**1. Introduction**

Several recent high-profile uses of chemical warfare agents (CWA) derived from organophosphorus (OP) compounds have brought them back to attention in the past few years. These include their use in the Syrian conflict (2013–2017) and three separate targeted attacks between 2017 and 2020 resulting in Kim Jong-Nam's death and the poisonings of Sergei Skripal and Alexei Navalny [1]. These events all took place more than two decades after the first documented uses of OP nerve agents (NA) during the Iran–Iraq armed

**Citation:** François, S.; Mondot, S.; Gerard, Q.; Bel, R.; Knoertzer, J.; Berriche, A.; Cavallero, S.; Baati, R.; Orset, C.; Dal Bo, G.; et al. Long-Term Anxiety-like Behavior and Microbiota Changes Induced in Mice by Sublethal Doses of Acute Sarin Surrogate Exposure. *Biomedicines* **2022**, *10*, 1167. https:// doi.org/ 10.3390/biomedicines10051167

Academic Editor: María Morell Hita

Received: 25 February 2022 Accepted: 16 May 2022 Published: 18 May 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

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conflict and in two terrorist attacks in Japan, for which long-term neurological sequelae are still emerging [2,3]. However, the NA dose exposure could not be quantified in either of these reports, and while the described NA exposure was combined with sulfur mustard in Talabani et al. [3], similar symptoms were observed particularly in visual (lacrimation, impaired ability to focus, ocular pain, etc.) and neuropathophysiological areas (headache, numbness, confusion, agitation, etc.) as well as in less-suspected organs (abdominal pain, nausea, and a higher prevalence of respiratory infection). Post-traumatic stress responses were also highly enhanced in sarin-exposed Tokyo victims, correlating with observations made in Gulf War (GW) veterans exposed to high doses of OP (dichlorvos) and carbamate (lindane, bendiocard) pesticides and pyridostigmine bromide (PB) pills and/or NA [4]. These reports therefore highlight the need to meticulously investigate the long-term effects of NA exposure.

Pathophysiologically, NA, OP and carbamate pesticides as well as PB inhibit cholinesterase (ChE), leading to toxic hypercholinergy throughout the body. At the cellular level, acetylcholine accumulation appears at neuromuscular junctions and in synapses, inducing any number of symptoms including fasciculation, hypersecretion, muscle contractions, tetany, tremors and convulsion [5]. Acute exposure to large doses of OP results in muscle paralysis, respiratory distress and ultimately death in only a few minutes postexposure. This can be avoided by providing an antidote therapy containing a muscarinic cholinergic receptor antagonist (atropine sulfate) and an oxime (pralidoxime, asoxime, etc.) with a strong nucleophile to reactivate the OP-inhibited acetylcholinesterase (AChE). Anticonvulsant medications are also added to the antidote therapy to avoid epileptic seizures and subsequent brain damage [6,7]. However, animal models have revealed that even if the pharmaceutical-based intervention stabilizes the convulsions and improves survival, it is still not enough to avoid long-term neurologic deficits [8]. In addition, other animal models exposed to lower doses of OP have also exhibited long-term neuropsychological dysfunctions, specifically anxiety, depression and cognitive deficits [8–10]. These results correlate with the delayed neurological consequences observed in humans after acute OP intoxication, specifically regarding anxiety-related behavior and cognitive deficits [4,11–13]. Nevertheless, clarification is needed in animal models, as a single low-dose exposure to OP induces only transient behavioral changes [14] and long-term deficits require concurrent stress [10] or repeated exposure to NA [14–16].

Restrictions regulating CWA use and storage are a major restraint to the study of OP-based NA neurotoxicity. Recently, using the sarin surrogate 4-nitrophenyl isopropyl methylphosphonate (NIMP), we developed a murine model to facilitate investigation in CWA-unauthorized laboratories [17]. NIMP is less toxic than sarin, but it has proven itself to be highly effective at inhibiting ChE in vitro and in vivo [18–22]. Furthermore, it can reproduce several features of sarin intoxication, including seizure-like behavior, cortical and hippocampal neuropathologies, neuroinflammatory processes and memory impairment in rodents [17,18,20,22]. These findings thus offer grea<sup>t</sup> promise for the study of the consequences of longer-term NIMP exposure in order to better anticipate neurologic sequelae induced by NA exposure.

The aim of this study was to characterize the long-term effects of a single NIMP exposure in mice by evaluating two different sublethal OP doses (0.5 and 0.9 LD50) for over 6 months. For this, we evaluated cerebral and blood ChE inhibition, neuroinflammation, systemic inflammation, behavioral modifications, anatomical and diffusion magnetic resonance imaging (MRI) and gu<sup>t</sup> microbiota impacts at different timepoints for up to 6 months post-exposure. Our results demonstrate that exposure to a low dose of NA has the potential to disrupt gu<sup>t</sup> microbiota and immune homeostasis as well as alter emotional behavior in the long-term.

### **2. Materials and Methods**
