Seizure suppression and neuroprotection in soman-exposed rats following delayed intramuscular treatment of adenosine A1 receptor agonist as an adjunct to standard medical treatment.

Soman produces excitotoxic effects by inhibiting acetylcholinesterase in the cholinergic synapses and neuromuscular junctions, resulting in soman-induced sustained status epilepticus (SSE). Our previous work showed delayed intramuscular (i.m.) treatment with A1 adenosine receptor agonist N-bicyclo-[2.2.1]-hept-2-yl-5'-chloro-5'-deoxyadenosine (ENBA) alone suppressed soman-induced SSE and prevented neuropathology. Using this same rat soman seizure model, we tested if delayed therapy with ENBA (60 mg/kg, i.m.) would terminate seizure, protect neuropathology, and aid in survival when given in conjunction with current standard medical countermeasures (MCMs): atropine sulfate, 2-PAM, and midazolam (MDZ). Either 15- or 30-min following soman-induced SSE onset, male rats received atropine and 2-PAM plus either MDZ or MDZ + ENBA. Electroencephalographic (EEG) activity, physiologic parameters, and motor function were recorded. Either 2- or 14-days following exposure surviving rats were euthanized and perfused for histology. All animals treated with MDZ + ENBA at both time points had 100% EEG seizure termination and reduced total neuropathology compared to animals treated with MDZ (2-day, p = 0.015 for 15-min, p = 0.002 for 30-min; 14-day, p < 0.001 for 15-min, p = 0.006 for 30-min), showing ENBA enhanced MDZ's anticonvulsant and neuroprotectant efficacy. However, combined MDZ + ENBA treatment, when compared to MDZ treatment groups, had a reduction in the 14-day survival rate regardless of treatment time, indicating possible enhancement of MDZ's neuronal inhibitory effects by ENBA. Based on our findings, ENBA shows promise as an anticonvulsant and neuroprotectant in a combined treatment regimen following soman exposure; when given as an adjunct to standard MCMs, the dose of ENBA needs to be adjusted.

Learning and memory function preserved by delayed A1 adenosine receptor agonist treatment following soman intoxication in rats and a humanized esterase mouse model.

Exposure to organophosphorus compounds, such as soman (GD), cause widespread toxic effects, sustained status epilepticus, neuropathology, and death. The A1 adenosine receptor agonist N-bicyclo-(2.2.1)-hept-2-yl-5'-chloro-5'-deoxyadenosine (ENBA), when given 1 min after GD exposure, provides neuroprotection and prevents behavioral impairments. Here, we tested the ability of ENBA at delayed treatment times to improve behavioral outcomes via a two-way active avoidance task in two male animal models, each consisting of saline and GD exposure groups. In a rat model, animals received medical treatments (atropine sulfate [A], 2-PAM [P], and midazolam [MDZ]) or AP + MDZ + ENBA at 15 or 30 min after seizure onset and were subjected to behavioral testing for up to 14 days. In a human acetylcholinesterase knock-in serum carboxylesterase knock-out mouse model, animals received AP, AP + MDZ, AP + ENBA, or AP + MDZ + ENBA at 15 min post seizure onset and were subjected to the behavioral task on days 7 and 14. In rats, the GD/AP + MDZ + ENBA group recovered to saline-exposed avoidance levels while the GD/AP + MDZ group did not. In mice, in comparison with GD/AP + MDZ group, the GD/AP + MDZ + ENBA showed decreases in escape latency, response latency, and pre-session crossings, as well as increases in avoidances. In both models, only ENBA-treated groups showed control level inter-trial interval crossings by day 14. Our findings suggest that ENBA, alone and as an adjunct to medical treatments, can improve behavioral and cognitive outcomes when given at delayed time points after GD intoxication.

Hypothermia as potential therapeutic approach to attenuating soman-induced seizure, neuropathology, and mortality with an adenosine A1 receptor agonist and body cooling.

Organophosphorus nerve agents, such as soman (GD), produce excitotoxic effects resulting in sustained status epilepticus (SSE) and brain damage. Previous work shows that neuronal inhibitory effects of A1 adenosine receptor (A1AR) agonists, such as N6- Bicyclo (2.2.1)-hept-2-yl-5'-chloro-5'-deoxyadenosine (Cl-ENBA), suppresses GD-induced SSE and improves neuropathology. Some other physiologic effects of these agonists are hypothermia, hypotension, and sedation. Hypothermia may also shield the brain from injury by slowing down chemical insults, lessening inflammation, and contributing to improved neurological outcomes. Therefore, we attempted to isolate the hypothermic effect from ENBA by assessing the neuroprotective efficacy of direct surface body cooling in a rat GD-induced SSE model, and comparing the effects on seizure termination, neuropathology, and survival. Male rats implanted with a body temperature (Tb) transponder and electroencephalographic (EEG) electrodes were primed with asoxime (HI-6), exposed to GD 30 min later, and then treated with Cl-ENBA or had Tb lowered directly via body cooling at 30 min after the onset of seizure activity. Afterwards, they were either allowed to develop hypothermia as expected, or received thermal support to maintain normothermic Tb for a period of 6-h. Neuropathology was assessed at 24 h. Regardless of Cl-ENBA or surface cooling, all hypothermic GD-exposed groups had significantly improved 24-h survival compared to rats with normothermic Tb (81% vs. 39%, p < 0.001). Cl-ENBA offered neuroprotection independently of hypothermic Tb. While hypothermia enhanced the overall efficacy of Cl-ENBA by improving survival outcomes, body cooling didn't reduce seizure activity or neuropathology following GD-induced SSE.

Soman-induced toxicity, cholinesterase inhibition and neuropathology in adult male Göttingen minipigs.

Animal models are essential for evaluating the toxicity of chemical warfare nerve agents (CWNAs) to extrapolate to human risk and are necessary to evaluate the efficacy of medical countermeasures. The Göttingen minipig is increasingly used for toxicological studies because it has anatomical and physiological characteristics that are similar to those of humans. Our objective was to determine whether the minipig would be a useful large animal model to evaluate the toxic effects of soman (GD). We determined the intramuscular (IM) median lethal dose (LD50) of GD in adult male Göttingen minipigs using an up-and-down dosing method. In addition to lethality estimates, we characterized the observable signs of toxicity, blood and tissue cholinesterase (ChE) activity and brain pathology following GD exposure. The 24 h LD50 of GD was estimated to be 4.7 µg/kg, with 95 % confidence limits of 3.6 and 6.3 µg/kg. As anticipated, GD inhibited ChE activity in blood and several tissues. Neurohistopathological analysis showed neurodegeneration and neuroinflammation in survivors exposed to 4.7 µg/kg of GD, including in the primary visual cortex and various thalamic nuclei. These findings suggest that the minipig will be a useful large animal model for assessing drugs to mitigate neuropathological effects of exposure to CWNAs.