Post-exposure administration of diazepam combined with soluble epoxide hydrolase inhibition stops seizures and modulates neuroinflammation in a murine model of acute TETS intoxication

https://doi.org/10.1016/j.taap.2014.10.001Get rights and content

Highlights

  • ā€¢

    Acute TETS intoxication causes delayed and persistent neuroinflammation.

  • ā€¢

    Diazepam given post-TETS prevents lethal tonic seizures but not neuroinflammation.

  • ā€¢

    A soluble epoxide hydrolase inhibitor alters TETS-induced neuroinflammation.

  • ā€¢

    Acute TETS intoxication may be more effectively treated by a combinatorial therapy.

Abstract

Tetramethylenedisulfotetramine (TETS) is a potent convulsant poison for which there is currently no approved antidote. The convulsant action of TETS is thought to be mediated by inhibition of type A gamma-aminobutyric acid receptor (GABAAR) function. We, therefore, investigated the effects of post-exposure administration of diazepam, a GABAAR positive allosteric modulator, on seizure activity, death and neuroinflammation in adult male Swiss mice injected with a lethal dose of TETS (0.15Ā mg/kg, ip). Administration of a high dose of diazepam (5Ā mg/kg, ip) immediately following the second clonic seizure (approximately 20Ā min post-TETS injection) effectively prevented progression to tonic seizures and death. However, this treatment did not prevent persistent reactive astrogliosis and microglial activation, as determined by GFAP and Iba-1 immunoreactivity and microglial cell morphology. Inhibition of soluble epoxide hydrolase (sEH) has been shown to exert potent anti-inflammatory effects and to increase survival in mice intoxicated with other GABAAR antagonists. The sEH inhibitor TUPS (1Ā mg/kg, ip) administered immediately after the second clonic seizure did not protect TETS-intoxicated animals from tonic seizures or death. Combined administration of diazepam (5Ā mg/kg, ip) and TUPS (1Ā mg/kg, ip, starting 1Ā h after diazepam and repeated every 24Ā h) prevented TETS-induced lethality and influenced signs of neuroinflammation in some brain regions. Significantly decreased microglial activation and enhanced reactive astrogliosis were observed in the hippocampus, with no changes in the cortex. Combining an agent that targets specific anti-inflammatory mechanisms with a traditional antiseizure drug may enhance treatment outcome in TETS intoxication.

Introduction

Tetramethylenedisulfotetramine (TETS; tetramine; TMDT) is a heteroadamantane compound originally synthesized as a condensation product of sulfamide and formaldehyde (Wood and Battye, 1933). It was subsequently found to be a highly toxic convulsant poison and was widely used as a rodenticide until banned in China in 1984 (Whitlow et al., 2005). TETS is, however, still produced illicitly and has been implicated in the accidental or intentional poisoning of as many as 14,000 individuals in China between 1991 and 2010, as well as more than 50 human poisonings in Western countries since 2002 (Li et al., 2012, Zhang et al., 2011). TETS meets the criteria for inclusion in the World Health Organization's list of ā€œextremely hazardous pesticidesā€ (Whitlow et al., 2005) and is considered a high priority chemical threat agent by the National Institutes of Health (Jett and Yeung, 2010).

Based on clinical reports, exposure to low doses of TETS causes dizziness and headaches while high dose exposures trigger acute seizures and status epilepticus (SE) and can lead to death. Exposure to sublethal doses of TETS can cause persistent neurologic sequelae in survivors, including epileptogenesis that lasts for years following the initial exposure to TETS (Bai et al., 2005, Barrueto et al., 2003, Li et al., 2012, Whitlow et al., 2005). Recently, our group characterized rodent models of acute TETS intoxication, which confirmed the high potency of TETS as a convulsant in mice and rats (Zolkowska et al., 2012). Our findings were confirmed in a study by Shakarjian et al. (2012), which demonstrated that treatment with high dose diazepam (5Ā mg/kg, ip), a GABAAR positive allosteric modulator, protected adult male C57BL/6 mice from further motor seizures and increased survival at 1Ā h following TETS exposure. This is consistent with experimental evidence indicating that TETS is a noncompetitive antagonist of the GABAAR [reviewed by Zhao et al., 2014]. However, this high dose of diazepam did not stop electrographic seizures, and the animals died several hours after treatment (Shakarjian et al., 2012). These observations are consistent with case reports indicating that benzodiazepine therapy is not always efficacious in treating humans poisoned with TETS (Lu et al., 2008, Poon et al., 2005). Collectively, these data suggest that benzodiazepines alone are insufficient to protect against acute TETS poisoning.

Emerging evidence implicates neuroinflammation, specifically reactive astrogliosis and microglial activation, in the pathogenesis of spontaneous recurrent seizures following SE (Clasadonte et al., 2013, Devinsky et al., 2013, Hunt et al., 2013, Pineda et al., 2013, Rossi et al., 2013). Interestingly, we found in our earlier study that exposure to sublethal convulsant doses of TETS significantly increased reactive astrogliosis and microglial activation in the cortex and hippocampus of Swiss mice several days after TETS exposure (Zolkowska et al., 2012). This suggests the possibility that combinatorial therapy with anticonvulsant and anti-inflammatory agents may better protect the brain following acute TETS intoxication. Epoxygenated fatty acids are produced in the brain and have been shown to be potent anti-inflammatory compounds (Iliff and Alkayed, 2009, Inceoglu et al., 2007, Wang et al., 2013). The beneficial effect of these naturally occurring epoxy fatty acids is severely limited by their rapid metabolism via soluble epoxide hydrolase (sEH) (Imig and Hammock, 2009, Newman et al., 2005). Pharmacologic inhibition of sEH stabilizes these bioactive molecules (Spector and Norris, 2007) and small molecule sEH inhibitors (sEHI) have been shown to have significant anti-inflammatory and neuroprotective activity in animal models of pain, inflammation and ischemic stroke (Iliff and Alkayed, 2009, Inceoglu et al., 2008, Wang et al., 2013). In addition, we recently demonstrated that sEHI pretreatment delays the onset of clonic seizures and prevents lethality in NIH Swiss mice exposed to a lethal dose of other GABAA receptor antagonists, including picrotoxin and pentylenetetrazole (PTZ) (Inceoglu et al., 2013).

The goal of this study was to characterize the effects of high dose diazepam administered alone or in combination with the sEHI TUPS on TETS-induced seizures, death and neuroinflammation. Neuroinflammation was assessed by quantifying GFAP and Iba-1 immunoreactivity as biomarkers of reactive astrogliosis and microglial activation, respectively, in the cortex and hippocampus, which are key brain regions implicated in the generation of seizures (Hunt et al., 2013). Our findings support the argument that a combinatorial approach targeting different pathogenic mechanisms of acute TETS intoxication may be more efficacious than using benzodiazepines alone to protect the brain following acute TETS intoxication.

Section snippets

Chemicals

Paraformaldehyde, sulfamide, hydrochloric acid, acetone, and hexane were obtained from Thermo Fisher Scientific (Waltham, MA). All chemicals were of the highest purity available. TETS was synthesized as previously described (Zolkowska et al., 2012). A final recrystallization step was performed to ensure no water remained in the crystals and characterization of the final product by gas chromatographyā€“mass spectrometry supported a purity of >Ā 98%. USP grade diazepam manufactured by Hospira (in 40%

Post-exposure administration of diazepam prevents lethality in TETS-intoxicated mice

Consistent with previous observations (Zolkowska et al., 2012), mice dosed with 0.15Ā mg/kg TETS displayed a brief period of hyperactivity followed by a brief period of quiescence, Straub tail, twitches, imbalance, clonic seizures, and subsequently, tonic seizures and death. The majority of animals (>Ā 80%) exhibited a period of wild running immediately prior to the onset of tonus. Mice intoxicated with this lethal dose of TETS exhibited a stereotypic pattern of seizure behavior consisting of two

Pharmacologic inhibition of soluble epoxide hydrolase (sEH) differentially modulates TETS-induced reactive astrogliosis and microglial activation

Since inhibition of sEH has been shown to exert potent anti-inflammatory action (Iliff and Alkayed, 2009, Inceoglu et al., 2008, Wang et al., 2013), we quantified the effects of administering TUPS, a small molecule inhibitor of sEH, on TETS-induced seizures and neuroinflammation. Initial experiments focused on determining whether pharmacologic inhibition of sEH was sufficient to protect against TETS-induced tonus and death in the absence of diazepam. Pretreatment with TUPS (1Ā mg/kg, ip) 1Ā h prior

Discussion

The present study supports and extends our previous characterization of TETS-induced seizures in adult male NIH Swiss mice (Zolkowska et al., 2012). We now show that the behavioral seizures are accompanied by electrographic seizure activity, and that ā€œrescue therapyā€ with a high dose of diazepam (5Ā mg/kg, ip) administered upon cessation of the second clonic seizure (approximately 20Ā min post-TETS) effectively stops both behavioral and electrographic seizures for at least 1Ā h. This extends the

Funding information

This research was supported by the CounterACT Program, the National Institutes of Health Office of the Director, and the National Institute of Neurological Disorders and Stroke [U54 NS079202]. The sponsor was involved in the study design but not in the collection, analysis, and interpretation of data, in the writing of the report or in the decision to submit the paper for publication. Support was also provided by training grants from the National Institute of General Medical Sciences [T32

Conflict of interest statement

The authors declare no conflict of interest.

Acknowledgments

We thank Mark McCoy (in the Hammock laboratory at UC Davis at the time of the synthesis, now at the University of Alaska) for the synthesis of TETS, Paul Jones (in the Hammock laboratory at UC Davis at the time of the synthesis, now at International Flavors and Fragrances, Union Beach, NJ) for the synthesis of the sEHI TUPs and Danielle Harvey (UC Davis) for assistance with statistical analyses and review of early versions of the manuscript.

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