Gulf War Agents Research Articles

Probiotic Treatment Ameliorates Exercise Intolerance, Deficits in Fear Learning and Systemic and Neuro-inflammation in a Mouse Model of Gulf War Illness

Veterans of the Persian Gulf War (GW) continue to suffer from Gulf War Illness (GWI) characterized by cognitive deficit and fatigue. Our group and others have suggested possible involvement of the microbiota-gut-brain axis in GWI pathology (Kozlova et a., 2021a; 2021b). The current study tested the hypothesis that probiotic treatment (P) prevents GWI symptoms, and systemic and neuro-inflammation. Adult male C57Bl/6N mice were separated into 4 groups (n=16/group): GW group was exposed to 8.7 mg/kg/d pyridostigmine bromide (PB) in saline (150uL/30g bw, oral gavage), 1.3 mg/kg PER in 100% DMSO (dermal), and 33% DEET in 70% EtOH (dermal) for 28 days (5 days/week). CON/S group was sham-treated. CON/S+P and GW+P groups were treated with a probiotic (P) cocktail of L. reuteri, L. rhamnosus, L. casei, B. longum (10 8 CFU/mL, oral gavage) 3 times/week for 2 weeks prior to and during sham/GW agent treatment and until sacrifice (post-treatment (PT) 150). All groups were subjected to restraint stress (5 min/d for 28 d). RT-qPCR on fecal pellets showed bacteria colonization of all strains except L. rhamnosus and B. longum after administration of 6-7 doses. At PT1, all strains showed increased colonization relative to baseline, which persisted to PT150, except B. longum. Body weight, fat, lean mass at PT150 showed no changes among groups. An exercise endurance (EE) test was performed to measure differences in fatigue. GW mice tired faster relative to CON/S at PT56 which persisted at PT150. Probiotic normalized exercise endurance to CON/S levels at PT56. On the passive avoidance test all groups behaved normally at PT50 but GW showed deficits on day 1 acquisition trial of fear learning when tested again 14 weeks later at PT150. GW+P behaved similarly to CON/S. Strength and depression-like behavior was examined before and after exercise stress to simulate post-exertional malaise. Mean percent time spent mobile on tail suspension test (TST) were significantly lower for GW but not GW+P at PT50. All groups were normal at PT150. There was no effect of GW agents or probiotics on the hanging wire test. Systemic and neuroinflammatory markers: C-reactive protein (CRP), high mobility group box 1 (HMGB1), and interleukin-6 (IL-6) were examined using ELISA. Liver and plasma CRP was increased in GW group but not GW+P in liver (p=0.06 in plasma). Plasma HMGB1 was reduced and brain IL-6 was elevated in GW mice but not GW+P. Immunofluorescence analysis in fixed hippocampal sections showed more Iba-1 positive cells in CA1 and DG in GW vs CON/S, while GW+P showed no significant changes vs CON/S. These results show that GW mice can be used to model long-lasting fatigue and other GWI symptoms as well as systemic and neuro-inflammation concurrently. After exercise stress GW agents produced depression. Importantly, probiotic treatment improved exercise endurance, depression, systemic and neuro-inflammation in GW mice, indicating its potential therapeutic use in GWI. Clara Berdasco and Elena Kozlova are co-first authors. Supported by DoD #W81XWH-19-1-0802 (MCC). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Impact of gulf war toxic exposures after mild traumatic brain injury

Chemical and pharmaceutical exposures have been associated with the development of Gulf War Illness (GWI), but how these factors interact with the pathophysiology of traumatic brain injury (TBI) remains an area of study that has received little attention thus far. We studied the effects of pyridostigmine bromide (an anti-nerve agent) and permethrin (a pesticide) exposure in a mouse model of repetitive mild TBI (r-mTBI), with 5 impacts over a 9-day period, followed by Gulf War (GW) toxicant exposure for 10 days beginning 30 days after the last head injury. We then assessed the chronic behavioral and pathological sequelae 5 months after GW agent exposure. We observed that r-mTBI and GWI cumulatively affect the spatial memory of mice in the Barnes maze and result in a shift of search strategies employed by r-mTBI/GW exposed mice. GW exposure also produced anxiety-like behavior in sham animals, but r-mTBI produced disinhibition in both the vehicle and GW treated mice. Pathologically, GW exposure worsened r-mTBI dependent axonal degeneration and neuroinflammation, increased oligodendrocyte cell counts, and increased r-mTBI dependent phosphorylated tau, which was found to colocalize with oligodendrocytes in the corpus callosum. These results suggest that GW exposures may worsen TBI-related deficits. Veterans with a history of both GW chemical exposures as well as TBI may be at higher risk for worse symptoms and outcomes. Subsequent exposure to various toxic substances can influence the chronic nature of mTBI and should be considered as an etiological factor influencing mTBI recovery.

Gulf War Agent Exposure Causes Dose‐Dependent Impairment of Memory Formation and Exercise Intolerance in a Mouse Model of Gulf War Illness

Gulf War Illness (GWI) is a chronic multi-symptom condition that still affects a third of veterans who served in the 1991 Persian Gulf War (GW). Latently-emerging and persistent symptoms fall into 5 general domains: neurological/cognition/mood, muscle/joint pain, respiratory, gastrointestinal disturbances and especially chronic fatigue. In particular, veterans exhibit CNS deficits in the neuropsychological domains of visuospatial abilities, attention/executive functioning, and learning/memory. GWI remains untreated due its poorly understood etiology and pathophysiology. It is suggested that GWI is a result of continuous exposure of military personnel to GW agents (pyridostigmine bromide, (PB), permethrin (PER), diethyltoluamide (DEET) and deployment/combat stress. To further investigate the cognitive manifestations of GWI pathophysiology, we studied the effects of chronic GW agent exposure in adult C57Bl/6N males utilizing a repeated measures design. The dosing paradigm consisted of 5 d/week for 28d of exposure to two doses of PB (6.5 mg/kg/d, 2x/d and 8.7 mg/kg/d, 1x/d; po), PER (1.3 mg/kg/d; top) in 70% ETOH, DEET (1650 mg/kg/d in DMSO) and stress 5 min/d (n=5–7/group). Two controls were included: sham (saline po, 70% ETOH and DMSO top) and sham/stress. Behavioral endpoints were measured at different timepoints post treatment (PT) up to PT160d. The 8.7 mg/kg/d dose was more effective in modeling cognitive deficits on the passive avoidance test since this group failed to avoid the aversive dark chamber at the 24h, and 7d and/or 3d retention timepoints at PT70. A repeat test at PT120 indicated that the 8.7 and sham/stress (P<.01) but not the 6.5 mg/kg/d group displayed retention of emotional reactivity memory at all test timepoints. In the novel object recognition test, performed at PT17, GW groupsbut not sham/stress displayed deficient memory retention at 24h (P<.01). On the Barnes Maze, no GW exposure effects on reference memory were noted during acquisition training nor a 24h acquisition probe trial. Since the neuropsychological deficits seen in GW chemical exposures can be exacerbated by comorbid mood-related conditions, animals were tested for anxiety and depression but no GW-specific effects were found. To model chronic fatigue, using a 5-day exercise endurance test we found that 8.7 mg/kg/d showed decreased latency to exhaustion at PT23 and PT161 compared to sham/stress (P<.05, P<.01). This effect could not be attributed to problems with motor coordination as measured on Suok and rotarod tests. In summary, the GW agent exposure paradigm produces specific cognitive deficits in associative aversive learning and novel object recognition memory but not visuospatial (reference) memory. GW agents also cause mice to tire faster during exercise without deficits in motor coordination. This animal model may be useful in examining common mechanisms underlying deficient cognitive performance and chronic fatigue in GWI.

Modeling the behavioral and metabolic phenotype of mice exposed to Gulf War toxicants

Gulf War Illness (GWI) is a chronic multi‐symptom condition that still affects a third of veterans who served in the 1991 Persian Gulf War (GW). Latently‐emerging and persistent symptoms fall into 5 general domains: neurological/cognition/mood (memory, concentration, anxiety, depression), muscle/joint pain, respiratory, gastrointestinal disturbances and especially chronic fatigue. GWI remains untreated due its poorly understood etiology and pathophysiology. It is suggested that GWI is a result of continuous exposure to GW agents administered to protect military personnel: 1) acetylcholinesterase inhibitor pyridostigmine bromide (PB) taken orally as prophylaxis against nerve agents, 2) topical insecticide permethrin (PER), and 3) topical insect repellent diethyltoluamide (DEET). Although GW agents were administered at safe doses, their chronic and combined use, in addition to stress, a critical component of GW deployment, may produce and/or intensify the pathophysiology of GWI for reasons not fully understood. Additionally, GW veterans report symptoms associated with delayed‐onset Type II diabetes mellitus, which may be associated with chronic fatigue. To further investigate the behavioral, metabolic manifestations of GWI pathophysiology, we studied the effects of acute (AE) and chronic (CE) GW toxicant exposure in adult C57Bl/6N males utilizing a repeated measures design. The AE model consisted of 1wk exposure to PB (2 mg/kg/d; ip), PER (200 mg/kg/d in 70% ETOH; top), DEET (40 mg/kg/d in ETOH; top) and 5 min restraint stress/d (n=4–7). The CE model consisted of 5 days/week for 28 days of exposure to PB (13 mg/kg/d; po), PER (1.3 mg/kg/d; top) in 70% ETOH, DEET (40 mg/kg/d in ETOH; top) and stress 5min/d (n=6–8). A sham control was also tested in CE experiments. Behavioral and metabolic endpoints were measured at one week post treatment (PT) and 60d PT (AE) and 90d PT (CE) to model the delayed and persistent effects seen in GW veterans. When compared to pre‐treatment, GW mice exposed to CE paradigm showed depressive‐like behavior on forced swim, tail suspension and hanging wire tests at post‐treatment. On elevated platform (SUOK) test AE and CE produced sensorimotor alterations as measured by an increased number segments crossed CE decreased missteps/segments crossed relative to pre‐treatment. Increased anxiety was observed as decreased risk assessment (CE) and increased vegetative behavior in AE on SUOK and in AE and CE as less percent time spent in open arm of an elevated plus maze. GW agent exposure had no effect on three‐chambered sociability test. Both CE and AE groups displayed transient but marked insulin insensitivity relative to pre‐treatment. These results show that GW agents can produce insulin insensitivity along with altered sensorimotor integration, anxiety and depressive‐like behavior. In comparison to GW effects on anxiety which persisted at 90d PT (with the exception of vegetative behavior), the depressive‐like effects normalized at 90d PT. Our findings will help inform studies that aim to elucidate the etiology and pathophysiology of GWI.Support or Funding InformationAmerican Physiological Society (AB) and UC Minigrant (AB and JT).

Oligodendrocyte involvement in Gulf War Illness.

Low level sarin nerve gas and other anti‐cholinesterase agents have been implicated in Gulf War illness (GWI), a chronic multi‐symptom disorder characterized by cognitive, pain and fatigue symptoms that continues to afflict roughly 32% of veterans from the 1990–1991 Gulf War. How disrupting cholinergic synaptic transmission could produce chronic illness is unclear, but recent research indicates that acetylcholine also mediates communication between axons and oligodendrocytes. Here we investigated the hypothesis that oligodendrocyte development is disrupted by Gulf War agents, by experiments using the sarin‐surrogate acetylcholinesterase inhibitor, diisopropyl fluorophosphate (DFP). The effects of corticosterone, which is used in some GWI animal models, were also investigated. The data show that DFP decreased both the number of mature and dividing oligodendrocytes in the rat prefrontal cortex (PFC), but differences were found between PFC and corpus callosum. The differences seen between the PFC and corpus callosum likely reflect the higher percentage of proliferating oligodendroglia in the adult PFC. In cell culture, DFP also decreased oligodendrocyte survival through a non‐cholinergic mechanism. Corticosterone promoted maturation of oligodendrocytes, and when used in combination with DFP it had protective effects by increasing the pool of mature oligodendrocytes and decreasing proliferation. Cell culture studies indicate direct effects of both DFP and corticosterone on OPCs, and by comparison with in vivo results, we conclude that in addition to direct effects, systemic effects and interruption of neuron–glia interactions contribute to the detrimental effects of GW agents on oligodendrocytes. Our results demonstrate that oligodendrocytes are an important component of the pathophysiology of GWI.

Gulf War agents pyridostigmine bromide and permethrin cause hypersensitive nociception that is restored after vagus nerve stimulation

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.

Complementary proteomic approaches reveal mitochondrial dysfunction, immune and inflammatory dysregulation in a mouse model of Gulf War Illness

PurposeLong‐term consequences of combined pyridostigmine bromide (PB) and permethrin (PER) exposure in C57BL6/J mice using a well‐characterized mouse model of exposure to these Gulf War (GW) agents were explored at the protein level.Experimental designWe used orthogonal proteomic approaches to identify pathways that are chronically impacted in the mouse CNS due to semiacute GW agent exposure early in life. These analyses were performed on soluble and membrane‐bound protein fractions from brain samples using two orthogonal isotopic labeling LC‐MS/MS proteomic approaches—stable isotope dimethyl labeling and iTRAQ.ResultsThe use of these approaches allowed for greater coverage of proteins than was possible by either one alone and revealed both distinct and overlapping datasets. This combined analysis identified changes in several mitochondrial, as well as immune and inflammatory pathways after GW agent exposure.Conclusions and clinical relevanceThe work discussed here provides insight into GW agent exposure dependent mechanisms that adversely affect mitochondrial function and immune and inflammatory regulation. Collectively, our work identified key pathways which were chronically impacted in the mouse CNS following acute GW agent exposure, this may lead to the identification of potential targets for therapeutic intervention in the future.Long‐term consequences of combined PB and PER exposure in C57BL6/J mice using a well‐characterized mouse model of exposure to these GW agents were explored at the protein level. Expanding on earlier work, we used orthogonal proteomic approaches to identify pathways that are chronically impacted in the mouse CNS due to semiacute GW agent exposure early in life. These analyses were performed on soluble and membrane‐bound protein fractions from brain samples using two orthogonal isotopic labeling LC‐MS/MS proteomic approaches—stable isotope dimethyl labeling and iTRAQ. The use of these approaches allowed for greater coverage of proteins than was possible by either one alone and revealed both distinct and overlapping datasets. This combined analysis identified changes in several mitochondrial, as well as immune and inflammatory pathways after GW agent exposure. The work discussed here provides insight into GW agent exposure dependent mechanisms that adversely affect mitochondrial function and immune and inflammatory regulation at 5 months postexposure to PB + PER.

Phospholipid profiling of plasma from GW veterans and rodent models to identify potential biomarkers of Gulf War Illness.

Gulf War Illness (GWI), which affects at least one fourth of the 700,000 veterans deployed to the Gulf War (GW), is characterized by persistent and heterogeneous symptoms, including pain, fatigue and cognitive problems. As a consequence, this illness remains difficult to diagnose. Rodent models have been shown to exhibit different symptomatic features of GWI following exposure to particular GW agents (e.g. pyridostigmine bromide, permethrin and DEET) and/or stress. Preclinical analyses have shown the activation of microglia and astroglia as a pathological hallmark in these mouse and rat models. Although much has been learned in recent years from these different rodent models and independent clinical studies, characterization studies to identify overlapping features of GWI in animals and humans have been missing. Thus, we aimed to identify biomarkers that co-occur in the plasma of rodent models of GWI and human GWI patients. We observed increases of multiple phospholipid (PL) species across all studied cohorts. Furthermore, these data suggested dysfunction within ether and docosahexaenoic acid and arachidonic acid containing PL species in relation to GWI. As these PL species play a role in inflammatory processes, these findings suggest a possible role for inflammatory imbalance in GWI. Overall, we show that the peripheral lipid disturbances are present both in human GWI patients and in the preclinical rodent models of GWI, highlighting the importance of lipidomics as a potential platform for further biomarker discovery and supporting the value of GW agent exposed models of GWI.

Translational potential of long-term decreases in mitochondrial lipids in a mouse model of Gulf War Illness

Gulf War Illness (GWI) affects 25% of veterans from the 1990-1991 Gulf War (GW) and is accompanied by damage to the brain regions involved in memory processing. After twenty-five years, the chronic pathobiology of GWI is still unexplained. To address this problem, we examined the long-term consequences of GW exposures in an established GWI mouse model to identify biological processes that are relevant to the chronic symptoms of GWI. Three-month old male C57BL6 mice were exposed for 10days to GW agents (pyridostigmine bromide and permethrin). Barnes Maze testing conducted at 15- and 16-months post-exposure revealed learning and memory impairment. Immunohistochemical analyses showed astroglia and microglia activation in the hippocampi of exposed mice. Proteomic studies identified perturbation of mitochondria function and metabolomics data showed decreases in the Krebs cycle compounds, lactate, β-hydroxybutyrate and glycerol-3 phosphate in the brains of exposed mice. Lipidomics data showed decreases in fatty acids, acylcarnitines and phospholipids, including cardiolipins in the brains of exposed mice. Pilot biomarker studies showed that plasma from exposed mice and veterans with GWI had increases in odd-chain, and decreases in long-chain, acylcarnitines compared to their respective controls. Very long-chain acylcarnitines were decreased in veterans with GWI compared to controls. These studies suggest that mitochondrial lipid disturbances might be associated with GWI and that further investigation is required to determine its role in the pathophysiology of this illness. Targeting mitochondrial function may provide effective therapies for GWI, and that lipid abnormalities could serve as biomarkers of GWI.

A Chronic Longitudinal Characterization of Neurobehavioral and Neuropathological Cognitive Impairment in a Mouse Model of Gulf War Agent Exposure.

Gulf War Illness (GWI) is a chronic multisymptom illness with a central nervous system component that includes memory impairment as well as neurological and musculoskeletal deficits. Previous studies have shown that in the First Persian Gulf War conflict (1990–1991) exposure to Gulf War (GW) agents, such as pyridostigmine bromide (PB) and permethrin (PER), were key contributors to the etiology of GWI. For this study, we used our previously established mouse model of GW agent exposure (10 days PB+PER) and undertook an extensive lifelong neurobehavioral characterization of the mice from 11 days to 22.5 months post exposure in order to address the persistence and chronicity of effects suffered by the current GWI patient population, 24 years post-exposure. Mice were evaluated using a battery of neurobehavioral testing paradigms, including Open Field Test (OFT), Elevated Plus Maze (EPM), Three Chamber Testing, Radial Arm Water Maze (RAWM), and Barnes Maze (BM) Test. We also carried out neuropathological analyses at 22.5 months post exposure to GW agents after the final behavioral testing. Our results demonstrate that PB+PER exposed mice exhibit neurobehavioral deficits beginning at the 13 months post exposure time point and continuing trends through the 22.5 month post exposure time point. Furthermore, neuropathological changes, including an increase in GFAP staining in the cerebral cortices of exposed mice, were noted 22.5 months post exposure. Thus, the persistent neuroinflammation evident in our model presents a platform with which to identify novel biological pathways, correlating with emergent outcomes that may be amenable to therapeutic targeting. Furthermore, in this work we confirmed our previous findings that GW agent exposure causes neuropathological changes, and have presented novel data which demonstrate increased disinhibition, and lack of social preference in PB+PER exposed mice at 13 months after exposure. We also extended upon our previous work to cover the lifespan of the laboratory mouse using a battery of neurobehavioral techniques.

Corticosterone primes the neuroinflammatory response to DFP in mice: potential animal model of Gulf War Illness

Gulf War Illness (GWI) is a multi‐symptom disorder with features characteristic of persistent sickness behavior. Among conditions encountered in the Gulf War (GW) theater were physiological stressors (e.g., heat/cold/physical activity/sleep deprivation), prophylactic treatment with the reversible AChE inhibitor, pyridostigmine bromide (PB), the insect repellent, N,N‐diethyl‐meta‐toluamide (DEET), and potentially the nerve agent, sarin. Prior exposure to the anti‐inflammatory glucocorticoid, corticosterone (CORT), at levels associated with high physiological stress, can paradoxically prime the CNS to produce a robust proinflammatory response to neurotoxicants and systemic inflammation; such neuroinflammatory effects can be associated with sickness behavior. Here, we examined whether CORT primed the CNS to mount neuroinflammatory responses to GW exposures as a potential model of GWI. Male C57BL/6 mice were treated with chronic (14 days) PB/ DEET, subchronic (7–14 days) CORT, and acute exposure (day 15) to diisopropyl fluorophosphate (DFP), a sarin surrogate and irreversible AChE inhibitor. DFP alone caused marked brain‐wide neuroinflammation assessed by qPCR of tumor necrosis factor‐α, IL6, chemokine (C‐C motif) ligand 2, IL‐1β, leukemia inhibitory factor, and oncostatin M. Pre‐treatment with high physiological levels of CORT greatly augmented (up to 300‐fold) the neuroinflammatory responses to DFP. Anti‐inflammatory pre‐treatment with minocycline suppressed many proinflammatory responses to CORT+DFP. Our findings are suggestive of a possible critical, yet unrecognized interaction between the stressor/environment of the GW theater and agent exposure(s) unique to this war. Such exposures may in fact prime the CNS to amplify future neuroinflammatory responses to pathogens, injury, or toxicity. Such occurrences could potentially result in the prolonged episodes of sickness behavior observed in GWI. Gulf War (GW) veterans were exposed to stressors, prophylactic medicines and, potentially, nerve agents in theater. Subsequent development of GW Illness, a persistent multi‐symptom disorder with features characteristic of sickness behavior, may be caused by priming of the CNS resulting in exaggerated neuroinflammatory responses to pathogens/insults. Nerve agent, diisopropyl fluorophosphate (DFP), produced a neuroinflammatory response that was exacerbated by pre‐treatment with levels of corticosterone simulating heightened stressor conditions. While prophylactic treatments reduced DFP‐induced neuroinflammation, this effect was negated when those treatments were combined with corticosterone.

Gulf War agent exposure causes impairment of long-term memory formation and neuropathological changes in a mouse model of Gulf War Illness.

Gulf War Illness (GWI) is a chronic multisymptom illness with a central nervous system component such as memory deficits, neurological, and musculoskeletal problems. There are ample data that demonstrate that exposure to Gulf War (GW) agents, such as pyridostigmine bromide (PB) and pesticides such as permethrin (PER), were key contributors to the etiology of GWI post deployment to the Persian GW. In the current study, we examined the consequences of acute (10 days) exposure to PB and PER in C57BL6 mice. Learning and memory tests were performed at 18 days and at 5 months post-exposure. We investigated the relationship between the cognitive phenotype and neuropathological changes at short and long-term time points post-exposure. No cognitive deficits were observed at the short-term time point, and only minor neuropathological changes were detected. However, cognitive deficits emerged at the later time point and were associated with increased astrogliosis and reduction of synaptophysin staining in the hippocampi and cerebral cortices of exposed mice, 5 months post exposure. In summary, our findings in this mouse model of GW agent exposure are consistent with some GWI symptom manifestations, including delayed onset of symptoms and CNS disturbances observed in GWI veterans.

Chronic elevation of phosphocholine containing lipids in mice exposed to Gulf War agents pyridostigmine bromide and permethrin

For two decades, 25% of the veterans who served in the 1991 Gulf War (GW) have been living with Gulf War Illness (GWI), a chronic multisymptom illness. Evidence suggests that brain structures involved in cognitive function may be affected in GWI. Gulf War agents such as the acetylcholinesterase (AChE) inhibitor pyridostigmine bromide (PB) and the pesticide permethrin (PER) are considered key etiogenic factors in GWI. We therefore developed a mouse model of GW agent exposure by co-administering PB and PER and showed that this model exhibits cognitive impairment and anxiety, and increased astrogliosis at chronic post-exposure time-points. Since GW agents inhibit AChE, we hypothesized that PB+PER exposure will modulate phosphatidylcholine (PC) and sphingomyelin (SM), which are reservoirs of phosphocholine required for endogenous ACh synthesis. Lipidomic analyses showed that PC and SM were elevated in the brains of exposed compared to control mice. Brain ether PC (ePC) species were increased but lyso-platelet activating factors (lyso-PAF) that are products of ePC were decreased in exposed animals compared to controls. Catalase expression (a marker for peroxisomes) was increased in GW agent exposed mice compared to controls. Ether PC and lyso-PAF modulation was also evident in the plasma of GW agent exposed mice compared to controls. These studies suggest peroxisomal and lysosomal dysfunction in the brain at a chronic post-exposure timepoint following GW agent exposure. Our studies provide a new direction for GWI research, which will be useful for developing suitable therapies for treating GWI.

Exposure to an organophosphate pesticide, individually or in combination with other Gulf War agents, impairs synaptic integrity and neuronal differentiation, and is accompanied by subtle microvascular injury in a mouse model of Gulf War agent exposure

Gulf War illness (GWI) is a currently untreatable multi-symptom disorder experienced by 1990-1991 Persian Gulf War (GW) veterans. The characteristic hallmarks of GWI include cognitive dysfunction, tremors, migraine, and psychological disturbances such as depression and anxiety. Meta-analyses of epidemiological studies have consistently linked these symptomatic profiles to the combined exposure of GW agents such as organophosphate-based and pyrethroid-based pesticides (e.g. chlorpyrifos (CPF) and permethrin (PER) respectively) and the prophylactic use of pyridostigmine bromide (PB) as a treatment against neurotoxins. Due to the multi-symptomatic presentation of this illness and the lack of available autopsy tissue from GWI patients, very little is currently known about the distinct early pathological profile implicated in GWI (including its influence on synaptic function and aspects of neurogenesis). In this study, we used preclinical models of GW agent exposure to investigate whether 6-month-old mice exposed to CPF alone, or a combined dose of CPF, PB and PER daily for 10 days, demonstrate any notable pathological changes in hippocampal, cortical (motor, piriform) or amygdalar morphometry. We report that at an acute post-exposure time point (after 3 days), both exposures resulted in the impairment of synaptic integrity (reducing synaptophysin levels) in the CA3 hippocampal region and altered neuronal differentiation in the dentate gyrus (DG), demonstrated by a significant reduction in doublecortin positive cells. Both exposures also significantly increased astrocytic GFAP immunoreactivity in the piriform cortex, motor cortex and the basolateral amygdala and this was accompanied by an increase in (basal) brain acetylcholine (ACh) levels. There was no evidence of microglial activation or structural deterioration of principal neurons in these regions following exposure to CPF alone or in combination with PB and PER. Evidence of subtle microvascular injury was demonstrated by the reduction of platelet endothelial cell adhesion molecule (PECAM)-1 levels in CPF+PB+PER exposed group compared to control. These data support early (subtle) neurotoxic effects on the brain following exposure to GW agents.

Lipidomic Profiling of Phosphocholine Containing Brain Lipids in Mice with Sensorimotor Deficits and Anxiety-Like Features After Exposure to Gulf War Agents

The central nervous system (CNS)-based symptoms of Gulf War Illness (GWI) include motor dysfunction, anxiety, and cognitive impairment. Gulf War (GW) agents, such as pyridostigmine bromide (PB), permethrin (PER), N,N-diethyl-meta-toluamide (DEET), and stress, are among the contributory factors to the pathobiology of GWI. This study characterizes disturbances in phosphocholine-containing lipids that accompany neurobehavioral and neuropathological features associated with GW agent exposure. Exposed mice received PB orally, dermal application of PER and DEET and restraint stress daily for 28 days, while controls received vehicle during this period. Neurobehavioral studies included the rotarod, open field, and Morris water maze tests. Histopathological assessments included glial fibrillary acid protein, CD45, and Nissl staining. Liquid chromatography/mass spectrometry with source collision-induced dissociation in negative and positive ionization scanning modes was performed to characterize brain phosphatidylcholine (PC) and sphingomyelin (SM). A significant increase in ether containing PC (ePC34:0, ePC36:2, and ePC36:1) or long-chain fatty acid-containing PC (38:1, 40:4, 40:2) was observed in exposed mice compared with controls. Among differentially expressed PCs, levels of those with monounsaturated fatty acids were more affected than those with saturated and polyunsaturated fatty acids. Sensorimotor deficits and anxiety, together with an increase in astrocytosis, were observed in exposed mice compared with controls. These lipid changes suggest that alterations in peroxisomal pathways and stearoyl-CoA desaturase activity accompany neurobehavioral and neuropathological changes after GW agent exposure and represent possible treatment targets for the CNS symptoms of GWI.

Proteomic CNS Profile of Delayed Cognitive Impairment in Mice Exposed to Gulf War Agents

Gulf War Illness (GWI) is a chronic multisymptom condition with a central nervous system (CNS) component, for which there is no treatment available. It is now believed that the combined exposure to Gulf War (GW) agents, including pyridostigmine bromide (PB) and pesticides, such as permethrin (PER), was a key contributor to the etiology of GWI. In this study, a proteomic approach was used to characterize the biomolecular disturbances that accompany neurobehavioral and neuropathological changes associated with combined exposure to PB and PER. Mice acutely exposed to PB and PER over 10 days showed an increase in anxiety-like behavior, psychomotor problems and delayed cognitive impairment compared to control mice that received vehicle only. Proteomic analysis showed changes in proteins associated with lipid metabolism and molecular transport in the brains of GW agent-exposed mice compared to controls. Proteins associated with the endocrine and immune systems were also altered, and dysfunction of these systems is a prominent feature of GWI. The presence of astrogliosis in the GW agent-exposed mice compared to control mice further suggests an immune system imbalance, as is observed in GWI. These studies provide a broad perspective of the molecular disturbances driving the late pathology of this complex illness. Evaluation of the potential role of these biological functions in GWI will be useful in identifying molecular pathways that can be targeted for the development of novel therapeutics against GWI.

Gulf War agents trigger discrete transcriptional changes in human neuronal cells

Reports on Gulf War illness (GWI) implicated the use of the pesticide permethrin (PER), and the insect repellent N,N-diethyl-m-toluamide (DEET), in its etiology, as well as pyridostigmine bromide (PB), which was given as a prophylactic treatment against nerve agent exposure. Using Affymetrix microarrays the genomic response of human neuronal SHSY-5Y cells to 10 days of exposure to these agents was characterized and profiles of gene modulation unique to each treatment were demonstrated. In particular, a significantly greater impact of PER was observed compared to the other treatments. The Ingenuity Pathway Analysis knowledgebase was used to analyze the genomic datasets and attribute functional consequences to the effects of related genes, which were significantly up- or down-regulated in response to different treatments. Canonical pathways shown to be significantly modulated at the genomic level in response to exposure included cellular mechanisms, which might contribute to the clinical presentation in GWI patients and thus be targeted for further investigation as potential targets for therapeutic intervention.

Proteomic Analysis of Human Neuronal Cells Treated with the Gulf War Agent Pyridostigmine Bromide

Gulf War Illness (GWI) is characterized by a wide array of symptomology, which is possibly linked to the prophylactic treatment with pyridostigmine bromide (PB) against neurotoxins. It is now hypothesized that the pathological origin of this multi-symptom illness lies within the central nervous system and is caused by irregular activation of neuronal signaling pathways. To investigate this possibility, a proteomic-based approach was applied to characterize cellular responses of neuronal cells to PB exposure. Protein extracts from cultured neuroblastoma cells treated with 700nM PB for 10 days, as well as extracts from control cells were separated using two-dimensional gel electrophoresis. Twenty two differentially-expressed proteins were identified by MALDI-TOF mass spectrometry (MS). Ingenuity Pathways Analysis (IPA) software was then used to determine the biological functions and canonical pathways associated with the PB-responsive proteins.