Antidote administration in mice exposed to sarin
resulted in brainwave stabilization without cognitive
protection

Mélanie Lagadec, Pauline Thiebot, Isabelle Malissin, Alexandre Champault, Julie Somkhit, Julie Knoertzer, Assia Belkebir, Bruno Megarbane, Karine Thibault, Gregory Dal Bo

► To cite this version:

Mélanie Lagadec, Pauline Thiebot, Isabelle Malissin, Alexandre Champault, Julie Somkhit, et al. Antidote administration in mice exposed to sarin resulted in brainwave stabilization without cognitive protection. Neuroscience Research, 2026, 223, pp.105013. 10.1016/j.neures.2025.105013. hal-05448241

HAL Id: hal-05448241

https://hal.science/hal-05448241v1

Submitted on 8 Jan 2026

**HAL** is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons CC BY 4.0 - Attribution - International License
<figure><img src="image_2.png" /></figure>

<figure><img src="image_3.png" /></figure>

# Antidote administration in mice exposed to sarin resulted in brainwave stabilization without cognitive protection

Mélanie Lagadec<sup>a,b,1</sup>, Pauline Thiebot<sup>a,c,1</sup> <sup>©</sup>, Isabelle Malissin<sup>c,d</sup>, Alexandre Champault<sup>a,e</sup> <sup>©</sup>, Julie Somkhit<sup>a,f</sup>, Julie Knoertzer<sup>a</sup>, Assia Belkebir<sup>a,e</sup>, Bruno Megarbane<sup>c,d</sup> <sup>©</sup>, Karine Thibault<sup>a,e,1</sup>, Gregory Dal Bo<sup>a,e,*1,2</sup>

## ARTICLE INFO

### Keywords:

sarin
organophosphate
antidote
atropine sulfate
pralidoxime
neuroprotection
cognitive impairment
brain oscillations

## ABSTRACT

Resurgence of sarin use in the Syrian conflict demonstrates that chemical weapons are a vivid threat. Like other organophosphate compounds, sarin inhibits acetylcholinesterase resulting in cholinergic crisis. The current antidote therapy includes atropine (a muscarinic cholinergic antagonist) + an oxime (e.g., pralidoxime) to reactivate the inhibited cholinesterases. Long-term sequelae reported in victims of the 1995 Tokyo subway sarin attack emphasize the need to improve neuroprotection and to delineate more precise biomarkers of intoxication severity. Previously, we proposed that brain oscillations could indicate exposure severity to a sublethal dose of a neurotoxic agent. In the present study, we evaluated the effects of atropine + pralidoxime in mice exposed to a sublethal dose of sarin (0.9 LD₅₀) on changes in short-term recovery, brainwaves and behavioral patterns. We demonstrated the benefits of the antidotes on alleviation of the sarin-induced encephalopathy, stabilization of brain oscillations, and attenuation of anhedonia behavior. Nevertheless, mice exhibited work memory impairments associated with the disruption of theta oscillations during the task, the disruption of sleep architecture and the anxiety-like behavior when exposed to the non-convulsive sarin dose. These outcomes were not mitigated by atropine + pralidoxime administration. Our findings suggest that the currently recommended antidote for sarin poisoning provides only partial neuroprotection. Despite stabilizing brain waves in resting state, current antidote is not effective in preventing long-term neurobehavioral complications.

## 1. Introduction

Organophosphate (OP) nerve agents (NA), ones of the most toxic chemical weapons, still represent a threat despite their ban in 1997 by the Organization for Prohibition of Chemical Weapons. Historically, sarin (GB, O-isopropyl methylphosphonofluoridate) has been the most

used NA in armed conflicts and terrorist attacks involving military and civilian populations (Abou-Donia et al., 2016; Shi et al., 2023). As is typical of all OP compounds, sarin inhibits cholinesterases (ChE), increasing neuronal cholinergic activities and leading to cholinergic manifestations. Usual toxidrome include miosis, cough, excessive bronchial secretions, dyspnea, headaches and muscle weakness.

Abbreviations: 2-PAM, pralidoxime; AChE, acetylcholinesterase; AS, atropine sulfate; AUC, area under the curve; ANT, antidote; BBB, blood brain barrier; ChE, cholinesterase; ECOG, electrocorticography; EPM, elevated plus maze; LD₅₀, median lethal dose; NA, nerve agent; NaCl, sodium chloride; NIMP, 4-nitrophenyl isopropyl methylphosphonate; NORT, novel object recognition test; QF, open-field; OP, organophosphate; SD, standard deviation.

E-mail address: gregory.dal-bo@def.gouv.fr (G. Dal Bo).

<footnote><sup>a</sup> Department of Toxicology and Chemical Risks, French Armed Forces Biomedical Research Institute, Bretigny-sur-Orge, France</footnote>

<footnote><sup>b</sup> Normandie University, UNICAEN, INSERM, GIP Cyeron, Institut Blood and Brain @Caen-Normandie (BB@C), UMR-S U1237, Physiopathology and Imaging of Neurological Disorders (PhIND), Caen, France</footnote>

<footnote><sup>c</sup> Université Paris-Cité, INSERM UMRS-1144, Paris, France</footnote>

<footnote><sup>d</sup> Department of Medical and Toxicological Critical Care, APHP, Lariboisière Hospital, Federation of Toxicology, Paris, France</footnote>

<footnote><sup>e</sup> Laboratoire d'Imagerie Biomédicale Multimodale (BioMaps), CEA, CNRS, Inserm, Service Hospitalier Frédéric Joliot, Université Paris-Saclay, Orsay, France</footnote>

<footnote><sup>f</sup> Laboratoire d'Etudes et de Recherches en Immunoanalyse, CEA, Département Médicaments et Technologies pour la Santé, Gif-sur-Yvette, France</footnote>

<footnote>* Correspondence to: Institut de Recherche Biomédicale des Armées, Département de Toxicologie et Risques Chimiques, 1 place du Général Valérie André, BP73, Brétigny-sur-Orge 91223, France</footnote>

<footnote n="1">¹ These authors contributed equally to this work.</footnote>

<footnote n="2">² ORCID # 0000-0001-7683-5854</footnote>
Interestingly, in sarin-poisoned victims following the two terrorist sarin attacks in Japan (Matsumoto in 1994 and Tokyo in 1995), these usual manifestations disappeared shortly after exposure (Okudera, 2002; Yanagisawa et al., 2006), whereas less usual long-term manifestations were reported in patients exposed to initially asymptomatic or non-convulsive doses (Miyaki et al., 2005; Sugiyama et al., 2020; Talabani et al., 2018).

Experimental studies have extensively contributed to the current understanding of sarin-attributed toxic manifestations including long-term persisting neurological complications (Burchfiel et al., 1976; Mach et al., 2008; Nieminen et al., 1990; Okudera, 2002; van Helden et al., 2004). Utilization of animal models also represent a long-standing practice to evaluate antidote effectiveness in OP exposure management (Bajgar et al., 1975; Hobbiger and Sadler, 1958; Shih et al., 2009). The currently recommended antidotal therapy combines a muscarinic re- ceptor antagonist (atropine) to limit acetylcholine hyperactivity and an oxime to reactivate the inhibited ChE (Abou-Donia et al., 2016; Jokanović et al., 2020; Narimatsu et al., 2010). Pralidoxime (2-PAM), the first synthesized oxime, remains most commonly used to counteract OP-induced ChE inhibition, despite the lack of clear demonstration of its therapeutic benefits (Kuca et al., 2005; Shih et al., 2009). In the Tokyo terrorist attack, atropine and 2-PAM, administered 2–6 h after sarin exposure, markedly improved serum ChE activity recovery (Yanagisawa et al., 2006). Although not crossing the blood-brain barrier (BBB), 2-PAM seems to have prevented the onset of sarin-related delayed neurological manifestations in the most severely poisoned survivors (Yanagisawa et al., 2006). By contrast, psychological issues were re- ported at a high prevalence in the mildly sarin-exposed patients (Murata et al., 1997; Ohtani et al., 2004; Yanagisawa et al., 2006). Since not all sarin-exposed victims received atropine and 2-PAM in Tokyo, it was retrospectively difficult to conclude whether antidotes, if more largely administered, would have limited the development of delayed psycho- logical disturbances.

In a previous study, we investigated the short- and long-term effects of sub-lethal doses of a sarin surrogate, 4-nitrophenyl isopropyl meth- ylphosphonate (NIMP) (Angrand et al., 2021; François et al., 2022). The use of electrocorticography (ECoG) revealed sustained disruption in the brain oscillations of mice exposed to a mild (0.5 of the median lethal dose, LD₅₀) or a convulsive (0.9 LD₅₀) dose of NIMP. Both NIMP doses were found to cause transient hippocampal edema, to impair short-term memory, and to induce long-term anxiety.

Therefore, given the unexpected onset of delayed psychological disturbances in some mildly sarin-exposed patients in Tokyo, who may have not received any antidote, we sought to investigate the protective effects of atropine sulfate (AS) + 2-PAM in mice exposed to a sub-lethal dose of sarin. Our objectives were to evaluate the antidote effects on sarin-induced brainwaves using ECoG at both short and long terms and to examine their influence on sarin-attributed mice behavioral modifications.

## 2. Material and methods

### 2.1. Animals

All experimental procedures were approved by the animal ethics committee of the Service de Santé des Armées according to the French legislation (Directive 2010/63/UE, décret 2013–118). All animal ex- periments complied with ARRIVE guidelines. 105 young adult male Swiss mice (Janvier Labs, France) were housed four per cage on a 12-h light /12-h dark cycle, with food and water *ad libitum*. After 7 days of acclimation, the animals commenced their dedicated experiment. All animals were 9 weeks old at the time of sarin challenge.

### 2.2. Sarin exposure

Sarin (93.5 % purity, determined by nuclear magnetic resonance)

was provided by the Direction Générale de l'Armement (France). The day of the experiment, mice received a single subcutaneous injection (10 mL/kg) of sarin (LD₅₀ = 0.63 mg/kg), diluted in sterile 0.9 % NaCl at 0.9 LD₅₀. The LD₅₀ was estimated using the improved method of Dixon's up-and-down procedure (Dixon, 1991). Control mice (0 LD₅₀) received a similar injection of sterile NaCl 0.9 % (vehicle).

### 2.3. AS + 2-PAM treatment

Atropine sulfate monohydrate (AS) (3 mg/kg) and 2-PAM (100 µmol/kg) were provided by Pharmacie Centrale des Armées (Orléans, France), and were diluted in sterile saline at a dose of 10 mL/kg and intraperitoneally administered two hours post-sarin exposure in the same solution [AS + 2-PAM = antidote (ANT)]. All solutions were freshly prepared and used on the same day. Four groups were designed: 0 LD₅₀ (NaCl – NaCl); 0 LD₅₀ + ANT (NaCl – antidote); 0.9 LD₅₀ (sarin – NaCl), and 0.9 LD₅₀ + ANT (sarin – antidote). To avoid any bias resulting from cage effects, intoxicated (sarin or control) and treated (ANT or NaCl) mice were randomized in each cage.

### 2.4. Behavioral observation

To avoid experimental biases, ‘treatment-blinded’ experimenters performed behavioral assessment and data analysis.

#### 2.4.1. Intoxication severity scale

Mice behavior was monitored in the hour following sarin exposure. Previous studies in our laboratory allowed the detection of several typical signs of cholinergic toxicity, for which a 13-grade scale was used to score the animals from normal (=0) to death (=12) (Angrand et al., 2021). The scale has been described as follows: 1- fasciculation; 2- chewing/yawning; 3- muscle weakness; 4- hunched posture; 5- head myoclonus; 6- muscular spasm; 7- body myoclonus; 8- short convulsions (< 10 s); 9- generalized convulsions; 10- long lasting convulsions (> 30 min); 11- eye bulging; 12- death.

#### 2.4.2. Animal monitoring and recovery

Mice weight was monitored in the hour preceding and the days following sarin exposure. Blind monitoring was carried out daily to assess a recovery score up to 6 days post-intoxication. Symptoms are scored from 0 to –2 according to severity and include: coat appearance, kyphosis, respiration, in-cage activity, lacrimation, tremors, aggres- siveness, muscle tone, and weight loss (Fig. 1A). The scale starts at 0, corresponding to an animal with no obvious symptoms. Each symptom listed above is subtracted by 1 or 2 points depending on its severity: the lower the score, the poorer the recovery from NA exposure.

### 2.5. Cognitive test: novel object recognition test (NORT)

Before and 1 month after sarin exposure, NORT was performed on non-surgical mice according to our protocol. Mice were placed in an empty open-field (OF) box for a 10 min habituation period. Mice were placed in the same OF box with 2 identical objects and were allowed to explore for 5 min before returning to their home cage. After 1 h, the mice were placed in the OF containing one of the familiar objects and a novel object, and allowed to explore for 5 min. The objects were placed at the same locations as the first session. All NORT sessions were vid- eotaped, and the exploration time was analyzed using EthoVision XT software (Noldus, NL). A border of 2 cm around each object was drawn to determine active investigation area. The cumulative time the mouse investigated each object was calculated by the presence of nose point in the area of interest. The discrimination index was calculated with the following formula:

$$ \frac{(\text{Time with the novel object})}{(\text{Time with novel object} + \text{Time with familiar object})} \times 100 $$
<table><thead><tr><th>Behaviour</th><th></th><th>Score</th></tr></thead><tbody><tr><td rowspan="3">Fur appearance</td><td>Normal</td><td>=0</td></tr><tr><td>Moderate care</td><td>=-1</td></tr><tr><td>No care</td><td>=-2</td></tr><tr><td rowspan="3">Kyphosis</td><td>Normal</td><td>=0</td></tr><tr><td>Moderate</td><td>=-1</td></tr><tr><td>Very pronounced</td><td>=-2</td></tr><tr><td rowspan="3">Breathing</td><td>Normal</td><td>=0</td></tr><tr><td>Moderate increase in frequency</td><td>=-1</td></tr><tr><td>Labored abdominal breathing</td><td>=-2</td></tr><tr><td rowspan="3">Activity in the cage</td><td>Normal</td><td>=0</td></tr><tr><td>Reduced mobility</td><td>=-1</td></tr><tr><td>Immobility</td><td>=-2</td></tr><tr><td rowspan="3">Lacrimation</td><td>Normal</td><td>=0</td></tr><tr><td>Moderate</td><td>=-1</td></tr><tr><td>Severe</td><td>=-2</td></tr><tr><td rowspan="3">Tremor</td><td>Normal</td><td>=0</td></tr><tr><td>Moderate</td><td>=-1</td></tr><tr><td>Severe</td><td>=-2</td></tr><tr><td rowspan="3">Reactivity (aggressivity)</td><td>Normal</td><td>=0</td></tr><tr><td>Moderate: freezing, tension, stiffness in the hand</td><td>=-1</td></tr><tr><td>Strong: torsion, attempted biting, vocalisations</td><td>=-2</td></tr><tr><td rowspan="3">Muscular tonus</td><td>Normal</td><td>=0</td></tr><tr><td>Reduced responses to stimuli</td><td>=-1</td></tr><tr><td>No responses to stimuli</td><td>=-2</td></tr><tr><td rowspan="3">Weight loss</td><td>Initial weight</td><td>=0</td></tr><tr><td>&lt;20%</td><td>=-1</td></tr><tr><td>&gt;20%</td><td>=-2</td></tr></tbody></table>

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Antidote treatment effects on recovery from sarin toxicity. (A) Symptoms scores recovery scale. (B) Body weight changes relative to baseline (n = 14 for 0 LD₅₀ animals; n = 19 for 0 LD₅₀ + ANT animals; n = 21 for 0.9 LD₅₀ animals and n = 22 for 0.9 LD₅₀ + ANT animals). Significant differences were determined by 2-way repeated measures ANOVA (F<sub>time</sub> = 7.2, p < 0.0001; F<sub>treatment</sub> = 50.2, p < 0.0001) followed with Tukey's post hoc test to compare treatments. (C) Mean recovery score during the first week post-sarin exposure. Significant differences were determined by Kruskal-Wallis test (F<sub>D1</sub> = 45.3, p < 0.0001; F<sub>D2</sub> = 19.3, p = 0.0002; F<sub>D3</sub> = 18.9, p = 0.0003; F<sub>D4</sub> = 13.9, p = 0.003; F<sub>D5</sub> = 16.1, p = 0.0011; F<sub>D6</sub> = 20.5, p = 0.0001) followed with Dunn's multiple comparisons test. Post hoc tests compare 0 LD₅₀ animals vs. 0.9 LD₅₀ animals (*) or 0 LD₅₀ + ANT animals vs. 0.9 LD₅₀ + ANT animals (#); *or # p < 0.05, ** or ## p < 0.01, *** or ### p < 0.001, **** or #### p < 0.0001.</figcaption></figure>

The same experimental conditions for NORT were used in ECoG telemetric implanted mice (see below). One month after sarin exposure, telemetric implanted mice were submitted to the NORT sessions paradigm and were recorded during each task by positioning the open field (OF) box on a Physiotel receiver (DSI, USA) and videotaped to analyze their behavior using the EthoVision XT software (Noldus, NL).

## 2.6. Anxiety-like behavior tests: elevated-plus-maze (EPM) test

The EPM apparatus consists of a raised maze (80 cm off the floor) with four arms in a cross shape: two arms are exposed to the open air, and the other two arms are enclosed. One month after sarin exposure, mice were placed in the maze for a 5-min period. The test was video-recorded and the activity of the animal over time was analyzed using EthoVision XT software. Time spent in the open arms was evaluated. The cumulative duration of body elongation was also assessed: this body shape was noted as a characteristic of the stretching posture, which is often seen as a response to an environment that induces both anxiety and fear.

## 2.7. Anhedonia behavior test: sucrose splash test

The protocol consists of spraying a 10 % sucrose solution onto the coat of a mouse. The mouse was first individually placed in a cage (40 x

20 x 15 cm) lighted by 35 lux. The dorsal coat of each mouse was then sprayed with a sucrose solution containing 10 % sucrose. Mice responses were videorecorded for 5 min. As an index of motivational behavior, latency (time between spraying and initiation of grooming) and total duration of grooming were manually coded. Each video was analyzed twice. Protocol was adapted from Ducottet and Belzung (Ducottet and Belzung, 2004). A reduction in grooming behavior is a depression-like behavior in mice.

## 2.8. Surgery

Two different ECoG procedures were used in this study. Seven-week-old animals were anesthetized in an induction box using isoflurane (5 % in O₂ at 0.5 L/min). Mice were injected with buprenorphine (Buprenorphine®, 0.1 mg/kg, s.c.; Produlab Pharma, NL) and prepared for surgery. Heads were shaved and lidocaine (Lurocaine®, 5 mg/kg; Vetoquinol, FR) was applied to scalps before placing the mice in a stereotaxic apparatus and maintaining them under isoflurane (2 %). Wired ECoG and telemetric ECoG surgeries were performed as previously described (Angrand et al., 2021).

## 2.9. ECoG recording and power spectra analysis

For wired ECoG recordings, 8 animals per experimental day were
connected via a cable to a data acquisition system and recorded for 24 h. Waveform cerebral activity was recorded using an MP160 System (Biopac, USA). Baseline cerebral activity was recorded for 1 h before sarin exposure.

For telemetric recordings, 16 animals were recorded simultaneously using the DataQuest acquisition software (DSI, USA). In addition to ECoG, subcutaneous temperature, animal activity and transmitter power were recorded. Baseline cerebral activity was recorded 1 h before sarin exposure. Mice were recorded for 24 h every week until one-month post-intoxication.

The ECoG power spectrum was divided into the following bands: delta (0.5–4 Hz), low theta (4–8 Hz), high theta (8–12 Hz), low beta (12–16 Hz) and high beta (16–24 Hz), using the Neuroscore software (DSI, St. Paul, MN, USA). Mean power was calculated for each band in 1-min epochs. For each animal, data from the telemetric recordings were normalized to the pre-intoxication baseline recording values.

Encephalopathy was stratified using Markand's classification (Markand, 1984), based on cerebral spontaneous activity analysis by a neurologist blinded to animal treatment. Spontaneous ECoG reactivity was defined as brainwave modifications in the background activity (acceleration or slowing) in relation to spontaneous fluctuations in arousal level.

## 2.10. Statistics

An a priori sample size calculation was made using SHADE application (Pasteur, Paris, France). The percentage of variability observed in our results or those in the literature in similar experiments. Thus, we will be able to detect a biological difference of at least 20 % between the treated and control groups with a variability of 20 % depending on the parameter studied and assuming that the risk of type 1 is equal to 5 % (p < 0.05), with a power of around 80 %. Data were expressed as the mean ± standard deviation (SD) and analyzed using PRISM 9 software (GraphPad, USA). Quantitative data are expressed as mean and standard deviation. The normality of the data distribution was confirmed by the Shapiro-Wilk test. For weight monitoring, splash test, NORT studies and ECoG analyses, we performed a two-way analysis of variance (ANOVA) repeated measure followed by Tukey's post hoc test. In these analyses, time and treatment were used as variables of interest. For ECoG analyses during NORT challenge, we performed a two-way analysis of variance (ANOVA) repeated measure followed by Tukey's post hoc test, where condition of test (OF empty, Familiar object and Novel object) and treatment were used as variables of interest. For recovery scoring analyses, we performed Kruskal-Wallis tests. To identify outliers, ROUT (Q = 1 %) was applied. The figure legends include the individual statistical tests used and the number of samples. Statistically significant differences were considered to be those with a probability value of p < 0.05.

# 3. Results

## 3.1. Physiological effects of ANT treatment and brainwaves changes management

The ANT treatment was injected 2 h after sarin exposure to mimic the delayed care. To avoid any bias, we insured that 0.9 LD₅₀ sarin-exposed mice groups presented similar poisoning severity levels before administering either the vehicle (intoxication score = 10.6 ± 1.0) or the antidote (intoxication score = 10.3 ± 1.3).

During the first week following sarin exposure, we noticed significant weight loss in both 0.9 LD₅₀ exposed mice groups, with no difference between these two groups (Fig. 1B). Six days after sarin exposure, weight loss was still observed in sarin-exposed mice compared to the control group. Moreover, ANT treatment did not improve the recovery score, and both sarin-exposed groups presented similar recovery patterns (Fig. 1C).

Telemetric ECoG recordings revealed marked encephalopathy

without seizure in all 0.9 LD₅₀ sarin-exposed mice (Table 1). In the 0.9 LD₅₀ group (without ANT), encephalopathy was moderate (grade 2) in two mice and severe (grade 4 A) in three mice, characterized by an early onset and lasting several hours (Table 1). Sarin also altered sleep activity, reducing the proportion of spindles during the N2 phase for a variable period (6 h in three mice and until the end of the recording in two mice) (Table 1). In the 0.9 LD₅₀ + ANT group, all mice exhibited an early-onset encephalopathy that was moderate (grade 2) in one animal and severe in four mice (grade 3 in one mouse and grade 4 A in three mice). ANT administration reduced the encephalopathy duration compared to the 0.9 LD₅₀ group (Fig. 2A-B and Table 1). However, sarin-attributed disruption of sleep architecture was not restored by ANT administration, and the proportion of spindles during N2 sleep was reduced (around 4 h in two mice and until the end of the recording in three mice) (Table 1). Interestingly, in the 0 LD₅₀ + ANT group, four mice exhibited mild encephalopathy (maximum grade, 1–2) peaking at 11 ± 6 min post-ANT administration lasting 68 ± 33 min (Fig. 2A-B and Table 1). Finally, no alterations in EEG activity or significant modifications to the awake/sleep architecture (particularly no reduction in the proportion of spindles) were observed in any group 7 days after sarin challenge or ANT administration.

During the 10 h post-intoxication, the mean encephalopathy grade improved in ANT animals, with a significant decrease in the AUC (area under the curve) for the post-treatment period in the 0.9 LD₅₀ + ANT group compared to the 0.9 LD₅₀ group (Fig. 2C).

The brainwave distribution analysis revealed a slight increase in delta rhythms (0.5–4 Hz) that persisted up to 28 days after sarin exposure (Fig. 2D). This increase was correlated with a significant decrease in the high theta rhythms (8–12 Hz) (Fig. 2F). Despite a slight decrease in low theta rhythms distribution, no statistically significant differences were observed in these brainwaves or in beta rhythms (Fig. 2E & 2H). Interestingly, the delta and high theta brainwaves distribution of 0.9 LD₅₀ + ANT treated animals had stabilized (Fig. 2D-H). Seven days after exposure, the low beta rhythms (12–16 Hz) in both sarin groups, showed no modification compared to control (Fig. 2G).

## 3.2. ANT treatment effect on sarin-induced behavior

It is noteworthy that a beneficial effect of ANT was observed in the depressive-like behavior test one month after exposure (Fig. 3A). Indeed, animals treated with 0.9 LD₅₀ exhibited a significant reduction in grooming duration and an increase in latency to groom in the splash test, which were not observed in animals treated with 0.9 LD₅₀ + ANT (Fig. 3B-C).

Considering the established link between depression-like and anxiety-like behaviors, the animals were tested on the elevated plus maze (EPM) one-month post-exposure (Fig. 3D). In a somewhat unexpected finding, animals exposed to sarin did not exhibit any notable signs of anxiety-like behavior. By contrast, the sarin-exposed animals exhibited a tendency towards an increase in open arm activity (cumulative duration), which is indicative of an anti-anxiety response (Fig. 3E). The observed increase in open arm duration appears to be positively correlated with risk assessment behavior (i.e., body elongation postures) in the EPM (Fig. 3F). The ANT treatment was unsuccessful in reinstating typical anxiety-like behavior (Fig. 3E-F).

In consideration of the impact of NA on memory and cognitive functions, the mice from each group were subjected to a NORT, which is designed to assess the degree of exploration exhibited towards novel objects. The initial NORT session was conducted one-month post-exposure on non-implanted animals (Fig. 3G) and on animals implanted for telemetric recording (Fig. 3H). In both instances, control mice (0 LD₅₀ and 0 LD₅₀ + ANT) demonstrated expected performance, whereas all sarin-exposed mice exhibited significantly reduced NORT performance. No velocity modification was observed in any group. It is noteworthy that the analysis of the brainwave recorded during the cognitive task revealed a lack of increase in low theta rhythms in both sarin-exposed
<table><caption>**Table 1**

Description of ECoG aspects in the different groups of mice according to sarin exposure and/or the antidote treatment.</caption><thead><tr><th></th><th>0 LD<sub>50</sub></th><th>0 LD<sub>50</sub> + ANT</th><th>0.9 LD<sub>50</sub></th><th>0.9 LD<sub>50</sub> + ANT</th></tr></thead><tbody><tr><td>Background activity</td><td>continuous</td><td>continuous</td><td>continuous</td><td>continuous</td></tr><tr><td>Spontaneous reactivity</td><td>preserved</td><td>preserved</td><td>preserved except during grade 4 A encephalopathy</td><td>preserved except during grade 4 A encephalopathy</td></tr><tr><td>Encephalopathy (n)</td><td>0/5</td><td>4/5</td><td>5/5</td><td>5/5</td></tr><tr><td>Maximal Grade</td><td>-</td><td>2 (n = 3), 1 (n = 1)</td><td>4 A (n = 3), 2 (n = 2)</td><td>4 A (n = 3), 3 (n = 1), 2 (n = 1)</td></tr><tr><td>Onset (min ± SD)</td><td>-</td><td>125.5 ± 1.3 post-ANT injection</td><td>10.2 ± 1.8 post-sarin exposure</td><td>6.2 ± 1.8 post-sarin exposure</td></tr><tr><td>Duration (min ± SD)</td><td>-</td><td>68.7 ± 33.3</td><td>437.2 ± 122.7</td><td>242.6 ± 115.6</td></tr><tr><td>Stage N1 proportion (%)</td><td>45 %</td><td>70 %</td><td>70 %</td><td>70 %</td></tr><tr><td>Stage N2 proportion (%)</td><td>40 %</td><td>20 %</td><td>25 %</td><td>25 %</td></tr><tr><td>% Spindles, compared to baseline</td><td>100 %</td><td>100 %</td><td>10 %</td><td>10 %</td></tr><tr><td>Duration of alteration of spindles (h ± SD)</td><td>0</td><td>0</td><td>12.2 ± 2.2</td><td>18.2 ± 6.7</td></tr></tbody></table>

groups during the novel object exploration phase (Fig. 3I). The findings revealed that, despite the stabilization of the electrical activity by ANT treatment to counteract the sarin-induced disruption, this treatment proved to be ineffective in restoring memory abilities.

## 4. Discussion

NA and pesticides OP-containing products represent a major threat worldwide. Poisoning management is highly challenging as no new pharmaceutical became available for human use since the Tokyo sub-way terrorist attack. While, in the US, France and Japan, atropine and 2-PAM remain the recommended antidotes in addition to supportive care (Abou-Donia et al., 2016; Aman et al., 2021; Eddleston et al., 2008), reports showed that OP-exposed survivors may develop long-term neurological complications (Miyaki et al., 2005; Okudera, 2002; Okumura et al., 2005; Sugiyama et al., 2020). The reactivation potency of 2-PAM against various NA has been evaluated in numerous studies, both *in vitro* and *in vivo* (Koplovitz and Stewart, 1994; Kuca et al., 2005; Shih et al., 2009; Sidell and Groff, 1974). However, studies did not analyze the outcome in relation to antidote administration and the benefits of AS + 2-PAM on the relief of OP-induced symptoms have been poorly investigated (Antonijevic and Stojiljkovic, 2007; Eddleston et al., 2002; Reddy et al., 1991; Smith, 1993).

To assess the effectiveness of the antidote therapy (ANT = AS + 2-PAM), we administered the treatment at a two-hour post-exposure interval, aligning with the timeframe of the emergency response following the Tokyo attack (Okumura et al., 2005, 1998). With this timeframe, ANT was effective at limiting the sarin-induced encephalopathy severity. In accordance with the NIMP results, at the dose of 0.9 LD₅₀ (Angrand et al., 2021), sarin did not induce epileptic seizures in any animal nor was there a recurrent seizure during the month following the exposure. Despite this absence of seizure, all sarin-exposed animals showed a weight loss that was not compensate by the administration of the antidotes. Our findings indicate that OP compounds were associated with weight loss regardless of the onset of epileptic seizures (Philippens et al., 1992; Shih et al., 2011). Additionally, our results demonstrated that current antidotes are not effective in improving recovery in animals exposed to sarin. Nevertheless, at long term, ANT normalized the brainwaves distribution. Indeed, the disturbances in delta and high theta rhythms persisted over the longer term (one month) following sarin exposure and was stabilized by ANT administration. Both atropine and 2-PAM have been reported to prevent altered spikes recorded in hippocampal slices (Ferchmin et al., 2014). However, evidence supporting AS contribution to limit seizures post-OP exposure exist (McDonough and Shih, 1997; Shih et al., 2007), by contrast to 2-PAM that poorly crosses the BBB and does not efficiently reactivate brain ChE (Bohnert et al., 2020; Chambers and Meek, 2020; Kuznetsova et al., 2022). Moreover, it has been demonstrated that the depression of field

excitatory postsynaptic potentials induced by the application of paraoxon on hippocampal slices was only inhibited by post-treatment with atropine (Narimatsu et al., 2010). Therefore, we hypothesize that stabilization of ECoG traces might be elicited by AS acting on brain muscarinic receptors. Despite the absence of homogeneity, a decrease in alpha wave (8–12 Hz) distribution has been reported in patients diagnosed with depression (Özçoban and Tan, 2025; Zhao et al., 2025). This finding is consistent with the observations reported in our study, which demonstrated that animals exposed to sarin exhibited a decline in high theta waves (8–12 Hz) and anhedonia. The prolonged stabilization of high theta waves in subjects treated with the antidote could provide a plausible explanation for the observed behavioral normalization. However, the NORT has been demonstrated to involve the hippocampal formation, where theta rhythms (4–8 Hz) predominate and which is influenced by the cholinergic innervation of the septum (Ma et al., 2020). The administration of ANT does not appear to stabilize low theta waves (4–8 Hz) during a cognitive task, highlighting a deficiency in working memory and suggesting an impact at the cellular level (e.g., expression of cholinergic receptors, changes in connectivity) (Liu and Pope, 1998; Schwab et al., 1981; Yagle and Costa, 1996), requiring further study. Mice exposed to sarin demonstrated increased exploratory activity in the open arms of the EPM test. This result was unexpected, given that in our previous study, mice exposed to the sarin analog NIMP, at the same dose (0.9 LD₅₀), exhibited anxious behavior one month after intoxication (François et al., 2022). Anxiety-like behaviors are the result of a complex interaction between the amygdala, the hippocampus, and the prefrontal cortex. Each component exerts specific functions, and their interplay contributes to the detection of threats and the regulation of emotions (Bannerman et al., 2004). Preliminary findings from ECoG results indicate that both NIMP and sarin induce disruption in the function of the hippocampus. However, these agents appear to exert divergent effects on emotional behavior. This finding indicates that NIMP was unable to replicate the sarin intoxication with complete fidelity. Consequently, the nature of the leaving group may be a contributing factor to the divergent behaviors observed during the EPM test. Moreover, the administration of the ANT did not mitigate the increased exploratory activity in the open-arms, indicating a behavioral phenotype of impulsivity (Kishikawa et al., 2014; Langen and Dost, 2011; Pawlak et al., 2012; Ueno et al., 2002), observed in animals exposed to sarin in the EPM task, revealing an inability to stabilize the development of anxiety disorders. In accordance with the results of the aforementioned studies, the increased exploratory behavior observed in the open arms of the EPM is accompanied by impaired performance in cognitive tasks, including the alternation test in the Y-Maze and the discrimination index in the NORT (Kishikawa et al., 2014; Langen and Dost, 2011; Ueno et al., 2002). These data suggest that the impulsivity exhibited by the mice exposed to sarin in the EPM is a potential indicator of an attention deficit (Desor et al., 2024). In addition, ANT
<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Antidote prevented encephalopathy and cortical activity induced by sarin exposure. (A) Mean grade reached in each treated group post-sarin exposure according to Markand's scale. The dotted line indicates ANT injection time (120 min post-sarin exposure). (B) Evolution of the encephalopathy represented individually for each mouse in the respective groups. (C) The area under the curve (AUC) is represented before and after ANT injection. Significant differences were determined by 2-way repeated measures ANOVA ($F_{time} = 4.4, p = 0.05; F_{treatment} = 10.1, p = 0.003$) followed with Tukey's post hoc test to compare treatments. 0 LD₅₀ + ANT animals vs. 0.9 LD₅₀ + ANT animals (#); 0.9 LD₅₀ vs. 0 LD₅₀ + ANT animals and vs. 0.9 LD₅₀ + ANT animals (+). (D-H) Changes in brainwave distributions at different time post-sarin exposure over 1-month: Delta (D), Low Theta (E), High Theta (F), Low Beta (G) and High Beta (H). (n = 7 for 0 LD₅₀ animals; n = 7 for 0 LD₅₀ + ANT animals; n = 6 for 0.9 LD₅₀ animals and n = 6 for 0.9 LD₅₀ + ANT animals). Significant differences were determined by 2-way repeated measures ANOVA (Delta: $F_{time} = 1.7, p = 0.19; F_{treatment} = 2.0, p = 0.14$; Low Theta: $F_{time} = 2.82, p = 0.04; F_{treatment} = 2.81, p = 0.06$; High Theta: $F_{time} = 0.3, p = 0.85; F_{treatment} = 2.96, p = 0.049$; Low Beta: $F_{time} = 1.5, p = 0.23; F_{treatment} = 0.8, p = 0.53$; High Beta: $F_{time} = 0.61, p = 0.61; F_{treatment} = 0.96, p = 0.42$) followed with Tukey's post hoc test to compare treatments. 0 LD₅₀ animals vs. 0.9 LD₅₀ animals (*), * p < 0.05, ** p < 0.01, *** p < 0.001.</figcaption></figure>
A

<figure><img src="image_6.png" /></figure>

B

<figure><img src="image_7.png" /></figure>

C

<figure><img src="image_8.png" /></figure>

D

<figure><img src="image_9.png" /></figure>

E

<figure><img src="image_10.png" /></figure>

F

<figure><img src="image_11.png" /></figure>

G

<figure><img src="image_12.png" /></figure>

H

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /></figure>

I

<figure><img src="image_16.png" /></figure>

Low Theta

$p=0.07$

J

<figure><img src="image_17.png" /></figure>

High Theta

Familiar

Novel

(caption on next page)
**Fig. 3.** Inconsistent outcomes provided by the antidote treatment on behavior and cognition recovery following sarin exposure. (A) Experimental representation of the splash test to assess anhedonia behavior in mice. (B-C) Exposure to sarin alters both grooming duration (B) and initiation latency (C). (n = 9 for 0 LD₅₀ animals; n = 9 for 0 LD₅₀ + ANT animals; n = 12 for 0.9 LD₅₀ animals and n = 12 for 0.9 LD₅₀ + ANT animals). Significant differences were determined by 2-way repeated measures ANOVA (Grooming duration: *F*<sub>time</sub> = 0.23, *p* = 0.63; *F*<sub>treatment</sub> = 5.2, *p* = 0.005; Latency to groom: *F*<sub>time</sub> = 3.2, *p* = 0.08; *F*<sub>treatment</sub> = 3.34, *p* = 0.049) followed with Tukey's post hoc test to compare treatments. (D) Experimental representation of the EPM test to assess anxiety behavior in mice one-month post-sarin exposure. (E) Time spent in open arms unchanged 1 month after sarin exposure. (F) The mice exposed to sarin present an abnormal posture (body elongation) during the EPM task. (n = 9 for 0 LD₅₀ animals; n = 9 for 0 LD₅₀ + ANT animals; n = 12 for 0.9 LD₅₀ animals and n = 12 for 0.9 LD₅₀ + ANT animals). Significant differences were determined by one-way ANOVA (Open arms duration: *F* = 1.4, *p* = 0.24; Body elongation duration: *F* = 3.7, *p* = 0.02) followed with Tukey's post hoc test to compare treatments. (G-H) Experimental representations of novel object recognition tests without (G) or with (H) telemetric recording, with their respective discrimination index (below) corresponding to the percentage of time spent to explore the familiar object or the novel object. Without telemetric recording: (n = 7 for 0 LD₅₀ animals; n = 7 for 0 LD₅₀ + ANT animals; n = 12 for 0.9 LD₅₀ animals and n = 11 for 0.9 LD₅₀ + ANT animals). Significant differences were determined by 2-way repeated measures ANOVA (*F*<sub>timextreatment</sub> = 4.3, *p* = 0.01); with telemetric recording: (n = 4 for 0 LD₅₀ animals; n = 4 for 0 LD₅₀ + ANT animals; n = 5 for 0.9 LD₅₀ animals and n = 5 for 0.9 LD₅₀ + ANT animals). Significant differences were determined by 2-way repeated measures ANOVA (*F*<sub>treatment</sub> = 7.0, *p* = 0.004) followed with Tukey's post hoc test to compare treatments. (I-J) Changes in brainwave distributions during the different sessions normalized to the open field session without any object. (n = 4 for 0 LD₅₀ animals; n = 4 for 0 LD₅₀ + ANT animals; n = 5 for 0.9 LD₅₀ animals and n = 5 for 0.9 LD₅₀ + ANT animals). Significant differences were determined by 2-way repeated measures ANOVA (Low Theta: *F*<sub>conditionxtreatment</sub> = 3.05, *p* = 0.027; High Theta: *F*<sub>conditionxtreatment</sub> = 1.5, *p* = 0.22) followed with Dunnett's post hoc test to compare treatments. Post hoc tests compare 0 LD₅₀ animals vs. 0.9 LD₅₀ animals (*) or 0 LD₅₀ + ANT animals vs. 0.9 LD₅₀ + ANT animals (#); \*or \# *p* < 0.05, \*\* or \#\# *p* < 0.01.

<figure><img src="image_18.png" /></figure>

administration failed to restore normal sleep architecture in the hours
following sarin exposure, and this transient sleep disruption could affect
cognitive performance and induce mood disorders in the longer term
(Trost Bobić et al., 2016; Yang et al., 2023). However, it is noteworthy
that the ANT prevented the development of anhedonia in subjects
exposed to sarin. The evidence presented herein clearly demonstrates
that the administration of ANT is an ineffective therapeutic strategy for
preventing long-term NA effects. As anticipated, the results demon-
strated the well-established drawbacks of 2-PAM, including incomplete
ChE reactivation, short plasma half-life, restricted spectrum, and poor
ability to cross the BBB (Da Silva et al., 2022; Kuca et al., 2005; Kuca and
Jun, 2006; Worek et al., 2004), while AS only counteracts muscarinic
receptor overstimulation alleviating muscarinic symptoms, and nico-
tinic receptor signaling remains upregulated after NA exposure
(Ferchmin et al., 2015, 2014). However, our study did not mimic the
dose regimen consisting in repeated AS injections and continuous
2-PAM infusion started as soon as possible and maintained as long as
needed (Jokanović et al., 2020). Although such experimental protocol
could be relevant but difficult to implement in mice, since their size
would restrict the health monitoring necessary to avoid side effects
(Bashyal et al., 2023; Eddleston et al., 2009). Regardless of these con-
siderations, our preclinical findings indicate that even when exposed to
a sub-lethal dose of sarin, the administration of ANT does not prevent
the development of long-term neurological disorders, despite the
beneficial effect observed on brainwaves.

In conclusion, our findings obtained in a rodent model highlight that ANT administration has limited effectiveness to counteract sarin-induced long-term impairments. Considering the difficulty to develop a broad-spectrum oxime able to cross the BBB, drugs targeting alternative targets should be considered in combination to the current antidotes. Finally, new therapeutic strategies improving BBB crossing of current available antidotes might be worthy of investigation in our models to assess possible long-term benefits.

Funding

This work was supported by the French Ministry of Armed Forces: Direction Générale de l'Armement (DGA) and the Service de Santé des Armées (SSA) NBC-5C-4211. This project is also part of the ANR-22-CE-39-0007 (NeuroTarget) and ANR-22-ASTR-0022 (B-BOP).

CRediT authorship contribution statement

**Mélanie LAGADEC:** Writing – review & editing, Writing – original draft, Investigation, Formal analysis. **Pauline THIEBOT:** Writing – review & editing, Writing – original draft, Investigation, Formal analysis. **Isabelle MALISSIN:** Writing – review & editing, Writing – original draft,

Validation, Formal analysis, Data curation. **Alexandre CHAMPAULT:** Writing – review & editing, Investigation, Formal analysis. **Julie SOM-KHIT:** Writing – review & editing, Investigation, Formal analysis. **Julie KNOERTZER:** Writing – review & editing, Supervision, Investigation, Formal analysis. **Assia BELKEBIR:** Writing – review & editing, Investigation, Formal analysis. **Bruno MEGARBANE:** Writing – review & editing, Writing – original draft, Validation, Supervision, Formal analysis. **Karine THIBAULT:** Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. **Dal Bo Gregory:** Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization.

Declaration of Competing Interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Acknowledgments

This work used the imagery and biological analysis platforms, and
the zootechnicians team of IRBA, supported by the Service de Santé des
Armées. Graphical abstract was created with BioRender.com. The
manuscript was proofread by Brandon Loveall, PhD (Improvence, FR).

ARRIVE Statement

All experimental procedures were approved by the animal ethics
committee of the Service de Santé des Armées according to the French
legislation (Directive 2010/63/UE, décret 2013–118).

Data availability

Data will be made available on request.

References

Abou-Donia, M.B., Siracuse, B., Gupta, N., Sobel Sokol, A., 2016. Sarin (GB, O-isopropyl methylphosphonofluoridate) neurotoxicity: critical review. Crit. Rev. Toxicol. 46, 845–875. https://doi.org/10.1080/10408444.2016.1220916.

Aman, S., Paul, S., Chowdhury, F.R., 2021. Management of organophosphorus poisoning: standard treatment and beyond. Crit. Care Clin. 37, 673–686. https://doi.org/10.1016/j.ccc.2021.03.011.
Angrand, L., Takillah, S., Malissin, I., Berriche, A., Cervera, C., Bel, R., Gerard, Q., Knoertzer, J., Baati, R., Kononchik, J.P., Megarbane, B., Thibault, K., Dal Bo, G., 2021. Persistent brainwave disruption and cognitive impairment induced by acute sarin surrogate sub-lethal dose exposure. Toxicology 456, 152787. https://doi.org/10.1016/j.tox.2021.152787.

Antonijevic, B., Stojiljkovic, M.P., 2007. Unequal efficacy of pyridinium oximes in acute organophosphate poisoning. Clin. Med. Res. 5, 71–82. https://doi.org/10.3121/cmr.2007.701.

Bajgar, J., Patocka, J., Jakl, A., Hrdina, V., 1975. Antidotal therapy and changes of acetylcholinesterase activity following isopropyl methylphosphofluoridate intoxication in mice. Acta Biol. Med. Ger. 34, 1049–1055.

Bannerman, D.M., Rawlins, J.N.P., McHugh, S.B., Deacon, R.M.J., Yee, B.K., Bast, T., Zhang, W.-N., Pothuizen, H.H.J., Feldon, J., 2004. Regional dissociations within the hippocampus—memory and anxiety. Neurosci. Biobehav. Rev. 28, 273–283. https://doi.org/10.1016/j.nebiuorev.2004.03.004.

Bashyal, B., Yadav, A., Deo, A.K., Kharel, K.K., Kharel, D., Panthi, B., 2023. Severe acute organophosphate poisoning managed with 2-month prolonged atropine therapy: a case report. Ann. Med. Surg. 85, 5179–5182. https://doi.org/10.1097/MS9.00000000000001207.

Bohnert, S., van den Berg, R.M., Mikler, J., Klaassen, S.D., Joosen, M.J.A., 2020. Pharmacokinetics of three oximes in a guinea pig model and efficacy of combined oxime therapy. Toxicol. Lett. 324, 86–94. https://doi.org/10.1016/j.toxlet.2020.01.013.

Burchfiel, J.L., Duffy, F.H., Van Sim, M., 1976. Persistent effects of sarin and dieldrin upon the primeate electroencephalogram. Toxicol. Appl. Pharmacol. 35, 365–379. https://doi.org/10.1016/0041-008x(76)90296-9.

Chambers, J.E., Meek, E.C., 2020. Novel centrally active oxime reactivators of acetylcholinesterase inhibited by surrogates of sarin and VX. Neurobiol. Dis. 133, 104487. https://doi.org/10.1016/j.nbd.2019.04.0478.

Da Silva, O., Probst, N., Landry, C., Hanak, A.-S., Warnault, P., Coisne, C., Calas, A.-G., Gosselet, F., Courageux, C., Gastellier, A.-J., Trancart, M., Baati, R., Dehouck, M.-P., Jean, L., Nachon, F., Renard, P.-Y., Dias, J., 2022. A new class of Bi- and triphenonal sugar oximes as antidotes against organophosphorus poisoning. J. Med. Chem. 65, 4649–4666. https://doi.org/10.1021/acs.jmedchem.1c01748.

Desor, F., El Hajj, A., Herzine, A., Djelti, F., Bombail, V., Denis, I., Oster, T., Malaplate, C., Lanhers, M.-C., Yen, F.T., Claudepierre, T., 2024. CRE mice exhibit hyperactive and impulsive behavior affecting their learning and retention performances. https://doi.org/10.1101/2024.02.27.582228.

Dixon, W.J., 1991. Staircase bioassay: the up-and-down method. Neurosci. Biobehav. Rev. 15, 47–50. https://doi.org/10.1016/s0149-7634(05)80090-9.

Ducottet, C., Belzung, C., 2004. Behaviour in the elevated plus-maze predicts coping after subchronic mild stress in mice. Physiol. Behav. 81, 417–426. https://doi.org/10.1016/j.physbeh.2004.01.013.

Eddleston, M., Buckley, N.A., Eye, P., Dawson, A.H., 2008. Management of acute organophosphorus pesticide poisoning. Lancet 371, 597–607. https://doi.org/10.1016/S0140-6736(07)61202-1.

Eddleston, M., Eye, P., Worek, F., Juszczak, E., Alder, N., Mohamed, F., Senarathna, L., Hittarage, A., Azher, S., Jeganathan, S., Jayamanne, S., von Meyer, L., Dawson, A.H., Sheriff, M.H.R., Buckley, N.A., 2009. Pralidoxime in acute organophosphorus insecticide poisoning—a randomised controlled trial. PLOS Med. 6, e100014. https://doi.org/10.1371/journal.pmed.1000104.

Eddleston, M., Roberts, D., Buckley, N., 2002. Management of severe organophosphorus pesticide poisoning. Crit. Care 6, 259. https://doi.org/10.1186/cc1499.

Ferchmin, P.A., Andino, M., Reyes Salaman, R., Alves, J., Velez-Roman, J., Cuadrado, B., Carrasco, M., Torres-Rivera, W., Segarra, A., Martins, A.H., Lee, J.E., Eterovic, V.A., 2014. 4R-cembranoid protects against diisopropylfluorophosphate-mediated neurodegeneration. Neurotoxicology 44, 80–90. https://doi.org/10.1016/j.neuro.2014.06.001.

Ferchmin, P.A., Pérez, D., Cuadrado, B.L., Carrasco, M., Martins, A.H., Eterović, V.A., 2015. Neuroprotection against diisopropylfluorophosphate in acute hippocampal slices. Neurochem. Res. 40, 2143–2151. https://doi.org/10.1007/s11064-015-1729-4.

François, S., Mondot, C., Gerard, Q., Bel, R., Knoertzer, J., Berriche, A., Cavallero, S., Baati, R., Orset, C., Dal Bo, G., Thibault, K., 2022. Long-term anxiety-like behavior and microbiota changes induced in mice by sublethal doses of acute sarin surrogate exposure. Biomedicines 10. https://doi.org/10.3390/biomedicines10051167.

van Helden, H.P.M., Vanwersch, R.A.P., Kuijpers, W.C., Trap, H.C., Philippens, I.H.C., Benschop, H.P., 2004. Low levels of sarin affect the EEG in marmoset monkeys: a pilot study. J. Appl. Toxicol. 24, 475–483. https://doi.org/10.1002/jat.1001.

HOBBIGER, F., SADLER, P.W., 1958. Protection of oximes of bis-pyridinium ions against lethal diisopropyl phosphonofluoridate poisoning. Nature 182, 1672–1673. https://doi.org/10.1038/1821672a0.

Jokanović, M., Stojiljković, M.P., Kovać, B., Ristić, D., 2020. Chapter 68 - Pyridinium oximes in the treatment of poisoning with organophosphorus compounds, in: In: Gupta, R.C. (Ed.), Handbook of Toxicology of Chemical Warfare Agents (Third Edition). Academic Press, Boston, pp. 1145–1159. https://doi.org/10.1016/B978-0-12-819090-6.00068-4.

Kishikawa, Y., Kawahara, Y., Yamada, M., Kaneko, F., Kawahara, H., Nishi, A., 2014. The spontaneously hypertensive rat/Izm (SHR/Izm) shows attention deficit/ hyperactivity disorder-like behaviors but without impulsive behavior: therapeutic implications of low-dose methylphenidate. Behav. Brain Res. 274, 235–242. https://doi.org/10.1016/j.bbr.2014.08.026.

Koplovitz, I., Stewart, J.R., 1994. A comparison of the efficacy of H16 and 2-PAM against soman, tabun, sarin, and VX in the rabbit. Toxicol. Lett. 70, 269–279. https://doi.org/10.1016/0378-4274(94)90121-x.

Kuca, K., Cabal, J., Kassa, J., 2005. In vitro reactivation of sarin-inhibited brain acetylcholinesterase from different species by various oximes. J. Enzym. Inhib. Med. Chem. 20, 227–232. https://doi.org/10.1080/14756360500043208.

Kuca, K., Jun, D., 2006. Reactivation of sarin-inhibited pig brain acetylcholinesterase using oxime antidotes. J. Med. Toxicol. 2, 141–146. https://doi.org/10.1007/BF03161181.

Kuznetsova, D.A., Gayanova, G.A., Vasilievia, E.A., Pavlov, R.V., Zueva, I.V., Babaev, V. M., Kuznetsov, D.M., Voloshina, A.D., Petrov, K.A., Zakharova, L.Y., Sinyashin, O.G., 2022. Oxime therapy for brain AChE reactivation and neuroprotection after organophosphate poisoning. Pharmaceutics 14. https://doi.org/10.3390/pharmaceutics14091950.

Langen, B., Dost, R., 2011. Comparison of SHR, WKY and Wistar rats in different behavioural animal models: effect of dopamine D1 and alpha2 agonists. Atten. Defic. Hyperact Disord. 3, 1–12. https://doi.org/10.1007/s12402-010-0034-y.

Liu, J., Pope, C., 1998. Comparative presynaptic neurochemical changes in rat striatum following exposure to chlorpyrifos or parathion. J. Toxicol. Environ. Health Part A 53, 531–544. https://doi.org/10.1080/009841098159123.

Ma, X., Zhang, Y., Wang, L., Li, N., Barkai, E., Zhang, X., Lin, L., Xu, J., 2020. The firing of theta state-related septal cholinergic neurons disrupt hippocampal ripple oscillations via muscarinic receptors. J. Neurosci. 40, 3591–3603. https://doi.org/10.1523/JNEUROSCI.1568-19.2020.

Mach, M., Grubbs, R.D., Price, W.A., Nagaoka, M., Dubovický, M., Lucot, J., 2008. Delayed behavioral and endocrine effects of sarin and stress exposure in mice. J. Appl. Toxicol. 28, 132–139. https://doi.org/10.1002/jat.1258.

Markand, O.N., 1984. Electroencephalography in diffuse encephalopathies. J. Clin. Neurophysiol. 1, 357–407.

McDonough, T.M., 1997. Neuropharmacological mechanisms of nerve agent-induced seizure and neuropathology. Neurosci. Biobehav. Rev. 21, 559–579. https://doi.org/10.1016/s0149-7634(96)00050-4.

Miyaki, K., Nishiwaki, Y., Maekawa, K., Ogawa, Y., Asukai, N., Yoshimura, K., Etoh, N., Matsumoto, Y., Kikuchi, Y., Kumagai, N., Omae, K., 2005. Effects of sarin on the nervous system of subway workers seven years after the Tokyo subway sarin attack. J. Occup. Health 47, 299–304. https://doi.org/10.1539/joh.47.299.

Murata, K., Araki, S., Yokoyama, K., Okumura, T., Ishimatsu, S., Takasu, N., White, R.F., 1997. Asymptomatic sequelae to acute sarin poisoning in the central and autonomic nervous system 6 months after the Tokyo subway attack. J. Neurol. 244, 601–606. https://doi.org/10.1007/s004150050153.

Narimatsu, E., Niiya, T., Kawamata, T., Kawamata, M., Yamakage, M., 2010. Effects of atropine and pralidoxime on neuronal actions of paraoxon in rat hippocampal slices. Neurosci. Res. 68, 276–284. https://doi.org/10.1016/j.neures.2010.08.008.

Nieminen, S.A., Lecklin, A., Heikkinen, O., Ylitalo, P., 1990. Acute behavioural effects of the organophosphates sarin and soman in rats. Pharmacol. Toxicol. 67, 36–40. https://doi.org/10.1111/j.1600-0773.1990.tb00778.x.

Ohtani, T., Iwanami, A., Kasai, K., Yamasue, H., Kato, T., Sasaki, T., Kato, N., 2004. Posttraumatic stress disorder symptoms in victims of Tokyo subway attack: a 5-year follow-up study. Psychiatry Clin. Neurosci. 58, 624–629. https://doi.org/10.1111/j.1440-1819.2004.01313.x.

Okudera, H., 2002. Clinical features on nerve gas terrorism in Matsumoto. J. Clin. Neurosci. 9, 17–21. https://doi.org/10.1054/jcnn.2001.1020.

Okumura, T., Hisaoka, T., Naito, T., Isonuma, H., Okumura, S., Miura, K., Maekawa, H., Ishimatsu, S., Takasu, N., Suzuki, K., 2005. Acute and chronic effects of sarin exposure from the Tokyo subway incident. Environ. Toxicol. Pharmacol. 19, 447–450. https://doi.org/10.1016/j.etap.2004.12.005.

Okumura, T., Suzuki, K., Fukuda, A., Kohama, A., Takasu, N., Ishimatsu, S., Hinohara, S., 1998. The Tokyo subway sarin attack: disaster management, Part 2: hospital response. Acad. Emerg. Med. 5, 618–624. https://doi.org/10.1111/j.1553-2712.1998.tb02471.x.

Özçoban, M.A., Tan, O., 2025. Electroencephalographic markers in major depressive disorder: insights from absolute, relative power, and asymmetry analyses. Front. Psychiatry 15, 1480228. https://doi.org/10.3389/fpsyt.2024.1480228.

Pawlak, C.R., Karrenbauer, B.D., Schneider, P., Ho, Y.-J., 2012. The elevated plus-maze test: differential psychopharmacology of anxiety-related behavior. Emot. Rev. 4, 98–115. https://doi.org/10.1177/1754073911421374.

Philippens, I.H., Melchers, B.P., de Groot, D.M., Wolthuis, O.L., 1992. Behavioral performance, brain histology, and EEG sequela after immediate combined atropine/diazepine treatment of soman-intoxicated rats. Pharmac. Biochem. Behav. 42, 711–719. https://doi.org/10.1016/0091-3057(92)90019-c.

Reddy, V.K., Deshpande, S.S., Cintra, W.M., Scoble, G.T., Albuquerque, E.X., 1991. Effectiveness of oximes 2-PAM and H-6 in recovery of muscle function depressed by organophosphate agents in the rat hemidiaphragm: an in vitro study. Fundam. Appl. Toxicol. 17, 746–760. https://doi.org/10.1016/0272-0590(91)90182-4.

Schwab, B.W., Hand, H., Costa, L.G., Murphy, S.D., 1981. Reduced muscarinic receptor binding in tissues of rats tolerant to the insecticide disulfuron. Neurotoxicology 2, 635–647.

Shi, J., Liu, D., Jin, Q., Chen, X., Zhang, R., Shi, T., Zhu, S., Zhang, Y., Zong, X., Wang, C., Li, L., 2023. Whole-transcriptome analysis of repeated low-level sarin-exposed rat hippocampus and identification of cerna networks to investigate the mechanism of sarin-induced cognitive impairment. Biology 12. https://doi.org/10.3390/biology12040627.

Shih, T.-M., Guarisco, J.A., Myers, T.M., Kan, R.K., McDonough, J.H., 2011. The oxime pro-2-PAM provides minimal protection against the CNS effects of the nerve agents sarin, cyclosarin, and VX in guinea pigs. Toxicol. Mech. Methods 21, 53–62. https://doi.org/10.3109/15376516.2010.529190.

Shih, T.-M., Rowland, T.C., McDonough, J.H., 2007. Anticonvulsants for nerve agent-induced seizures: The influence of the therapeutic dose of atropine. J. Pharmacol. Exp. Ther. 320, 154–161. https://doi.org/10.1124/jpet.106.1111252.
Shih, T.-M., Skovira, J.W., O'Donnell, J.C., McDonough, J.H., 2009. Evaluation of nine oximes on in vivo reactivation of blood, brain, and tissue cholinesterase activity inhibited by organophosphorus nerve agents at lethal dose. Toxicol. Mech. Methods 19, 386–400. https://doi.org/10.1080/15376510903213892.

Sidell, F.R., Groff, W.A., 1974. The reactivability of cholinesterase inhibited by VX and sarin in man. Toxicol. Appl. Pharmacol. 27, 241–252. https://doi.org/10.1016/0041-008x(74)90195-1.

Smith, A.P., 1993. Long-term effects of the anticholinesterases sarin and soman on latencies of muscle action potentials in mouse diaphragm muscle. J. Pharm. Pharmacol. 45, 176–181. https://doi.org/10.1111/j.2042-7158.1993.tb05528.x.

Sugiyama, A., Matsuoka, T., Sakamune, K., Akita, T., Makita, R., Kimura, S., Kuroiwa, Y., Nagao, M., Tanaka, J., 2020. The Tokyo subway sarin attack has long-term effects on survivors: a 10-year study started 5 years after the terrorist incident. PLOS One 15, e0234967. https://doi.org/10.1371/journal.pone.0234967.

Talabani, J.M., Ali, A.I., Kadir, A.M., Rashid, R., Samin, F., Greenwood, D., Hay, A., 2018. Long-term health effects of chemical warfare agents on children following a single heavy exposure. Hum. Exp. Toxicol. 37, 836–847. https://doi.org/10.1177/0960327117734620.

Trošt Bobić, T., Sečić, A., Zavoreo, I., Matijević, V., Filipović, B., Kolak, Ž., Basić Kes, V., Ciliga, D., Sajković, D., 2016. The impact of sleep deprivation on the brain. Acta Clin. Croat. 55 469–473. https://doi.org/10.20471/acc.2016.55.03.17.

Ueno, K.-i, Togashi, H., Mori, K., Matsumoto, M., Ohashi, S., Hoshino, A., Fujita, T., Saito, H., Minami, M., Yoshioka, M., 2002. Behavioural and pharmacological relevance of stroke-prone spontaneously hypertensive rats as an animal model of a developmental disorder. Behav. Pharmacol. 13, 1–13. https://doi.org/10.1097/00008877-200202000-00001.

Worek, F., Thiermann, H., Szinicz, L., Eyer, P., 2004. Kinetic analysis of interactions between human acetylcholinesterase, structurally different organophosphorus compounds and oximes. Biochem. Pharmacol. 68, 2237–2248. https://doi.org/10.1016/j.bcp.2004.07.038.

Yagle, K., Costa, L.G., 1996. Effects of organophosphate exposure on muscarinic acetylcholine receptor subtype mRNA levels in the adult rat. Neurotoxicology 17, 523–530.

Yanagisawa, N., Morita, H., Nakajima, T., 2006. Sarin experiences in Japan: acute toxicity and long-term effects. J. Neurol. Sci. 249, 76–85. https://doi.org/10.1016/j.jns.2006.06.007.

Yang, D.-F., Huang, W.-C., Wu, C.W., Huang, C.-Y., Yang, Y.-C.S.H., Tung, Y.-T., 2023. Acute sleep deprivation exacerbates systemic inflammation and psychiatry disorders through gut microbiota dysbiosis and disruption of circadian rhythms. Microbiol. Res. 268, 127292. https://doi.org/10.1016/j.micres.2022.127292.

Zhao, Y., Guo, Y., Wu, D., Zhang, J., Zheng, S., Xie, W., Wang, K., Tian, Y., 2025. Alpha oscillatory dysregulation: mapping EEG oscillatory in suicidal depression. Front. Hum. Neurosci. 19, 1582330. https://doi.org/10.3389/fnhum.2025.1582330.