Inhibition of the prostaglandin EP2 receptor is neuroprotective and accelerates functional recovery in a rat model of organophosphorus induced status epilepticus
Introduction
Organophosphorus pesticides and nerve agents potently inhibit acetylcholinesterase (AChE), leading to the accumulation of acetylcholine. Exposure to high levels of organophosphorus compounds induces status epilepticus (SE, an unremitting seizure lasting longer than 5–30 min, or a series of seizures without intervening regain of consciousness) in humans and rodents, leading to brain injury and long-term cognitive deficits. The current treatments following toxic OP exposure in man such as pralidoxime (2-PAM) and atropine, must be given soon after OP exposure for optimal effectiveness (Jett, 2007). Delay in atropine administration results in decreased heart rate, increased bronchial secretions, lacrimation, and other features of cholinergic stimulation. The administration of an oxime such as 2-PAM helps to remove OP agents from acetylcholinesterase and prevent aging in which the bond between the OP and AChE is strengthened thus destroying the enzyme (Jett, 2007). Diazepam is administered to terminate seizures that may develop following OP exposure (Jett, 2007). The current treatments are not logistically possible within a therapeutic timeframe for most civilian populations exposed to OPs and thus therapies that are effective hours to days after OP exposure are needed to mitigate the ensuing brain injury. One prominent feature of evolving brain injury is an inflammatory response mediated partly by cyclooxygenase-2 (COX-2), a rate limiting enzyme critical for the production of prostaglandins. COX-2 inhibitors can be helpful or harmful in various animal models of brain injury including epilepsy (Rojas et al., 2014). Prolonged treatment with COX-2 inhibitors can lead to deleterious side effects, recalling the problems with Vioxx (Rojas et al., 2014). Prostanoid receptors that bind prostaglandins are the main downstream effectors of COX-2 and offer alternative, more selective targets for drug development and disease modification. The prostaglandin EP2 receptor plays an important role in neuroinflammatory conditions (Andreasson, 2010, Bilak et al., 2004, Jiang et al., 2012, Jiang et al., 2013, Johansson et al., 2013, Liang et al., 2005, Liang et al., 2008, Liu et al., 2005, McCullough et al., 2004, Taniguchi et al., 2011). Recent studies demonstrated that inhibition of the EP2 receptor with a selective small molecule antagonist (TG6-10-1) affords neuroprotection after pilocarpine induced status epilepticus in mice, associated with reduced inflammation and accelerated functional recovery (Jiang et al., 2013). Here, we tested the hypothesis that EP2 receptor inhibition by TG6-10-1 reduces DFP induced neuropathologies. We ask the questions: What is the time course of up-regulation of COX-2, brain inflammation and neurodegeneration after DFP? Does EP2 inhibition by a selective small molecule antagonist oppose such neuropathologies, and what is the optimal timing of antagonist administration? To address these questions we optimized an adult rat model of status epilepticus following DFP exposure. Prior to or following DFP exposure rats were injected with either the EP2 receptor antagonist TG6-10-1 or its vehicle. The rats were allowed to recover and were examined for seizure associated neuropathologies within one week of the initial insult. The results support further investigation into therapeutic modalities for the inhibition of EP2 following exposure to organophosphorus nerve agents or pesticides.
Section snippets
Organophosphorus-induced status epilepticus (SE)
All procedures and experiments conformed to the guidelines of the Animal Care and Use Committee of Emory University. Adult male Sprague–Dawley rats (200–220 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 organophosphorus exposure the rats were weighed, placed individually into a
DFP-induced status epilepticus and pharmacokinetics of TG6-10-1 in rats
Experiments were performed to adapt a rat organophosphorus seizure model to investigate the importance of the EP2 receptor in seizure induced neuropathologies. Adult male Sprague–Dawley rats were injected subcutaneously with pyridostigmine bromide and atropine methylbromide followed 10 min later by a single dose of DFP (9.5 mg/kg, ip) according to the protocol in Fig. 1A. This dose of DFP was optimized to effectively obtain status epilepticus in adult male rats. DFP administration has been
Discussion
We optimized a model of organophosphorus exposure involving status epilepticus in adult male rats and investigated the role of the EP2 receptor in the associated neuropathologies. In the rat DFP model of status epilepticus, TG6-10-1 does not act as a conventional acute anticonvulsant like benzodiazepines, as judged by latency to enter status, temporal evolution of seizure behavior, and proportion of rats that enter status epilepticus. However, multiple doses of TG6-10-1 beginning 80–150 min
Conclusion
DFP causes a rapid induction of cyclooxygenase-2 in hippocampal neurons, which is expected to result in rapid synthesis of PGE2 and consequent activation of prostaglandin receptors on neurons and glia, including EP2. The EP2 antagonist, TG6-10-1, reduced morbidity in rats exposed to DFP, as manifested by reduced neuroinflammation, neurodegeneration and microgliosis, and accelerated weight regain within 4 days after DFP exposure. One somewhat trivial explanation for the beneficial effect of
Acknowledgments
This work is supported by NIH UO1 NS058158-08 (RD), T32 DA15040 (AR) and P30 NS055077.
Participated in research design: Rojas and Dingledine.
Conducted experiments: Rojas, Gueorguieva and Lelutiu.
Performed data analysis: Rojas, Gueorguieva, Lelutiu and Dingledine.
Wrote or contributed to the writing of the manuscript: Rojas and Dingledine.
Synthesized TG6-10-1 and participated in design of pharmacokinetics: Ganesh.
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