Neuroprotectant Activity of Novel Water-Soluble Synthetic Neurosteroids on Organophosphate Intoxication and Status Epilepticus-Induced Long-Term Neurological Dysfunction, Neurodegeneration, and Neuroinflammation

Abstract

Organophosphates (OPs) and nerve agents are potent neurotoxic compounds that cause seizures, status epilepticus (SE), brain injury, or death. There are persistent long-term neurologic and neurodegenerative effects that manifest months to years after the initial exposure. Current antidotes are ineffective in preventing these long-term neurobehavioral and neuropathological changes. Additionally, there are few effective neuroprotectants for mitigating the long-term effects of acute OP intoxication. We have pioneered neurosteroids as novel anticonvulsants and neuroprotectants for OP intoxication and seizures. In this study, we evaluated the efficacy of two novel synthetic, water-soluble neurosteroids, valaxanolone (VX) and lysaxanolone (LX), in combating the long-term behavioral and neuropathological impairments caused by acute OP intoxication and SE. Animals were exposed to the OP nerve agent surrogate diisopropylfluorophosphate (DFP) and were treated with VX or LX in addition to midazolam at 40 minutes postexposure. The extent of neurodegeneration, along with various behavioral and memory deficits, were assessed at 3 months postexposure. VX significantly reduced deficits of aggressive behavior, anxiety, memory, and depressive-like traits in control (DFP-exposed, midazolam-treated) animals; VX also significantly prevented the DFP-induced chronic loss of NeuN(+) principal neurons and PV(+) inhibitory neurons in the hippocampus and other regions. Additionally, VX-treated animals exhibited a reduced inflammatory response with decreased GFAP(+) astrogliosis and IBA1(+) microgliosis in the hippocampus, amygdala, and other regions. Similarly, LX showed significant improvement in behavioral and memory deficits, and reduced neurodegeneration and cellular neuroinflammation. Together, these results demonstrate the neuroprotectant effects of the novel synthetic neurosteroids in mitigating the long-term neurologic dysfunction and neurodegeneration associated with OP exposure.

SIGNIFICANCE STATEMENT

Survivors of nerve agents and organophosphate (OP) exposures suffer from long-term neurological deficits. Currently, there is no specific drug therapy for mitigating the impact of OP exposure. However, novel synthetic neurosteroids that activate tonic inhibition provide a viable option for treating OP intoxication. The data from this study indicates the neuroprotective effects of synthetic, water-soluble neurosteroids for attenuation of long-term neurological deficits after OP intoxication. These findings establish valaxanolone and lysaxanolone as potent and efficacious neuroprotectants suitable for injectable dosing.:

Introduction

Acute intoxication with organophosphates (OPs) and nerve agents triggers a life-threatening cholinergic toxidrome associated with rapid onset of seizures and status epilepticus (SE) (Reddy and Colman, 2017; Reddy, 2019a). These early-onset seizures result from excessive cholinergic activation and excessive glutamate release, leading to the development of epilepsy and SE (Myhrer, 2007; Todorovic et al., 2012). Currently, the standard-of-care treatment of OP poisoning consists of atropine sulfate, pralidoxime, and midazolam. Midazolam has recently been approved for the treatment of acute SE, including those caused by nerve agents. Midazolam is an effective anticonvulsant if administered within 10–20 minutes (i.e., early treatment) after the initiation of seizures but becomes ineffective in terminating established SE if given 40 minutes or later (i.e., delayed treatment) after seizure onset (Wu et al., 2018; Reddy, 2019a). However, current antidote regimens do not adequately protect survivors against long-term neurologic deficits, including neuronal damage and persistent neurologic dysfunction, which are hallmark features of OP intoxication (Reddy and Reddy, 2015; Reddy et al., 2020a; Supasai et al., 2020). This is evident from the chronic health issues present in survivors of nerve agent incidents in Japan, Syria, and other countries (Savage et al., 1988; Murata et al., 1997; Eskenazi et al., 1999; Delgado et al., 2004; Ohtani et al., 2004; Yanagisawa et al., 2006). Controlling persistent SE at an early stage is critical for promoting survival, reducing brain damage, and preventing long-term neurologic dysfunction in cases of a terrorist attack with nerve agents. However, there is currently no approved drug therapy for preventing the long-term effects of acute OP intoxication.

Neurosteroids are a new class of powerful anticonvulsants useful in protecting against OP intoxication and seizures (Reddy and Woodward, 2004; Reddy, 2009; Reddy et al., 2012; 2019a). They elicit broad-spectrum anticonvulsant effects conferring seizure protection (Reddy and Rogowski, 2002; Carver and Reddy, 2013; Wu et al., 2013; Reddy and Estes, 2016). We have pioneered neurosteroids as novel anticonvulsants and neuroprotectants for acute seizures and SE (Reddy, 2016, 2019a). Specifically, neurosteroids are positive allosteric modulators and direct activators of both synaptic and extrasynaptic GABA-A receptors in the brain (Reddy and Estes, 2016). Unlike benzodiazepines, neurosteroids directly open the GABA-A receptor chloride channels at low micromolar concentrations and act on all GABA-A receptor isoforms, specifically extrasynaptic GABA-A receptors (Chuang and Reddy, 2018). Furthermore, neurosteroids target extrasynaptic receptors that do not undergo internalization during SE. In addition, they shunt hypersynchronous discharges that lead to prolonged SE, consequently lessening neuronal damage and the occurrence of neuroinflammation (Reddy and Estes, 2016; Reddy, 2018, 2023a). Moreover, neurosteroids offer a unique advantage in that they do not appear to induce tolerance even with prolonged use (Reddy and Rogawski, 2000). Lately, the Food and Drug Administration (FDA) has granted approval for two neurosteroids, brexanolone and ganaxolone, to address brain disorders, which emphasizes the evident clinical importance of neurosteroids. Notably, ganaxolone has received FDA approval specifically for managing seizures linked to CDKL5 deficiency disorder (Knight et al., 2022). Hence, neurosteroids are clinically viable therapeutic agents for SE.

Neurosteroids are novel medical countermeasures for nerve agents (Reddy, 2016). Neurosteroids such as allopregnanolone (brexanolone), pregnanolone, and ganaxolone have demonstrated efficacy as anticonvulsants in animal models of cholinergic SE induced by pilocarpine (Briyal and Reddy, 2008; Reddy et al., 2008, 2019; Kuruba and Reddy, 2011; Rogawski et al., 2013; Saporito et al., 2019; Neff and Reddy, 2024), diisopropylfluorophosphate (DFP) (Reddy, 2019a), or soman (Althaus et al., 2017; Reddy, 2019b). These neurosteroids rapidly and effectively mitigate SE and neuronal damage in OP models (Reddy, 2019b; Reddy et al., 2019; Barker et al., 2020). Neurosteroids have also exhibited efficacy in diverse non-OP seizure models (Reddy and Woodward, 2004; Reddy et al., 2012, 2019; Zolkowska et al., 2018; Saporito et al., 2019). Specifically, ganaxolone is a synthetic neurosteroid analog with more desirable features than its natural neurosteroid counterparts (Reddy and Woodward, 2004). We have extensively tested neuronal mechanisms, the anticonvulsant profile, pharmacokinetics, and the safety profile of ganaxolone (Reddy and Rogowski, 2010; Reddy, 2016; Chuang and Reddy, 2018; Reddy et al., 2019). Based on these studies, ganaxolone has advanced to clinical trials for the treatment of SE and nerve agent seizures (Vaitkevicius et al., 2022). Despite their robust antiseizure and neuroprotectant effects, ganaxolone and brexanolone have certain limitations, including poor bioavailability, low aqueous solubility, short half-life, and rapid metabolic inactivation (Reddy and Rogawski, 2012). However, there is an unfulfilled need for synthetic neurosteroids that surpass these limitations. In general, water solubility is a crucial characteristic when formulating intravenous treatments for SE. Neurosteroids with enhanced bioavailability provide a more advantageous therapeutic profile for the treatment of SE. Recently, we developed novel water-soluble neurosteroids with improved biopharmaceutical properties (Reddy, 2023b). Among the water-soluble class, valaxanolone (VX) and lysaxanolone (LX) emerge as the most promising neurosteroid analogs. These neurosteroids are prepared as a dry powder for injection, requiring them to be mixed with an aqueous buffer before administration. In contrast, brexanolone and ganaxolone require complex formulations, such as β-cyclodextrin, that carry potential side effects and limits daily administration. Newer synthetic neurosteroids (super-ganaxolones) have been designed to retain their protective activity while increasing hydrophilic properties and enhancing drug delivery (Reddy, 2019b). Therefore, these analogs have promising potential to provide improved options for developing injectable medical countermeasures for OP intoxication.

Here, we sought to characterize the protective effects of two novel water-soluble synthetic neurosteroids, VX and LX, in mitigating the long-term neurologic dysfunction and neuropathological changes caused by acute OP intoxication. Our results show a striking neuroprotectant ability of both neurosteroids in attenuating the long-lasting neurologic deficits and preventing neurodegeneration in the rat model of OP intoxication. Together, these outcomes establish VX and LX as potent and efficacious neuroprotectant lead compounds suitable for clinic- and field-ready injectable dosing.

Materials and Methods

Animals

Adult male Sprague-Dawley rats (225–350 g) were purchased at 8–10 weeks of age from Taconic Farms in Rockville, MD. Animals were housed in standard plastic cages under controlled environmental conditions in our vivarium facility, with access to food and water provided ad libitum. All experimental procedures were carried out in accordance with National Institutes of Health guidelines and were approved by the Institutional Animal Care and Use Committee.

Drugs and Reagents

DFP, atropine methyl nitrate (AMN), and pralidoxime chloride (2-PAM) were purchased from Sigma-Aldrich (St. Louis, MO). A commercially available formulation of midazolam (MDZ; 5 mg/ml) was obtained from Hospira Inc. (Lake Forest, IL). VX and LX were synthesized as outlined previously (Reddy, 2023b) and were dissolved in saline. Drug solutions were administered subcutaneously or intramuscularly in a volume equaling 0.1% of the animal’s body weight.

DFP Exposure Protocol

The overall experimental protocol for DFP exposure and behavior testing are depicted in Fig. 1. DFP (3.2 mg/kg, s.c.) was administered to induce OP intoxication and persistent SE. Animals were pretreated with pyridostigmine bromide (0.026 mg/kg, i.m.) 30 minutes before DFP injection. One minute following the DFP injection, rats were given 2-PAM (25 mg/kg, i.m.) and AMN (2 mg/kg, i.m.) in an attempt to increase their chances of survival (Wu et al., 2018; Reddy et al., 2021). Animals were allowed to recover and were continuously monitored in the animal facility while subsequently cared for by a veterinarian for a period of 3 months. Three months after DFP exposure, animals were evaluated in an array of behavior tests reminiscent of aggression, object recognition memory, spatial learning and memory, anxiety, and depression. Immediately after completing the behavioral assessments, animal brains were collected and processed for histology (Fig. 1).

Fig. 1.

Experimental protocol for DFP exposure model of SE in rats. Time “0” is the time of chemical exposure. In the DFP model, rats were pretreated with pyridostigmine bromide (PB; 0.026 mg/kg, i.m.) 30 minutes before DFP (3.2 mg/kg, s.c.). They were given an antidote regimen of AMN (2 mg/kg, i.m.) and 2-PAM (25 mg/kg, i.m.). These treatments are standard antidotes for improving survival and enabling the induction of SE. Tested synthetic neurosteroid analogs (LX and VX) were administered 40 minutes after DFP exposure, along with midazolam. Animals were screened in a battery of behavior assessments 3 months after DFP exposure, and they were perfused to observe histopathological alterations. Open field and plus maze testing was done first, followed by the novel object recognition test and water maze memory evaluations. 2-PAM, pralidoxime chloride; AMN, atropine methyl nitrate; PB, pyridostigmine bromide.

Drug Treatment

The overall summary of the animal cohorts is outlined in Table 1. All treatments were administered intramuscularly. The animals were divided into various cohorts using a random selection process (n = 8–10/group). Group 1 (sham) consists of animals that were not exposed to DFP but were administered pyridostigmine bromide, 2-PAM, AMN, and vehicle. Group 2 (DFP plus MDZ as control) received the standard anticonvulsant MDZ (2 mg/kg) at 40 minutes post DFP exposure. Groups 3 and 4 (VX groups) were subjected to DFP exposure followed by administration of VX at 5 and 10 mg/kg, respectively, along with MDZ (2 mg/kg). Groups 5 and 6 (LX groups) were subjected to DFP exposure followed by treatment with LX at 5 and 10 mg/kg, respectively, along with MDZ (2 mg/kg).

TABLE 1.

Experimental design for the evaluation of novel synthetic neurosteroids in the DFP exposure model

Group	Treatment
Sham	No DFP, vehicle, and solutions (with PB, AMN, and 2-PAM)
Control	MDZ (2 mg/kg, i.m.) at 40 min after DFP challenge
VX (5 mg)	MDZ (2 mg/kg, i.m.) + VX (5 mg/kg, i.m.) at 40 min after DFP challenge
VX (10 mg)	MDZ (2 mg/kg, i.m.) + VX (10 mg/kg, i.m.) at 40 min after DFP challenge
LX (5 mg)	MDZ (2 mg/kg, i.m.) + LX (5 mg/kg, i.m.) at 40 min after DFP challenge
LX (10 mg)	MDZ (2 mg/kg, i.m.) + LX (10 mg/kg, i.m.) at 40 min after DFP challenge

2-PAM, pralidoxime chloride; AMN, atropine methyl nitrate; LX, lysaxanolone; MDZ, midazolam; PB, pyridostigmine bromide.

Aggression Test

Following the procedure described previously, rats were evaluated for aggressive behaviors and startle responses by tapping on the exterior of the cage, followed by the animals being gently handled by investigators (Wu et al., 2021). The responses of the rat were assessed using a five-point scale: 1, no startle with the minimum response; 2, some response without startle; 3, startle with hyper-response; 4, jumpy and aggressive, showing aversion for handling; and 5, tail up, biting the edge of the cage when lifted.

Depressive Behavior in the Social Interaction Test

Social interaction test (SIT) measures depressive-like behavior in rodents (Wu et al., 2021). This test is based on ethologically relevant ideas, which involve introducing an experimental rat into a field and allowing it to explore for 5 minutes. After the exploration period, a nonexperimental rat is introduced into a box and placed as far away from the experimental rat as possible. The two rats were then allowed to interact for 5 minutes, during which the contact times were recorded. Contact time was measured when the experimental rat faced the nonexperimental rat and explored its head, behind, or other body parts with its nose and/or forepaws.

Anxiety-Like Behavior in the Elevated Plus Maze Test

The elevated plus maze (EPM) is a widely used experimental tool for measuring rodent anxiety-like behavior (Reddy and Kulkarni, 1996; Wu et al., 2021). It consists of a cross-shaped apparatus made of fiberglass with four arms that are 50-cm long and 10-cm wide, elevated 50 cm from the floor. Two opposing arms are enclosed with 40-cm-high walls (closed arms), whereas the other two arms are open (open arms). The central area where the arms cross measures 10 × 10 cm. During the test, each rat is placed in the central zone of the EPM with its head facing a closed arm and allowed to explore the maze freely for 5 minutes. The amount of time spent in the open and closed arms, as well as the number of entries into the open and closed arms, are then used as measures of anxiety-like behavior. Rats exhibiting increased anxiety-like behavior tend to spend more time in the closed arms and reduced time and fewer entries into the open arms.

Exploratory and Anxiety Behavior in the Open Field Test

Following the procedure described previously, animals were assessed for their anxiety levels and general motor ability through an open field test (Gould et al., 2009; Wu et al., 2021). The open field arena is a 100 × 100–cm Plexiglas box divided into 16 squares (25 × 25 cm each) and utilizes a computer tracking system (Noldus-Ethovision, Inc.). The four squares located in the central area were defined as the central zone, the four squares located at each corner were defined as corners, and the remaining squares in the periphery were defined as sides. Each rat was placed in one of the corners of a brightly lit open field, and its movement was recorded for 10 minutes.

Object Recognition Memory in the Novel Object Recognition Test

As described previously, the novel object recognition test assesses recognition memory abilities by observing how an animal interacts with two objects placed in an open field box (Leger et al., 2013; Wu et al., 2021). This test involved three successive trials for each rat, with an intertrial interval of 60 minutes. During the first two trials, the rat is placed in an empty open field box for 5 minutes (habituation phase), then in the same box with two identical objects on opposite sides for another 5 minutes (sample phase); in the third trial (object recognition memory testing phase), the rat is placed again in the box, but this time with one familiar object as well as a novel object. The rat’s exploratory behavior is recorded when the rat’s nose is pointed toward the object and is within ∼2 cm, indicating object exploration. The time spent exploring the novel object, familiar object, and both objects is recorded, and the percentage of time spent with the novel object versus time spent with both objects (i.e., discrimination index) is calculated. A higher preference for exploring the novel object indicates better learning and recognition memory function.

Spatial Learning and Memory Function in the Morris Water Maze

The hippocampus-dependent spatial learning and memory functions were assessed using the Morris water maze test (Morris, 1984; Vorhees and Williams, 2006; Wu et al., 2021). Rats were allowed 90 seconds to locate the platform using spatial cues in four acquisition trials per day, conducted over 7 days. The average latency to reach the platform was plotted to measure the learning process. Memory retrieval and memory retention were assessed on day 8 (probe 1) and day 15 (probe 2), respectively. During these probe days, the platform was removed, and the rats’ performance in locating the platform quadrant was observed for 45 seconds. The time spent in the platform quadrant was measured in all groups to assess the effect of neurosteroids on spatial learning and memory dysfunction following DFP exposure.

Brain Histology and Immunohistochemistry

Regarding immunohistochemistry, rats were anesthetized with a ketamine-xylazine mixture and transcardially perfused with a 4% paraformaldehyde solution in sodium phosphate buffer (pH 7.4). Brains were removed and postfixed in the 4% paraformaldehyde solution as described previously (Kuruba et al., 2018; Wu et al., 2018). Serial coronal sections (30-μm thick) were cut from the anterior to posterior parts of the brain containing the somatosensory cortex, hippocampus, and amygdala (AMY), approximately from bregma. These sections were preserved in 24-well plates containing sodium phosphate buffer, with sections taken at every 600 μm. Following preservation, sections were stained for neuronal nuclei antigen (NeuN), parvalbumin (PV), glial fibrillary acidic protein (GFAP), and ionized calcium-binding adapter molecule-1 (IBA1) (Wu et al., 2018; Reddy et al., 2020a).

Immunochemistry for Neurodegeneration and Cellular Neuroinflammation

Analyses of neurodegeneration and neuroinflammation in the brain slices were conducted using established methods as described previously (Kuruba et al., 2018; Wu et al., 2018). Principal neurons were identified via NeuN(+) immunostaining to identify neuronal loss and total neuron cell population (Mullen et al., 1992). PV(+) immunostaining identified GABAergic inhibitory interneuron populations to evaluate the damage to the neural network’s regulatory ability (Godoy et al., 2022). GFAP(+) was used to examine astrogliosis, a protein marker that is rapidly expressed on astrocytes following central nervous system injury (Jeromin and Bowser, 2017). IBA1(+) staining visualized the population of activated ameboid microglia within the central nervous system as protein expression is coupled with the activation of microglia cells (Wittekindt et al., 2022).

Stereological Quantification

A stereology protocol was used to quantify the total number of positively stained cells for the NeuN and PV stains (Golub et al., 2015). Cell counts were specified for different regions of the hippocampus: cornus ammonis (CA)-1 and CA3, dentate gyrus (DG), and the dentate hilus (DH). Newcast software (Visiopharm, Hørsholm, Denmark) was used in conjunction with an Olympus BX53 microscope and a mounted colored camera. For each specific region, 5% of the total area with a 60× objective lens was used to quantify the number of stained neurons in the CA1, CA3, and DG regions, and 10% of the total area using a 60× objective lens was used to quantify the DH subregions. To ensure optimal sampling for stereological counts, the total area selection was based on the relative density of cells in these regions. For the volume of each specific region, a 10× objective lens used at least 200 points, which used 100% of each tissue region (Boyce et al., 2010; Golub et al., 2015). All procedures described were accomplished through a blinded analysis to eliminate subjective bias.

Area Fractionation Densitometry

Quantification of GFAP(+) and IBA1(+) immunostaining was evaluated by area fractional densitometry using National Institutes of Health ImageJ software as previously described (Kuruba et al., 2018). For each rat, at least five images were captured at the same anatomic location. Subsequently, each of these images was converted to a 16-bit greyscale format. A threshold value was then selected to highlight the cells expressing the stain but not the background. This highlighted area was then analyzed to assess the area fraction of the particles compared with the total examined area.

Statistical Analysis

Statistical tests used during the analysis were performed utilizing OriginPro 2020 software (OriginLab Corporation, Northampton, MA). All tests depicted that statistical differences were set at P < 0.05 unless otherwise specified. Values are expressed as the mean ± S.E.M. Parametric outcomes, such as behavior and neuropathological measures, were compared between groups using the one-way ANOVA, followed by Tukey’s test. The nonparametric data, such as the aggression score, were analyzed using the nonparametric Kruskal-Wallis test, followed by a Mann-Whitney U test.

Results

Synthetic Hydrophilic Neurosteroids Reduce Aggressive Traits in DFP-Exposed Rats

To investigate the effects of synthetic neurosteroids on aggressive behavior, we assessed the extent of aggression in OP-exposed animals. Control animals exhibited a significant level of aggression toward the handler (Fig. 2A; Fig. 3A). VX-treated (5 or 10 mg/kg) animals showed a significant decrease in aggression score compared with the control group (Fig. 2A). Similarly, a significant reduction in aggression score was evident in LX-treated (5 or 10 mg/kg) animals (Fig. 3A). These results suggest that these neurosteroid analogs effectively mitigated aggressive characteristics developed after OP exposure.

Fig. 2.

Effect of VX on aggression, depression, and anxiety-like behaviors in DFP-exposed rats. (A) Aggressive behavior in VX-treated groups. (B) Depression-like behavior in the social interaction test was assessed as total time in contact with another rat. (C) Anxiety behavior was evident from reduced time spent in open arms in the EPM. (D) Anxiety behavior is evident from increased time spent in closed arms in EPM. (E) Number of entries in open arms of the EPM. (F) Number of entries in closed arms of the EPM. The aggression data in (A) was analyzed by the nonparametric Kruskal-Wallis test, followed by the Mann-Whitney U test. Data in all other panels were analyzed by one-way ANOVA followed by Tukey’s test. Data represent means ± S.E.M. (n = 7–9 per group). *P < 0.05 versus sham group; ^#P < 0.05 versus control group (DFP exposed, midazolam treated).

Fig. 3.

Effect of LX on aggression, depression, and anxiety-like behaviors in DFP-exposed rats. (A) Aggressive behavior in LX-treated groups. (B) Depression-like behavior in the social interaction test was assessed as total time in contact with another rat. (C) Anxiety behavior was evident from reduced time spent in open arms in the EPM. (D) Anxiety behavior is evident from increased time spent in closed arms in EPM. (E) Number of entries in open arms in EPM. (F) Number of entries in closed arms in EPM. The aggression data in (A) was analyzed by the nonparametric Kruskal-Wallis test, followed by the Mann-Whitney U test. Data in all other panels were analyzed by one-way ANOVA followed by Tukey’s test. Data represent means ± S.E.M. (n = 8–10 per group). *P < 0.05 versus sham group; ^#P < 0.05 versus control group (DFP exposed, midazolam treated).

Synthetic Hydrophilic Neurosteroids Reduce Depression-Like Behavior in DFP-Exposed Rats

To assess the effects of synthetic neurosteroids on depression-like behavior, a social interaction test was performed. Control animals showed a significant decrease in total time in contact compared with sham animals, indicating severe depressive-like behavior (Fig. 2B; Fig. 3B). VX-treated (10 mg/kg) animals showed a significant improvement in depressive-like behavior (Fig. 2B). Similarly, social interaction was significantly improved in the LX-treated (10 mg/kg) group (Fig. 3B). These results demonstrate that the neurosteroid analogs reduced depression-like behaviors following OP exposure.

Synthetic Hydrophilic Neurosteroids Reduce Anxiety-Like Behavior in DFP-Exposed Rats

To investigate the effects of synthetic neurosteroids on anxiety-like behavior, we used the EPM test. Control animals exhibited a significant increase in anxiety, displayed by the reduced time in open arms and increased time spent in closed arms (Fig. 2, C and D; Fig. 3, C and D). VX (10 mg/kg) treatment significantly lessened anxiety-like behaviors caused by OP exposure (Fig. 2, C and D). Similar effects were observed in LX-treated (10 mg/kg) animals (Fig. 3 C and D). No notable differences were observed in the number of entries made in both open and closed arms for either neurosteroid (Fig. 2, E and F; Fig. 3, E and F). These results show that neurosteroid treatment reduced anxiety-like behavior caused by OP exposure.

Synthetic Hydrophilic Neurosteroids Reduce Anxiety-Related Exploratory Deficits in DFP-Exposed Rats

To assess the effects of synthetic neurosteroids on anxiety-related exploratory deficits, an open field test was performed. Control animals spent significantly less time (∼60% of sham, P < 0.05) in the center of the arena compared with sham animals, indicating anxious behavior (Fig. 4A; Fig. 5A). VX (10 mg/kg) treatment significantly increased the time spent in the center arena compared with control animals (Fig. 4A). Similarly, LX-treated (10 mg/kg) animals spent a significantly longer time in the center arena, indicating improved exploratory traits (Fig. 5A). However, no significant effect was observed on the number of entries into the central arena, suggesting that animals spent more time in the central arena with each entry rather than making multiple entries in neurosteroid-treated groups (Fig. 4B; Fig. 5B). These results show that neurosteroid treatment reduced anxiety-related exploratory deficits present due to OP exposure.

Fig. 4.

Effect of VX on open-field exploration, spatial learning, and memory deficits and object recognition memory impairment in DFP-exposed rats. (A) Anxiety-like behavior was observed in the open field test. (B) Number of inner crossings in the open field test. (C–G) The learning and memory evaluations in sham, control, and VX-treated rats in the novel object recognition test (NORT) and the Morris water maze (MWM). (C and D) Object recognition memory in the NORT. (C) Total time spent with the novel object by sham and control animals. (D) Percentage of time spent with the novel object relative to the total time spent with both novel and familiar objects. (E–G) Spatial learning and memory test in the MWM test. (E) Mean latency to reach the platform during the 7-day learning/training trials. (F) Short-term memory (probe 1) was evaluated on day 1 after the training session. (G) Memory retention and recall tests (probe 2) were evaluated on day 8 after the training session. Data in all panels were analyzed by one-way ANOVA followed by Tukey’s test. Data represent means ± S.E.M. (n = 7 to 9 per group). *P < 0.05 versus sham group; ^#P < 0.05 versus control group (DFP exposed, midazolam treated).

Fig. 5.

Effect of LX on open-field exploration, object recognition memory impairment, and spatial learning and memory deficits in DFP-exposed rats. (A) Anxiety-like behavior was observed in the open field test. (B) Number of inner crossings in the open field. (C–G) The learning and memory evaluations in sham, control, and VX-treated rats in the novel object recognition test (NORT) and the Morris water maze (MWM). (C and D) Object recognition memory in the NORT test. (C) Total time spent with the novel object by sham and control animals. (D) Percentage of time spent with the novel object relative to the total time spent with both novel and familiar objects. (E–G) Spatial learning and memory test in the MWM test. (E) Mean latency to reach the platform during the 7-day learning/training trials. (F) Short-term memory (probe 1) was evaluated on day 1 after the training session. (G) Memory retention and recall tests (probe 2) were evaluated on day 8 after the training session. Data in all panels were analyzed by one-way ANOVA followed by Tukey’s test. Data represent means ± S.E.M. (n = 6–10 per group). *P < 0.05 versus sham group; ^#P < 0.05 versus control group (DFP-exposed, midazolam-treated).

Synthetic Hydrophilic Neurosteroids Diminish Object Recognition Memory Deficits in DFP-Exposed Rats

To evaluate the effects of synthetic neurosteroids on short-term memory recognition and cognitive impairments, a novel object recognition test was performed. Control animals spent significantly less time with the novel object than sham animals, indicating a severely impaired memory (Fig. 4C). VX (5 and 10 mg/kg) treatment significantly improved short-term memory deficits in control animals (Fig. 4C). The discrimination index is the percentage of time spent with a novel object to the total time spent on both objects by the animal. Control animals exhibited a significantly low (∼37% of sham, P < 0.05) discrimination index, revealing a loss of object recognition memory (Fig. 4D). VX (5 and 10 mg/kg) significantly increased (∼152% of control, P < 0.05) the discrimination index, indicating an improvement in recognition memory and cognition (Fig. 4D). Likewise, LX (10 mg/kg) treatment also significantly improved short-term memory deficits caused by OP exposure (Fig. 5, C and D). These findings show that the neurosteroid analogs ameliorate memory and cognitive impairments inflicted by DFP exposure.

Synthetic Hydrophilic Neurosteroids Reduce Spatial Learning and Memory Deficits in DFP-Exposed Rats

To study the effects of synthetic neurosteroids on hippocampal-dependent spatial learning and long-term memory recall impairments, a Morris water maze test was performed. During the 7-day learning (i.e., training) period, control animals consistently exhibited significantly higher latencies to reach the platform than sham animals, indicating a depreciated learning ability (Fig. 4E). Control animals showed a ∼35% decrease in average latency to the platform from the first to the seventh day (Fig. 4E). VX-treated (5 and 10 mg/kg) animals exhibited significantly lower latencies to the platform during the 7-day learning phase, with a striking 80% decrease on day 7 compared to control (Fig. 4E). Similar learning outcomes were observed in LX-treated (5 and 10 mg/kg) animals after DFP exposure (Fig. 5E). On day 8 (24 hours after the last learning session, probe 1), control animals spent ∼40% less time in the target quadrant than sham animals (Fig. 4F; Fig. 5F). At day 15 (probe 2), control animals spent ∼50% less time compared with sham animals in the target quadrant, indicating impaired memory retrieval (Fig. 4G; Fig. 5G). The VX-treated (5 and 10 mg/kg) group spent significantly more time in the target quadrant than control animals (∼35%, P < 0.05) on probe 1 (Fig. 4F). On probe 2, the VX-treated (10 mg/kg) group showed a significant increase in the time spent in the target quadrant (∼40%, P < 0.05) compared with control animals (Fig. 4G). Similarly, the LX-treated (10 mg/kg) group showed a significant increase in the time spent in the target quadrant (probe 1, ∼37%; probe 2, ∼44%; P < 0.05) compared with control animals (Fig. 5, F and G). These findings demonstrate that the neurosteroid analogs alleviate spatial learning and memory retention deficits caused by OP exposure.

Synthetic Hydrophilic Neurosteroids Prevent the Loss of Principal Neurons in DFP-Exposed Rats

To investigate the effects of synthetic neurosteroids on the DFP-induced loss of principal neurons in the hippocampus, NeuN(+) immunostaining was performed in brain sections. Figure 6 provided representative images of NeuN(+) principal neuron distribution in various subfields of the hippocampus for sham, control, and synthetic neurosteroid–treated groups. The top panel (Fig. 6, A1–4) showed images of the entire hippocampus at 1.25× objective lens. The bottom panels (Fig. 6, B–D) displayed magnified images at 20× objective lens for the designated hippocampal regions: CA1, CA3, and DG (DH included). The control rat brain exhibited a significant loss of NeuN(+)-stained principal neurons in the CA1 and DG regions compared with the sham rat brain. In contrast, the VX- and LX-treated rat brain sections showed a less prominent loss in principal neurons, indicating the potential protective effects of these novel neurosteroid treatments.

Fig. 6.

Protective effects of VX and LX on NeuN(+) principal neuron loss in the hippocampus (HPC) and its subfields in DFP-exposed rats. Three months after DFP exposure, representative sections of NeuN immunostaining were examined in the various brain regions of sham, control, VX-treated (10 mg/kg), and LX-treated (10 mg/kg) animals. In the control group, NeuN staining revealed a significant loss of principal neurons and a reduction in nuclear staining in the PL of CA1 and GCL of DG subfields compared with the other groups. GCL, granule cell layer; ML, molecular layer; PL, pyramidal layer; SO, stratum oriens; SR, stratum radiatum.

Stereological quantification of the total cell number and the percentage of damage of cells revealed a significant loss of NeuN(+) cells throughout the whole hippocampus (∼25% damage versus sham, P < 0.05) in control animals compared with sham animals (Fig. 7, A and B). This loss mainly occurred in the CA1 and DG subfields of the hippocampus (Fig. 7, C and G). The percentages of neuronal damage in the CA1 and DG subfields were ∼25% and ∼22%, respectively (Fig. 7, D and H). VX (10 mg/kg) treatment significantly rescued (∼95% protected, P < 0.05) OP-induced loss of NeuN(+) neurons across the hippocampus compared with control animals (Fig. 7, A and B). Additionally, this treatment showed a significant increase in total NeuN(+) cells in the CA1 (Fig. 7C) subregion, exhibiting remarkable neuroprotection (CA1, ∼99%; DG, ∼97%) as shown in Fig. 7D. No significant loss of NeuN(+) neurons were observed in the CA3 and DH subfields of the hippocampus (Fig. 7, E, F, I, and J). Similarly, LX (10 mg/kg) treatment reduced OP-induced loss of NeuN(+) neurons across the hippocampus (Fig. 7, A–J). These results suggest that delayed treatment with neurosteroid analogs provide robust long-term neuroprotection from OP-induced principal neuron damage.

Fig. 7.

Protective effects of VX and LX on the degeneration of NeuN(+) principal neurons in the hippocampus (HPC) subfields in DFP-exposed rats. (A) Stereological quantification of the absolute NeuN(+) cells in the HPC. (B) Percentage of damage of NeuN(+) cells in the hippocampus (versus sham). (C) Stereological quantification of the absolute NeuN(+) cells in CA1. (D) Percentage of damage of NeuN (+) cells in CA1 (versus sham). (E) Stereological quantification of the absolute NeuN(+) cells in CA3. (F) Percentage of damage of NeuN (+) cells in CA3 (versus sham). (G) Stereological quantification of the absolute NeuN(+) cells in DG. (H) Percentage of damage of NeuN (+) cells in the dentate gyrus (DG versus sham). (I) Stereological quantification of the absolute NeuN(+) cells in DH. (J) Percentage of damage of NeuN (+) cells in DH (versus sham). Data in all panels were analyzed by one-way ANOVA followed by Tukey’s test. Data represent means ± S.E.M. (n = 6–10 per group). *P < 0.05 versus sham group; ^#P < 0.05 versus control group (DFP exposed, midazolam treated).

Synthetic Hydrophilic Neurosteroids Reduce the Loss of Inhibitory Interneurons in DFP-Exposed Rats

To investigate the effect of synthetic neurosteroids on the DFP-induced loss of GABAergic inhibitory interneurons in the hippocampus, brain sections were immunostained for PV(+). Figure 8 provided representative images of PV(+) interneuron distribution in different subfields of the hippocampus for sham, control, and synthetic neurosteroid–treated groups. The top panel (Fig. 8, A1–4) displayed images of the entire hippocampus at 1.25× objective lens. The bottom panels (Fig. 8, B–D) showed magnified images at 20× objective lens for the designated hippocampal regions: CA1, CA3, and DG. The loss of PV(+)-stained cell distribution was markedly visible in the CA1 and DG regions in the control rats as compared with the sham rats. In comparison, the VX- and LX-treated groups showed a less prominent interneuron loss in these regions, indicating the potential protective effects offered by these novel neurosteroid treatments.

Fig. 8.

Protective effects of VX and LX on PV(+) interneuron loss in the hippocampus (HPC) and its subfields in DFP-exposed rats. Three months after DFP exposure, representative sections of PV(+) immunostaining were examined in various brain regions of the sham, control, VX-treated (10 mg/kg), and LX-treated (10 mg/kg) animals. In the control group, PV(+) staining revealed a significant loss of interneurons in the PL of CA1 and GCL of DG subfields. GCL, granule cell layer; ML, molecular layer; PL, pyramidal layer; SO, stratum oriens; SR, stratum radiatum.

Stereological quantification shows that control animals exhibited significant (∼25% of sham, P < 0.05) loss of PV(+) interneurons throughout the whole hippocampus (Fig. 9, A and B). This loss mainly occurred in the CA1 (Fig. 9, C and D) and DG (Fig. 9, G and H) regions but not in the CA3 subregion (Fig. 9, E and F). The percentages of PV(+) neuron damage in the CA1 and DG subfields were ∼23% and ∼27%, respectively (Fig. 9, D and H). Treatment with VX (10 mg/kg) offered significant protection in preventing the loss of inhibitory interneurons in the whole hippocampus (Fig. 9, A and B). A similar neuroprotective effect was observed in the LX-treated (10 mg/kg) group (Fig. 9, A and B). LX-treated (10 mg/kg) animals showed a significant increase in total PV(+) cells in CA1 (Fig. 9C) and DG (Fig. 9G) subfields, exhibiting substantial neuroprotection (CA1, ∼99%; DG, ∼97%) as shown in Fig. 9, D and H. These results suggest that these synthetic neurosteroids provide strong long-term neuroprotection from OP-induced inhibitory interneuron damage.

Fig. 9.

Protective effects of VX and LX on the degeneration of PV(+) inhibitory neurons in the hippocampus (HPC) subfields in DFP-exposed rats. (A) Stereological quantification of the absolute PV(+) cells in the HPC. (B) Percentage of damage of PV(+) cells in the hippocampus (versus sham). (C) Stereological quantification of the absolute PV(+) cells in CA1. (C) Percentage of damage of PV(+) cells in CA1 (versus sham). (E) Stereological quantification of the absolute PV(+) cells in CA3. (F) Percentage of damage of PV(+) cells in CA3 (versus sham). (G) Stereological quantification of the absolute PV(+) cells in DG. (H) Percentage of damage of PV(+) cells in DG (versus sham). Data in all panels were analyzed by one-way ANOVA followed by Tukey’s test. Data represent means ± S.E.M. (n = 6–10 per group). *P < 0.05 versus sham group; ^#P < 0.05 versus control group (DFP exposed, midazolam treated).

Synthetic Hydrophilic Neurosteroids Reduce Astrogliosis in the Hippocampus and Amygdala in DFP-Exposed Rats

To evaluate the neuroprotective effect of synthetic neurosteroids on the OP-induced activation of astrocytes and inflammatory response in the hippocampus and AMY, brain slices were immunostained for GFAP(+). Figure 10 presented representative images of astrocyte distribution in different subfields of the hippocampus for sham, control, and synthetic neurosteroid–treated groups. The top panel (Fig. 10, A1–4) displayed images of the entire hippocampus at 1.25× objective lens. The bottom panels (Fig. 10, B–E) showed magnified images at 60× objective lens for the designated hippocampal subfields (CA1, CA3, and DG) and the AMY region. Astrogliosis, as seen in the control group, was visualized by a distinct morphologic shape in response to neuroinflammation; this distinct shape included noticeably swollen somas and thickened and extensive branching of the cell processes. Astrogliosis was markedly visible in the hippocampal (i.e., CA1, CA3, and DG) and AMY regions in the control rat brain compared to the sham rat brain. In comparison, the VX- and LX-treated groups showed far less astrogliosis in all regions, indicating the potential protective effects of these synthetic neurosteroid treatments.

Fig. 10.

Protective effects of VX and LX on GFAP(+) astrocyte neuroinflammation in the hippocampus (HPC) and AMY in DFP-exposed rats. Three months after DFP exposure, representative sections of GFAP(+) immunostaining were examined in the various brain regions of sham, control, VX-treated (10 mg/kg), and LX-treated (10 mg/kg) animals. In the control group, GFAP(+) staining revealed a significant reactive astrocyte image, including swollen somas with thickened and extended processes. Amygdala images are taken from the basolateral amygdala.

Area fractional densitometric analysis indicated a significant increase in GFAP expression (∼122% of sham, P < 0.05) throughout the whole hippocampus (Fig. 11, A and B), including subfields of the CA1 (∼115% of sham, P < 0.05; Fig. 11, C and D), CA3 (∼122% of sham, P < 0.05; Fig. 11, E and F), DG (∼130% of sham, P < 0.05; Fig. 11, G and H), and amygdala (∼158% of sham, P < 0.05; Fig. 11, I and J), indicating activation of astrocytes and neuroinflammation caused by OP exposure. VX (10 mg/kg) treatment significantly decreased GFAP(+) expression across the hippocampus and amygdala compared with control animals (Fig. 11, A–J). This treatment showed a significant reduction in astrogliosis in the hippocampus and amygdala (CA1, ∼101%; CA3, ∼102%; DG, ∼101%; AMY, ∼102% of sham, respectively), suggesting a potential neuroprotective effect (Fig. 11, D–J). Similar neuroprotective effects were observed in the LX-treated (10 mg/kg) group (Fig. 11, A–J). These findings suggest that synthetic neurosteroids modify immune responses by reducing astrocyte activation after DFP exposure.

Fig. 11.

Protective effects of VX and LX on GFAP(+) astrogliosis and inflammation in the hippocampus (HPC) subfields and amygdala in DFP-exposed rats. (A) Area fractionation (AF) densitometric quantification of GFAP(+) expression in HPC. (B) Percentage of damage of GFAP(+) microgliosis in HPC (versus sham animals). (C) AF densitometric quantification of GFAP(+) expression in CA1. (D) Percentage of damage of GFAP(+) microgliosis in CA1 (versus sham animals). (E) AF densitometric quantification of GFAP(+) expression in CA3. (F) Percentage of damage of GFAP(+) microgliosis in CA3 (versus sham animals). (G) AF densitometric quantification of GFAP(+) expression in DG. (H) Percentage of damage of GFAP(+) microgliosis in DG (versus sham animals). (I) AF densitometric quantification of GFAP(+) expression in amygdala. (J) Percentage of damage of GFAP(+) microgliosis in the amygdala (versus sham animals). Data in all panels were analyzed by one-way ANOVA followed by Tukey’s test. Data represent means ± S.E.M. (n = 6–10 per group). *P < 0.05 versus sham group; ^#P < 0.05 versus control group (DFP exposed, midazolam treated).

Synthetic Hydrophilic Neurosteroids Reduce Activated Microgliosis in the Hippocampus and Amygdala in DFP-Exposed Rats

To evaluate the effect of synthetic neurosteroids on the DFP-induced activation of microglia and inflammatory response, we immunostained for IBA1(+). Figure 12 presents images of microglia distribution in different subfields of the hippocampus for sham, control, and synthetic neurosteroid–treated groups. The top panel (Fig. 12, A1–4) displayed images of the entire hippocampus at 1.25× objective lens. The bottom panels (Fig. 12, B–E) showed magnified images at 60× objective lens for the designated hippocampal subfields (CA1, CA3, and DG) and the AMY region. Microgliosis, as seen in the control group, was visualized by its distinct morphologic shape in response to neuroinflammation; this distinct shape was marked by an increase in soma size and ramified processes. Microgliosis was markedly visible in the hippocampal (i.e., CA1, CA3, and DG) and AMY regions in the control rat brain compared with the sham rat brain. In comparison, the VX- and LX-treated brains showed far less microgliosis in all regions, indicating the potential protective effects of these synthetic neurosteroid treatments.

Fig. 12.

Protective effects of VX and LX on IBA1(+) microglial neuroinflammation in the hippocampus (HPC) and AMY in DFP-exposed rats. Three months after DFP exposure, representative sections of IBA1(+) immunostaining were examined in the various brain regions of sham, control, VX-treated (10 mg/kg), and LX-treated (10 mg/kg) animals. In the control group, IBA1(+) staining revealed a significant reactive microglial image, including enlarged somas and extended processes. Amygdala images are taken from the basolateral amygdala.

Area fractional densitometric analysis indicated a significant increase in IBA1(+) expression (∼135% of sham, P < 0.05) throughout the whole hippocampus (Fig. 13, A and B), including the CA1 (∼133% of sham, P < 0.05; Fig. 13, C and D), CA3 (∼171% of sham, P < 0.05; Fig. 13, E and F), DG (∼113% of sham, P < 0.05; Fig. 13, G and H), and AMY regions (∼125% of sham, P < 0.05; Fig. 13, I and J), indicating microglial recruitment and severe inflammation caused by OP exposure. VX (10 mg/kg) treatment significantly decreased IBA1(+) expression across the hippocampus and amygdala compared with control animals (Fig. 13, A–J). This treatment showed a significant reduction in microgliosis in the hippocampus and amygdala (CA1, ∼117%; CA3, ∼121% DG, ∼105%; AMY, ∼108% of sham, respectively), suggesting a potential neuroprotective effect (Fig. 13, D–J). Similar neuroprotective effects were observed in the LX-treated (10 mg/kg) group (Fig. 13, A–J). These findings suggest that synthetic neurosteroids modify immune responses by reducing microglial activation after DFP exposure.

Fig. 13.

Protective effect of VX and LX on IBA1(+) astrogliosis and inflammation in the hippocampus (HPC) subfields and amygdala in DFP-exposed rats. (A) Area fractionation (AF) densitometric quantification of IBA1(+) expression in HPC. (B) Percentage of damage of IBA1(+) microgliosis in HPC (versus sham). (C) AF densitometric quantification of IBA1(+) expression in CA1. (D) Percentage of damage of IBA1(+) microgliosis in CA1 (versus sham animals). (E) AF densitometric quantification of IBA1(+) expression in CA3. (F) Percentage of damage of IBA1(+) microgliosis in CA3 (versus sham). (G) AF densitometric quantification of IBA1(+) expression in DG. (H) Percentage of damage of IBA1(+) microgliosis in DG (versus sham). (I) AF densitometric quantification of IBA1(+) expression in the amygdala. (J) Percentage of damage of IBA1(+) microgliosis in the amygdala (versus sham). Data in all panels were analyzed by one-way ANOVA followed by Tukey’s test. Data represent means ± S.E.M. (n = 6–10 per group). *P < 0.05 versus sham group; ^#P < 0.05 versus control group (DFP exposed, midazolam treated).

Discussion

In this study, we show for the first time that delayed treatments with the novel, water-soluble synthetic neurosteroids VX and LX attenuate long-term neurologic dysfunction and neuropathological damage after acute DFP exposure in rats. The key findings of this study are as follows: 1) synthetic neurosteroids effectively reduced aggression-like behavior; 2) groups treated with synthetic neurosteroids exhibited a significant reduction in anxiety and depression-like behavior; 3) synthetic neurosteroids effectively alleviated short-term, long-term, and spatial memory impairment; 4) synthetic neurosteroids demonstrated robust neuroprotection by preventing the loss of principal neurons and interneurons in the hippocampus; and 5) the application of synthetic neurosteroids resulted in a reduction in inflammatory astrogliosis and microgliosis in the hippocampus and amygdala. The observed improvements in neuropathological defects align with the improvement of neurologic functional deficits. As projected, VX and LX elicited protection against OP-induced SE, chronic epileptic seizures, and ictal biomarkers in the DFP model (Ramakrishnan et al., 2024). Together, these findings establish VX and LX as potent and efficacious synthetic neurosteroids with drug-like properties suitable for counteracting the long-term neuropsychiatric sequel following OP exposure.

Neurosteroids have emerged as an effective treatment of acute and long-term impact of OP intoxication (Reddy, 2016). Mechanistically, neurosteroids exhibit superior features compared to benzodiazepines for activating both synaptic and extrasynaptic GABA-A receptors and confering robust seizure protection (Reddy and Rogawski, 2010; Wu et al., 2013; Reddy et al., 2015; Carver and Reddy, 2016; Chuang and Reddy, 2018; 2020). Neurosteroids have demonstrated effectiveness in terminating SE (Chuang and Reddy, 2018). Unlike benzodiazepines, neurosteroids do not lead to anticonvulsant tolerance (Reddy, 2010). These neurosteroids have been reported to potentiate inhibitory currents through a novel mechanism of action that activates all isoforms of GABA-A receptors, including extrasynaptic GABA-A receptors (Scimemi et al., 2005; Zolkowska et al., 2018; Reddy, 2019; Saporito et al., 2019). Due to its broad impact on various types of GABA-A receptors, these synthetic hydrophilic neurosteroids with improved bioavailability demonstrated greater efficacy compared with midazolam, even when administrated at 40 minutes after DFP. This is attributed to its ability to generate inhibitory tonic currents, which remain unaffected even when synaptic GABA-A receptors undergo internalization during prolonged SE (Naylor et al., 2005; Goodkin et al., 2008; Carver and Reddy, 2013; Wu et al., 2013). Thus, neurosteroid intervention after OP exposure would be a powerful adjunct to benzodiazepine treatment that offers mechanistic advantages in mitigating the long-term impact of OP neurotoxicity because of the pharmacological synergism between neurosteroids and benzodiazepines (Chuang and Reddy, 2020).

DFP is used as an experimental tool and nerve agent surrogate for the modeling of OP intoxication. It produces consistent seizures, neuronal damage, and long-term neurologic effects reminiscent of humans exposed to OP chemicals and nerve agents (Deshpande et al., 2010; Wright et al., 2010; Pouliot et al., 2016; Kuruba et al., 2018; Wu et al., 2018; Reddy et al., 2019; 2021). Prolonged SE, caused by OP exposure, poses a substantial risk of long-term behavioral and cognitive effects associated with neuropathological changes later in life (Brewer et al., 2013; Abou-Donia et al., 2016; Rojas et al., 2016; Lumley et al., 2019). These findings closely resemble the manifestation of neurologic symptoms observed in both humans and rats following acute DFP exposure (Ricceri et al., 2006; Venerosi et al., 2008; Langston et al., 2012; Muñoz-Quezada et al., 2013; Talabani et al., 2018).

Brexanolone and ganaxolone have shown superior anticonvulsant properties compared with midazolam in controlling persistent SE and mitigating neuronal damage caused by DFP and soman (Reddy, 2019a,b). However, the clinical use of ganaxolone-like neurosteroids faces some obstacles, including poor water solubility, limited bioavailability, rapid hepatic inactivation, and short plasma half-life, which complicates the formulation of stable aqueous injection products. To overcome these limitations, we have synthesized various water-soluble analogs around the ganaxolone scaffold that exhibit improved anticonvulsant properties (Reddy, 2019b). This breakthrough has resulted in the development of hydrophilic analogs named VX and LX, which overcome the challenges associated with FDA-approved brexanolone and ganaxolone. They are potent modulators of GABA-A receptors with robust anticonvulsant activity. They possess water solubility and favorable biopharmaceutical properties. Both VX and LX are formulated as dry powders, allowing for convenient mixing before parenteral administration and ensuring stability for long-term storage.

We observed strong protective effects of neurosteroid analogs in the DFP model. Limbic areas, such as the amygdala and hippocampus, have been implicated in mediating the expression of anxiety and aggressive behaviors (Blanchard et al., 1970, 1978; Davis et al., 1994; Price and Drevets, 2010). In this study, the synthetic neurosteroids VX and LX were found to decrease the severity of anxiety and depressive symptoms associated with DFP exposure. These enduring manifestations of anxiety and depression are frequently observed in cases of human OP poisonings (Eskenazi et al., 1999; Chen, 2012; Supasai et al., 2020; Calsbeek et al., 2021). As expected, the neurodegeneration observed in these limbic regions may contribute to the emergence of mood, anxiety, and memory deficits. The anxiety-like behavior observed in control animals could potentially be attributed to neuronal damage occurring in crucial regions such as the frontal lobe, cingulate cortex, amygdala, and hippocampus (Brandt et al., 2003; Ohman, 2005; Kennedy et al., 2009; Adolphs, 2010; Flannery et al., 2016; LaGrant et al., 2020). Thus, it is likely that neuronal injury or neurodegeneration in specific brain regions is manifested as chronic behavioral deficits and poorer cognitive performance in DFP-exposed animals.

Neurosteroids have garnered significant interest in the field of neuroinflammation and neurodegenerative diseases due to their ability to regulate inflammation in the brain (Maguire and Mennerick, 2023). Neuroinflammation has been implicated in cognitive dysfunctions (Kofman et al., 2006; Padurariu et al., 2012; Flannery et al., 2016). Astrocytes play a crucial role in promoting neuroprotection by mitigating the inflammatory response through various mechanisms (Zhang et al., 2018). Neuroinflammation often triggers microgliosis, leading to various immune functions attempting to restore tissue balance through simultaneous tissue breakdown and repair processes (Woodburn et al., 2021). However, unregulated microglial inflammatory responses can negatively impact neuronal function and survival, contributing to neurodegenerative diseases. Additionally, activated astrocytes can contribute to inflammation by releasing cytokines, further stimulating microglial activation and neurodegeneration (Sofroniew, 2009; Glass et al., 2010). Both microglia and astrocytes secrete neurotrophic factors, including neural growth factor, brain-derived growth factor, and glial-derived growth factor, supporting neuronal survival (Frade and Barde, 1998; Batchelor et al., 1999; Troncoso-Escudero et al., 2018). Studies have shown that neurosteroids can modulate the activity of microglia and astrocytes, exerting anti-inflammatory effects by reducing the production and release of proinflammatory molecules such as cytokines and inhibiting the activation of these glial cells (Cutler et al., 2007; Hua et al., 2011; Lei et al., 2014; Aryanpour et al., 2017). Astrocytes are also activated by progesterone, leading to the release of a brain-derived neurotrophic factor (Kaur et al., 2007; Jodhka et al., 2009; Su et al., 2012; Atif et al., 2013; Sun et al., 2016). Allopregnanolone modulates microglia and astrocytes, exerting anti-inflammatory effects by inhibiting NFκB (Lambert et al., 2003; Lee et al., 2011; Singh et al., 2013). These reports are consistent with the protective potential of the synthetic neurosteroids VX and LX in the DFP model.

Neurosteroids possess strong neuroprotectant and anti-inflammatory properties. The test synthetic neurosteroids are active at synaptic and extrasynaptic GABA-A receptors (Chuang and Reddy, 2018). They exhibited a broad spectrum of antiseizure activity in diverse models of acute seizures and SE (Reddy, 2023b). The enhancement of tonic inhibition could likely contribute to their disease-modifying effects in the DFP model (Carver and Reddy, 2013; Wu et al., 2013). Neurosteroid treatment reduced acute neuronal loss following DFP-induced SE by significantly attenuating excitotoxicity caused by the excessive activation of the glutamatergic pathway (Reddy, 2016; Neff and Reddy, 2024). Neurosteroid analogs decreased degeneration of neurons, specifically inhibitory interneurons in the hippocampus and other regions. Moreover, they exhibit striking disease-modifying effects on DFP-induced long-term epileptic seizures and discharges (Ramakrishnan et al., 2024). These mechanistic and neuropathological improvements likely led to the protective effects observed in seizure outcomes. Prior studies have also suggested that neurosteroids possess the ability to directly regulate the immune response, leading to a decrease in neuroinflammation (Müller and Kerschbaum, 2006). Overall, due to their multimodal actions at tonic inhibition, inhibitory networks, and anti-inflammatory properties, synthetic neurosteroids may offer an adjunct option in mitigating the long-term deficits of acute OP exposure.

Despite the strong neuroprotectant potential of novel neurosteroids in the DFP model, certain caveats should be considered when interpreting these findings. First, the tested drugs were administered in two different doses in a delayed protocol, 40 minutes after the DFP challenge, and in combination with midazolam. The study design required the use of midazolam to improve survival after DFP exposure (Wu et al., 2018; Reddy et al., 2020b). However, it is likely that these neurosteroids alone also could elicit neuroprotection, as evident from ganaxolone (Neff and Reddy, 2024). Second, the precise progression of neuronal damage is not estimated directly. This lack of longitudinal evaluation of efficacy affects the interpretation of the outcomes of neuroprotection. Furthermore, the studies were conducted using male rats. Future studies conducted in females will evaluate the sex differences of neurosteroids in OP models. Additionally, ongoing pharmacokinetic studies on synthetic neurosteroids allow the correlation between neurosteroid levels and their protective responses. Regarding dose-related protection, we observed a distinct pattern where higher doses consistently provided greater protection. Notably, this pattern was evident in outcomes such as the open field test, plus maze behavior, aggression scores, and the object recognition test. The lack of such distinction in the water maze test and histology is likely to be attributed to a ceiling effect on these particular parameters. Nevertheless, the use of rigorous experimental protocols and outcome parameters in assessing the effects of neurosteroids on behavior deficits and histopathological changes strongly supports the neuroprotectant potential of tested synthetic neurosteroids in the OP model.

In conclusion, these results indicate that the administration of the synthetic water-soluble neurosteroids VX and LX effectively attenuates chronic neurologic dysfunction and memory deficits associated with OP exposure. Neurosteroid treatment also mitigates DFP-induced chronic neurodegeneration and neuroinflammation. The neuroprotective responses in neuropathological analysis consistently correlates with their beneficial effect on improving neurobehavioral outcomes, providing pilot mechanistic insights. VX and LX were designed to improve hydrophilic properties for better bioavailability and tissue distribution than ganaxolone. In addition, they decreased the occurrence of chronic epileptic seizures and ictal biomarkers in the DFP-induced refractory SE model (Ramakrishnan et al., 2024). Collectively, these findings demonstrate the application of these synthetic neurosteroids for ameliorating the long-term neurologic consequences after OP intoxication with drug-like properties for hospital and field-ready injectable dosing.

Abbreviations

AMN

atropine methyl nitrate

AMY

amygdala

CA

cornus ammonis

DFP

diisopropylfluorophosphate

DG

dentate gyrus

DH

dentate hilus

EPM

elevated plus maze

FDA

Food and Drug Administration

GFAP

glial fibrillary acidic protein

IBA1

ionized calcium binding adaptor molecule 1

LX

lysaxanolone

MDZ

midazolam

NeuN

neuronal nuclei antigen

OP

organophosphate

2-PAM

pralidoxime chloride

PV

parvalbumin

SE

status epilepticus

VX

valaxanolone

Authorship Contributions

Participated in research design: Reddy.

Contributed research reagents: Reddy.

Conducted experiments: Reddy, Singh, Huber, Wu.

Performed data analysis: Reddy, Singh, Ramakrishnan, Wu.

Wrote or contributed to the writing of the manuscript: Reddy, Singh, Ramakrishnan, Huber, Wu.