# Enhancing oxime efficacy into brain using ultrasound to counteract nerve agent exposure

Lépinard Lucie, Leterrier Sarah, Jourdain Laurène, Turri Louise, Belkebir Assia, Knoertzer Julie, Champault Alexandre, Bel Rosalie, Selingue Erwan, Mériaux Sébastien, et al.

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## ► To cite this version:

Lépinard Lucie, Leterrier Sarah, Jourdain Laurène, Turri Louise, Belkebir Assia, et al.. Enhancing oxime efficacy into brain using ultrasound to counteract nerve agent exposure. Biomedicine and Pharmacotherapy, 2025, 187, pp.118120. 10.1016/j.biopha.2025.118120. hal-05088653

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Distributed under a Creative Commons Attribution 4.0 International License
Enhancing oxime efficacy into brain using ultrasound to counteract nerve agent exposure

Lépinard Lucie<sup>a,b</sup>, Leterrier Sarah<sup>b</sup>, Jourdain Laurène<sup>b</sup>, Turri Louise<sup>a</sup>, Belkebir Assia<sup>a,b</sup>, Knoertzer Julie<sup>a</sup>, Champault Alexandre<sup>a,b</sup>, Bel Rosalie<sup>a</sup>, Selingue Erwan<sup>c</sup>, Mériaux Sébastien<sup>c</sup>, Larrat Benoit<sup>c</sup>, Tournier Nicolas<sup>b</sup>, Dal Bo Grégory<sup>a,b,1</sup>, Thibault Karine<sup>a,b,1</sup>, Novell Anthony<sup>b,e,1</sup>

ARTICLE INFO

Keywords:

Focused ultrasound

Nerve agent

Acetylcholinesterase

Oximes

Blood-brain barrier opening

A B S T R A C T

Organophosphates (OP) found in pesticides and chemical weapons irreversibly inhibit acetylcholinesterases (AChE) and cause toxic accumulation of acetylcholine throughout the organism. Due to their lipophilicity, OP easily cross the blood-brain barrier (BBB) and affect the central nervous system (CNS), resulting in epileptic seizures and long-term cognitive impairment. The antidote includes oximes which reactivate inhibited AChE. Unfortunately, oximes have limited BBB penetration and therefore fail to prevent neurological damage. Improving the penetration of oximes through the CNS and their therapeutic effect on the brain, is a major challenge. Recent studies have demonstrated the efficacy of transcranial focused ultrasound (FUS), in combination to intravenously injected microbubbles, to transiently disrupt the BBB for drug delivery. We assessed the efficacy of FUS to deliver two known oximes (2-PAM, HI-6) into the brain and reactivate AChE following an exposure to VX in a mouse model. After both sub-lethal and supra-lethal exposure, HI-6 + FUS treatment reactivated nearly 30% more AChE in the hippocampus than HI-6 alone. In contrast, 2-PAM+FUS was not effective. Furthermore, animals treated with HI-6 + FUS following an exposure to a supra-lethal dose of VX exhibited enhanced short-term recovery and an increased 24 hours survival rate. Finally, up to 7 days after exposure to a supra-lethal dose of VX, HI-6 + FUS showed a significant reduction of pro-inflammatory cytokines IL-6 and MIP-1α expression levels in the hippocampus. Thus, the use of FUS is very promising for improving the medical care of OP exposure because it enables antidotes to treat central symptoms and it may reduce brain damage.

# 1. Introduction

Organophosphate (OP) compounds were originally synthesized for pesticide use (chlorpyrifos, malathion). Despite a collective effort to restrict OPs since the Chemical Weapons Convention was implemented in 1997, they are still employed in some regions of the world. Several million people are exposed each year, resulting in 100000 deaths [18]. Furthermore, some OPs have been developed and used as chemical weapons since the mid-20th century and represent an additional threat. For example, sarin and tabun were used for military purposes during the

armed conflict between Iran and Iraq in the 1980s and during the civil war in Syria [51]. Sarin was also used in a chemical attack on a Tokyo subway train in 1995 [43] resulting in 13 fatalities and affecting over 6000 individuals [60]. More recently, Kim Jong-Nam was assassinated by VX exposure. The threat posed by OP is real and current, affecting both civilian and military populations.

Chemical warfare nerve agents are lipophilic, which facilitates their crossing of the blood-brain barrier (BBB). Consequently, intoxication impacts both the central nervous system (CNS) and peripheral system. The neurotoxicity of these compounds is due to their irreversible

<footnote><sup>a</sup> IRBA, Département Toxicologie et Risques Chimiques, Brétigny-sur-Orge, France</footnote>

<footnote><sup>b</sup> Université Paris-Saclay, CEA, CNRS, Inserm, BioMaps, SHFJ, Orsay, France</footnote>

<footnote><sup>c</sup> Université Paris-Saclay, CEA, CNRS, BAOBAB, Neurospin, Gif-sur-Yvette, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: anthony.novell@universite-paris-saclay.fr (N. Anthony).</footnote>

<footnote n="1">¹ equal contribution</footnote>
inhibitory activity on acetylcholinesterase (AChE). This enzyme normally degrades acetylcholine (ACh) in the synaptic cleft. When the cholinesterase (ChE) is inhibited by OPs, ACh is no longer degraded and accumulates in the synapse [13]. This results in hyperactivity of muscarinic and nicotinic ACh receptors, leading to peripheral and central hypercholinergia. An exposed individual will present muscarinic, nicotinic and central syndromes. The muscarinic syndrome is characterised by hypersecretions while nicotinic syndrome presents with fasciculation, tachycardia and convulsions [4]. At the central level, cholinergic hyperactivity induces hyperexcitation of glutamatergic neurons which may results in developing epileptic seizures that may become recurrent over time [4]. A previous study reported the onset of cognitive impairment one month after exposure to a sarin surrogate, the 4-nitrophenyl isopropyl methylphosphonate [2]. Sugiyama et al. collected and analysed data from 747 victims of the Tokyo sarin attack up to 15 years after exposure. Authors reported long-term fatigue, headache and visual symptoms, post-traumatic stress disorder, and numbness of a limb suggesting the onset of long-term distal neuropathy [60]. OP exposure therefore induces not only severe symptoms in the acute-phase, but also persistent symptoms for years afterwards, even after exposure to a non-lethal dose.

The current medical countermeasure for OPs combines an anti-muscarinic, an anticonvulsant, and an oxime to reactivate AChE [19]. The anti-muscarinic employed is atropine sulfate [48] which competitively opposes the muscarinic effects of ACh by antagonizing its receptors, thereby preventing the development of cholinergic syndrome. Oximes reactivate the AChE by breaking the bond between the OP and the enzyme and release the AChE catalytic site. The first synthesized oxime is pralidoxime (2-PAM) but many other oximes such as asoxime (HI-6) or trimedoxime bromide (TMB-4) were subsequently developed [61]. While the oximes effectiveness varies according to the toxicant [3], their main limitation is their inability to effectively cross the BBB [41]. Consequently, while these therapeutic agents exert a beneficial effect at the peripheral level, they are unable to reactivate AChE within the CNS. This does not prevent the onset of serious neurological damage or epileptic seizures at a later stage. Therefore, anticonvulsant like diazepam is needed to limit epileptic seizures [38]. It is therefore essential to develop an alternative therapeutic strategy to avoid the consequences of OP exposure in the CNS.

Two decades ago, focused ultrasound (FUS) has emerged as a relevant approach for transient BBB disruption and drug delivery in the brain [30]. Basically, low-intensity FUS are applied to the brain subsequent to the intravenous injection of microbubbles [14,29]. Then, the microbubbles oscillate under the effect of the ultrasound and transiently disrupt the BBB by exerting mechanical stress on the endothelial cells [57]. A large number of studies have demonstrated the effectiveness of this method in recent years, in both preclinical and clinical settings [26, 47,55]. Indeed, FUS is currently being investigated in clinical trials for delivery of an anticancer agent to the brain in glioblastoma [6,9,11,58], for the treatment of Alzheimer's disorder [39,54] or the treatment of amyotrophic lateral sclerosis [1]. Furthermore, the safety of this technique has also been rigorously assessed, and no major adverse effects have been reported as long as acoustic parameters are controlled [10,46, 49]. The opening of the BBB is transient, and integrity is restored within 24 hours [1,15,42]. Therefore, FUS could be used in the context of treatment of OP intoxication to facilitate the passage of oxime into the brain.

In the context of OP exposure, which affects all cerebral structures, the opening of the BBB induced by FUS will require to cover a large area of the brain. Therefore, the objective of this study was to perform a non-invasive ultrasound protocol that permits an extensive BBB opening [22] to deliver oximes into the brain of mice following VX exposure. To assess the approach's efficacy in preventing cerebral damage, we first measured AChE enzymatic activity in both peripheral and cerebral structures following exposure to VX at both sub-lethal (0.5 LD₅₀) and supra-lethal (17 LD₅₀) doses. Then, we investigated whether a

combination treatment of oxime and FUS after supra-lethal exposure to VX improved short-term recovery and reduced neuroinflammation.

## 2. Materials and methods

### 2.1. Animals

All animal procedures were performed in accordance with the European Directive 2010/63/EU on the protection of animals used for scientific purposes. The experimental protocols were approved by the French "Service de Santé des Armées" animal ethics committee and the local ethic committee n°44. A total of 162 male Swiss mice (Janvier Labs, Le Genest-Saint-Isle, France), 9 weeks old (mean weight: 35-40 g) at the time of the experimentation, were used. Male-only animals were used as they are more sensitive to OP, therefore provide the most optimal assessment of the effects of poisoning and treatment effects [44, 50,64]. The animals were housed 3-4 per cage in an enriched environment (nesting material, tunnel) in a 12 h/12 h light/dark cycle temperature-controlled room (22 ± 2 °C). The food and the water were available *ad libitum*. All mice were acclimated 14 days before experimentation.

### 2.2. FUS-induced BBB opening

All interventions on animals were performed under isoflurane anaesthesia (2-2.5 % isoflurane in a mixture of air/O₂, v/v). The mice's head was shaved with a trimmer and depilatory cream to allow acoustic coupling. A catheter was placed in the tail vein before positioning the animal in a stereotaxic frame. A 1.5 MHz ultrasound probe (5 mm axial and 1 mm focal spot, Imasonic, Voray sur l'Ognon, France), focused at 20 mm depth, was mounted on an XYZ motorized stage and connected to a single-channel programmable generator (Image Guided Therapy, Pessac, France). The probe was placed above the mouse's head. The acoustic coupling was ensured by a latex balloon filled with deionized and degassed water and ultrasound gel spread on the top of the head. A few seconds before initiating the ultrasound sequence, the microbubbles (50 µL; SonoVue, Bracco, Italy) were intravenously injected. A continuous XY raster scan (6 mm × 6 mm, duration 5 s) was repeated 25 times for a total exposure of 127 seconds. The acoustic pressure transmitted in the mouse brain is estimated at 340 kPa, considering a skull attenuation of 40 % at 1.5 MHz measured on *ex vivo* samples of Swiss mouse skulls (Fig. 1A).

### 2.3. Validation of the FUS protocol using MRI

A total of 4 Swiss mice (1 control without FUS and 3 FUS) were subjected to magnetic resonance imaging (MRI) to confirm the efficacy of the FUS protocol in opening the BBB for drug delivery. Three mice underwent MRI following FUS-induced BBB opening, and one mouse underwent the same protocol without ultrasound (sham procedure). To do this, mice were i.v. injected with 100 µL of MR contrast agent, gadolinium, (Dotarem, Molecular Weight: 753.9 g/mol, Guerbet, France) and microbubbles. Approximately 5 min after FUS sequence, contrast-enhanced T₁-weighted MRI were acquired with a 7 T/90 mm MRI scanner (Pharmascan scanner, Bruker, Germany) using a MSME sequence (TE/TR = 5/300 ms, matrix = 128 × 128 × 20, resolution = 0.15 × 0.15 × 0.4 mm³, 12 averages, acquisition time = 7 min 41 sec) to evaluate the efficiency of BBB disruption. A day after FUS exposure, T₂-weighted MRI were performed on the same mice using a RARE sequence (TE/TR = 22/3000 ms, matrix = 128 × 128 × 14, resolution = 0.15 × 0.15 × 0.6 mm³, 6 averages, acquisition time = 5 min 48 sec) to validate the safety of FUS protocol (Fig. 1C-E).

### 2.4. VX challenges and treatments

VX (LD₅₀ = 13 µg/kg; 97 % purity determined by NMR) was provided
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<figure><img src="image_7.png" /><figcaption>**Fig. 1.** (A) Protocol design for VX challenge and FUS-induced BBB opening. The FUS trajectory (6 × 6 mm) on the mouse brain is displayed (blue circle: starting point, red circle: ending point). (B) Flowchart of mice distribution in each experimental group. (C) T₁-weighted MRI image acquired 10 min after the injection of gadolinium without FUS. (D) T₁-weighted MRI image acquired 10 min after the gadolinium injection and the FUS-induced BBB opening. (E) T₂-weighted MRI image acquired 24 h after FUS-induced BBB opening.</figcaption></figure>
by DGA maîtrise NRBC (Vert-le-Petit, France). Two protocols using different doses of VX were performed to evaluate the benefit of FUS-induced BBB opening in exposed mice. The comparison of the antidote efficiency was evaluated on an association of atropine sulfate monohydrate [21] (3 mg/kg diluted in NaCl) with either asoxime dimethane sulfonate (HI-6 DMS, Molecular Weight = 356.9 g/mol, 99 % purity; NOAEL dose = 68 µmol/kg equivalent to a 6.8 mM solution injected at 10 mL/kg) or pralidoxime methyl sulfate (2-PAM MS, Molecular Weight: 137.2 g/mol, 99 % purity; NOAEL dose = 248.25 µmol/kg equivalent to 24.8 mM solution injected at 10 mL/kg, both doses has been determined with the same method used in [63] (Fig. 1A). Treatments were provided by the Pharmacie Centrale des Armées (Orléans, France).

### 2.4.1. Sub-lethal VX challenge

A total of 60 mice were randomly assigned to one of the 8 groups. Group 1: no VX nor an antidote, but with FUS (NaCl-NaCl-FUS, n = 9); Group 2: with VX exposure, without antidote, and with FUS (VX-NaCl-FUS, n = 9); Groups 3 and 5: with VX and one of the antidotes (HI-6 or 2-PAM, respectively) without FUS (VX-HI-6/2-PAM-no FUS, n = 11 and 10, respectively); Groups 4 and 6: with VX and one of the antidotes (HI-6 or 2-PAM, respectively), and with FUS (VX-HI-6/2-PAM-FUS, n = 11 and 10, respectively). Additionally, 16 mice (NaCl-NaCl, n = 8 and VX-NaCl, n = 8) not subjected to FUS protocol (i.e., no ultrasound, no microbubbles) were used to evaluate the impact of FUS in intoxicated mice. The mice received a single sub-cutaneous injection (10 mL/kg) of VX freshly diluted in NaCl 0.9 % at 0.5 LD₅₀ dose. The control mice were injected with the same volume of NaCl without VX. The antidote (10 mL/kg) was intraperitoneally injected 1 h following the VX exposure, corresponding to the maximal inhibition of blood ChE [33]. Approximately 75 min after VX exposure and 15 min after antidote injection, the animals were exposed to FUS for BBB opening. Protocols and groups are summarized in Fig. 1A and 1B. Then, the animals were placed in individual cage under a heat lamp until awakening. The control mice without FUS followed the identical protocol including shaving, stereotactic placement and microbubble injection, and received a sham ultrasound sequence (pressure = 0 kPa).

### 2.4.2. Supra-lethal VX challenge

A total of 82 mice were randomly assigned in one of the 4 groups. Group 1: no VX nor antidote but with FUS (NaCl-NaCl-FUS, n = 14); Group 2: no VX, with HI-6 and with FUS (NaCl-HI-6-FUS, n = 15); Groups 3: with VX and HI-6 without FUS (VX-HI-6-no FUS, n = 26); Group 4: with VX and HI-6, and with FUS (VX-HI-6-FUS, n = 27). Under anaesthesia, the mice received a single sub-cutaneous injection (10 mL/kg) of VX diluted in 0.9 % NaCl at a dose corresponding to a fraction (0.7) of HI-6-protective index (PI = 25.21; PI determined with the same method used in [8] and this formula:

$$PI(HI-6) = \frac{LD_{50}(VX + HI-6)}{LD_{50}(VX)}$$

0.7 PI<sub>H1-6</sub> corresponding to 17 LD₅₀ of VX. Control mice were injected with the same volume of NaCl without VX. The antidote (10 mL/kg) was intraperitoneally injected 1 minute after VX exposure. At this supra-lethal dose of VX, oxime must be administered very rapidly to avoid the certain death of the animal within minutes of intoxication. Thus, the effect of ultrasound alone (without oximes) cannot be tested on these high-dose intoxicated animals. Approximately 3 minutes after the VX exposure and 2 minutes after the antidote injection, animals were

exposed to FUS for BBB opening. Protocols and groups are summarized in Fig. 1A and B. Then, the animals were placed in individual cages under a heat lamp until awakening. The days following VX challenge, all animals were daily weighted and behaviourally monitored (blind scoring) to evaluate their recovery. Behavioural parameters including: fur aspect, kyphosis, respiratory difficulty, activity, lacrimation, tremor, aggressiveness, muscle tone, weight loss, are scored according their severity. The scale starts at 0, corresponding to an animal with no apparent symptoms. Each symptom listed above is deducted 1 or 2 points, depending on its severity, meaning that the lower the score (negative value), the poorer the recovery after VX exposure (Supp Table 1).

### 2.5. Tissue collection

In the VX sub-lethal study, all animals were euthanized 2 h after exposure to the toxicant. In the supra-lethal VX study, animals were euthanized 24 h [(NaCl-NaCl-FUS, n = 5); (NaCl-HI-6-FUS, n = 5); (VX-HI-6-no FUS, n = 6); (VX-HI-6-FUS, n = 8)], 72 h [(NaCl-NaCl-FUS, n = 4); (NaCl-HI-6-FUS, n = 5); (VX-HI-6-no FUS, n = 8); (VX-HI-6-FUS, n = 9)] or 7 d [(NaCl-NaCl-FUS, n = 5); (NaCl-HI-6-FUS, n = 5); (VX-HI-6-no FUS, n = 8); (VX-HI-6-FUS, n = 9)] after exposure. For euthanasia, the mice received a subcutaneous injection of buprenorphine (Bupredine®, 0.1 mg/kg) 40 min before being deeply anesthetized with isoflurane (5 %). Following a thoracic surgery, a sample of blood was collected directly from the left ventricle and directly frozen. Then, the mice were transcardially perfused with 15 mL of cold 0.9 % NaCl. The mice brain was quickly removed and micro-dissected to collect the motor cortex, the somatosensory cortex, the combined zone including piriform cortex and amygdala, and the hippocampus. Additionally, the diaphragm and lumbar spinal cord were collected. Each structure was placed into microcentrifuge tubes and stored at -80 °C.

### 2.6. Tissue grinding

The samples were homogenized in 300 µL (or 500 µL for hippocampus and diaphragm) of 0.5 M phosphate buffer (pH 7.4) diluted in ultrapure milliQ water + 0.5 % Tween 20 using a bead mill homogenizer (OMNI Bead Ruptor Elite, Dutscher, France) with 1.4 mm ceramic beads. The lysates were kept on ice for 15 min and centrifuged at 10,000 G (4 °C) for 10 min. The supernatants were stored at -80 °C.

### 2.7. Cholinesterase inhibition assay

The total protein concentration was determined using DC Protein Assay kit (Bio-Rad) according to the manufacturer's protocol. The AChE activity was determined using the Ellman method [20]. Briefly, 5 µL from each sample was added to 175 µL of Ellman buffer (0.1 mg DTNB/mL phosphate buffer 0.1 M pH 7.4) supplemented with ethoxypropazine hydrochloride (0.1 mM final concentration). After a 15 min baseline reading to account for the thiols present in the samples, 20 µL of acetylthiocholine (1 mM final concentration, Sigma Aldrich, USA) was added to each well and the reaction between thiocholine and DTNB was monitored for 30 min at 412 nm (or 437 nm for blood samples) and at 25 °C in a microplate reader (Tecan Spark, Switzerland). All samples were assayed in duplicate. The enzyme activity of each sample was related to the total protein concentration previously determined and expressed as percentage of the average control (NaCl-NaCl-FUS) activity in this way:

$$ \text{Percentage of enzymatic activity} = \frac{\text{Individual enzymatic activity}}{\text{Average enzyme activity of control group}} \times 100 $$
Final results are expressed as the mean percentage and standard
deviation of activity for each group.

## 2.8. Multiplex cytokine assay

The hippocampal concentration of IL-6, IL-10 and MIP-1α was quantified with the Milliplex Mouse Cytokine/Chemokine Magnetic Bead Panel kit MCYTMAG specially designed containing 3 cytokines (Millipore, Sigma Aldrich, USA) and a MagPix reader (Luminex, Thermo Fisher, USA).

## 2.9. Statistics

The data were expressed as means ± standard deviation (SD), and analysed using PRISM 9 software (GraphPad, USA). The results are illustrated with whisker boxes. One-way ANOVA with Tukey's post hoc test was employed to ascertain significant differences in AChE activities and cytokine expression levels between groups. A non-parametric one-way ANOVA (Kruskal-Wallis) with Dunn's post hoc test was employed to determine significant differences in recovery scores between groups. For the analysis of weight evolution, a two-way repeated measures ANOVA with Tukey's post hoc test was employed to determine significant differences between groups. All tests were performed with an alpha significance level of 0.05. Significance between groups is denoted by \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001, or \*\*\*\*p < 0.0001.

# 3. Results

Prior to this study, the efficacy of the FUS protocol was confirmed by MRI on 4 mice (1 control + 3 FUS). An example is given for a sham mouse without FUS (Fig. 1C), and for a mouse subjected to the FUS protocol (Fig. 1D). MRI contrast is increased along the ultrasound trajectory (6 mm × 6 mm) and throughout the depth of the brain (approx. 7 mm). Control MRI, performed 24 h after the FUS protocol, confirmed the absence of visible brain damage (Fig. 1E).

## 3.1. Sub-lethal VX challenge

The AChE activity was assessed 2 h after sub-lethal VX challenge in CNS structures (hippocampus, somatosensory cortex, motor cortex, piriform cortex/amygdala, spinal cord), in blood and in diaphragm (Fig. 2A). As expected, the AChE residual activity in the VX-NaCl-FUS group was significantly reduced in comparison to the VX-free control group across all CNS structures (p < 0.0001 for all structures) (Fig. 2B-F and Supp Table 2). In the blood, the inhibition of AChE was almost total (p < 0.0001) (Fig. 2G and Supp Table 2). Additionally, AChE inhibition was quantified in peripheral structure (diaphragm, p < 0.0001) (Fig. 2H). As expected, the oximes induced a significant reactivation of AChE in blood and diaphragm (Fig. 2G and H), with a higher efficiency for HI-6 in diaphragm than for 2-PAM (p < 0.0001) (Fig. 2H and Supp Table 2). However, without FUS exposure, no significant AChE reactivation was noticed in any CNS structure despite the treatment with an oxime (2-PAM or HI-6) (Fig. 2B-F and Supp Table 2).

Nonetheless, a significant reactivation of cerebral AChE was quantified in VX-HI-6-FUS group in comparison to VX-HI-6-no FUS group in all cerebral areas: p = 0.0015 in the hippocampus (Fig. 2B and Supp Table 2), p = 0.0006 in the somatosensory cortex (Fig. 2C and Supp Table 2), p = 0.0218 in the motor cortex (Fig. 2D and Supp Table 2), and p = 0.0310 in the piriform cortex/amygdala (Fig. 2E and Supp Table 2). However, the FUS did not significantly enhance AChE residual activity when 2-PAM was administered (Fig. 2B-E). It is noteworthy that the combination of FUS (ultrasonic waves were transmitted in the brain) and HI-6 did not improve AChE activity in the spinal cord in comparison to HI-6 without FUS (p = 0.4073) (Fig. 2F and Supp Table 2). Moreover, as expected, the FUS protocol did not enhance blood AChE reactivation in comparison to the HI-6 treated mice without FUS (p > 0.9999),

neither in diaphragm (p > 0.9999) (Fig. 2G and H and Supp Table 2). Finally, the FUS protocol did not increased VX penetration into the brain. Indeed, AChE inhibitions in all cerebral structures quantified in VX-NaCl-FUS control group were similar to AChE inhibition observed after 0.5 LD₅₀ VX exposure without oxime nor FUS protocol (Fig. 2B-H and Supp Table 2). No statistical difference was observed between the two groups (p = 0.9671 in the hippocampus, p > 0.9999 in the somatosensory cortex, p = 0.6443 in the motor cortex, and p = 0.9998 in the piriform cortex/amygdala).

## 3.2. Supra-lethal VX challenge

### 3.2.1. Short-term recovery

A daily evaluation of the behavioural recovery was scored by a treatment-blinded experimenter, along with the weight measurement during 7 d following the VX exposure (Fig. 3A). A significantly lower recovery score was observed in the VX-HI-6-no FUS group compared with the NaCl-NaCl-FUS group at day 1 (p = 0.0187), day 2 (p = 0.0302), day 4 (p = 0.0042), and day 7 (p = 0.0416) post-exposure (Fig. 3B and Supp Table 3). In contrast, no significant difference was observed between the NaCl-NaCl-FUS and VX-HI-6-FUS groups (p = 0.0978 on day 1; p = 0.1973 on day 2; p > 0.9999 at day 3; p > 0.9999 on day 4 and p = 0.7741 on day 7 post-exposure) (Supp Table 3). This means that HI-6 +FUS treatment resulted in better recovery from poisoning than HI-6 alone. Regarding the animals' weight, a significant loss was observed in both VX-HI-6 groups compared to the NaCl-NaCl-FUS group on day 1 (p = 0.0052 and p < 0.0001 for VX-HI-6-no FUS and VX-HI-6-FUS, respectively), and day 2 (p = 0.0293 and p = 0.0430 for VX-HI-6-no FUS and VX-HI-6-FUS, respectively) (Supp Table 4). However, at day 7, no significant difference was observed in the VX-HI-6-FUS group compared to the NaCl-NaCl-FUS animals (p = 0.7803) while a significant loss was still observed in the VX-HI-6-no FUS group compared to NaCl-NaCl-FUS group (p = 0.0325) (Fig. 3C and Supp Table 4). This means that weight regain was faster in HI-6 +FUS-treated animals than in HI-6-treated animals. In addition, we observed a better 24 h survival rate in the VX-HI-6-FUS group compared to the VX-HI-6-no FUS group (96 % vs 81 %, respectively, corresponding to 1 death out of a total of 27 animals for the VX-HI-6-FUS group and 5 deaths out of a total of 26 animals for the VX-HI-6-no FUS group).

### 3.2.2. AChE reactivation

The AChE activity was quantified at 24 h, 72 h and 7 d following VX exposure in the somatosensory cortex and the hippocampus (Fig. 4A). As expected, the AChE activity was significantly reduced in the VX-HI-6-no FUS group compared to the control NaCl-NaCl-FUS 24 h following the VX exposure in both the hippocampus (p = 0.0001) and the somatosensory cortex (p < 0.0001) (Fig. 4B and C and Supp Table 5). The residual AChE activity was significantly enhanced in the HI-6 with FUS group in comparison to the HI-6 without FUS animals, in the hippocampus (p = 0.0022) and in the somatosensory cortex (p = 0.0051) 24 h after VX exposure (Fig. 4B and C and Supp Table 5). At the 72 h post-exposure time-point, no difference of AChE activity levels was observed between the different treatment groups (Fig. 4B and C and Supp Table 5).

### 3.2.3. Neuroinflammation

Additionally, the concentrations of cytokines were quantified in the hippocampus. Twenty-four hours after VX exposure, a 30-fold increase was observed in hippocampal IL-6 levels in the VX-HI-6-no FUS group compared to the control (no VX) group (p = 0.0488) and to the VX-HI-6-FUS group (p = 0.0317) (Fig. 4D and Supp Table 6). Indeed, no significant difference was observed between the VX-HI-6-FUS group and the control group, suggesting that FUS+HI-6 prevents the neuroinflammation. Furthermore, a 9-fold increase in MIP-1α hippocampal levels was observed in the VX-HI-6-no FUS group compared to the control group (p = 0.0502) (Fig. 4E and Supp Table 6). At 24 h, the
A

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B

Hippocampus

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C

Somatosensory cortex

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D

Motor cortex

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E

Piriform cortex/amygdala

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F

Spinal cord

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G

Blood

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H

Diaphragm

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

(caption on next page)
**Fig. 2.** (A) Timeline illustration of the sub-lethal dose (0.5LD₅₀) of VX protocol. (B-H) Diagrams of AChE reactivation percentage normalized to the control (NaCl-NaCl or NaCl-NaCl-FUS) in the different experimental groups in the hippocampus (B), the somatosensory cortex (C), the motor cortex (D), the piriform cortex/amygdala (E), the spinal cord (F), the blood (G) and the diaphragm (H). Data were analyzed using one-way ANOVA followed by Tukey's post-hoc tests. † represents significant difference with NaCl-NaCl control group and # represents significant difference with NaCl-NaCl-FUS control group. * or # p < 0.05; ** or ## p < 0.01; *** or ### p < 0.001; †††† or * * * * or #### p < 0.0001.

difference was not statistically significant between VX-HI-6-no FUS group and VX-HI-6-FUS group, although a slight difference was observed (p = 0.0589). The expression levels of IL-6 and MIP-1α were significantly reduced for the VX-HI-6-FUS group compared to the VX-HI-6-no FUS group at 72 h (IL-6: p = 0.0311; and MIP-1α: p = 0.0060, respectively) and at 7 d (IL-6: p = 0.0335; and MIP-1α: p = 0.0367, respectively) (Fig. 4D and E and Supp Table 6) highlighting the benefit of the FUS protocol. Finally, a slight decline in IL-10 levels was observed at 72 h and 7 d in the VX-HI-6 group compared with the NaCl-NaCl-FUS group (Fig. 4F). No significant difference was observed in IL-10 level between the VX-HI-6-FUS animals compared to NaCl-NaCl-FUS animals (Supp Table 6).

## 4. Discussion

In the event of exposure to an OP such as VX, prompt and effective treatment administration is essential to prevent brain damage. The oximes currently used as antidotes to reactivate OP-inhibited ChE enhance survival, however they do not prevent immediate or long-term neurological impairment. Indeed, oximes poorly cross the BBB, mainly due to their quaternary structure [31]. For example, it was reported that only 10 % of 2-PAM would cross the BBB [56], whereas the passage of HI-6 across the BBB is strongly limited [41]. To overcome this limitation, we sought to evaluate the feasibility of using FUS technique to disrupt the BBB, thereby facilitating the passage of 2-PAM and HI-6 oximes, two small molecules, into the brain. Our objective was to ascertain whether this approach could counteract the onset of immediate cerebral damage

following sub-lethal or supra-lethal VX exposure.

To assess the oximes delivery enhancement into the brain induced by FUS, reactivation of AChE enzyme activity was quantified in different brain regions, following a single exposure to a sub-lethal or supra-lethal dose of VX. Although it is acknowledged that OP irreversibly inhibit both AChE and butyrylcholinesterase (BChE) [5,67], the focus of this study is the measurement of AChE activity, as AChE is mainly present in synapses and neuromuscular junctions, while BChE is mainly localized in blood plasma [40]. Therefore, analysis of BChE enzymatic activity is less relevant for assessing the benefit of FUS BBB-opening on cerebral ChE reactivation by oximes.

Following a single sub-lethal VX exposure, the combination of FUS and HI-6 enhanced cerebral AChE reactivation. It is noteworthy that FUS did not exacerbate central AChE inhibition subsequent to exposure to a sub-lethal dose of VX (Supp Fig. 2). However, the BBB opening induced by FUS did not improve brain AChE reactivation by 2-PAM treatment. One potential explanation for this phenomenon is the facilitation of BBB passage of 2-PAM induced by FUS is not sufficient to compensate the lack of efficacy of 2-PAM itself. Indeed, the results obtained in blood or in peripheral tissue (diaphragm) highlight the lower efficiency of 2-PAM compared to HI-6 in VX-inhibited AChE reactivation. This finding is consistent with other data in the literature indicating that HI-6 is more efficient than 2-PAM in reactivating AChE inhibited by the majority of OP, including VX [36]. Nevertheless, further experimentation will be required to demonstrate that FUS does, in fact, facilitate the passage of 2-PAM across the BBB. However, for the subsequent demonstration of the FUS utility to enhance oxime passage through the BBB, we focused

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

**Fig. 3.** (A) Timeline illustration of the supra-lethal dose (17 LD₅₀) of VX protocol, including the daily-basis monitoring of mice during the week following VX challenge (weighing and behavioural scoring). (B) Recovery score in each experimental group on days 1, 2, 3, 4 and 7 following supra-lethal exposure to VX. Data were analyzed using Kruskal-Wallis and Dunn's post-hoc tests. # represents significant difference with NaCl-NaCl-FUS control group. * or # p < 0.05, ** or ## p < 0.01; *** or ### p < 0.001, * * * or #### p < 0.0001. (C) Percentage of weight loss in each experimental group on days 1, 2, 3, 4 and 7 following supra-lethal exposure to VX. Data were analyzed using two-ways ANOVA with repeated measures and Tukey's post-hoc tests. # represents significant difference with NaCl-NaCl-FUS control group. * or # p < 0.05, ** or ## p < 0.01; *** or ### p < 0.001, * * * or #### p < 0.0001.
<figure><img src="image_18.png" /><figcaption>**Fig. 4.** (A) Timeline illustration of the supra-lethal dose (17 LD₅₀) of VX protocol, including the euthanasia time points at 24 hours, 72 hours or 7 days post-VX challenge. (B-C) Diagrams of AChE activity percentage normalized to the control (NaCl-NaCl-FUS) in each experimental group in the hippocampus (B) and in the somatosensory cortex (C), at 24 h, 72 h and 7 d following VX exposure. (D-F) Diagrams of hippocampal IL-6 (D), MIP-1α (E), and IL-10 (F) cytokines levels normalized to the control (NaCl-NaCl-FUS) in each experimental group, at 24 h, 72 h and 7 d following VX exposure. All data were analyzed using one-way ANOVA and Tukey's post-hoc tests. # represents significant difference with NaCl-NaCl-FUS control group. * or # p < 0.05, ** or ## p < 0.01; *** or ### p < 0.001, **** or #### p < 0.0001.</figcaption></figure>
on HI-6, as its efficacy was confirmed by our results with a sub-lethal dose of VX.

It is noteworthy that the reactivation of brain AChE enhanced by HI-6 +FUS did not exhibit homogeneity across the different brain regions. Following the sub-lethal VX exposure, AChE reactivation induced by HI-6 +FUS treatment was up to 17.59 % in the piriform cortex, 23.14 % in the hippocampus, 25.86 % in the motor cortex and 30.86 % in the somatosensory cortex. The FUS protocol used in our study is a well-known and has been extensively validated and applied in previous work for the delivery of therapeutic molecules (antibodies, AAV9) [12,22,52]. However, this protocol must be further improved to achieve whole-brain opening and to reduce the heterogeneity of AChE reactivation within the rodent brain.

In addition, our results indicate that HI-6 +FUS did not exert any beneficial effect on AChE reactivation in the spinal cord. In 1988, Ligtenstein et al. revealed by autoradiography the presence of HI-6 in the choroid plexus but not in the spinal cord [37], suggesting that HI-6 does not diffuse from the choroid plexus into the cerebrospinal fluid that circulates throughout the CNS, including the spinal cord. Our results concur with this study, showing no reactivation of HI-6 in the spinal cord either with or without FUS. Furthermore, in a previous PET imaging study, we reported that an antibody administered with FUS-induced BBB permeabilization diffused extremely slowly within the brain parenchyma from the sonicated area using a similar FUS protocol [62]. The diffusion of molecules within the brain is inversely proportional to the molecular weight [66]. Despite its smaller size, the concentration of HI-6 that diffuses into the brain parenchyma would likely be insufficient to reach spinal cord. One hypothesis for the lack of efficacy of 2-PAM in the brain is that it is rejected by transporters expressed at the BBB. However, it is highly improbable that the levels of oximes (pralidoxime and HI-6) in the brain are modified by P-gp activity, given that neither of the two oximes is a substrate for this transporter [25,32]. The beneficial effect of HI-6, as demonstrated using a P-gp inhibitor (tariquidar, [32]), remains a subject of debate [34]. Further work will be needed to adapt our FUS protocol to improve the delivery of oximes to deep brain regions and to treat the spinal cord to prevent any neurological damage caused by VX poisoning.

FUS is a transient, non-ionizing, safe and effective protocol for BBB opening that can be performed in just a few minutes. Beyond the development of a translational protocol for global opening of the BBB and given the problematic targeted in this study, the main current limitation of FUS technology is its deployment in the operational field. Recent developments point the way to compact, transportable FUS devices. The approach envisaged in the first instance would be to treat the patient in a medical care unit. FUS would provide a secondary treatment in hospitals for victims of OP poisoning and complementary treatment to prevent neurological complications.

The majority of preclinical studies on neuroprotection against OP intoxication have concentrated on supra-lethal doses [16,21,24,27,45]. In light of these considerations, the efficacy of the HI-6 + FUS protocol was evaluated on a supra-lethal dose of VX (17 LD₅₀). The results showed a significantly elevated reactivation of cerebral AChE following FUS+HI-6 treatment in comparison to HI-6 administration alone during the first 24 h. In the subsequent days (3 and 7 d) following VX exposure, a spontaneous reactivation of AChE was observed in mice treated with HI-6 alone, indicating no further difference between the FUS and no-FUS groups. This phenomenon of spontaneous reactivation several days after exposure to VX is well-documented, and ChE activity is restored to control levels even after prolonged exposure to VX [7]. Nevertheless, the results of our study indicate that the HI-6 + FUS treatment considerably accelerates animal recovery and improves long-term recovery outcomes. In contrast, animals treated with HI-6 alone did not recover within a week, despite full recuperation of AChE activity by day 7. This phenomenon may be explained by the initial inhibition of AChE, which triggers a cascade of neuropathological events that are no longer linked to cholinergic activity rate. These include severe neuroinflammation,

which is a well-characterized consequence of OP exposure [17,28,59]. The results demonstrate that HI-6 + FUS treatment resulted in a significant reduction in the levels of the pro-inflammatory cytokines IL-6 and MIP-1α in the hippocampus. It is noteworthy that the facilitated passage of HI-6 across the BBB induced by FUS, not only enables the reactivation of VX-inhibited AChE, but also confers a neuroprotective effect by reducing levels of pro-inflammatory cytokines. In addition, the combination of HI-6 + FUS treatment prevented a decrease of IL-10 levels a few days following VX exposure, which could improve neuroprotection given the anti-inflammatory role of IL-10 [65]. Modulating neuroinflammation represents a promising avenue for therapeutic intervention in limiting brain damage induced by OP exposure [23,35,53]. To ascertain the additional benefits of HI-6 + FUS treatment on long-term recovery from VX exposure, further work will determine whether this approach can prevent the onset of epileptic seizures and cognitive impairment.

## 5. Conclusion

Our findings indicate that transcranial FUS used to facilitate HI-6 passage through the BBB enables cerebral AChE reactivation and mitigates the acute neurological effects of both sub-lethal and supra-lethal exposure to VX. Therefore, this approach is particularly promising for effective treatment of OP exposure in a second-line hospital setting after immediate emergency treatment.

## CRediT authorship contribution statement

**Lépinard Lucie:** Writing – original draft, Investigation, Formal analysis, Data curation. **Leterrier Sarah:** Writing – review & editing, Investigation. **Jourdain Laurène:** Writing – review & editing, Investigation. **Turri Louise:** Writing – review & editing, Investigation. **Belkebir Assia:** Writing – review & editing, Investigation. **Knoertzer Julie:** Writing – review & editing, Investigation. **Champault Alexandre:** Writing – review & editing, Investigation. **Bel Rosalie:** Writing – review & editing, Investigation. **Selingue Erwan:** Writing – review & editing, Investigation. **Mériaux Sébastien:** Writing – review & editing, Methodology. **Larrat Benoit:** Writing – review & editing, Methodology, Conceptualization. **Tournier Nicolas:** Writing – review & editing, Conceptualization. **Dal Bo Grégory:** Writing – review & editing, Supervision, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. **Thibault Karine:** Writing – review & editing, Supervision, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. **Novell Anthony:** Writing – review & editing, Supervision, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization.

## Data linking

https://doi.org/10.57745/V2BPSY

## Funding sources

This work was supported by the AID/CEA grant, internal laboratory funding and the ANR grant B-BOP (ANR-22-ASTR-0022).

## Declaration of Competing Interest

Benoit Larrat and Anthony Novell are stockholders of a company developing an ultrasound device for blood-brain barrier opening.

Other authors declare that they have no conflicts of interest.

## Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.biopha.2025.118120.
# Data availability

Data will be made available on request.

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