Article, Psychiatry

Naltrexone prevents delayed encephalopathy in rats poisoned with the sarin analogue diisopropylflurophosphate

a b s t r a c t

Background: Acute poisoning with organophosphate compounds can cause chronic neuropsychological disabilities not prevented by standard antidotes of atropine and pralidoxime. We determine the efficacy of naltrexone in preventing Delayed encephalopathy after poisoning with the sarin analogue diisofluoropho- sphate (DFP) in rats.

Methods: A randomized controlled experiment was conducted. Rats were randomly assigned to receive a single intraperitoneal (IP) injection of 5 mg/kg DFP (n = 12) or vehicle control (Isopropyl alcohol, n = 5). Rats were observed for cholinesterase toxicity and treated with IP atropine (2 mg/kg) and pralidoxime (25 mg/kg) as needed. After resolution of acute toxicity, rats injected with DFP were again randomized to receive daily injections of naltrexone (5 mg/kg per day) or saline (vehicle control). Control animals also received daily injections of saline. For 4 weeks after acute poisoning, rats underwent neurologic testing with the Morris Water Maze for assessment of spatial learning and reference memory. Comparisons on each test day were made across groups using analysis of variance followed by Fisher’s least significant difference. Comparisons of changes in performance between first and last test day within each group were made using a paired t test. Significance was determined at P b .05.

Results: All rats receiving DFP developed toxicity requiring rescue. Spatial learning was significantly worse in the DFP-only group compared with the naltrexone-treated and control groups at day 10 (P = .0078), day 13 (P = .01), day 24 (P = .034), and day 31 (P = .03). No significant differences in reference memory were detected at any time point.

Conclusion: Naltrexone protected against impairment of spatial learning from acute poisoning with DFP in rats.

(C) 2013

Introduction

Acute poisoning with cholinesterase inhibitors are known to have lasting effects on the brain, both in humans and rats, which are not prevented by treatment with the standard antidotes of atropine and pralidoxime. Emergency physicians need additional antidotes to prevent delayed neurotoxicity characterized by Memory loss, anxiety, and cognitive decline [1,2]. Exposure to sarin from scud missile attacks and demolition of ammunition dumps during the 1991 Gulf War has been suggested to have caused the neurologic disabilities associated with Gulf War Illness [3]. The sarin terrorist attacks on the Tokyo subway system resulted in over 5500 exposures, more than 1000 of which were symptomatic and 12 of which were fatal [4]. Many survivors experienced a decline in memory function that was not prevented by standard antidotal therapy and persisted for more

? Presented at the 2012 Society for Academic Emergency Medicine Meeting, May 2012.

* Corresponding author. Tel.: +1 252 744 2954; fax: +1 252 744 3589.

E-mail address: [email protected] (W.J. Meggs).

than 7 years after the attack [4]. To date, no therapies have been demonstrated to prevent delayed encephalopathy. It has been proposed that this condition may reflect an inflammatory cycle involving the brain, which may be a common mechanism of many neurologic conditions [5]. This suggests that novel anti-inflammatory drugs may be of benefit in symptom-defined illnesses related to a cycle of inflammation. The opioid antagonists naloxone and naltrex- one are neuroprotective against inflammatory mediated neurodegen- eration [6,7] and are, therefore, candidates to prevent neurologic sequela from organophosphate (OP) poisoning.

The American Association of Poison control centers‘ reported 91940 calls (3.3% of all human exposures) related to pesticide exposures in 2010, including exposures to OP agents [8]. This places pesticides as the 10th most common category of substances involved in human exposures. Although the incidence of OP exposure has been declining in North America, it is very significant internationally [9]. Although the true incidence of OP-induced neurologic abnormalities is unknown, the potential significance of these sequelae warrants investigation of novel therapies. Using a Rodent model, we performed a controlled trial of the efficacy of naltrexone to prevent long-term

0735-6757/$ – see front matter (C) 2013 http://dx.doi.org/10.1016/j.ajem.2012.12.003

K.L. Brewer et al. / American Journal of Emergency Medicine 31 (2013) 676679 677

deficits in working memory in rats acutely poisoned with the sarin analogue diisopropylfluorophosphate (DFP).

Methods

Study design

This was a 3-arm randomized controlled study of the ability of naltrexone to prevent long-term deficits in spatial learning and reference memory after an acute poisoning with DFP. All experimen- tal procedures were reviewed and approved by the Institutional Animal Care and Use Committee.

Subjects

Female, Long-Evans rats (200-225 g; Charles River Laboratories, Wilmington, MA) were individually housed with normal 12-hour light/dark cycles with ad lib access to normal rat chow and water.

Acute poisoning with DFP

Rats were randomly assigned to receive either a single intraper- itoneal (IP) injection of DFP (Sigma Aldrich, St Louis MO, n = 12) or vehicle control (isopropyl alcohol, n = 5). Diisopropylfluoropho- sphate was used instead of sarin because it is less volatile and, thereby, less hazardous to investigators. Diisopropylfluorophosphate was dissolved in isopropyl alcohol (20 mg/mL) and injected at a dose of 5 mg/kg. After injection, rats were monitored for signs and symptoms of cholinesterase toxicity, including excessive salivation, lacrimation, muscle fasciculations, or seizures. If toxicity developed, antidotal therapy was provided with IP atropine (2 mg/kg) and pralidoxime (25 mg/kg). Treatment was repeated every 10 minutes until signs of acute toxicity resolved.

Treatment with naltrexone

After the resolution of all signs of acute toxicity (within 1 hour of poisoning), rats injected with DFP were again randomized to receive daily subcutaneous injections of naltrexone (5 mg/kg per day, n = 6) or saline as naltrexone vehicle control (volume matched for weight, n = 6). Nonpoisoned control animals also received daily injections of saline (n = 5). Animals were allowed to survive for 4 weeks post poisoning.

Cognitive outcome measures

Spatial learning and reference memory of all rats was measured over time using the Morris Water Maze [10]. In this procedure, a round swim tank 6 ft in diameter is partially filled with water (Room temperature) and equal quadrants of the tank are denoted with string attached to the edge of the tank above the surface of the water. A clear, round platform (10 cm in diameter) is placed in the water with the top of the platform being 1 cm below the surface of the water. The water is made opaque with white tempura paint so that the platform is not visible from the surface of the water. Before injections, animals were habituated to the testing room for 3 days by being placed in the room with the Morris Water Maze and handled extensively by the person performing the testing. All testing was performed by trained personnel who were blinded to group assignments. Starting at 10 days after injection, animals were tested for spatial learning and reference memory. Testing began at day 10 after poisoning (to allow for resolution of any acute effects of the DFP) and was performed every other week for the 4-week survival period.

Spatial learning

Each animal underwent 4 trials on each day of testing. A trial began with the animal being placed in the water at a predetermined starting

position and ended once the animal found the platform. If the animal did not find the platform within 120 seconds, it was guided there by hand. After 30 seconds on the platform, the animal was placed back in the water at a second start site. This was repeated until the animal had started from each of 4 quadrants in the tank. The latency to find the platform from each starting position was recorded. This protocol was repeated for 4 consecutive days, with the platform in the same position each day.

Reference memory

On the fifth day of the first and last test week, a probe trial was performed in which the platform was removed from the tank and the rat placed in one of the previous start positions. All rats were placed in the same start position on each probe trial. The time spent in the quadrant where the platform had previously been located was recorded. Rats were removed after 60 seconds of searching during the probe trial.

Statistical analysis

The time to find the platform on each training day and probe trial day was compared across the 3 groups using analysis of variance followed by Fisher’s least significant difference post hoc test. Time to find the platform on day 10 (first trial after acute poisoning) was compared with day 31 (last trial after acute poisoning) within each group using a paired test. Significance was determined at P b .05.

Results

All rats receiving DFP developed significant toxicity within 5 minutes of injection and required antidotal rescue. Eleven of the rats required only a single dose of Rescue medications, with signs of acute toxicity resolving completely within 30 minutes. One rat receiving DFP did not respond to rescue attempts and died. The remaining 11 rats that received DFP were randomized into treatment groups (naltrexone, n = 6, vs saline, n = 5). No rats in the vehicle group (n = 5) developed acute toxicity after injection.

Over the 31-day survival period, 2 of the vehicle rats, 1 of the DFP alone rats (in addition to the one that died acutely), and 1 of the naltrexone treated rats developed Gastrointestinal complications unrelated to the poisoning (as determined by veterinarian-performed necropsy) and either died or were unable to continue with behavioral testing. All of these events occurred between days 13 and 29 after poisoning. The final number of animals included in analysis was 3 for control group, 4 for the DFP + vehicle group, and 5 for the DFP + naltrexone group.

Fig. 1. Time to find platform using Morris Water Maze at multiple time points after acute poisoning. Performance on the test of spatial learning as determined by the time in seconds to find a hidden platform was significantly worse in rats poisoned with DFP alone compared with rats treated with naltrexone after DFP poisoning and control rats. Asterisk indicates a significant difference at P b .05 compared with control animals.

678 K.L. Brewer et al. / American Journal of Emergency Medicine 31 (2013) 676679

system sites, brain regions associated with learning and memory [17- 19]. In addition, Acute OP poisoning increases secretion of proin- flammatory cytokines and chemokines in these same regions [20-22]. Outside of their prototypic role as pain control agents, opioids have been shown to have a role in the regulation of cytokine and cytokine receptor expression, generally promoting microglial activation and inflammation [23-26]. Mounting evidence demonstrates that opioid antagonists can have potent anti-inflammatory actions in several Disease states, including hepatic fibrosis, Crohn disease, and neurogenic pain [27-30]. However, the ability of opioid antagonists to combat inflammation and restore Cognitive function after a chemical exposure has not been tested. Naltrexone is an opioid antagonist whose safety

and availability as a generic formulation are already proven.

Fig. 2. Reference memory as indicated by time spent in the quadrant in which a platform was previously hidden. Performance on the test of reference memory was similar between rats poisoned with DFP alone, rats treated with naltrexone after DFP poisoning, and control (vehicle) rats.

Because these complications also occurred in the vehicle alone group, it was concluded that they were not due to exposure to the poison but more likely due to traumatic IP injection.

Using time to find a submerged platform in the Morris Water Maze as a measure of spatial learning and memory, all rats significantly improved their performance from day 10 after exposure to day 31 after exposure (Table 1). However, 10 days after exposure, rats poisoned with DFP alone took significantly longer than naltrexone- treated rats and control rats to find a submerged platform using the Morris Water Maze (42.8 +- 3.4 vs 31.5 +- 2.6 vs 30.6 +- 3.9 seconds, respectively; P = .03 vs naltrexone rats; P = .03 vs control rats). There was no significant difference in the time to platform between vehicle and naltrexone-treated rats (P = .78). This relationship persisted throughout the 31-day survival period (see Fig. 1), indicating a detrimental impact of DFP on spatial learning that was mitigated with long-term naltrexone treatment.

No significant differences existed in reference memory across groups on any of the probe trial days (Fig. 2). However, the naltrexone-treated animals tended to spend more time in the correct quadrant than the DFP alone animals, particularly in the early time points. A power analysis indicated that the trend toward DFP alone rats spending less time in the correct quadrant would reach statistical significance at 80% power with an additional 12 animals in each of these groups.

Discussion

The choice of DFP for this study was motivated by its similarity to sarin, an important military nerve gas and terrorist agent. Sarin was not used because it is much more volatile than DFP, poses a much greater hazard to investigators, and is not available. Both DFP and sarin are generally of greater potency and quicker aging (ie, time to irreversible binding to cholinesterases) than most insecticides. Hence, generalization of our results to other OP compounds must be done with caution.

The widely accepted primary mechanism underlying the neuro- toxic effects of organophosphorus agents is inhibition of acetylcho- linesterase activity and subsequent overstimulation of nicotinic and muscarinic receptors. However, recent studies have demonstrated that OP neurotoxicity is not entirely due to cholinergic mechanisms, especially with regard to neurobehavioral outcomes [11]. Based on observations that Anti-inflammatory agents are neuroprotective against acute OP poisoning [12] and that markers of inflammation correlate with neurobehavioral deficits secondary to neurodegener- ative diseases [13-16], inflammation has been proposed to play a role in the delayed neurodegenerative processes and behavioral changes associated with OP exposure [5]. Exposure to OP agents induces activation of microglia and astrocytes in, among other central nervous

The clinical significance of this pilot study is that a treatment initiated after resolution of acute OP poisoning reduced a component of the long-term Neurologic sequelae of the poisoning. The ability of naltrexone to reduce the neurologic impact of acute DFP poisoning may be due to its anti-inflammatory properties, although inflammatory markers were not directly measured. Previous studies have shown that OP pesticides, including DFP, cause direct neuronal damage as well as secondary, delayed neuronal injury through inflammatory effects mediated by astrocytes and oligodendrocytes [31-33].

Interestingly, not all OP agents elicit the same inflammatory response, with DFP specifically decreasing markers for activated astrocytes initially, with a subsequent increase 5 to 20 days later in the cerebrum [34]. Rats exposed to sarin develop significant but transient neuronal inflammation within 24 hours of exposure [35]. This inflammation returned by 1 month and continued to worsen for up to 6 months after exposure. During this time frame, exposed rats showed impaired working and reference memory with no recovery of function over time [35]. Our results showed that, using the Morris Water Maze, all rats decreased their time to find a hidden platform over a 4-week survival period. However, those rats that received DFP alone showed less improvement and consistently took longer to locate the platform than did the control animals or the DFP-poisoned animals that were treated with naltrexone. These results confirm that rats exposed to DFP provide a viable model of delayed neuronal damage induced by OPs, as previously suggested [32,33], and provide evidence that naltrexone has the ability to reduce the early brain inflammation to protect against loss of memory function. Based on the findings of Grauer et al [35], if naltrexone is having effects on the inflammatory response of the brain to DFP, we would expect to see a more pronounced effect of both the acute poisoning and the treatment with naltrexone on behaviors tested with a survival time beyond 1-month postexposure.

In the current study, naltrexone therapy was initiated at the time

of poisoning and continued throughout the survival time. This early intervention may have served to reduce the immediate inflammation known to occur with OP exposure [33,35]. However, other mecha- nisms of OP-induced neuronal damage also exist, including choliner- gic excitotoxicity [36], impaired microtubule function [37], and oxidative stress [38]. Some evidence exists for the ability of naltrexone to modulate oxidative stress in certain disease states [39,40]. Future studies will require additional outcome measures of central nervous system function as well as histologic and biochemical analyses of tissue from animals to determine the cellular and molecular mechanisms of the Cognitive impairments as well as the impacts of naltrexone therapy on these outcomes. In addition, because the inflammatory state induced by OP exposure appears to be dynamic, future studies will investigate the therapeutic window for naltrexone, including a determination of whether naltrexone can reverse the neurologic disabilities after they have developed.

The study is limited by the loss of animals over time, which reduced the power and ability to detect differences in performance. Veterinary staff indicated that the peritonitis that developed in some animals was likely due to the route of IP administration of the study

K.L. Brewer et al. / American Journal of Emergency Medicine 31 (2013) 676679 679

drugs (DFP and vehicle). Future studies will be designed with an alternative route such as subcutaneous. Despite these losses, the effect of naltrexone on spatial learning persisted throughout the survival period. As noted above, adding 12 animals to each group will provide the power needed to detect a significant difference in reference memory as well.

A second limitation may be the short survival time chosen. Published literature demonstrates that the inflammation induced by acute OP poisoning worsens after the 1-month period [35]. Therefore, the deficits in learning and memory in animals poisoned with DFP could potentially worsen over time. If this is the case, the effect of naltrexone will be more pronounced at later time points.

Lastly, the study did not include a DFP vehicle plus naltrexone arm. This may be necessary to determine that the naltrexone is not affecting performance by a mechanism other than reducing inflam- mation post-OP exposure. This addition would control for effects such as reduced glucocorticoid impairment of spatial memory as reported previously [41]. In conclusion, naltrexone therapy initiated at the time of an acute poisoning provided protection against impairment of spatial learning from acute poisoning with DFP in rats. Although this result is promising, further work is needed before recommendations for human poisonings with OPs can be given.

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