflammatory cytokines in veterans with Gulf War Illness (GWI): A pilot study.

AIM: To examine the effects of the low glutamate diet on inflammatory cytokines in veterans with Gulf War Illness (GWI). MAIN METHODS: Forty veterans with GWI were recruited from across the country. Anthropometric measurements and blood samples were collected at baseline and after one month on the low glutamate diet. Dietary adherence was measured with a glutamate food frequency questionnaire (FFQ). Inflammatory cytokines (IL-1ß, IL-6, IFN-γ, and TNF-α) were measured in pre- and post-diet serum (N = 34). Improvement was defined as being "much" or "very much" improved on the patient global impression of change scale (PGIC), or as having ≥30% of their symptoms remit. Correlations of the FFQ and the cytokines were calculated, followed by multivariable linear regression for significant findings. Mann Whitney U tests were used to compare cytokine levels according to improvement on the diet, and then logistic regression was used to estimate the association after adjustment for potential confounders. Classification trees were also produced to determine the ability of change in the inflammatory cytokines to predict improvement on the diet. KEY FINDINGS: Dietary adherence was significantly associated with reduction in TNF-α, and PGIC improvement was significantly associated with reduced IL-1ß, after adjustment for potential confounders. Classification trees demonstrated that IL-1ß, TNF-α, and IL-6 can predict improvement on the diet with 76.5% accuracy. SIGNIFICANCE: Findings suggest that the low glutamate diet may be able to reduce systemic inflammation in veterans with GWI.

The peroxisome proliferator-activated receptor gamma (PPARγ) agonist, rosiglitazone, ameliorates neurofunctional and neuroinflammatory abnormalities in a rat model of Gulf War Illness.

BACKGROUND: Gulf War (GW) Illness (GWI) is a debilitating condition with a complex constellation of immune, endocrine and neurological symptoms, including cognitive impairment, anxiety and depression. We studied a novel model of GWI based on 3 known common GW exposures (GWE): (i) intranasal lipopolysaccharide, to which personnel were exposed during desert sand storms; (ii) pyridostigmine bromide, used as prophylaxis against chemical warfare; and (iii) chronic unpredictable stress, an inescapable element of war. We used this model to evaluate prophylactic treatment with the PPARγ agonist, rosiglitazone (ROSI). METHODS: Rats were subjected to the three GWE for 33 days. In series 1 and 2, male and female GWE-rats were compared to naïve rats. In series 3, male rats with GWE were randomly assigned to prophylactic treatment with ROSI (GWE-ROSI) or vehicle. After the 33-day exposures, three neurofunctional domains were evaluated: cognition (novel object recognition), anxiety-like behaviors (elevated plus maze, open field) and depression-like behaviors (coat state, sucrose preference, splash test, tail suspension and forced swim). Brains were analyzed for astrocytic and microglial activation and neuroinflammation (GFAP, Iba1, tumor necrosis factor and translocator protein). Neurofunctional data from rats with similar exposures were pooled into 3 groups: naïve, GWE and GWE-ROSI. RESULTS: Compared to naïve rats, GWE-rats showed significant abnormalities in the three neurofunctional domains, along with significant neuroinflammation in amygdala and hippocampus. There were no differences between males and females with GWE. GWE-ROSI rats showed significant attenuation of neuroinflammation and of some of the neurofunctional abnormalities. CONCLUSION: This novel GWI model recapitulates critical neurofunctional abnormalities reported by Veterans with GWI. Concurrent prophylactic treatment with ROSI was beneficial in this model.

Neurotoxicity in Gulf War Illness and the potential role of glutamate.

Shortly after the Gulf War in 1990-1991, service men and women began reporting multiple symptoms ranging from persistent headaches, widespread pain, chronic fatigue, cognitive dysfunction, mood dysregulation, gastrointestinal issues, skin abnormalities, and respiratory problems. This prompted the Centers for Disease Control and Prevention (CDC) to initially classify the disorder as chronic multi-symptom illness (CMI), where it later became known as Gulf War Illness (GWI). Researchers and healthcare professionals since the early 1990s have been working extensively on alleviating the symptoms expressed in GWI as well as attempting to understand the mechanisms behind this illness. Scientific literature as well as reports from GWI veterans indicate that the toxic exposures during deployment may be responsible for the symptoms. These toxic exposures potentially include nerve agents, pyridostigmine bromide pills, pesticides, munitions with depleted uranium, and burning oil well fires. GWI currently affects 25-32 % of the 697,000 American troops who were stationed overseas during the short conflict. The purpose of this paper is to review the literature on neurotoxic exposures in Gulf War Illness, to explain how these exposures may lead to glutamate excitotoxicity, which has been implicated in the majority of the symptoms characterizing the illness, and to propose a novel treatment option for GWI.

Molecular mechanisms for the antidepressant-like effects of a low-dose ketamine treatment in a DFP-based rat model for Gulf War Illness.

Exposure to organophosphates (OP) during the First Gulf War is among one of the factors for Gulf War Illness (GWI) development in veterans and it has been challenging to treat GWI symptoms with existing therapies. Ketamine produces a rapid-onset and sustained antidepressant response, but there is no evidence whether ketamine treatment is effective for GWI depression. Repeated, low-dose exposure to diisopropyl fluorophosphate (DFP) mimic Gulf War related OP exposures and produces a chronic depressive state in rats. In this study, DFP-exposed rats treated with ketamine (10 mg/kg, i.p.) exhibited antidepressant-like effect on the Forced Swim Test at 1-h. This effect persisted at 24-h post ketamine, a time-point by which it is eliminated from the brain suggesting involvement of mechanisms that affect long-term synaptic plasticity. Western blot analysis showed significantly lower Brain-Derived Neurotrophic Factor (BDNF) levels in DFP rat brains. Ketamine produced a nonsignificant increase in BDNF expression at 1-h but produced a larger, significant (2.2-fold) increase at 24-h in DFP rats. We previously reported chronic hippocampal calcium elevations ([Ca2+]i) in DFP rats. Ketamine-treated DFP rats exhibited significantly lower [Ca2+]i at 1-h but not at 24-h. Interestingly, treatment with ANA-12, a TrkB-BDNF receptor antagonist, in DFP rats blunted ketamine's antidepressant-like effect at 24-h but not at 1-h. These experiments suggest that in a rat model of DFP-induced depression, inhibition of the NMDAR-Ca2+ contributes to the rapid-onset antidepressant effects of ketamine while the antidepressant actions that persisted at 24-h post ketamine administration involve upregulation of BDNF signaling.

Monosodium luminol reinstates redox homeostasis, improves cognition, mood and neurogenesis, and alleviates neuro- and systemic inflammation in a model of Gulf War Illness.

Enduring brain dysfunction is amid the highly manifested symptoms in veterans with Gulf War Illness (GWI). Animal studies have established that lasting brain dysfunction in GWI is concomitant with augmented oxidative stress, inflammation, and declined neurogenesis in the brain, and systemic inflammation. We hypothesize that drugs capable of restoring redox homeostasis in GWI will improve cognitive and mood function with modulation of neuroinflammation and neurogenesis. We examined the efficacy of monosodium luminol-GVT (MSL), a drug that promotes redox homeostasis, for improving cognitive and mood function in GWI rats. Young rats were exposed to GWI-related chemicals and moderate restraint stress for four weeks. Four months later, GWI rats received different doses of MSL or vehicle for eight weeks. Behavioral analyses in the last three weeks of treatment revealed that GWI rats receiving higher doses of MSL displayed better cognitive and mood function associated with reinstatement of redox homeostasis. Such restoration was evident from the normalized expression of multiple genes encoding proteins involved in combating oxidative stress in the brain and the return of several oxidative stress markers to control levels in the brain and the circulating blood. Sustained redox homeostasis by MSL also resulted in antiinflammatory and pro-neurogenic effects, which were apparent from reduced densities of hypertrophied astrocytes and activated microglia, and increased neurogenesis with augmented neural stem cell proliferation. Moreover, MSL treatment normalized the concentration of multiple proinflammatory markers in the circulating blood. Thus, MSL treatment reinstated redox homeostasis in an animal model of GWI, which resulted in alleviation of both brain and systemic inflammation, improved neurogenesis, and better cognitive and mood function.

Neurochemical and neuroinflammatory perturbations in two Gulf War Illness models: Modulation by the immunotherapeutic LNFPIII.

Gulf War Illness (GWI) manifests a multitude of symptoms, including neurological and immunological, and approximately a third of the 1990-1991 Gulf War (GW) veterans suffer from it. This study sought to characterize the acute neurochemical (monoamine) and neuroinflammatory profiles of two established GWI animal models and examine the potential modulatory effects of the novel immunotherapeutic Lacto-N-fucopentaose III (LNFPIII). In Model 1, male C57BL/6 J mice were treated for 10 days with pyridostigmine bromide (PB) and permethrin (PM). In Model 2, a separate cohort of mice were treated for 14 days with PB and N,N-Diethyl-methylbenzamide (DEET), plus corticosterone (CORT) via drinking water on days 8-14 and diisopropylfluorophosphate (DFP) on day 15. LNFPIII was administered concurrently with GWI chemicals treatments. Brain and spleen monoamines and hippocampal inflammatory marker expression were examined by, respectively, HPLC-ECD and qPCR, 6 h post treatment cessation. Serotonergic (5-HT) and dopaminergic (DA) dyshomeostasis caused by GWI chemicals was apparent in multiple brain regions, primarily in the nucleus accumbens (5-HT) and hippocampus (5-HT, DA) for both models. Splenic levels of 5-HT (both models) and norepinephrine (Model 2) were also disrupted by GWI chemicals. LNFPIII treatment prevented many of the GWI chemicals induced monoamine alterations. Hippocampal inflammatory cytokines were increased in both models, but the magnitude and spread of inflammation was greater in Model 2; LNFPIII was anti-inflammatory, more so in the apparently milder Model 1. Overall, in both models, GWI chemicals led to monoamine disbalance and neuroinflammation. LNFPIII co-treatment prevented many of these disruptions in both models, which is indicative of its promise as a potential GWI therapeutic.

A permethrin metabolite is associated with adaptive immune responses in Gulf War Illness.

Gulf War Illness (GWI), affecting 30% of veterans from the 1991 Gulf War (GW), is a multi-symptom illness with features similar to those of patients with autoimmune diseases. The objective of the current work is to determine if exposure to GW-related pesticides, such as permethrin (PER), activates peripheral and central nervous system (CNS) adaptive immune responses. In the current study, we focused on a PER metabolite, 3-phenoxybenzoic acid (3-PBA), as this is a common metabolite previously shown to form adducts with endogenous proteins. We observed the presence of 3-PBA and 3-PBA modified lysine of protein peptides in the brain, blood and liver of pyridostigmine bromide (PB) and  PER (PB+PER) exposed mice at acute and chronic post-exposure timepoints. We tested whether 3-PBA-haptenated albumin (3-PBA-albumin) can activate immune cells since it is known that chemically haptenated proteins can stimulate immune responses. We detected autoantibodies against 3-PBA-albumin in plasma from PB + PER exposed mice and veterans with GWI at chronic post-exposure timepoints. We also observed that in vitro treatment of blood with 3-PBA-albumin resulted in the activation of B- and T-helper lymphocytes and that these immune cells were also increased in blood of PB + PER exposed mice and veterans with GWI. These immune changes corresponded with elevated levels of infiltrating monocytes in the brain and blood of PB + PER exposed mice which coincided with alterations in the markers of blood-brain barrier disruption, brain macrophages and neuroinflammation. These studies suggest that pesticide exposure associated with GWI may have resulted in the activation of the peripheral and CNS adaptive immune responses, possibly contributing to an autoimmune-type phenotype in veterans with GWI.

Metabolic features of Gulf War illness.

BACKGROUND: More than 230,000 veterans-about 1/3 of US personnel deployed in the 1990-1991 Persian Gulf War-developed chronic, multi-symptom health problems now called "Gulf War illness" (GWI), for which mechanisms and objective diagnostic signatures continue to be sought. METHODS: Targeted, broad-spectrum serum metabolomics was used to gain insights into the biology of GWI. 40 male participants, included 20 veterans who met both Kansas and CDC diagnostic criteria for GWI and 20 nonveteran controls without similar symptoms that were 1:1 matched to GWI cases by age, sex, and ethnicity. Serum samples were collected and archived at -80° C prior to testing. 358 metabolites from 46 biochemical pathways were measured by hydrophilic interaction liquid chromatography and tandem mass spectrometry. RESULTS: Veterans with GWI, compared to healthy controls, had abnormalities in 8 of 46 biochemical pathways interrogated. Lipid abnormalities accounted for 78% of the metabolic impact. Fifteen ceramides and sphingomyelins, and four phosphatidylcholine lipids were increased. Five of the 8 pathways were shared with the previously reported metabolic phenotype of males with Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS). However, 4 of the 5 shared pathways were regulated in opposite directions; key pathways that were up-regulated in GWI were down-regulated in ME/CFS. The single pathway regulated in the same direction was purines, which were decreased. CONCLUSIONS: Our data show that despite heterogeneous exposure histories, a metabolic phenotype of GWI was clearly distinguished from controls. Metabolomic differences between GWI and ME/CFS show that common clinical symptoms like fatigue can have different chemical mechanisms and different diagnostic implications. Larger studies will be needed to validate these findings.

Targeting Intracellular Calcium Stores Alleviates Neurological Morbidities in a DFP-Based Rat Model of Gulf War Illness.

Gulf War Illness (GWI) is a chronic multi-symptom disorder afflicting the veterans of the First Gulf War, and includes neurological symptoms characterized by depression and memory deficits. Chronic exposure to organophosphates (OPs) is considered a leading cause for GWI, yet its pathobiology is not fully understood. We recently observed chronic elevations in neuronal Ca2+ levels ([Ca2+]i) in an OP-diisopropyl fluorophosphate (DFP)-based rat model for GWI. This study was aimed at identifying mechanisms underlying elevated [Ca2+]i in this DFP model and investigating whether their therapeutic targeting could improve GWI-like neurological morbidities. Male Sprague-Dawley rats (9 weeks) were exposed to DFP (0.5 mg/kg, s.c., 1×-daily for 5 days) and at 3 months postDFP exposure, behavior was assessed and rats were euthanized for protein estimations and ratiometric Fura-2 [Ca2+]i estimations in acutely dissociated hippocampal neurons. In DFP rats, a sustained elevation in intracellular Ca2+ levels occurred, and pharmacological blockade of Ca2+-induced Ca2+-release mechanisms significantly lowered elevated [Ca2+]i in DFP neurons. Significant reductions in the protein levels of the ryanodine receptor (RyR) stabilizing protein Calstabin2 were also noted. Such a posttranslational modification would render RyR "leaky" resulting in sustained DFP [Ca2+]i elevations. Antagonism of RyR with levetiracetam significantly lower elevated [Ca2+]i in DFP neurons and improved GWI-like behavioral symptoms. Since Ca2+ is a major second messenger molecule, such chronic increases in its levels could underlie pathological synaptic plasticity that expresses itself as GWI morbidities. Our studies show that treatment with drugs targeted at blocking intracellular Ca2+ release could be effective therapies for GWI neurological morbidities.

Inhibition of microRNA-124-3p as a novel therapeutic strategy for the treatment of Gulf War Illness: Evaluation in a rat model.

Gulf War Illness (GWI) is a chronic, multisymptom illness that continues to affect up to 30% of veterans deployed to the Persian Gulf during the 1990-1991 Gulf War. After nearly 30 years, useful treatments for GWI are lacking and underlying cellular and molecular mechanisms involved in its pathobiology remain poorly understood, although exposures to pyridostigmine bromide (PB) and pesticides are consistently identified to be among the strongest risk factors. Alleviation of the broad range of symptoms manifested in GWI, which involve the central nervous system, the neuroendocrine system, and the immune system likely requires therapies that are able to activate and inactivate a large set of orchestrated genes. Previous work in our laboratory using an established rat model of GWI identified persistent elevation of microRNA-124-3p (miR-124) levels in the hippocampus whose numerous gene targets are involved in cognition-associated pathways and neuroendocrine function. This study aimed to investigate the broad effects of miR-124 inhibition in the brain 9 months after completion of a 28-day exposure regimen of PB, DEET (N,N-diethyl-3-methylbenzamide), permethrin, and mild stress by profiling the hippocampal expression of genes known to play a critical role in synaptic plasticity, glucocorticoid signaling, and neurogenesis. We determined that intracerebroventricular infusion of a miR-124 antisense oligonucleotide (miR-124 inhibitor; 0.05-0.5 nmol/day/28 days), but not a negative control oligonucleotide, into the lateral ventricle of the brain caused increased protein expression of multiple validated miR-124 targets and increased expression of downstream target genes important for cognition and neuroendocrine signaling in the hippocampus. Off-target cardiotoxic effects were revealed in GWI rats receiving 0.1 nmol/day as indicated by the detection in plasma of 5 highly elevated protein cardiac injury markers and 6 upregulated cardiac-enriched miRNAs in plasma exosomes determined by next-generation sequencing. Results from this study suggest that in vivo inhibition of miR-124 function in the hippocampus is a promising, novel therapeutic approach to improve cognition and neuroendocrine dysfunction in GWI. Additional preclinical studies in animal models to assess feasibility and safety by developing a practical, noninvasive drug delivery system to the brain and exploring potential adverse toxicologic effects of miR-124 inhibition are warranted.

Oleoylethanolamide treatment reduces neurobehavioral deficits and brain pathology in a mouse model of Gulf War Illness.

There are nearly 250,000 Gulf War (GW) veterans who suffer from Gulf War Illness (GWI), a multi-symptom condition that remains untreatable. The main objective was to determine if targeting peroxisomal function could be of therapeutic value in GWI. We performed a pilot study that showed accumulation of very long chain fatty acids (VLCFA), which are metabolized in peroxisomes, in plasma from veterans with GWI. We then examined if targeting peroxisomal ß-oxidation with oleoylethanolamide (OEA) restores these lipids to the normal levels and mitigates neuroinflammation and neurobehavioral deficits in a well-established mouse model of GWI. In GWI mice, treatment with OEA corresponded with cognitive benefits and reduced fatigue and disinhibition-like behavior in GWI mice. Biochemical and molecular analysis of the brain tissue showed reduced astroglia and microglia staining, decreased levels of chemokines and cytokines, and decreased NFκB phosphorylation. Treatment with OEA reduced accumulation of peroxisome specific VLCFA in the brains of GWI mice. These studies further support the translational value of targeting peroxisomes. We expect that OEA may be a potential therapy for treating neurobehavioral symptoms and the underlying lipid dysfunction and neuroinflammation associated with GWI. Oleoylethanolamide is available as a dietary supplement, making it appealing for human translational studies.