Quantitative T2 mapping-based longitudinal assessment of brain injury and therapeutic rescue in the rat following acute organophosphate intoxication.

Acute intoxication with organophosphate (OP) cholinesterase inhibitors poses a significant public health risk. While currently approved medical countermeasures can improve survival rates, they often fail to prevent chronic neurological damage. Therefore, there is need to develop effective therapies and quantitative metrics for assessing OP-induced brain injury and its rescue by these therapies. In this study we used a rat model of acute intoxication with the OP, diisopropylfluorophosphate (DFP), to test the hypothesis that T2 measures obtained from brain magnetic resonance imaging (MRI) scans provide quantitative metrics of brain injury and therapeutic efficacy. Adult male Sprague Dawley rats were imaged on a 7T MRI scanner at 3, 7 and 28 days post-exposure to DFP or vehicle (VEH) with or without treatment with the standard of care antiseizure drug, midazolam (MDZ); a novel antiseizure medication, allopregnanolone (ALLO); or combination therapy with MDZ and ALLO (DUO). Our results show that mean T2 values in DFP-exposed animals were: (1) higher than VEH in all volumes of interest (VOIs) at day 3; (2) decreased with time; and (3) decreased in the thalamus at day 28. Treatment with ALLO or DUO, but not MDZ alone, significantly decreased mean T2 values relative to untreated DFP animals in the piriform cortex at day 3. On day 28, the DUO group showed the most favorable T2 characteristics. This study supports the utility of T2 mapping for longitudinally monitoring brain injury and highlights the therapeutic potential of ALLO as an adjunct therapy to mitigate chronic morbidity associated with acute OP intoxication.

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.

Long-Term Neuropsychiatric Developmental Defects after Neonatal Organophosphate Exposure: Mitigation by Synthetic Neurosteroids.

Children are much more susceptible to the neurotoxic effects of organophosphate (OP) pesticides and nerve agents than adults. OP poisoning in children leads to acute seizures and neuropsychiatric sequela, including the development of long-term disabilities and cognitive impairments. Despite these risks, there are few chronic rodent models that use pediatric OP exposure for studying neurodevelopmental consequences and interventions. Here, we investigated the protective effect of the neurosteroid ganaxolone (GX) on the long-term developmental impact of neonatal exposure to the OP compound, diisopropyl-fluorophosphate (DFP). Pediatric postnatal day-28 rats were acutely exposed to DFP, and at 3 and 10 months after exposure, they were evaluated using a series of cognitive and behavioral tests with or without the postexposure treatment of GX. Analysis of the neuropathology was performed after 10 months. DFP-exposed animals displayed significant long-term deficits in mood, anxiety, depression, and aggressive traits. In spatial and nonspatial cognitive tests, they displayed striking impairments in learning and memory. Analysis of brain sections showed significant loss of neuronal nuclei antigen(+) principal neurons, parvalbumin(+) inhibitory interneurons, and neurogenesis, along with increased astrogliosis, microglial neuroinflammation, and mossy fiber sprouting. These detrimental neuropathological changes are consistent with behavioral dysfunctions. In the neurosteroid GX-treated cohort, behavioral and cognitive deficits were significantly reduced and were associated with strong protection against long-term neuroinflammation and neurodegeneration. In conclusion, this pediatric model replicates the salient features of children exposed to OPs, and the protective outcomes from neurosteroid intervention support the viability of developing this strategy for mitigating the long-term effects of acute OP exposure in children. SIGNIFICANCE STATEMENT: An estimated 3 million organophosphate exposures occur annually worldwide, with children comprising over 30% of all victims. Our understanding of the neurodevelopmental consequences in children exposed to organophosphates is limited. Here, we investigated the long-term impact of neonatal exposure to diisopropyl-fluorophosphate in pediatric rats. Neurosteroid treatment protected against major deficits in behavior and memory and was well correlated with neuropathological changes. Overall, this pediatric model is helpful to screen novel therapies to mitigate long-term developmental deficits of organophosphate exposure.

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 Il1ß, 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.

Time-dependent neuropathology in rats following organophosphate-induced status epilepticus.

Exposure to high levels of a cholinesterase inhibiting organophosphorus (OP) agent often results in seizures that progress to status epilepticus (SE). Survivors of OP-induced SE often display neuropathological consequences the days following SE. In the current study, the temporal profile of neuropathology after SE was investigated in a rat model of diisopropylfluorophosphate (DFP)-induced SE. Adult Sprague-Dawley rats were injected with DFP to induce SE for one hour. Following termination of electrographic SE with urethane (0.8 g/kg, sc), cohorts of rats were euthanized 3, 24 and 48 h later and brain tissue was processed to determine immediate early gene and inflammatory mediator expression as well as blood-brain barrier changes and neurodegeneration. After SE rats displayed a time-dependent upregulation of immediate early genes such as cFos and ΔFosB as well as pro-inflammatory mediators COX-2, IL-1ß and IL-6. The profile of positive cFos staining, but not ΔFosB, coincided temporally with heightened brain activity measured by cortical electroencephalography (EEG). Neurodegeneration in limbic brain regions was absent 3 h after SE, but prominent 24 h later and continued to increase 48 h after SE. Serum albumin was detected in the cortex 3 h after SE suggesting early loss of blood brain barrier integrity. However, the blood-brain barrier appeared repaired 48 h after SE. This study demonstrates that following OP-poisoning in rats, immediate early gene expression in the brain precedes neuroinflammation followed by erosion of the blood-brain barrier and neurodegeneration. The study also demonstrates that seizure activity in brain nuclei coincides with cFos expression. Together, these studies give insight into the temporal molecular changes in the brain following organophosphate-induced status epilepticus.

Diisopropylfluorophosphate (DFP) volatizes and cross-contaminates wells in a common 96-well plate format used in zebrafish larvae toxicology studies.

Diisopropylfluorophosphate (DFP) is an organophosphate (OP) that is commonly used as a surrogate of OP nerve agents to study the neurotoxic effects of acute OP intoxication. In preliminary studies, we discovered abnormally high incidence of deaths in DMSO control zebrafish larvae housed in the same 96-well plate as DFP-exposed larvae and hypothesized that DFP volatilizes and cross-contaminates wells when using static waterborne exposures. Survivability and acetylcholinesterase activity assays were indicative of the presence of DFP in the tissues of zebrafish ostensibly exposed to DMSO only. These findings are consistent with DFP cross-contamination, which raises concerns for the experimental design of studies evaluating the toxicity of volatile and semi-volatile substances in zebrafish using medium-to-high throughput approaches.

Persistent neuropathology and behavioral deficits in a mouse model of status epilepticus induced by acute intoxication with diisopropylfluorophosphate.

Organophosphate (OP) nerve agents and pesticides are a class of neurotoxic compounds that can cause status epilepticus (SE), and death following acute high-dose exposures. While the standard of care for acute OP intoxication (atropine, oxime, and high-dose benzodiazepine) can prevent mortality, survivors of OP poisoning often experience long-term brain damage and cognitive deficits. Preclinical studies of acute OP intoxication have primarily used rat models to identify candidate medical countermeasures. However, the mouse offers the advantage of readily available knockout strains for mechanistic studies of acute and chronic consequences of OP-induced SE. Therefore, the main objective of this study was to determine whether a mouse model of acute diisopropylfluorophosphate (DFP) intoxication would produce acute and chronic neurotoxicity similar to that observed in rat models and humans following acute OP intoxication. Adult male C57BL/6J mice injected with DFP (9.5 mg/kg, s.c.) followed 1 min later with atropine sulfate (0.1 mg/kg, i.m.) and 2-pralidoxime (25 mg/kg, i.m.) developed behavioral and electrographic signs of SE within minutes that continued for at least 4 h. Acetylcholinesterase inhibition persisted for at least 3 d in the blood and 14 d in the brain of DFP mice relative to vehicle (VEH) controls. Immunohistochemical analyses revealed significant neurodegeneration and neuroinflammation in multiple brain regions at 1, 7, and 28 d post-exposure in the brains of DFP mice relative to VEH controls. Deficits in locomotor and home-cage behavior were observed in DFP mice at 28 d post-exposure. These findings demonstrate that this mouse model replicates many of the outcomes observed in rats and humans acutely intoxicated with OPs, suggesting the feasibility of using this model for mechanistic studies and therapeutic screening.

Diisopropylfluorophosphate-induced status epilepticus drives complex glial cell phenotypes in adult male mice.

Organophosphate pesticides and nerve agents (OPs), are characterized by cholinesterase inhibition. In addition to severe peripheral symptoms, high doses of OPs can lead to seizures and status epilepticus (SE). Long lasting seizure activity and subsequent neurodegeneration promote neuroinflammation leading to profound pathological alterations of the brain. The aim of this study was to characterize neuroinflammatory responses at key time points after SE induced by the OP, diisopropylfluorophosphate (DFP). Immunohistochemistry (IHC) analysis and RT-qPCR on cerebral tissue are often insufficient to identity and quantify precise neuroinflammatory alterations. To address these needs, we performed RT-qPCR quantification after whole brain magnetic-activated cell-sorting (MACS) of CD11B (microglia/infiltrated macrophages) and GLAST (astrocytes)-positive cells at 1, 4, 24 h and 3 days post-SE. In order to compare these results to those obtained by IHC, we performed, classical Iba1 (microglia/infiltrated macrophages) and GFAP (astrocytes) IHC analysis in parallel, focusing on the hippocampus, a brain region affected by seizure activity and neurodegeneration. Shortly after SE (1-4 h), an increase in pro-inflammatory (M1-like) markers and A2-specific markers, proposed as neurotrophic, were observed in CD11B and GLAST-positive isolated cells, respectively. Microglial cells successively expressed immuno-regulatory (M2b-like) and anti-inflammatory (M2a-like) at 4 h and 24 h post-SE induction. At 24 h and 3 days, A1-specific markers, proposed as neurotoxic, were increased in isolated astrocytes. Although IHC analysis presented no modification in terms of percentage of marked area and cell number at 1 and 4 h after SE, at 24 h and 3 days after SE, microglial and astrocytic activation was visible by IHC as an increase in Iba1 and GFAP-positive area and Iba1-positive cells in DFP animals when compared to the control. Our work identified sequential microglial and astrocytic phenotype activation. Although the role of each phenotype in SE cerebral outcomes requires further study, targeting specific markers at specific time point could be a beneficial strategy for DFP-induced SE treatment.

Organophosphorus diisopropylfluorophosphate (DFP) intoxication in zebrafish larvae causes behavioral defects, neuronal hyperexcitation and neuronal death.

With millions of intoxications each year and over 200,000 deaths, organophosphorus (OP) compounds are an important public health issue worldwide. OP poisoning induces cholinergic syndrome, with respiratory distress, hypertension, and neuron damage that may lead to epileptic seizures and permanent cognitive deficits. Existing countermeasures are lifesaving but do not prevent long-lasting neuronal comorbidities, emphasizing the urgent need for animal models to better understand OP neurotoxicity and identify novel antidotes. Here, using diisopropylfluorophosphate (DFP), a prototypic and moderately toxic OP, combined with zebrafish larvae, we first showed that DFP poisoning caused major acetylcholinesterase inhibition, resulting in paralysis and CNS neuron hyperactivation, as indicated by increased neuronal calcium transients and overexpression of the immediate early genes fosab, junBa, npas4b, and atf3. In addition to these epileptiform seizure-like events, DFP-exposed larvae showed increased neuronal apoptosis, which were both partially alleviated by diazepam treatment, suggesting a causal link between neuronal hyperexcitation and cell death. Last, DFP poisoning induced an altered balance of glutamatergic/GABAergic synaptic activity with increased NR2B-NMDA receptor accumulation combined with decreased GAD65/67 and gephyrin protein accumulation. The zebrafish DFP model presented here thus provides important novel insights into the pathophysiology of OP intoxication, making it a promising model to identify novel antidotes.

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 20 minutes. Lasting >8 hours, 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.

The chemical convulsant diisopropylfluorophosphate (DFP) causes persistent neuropathology in adult male rats independent of seizure activity.

Organophosphate (OP) threat agents can trigger seizures that progress to status epilepticus, resulting in persistent neuropathology and cognitive deficits in humans and preclinical models. However, it remains unclear whether patients who do not show overt seizure behavior develop neurological consequences. Therefore, this study compared two subpopulations of rats with a low versus high seizure response to diisopropylfluorophosphate (DFP) to evaluate whether acute OP intoxication causes persistent neuropathology in non-seizing individuals. Adult male Sprague Dawley rats administered DFP (4 mg/kg, sc), atropine sulfate (2 mg/kg, im), and pralidoxime (25 mg/kg, im) were monitored for seizure activity for 4 h post-exposure. Animals were separated into groups with low versus high seizure response based on behavioral criteria and electroencephalogram (EEG) recordings. Cholinesterase activity was evaluated by Ellman assay, and neuropathology was evaluated at 1, 2, 4, and 60 days post-exposure by Fluoro-Jade C (FJC) staining and micro-CT imaging. DFP significantly inhibited cholinesterase activity in the cortex, hippocampus, and amygdala to the same extent in low and high responders. FJC staining revealed significant neurodegeneration in DFP low responders albeit this response was delayed, less persistent, and decreased in magnitude compared to DFP high responders. Micro-CT scans at 60 days revealed extensive mineralization that was not significantly different between low versus high DFP responders. These findings highlight the importance of considering non-seizing patients for medical care in the event of acute OP intoxication. They also suggest that OP intoxication may induce neurological damage via seizure-independent mechanisms, which if identified, might provide insight into novel therapeutic targets.

Urethane attenuates early neuropathology of diisopropylfluorophosphate-induced status epilepticus in rats.

Seizures can be evident within minutes of exposure to an organophosphorus (OP) agent and often progress to status epilepticus (SE) resulting in a high mortality if left untreated. Effective medical countermeasures are necessary to sustain patients suffering from OP poisoning and to mitigate the ensuing brain injury. Here, the hypothesis was tested that a single subanesthetic dose of urethane prevents neuropathology measured 24 h following diisopropylfluorophosphate (DFP)-induced SE. Adult Sprague-Dawley rats were injected with DFP to induce SE. During SE rats displayed increased neuronal activity in the hippocampus and an upregulation of immediate early genes as well as pro-inflammatory mediators. In additional experiments rats were administered diazepam (10 mg/kg, ip) or urethane (0.8 g/kg, sc) 1 h after DFP-induced SE and compared to rats that experienced uninterrupted SE. Cortical electroencephalography (EEG) and power analysis strengthen the conclusion that urethane effectively terminates SE and prevents the overnight return of seizure activity. Neurodegeneration in limbic brain regions and the seizure-induced upregulation of key inflammatory mediators present 24 h after DFP-induced SE were strongly attenuated by administration of urethane. A trivial explanation for these beneficial effects, that urethane simply reactivates acetylcholinesterase, has been ruled out. These findings indicate that, by contrast to rats administered diazepam or rats that experience uninterrupted SE, the early neuropathology after SE is prevented by subanesthetic urethane, which terminates rather than interrupts, SE.

Sex as a biological variable in the rat model of diisopropylfluorophosphate-induced long-term neurotoxicity.

Sex differences in response to neurotoxicant exposure that initiates epileptogenesis are understudied. We used telemetry-implanted male and female adult rats exposed to an organophosphate (OP) neurotoxicant, diisopropylflourophosphate (DFP), to test sex differences in the severity of status epilepticus (SE) and the development of spontaneous recurrent seizures (SRS). Females had significantly less severe SE and decreased epileptiform spikes compared with males, although females received a higher dose of DFP than males. The estrous stages had no impact on seizure susceptibility, but rats with severe SE had a significantly prolonged diestrus. A previously demonstrated disease-modifying agent, an inducible nitric oxide synthase inhibitor, 1400W, was tested in both sexes. None of the eight males treated with 1400W developed convulsive SRS during 4 weeks post-DFP exposure, while two of seven females developed convulsive SRS. Concerning gliosis and neurodegeneration, there were region-specific differences in the interaction between sex and SE severity. As SE severity influences epileptogenesis, and as females had significantly less severe SE, sex as a biological variable should be factored into the design of future OP nerve agent experiments while evaluating neurotoxicity and optimizing potential disease-modifying agents.

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, six doses, 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.

Multifunctional compounds lithium chloride and methylene Blue attenuate the negative effects of diisopropylfluorophosphate on axonal transport in rat cortical neurons.

Organophosphates (OPs) are valuable as pesticides in agriculture and for controlling deadly vector-borne illnesses; however, they are highly toxic and associated with many deleterious health effects in humans including long-term neurological impairments. Antidotal treatment regimens are available to combat the symptoms of acute OP toxicity, which result from the irreversible inhibition of acetylcholinesterase (AChE). However, there are no established treatments for the long-term neurological consequences of OP exposure. In addition to AChE, OPs can negatively affect multiple protein targets as well as biological processes such as axonal transport. Given the fundamental nature of axonal transport to neuronal health, we rationalized that this process might serve as a general focus area for novel therapeutic strategies against OP toxicity. In the studies described here, we employed a multi-target, phenotypic screening, and drug repurposing strategy for the evaluations of potential novel OP-treatments using a primary neuronal culture model and time-lapse live imaging microscopy. Two multi-target compounds, lithium chloride (LiCl) and methylene blue (MB), which are FDA-approved for other indications, were evaluated for their ability to prevent the negative effects of the OP, diisopropylfluorophosphate (DFP) on axonal transport. The results indicated that both LiCl and MB prevented DFP-induced impairments in anterograde and retrograde axonal transport velocities in a concentration dependent manner. While in vivo studies will be required to confirm our in vitro findings, these experiments support the potential of LiCl and MB as repurposed drugs for the treatment of the long-term neurological deficits associated with OP exposure (currently an unmet medical need).

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-1ß, 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.

A rat model of organophosphate-induced status epilepticus and the beneficial effects of EP2 receptor inhibition.

This review describes an adult rat model of status epilepticus (SE) induced by diisopropyl fluorophosphate (DFP), and the beneficial outcomes of transient inhibition of the prostaglandin-E2 receptor EP2 with a small molecule antagonist, delayed by 2-4 h after SE onset. Administration of six doses of the selective EP2 antagonist TG6-10-1 over a 2-3 day period accelerates functional recovery, attenuates hippocampal neurodegeneration, neuroinflammation, gliosis and blood-brain barrier leakage, and prevents long-term cognitive deficits without blocking SE itself or altering acute seizure characteristics. This work has provided important information regarding organophosphate-induced seizure related pathologies in adults and revealed the effectiveness of delayed EP2 inhibition to combat these pathologies.

Persistent behavior deficits, neuroinflammation, and oxidative stress in a rat model of acute organophosphate intoxication.

Current medical countermeasures for organophosphate (OP)-induced status epilepticus (SE) are not effective in preventing long-term morbidity and there is an urgent need for improved therapies. Rat models of acute intoxication with the OP, diisopropylfluorophosphate (DFP), are increasingly being used to evaluate therapeutic candidates for efficacy in mitigating the long-term neurologic effects associated with OP-induced SE. Many of these therapeutic candidates target neuroinflammation and oxidative stress because of their implication in the pathogenesis of persistent neurologic deficits associated with OP-induced SE. Critical to these efforts is the rigorous characterization of the rat DFP model with respect to outcomes associated with acute OP intoxication in humans, which include long-term electroencephalographic, neurobehavioral, and neuropathologic effects, and their temporal relationship to neuroinflammation and oxidative stress. To address these needs, we examined a range of outcomes at later times post-exposure than have previously been reported for this model. Adult male Sprague-Dawley rats were given pyridostigmine bromide (0.1 mg/kg, im) 30 min prior to administration of DFP (4 mg/kg, sc), which was immediately followed by atropine sulfate (2 mg/kg, im) and pralidoxime (25 mg/kg, im). This exposure paradigm triggered robust electroencephalographic and behavioral seizures that rapidly progressed to SE lasting several hours in 90% of exposed animals. Animals that survived DFP-induced SE (~70%) exhibited spontaneous recurrent seizures and hyperreactive responses to tactile stimuli over the first 2 months post-exposure. Performance in the elevated plus maze, open field, and Pavlovian fear conditioning tests indicated that acute DFP intoxication reduced anxiety-like behavior and impaired learning and memory at 1 and 2 months post-exposure in the absence of effects on general locomotor behavior. Immunohistochemical analyses revealed significantly increased expression of biomarkers of reactive astrogliosis, microglial activation and oxidative stress in multiple brain regions at 1 and 2 months post-DFP, although there was significant spatiotemporal heterogeneity across these endpoints. Collectively, these data largely support the relevance of the rat model of acute DFP intoxication as a model for acute OP intoxication in the human, and support the hypothesis that neuroinflammation and/or oxidative stress represent potential therapeutic targets for mitigating the long-term neurologic sequelae of acute OP intoxication.

Lipidomes of brain from rats acutely intoxicated with diisopropylfluorophosphate identifies potential therapeutic targets.

Organophosphates (OPs), a class of phosphorus-containing chemicals that act by disrupting cholinergic transmission, include both toxic and fast-acting chemical warfare agents as well as less toxic but more easily accessible OP pesticides. The classical atropine/2-PAM antidote fails to protect against long-term symptoms following acute intoxication with OPs at levels that trigger status epilepticus. Acute OP intoxication also causes a robust neuroinflammatory response, which is implicated in the pathogenesis of long-term effects. In this study, we characterized the profiles of lipid mediators, important players in neuroinflammation, in the rat model of acute DFP intoxication. The profiles of lipid mediators were monitored in three different regions of the brain (cortex, hippocampus, and cerebellum) at 0, 1, 3, 7, 14, and 28 days post-exposure. The distribution pattern of lipid mediators was distinct in the three brain regions. In the cerebellum, the profile is dominated by LOX metabolites, while the lipid mediator profiles in cortex and hippocampus are dominated by COX metabolites followed by LOX and CYP 450 metabolites. Following acute DFP intoxication, most of the pro-inflammatory lipid mediators (e.g., PGD2 and PGE2) increased rapidly from day 1, while the concentrations of some anti-inflammatory lipid mediators (e.g. 14,15 EpETrE) decreased after DFP intoxication but recovered by day 14 post-exposure. The lipidomics results suggest two potential treatment targets: blocking the formation of prostaglandins by inhibiting COX and stabilizing the anti-inflammatory lipid mediators containing epoxides by inhibiting the enzyme soluble epoxide hydrolase (sEH).