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Neurobiology of Disease Jan 2020Current medical countermeasures for organophosphate (OP)-induced status epilepticus (SE) are not effective in preventing long-term morbidity and there is an urgent need... (Review)
Review
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.
Topics: Animals; Behavior, Animal; Brain; Disease Models, Animal; Inflammation; Isoflurophate; Male; Neurotoxicity Syndromes; Organophosphate Poisoning; Oxidative Stress; Rats; Rats, Sprague-Dawley; Status Epilepticus
PubMed: 30905768
DOI: 10.1016/j.nbd.2019.03.019 -
Neurobiology of Disease May 2021Organophosphate pesticides and nerve agents (OPs), are characterized by cholinesterase inhibition. In addition to severe peripheral symptoms, high doses of OPs can lead...
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.
Topics: Animals; Cholinesterase Inhibitors; Isoflurophate; Male; Mice; Neuroglia; Neurotoxicity Syndromes; Phenotype; Status Epilepticus
PubMed: 33529768
DOI: 10.1016/j.nbd.2021.105276 -
Neurotoxicology Jul 2022Exposure to high levels of a cholinesterase inhibiting organophosphorus (OP) agent often results in seizures that progress to status epilepticus (SE). Survivors of...
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-1β 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.
Topics: Animals; Brain; Disease Models, Animal; Isoflurophate; Organophosphate Poisoning; Organophosphates; Rats; Rats, Sprague-Dawley; Status Epilepticus
PubMed: 35500718
DOI: 10.1016/j.neuro.2022.04.010 -
International Journal of Molecular... Jul 2022Organophosphate (OP) compounds include highly toxic chemicals widely used both as pesticides and as warfare nerve agents. Existing countermeasures are lifesaving, but do...
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 , , , and to a lesser extent, immuno-modulatory cytokine , 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.
Topics: Acetylcholinesterase; Animals; Antidotes; Brain; Cholinesterase Inhibitors; Cytokines; Humans; Isoflurophate; Microglia; Neuroinflammatory Diseases; Organophosphate Poisoning; Organophosphates; Rats; Rats, Sprague-Dawley; Zebrafish
PubMed: 35897817
DOI: 10.3390/ijms23158240 -
Annals of the New York Academy of... Aug 2016Exposures to seizure-inducing chemical threat agents are a major public health concern. Of particular need is improved treatment to terminate convulsions and to prevent... (Review)
Review
Exposures to seizure-inducing chemical threat agents are a major public health concern. Of particular need is improved treatment to terminate convulsions and to prevent the long-term neurological sequelae in survivors. We are studying the organophosphorus cholinesterase inhibitor diisopropyl fluorophosphate (DFP) and the GABA receptor inhibitor tetramethylenedisulfotetramine (TETS), which arguably encompass the mechanistic spectrum of seizure-inducing chemical threats, with the goal of identifying therapeutic approaches with broad-spectrum efficacy. Research efforts have focused on developing translational models and translational diagnostic approaches, including (1) in vivo models of DFP- and TETS-induced seizures for studying neuropathological mechanisms and identifying treatment approaches; (2) in vivo imaging modalities for noninvasive longitudinal monitoring of neurological damage and response to therapeutic candidates; and (3) higher-throughput in vitro platforms for rapid screening of compounds to identify potential antiseizure and neuroprotective agents, as well as mechanistically relevant novel drug targets. This review summarizes our progress toward realizing these goals and discusses best practices and mechanistic insights derived from our modeling efforts.
Topics: Animals; Anticonvulsants; Brain; Bridged-Ring Compounds; Cholinesterase Inhibitors; Disease Models, Animal; Humans; Isoflurophate; Neuroprotective Agents; Organophosphate Poisoning; Seizures; Treatment Outcome
PubMed: 27467073
DOI: 10.1111/nyas.13137 -
Neurobiology of Disease Jan 2020This review describes an adult rat model of status epilepticus (SE) induced by diisopropyl fluorophosphate (DFP), and the beneficial outcomes of transient inhibition of... (Review)
Review
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-E 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.
Topics: Animals; Cholinesterase Inhibitors; Disease Models, Animal; Indoles; Isoflurophate; Organophosphate Poisoning; Rats; Receptors, Prostaglandin E, EP2 Subtype; Status Epilepticus
PubMed: 30818067
DOI: 10.1016/j.nbd.2019.02.010 -
Neurobiology of Disease Jan 2020Chemical 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...
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.
Topics: Amidines; Animals; Benzylamines; Brain; Disease Models, Animal; Enzyme Inhibitors; Isoflurophate; Male; Nerve Agents; Nerve Degeneration; Neuroprotective Agents; Neurotoxicity Syndromes; Nitric Oxide Synthase Type II; Rats; Rats, Sprague-Dawley
PubMed: 30940499
DOI: 10.1016/j.nbd.2019.03.031 -
The Journal of Pharmacology and... Jan 2024Children are much more susceptible to the neurotoxic effects of organophosphate (OP) pesticides and nerve agents than adults. OP poisoning in children leads to acute...
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.
Topics: Humans; Child; Rats; Animals; Organophosphates; Neurosteroids; Neuroinflammatory Diseases; Organophosphorus Compounds; Brain; Isoflurophate; Fluorides; Phosphates
PubMed: 37863488
DOI: 10.1124/jpet.123.001763 -
Molecules (Basel, Switzerland) Oct 2011Organophosphorus (OP) compounds are a diverse chemical group that includes nerve agents and pesticides. They share a common chemical signature that facilitates their...
Organophosphorus (OP) compounds are a diverse chemical group that includes nerve agents and pesticides. They share a common chemical signature that facilitates their binding and adduction of acetylcholinesterase (AChE) within nerve synapses to induce cholinergic toxicity. However, this group diversity results in non-uniform binding and inactivation of other secondary protein targets, some of which may be adducted and protein activity influenced, even when only a relatively minor portion of tissue AChE is inhibited. The determination of individual OP protein binding targets has been hampered by the sensitivity of methods of detection and quantification of protein-pesticide adducts. We have overcome this limitation by the employment of a microchannel plate (MCP) autoradiographic detector to monitor a radiolabelled OP tracer compound. We preincubated rat thymus tissue in vitro with the OP pesticides, azamethiphos-oxon, chlorfenvinphos-oxon, chlorpyrifos-oxon, diazinon-oxon, and malaoxon, and then subsequently radiolabelled the free OP binding sites remaining with 3H-diisopropylfluorophosphate (3H-DFP). Proteins adducted by OP pesticides were detected as a reduction in 3H-DFP radiolabelling after protein separation by one dimensional polyacrylamide gel electrophoresis and quantitative digital autoradiography using the MCP imager. Thymus tissue proteins of molecular weights -28 kDa, 59 kDa, 66 kDa, and 82 kDa displayed responsiveness to adduction by this panel of pesticides. The 59 kDa protein target (previously putatively identified as carboxylesterase I) was only significantly adducted by chlorfenvinphos-oxon (p < 0.001), chlorpyrifos-oxon (p < 0.0001), and diazinon-oxon (p < 0.01), the 66 kDa protein target (previously identified as serum albumin) similarly only adducted by the same three pesticides (p < 0.0001), (p < 0.001), and (p < 0.01), and the 82 kDa protein target (previously identified as acyl peptide hydrolase) only adducted by chlorpyrifos-oxon (p < 0.0001) and diazinon-oxon (p < 0.001), when the average values of tissue AChE inhibition were 30%, 35%, and 32% respectively. The -28 kDa protein target was shown to be heterogeneous in nature and was resolved to reveal nineteen 3H-DFP radiolabelled protein spots by two dimensional polyacrylamide gel electrophoresis and MCP autoradiography. Some of these 3H-DFP proteins spots were responsive to adduction by preincubation with chlorfenvinphos-oxon. In addition, we exploited the useful spatial resolution of the MCP imager (-70 mm) to determine pesticide micolocalisation in vivo, after animal dosing and autoradiography of brain tissue sections. Collectively, MCP autoradiographic imaging provided a means to detect targets of OP pesticides, quantify their sensitivity of adduction relative to tissue AChE inhibition, and highlighted that these common pesticides exhibit specific binding character to protein targets, and therefore their toxicity will need to be evaluated on an individual compound basis. In addition, MCP autoradiography afforded a useful method of visualisation of the localisation of a small radiolabelled tracer within brain tissue.
Topics: Animals; Autoradiography; Binding Sites; Isoflurophate; Isotope Labeling; Mice; Mice, Inbred C57BL; Neurotoxicity Syndromes; Organophosphorus Compounds; Pesticides; Proteomics; Rats; Thymus Gland; Tritium
PubMed: 21989313
DOI: 10.3390/molecules16108535 -
Neurochemical Research Oct 2015Diisopropylfluorophosphate (DFP) is an irreversible inhibitor of acetylcholine esterase and a surrogate of the organophosphorus (OP) nerve agent sarin. The neurotoxicity...
Diisopropylfluorophosphate (DFP) is an irreversible inhibitor of acetylcholine esterase and a surrogate of the organophosphorus (OP) nerve agent sarin. The neurotoxicity of DFP was assessed as a reduction of population spike (PS) area elicited by synaptic stimulation in acute hippocampal slices. Two classical antidotes, atropine, and pralidoxime, and two novel antidotes, 4R-cembranotriene-diol (4R) and a caspase nine inhibitor, were tested. Atropine, pralidoxime, and 4R significantly protected when applied 30 min after DFP. The caspase inhibitor was neuroprotective when applied 5-10 min before or after DFP, suggesting that early synaptic apoptosis is responsible for the loss of PSs. It is likely that apoptosis starts at the synapses and, if antidotes are not applied, descends to the cell bodies, causing death. The acute slice is a reliable tool for mechanistic studies, and the assessment of neurotoxicity and neuroprotection with PS areas is, in general, pharmacologically congruent with in vivo results and predicts the effect of drugs in vivo. 4R was first found to be neuroprotective in slices and later we demonstrated that 4R is neuroprotective in vivo. The mechanism of neurotoxicity of OPs is not well understood, and there is a need for novel antidotes that could be discovered using acute slices.
Topics: Acetylcholinesterase; Animals; Atropine; Cholinesterase Inhibitors; Hippocampus; Isoflurophate; Male; Neuroprotection; Neuroprotective Agents; Pralidoxime Compounds; Rats, Sprague-Dawley; Synapses
PubMed: 26438150
DOI: 10.1007/s11064-015-1729-4