Diisopropylfluorophosphate Exposure Research Articles

Nerve agent exposure and physiological stress alter brain microstructure and immune profiles after inflammatory challenge in a long-term rat model of Gulf War Illness

Gulf War Illness (GWI) is a disorder experienced by many veterans of the 1991 Gulf War, with symptoms including fatigue, chronic pain, respiratory and memory problems. Exposure to toxic chemicals during the war, such as oil well fire smoke, pesticides, physiological stress, and nerve agents, is thought to have triggered abnormal neuroinflammatory responses that contribute to GWI. Previous studies have examined the acute effects of combined physiological stress and chemical exposures using GWI rodent models and presented findings related to neuroinflammation and changes in diffusion magnetic resonance imaging (MRI) measures, suggesting a neuroimmune basis for GWI. In the current study, using ex vivo MRI, cytokine mRNA expression, and immunohistological analyses of brain tissues, we examined the brain structure and immune function of a chronic rat model of GWI. Our data showed that a combination of long-term corticosterone treatment (to mimic high physiological stress) and diisopropyl fluorophosphate exposure (to mimic sarin exposure) primed the response to subsequent systemic immune challenge with lipopolysaccharide resulting in elevations of multiple cytokine mRNAs, an increased activated glial population, and disrupted brain microstructure in the cingulate cortex and hippocampus compared to control groups. Our findings support the critical role of neuroinflammation, dysregulated glial activation, and their relationship to disrupted brain microstructural integrity in the pathophysiology of GWI and highlight the unique consequences of long-term combined exposures on brain biochemistry and structural connectivity.

Protective Activity of Novel Hydrophilic Synthetic Neurosteroids on Organophosphate Status Epilepticus-induced Chronic Epileptic Seizures, Non-Convulsive Discharges, High-Frequency Oscillations, and Electrographic Ictal Biomarkers.

Nerve agents and organophosphates (OP) are neurotoxic chemicals that induce acute seizures, status epilepticus (SE), and mortality. Long-term neurologic and neurodegenerative effects manifest months to years after OP exposure. Current benzodiazepine anticonvulsants are ineffective in preventing such long-term neurobehavioral and neuropathological changes. New and effective anticonvulsants are needed for OP intoxication, especially for mitigating the long-term sequelae after acute exposure. We developed neurosteroids as novel anticonvulsants and neuroprotectants in OP exposure models. In this study, we evaluated the long-term efficacy of novel synthetic neurosteroids in preventing the development of chronic epilepsy and hyperexcitable ictal events in a rat OP model of SE. Rats were exposed to the OP nerve agent surrogate diisopropylfluorophosphate (DFP), and the experimental groups were treated with the synthetic neurosteroid valaxanolone (VX) or lysaxanolone (LX) 40 minutes post-exposure in conjunction with midazolam. Video-electroencephalography was monitored for two months to assess spontaneous recurrent seizures (SRS), epileptiform discharges, interictal spikes, and high-frequency oscillations (HFOs). Within 60 days of DFP exposure, rats developed chronic epilepsy characterized by frequent SRS, epileptiform discharges, and HFOs. LX treatment was associated with a dose-dependent reduction of epilepsy occurrence and overall seizure burden with a significant decrease in SRS and epileptiform discharges. It also significantly reduced the occurrence of epileptic biomarkers of HFOs and interictal spikes, indicating potential disease-modifying activity. Similarly, the neurosteroid analog VX also significantly attenuated SRS, discharges, HFOs, and ictal events. These results demonstrate the long-term protective effects of synthetic neurosteroids in the OP-exposed post-SE model, indicating their disease-modifying potential to prevent epilepsy and ictal abnormalities. SIGNIFICANCE STATEMENT: The effects of nerve agents and organophosphate (OP) exposure are persistent, and survivors suffer from a number of devastating, chronic neurological dysfunctions. Currently, there is no specific therapy for preventing this disastrous impact of OP exposure. We propose synthetic neurosteroids that activate tonic inhibition provide viable options for preventing the long-term neurological effects of OP intoxication. The results from this study reveal the disease-modifying potential of two novel synthetic neurosteroids in preventing epileptogenesis and chronic epileptic seizures after OP-induced SE.

A Pediatric Rat Model of Organophosphate-Induced Refractory Status Epilepticus: Characterization of Long-Term Epileptic Seizure Activity, Neurologic Dysfunction and Neurodegeneration.

Children are highly vulnerable to the neurotoxic effects of organophosphates (OPs), which can cause neuronal developmental defects, including intellectual disability, autism, epilepsy, and related comorbidities. Unfortunately, no specific pediatric OP neurotoxicity model currently exists. In this study, we developed and characterized a pediatric rat model of status epilepticus (SE) induced by the OP diisopropylfluorophosphate (DFP) and examined its impact on long-term neurological outcomes. Postnatal day 21 rats were exposed to a DFP regimen with standard antidotes. Progressive behavioral deteriorations were assessed over a three-month period. Development of epileptic seizures, ictal discharges, high-frequency oscillations (HFOs), and interictal spikes were monitored by video-electroencephalography recordings. Histology-stereology analysis was performed to assess neurodegeneration, neuroinflammation, and morphologic abnormalities. DFP-exposed, post-SE animals exhibited significantly elevated levels of anxiety and depression than age-matched controls at 1, 2, and 3 months post-exposure. DFP-exposed animals displayed aggressive behavior and a marked decline in object recognition memory, as well as prominent impairment in spatial learning and memory. DFP-exposed animals had striking electrographic abnormalities with the occurrence of displayed epileptic seizures, ictal discharges, HFOs, and interictal spikes, suggesting chronic epilepsy. Neuropathological analysis showed substantially fewer principal neurons and inhibitory interneurons with a marked increase in reactive microglia and neuroinflammation in the hippocampus and other brain regions. DFP-exposed animals also exhibited mossy fiber sprouting indicating impaired network formations. Long-term epileptic seizures and neuropsychiatric functional deficits induced by DFP were consistent with neuropathological defects. Collectively, this pediatric model displays many hallmarks of chronic sequelae reminiscent of children exposed to OPs, suggesting that it will be a valuable tool for investigating pathologic mechanisms and potential treatment strategies to attenuate long-term OP neurotoxicity. SIGNIFICANCE STATEMENT: Millions of children are exposed to organophosphates (OPs) used in agriculture or chemical incidents. This study investigated the long-term impact of neonatal exposure to the OP chemical diisopropylfluorophosphate (DFP) on neurobehavioral and neurodevelopmental outcomes in adulthood. DFP exposure caused long-lasting behavioral abnormalities, epileptic seizures, and bilateral brain defects with an array of neurological sequelae seen in children's OP neurotoxicity. Thus, this model provides a novel tool to explore therapeutic interventions that mitigate long-term neurotoxic effects of children exposed to OP-induced seizures and status epilepticus.

Sex Differences in Organophosphate Model of Benzodiazepine-Refractory Status Epilepticus and Neuronal Damage.

Sex differences are common in human epilepsy. Although men are more susceptible to seizure than women, the mechanisms underlying sex-specific vulnerabilities to seizure are unclear. The organophosphate (OP) diisopropylfluorophosphate (DFP) is known to cause neurotoxicity and status epilepticus (SE), a serious neurologic condition that causes prolonged seizures and brain damage. Current therapies for OP poisoning and SE do not consider neuronal variations between male and female brains. Therefore, we investigated sex-dependent differences in electrographic seizure activity and neuronal injury using the DFP model of refractory SE in rats. Electroencephalogram recordings were used to monitor DFP-induced SE, and the extent of brain injury was determined using fluoro-jade-B staining to detect cellular necrosis. After DFP exposure, we observed striking sex-dependent differences in SE and seizure activity patterns as well as protective responses to midazolam treatment. Following acute DFP exposure, male animals displayed more severe SE with intense epileptiform spiking and greater mortality than females. In contrast, we observed significantly more injured cells and cellular necrosis in the hippocampus and other brain regions in females than in males. We also observed extensive neuronal injury in the somatosensory cortex of males. The anticonvulsant effect of midazolam against SE was limited in this model and found to be similar in males and females. However, unlike males, females exhibited substantially more protection against neuronal damage after midazolam treatment. Overall, these results demonstrate significant sex-dependent differences in DFP-induced refractory SE and neuronal damage patterns, suggesting that it may be possible to develop sex-specific neuroprotective strategies for OP intoxication and refractory SE. SIGNIFICANCE STATEMENT: Sex-dependent differences in neurotoxicity and status epilepticus (SE) are key biological variables after organophosphate (OP) exposure. Here, we investigated sex-dependent differences in SE and brain injury after acute diisopropylfluorophosphate exposure. Male rats had more severe SE and less survival than females, while females had more neuronal damage. Females had more neuroprotection to midazolam than males, while both sexes had similar but partial anticonvulsant effects. These findings suggest that a sex-specific therapeutic approach may prevent neurological complications of OP-induced SE.

1400 W, a selective inducible nitric oxide synthase inhibitor, mitigates early neuroinflammation and nitrooxidative stress in diisopropylfluorophosphate-induced short-term neurotoxicity rat model.

Organophosphate nerve agent (OPNA) exposure induces acute and long-term neurological deficits. OPNA exposure at sub-lethal concentrations induces irreversible inhibition of acetylcholinesterase and cholinergic toxidrome and develops status epilepticus (SE). Persistent seizures have been associated with increased production of ROS/RNS, neuroinflammation, and neurodegeneration. A total of 1400W is a novel small molecule, which irreversibly inhibits inducible nitric oxide synthase (iNOS) and has been shown to effectively reduce ROS/RNS generation. In this study, we investigated the effects of 1400W treatment for a week or two weeks at 10 mg/kg or 15 mg/kg per day in the rat diisopropylfluorophosphate (DFP) model. 1400W significantly reduced the number of microglia, astroglia, and NeuN+FJB positive cells compared to the vehicle in different regions of the brain. 1400W also significantly reduced nitrooxidative stress markers and proinflammatory cytokines in the serum. However, neither of the two concentrations of 1400W for two weeks of treatment had any significant effect on epileptiform spike rate and spontaneous seizures during the treatment period in mixed sex cohorts, males, or females. No significant sex differences were found in response to DFP exposure or 1400W treatment. In conclusion, 1400W treatment at 15 mg/kg per day for two weeks was more effective in significantly reducing DFP-induced nitrooxidative stress, neuroinflammatory and neurodegenerative changes.

Succinate prodrugs in combination with atropine and pralidoxime protect cerebral mitochondrial function in a rodent model of acute organophosphate poisoning

Pesticides account for hundreds of millions of cases of acute poisoning worldwide each year, with organophosphates (OPs) being responsible for the majority of all pesticide-related deaths. OPs inhibit the enzyme acetylcholinesterase (AChE), which leads to impairment of the central- and peripheral nervous system. Current standard of care (SOC) alleviates acute neurologic-, cardiovascular- and respiratory symptoms and reduces short term mortality. However, survivors often demonstrate significant neurologic sequelae. This highlights the critical need for further development of adjunctive therapies with novel targets. While the inhibition of AChE is thought to be the main mechanism of injury, mitochondrial dysfunction and resulting metabolic crisis may contribute to the overall toxicity of these agents. We hypothesized that the mitochondrially targeted succinate prodrug NV354 would support mitochondrial function and reduce brain injury during acute intoxication with the OP diisopropylfluorophosphate (DFP). To this end, we developed a rat model of acute DFP intoxication and evaluated the efficacy of NV354 as adjunctive therapy to SOC treatment with atropine and pralidoxime. We demonstrate that NV354, in combination with atropine and pralidoxime therapy, significantly improved cerebral mitochondrial complex IV-linked respiration and reduced signs of brain injury in a rodent model of acute DFP exposure.

Microglia Remodelling and Neuroinflammation Parallel Neuronal Hyperactivation Following Acute Organophosphate Poisoning.

Organophosphate (OP) compounds include highly toxic chemicals widely used both as pesticides and as warfare nerve agents. Existing countermeasures are lifesaving, but do not alleviate all long-term neurological sequelae, making OP poisoning a public health concern worldwide and the search for fully efficient antidotes an urgent need. OPs cause irreversible acetylcholinesterase (AChE) inhibition, inducing the so-called cholinergic syndrome characterized by peripheral manifestations and seizures associated with permanent psychomotor deficits. Besides immediate neurotoxicity, recent data have also identified neuroinflammation and microglia activation as two processes that likely play an important, albeit poorly understood, role in the physiopathology of OP intoxication and its long-term consequences. To gain insight into the response of microglia to OP poisoning, we used a previously described model of diisopropylfluorophosphate (DFP) intoxication of zebrafish larvae. This model reproduces almost all the defects seen in poisoned humans and preclinical models, including AChE inhibition, neuronal epileptiform hyperexcitation, and increased neuronal death. Here, we investigated in vivo the consequences of acute DFP exposure on microglia morphology and behaviour, and on the expression of a set of pro- and anti-inflammatory cytokines. We also used a genetic method of microglial ablation to evaluate the role in the OP-induced neuropathology. We first showed that DFP intoxication rapidly induced deep microglial phenotypic remodelling resembling that seen in M1-type activated macrophages and characterized by an amoeboid morphology, reduced branching, and increased mobility. DFP intoxication also caused massive expression of genes encoding pro-inflammatory cytokines Il1β, Tnfα, Il8, and to a lesser extent, immuno-modulatory cytokine Il4, suggesting complex microglial reprogramming that included neuroinflammatory activities. Finally, microglia-depleted larvae were instrumental in showing that microglia were major actors in DFP-induced neuroinflammation and, more importantly, that OP-induced neuronal hyperactivation was markedly reduced in larvae fully devoid of microglia. DFP poisoning rapidly triggered massive microglia-mediated neuroinflammation, probably as a result of DFP-induced neuronal hyperexcitation, which in turn further exacerbated neuronal activation. Microglia are thus a relevant therapeutic target, and identifying substances reducing microglial activation could add efficacy to existing OP antidote cocktails.

IL-17 and IL-17C Signaling Protects the Intestinal Epithelium against Diisopropyl Fluorophosphate Exposure in an Acute Model of Gulf War Veterans' Illnesses.

Gulf War Veterans' Illnesses (GWI) encompasses a broad range of unexplained symptomology specific to Veterans of the Persian Gulf War. Gastrointestinal (GI) distress is prominent in veterans with GWI and often presents as irritable bowel syndrome (IBS). Neurotoxins, including organophosphorus pesticides and sarin gas, are believed to have contributed to the development of GWI, at least in a subset of Veterans. However, the effects of such agents have not been extensively studied for their potential impact to GI disorders and immunological stability. Here we utilized an established murine model of GWI to investigate deleterious effects of diisopropyl fluorophosphate (DFP) exposure on the mucosal epithelium in vivo and in vitro. In vivo, acute DFP exposure negatively impacts the mucosal epithelium by reducing tight junction proteins and antimicrobial peptides as well as altering intestinal microbiome composition. Furthermore, DFP treatment reduced the expression of IL-17 in the colonic epithelium. Conversely, both IL-17 and IL-17C treatment could combat the negative effects of DFP and other cholinesterase inhibitors in murine intestinal organoid cells. Our findings demonstrate that acute exposure to DFP can result in rapid deterioration of mechanisms protecting the GI tract from disease. These results are relevant to suspected GWI exposures and could help explain the propensity for GI disorders in GWI Veterans.

Characterization of organophosphate‐induced brain injuries in a convulsive mouse model of diisopropylfluorophosphate exposure

Organophosphate (OP) compounds constitute a class of highly toxic molecules, characterized by irreversible cholinesterase (ChE) inhibition. Being either pesticides or chemical warfare agents, they present a major health issue in some countries, as well as a terrorist or military threat. Prompted by the need for suitable animal models to test novel medical countermeasures, we developed a new convulsive mouse model of OP poisoning using diisopropylfluorophosphate (DFP). Using electrocorticography (ECoG), we analyzed seizure and status epilepticus (SE) occurrences, as well as relative power of ECoG frequency band modifications after DFP injection in male Swiss mice. Next, we investigated DFP effect on ChE inhibition. Histological changes on neuronal activity and neuronal damage were examined by c-Fos immunolabeling and Fluoro-Jade C staining. We showed that mice exposed to DFP presented electrocorticographic seizures that rapidly progressed to SE within 20minutes. Lasting >8hours, DFP-induced SE was associated with major power spectrum modifications in seizing DFP animals compared to control animals. Seizures and SE development were concomitant with profound ChE inhibition and induced massive neuronal degeneration. Presenting all hallmarks of convulsive OP poisoning, we showed that our mouse model is valuable for studying pathophysiological mechanisms and preclinical testing of newly available therapeutic molecules.

Diapocynin, an NADPH oxidase inhibitor, counteracts diisopropylfluorophosphate-induced long-term neurotoxicity in the rat model.

Organophosphate (OP) nerve agents are a threat to both the military and civilians. OP exposure causes cholinergic crisis and status epilepticus (SE) because of irreversible inhibition of acetylcholinesterase that can be life-threatening if left untreated. OP survivors develop long-term morbidity, such as cognitive impairment and motor dysfunction, because of oxidative stress and progressive neuroinflammation and neurodegeneration, which act as disease promoters. Current medical countermeasures (MCMs) do not mitigate these pathologies. Therefore, our goal was to target these disease promoters using diapocynin (DPO), an NADPH oxidase inhibitor, in addition to MCMs, in a rat diisopropylfluorophosphate (DFP) model. The DFP-intoxicated rats were treated with DPO (300 mg/kg,oral,sixdoses,12-h intervals) or vehicle 2 h following behavioral SE termination with diazepam. The DPO treatment significantly rescued DFP-induced motor impairment and attenuated epileptiform spiking during the first 72 h after DFP exposure in severely seizing rats despite no difference in epileptiform spike rate between the vehicle and DPO groups in mild SE rats. DPO significantly reduced DFP-induced reactive astrogliosis, neurodegeneration, GP91phox , glutathiolated protein, serum nitrite, and proinflammatory cytokines and chemokines, such as interleukins (ILs) IL-1α, IL-6, IL-2, IL-17A, leptin, and IP-10, in the hippocampus. Collectively, these data support a neuroprotective role of DPO in an OP-induced neurotoxicity model.

Dorsoventral-Specific Effects of Nerve Agent Surrogate Diisopropylfluorophosphate on Synaptic Transmission in the Mouse Hippocampus.

Although there has been an increasing appreciation for functional differences between the dorsal (dH) and ventral (vH) hippocampal sectors, there is a lack of information characterizing the cholinergic and noncholinergic mechanisms of acetylcholinesterase inhibitors on synaptic transmission along the hippocampal dorsoventral axis. Diisopropylfluorophosphate (DFP) is an organophosphate (OP) that is commonly employed as a nerve agent surrogate in vitro as well as in rodent models of disease states, such as Gulf War Illness. The present study investigated the cholinergic and noncholinergic mechanisms responsible for the effects of acute DFP exposure on dH and vH synaptic transmission in a hippocampal slice preparation. A paired-pulse extracellular recording protocol was used to monitor the population spike (PS) amplitude as well as the PS paired-pulse ratio (PS-PPR) in the CA1 subfield of the dH and the vH. We observed that DFP-induced PS1 inhibition was produced by a cholinergic mechanism in the dH, whereas a noncholinergic mechanism was indispensable in mediating the inhibitory effect of DFP on the PS1 in the vH. PS-PPR in both dH and vH sectors was increased by acute DFP exposure, an effect that was blocked by an N-methyl-D-aspartate receptor antagonist but not by cholinergic antagonists. Clinical reports have indicated dorsoventral-specific hippocampal abnormalities in cases of OP intoxications. Therefore, the observed dorsoventral-specific noncholinergic mechanisms underlying the effects of DFP on hippocampal synaptic transmission may have important implications for the treatment of OP overexposures. SIGNIFICANCE STATEMENT: It is unknown if acetylcholinesterase inhibitors differentially impact dorsal and ventral hippocampal synaptic transmission. The data in the present study show that an organophosphate, diisopropylfluorophosphate, impacts glutamatergic transmission along the dorsoventral axis in a hippocampal slice preparation via distinct cholinergic and noncholinergic mechanisms. These findings may provide insight into investigations of therapeutic agents that target noncholinergic mechanisms in cases of organophosphate overexposures.

TSPO PET Using [18F]PBR111 Reveals Persistent Neuroinflammation Following Acute Diisopropylfluorophosphate Intoxication in the Rat.

Acute intoxication with organophosphates (OPs) can trigger status epilepticus followed by persistent cognitive impairment and/or electroencephalographic abnormalities. Neuroinflammation is widely posited to influence these persistent neurological consequences. However, testing this hypothesis has been challenging, in part because traditional biometrics preclude longitudinal measures of neuroinflammation within the same animal. Therefore, we evaluated the performance of noninvasive positron emission tomography (PET), using the translocator protein (TSPO) radioligand [18F]PBR111 against classic histopathologic measures of neuroinflammation in a preclinical model of acute intoxication with the OP diisopropylfluorophosphate (DFP). Adult male Sprague Dawley rats administered pyridostigmine bromide (0.1 mg/kg, im) 30 min prior to administration of DFP (4 mg/kg, sc), atropine sulfate (2 mg/kg, im) and 2-pralidoxime (25 mg/kg, im) exhibited moderate-to-severe seizure behavior. TSPO PET performed prior to DFP exposure and at 3, 7, 14, 21, and 28 days postexposure revealed distinct lesions, as defined by increased standardized uptake values (SUV). Increased SUV showed high spatial correspondence to immunohistochemical evidence of neuroinflammation, which was corroborated by cytokine gene and protein expression. Regional SUV metrics varied spatiotemporally with days postexposure and correlated with the degree of neuroinflammation detected immunohistochemically. Furthermore, SUV metrics were highly correlated with seizure severity, suggesting that early termination of OP-induced seizures may be critical for attenuating subsequent neuroinflammatory responses. Normalization of SUV values to a cerebellar reference region improved correlations to all outcome measures and seizure severity. Collectively, these results establish TSPO PET using [18F]PBR111 as a robust, noninvasive tool for longitudinal monitoring of neuroinflammation following acute OP intoxication.

Septotemporal‐specific Effects of a Gulf War Illness Sarin Surrogate, Diisopropylfluorophosphate, on Synaptic Transmission in the Mouse Hippocampus

Diisopropylfluorophosphate (DFP) is a common surrogate for nerve agents used in rodent models of Gulf War Illness (GWI) and is known to alter hippocampal synaptic transmission. Although there has been an increased appreciation for functional differences in the hippocampus between the dorsal (dH) and ventral (vH) sectors, there is a paucity of comparative information characterizing the effects of GWI‐relevant chemicals in the dH and the vH. Our objective was to investigate the effects of acute DFP exposure on hippocampal synaptic transmission using extracellular recording from mouse dH and vH slice preparations. DFP (30 μM) was washed‐in for 30 min while population spike (PS) amplitude was monitored. PS amplitude in the dH and vH was decreased by DFP. Following pretreatment with the nicotinic antagonist mecamylamine (MEC, 30 μM), the DFP‐induced PS inhibition was enhanced in both the dH and vH. In contrast, pretreatment with the muscarinic antagonist atropine (ATR, 3 μM) resulted in DFP enhancement of the PS amplitude in the dH whereas PS inhibition was still observed in the vH. Coapplication of ATR and MEC prevented DFP‐mediated modulation of PS amplitude in the dH but the cholinergic antagonists had no effect on DFP‐mediated PS inhibition in the vH. Similar to ATR pretreatment experiments, dH slices preexposed to the M2 selective antagonist AFDX‐116 (300 nM) exhibited enhanced PS amplitude whereas AFDX‐116 had no effect in the vH. Regarding possible non‐cholinergic mechanisms, DFP has been shown to directly interact with the NMDA receptor (NMDAR). Combined pretreatment with AFDX‐116, MEC, and the competitive NMDAR antagonist D‐APV (25 μM) prevented all DFP‐induced modulation of the PS in both the dH and vH. The AFDX‐116 blockade of the DFP‐mediated PS inhibition in the dH suggests an M2R‐dependent inhibition of excitation in CA1; however, in the vH this DFP effect is primarily mediated by a non‐cholinergic mechanism. Notably, pretreatment with D‐APV alone prevented a decrease in PS amplitude in the dH and the vH, suggesting a common involvement of NMDARs in DFP actions in both hippocampal sectors. Population spike paired‐pulse ratio (PS PPR) was also monitored to assess the impact of DFP exposure on recurrent inhibition. DH and vH slices exposed to DFP exhibited increased PS PPR, consistent with a reduction in inhibitory influence in CA1. Interestingly, in dH and vH slices pretreated with cholinergic antagonists, we still observed a DFP‐mediated increase of PS PPR whereas D‐APV pretreatment alone prevented this increase. Previous reports have demonstrated impairments in hippocampal‐dependent behavioral performance in both animal models and GWI patient populations. Mechanistic distinctions along the septotemporal axis that underlie the actions of DFP on synaptic activity may have important implications for potential therapeutic approaches to the treatment of GWI.Support or Funding InformationDepartment of Defense (to NMF and JJW [Award: W81XWH‐16‐1‐0586]) and UGA Interdisciplinary Toxicology Program (to KAB)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Inducible nitric oxide synthase inhibitor, 1400W, mitigates DFP-induced long-term neurotoxicity in the rat model

Chemical nerve agents (CNA) are increasingly becoming a threat to both civilians and military personnel. CNA-induced acute effects on the nervous system have been known for some time and the long-term consequences are beginning to emerge. In this study, we used diisopropylfluorophosphate (DFP), a seizurogenic CNA to investigate the long-term impact of its acute exposure on the brain and its mitigation by an inducible nitric oxide synthase (iNOS) inhibitor, 1400W as a neuroprotectant in the rat model. Several experimental studies have demonstrated that DFP-induced seizures and/or status epilepticus (SE) causes permanent brain injury, even after the countermeasure medication (atropine, oxime, and diazepam). In the present study, DFP-induced SE caused a significant increase in iNOS and 3-nitrotyrosine (3-NT) at 24 h, 48 h, 7d, and persisted for a long-term (12 weeks post-exposure), which led to the hypothesis that iNOS is a potential therapeutic target in DFP-induced brain injury. To test the hypothesis, we administered 1400W (20 mg/kg, i.m.) or the vehicle twice daily for the first three days of post-exposure. 1400W significantly reduced DFP-induced iNOS and 3-NT upregulation in the hippocampus and piriform cortex, and the serum nitrite levels at 24 h post-exposure. 1400W also prevented DFP-induced mortality in <24 h. The brain immunohistochemistry (IHC) at 7d post-exposure revealed a significant reduction in gliosis and neurodegeneration (NeuN+ FJB positive cells) in the 1400W-treated group. 1400W, in contrast to the vehicle, caused a significant reduction in the epileptiform spiking and spontaneous recurrent seizures (SRS) during 12 weeks of continuous video-EEG study. IHC of brain sections from the same animals revealed a significant reduction in reactive gliosis (both microgliosis and astrogliosis) and neurodegeneration across various brain regions in the 1400W-treated group when compared to the vehicle-treated group. A multiplex assay from hippocampal lysates at 6 weeks post-exposure showed a significant increase in several key pro-inflammatory cytokines/chemokines such as IL-1α, TNFα, IL-1β, IL-2, IL-6, IL-12, IL-17a, MCP-1, LIX, and Eotaxin, and a growth factor, VEGF in the vehicle-treated animals. 1400W significantly suppressed IL-1α, TNFα, IL-2, IL-12, and MCP-1 levels. It also suppressed DFP-induced serum nitrite levels at 6 weeks post-exposure. In the Morris water maze, the vehicle-treated animals spent significantly less time in the target quadrant in a probe trial at 9d post-exposure compared to their time spent in the same quadrant 11 days previously (i.e., 2 days prior to DFP exposure). Such a difference was not observed in the 1400W and control groups. However, learning and short-term memory were unaffected when tested at 10-16d and 28-34d post-exposure. Accelerated rotarod, horizontal bar test, and the forced swim test revealed no significant changes between groups. Overall, the findings from this study suggest that 1400W may be considered as a potential therapeutic agent as a follow-on therapy for CNA exposure, after controlling the acute symptoms, to prevent mortality and some of the long-term neurotoxicity parameters such as epileptiform spiking, SRS, neurodegeneration, reactive gliosis in some brain regions, and certain key proinflammatory cytokines and chemokine.

Midazolam-Resistant Seizures and Brain Injury after Acute Intoxication of Diisopropylfluorophosphate, an Organophosphate Pesticide and Surrogate for Nerve Agents.

Organophosphates (OP) such as the pesticide diisopropylfluorophosphate (DFP) and the nerve agent sarin are lethal chemicals that induce seizures, status epilepticus (SE), and brain damage. Midazolam, a benzodiazepine modulator of synaptic GABA-A receptors, is currently considered as a new anticonvulsant for nerve agents. Here, we characterized the time course of protective efficacy of midazolam (0.2-5 mg/kg, i.m.) in rats exposed to DFP, a chemical threat agent and surrogate for nerve agents. Behavioral and electroencephalogram (EEG) seizures were monitored for 24 hours after DFP exposure. The extent of brain injury was determined 3 days after DFP exposure by unbiased stereologic analyses of valid markers of neurodegeneration and neuroinflammation. Seizures were elicited within ∼8 minutes after DFP exposure that progressively developed into persistent SE lasting for hours. DFP exposure resulted in massive neuronal injury or necrosis, neurodegeneration of principal cells and interneurons, and neuroinflammation as evident by extensive activation of microglia and astrocytes in the hippocampus, amygdala, and other brain regions. Midazolam controlled seizures, neurodegeneration, and neuroinflammation when given early (10 minutes) after DFP exposure, but it was less effective when given at 40 minutes or later. Delayed therapy (≥40 minutes), a simulation of the practical therapeutic window for first responders or hospital admission, was associated with reduced seizure protection and neuroprotection. These results strongly reaffirm that the DFP-induced seizures and brain damage are progressively resistant to delayed treatment with midazolam, confirming the benzodiazepine refractory SE after OP intoxication. Thus, novel anticonvulsants superior to midazolam or adjunct therapies that enhance its efficacy are needed for effective treatment of refractory SE.

Benzodiazepine-refractory status epilepticus, neuroinflammation, and interneuron neurodegeneration after acute organophosphate intoxication

Nerve agents and some pesticides such as diisopropylfluorophosphate (DFP) cause neurotoxic manifestations that include seizures and status epilepticus (SE), which are potentially lethal and carry long-term neurological morbidity. Current antidotes for organophosphate (OP) intoxication include atropine, 2-PAM and diazepam (a benzodiazepine for treating seizures and SE). There is some evidence for partial or complete loss of diazepam anticonvulsant efficacy when given 30 min or later after exposure to an OP; this condition is known as refractory SE. Effective therapies for OP-induced SE are lacking and it is unclear why current therapies do not work. In this study, we investigated the time-dependent efficacy of diazepam in the nerve agent surrogate DFP model of OP intoxication on seizure suppression and neuroprotection in rats, following an early and late therapy. Diazepam (5 mg/kg, IM) controlled seizures when given 10 min after DFP exposure (“early”), but it was completely ineffective at 60 or 120 min (“late”) after DFP. DFP-induced neuronal injury, neuroinflammation, and neurodegeneration of principal cells and GABAergic interneurons were significantly reduced by early but not late therapy. These findings demonstrate that diazepam failed to control seizures, SE and neuronal injury when given 60 min or later after DFP exposure, confirming the benzodiazepine-refractory SE and brain damage after OP intoxication. In addition, this study indicates that degeneration of inhibitory interneurons and inflammatory glial activation are potential mechanisms underlying these morbid outcomes of OP intoxication. Therefore, novel anticonvulsant and neuroprotectant antidotes, superior to benzodiazepines, are desperately needed for controlling nerve agent-induced SE and brain injury.

Cardiac function in a chemical toxin (organophosphate) induced model of gulf war Illness: benefit of exercise training

BackgroundGulf War Illness (GWI) is a debilitating disease characterized by a cadre of neural, immunological and autonomic symptoms. We show that exposure to a sub‐lethal dose of Sarin lead to autonomic imbalance, cardiomyopathy and central nervous system changes. Diisopropyl fluorophosphate (DFP), an analogue of Sarin (GB), can be used as surrogate agent to produce neurotoxicity and neuro‐inflammation. There is limited information on the use of DFP to produce model of GWI with emphasis on the study of cardiovascular‐autonomic dysfunctions. Consideration was given to the inclusion of exercise training (ET) as part of the treatment of cardiac dysfunction induced by organophosphate exposure.ObjectiveThe aim was to determine the effect of chronic exercise training to the response to DFP on autonomic and cardiovascular function.MethodsMale C57BL/6J mice were divided into two groups (n=8/group): Sedentary (S) and trained (T) before and after DFP exposure. ET was performed on an electronic wheel at low intensity for 1 h/day, 5 days/wk. GWI model was induced by corticosterone (CORT) administered for 7 days (200 mg/l) followed by DFP (1.5 mg/kg, s.c). Cardiac function and ECG were assessed by transthoracic echocardiography (echo) under isoflurane anesthesia (2 – 2.5% on 0.8 l/min of O2).ResultsThe low dose of DFP produced left ventricular dilation on sedentary group, specifically, LV dilation of end systolic area (ESA) (S: 10.34±0.86 vs S+DFP: 6.69 ±0.41 cm2). The ET ameliorated DFP induced toxicity (T: 7.99 ±0.40 vs T+DFP 7.66±0.96cm2). Cardiac Output (CO) was reduced in sedentary group after DFP (S: 20.61±2.63 vs S+DFP: 14.78±0.85 ml/min), not change was observed in the ET group, revealing the efficacy of exercise protocol (T: 16.99±1.69 vs T+DFP: 17.90±1.42 ml/min). Myocardial performance index (MPI) and E/A ratio were significantly increased in S group after DFP exposure (MPI: S: 0.52±0.03 vs S+DFP: 0.60±0.02; E/A: S: 1.29±0.09 vs S+DFP: 1.64±0.07) changes that were not observed on T group (MPI: T: 0.50±0.04 vs T+DFP: 0.48±0.03; E/A: S: 1.35±0.09 vs S+DFP: 1.56±0.10) suggesting protection against DFP. Finally sympathetic modulation (LF) was increased in S group compared initial values and T group, as well as LF/HF ratio. Vagal modulation was similar between both groups.ConclusionIn conclusion, results demonstrate that DFP exerts a negative impact on cardiac/autonomic function. Our critical finding is that chronic exercise prevented most of the cardiovascular dysfunctions elicited by DFP, documenting the positive effects of exercise. This treatment protocol maybe equally applicable to other animal models or even humans.Support or Funding InformationThis work was funded under U.S. Department of Defense grant W81XWH‐13‐2‐0085This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

From the Cover: MagneticResonance Imaging Reveals Progressive Brain Injury in Rats Acutely Intoxicated With Diisopropylfluorophosphate.

Acute intoxication with organophosphates (OPs) can trigger seizures that progress to status epilepticus, and survivors often exhibit chronic neuropathology, cognitive impairment, affective disorders, and/or electroencephalographic abnormalities. Understanding how acute injury transitions to persistent neurological sequelae is critical to developing medical countermeasures for mitigating damage following OP-induced seizures. Here, we used in vivo magnetic resonance imaging (MRI) to monitor the spatiotemporal patterns of neuropathology for 1 month after acute intoxication with diisopropylfluorophosphate (DFP). Adult male Sprague Dawley rats administered pyridostigmine bromide (0.1 mg/kg, im) 30 min prior to successive administration of DFP (4 mg/kg, sc), atropine sulfate (2 mg/kg, im), and 2-pralidoxime (25 mg/kg, im) exhibited moderate-to-severe seizure behavior. T2-weighted and diffusion-weighted MR imaging prior to DFP exposure and at 3, 7, 14, 21, or 28 days postexposure revealed prominent lesions, tissue atrophy, and ventricular enlargement in discrete brain regions. Lesions varied in intensity and/or extent over time, with the overall magnitude of injury strongly influenced by seizure severity. Importantly, lesions detected by MRI correlated spatially and temporally with histological evidence of brain pathology. Analysis of histogram parameters extracted from frequency distributions of regional apparent diffusion coefficient (ADC) values identified the standard deviation and 90th percentile of the ADC as robust metrics for quantifying persistent and progressive neuropathological changes. The interanimal and interregional variations observed in lesion severity and progression, coupled with potential reinjury following spontaneous recurrent seizures, underscore the advantages of using in vivo imaging to longitudinally monitor neuropathology and, ultimately, therapeutic response, following acute OP intoxication.

Inhibition of the prostaglandin E2 receptor EP2 prevents status epilepticus-induced deficits in the novel object recognition task in rats

Survivors of exposure to an organophosphorus nerve agent may develop a number of complications including long-term cognitive deficits (Miyaki et al., 2005; Nishiwaki et al., 2001). We recently demonstrated that inhibition of the prostaglandin E2 receptor, EP2, attenuates neuroinflammation and neurodegeneration caused by status epilepticus (SE) induced by the soman analog, diisopropylfluorophosphate (DFP), which manifest within hours to days of the initial insult. Here, we tested the hypothesis that DFP exposure leads to a loss of cognitive function in rats that is blocked by early, transient EP2 inhibition. Adult male Sprague-Dawley rats were administered vehicle or the competitive EP2 antagonist, TG6-10-1, (ip) at various times relative to DFP-induced SE. DFP administration resulted in prolonged seizure activity as demonstrated by cortical electroencephalography (EEG). A single intraperitoneal injection of TG6-10-1 or vehicle 1 h prior to DFP did not alter the development of seizures, the latency to SE or the duration of SE. Rats administered six injections of TG6-10-1 starting 90 min after the onset of DFP-induced SE could discriminate between a novel and familiar object 6–12 weeks after SE, unlike vehicle treated rats which showed no preference for the novel object. By contrast, behavioral changes in the light-dark box and open field assays were not affected by TG6-10-1. Delayed mortality after DFP was also unaffected by TG6-10-1. Thus, selective inhibition of the EP2 receptor may prevent SE-induced memory impairment in rats caused by exposure to a high dose of DFP.

Repeated low-dose organophosphate DFP exposure leads to the development of depression and cognitive impairment in a rat model of Gulf War Illness

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.