Paraoxon (POX) is an active metabolite of organophosphate (OP) pesticide parathion that has been weaponized and used against civilian populations. Exposure to POX produces high mortality. OP poisoning is often associated with chronic neurological disorders. In this study, we optimize a rat survival model of lethal POX exposures in order to mimic both acute and long-term effects of POX intoxication. Male Sprague-Dawley rats injected with POX (4 mg/kg, ice-cold PBS, s.c.) produced a rapid cholinergic crisis that evolved into status epilepticus (SE) and death within 6–8 min. The EEG profile for POX induced SE was characterized and showed clinical and electrographic seizures with 7–10 Hz spike activity. Treatment of 100% lethal POX intoxication with an optimized three drug regimen (atropine, 2 mg/kg, i.p., 2-PAM, 25 mg/kg, i.m. and diazepam, 5 mg/kg, i.p.) promptly stopped SE and reduced acute mortality to 12% and chronic mortality to 18%. This model is ideally suited to test effective countermeasures against lethal POX exposure. Animals that survived the POX SE manifested prolonged elevations in hippocampal [Ca2+]i (Ca2+ plateau) and significant multifocal neuronal injury. POX SE induced Ca2+ plateau had its origin in Ca2+ release from intracellular Ca2+ stores since inhibition of ryanodine/ IP3 receptor lowered elevated Ca2+ levels post SE. POX SE induced neuronal injury and alterations in Ca2+ dynamics may underlie some of the long term morbidity associated with OP toxicity.

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.

Organophosphate (OP) compounds, including paraoxon (POX), are similar to nerve agents such as sarin. There is a growing concern that OP agents could be weaponized to cause mass civilian causalities. We have developed a rodent survival model of POX toxicity that is being used to evaluate chronic morbidity and to screen for medical countermeasures against severe OP exposure. It is well known that the survivors of nerve gas and chronic OP exposure exhibit neurobehavioral deficits such as mood changes, depression, and memory impairments. In this study we investigated whether animals surviving severe POX exposure exhibited long-term neurological impairments. POX exposure produced overt signs of cholinergic toxicity. Rats were rescued using an optimized atropine, 2-PAM and diazepam therapy. Surviving rats were studied using established behavioral assays for identifying symptoms of depression and memory impairment 3-months after POX exposure. In the forced swim test, POX rats exhibited increased immobility time indicative of a despair-like state. In the sucrose preference test, POX rats consumed significantly less sucrose water indicating anhedonia-like condition. POX rats also displayed increased anxiety as characterized by significantly lower performance in the open arm of the elevated plus maze. Further, when tested with a novel object recognition paradigm, POX rats exhibited a negative discrimination ratio indicative of impaired recognition memory. The results indicate that this model of survival from severe POX exposure can be employed to study some of the molecular bases for OP-induced chronic behavioral and cognitive comorbidities and develop therapies for their treatment.

Gulf War Illness (GWI) is a chronic multi-symptom disorder afflicting the veterans of the First Gulf War, and includes neurological symptoms characterized by depression and memory deficits. Chronic exposure to organophosphates (OPs) is considered a leading cause for GWI, yet its pathobiology is not fully understood. We recently observed chronic elevations in neuronal Ca 2+ levels ([Ca 2+ ] i ) in an OP-diisopropyl fluorophosphate (DFP)-based rat model for GWI. This study was aimed at identifying mechanisms underlying elevated [Ca 2+ ] i in this DFP model and investigating whether their therapeutic targeting could improve GWI-like neurological morbidities. Male Sprague-Dawley rats (9 weeks) were exposed to DFP (0.5 mg/kg, s.c . , 1×-daily for 5 days) and at 3 months postDFP exposure, behavior was assessed and rats were euthanized for protein estimations and ratiometric Fura-2 [Ca 2+ ] i estimations in acutely dissociated hippocampal neurons. In DFP rats, a sustained elevation in intracellular Ca 2+ levels occurred, and pharmacological blockade of Ca 2+ -induced Ca 2+ -release mechanisms significantly lowered elevated [Ca 2+ ] i in DFP neurons. Significant reductions in the protein levels of the ryanodine receptor (RyR) stabilizing protein Calstabin2 were also noted. Such a posttranslational modification would render RyR “leaky” resulting in sustained DFP [Ca 2+ ] i elevations. Antagonism of RyR with levetiracetam significantly lower elevated [Ca 2+ ] i in DFP neurons and improved GWI-like behavioral symptoms. Since Ca 2+ is a major second messenger molecule, such chronic increases in its levels could underlie pathological synaptic plasticity that expresses itself as GWI morbidities. Our studies show that treatment with drugs targeted at blocking intracellular Ca 2+ release could be effective therapies for GWI neurological morbidities.

Over 20 yr have elapsed since the end of the First Gulf War, yet approximately one-third of the veterans exhibit Gulf War Illness (GWI) symptoms, particularly depression and memory impairments. Exposure to organophosphate (OP) compounds is implicated for GWI development. The role of calcium (Ca2+) signaling in learning, memory, and mood is well established and disruptions in Ca2+ homeostasis are observed in many neurological disorders. However, the status of Ca2+ homeostasis in the development of GWI behavioral impairments is not known. Male Sprague-Dawley rats were exposed to OP agent diisopropyl fluorophosphate (DFP; 0.5 mg/kg, s.c. 5 days), and at 6 mo post-DFP exposure, rats were subjected to behavioral assays for the determination of GWI neurological morbidities. Fura-2AM loaded acutely isolated hippocampal neurons were used for [Ca2+]i estimations. We observed chronic depressive symptoms and cognitive deficits in rats exposed to repeated low-dose DFP. The GWI rats also manifested elevations in hippocampal [Ca2+]i along with a significant increase in the number of neurons displaying these elevations. As Ca2+ is a major second-messenger molecule, such sustained increases in its levels could activate multiple signaling cascades and alter gene expression of proteins involved in synaptic plasticity and possibly underlie the neuronal injury and chronic morbidities in GWI.