Inhibition of the prostaglandin E2 receptor EP2 prevents status epilepticus-induced deficits in the novel object recognition task in rats
Introduction
Exposure to organophosphorus based nerve agents and pesticides poses a major public health concern. Organophosphorus agents bind and potently inhibit acetylcholinesterase (AChE), leading to an irreversible structural change of the enzyme termed “aging”, accompanied by the rapid accumulation of acetylcholine (Colovic et al., 2013, Costa, 2006), and the consequent clinical presentation of cholinergic symptoms following organophosphorus poisoning. Exposure to high levels of an organophosphorus agent often leads to seizures that can evolve into status epilepticus (SE, an unremitting seizure lasting longer than 5 min, or a series of seizures without intervening regain of consciousness). In the event of an exposure to high levels of organophosphorus agents the response time and treatment measures carried out by first responders is critical to minimize casualties, so it would be desirable to develop treatments that can mitigate long-term consequences. In animal models of organophosphorus agent exposure, survivors exhibit a number of consequences such as weight loss, neuroinflammation, neurodegeneration, long-term cognitive deficits and the development of spontaneous recurrent seizures (SRS) (Binukumar et al., 2011, Chen, 2012, Gilat et al., 2005, Joosen et al., 2009, Pan et al., 2012, Raveh et al., 2003, Reddy and Kuruba, 2013, Rojas et al., 2015, Tattersall, 2009, Tilson et al., 1990). Short-term inhibition of the EP2 receptor following SE in rats and mice induced by DFP or the muscarinic receptor agonist pilocarpine attenuates the consequences of status epilepticus that manifest within hours to days following the initial insult, including neuroinflammation, neuronal injury and breakdown of the blood-brain barrier (Jiang et al., 2013, Jiang et al., 2015, Rojas et al., 2015). Whether EP2 inhibition also ameliorates the long-term consequences of DFP-induced SE such as cognitive deficits has not been investigated and is the focus of the current study. Here we ask the questions: Does DFP-induced SE lead to long term cognitive deficits in rats? And, does EP2 inhibition by TG6-10-1 alter DFP-induced seizure characteristics or improve cognitive function after DFP? To address these questions we used a model of SE in adult rats involving exposure to a high dose of DFP. Following exposure to DFP rats were injected with either the EP2 receptor antagonist TG6-10-1 or its vehicle using two dosing paradigms. The rats were allowed to recover and were examined functionally 4 weeks and 6–12 weeks after the initial insult. The results shown here promote a better understanding of the pathophysiology of organophosphorus poisoning and further support therapies targeting the EP2 receptor to combat neuropathologies following exposure to organophosphorus based agents.
Section snippets
Ethics statement
All procedures concerning animals were approved by Emory University Institutional Animal Care and Use Committee and conformed to NIH guidelines.
Diisopropylfluorophosphate (DFP)-induced status epilepticus (SE)
Adult male Sprague–Dawley rats (200–240 g body weight) were purchased from Charles River Labs (Wilmington, MA, USA) and housed in standard plastic cages (2 rats/cage) in a temperature controlled room (22 ± 2 °C) on a 12 h reverse light–dark cycle. Food and water were provided ad libitum. On the day of DFP exposure the rats were weighed, placed
Pharmacokinetics of TG6-10-1 in rats and DFP-induced SE characteristics
The pharmacokinetic studies were carried out on normal rather than DFP-treated rats for logistical reasons. Administration of TG6-10-1 (10 mg/kg ip) dissolved in 10% DMSO, 50% PEG400 and 40% water in adult male rats revealed a brain to plasma concentration ratio in the range of 0.3–0.4 from 1 to 4 h after injection, and an average plasma concentration of 138 ng/ml (308 nM) at 4 h (Fig. 1B, C and Table 1). The TG6-10-1 exposure profile found here with DMSO, PEG400 and water is similar to that in
Discussion
Status epilepticus induced by nerve agents in animals and humans leads to the development of spontaneous recurrent seizures and long-term cognitive deficits (Chen, 2012, de Araujo Furtado et al., 2012, Joosen et al., 2009, Miyaki et al., 2005, Nishiwaki et al., 2001). In animal studies investigating learning and memory, these long term cognitive deficits are associated with the early neurodegeneration and the neuronal plasticity that occurs in the brain after status epilepticus (Chen, 2012,
Conclusion
Inhibition of the EP2 receptor in rats exposed to DFP blocks long term memory impairment, but does not affect anxiety behaviors. One somewhat trivial explanation for the beneficial effect of TG6-10-1 in the DFP model, that the compound simply aborts status epilepticus, has been ruled out. We conclude that short-term treatment with an EP2 receptor inhibitor beginning ≥90 min after DFP exposure produces long-term cognitive benefits. These studies give insight into therapeutic modalities for the
Author contributions
Participated in research design: Rojas and Dingledine.
Conducted experiments: Rojas, Manji and O'neill.
Performed data analysis: Rojas, Ganesh, Manji, O'neill and Dingledine.
Wrote or contributed to the writing of the manuscript: Rojas and Dingledine.
Designed pharmacokinetics experiments: Rojas, Ganesh and Dingledine.
Synthesized TG6-10-1: Ganesh.
Acknowledgements
This work is supported by NIH UO1 NS058158-08, R01 NS097776 (RD) and T32 DA15040 (AR). We thank Dr. Jason Schroeder of the Emory rodent behavioral core facility for help with light-dark box preference and novel object recognition experiments.
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