Galantamine counteracts development of learning impairment in guinea pigs exposed to the organophosphorus poison soman: Clinical significance

Abstract

Galantamine, a drug used to treat Alzheimer's disease, protects guinea pigs against the acute toxicity and lethality of organophosphorus (OP) compounds, including soman. Here, we tested the hypothesis that a single exposure of guinea pigs to 1xLD50 soman triggers cognitive impairments that can be counteracted by galantamine. Thus, animals were injected intramuscularly with saline (0.5 ml/kg) or galantamine (8 mg/kg) and 30 min later injected subcutaneously with soman (26.3 μg/kg) or saline. Cognitive performance was analyzed in the Morris water maze (MWM) four days or three months after the soman challenge. Fifty percent of the saline-injected animals that were challenged with soman survived with mild-to-moderate signs of acute toxicity that subsided within a few hours. These animals showed no learning impairment and no memory retention deficit, when training in the MWM started four days post-soman challenge. In contrast, animals presented significant learning impairment when testing started three months post-challenge. Though the magnitude of the impairment correlated with the severity of the acute toxicity, animals that presented no or only mild signs of toxicity were also learning impaired. All guinea pigs that were treated with galantamine survived the soman challenge with no signs of acute toxicity and learned the MWM task as control animals, regardless of when testing began. Galantamine also prevented memory extinction in both saline- and soman-challenged animals. In conclusion, learning impairment develops months after a single exposure to 1xLD50 soman, and galantamine prevents both the acute toxicity and the delayed cognitive deficits triggered by this OP poison.

Introduction

Organophosphorus (OP) compounds, including pesticides and the nerve agents soman, sarin and VX, are among the most toxic man-made chemicals. Although different OPs interact with specific targets in the peripheral and central nervous systems (Albuquerque et al., 1985), their acute toxicity is characterized by overstimulation followed by desensitization of cholinergic muscarinic and nicotinic receptors that results in part from the irreversible inhibition of acetylcholinesterase (AChE) – the enzyme that hydrolyzes acetylcholine (Newmark, 2007).

Some of the nerve agents have been used with catastrophic results in wars and terrorist attacks (Coupland and Leins, 2005, Romano and King, 2001). The 1995 terrorist attack with sarin in the Tokyo subway is the largest documented exposure of a civilian population to a nerve gas. Approximately 95% of the victims who were admitted to hospitals and diagnosed as moderately or severely intoxicated were treated intravenously with atropine to block the muscarinic receptors and pralidoxime to reactivate OP-inhibited AChE; diazepam was used as needed to control the convulsions (Okumura et al., 1996). Extended follow-up studies of six months to ten years reported an increased incidence of post-traumatic stress disorder (Ohtani et al., 2004) and chronic memory decline (Hood, 2001, Nishiwaki et al., 2001) among victims of the sarin attack, suggesting that the approved treatments of OP poisoning did not effectively prevent the delayed development of neurological disorders.

Studies from multiple laboratories have successfully identified neurobehavioral deficits in rats, guinea pigs, and non-human primates following a single exposure to nerve agents. For instance, following low-level inhalation exposure to sarin, rats showed significant impairment in spatial discrimination in the Y-Maze (Kassa et al., 2002). In that study the levels of sarin were sufficiently low to trigger no or only mild signs of cholinergic hyperstimulation. Other studies reported that rats and mice that developed severe signs of acute toxicity, including convulsions, following a single subcutaneous (sc) exposure to 1–1.2xLD50 soman presented acute and delayed cognitive impairments in the Morris water maze (MWM) (Filliat et al., 1999, Filliat et al., 2007, Raveh et al., 2002). Of particular interest is a report that in asymptomatic, soman-challenged mice cognitive deficits could be detected at three months, but not one month following the challenge (Filliat et al., 2007).

Reports that the intensity and duration of convulsions in rodents exposed to soman correlate with the magnitude of neuropathology scores and behavioral deficits (McDonough and Shih, 1997, Raveh et al., 2002) suggested that early management of soman-induced convulsions would be sufficient to reduce the neuropathology and the accompanying cognitive impairments. However, neurodegeneration and the resulting cognitive deficits observed in soman-intoxicated rodents can be significantly reduced by therapeutic interventions that, although unable to control the seizures, effectively decrease glutamate excitotoxicity (Filliat et al., 1999). Identifying an antidote capable of counteracting the delayed neurotoxic effects of an exposure to nerve agents is crucial for management of a population exposed to these agents either during military operations or in the event of a terrorist attack.

It is well accepted that guinea pigs are the best non-primate model for predicting the effectiveness of antidotal therapies for OP poisoning in humans (Maxwell et al., 1987). We have demonstrated that galantamine, a drug approved for treatment of Alzheimer's disease (Corey-Bloom, 2003), effectively counteracts the lethality and the acute toxicity of OPs in guinea pigs (Albuquerque et al., 2006). Functional analyses of synaptic transmission and plasticity, histological evaluation of neuronal viability, and magnetic resonance imaging (MRI) analysis of the structural integrity of the brains of nerve agent-challenged guinea pigs subjected to different treatments provided additional evidence that galantamine is an effective and safe antidote against the acute toxicity induced by OPs (Alexandrova et al., 2010, Alkondon et al., 2009, Gullapalli et al., 2010).

The present study was designed to test the hypothesis that a single exposure of guinea pigs to soman triggers cognitive deficits that can be prevented by galantamine. To test this hypothesis, guinea pigs were tested in the MWM at four days or three months after their challenge with 1xLD50 soman and/or treatment with galantamine. Earlier studies have reported that AChE activity is quickly inhibited in guinea pigs injected with 1xLD50 soman (Lintern et al., 1998, Shih et al., 2005). Recovery of the enzyme activity is very slow, and seven days following the soman challenge AChE activity remains significantly inhibited in some brain regions of guinea pigs (Lintern et al., 1998). Thus, studying the guinea pigs four days and three months after the initial soman challenge is essential to delineate the contribution of AChE inhibition to behavioral deficits induced by the nerve agent. The MWM, which was originally developed to assess spatial learning in rats (Morris et al., 1986), has proven to be a valuable task to assess spatial behavior, learning, and memory processes in guinea pigs (Byrnes et al., 2004, de Groot et al., 2001, Dringenberg et al., 2001, Filliat et al., 2002, Lewejohann et al., 2010). The results presented here strongly support the hypothesis and demonstrate that galantamine is a highly effective medical countermeasure to prevent the delayed learning impairment that develops as a result of an acute exposure to soman.