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• Gulf War Illness (GWI) is a multi-symptom chronic condition associated with neurotoxic exposures. • Troops were exposed to burning munitions & oil fires, pesticides, and uranium. • Multiple of these toxins can increase glutamate levels leading to excitotoxicity. • Toxic particulates may lead to glutamate excitotoxicity induced oxidative stress. • Future research should examine glutamate dysregulation and oxidative stress in GWI. 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. [ABSTRACT FROM AUTHOR]

• Blood NAD and Sirt1 are low in GW veterans with GWI. • Treatment with NR increased NAD and alleviated fatigue-like behavior in a mouse model of GWI. • Consistent with human GWI, blood NAD and Sirt1 are low in our mouse model of GWI. • Brain NAD levels were normal in GWI mice but are increased in both GWI and control mice after NR treatment. • Treatment with NR increased Sirt1 activity and reduced neuroinflammation in the brains of GWI mice. • Targeting sirtuin activity with NR may represent a new avenue for treating GWI. Gulf War Illness (GWI) affects 30% of veterans from the 1991 Gulf War (GW), who suffer from symptoms that reflect ongoing mitochondria dysfunction. Brain mitochondria bioenergetics dysfunction in GWI animal models corresponds with astroglia activation and neuroinflammation. In a pilot study of GW veterans (n = 43), we observed that blood nicotinamide adenine dinucleotide (NAD) and sirtuin 1 (Sirt1) protein levels were decreased in the blood of veterans with GWI compared to healthy GW veterans. Since nicotinamide riboside (NR)-mediated targeting of Sirt1 is shown to improve mitochondria function, we tested whether NR can restore brain bioenergetics and reduce neuroinflammation in a GWI mouse model. We administered a mouse diet supplemented with NR at 100μg/kg daily for 2-months to GWI and control mice (n = 27). During treatment, mice were assessed for fatigue-type behavior using the Forced Swim Test (FST), followed by euthanasia for biochemistry and immunohistochemistry analyses. Fatigue-type behavior was elevated in GWI mice compared to control mice and lower in GWI mice treated with NR compared to untreated GWI mice. Levels of plasma NAD and brain Sirt1 were low in untreated GWI mice, while GWI mice treated with NR had higher levels, similar to those of control mice. Deacetylation of the nuclear-factor κB (NFκB) p65 subunit and peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α) was an increase in the brains of NR-treated GWI mice. This corresponded with a decrease in pro-inflammatory cytokines and lipid peroxidation and an increase in markers of mitochondrial bioenergetics in the brains of GWI mice. These findings suggest that targeting NR mediated Sirt1 activation restores brain bioenergetics and reduces inflammation in GWI mice. Further evaluation of NR in GWI is warranted to determine its potential efficacy in treating GWI. [ABSTRACT FROM AUTHOR]

• Gulf War Illness (GWI) chemicals effects in two different rodent models were tested. • GWI chemicals caused broad and model/brain region-specific monoamine disruption. • Neuroinflammation was present in the ventral hippocampus in both GWI models. • Splenic serotonin was disrupted in both GWI models, but norepinephrine only in one. • LNFPIII, a novel immunotherapeutic, reduced or prevented effects of GWI treatments. 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. [ABSTRACT FROM AUTHOR]

Highlights • Neurotoxic chemicals and stress caused persistent elevation in hippocampal miR-124-3p. • miR-124 inhibition increased synaptic plasticity and neuroendocrine gene expression. • Off-target cardiotoxic effects may occur at high doses of miR-124 inhibitor in brain. • Inhibition of miR-124 in the hippocampus is a promising therapeutic approach for GWI. Abstract 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. [ABSTRACT FROM AUTHOR]

Highlights • Sarin surrogate, DFP, causes minimal increases in cytokines in liver and blood. • Unlike results for the brain, CORT does not prime peripheral cytokine responses. • PB/DEET generally suppresses cytokine levels in our model of Gulf War Illness. • Lack of CORT priming in periphery supports a neuroimmune basis of Gulf War Illness. Abstract Gulf War Illness (GWI) is a chronic multi-symptom disorder experienced by as many as a third of the veterans of the 1991 Gulf War; the constellation of "sickness behavior" symptoms observed in ill veterans is suggestive of a neuroimmune involvement. Various chemical exposures and conditions in theater have been implicated in the etiology of the illness. Previously, we found that GW-related organophosphates (OPs), such as the sarin surrogate, DFP, and chlorpyrifos, cause neuroinflammation. The combination of these exposures with exogenous corticosterone (CORT), mimicking high physiological stress, exacerbates the observed neuroinflammation. The potential relationship between the effects of OPs and CORT on the brain versus inflammation in the periphery has not been explored. Here, using our established GWI mouse model, we investigated the effects of CORT and DFP exposure, with or without a chronic application of pyridostigmine bromide (PB) and N ,N- diethyl-meta-toluamide (DEET), on cytokines in the liver and serum. While CORT primed DFP-induced neuroinflammation, this effect was largely absent in the periphery. Moreover, the changes found in the peripheral tissues do not correlate with the previously reported neuroinflammation. These results not only support GWI as a neuroimmune disorder, but also highlight the separation between central and peripheral effects of these exposures. [ABSTRACT FROM AUTHOR]

Abstract Gulf war illness (GWI) is a chronic multi-symptom disease that afflicts 25–33% of troops that were deployed in the 1990–1991 Gulf War. GWI symptoms include cognitive, behavioral and emotional deficits, as well as migraines and pain. It is possible that exposure to Gulf War agents and prophylactics contributed to the reported symptomology. Pyridostigmine bromide (PB) and permethrin (PER) were given to protect from nerve gas attacks and insect vector born disease, respectively. Previous studies have demonstrated that 10 days of exposure to these chemicals can cause symptoms analogous to those observed in GWI, including impairment of long-term memory in mice. Other studies using this model have shown chronic neuroinflammation, and chronic neuroinflammation can lead to altered nociceptive sensitivity. At 10-weeks after the 10-day PB and PER exposure paradigm, we observed lowered nociceptive threshold on the Von Frey test that was no longer evident at 28 weeks and 38 weeks post-exposure. We further determined that vagus nerve stimulation, initiated at 38 weeks after exposure, restores the lowered nociceptive sensitivity. Therefore, stimulating the vagus nerve appears to influence nociception. Future studies are need to elucidate possible mechanisms of this effect. [ABSTRACT FROM AUTHOR]

Chlorpyrifos (CPF) is an extensively used organophosphorus pesticide that has recently come under increasing scrutiny due to environmental health concerns particularly its association with neurodevelopmental defects. While the insecticidal actions and acute toxicity of CPF are attributed to its oxon metabolite (CPO) which potently inhibits the cholinergic enzyme acetylcholinesterase (AChE), there is significant evidence that CPF, CPO, and other organophosphates may affect a variety of neuronal targets and processes that are not directly related to AChE. Previously, in adult rat sciatic nerves ex vivo and postnatal neurons from rats in vitro we observed that CPF and CPO impaired the movements of vesicles and mitochondria in axons. Here, in embryonic neurons from rats in culture, we evaluated 24 h exposures to CPF and CPO across picomolar to micromolar concentrations for effects on fast axonal transport of membrane bound organelles (MBOs) that contained the amyloid precursor protein (APP) tagged with the fluorescent marker, Dendra2 (APPDendra2). The most notable observations of this study were concentration-dependent decreases in the velocity and percentage of MBOs moving in the anterograde direction, an increase in the number of stationary MBOs, and an increased frequency of pauses associated with both CPF and CPO. These effects occurred at concentrations that did not significantly inhibit AChE activity, they were not blocked by cholinergic receptor antagonists, and they were not associated with compromised cell viability. These effects of CPF and CPO may be significant given the importance of axonal transport to neuronal development as well the function of fully developed neurons. [ABSTRACT FROM AUTHOR]

Gulf War Illness (GWI) is a chronic, multisymptom illness that affects 25% of the 700,000 US veterans deployed to the Persian Gulf during the 1990–1991 Gulf War. Central nervous system impairments are among the most common symptoms reported, including memory dysfunction and depression. After 25 years, the diagnosis remains elusive, useful treatments are lacking, and the cause is poorly understood, although exposures to pyridostigmine bromide (PB) and pesticides are consistently identified to be among the strongest risk factors. Epigenetic changes including altered microRNA (miRNA) expression and DNA methylation play an important role in learning, memory, and emotion regulation and have been implicated in various neurological disorders. In this study, we used an established rat model of GWI to determine whether 1) chronic alterations in miRNA expression and global DNA methylation and DNA hydroxymethylation are mechanisms involved in the pathobiology of GWI, and 2) plasma exosome small RNAs may serve as potential noninvasive biomarkers of this debilitating disease. One year after a 28-day exposure regimen of PB, DEET ( N,N -diethyl-3-methylbenzamide), permethrin, and mild stress, expression of 84 mature miRNAs and global 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) content were analyzed in the brains of GWI rats and vehicle controls by PCR array and enzyme-linked immunosorbent assay, respectively. Plasma exosome RNA next-generation sequencing analysis was performed in pooled samples to discover potential noninvasive biomarkers. We found that combined exposure to low doses of GW-related chemicals and mild stress caused epigenetic modifications in the brain that persisted one year after exposure, including increased expression of miR-124-3p and miR-29b-3p in the hippocampus and regional alterations in global 5mC and 5hmC content. GW-relevant exposures also induced the differential expression of two piwi-interacting RNAs (piRNAs) in circulation (piR-007899 and piR-019162). Results from this study implicate a role for epigenetic alterations in GWI. Evaluation of the diagnostic potential of plasma exosome RNAs in veterans with GWI is warranted. [ABSTRACT FROM AUTHOR]

Approximately 175,000–250,000 of the returning veterans from the 1991 Persian Gulf War exhibit chronic multi-symptom illnesses that includes neurologic co-morbidities such as depression, anxiety and cognitive impairments. Amongst a host of causative factors, exposure to low levels of the nerve agent Sarin has been strongly implicated for expression of Gulf War Illness (GWI). Nerve agents similar to pesticides are organophosphate (OP) compounds. There is evidence from civilian population that exposure to OPs such as in agricultural workers and nerve agents such as the survivors and first-responders of the Tokyo subway Sarin gas attack suffer from chronic neurological problems similar to GWI symptoms. Given this unique chemical profile, OPs are ideal to study the effects of nerve agents and develop models of GWI in civilian laboratories. In this study, we used repeated low-dose exposure to OP agent diisopropyl fluorophosphate (DFP) over a 5-day period to approximate the duration and level of Sarin exposure during the Persian Gulf War. We tested the rats at 3-months post DFP exposure. Using a battery of behavioral assays, we observed the presence of symptoms of chronic depression, anxiety and memory problems as characterized by increased immobility time in the Forced Swim Test, anhedonia in the Sucrose Preference Test, anxiety in the Elevated Plus Maze, and spatial memory impairments in the Object Location Test, respectively. Chronic low dose DFP exposure was also associated with hippocampal neuronal damage as characterized by the presence of Fluoro-Jade staining. Given that OP exposure is considered a leading cause of GWI related morbidities, this animal model will be ideally suited to study underlying molecular mechanisms for the expression of GWI neurological symptoms and identify drugs for the effective treatment of GWIs. [ABSTRACT FROM AUTHOR]

The toxicity of the class of chemicals known as the organophosphates (OP) is most commonly attributed to the inhibition of the enzyme acetylcholinesterase. However, there is significant evidence that this mechanism may not account for all of the deleterious neurologic and neurobehavioral symptoms of OP exposure, especially those associated with levels that produce no overt signs of acute toxicity. In the study described here we evaluated the effects of the commonly used OP-pesticide, chlorpyrifos (CPF) on axonal transport in the brains of living rats using manganese (Mn 2+ )-enhanced magnetic resonance imaging (MEMRI) of the optic nerve (ON) projections from the retina to the superior colliculus (SC). T1-weighted MEMRI scans were evaluated at 6 and 24 h after intravitreal injection of Mn 2+ . As a positive control for axonal transport deficits, initial studies were conducted with the tropolone alkaloid colchicine administered by intravitreal injection. In subsequent studies both single and repeated exposures to CPF were evaluated for effects on axonal transport using MEMRI. As expected, intravitreal injection of colchicine (2.5 μg) produced a robust decrease in transport of Mn 2+ along the optic nerve (ON) and to the superior colliculus (SC) (as indicated by the reduced MEMRI contrast). A single subcutaneous (s.c.) injection of CPF (18.0 mg/kg) was not associated with significant alterations in the transport of Mn 2+ . Conversely, 14-days of repeated s.c. exposure to CPF (18.0 mg/kg/day) was associated with decreased transport of Mn 2+ along the ONs and to the SC, an effect that was also present after a 30-day (CPF-free) washout period. These results indicate that repeated exposures to a commonly used pesticide, CPF can result in persistent alterations in axonal transport in the living mammalian brain. Given the fundamental importance of axonal transport to neuronal function, these observations may (at least in part) explain some of the long term neurological deficits that have been observed in humans who have been repeatedly exposed to doses of OPs not associated with acute toxicity. [ABSTRACT FROM AUTHOR]

In a companion paper we examined whether combinations of K v 7 channel openers (Retigabine and Diclofenac; RET, DIC) could be effective modifiers of deep tissue nociceptor activity; and whether such combinations could then be optimized for use as safe analgesics for pain-like signs that developed in a rat model of GWI (Gulf War Illness) pain. In the present report, we examined the combinations of Retigabine/Meclofenamate (RET/MEC) and Meclofenamate/Diclofenac (MEC/DIC). Voltage clamp experiments were performed on deep tissue nociceptors isolated from rat DRG (dorsal root ganglion). In voltage clamp studies, a stepped voltage protocol was applied (-55 to -40 mV; V h =-60 mV; 1500 msec) and K v 7 evoked currents were subsequently isolated by Linopirdine subtraction. MEC greatly enhanced voltage dependent conductance and produced exceptional maximum sustained currents of 6.01 ± 0.26 pA/pF (EC 50 : 62.2 ± 8.99 μM). Combinations of RET/MEC, and MEC/DIC substantially amplified resting currents at low concentrations. MEC/DIC also greatly improved voltage dependent conductance. In current clamp experiments, a cholinergic challenge test (Oxotremorine-M, 10 μM; OXO), associated with our GWI rat model, produced powerful action potential (AP) bursts (85 APs). Optimized combinations of RET/MEC (5 and 0.5 μM) and MEC/DIC (0.5 and 2.5 μM) significantly reduced AP discharges to 3 and 7 Aps, respectively. Treatment of pain-like ambulatory behavior in our rat model with a RET/MEC combination (5 and 0.5 mg/kg) successfully rescued ambulation deficits, but could not be fully separated from the effect of RET alone. Further development of this approach is recommended., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Highlights: [•] Permethrin interacted differently with skin, muscle and vascular nociceptors. [•] Permethrin altered the voltage dependent activation and deactivation of Nav1.8. [•] Permethrin activated and increased the excitability of muscle nociceptors only. [•] Permethrin had no influence on Kv7 amplitude or voltage dependence. [•] Increasing the temperature to 35°C reduced but did not eliminate permethrin effects. [ABSTRACT FROM AUTHOR]

Abstract: An exaggerated response to emotional stimuli is one of the several symptoms widely reported by veterans of the 1991 Persian Gulf War. Many have attributed these symptoms to post-war stress; others have attributed the symptoms to deployment-related exposures and associated damage to cholinergic, dopaminergic, and white matter systems. We collected event-related potential (ERP) data from 20 veterans meeting Haley criteria for Gulf War Syndromes 1–3 and from 8 matched Gulf War veteran controls, who were deployed but not symptomatic, while they performed an auditory three-condition oddball task with gunshot and lion roar sounds as the distractor stimuli. Reports of hyperarousal from the ill veterans were significantly greater than those from the control veterans; different ERP profiles emerged to account for their hyperarousability. Syndromes 2 and 3, who have previously shown brainstem abnormalities, show significantly stronger auditory P1 amplitudes, purported to indicate compromised cholinergic inhibitory gating in the reticular activating system. Syndromes 1 and 2, who have previously shown basal ganglia dysfunction, show significantly weaker P3a response to distractor stimuli, purported to indicate dysfunction of the dopaminergic contribution to their ability to inhibit distraction by irrelevant stimuli. All three syndrome groups showed an attenuated P3b to target stimuli, which could be secondary to both cholinergic and dopaminergic contributions or disruption of white matter integrity. [Copyright &y& Elsevier]

Abstract: Many veterans chronically ill from the 1991 Gulf War exhibit symptoms of altered sensation, including chronic pain. In this study of 55 veterans of a Construction Battalion previously examined in 1995–1996 and 1997–1998, brain activation to innocuous and noxious heat stimuli was assessed in 2008–2009 with a quantitative sensory testing fMRI protocol in control veterans and groups representing three syndrome variants. Testing outside the scanner revealed no significant differences in warm detection or heat pain threshold among the four groups. In the fMRI study, Syndrome 1 and Syndrome 2, but not Syndrome 3, exhibited hypo-activation to innocuous heat and hyper-activation to noxious heat stimuli compared to controls. The results indicate abnormal central processing of sensory and painful stimuli in 2 of 3 variants of Gulf War illness and call for a more comprehensive study with a larger, representative sample of veterans. [Copyright &y& Elsevier]

Abstract: A highly plausible etiology for Gulf War Illness (GWI) is that the neural damage and cognitive deficits are associated with excessive exposure to cholinesterase-inhibiting cholinergic stimulants. Our previous SPECT study provided strong indication that cerebral blood flow (CBF) in veterans with GWI may be different from those of unaffected control veterans. The present study confirmed and extended previous findings that patients with GWI have abnormal response to an inhibitory cholinergic challenge, physostigmine infusion, when compared to age–gender–education matched control veterans. The MRI-based arterial spin labeling (ASL) and phase-contrast techniques have several key advantages over SPECT, including shorter experiment duration, complete non-invasiveness, and higher spatial and temporal resolutions, and therefore may provide a cost-effective biomarker for characterization of GWI. [Copyright &y& Elsevier]