Elsevier

Epilepsy Research

Volume 101, Issue 3, September 2012, Pages 268-276
Epilepsy Research

Characterization of status epilepticus induced by two organophosphates in rats

https://doi.org/10.1016/j.eplepsyres.2012.04.014Get rights and content

Summary

Organophosphates (OPs) inhibit the enzyme cholinesterase and cause accumulation of acetylcholine, and are known to cause seizures and status epilepticus (SE) in humans. The animal models of SE caused by organophosphate analogs of insecticides are not well characterized. SE caused by OPs paraoxon and diisopropyl fluorophosphate (DFP) in rats was characterized by electroencephalogram (EEG), behavioral observations and response to treatment with the benzodiazepine diazepam administered at various stages of SE. A method for SE induction using intrahippocampal infusion of paraoxon was also tested. Infusion of 200 nmol paraoxon into the hippocampus caused electrographic seizures in 43/52 (82.7%) animals tested; and of these animals, 14/43 (30%) had self-sustaining seizures that lasted 4–18 h after the end of paraoxon infusion. SE was also induced by peripheral subcutaneous injection of diisopropyl fluorophosphate (DFP, 1.25 mg/kg) or paraoxon (1.00 mg/kg) to rats pretreated with atropine (2 mg/kg) and 2-pralidoxime (2-PAM, 50 mg/kg) 30 min prior to OP injection. SE occurred in 78% paraoxon-treated animals and in 79% of DFP-treated animals. Diazepam (10 mg/kg) was administered 10 min and 30 min after the onset of continuous EEG seizures induced by paraoxon and it terminated SE in a majority of animals at both time points. DFP-induced SE was terminated in 60% animals when diazepam was administered 10 min after the onset of continuous EEG seizure activity but diazepam did not terminate SE in any animal when it was administered 30 min after the onset of continuous seizures. These studies demonstrate that both paraoxon and DFP can induce SE in rats but refractoriness to diazepam is a feature of DFP induced SE.

Introduction

Status epilepticus (SE) is a neurological emergency characterized by recurrent or continuous self-sustaining seizures. SE can contribute to morbidity, sustained neuronal injury and contribute to mortality (Fujikawa et al., 2000, Fujikawa, 2005). Understanding mechanisms of SE in humans is a particular challenge because patients have to be treated promptly and underlying neurological insult contributes to the pathology. Animal models have been used to understand the pathophysiology of SE and test novel therapies (Chen et al., 2007, Wasterlain et al., 1993). Current animals models of SE are based on chemical stimulation of cholinergic system by muscarinic agonists such as pilocarpine, agonists of glutamate receptors such as kainate and electrical stimulation of limbic structures (Turski et al., 1983, Honchar et al., 1983, Lothman et al., 1981, Lothman et al., 1989, Mazarati et al., 1998b). However, there are few instances of these toxins causing human SE.

Organophosphates (OPs) are potent inhibitors of the enzyme cholinesterase, and several of these cause SE in humans and experimental animals. OPs such as parathion and malathion are used as insecticides and there are numerous reports of SE in humans induced by these insecticides (Garcia et al., 2003, Hoffmann and Papendorf, 2006). Extremely potent OPs, such as soman, sarin and VX are also used as nerve agents for chemical warfare and in civilian terrorist attacks, and they cause SE (McDonough and Shih, 1997, Morita et al., 1995, Nozaki et al., 1995). However, OP nerve agents sarin, soman, VX, etc. are restricted use chemicals and SE induced by these agents has been studied in defense labs. Therefore, acceptable surrogate OP agents must be used for civilian research to understand the mechanisms, pathophysiology and treatment of OP induced SE.

Several organophosphates are available for civilian use including diisopropyl fluorophosphate (DFP), paraoxon, chlorfenvinphos or dichlorvos. Chlorfenvinphos does not appear to cause seizures, and dichlorvos primarily causes fatalities by central respiratory depression (Bird et al., 2003, Gralewicz et al., 1989). On the other hand DFP and paraoxon models have been have been used to study neuropathology, drug response and calcium homeostasis neuropathology associated with OPs (Deshpande et al., 2010, Harrison et al., 2004, Kadriu et al., 2011, Li et al., 2011, Zaja-Milatovic et al., 2009, Zhu et al., 2010). However, these studies did not characterize the evolution SE with EEG. Thus the time to initiation of seizures, duration of seizures and their EEG characteristics remain unknown. Furthermore, they did not test responsiveness to benzodiazepines, such as diazepam. Benzodiazepines are the mainstay of treatment of seizures and OP induced seizures (Alldredge et al., 2001, Treiman et al., 1998, Treiman, 2007). These drugs fail in 35–45% of cases and better treatments are needed.

We characterized SE induced by paraoxon and DFP by means of EEG and behavior. We also characterized the response to treatment with benzodiazepine diazepam at various stages of SE.

Section snippets

Surgery

All procedures on animals were performed according to a protocol approved by the institutional Animal Care and Use Committee. Adult male Sprague-Dawley rats (Taconic) weighing 175–300 g were housed with food and water ad libitum. The animals were anesthetized with ketamine (50 mg/kg) and xylazine (10 mg/kg) for implantation. For animals undergoing intrahippocampal infusion, a bipolar electrode was implanted in the left ventral hippocampus (AP −5.3, ML −4.9, DV −5.0 to dura; incisor bar −3.3). A

Seizures and SE caused by paraoxon infusion

Infusion of 100 nmol paraoxon into the hippocampus caused electrographic seizures in 2/9 (22.2%) animals tested. None of the animals had seizures lasting beyond the end of infusion and 2 animals displayed intermittent seizures during paraoxon infusion which were not self-sustaining. No change in baseline EEG, and no behavioral seizures occurred in the remaining 7 animals (Fig. 1A).

Infusion of 200 nmol paraoxon into the hippocampus caused electrographic seizures in 43/52 (82.7%) animals tested. In

Discussion

We have characterized SE induced by two different organophosphates, intrahippocampal infusion of paraoxon and peripheral injection of DFP or paraoxon following treatment of 2-PAM and atropine. Peripheral injection of OP agents has a higher incidence of producing self-sustaining seizures, while intrahippocampal infusion has the benefit of not requiring pretreatment with 2-PAM and atropine.

Direct injection of 200 nmol paraoxon into the hippocampus caused self-sustaining seizures without killing

Acknowledgments

The research is supported by the CounterACT Program, National Institutes of Health Office of the Director, and the National Institute Neurological Disorders and Stroke, Grant Numbers NIH-NINDS UO1 NS58204 and RO1 NS040337 and also by the Department of Defense grant PR093963.

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