Abstract
Children are especially vulnerable to the neurotoxic effects of nerve agents, which can cause lasting neuronal dysfunction, including cognitive impairments, epilepsy, and related comorbidities. Current benzodiazepine anticonvulsants often fail to prevent long-term neuropathology following neurotoxic chemical exposure, underscoring the urgent need for more effective treatments. This study investigated the therapeutic potential of the synthetic neurosteroid ganaxolone (GX) in a pediatric rat model of the nerve agent soman exposure. Pediatric (postnatal day 21) rats were acutely exposed to soman and received GX alone or in combination with midazolam treatment 40 minutes later. Continuous video-EEG monitoring, behavioral assessments and MRI imaging were conducted over a 3-month period after the acute challenge. Soman exposure led to persistent epileptic seizures, electrographic ictal biomarkers, cognitive dysfunction, behavioral impairments, hippocampal atrophy, neuroinflammation and neurodegeneration. Midazolam alone had minimal neuroprotective effects. In both monotherapy and combination regimen, GX significantly reduced memory deficits, anxiety, and depressive-like behaviors. GX attenuated spontaneous seizures and suppressed non-convulsive epileptiform discharges, interictal spikes, and high-frequency oscillations, suggesting disease-modifying effects. Mechanistic immunohistology analyses showed that GX preserved parvalbumin-positive inhibitory interneurons and NeuN-positive principal neurons, enhanced doublecortin-positive neurogenesis in the hippocampus, and reduced inflammatory microgliosis as indicated by IBA1 expression. MRI scans confirmed reduced neuropathological changes in GX-treated animals. Collectively, these findings demonstrate that GX alone or in combination with midazolam offers strong neuroprotective, antiepileptogenic, and anti-inflammatory benefits, highlighting its promise as a robust pediatric anticonvulsant for nerve agent-induced seizures and long-term neurologic dysfunction.
Keywords: Epilepsy, Epileptogenesis, Neuroinflammation, Nerve agent, Soman, Pediatric
1. Introduction
Nerve agents such as sarin, cyclosarin, and soman (GD) are extremely lethal chemical agents that are used in military operations or terrorist attacks on civilians. Nerve agents are highly potent neurotoxic agents that irreversibly inhibit acetylcholinesterase (AChE), leading to high acetylcholine levels (Tafuri and Roberts, 1987; de Araujo Furtado et al., 2012). Excessive synaptic acetylcholine levels overstimulate cholinergic receptors, subsequently activating glutamatergic pathways, potentially leading to prolonged muscle twitches, seizures, status epilepticus (SE) and even death (Reddy, 2024). The devastating effects of nerve agents have been shown in the large-scale injury and death caused by the sarin attacks in Syria and Tokyo (Reddy and Colman, 2017; Rosman et al., 2014; Yanagisawa et al., 2006). Current standard treatment for nerve agent exposure involves the use of a three-drug combination regime: (a) a muscarinic antagonist such as atropine that preserves vital functions by blocking the peripheral deleterious effects of excess acetylcholine, (b) an oxime reactivator such as pralidoxime to reactivate AChE, and (c) a benzodiazepine anticonvulsant such as diazepam or midazolam (Reddy, 2024; Reddy and Reddy, 2015). While benzodiazepines can help control seizures, these medications must be given less than 40 minutes after nerve agent exposure to have a significant effect (Apland et al., 2014; Reddy and Reddy, 2015; Singh et al., 2024a; Wu et al., 2018), making benzodiazepines impractical in many instances of exposure where victims are out of vicinity from treatment centers. Additionally, because benzodiazepines do not act on extrasynaptic GABA-A receptors, they may have limited efficacy once synaptic inhibition is compromised (Reddy and Reddy, 2015; Wu et al., 2018). Thus, there is a critical need for novel neuroprotectants to prevent dire long-term neurological complications in those exposed to nerve agents.
Children are more vulnerable to the neurotoxic effects of nerve agents and organophosphorus (OP) pesticides. Every year, around 3 million exposures to OPs occur, resulting in ~300,000 deaths (Amir et al., 2020; Gunnell and Eddleston, 2003; Neff and Reddy, 2024). Children represent about one-third of all victims affected by early life OP exposure (Dharmani and Jaga, 2005; Gummin et al., 2021). Moreover, children who survive the initial OP poisoning risk life-long neurological deficits, including anxiety, depression, memory loss and post-traumatic stress disorder (Cavalheiro et al., 1987; Cilio et al., 2003; Rice et al., 1998). This exposure can also severely impair development causing learning disabilities, attention disorders, diminished short-term visual and verbal memory, and even movement disorders (Eskenazi et al., 2007; Joosen et al., 2009; Neff and Reddy, 2024; Pulkrabkova et al., 2023; Wright et al., 2016). The devastating effects of OPs in children emphasizes the urgent necessity for effective treatments to mitigate the long-term consequences associated with OP pesticides and nerve agent exposure.
Our previous research has introduced neurosteroids as a promising new approach to treat seizures and SE, particularly those caused by OP poisoning and nerve agents (Ramakrishnan et al., 2025; Reddy, 2024, 2016; D. S. Reddy, 2019; Reddy et al., 2024b; Reddy and Estes, 2016). Neurosteroids are positive allosteric modulators and direct activators of both synaptic and extrasynaptic GABA-A receptors (D. S. Reddy, 2019; Reddy, 2016; Reddy and Estes, 2016). GABA-A receptors are pentameric chloride channels composed of various α, β, γ, δ, and other subunits and mediate synaptic (phasic) and extrasynaptic (tonic) inhibition. Neurosteroids directly open the GABA-A receptor chloride channels, including extrasynaptic GABA-A receptors which are crucial for controlling seizures (Carver and Reddy, 2013; D. S. Reddy, 2019; Reddy, 2016, 2002; Reddy et al., 2019; Reddy and Rogawski, 2002; Wu et al., 2013). Extrasynaptic receptors do not internalize during SE and can shunt hypersynchronous discharges that cause SE (D. Reddy, 2019; Reddy, 2024, 2018; Reddy and Estes, 2016). The use of neurosteroids shows promise, with recent approvals for three neurosteroids (brexanolone, ganaxolone and zuranolone) for treating different brain disorders. Ganaxolone (GX) is FDA-approved for the treatment of seizures associated with CDKL5-deficiency disorder (Knight et al., 2022; D. Reddy, 2019). In experimental models of SE, neurosteroids including allopregnanolone and GX showed great promise in preventing seizures and brain damage (Althaus et al., 2020, 2017; Andrew et al., 2025; Nguyen et al., 2024; D. Reddy, 2019; Reddy, 2024; Rogawski et al., 2013; Saporito et al., 2019). The neurosteroids halted seizures and reduced neuronal damage even when given 40 minutes post OP (Barker et al., 2020; Reddy, 2024, 2004; Saporito et al., 2019; Zolkowska et al., 2018). GX, a synthetic neurosteroid, possesses even better properties for potential human use for effective treatment of acute seizure and SE post nerve agent exposure (Reddy and Woodward, 2004). We have characterized GX’s anticonvulsant effects, pharmacokinetics, and its safety profile (Chuang and Reddy, 2018; Reddy, 2024, 2016; Reddy et al., 2019). These outcomes have led GX to be tested in clinical trials as a treatment of refractory SE (Vaitkevicius et al., 2022).
Emerging evidence highlights important age-dependent differences in the pathophysiology and treatment responsiveness of nerve agent-induced seizures. Indeed, studies in juvenile and infant rat models demonstrate that while benzodiazepines can terminate seizures, they fail to confer sustained neuroprotection and are associated with long-term behavioral deficits (Apland et al., 2018a; De Araujo Furtado et al., 2024). In contrast, antiglutamatergic strategies targeting AMPA, kainate, and NMDA receptors have shown markedly superior efficacy. Notably, the combined administration of the AMPA/GluK1 receptor antagonist LY293558 (tezampanel) with the NMDA/muscarinic antagonist caramiphen (CRM) provides rapid seizure termination and prevents long-term neuropathology in pediatric models (Apland et al., 2018a, 2018b; De Araujo Furtado et al., 2024). These findings are consistent with the mechanistic understanding that glutamatergic hyperexcitation, rather than cholinergic overdrive alone, sustains and propagates SE following soman exposure. Furthermore, sex-dependent differences in treatment response and disease progression have also been reported, further emphasizing the complexity of pediatric soman toxicity (Figueiredo et al., 2025). Therefore, neurosteroids represent a promising therapeutic approach due to their ability to modulate both synaptic and extrasynaptic GABA-A receptors, offering potential advantages in refractory and pediatric nerve agent-induced seizures.
In this study, we investigated the comprehensive protective potential of the synthetic neurosteroid GX in preventing nerve agent-induced chronic epilepsy, mitigating chronic neurological impairments and neuropathological changes resulting from acute soman exposure-induced SE in pediatric (early childhood) rat model of nerve agent exposure. Our findings reveal a remarkable neuroprotective efficacy of GX monotherapy and its combination with midazolam in reducing epileptogenesis, alleviating neurological deficits and preventing long-term neurodegeneration and neuroinflammation in the pediatric soman model.
2. Materials and methods
2.1. Animals
Postnatal day 21 (P21) male Sprague-Dawley rat pups (45-60 g) were utilized in this study. Rats at P21 age corresponds broadly to early childhood in humans (about 2–3 years of age) based on comparative neurodevelopmental milestones. Rat pups were co-housed with their dams in individual standard cages within an environmentally controlled vivarium and provided with ad libitum access to food and water. Experiments were conducted after 1 week of acclimation to the vivarium. Drug solutions were administered subcutaneously (1 ml/kg) or intramuscularly (0.5 ml/kg) according to the animal’s body weight. All procedures followed the guidelines outlined in the NIH Guide for the Care and Use of Laboratory Animals and were carried out under a protocol approved by the university’s Institutional Animal Care and Use Committee.
2.2. Soman exposure and experimental design
The experimental procedure for soman (GD) exposure and SE induction is depicted in Figure 1. Soman was administered subcutaneously to P21 rats as described previously (Miller et al., 2015; Reddy et al., 2021; Wright et al., 2016). Soman exposure studies were conducted at the MRI Global facility (Kansas City, MO). Asoxime chloride (HI-6; 125 mg/kg), an oxime reactivator of cholinesterase, was administered intraperitoneally 30 minutes before soman exposure to improve immediate survival. A pediatric dose of soman (86.8 μg/kg, 1.4 x LD50) was administered subcutaneously to induce persistent intoxication and SE. One minute post soman injection, atropine methylnitrate (AMN, 2 mg/kg) was administered intramuscularly to minimize peripheral cholinergic toxic effects. AMN, which crosses the blood-brain barrier more poorly than atropine sulfate, was selected to minimize brain penetration and limit its neurological effects. Test drug GX was administered intramuscularly 40 minutes post soman exposure, with or without intramuscular midazolam (MDZ). The rat pups were monitored frequently during a recovery period for 7 days after soman exposure. All surviving rat pups were shipped to Texas A&M facility (Table 1) for chronic monitoring studies as outlined in Figure 1.
Fig. 1. Experimental protocol for Soman exposure model in pediatric postnatal day 21 (P21) rats.
In the soman (GD) model, P21 male rats were exposed to GD (86.8 μg/kg, s.c.). Asoxime chloride (HI-6, 125 mg/kg, i.p.) was administered 30 min before GD to increase the survival rate. Within 1 min of GD exposure, they were treated with atropine methylnitrate (atropine, 2 mg/kg, i.m.). Midazolam (1 mg/kg, i.m.) and/or Ganaxolone (10 - 20 mg/kg, i.m.) were given at 40 minutes after soman exposure. Rats were monitored longitudinally for behavioral deficits and EEG seizures, and biochemical and neuropathological assessments were performed 4 months after GD exposure
Table 1.
Experimental design for evaluation of GX therapy in the chronic pediatric GD model.
| Treatment | Group size (N) in chronic studies | Description |
|---|---|---|
| Control (age-matched) | 12 | Unexposed group (with AMN and HI-6) |
| GD | 12 | GD exposure; received vehicle injection |
| GD + MDZ | 10 | GD exposure; received MDZ (1 mg/kg, i.m.) at 40 min |
| GD + GX-10 | 10 | GD exposure; received GX (10 mg/kg, i.m.) at 40 min |
| GD + MDZ + GX-10 | 10 | GD exposure; received MDZ (1 mg/kg, i.m.) + GX (10 mg/kg, i.m.) at 40 min |
| GD + GX 20 | 10 | GD exposure; received GX (20 mg/kg, i.m.) at 40 min |
AMN, atropine methylnitrate; GD, soman; GX, ganaxolone; HI-6, Asoxime chloride; MDZ, midazolam.
2.3. Drug treatment
The overall experimental design for drug treatment, study groups and group sizes is presented in Table 1. Rat pups were randomized into various groups. Group 1 (control) was age-matched, vehicle-treated non-exposed animals. This group received HI-6 and AMN regimens but did not receive GD or GX. Group 2 (GD) received only soman without any anticonvulsant drug treatment. Group 3 (GD + MDZ) received soman followed by the standard anticonvulsant MDZ (1 mg/kg, i.m.) at 40 minutes. Group 4 (GD + GX 10) received GD followed by GX (10 mg/kg, i.m.) at 40 minutes. Group 5 (GD + MDZ + GX 10) received soman followed by MDZ (1 mg/kg) and GX (10 mg/kg) at 40 minutes. Group 6 (GD + GX 20) received soman followed by GX (20 mg/kg) at 40 minutes. All groups received the standard regimen of HI-6 and AMN injections. Atropine methylnitrate and asoxime chloride (HI-6) were purchased from Sigma-Aldrich (St. Louis, MO). A commercially available midazolam (MDZ; 5 mg/ml) injection product was obtained from Hospira Inc. (Lake Forest, IL).
The overall experimental timeline for behavioral and MRI assessments is illustrated in Figure 1. Behavioral observations were conducted at 1-, 2-, and 3-months following soman exposure, encompassing assessments of aggression, memory, anxiety, exploratory behavior, and depression. At the 3-month timepoint, cognitive performance was evaluated using the Morris water maze test. Deep T2-weighted MRI was performed to detect neuroanatomical abnormalities. For EEG recordings, stainless steel electrodes were surgically implanted in the hippocampus to monitor seizure activity post-soman exposure. After completing behavioral testing, animals were deeply anesthetized, and blood was collected via cardiac puncture. Plasma was separated for biochemical analyses, including cytokine and inflammatory biomarker quantification. Subsequently, animals were transcardially perfused with 4% paraformaldehyde, and brains were harvested and processed for histological evaluation, as detailed below (Ramakrishnan et al., 2025; Singh et al., 2024b).
2.4. EEG Recording and analysis
Rats were implanted with EEG electrodes (Plastics One, Roanoke, VA) in the hippocampus and cortex as previously described (Wu et al., 2018; Reddy et al., 2021). Electrode implantation was performed at 40 days post-soman. Briefly, rats were anesthetized with ketamine (100 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.). A stainless-steel surface EEG electrode (Plastics One, Roanoke, VA) was placed over the right frontoparietal cortex, and an intracranial depth electrode was inserted into the right dentate gyrus. A reference electrode was positioned over the left cerebellum. Following surgery, animals were allowed to recover and acclimate for two weeks. Continuous (24/7) video-EEG recordings were then obtained for 120 days using pCLAMP software (Molecular Devices, San Jose, CA) interfaced with Grass Technologies hardware. EEG signals were acquired at a sampling rate of 2000 Hz from two channels (hippocampus and cortex) and recorded continuously. A validated MATLAB-based detection pipeline was used to identify and quantify spontaneous recurrent seizures (SRS), epileptiform discharges, high-frequency oscillations (HFOs), and interictal spikes, as previously described (Ramakrishnan et al., 2024).
2.5. Assessment of aggressive reactivity
The evaluations of the rats for aggressive reactivity and startle responses involved tapping on the exterior of the rat’s cage and gently handling them with a blinded investigator (Ramakrishnan et al., 2025; Reddy et al., 2024a). Responses 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, inhibits handling; 5) tail up, biting the edge of the cage when attempting to handle. This test was completed in the home cages within the vivarium facility.
2.6. Assessment of depressive behavior
The social interaction test (SIT) in rats is a widely used behavioral assay to evaluate sociability, social preference, and social avoidance/anxiety-like behavior, in models of neuropsychiatric disorders, brain injury, and neurodevelopmental conditions (File and Hyde, 1978; Reddy et al., 2024a; Wilson and Koenig, 2014). This test measures the natural tendency of rats to seek and engage in social contact with a conspecific. Reduced interaction is interpreted as social withdrawal or increased anxiety, whereas increased interaction reflects normal or enhanced sociability, making it a sensitive behavioral readout for therapeutic efficacy. In this test, an experimental rat is introduced into a designated area and allowed to explore this area for 5 min. After the exploration period, a non-experimental rat is introduced and placed at a distance from the experimental rat. The two rats are then allowed to interact for 5 min. In this time frame, instances where the experimental rat faces the non-experimental rat and explores its head, behind, or other body parts are meticulously observed.
2.7. Assessment of anxiety-like behavior
The elevated plus maze (EPM) is a widely employed experimental method for assessing anxiety-like phenotypes in animals (Reddy et al., 2024a; Reddy and Kulkarni, 1996). It consists of a cross-shaped apparatus made from perspex walls with four arms that are 50 cm long and 10 cm wide, elevated 50 cm above the floor (Harvard Apparatus). Two opposing arms are enclosed with 40 cm high walls (closed arms), while the other two arms are open (open arms). The central area where the arms intersect consists of 10 x 10 cm block. In the test, each rat is positioned in the central zone of the EPM with its head facing a closed arm and is permitted to freely explore the maze for 5 min. Anxiety-like phenotype is estimated by measuring the time spent in open and closed arms, where heightened anxiety-like phenotypes correlate with more time in the closed arms.
The open field test (OFT) is a commonly used method in behavioral research, providing insights into anxiety-related exploratory behavior, and emotion in rodents (Hall and Ballachey, 1932; Reddy et al., 2024a). The open field arena consists of a 100 × 100 cm Plexiglass box divided into 16 squares (25 × 25 cm each). The four squares located in the central area are defined as the central zone, the four squares at each corner are designated as corners, and the remaining squares in the periphery are defined as sides. Each rat is placed in one of the corners of a brightly lit open field, and its movement is recorded for 10 min. The apparatus is cleaned with 70% alcohol and air-dried prior to each trial. The time spent in the central zone and the number of entries into the central zone are measured to assess anxiety-like behavior. Rats displaying heightened anxiety-like phenotypes tend to prefer the corners or walls and avoid spending time in the open central area, perceiving it as an unsafe zone.
2.8. Object recognition memory test
The novel object recognition test (NORT) evaluates recognition memory abilities by observing an animal’s interaction with two objects placed in an open field box (Leger et al., 2013; Pascoli et al., 2009; Reddy et al., 2024a). This test involves placing two objects in an open field box, with three successive trials for each rat with an inter-trial interval of 60 min. During the first two trials, the rat is placed in an empty open field box for five minutes to acclimate (habituation phase). In the same box, two identical objects on opposite sides, and the rat is allowed to explore for five minutes (sample phase). In the third trial, one familiar object and a novel object replace the two identical objects. The rat’s exploration behavior, indicated by a nose proximity of ~ 2 cm to the object, is recorded. The time spent exploring objects is recorded, and the percentage of time spent with the novel object is calculated. A discrimination index (DI) is calculated as: DI = [novel (sec) – familiar (sec)] / [novel (sec) + familiar (sec)]).
2.9. Spatial learning and memory testing
The hippocampus-dependent spatial learning and memory function is evaluated using the Morris water maze (MWM) test (Morris, 1984; Reddy et al., 2024a). Rats are allowed 90 sec to locate the platform using spatial cues in four acquisition trials per day, conducted for seven days. The average latency to reach the platform is plotted to measure the learning process. Additionally, memory retrieval and retention are assessed on day 8 (probe 1) and day 15 (probe 2), respectively. On these probe days, the platform is removed, and the rats’ performance in locating the platform quadrant is observed for 45 seconds. The time spent in the platform quadrant is then measured to assess the effect of GX on spatial learning and memory dysfunction after GD exposure. Seizures were not observed during the learning and probe testing.
2.10. MRI Scanning and analysis
MRI scans were performed three months following soman exposure as per the methodology used previously (Reddy et al., 2023; Wu et al., 2021). Scans were acquired using a 3-Tesla Siemens MAGNETOM B17 Verio scanner equipped with a 15-channel coil. To minimize air gaps during imaging, the scanner was modified with PVC tubing specifically designed for rats. The MRI protocol included T2-weighted coronal dual-echo fast spin-echo and sagittal spin-echo sequences, optimized for high-resolution anatomical imaging using a small animal coil for transmission and a surface coil for signal detection. T2-weighted imaging was utilized to detect structural brain changes, particularly region-specific neuronal damage. Variations in T1 and T2 relaxation times were leveraged to characterize tissue damage, with brain lesion T2 values typically ranging from 170–200 ms, making this approach well-suited for identifying subtle pathologies. Quantitative analyses of brain tissue volumes and T2 signal intensities were conducted using Inveon Research Workplace software, which facilitates reproducible MRI evaluations. Regions of interest (ROIs) were identified in coronal slices to calculate T2 signal intensity for areas such as the hippocampus, thalamus, and lateral ventricles. Additionally, whole-brain ROIs were defined in sagittal slices to measure total brain volume, guided by the Paxinos and Watson Rat Brain Atlas (Paxinos and Watson, 2007). T2 relaxation times within dual-echo coronal slice ROIs were computed using the formula (TE2 – TE1) / ln(SI1 / SI2), where TE1 (27 ms) and TE2 (123 ms) indicate the respective echo times, and SI1 and SI2 represent the signal intensities at those times (Reddy et al., 2020; Wu et al., 2021).
2.11. Brain fixation and histology processing
Three months after soman exposure, P21 rats were anesthetized with ketamine and xylazine and underwent transcardial perfusion with heparinized saline followed by 4% paraformaldehyde in phosphate buffered saline (PBS) (Reddy et al., 2023; Reddy and Abeygunaratne, 2022). The procedure began with perfusion using 150 ml of saline containing heparin (10 IU/ml), followed by 100 ml of 4% paraformaldehyde. After perfusion, brains were extracted, post-fixed in 4% paraformaldehyde overnight at 4°C, and transferred to PBS for 24 hours. Cryoprotection was achieved by sequential immersion in 10%, 20%, and 30% sucrose solutions. The brains were then stored in 20% sucrose in 0.1 M phosphate buffer (pH 7.4) for 72 hours, rapidly frozen in pre-cooled isopentane (−70°C), and stored at −80°C. Coronal sections (30 μm thick) were prepared from the forebrain, encompassing the hippocampus and amygdala, using coordinates relative to bregma (−0.24 mm to −7.44 mm) (Paxinos and Watson, 2007). Sections were collected in 24-well plates containing cold PBS, and every 20th section (600 μm apart) was selected for immunohistochemistry (10 sections per animal, n = 5–11 rats per group). All sections were preserved in a storage solution at −20°C until further processing.
2.12. Immunohistochemistry for neurodegeneration, neurogenesis, and neuroinflammation
Neurodegeneration, neurogenesis, and neuroinflammation in brain sections were assessed using established immunohistochemical protocols (Reddy et al., 2024c; Singh et al., 2024a). Neuronal loss and overall neuronal populations were evaluated through immunostaining for neuronal nuclei antigen (NeuN(+)). Damage to GABAergic inhibitory interneurons, indicative of disrupted neural network regulation, was assessed using parvalbumin (PV(+)) immunostaining. Neuroinflammation, characterized by activated microglia, was detected through ionized calcium-binding adaptor molecule 1 (IBA1(+)) immunostaining. Hippocampal neurogenesis and migration of immature neurons were analyzed via doublecortin (DCX(+)) immunostaining (Godoy et al., 2022; Golub and Reddy, 2022; Mullen et al., 1992; Neff and Reddy, 2024; Rao and Shetty, 2004; Wittekindt et al., 2022).
2.13. Stereology quantification of tissue volume and cell numbers
A rigorous stereology protocol was employed to quantify the total number of NeuN(+), PV(+), and DCX(+) cells, as described previously (Golub et al., 2015; Reddy et al., 2024c). All stereological analyses were conducted under blinded conditions to eliminate observer bias and ensure the integrity of the quantitative data. Cell counting was performed across well-defined hippocampal subregions, including cornu ammonis-1 (CA1), CA2, CA3, the dentate gyrus (DG), and the dentate hilus (DH), each of which was delineated according to established anatomical boundaries. Quantification was carried out using the newCAST stereology software package (version VIS 4.0; Visiopharm, Hørsholm, Denmark) in conjunction with an Olympus BX53 research-grade microscope and a high-resolution DP73 color digital camera.
Tissue volume estimations were obtained using a 10× objective lens in combination with a point grid containing at least 200 individual points, thereby ensuring a complete (100%) volumetric coverage of each designated hippocampal region (Boyce et al., 2010; Golub et al., 2015; Reddy et al., 2024c). Sampling fractions were determined according to both the marker type and the expected cell density within each region. For NeuN(+), PV(+), and DCX(+) immunostaining in CA1, CA3, and DG, a systematic random sampling approach was implemented in which 5% of the total area was analyzed using a 60× objective lens. In contrast, for NeuN and PV immunostaining in CA2 and DH, a 10% area sampling fraction was adopted to compensate for lower neuronal densities and to enhance statistical precision. These sampling strategies were carefully selected to optimize the balance between efficiency, and the accuracy of unbiased stereological cell counts.
2.14. Neuropathology scoring of PV(+) Interneuron in extra-hippocampal regions
Parvalbumin-positive (PV(+)) interneurons were evaluated in extra-hippocampal brain regions, including the thalamus, hypothalamus, amygdala, piriform cortex, somatosensory cortex, and entorhinal cortex, using a neuropathology scoring system (Apland et al., 2010; Myhrer et al., 2005; Wu et al., 2018). Nissl-stained tracings were superimposed onto PV(+) stained sections, and scores were assigned based on the severity of PV(+) immunostaining loss: 0 = no neuropathology (0% affected), 1 = minimal (1–10%), 2 = mild (11–25%), 3 = moderate (26–45%), 4 = severe (>45%). This approach has been shown to yield results consistent with quantitative stereological assessments (Qashu et al., 2010; Wu et al., 2018).
2.15. Area fractionation densitometry for cellular inflammation
IBA1(+) immunostaining was quantified through area fractionation densitometry using NIH ImageJ software, in accordance with previously established protocols (Wu et al., 2018). Images were first processed by applying an intensity threshold to clearly differentiate IBA1-positive cellular staining from background signal. The thresholded images were then analyzed to calculate the proportion of stained area relative to the total measured field or defined region of interest, thereby providing a quantitative assessment of microglial activation levels.
2.16. Multiplex assay for plasma cytokine levels
Blood samples were collected at the study endpoint by cardiac puncture under deep anesthesia into EDTA-coated tubes. Samples were centrifuged at 3000 rpm for 15 minutes, and plasma was collected from the supernatant and stored at −80 °C until further analysis. Simultaneous quantification of pro- and anti-inflammatory cytokines and chemokines in plasma was performed using the Bio-Plex Pro™ Rat Cytokine multiplex assay kit (Bio-Rad, Hercules, CA, USA; Cat. #12005641), a magnetic bead-based immunoassay, according to the manufacturer’s instructions. Briefly, rat cytokine standards were serially diluted to generate standard curves. Magnetic capture beads were added to 96-well plates, washed, and incubated with plasma samples and standards in triplicate at room temperature. Wells were then washed and incubated with detection antibodies, followed by streptavidin-phycoerythrin. After the final washes, assay buffer was added, and the plates were read on a Bio-Plex 200 system (Bio-Rad) using the manufacturer’s default detection settings. A standard curve for all the cytokines was calculated using the standard cytokines, and the concentrations detected in the samples were expressed as pg/mL.
2.17. Statistical analysis
Statistical tests used during the analysis were performed with OriginPro 2020 software (OriginLab Corporation, Northampton, MA). All data were expressed as the mean ± SEM, with individual data points when feasible. In the NORT test, the discrimination index was expressed as the median and interquartile range. Data normality was assessed using the Shapiro-Wilk test to determine whether parametric or nonparametric tests were appropriate. Longitudinal EEG outcomes, including cumulative discharge counts, were analyzed using two-way repeated-measures ANOVA followed by appropriate post hoc comparisons. Incidence curves were analyzed using log-rank tests, and endpoint proportions were analyzed using Fisher’s exact test. Nonparametric data, such as aggression, SRS, or discharges per animal, mean ripple rate, were analyzed using the Kruskal-Wallis test, followed by the Mann-Whitney U test or Dunn’s post hoc test. Parametric outcomes from behavioral tests and neuropathology assays were analyzed using one-way ANOVA followed by Tukey’s post hoc test. Statistical significance was set at p<0.05 unless otherwise specified.
3. Results
3.1. Reduced incidence of epilepsy and seizure frequency in GX-treated pediatric rats
To progressively assess the effect of GX on the development of epilepsy with SRS, we monitored animals for seizure activity for 120 days following soman exposure. Animals were either untreated or treated with MDZ alone, GX alone or GX + MDZ combination. The EEG data were analyzed for two key seizure activity outcomes: (i) SRS and (ii) epileptiform discharges. Representative EEG traces of typical SRS from each group are shown in Figure 2A. As expected, when pediatric rats were exposed to soman, they developed epilepsy characterized by an increased frequency of SRS in the untreated group (Fig. 2B). At 110 days post-exposure, epileptic activity was present in all untreated animals, with no individuals remaining seizure-free. There was a significant reduction in the incidence of epilepsy development in GX + MDZ combination group as compared to MDZ alone, as indicated by a substantial increase in percent seizure free (Fig. 2B). At the end of the study period, seizure freedom rates were highest in the GX + MDZ cohort (86%), compared to 43% in the MDZ-alone group and 57% in the GX-alone group, confirming the enhanced protective efficacy of GX when co-administered with MDZ (Fig. 2C). A further evaluation of seizure burden was ascertained by analysis of cumulative number of seizures in each of these treatment cohorts (Fig. 2D) and total number of seizures in each animal (Fig. 2E). In MDZ + GX combination group, there were significantly reduced cumulative seizures than soman control and MDZ alone groups (p < 0.05; Fig. 2D and Fig. 2E). Collectively, these results demonstrate that GX combined with MDZ effectively prevents epileptogenesis and reduces seizure burden in a pediatric rat model of soman exposure.
Fig. 2. Protective effect of GX monotherapy and its combination with MDZ on the incidence of epilepsy with spontaneous recurrent seizures (SRS) and seizure burden in soman (GD)-exposed rats.
(A) Representative EEG traces illustrate the effect of GX on SRS in a rat on day 108 post-exposure. Traces represent 1-minute EEG epochs in the hippocampus of one randomly selected animal from each cohort. (B) Incidence of SRS frequency in all animals over the 120-day recording period. (C) Percentage of all animals that are seizure-free on day 120 post-exposure. (D) Cumulative number of seizures in all animals over the 120-day recording period. (E) Total number of SRS per animal in each group. Panels B and C were analyzed using log-rank and Fisher’s exact test, respectively. Panels D and E were analyzed using two-way repeated-measures ANOVA followed by post hoc comparisons and the Kruskal-Wallis test, respectively. *p<0.05 versus age-matched control group; #p<0.05 versus GD-exposed animals; &p<0.05 versus GD + MDZ animals (n = 6-7 per group).
3.2. Reduced epileptiform discharges in GX-treated pediatric rats
To evaluate the effect of GX on epileptiform discharges activity, we quantified epileptiform discharges for 120 days following soman exposure. Representative EEG traces of typical discharges from each group are shown in Figure 3A. Soman-exposed animals exhibited a rapid decline in discharge-free status, with all untreated animals developing epileptiform activity by the end of the study period (Fig. 3B). In contrast, the GX + MDZ combination group showed a marked increase in epileptiform discharge-free animals over time compared to MDZ alone and soman controls. (Fig. 3B). At the endpoint, discharge freedom rates were highest in the GX + MDZ cohort (86%), compared to 29% in the MDZ-alone group and 43% in the GX-alone group, confirming the enhanced protective efficacy of GX when co-administered with MDZ (Fig. 3C). Cumulative number of discharges and discharges per animal were calculated to assess overall epileptiform discharge burden (Fig 3D and 3E). In MDZ + GX combination group, there were significantly reduced cumulative discharges than soman control and MDZ alone groups (p < 0.05; Fig. 3D and 3E). Collectively, these results demonstrate that GX combined with MDZ effectively reduces the incidence and burden of epileptiform discharges in a pediatric rat model of soman exposure.
Fig. 3. Effect of GX monotherapy and its combination with MDZ on the incidence of non-convulsive epileptic discharges and discharge burden in soman (GD)-exposed rats.
(A) Representative EEG traces illustrate the effect of GX on the progression of epileptiform discharges in a rat on day 112 post-GD exposure. Traces represent 20-second EEG epochs in the hippocampus of one randomly selected animal from each cohort. (B) Incidence of discharge frequency in all animals over the 120-day recording period. (C) Percentage of all animals that are discharge-free on day 120 post-GD. (D) Cumulative number of discharges in all animals over the 120-day recording period. (E) Total number of discharges per animal in each group. Panels B and C were analyzed using log-rank and Fisher’s exact test. Panels D and E were analyzed using two-way repeated-measures ANOVA followed by post hoc comparisons and the Kruskal-Wallis test, respectively. *p<0.05 versus age-matched control group; #p < 0.05 versus GD-exposed animals; &p<0.05 versus GD + MDZ animals (n = 6-7 per group).
3.3. Reduced HFOs in GX-treated pediatric rats
To investigate the effects of GX on the occurrence of epileptic HFOs, we quantified the average event frequency per minute within two distinct frequency bands: ripples (80 – 250 Hz) and fast ripples (250 – 500 Hz). A computational algorithm was employed for automatic detection of these events. Consistent with previous reports (Ramakrishnan et al., 2024), sham animals exhibited ripples but lacked fast ripples. Animals exposed to soman alone demonstrated a significant elevation in average ripple rate relative to sham controls, indicating a pathological electrophysiological state (Fig. 4A and 4B). Treatment with GX significantly decreased ripple frequency compared to both the GD-only and GD + MDZ groups, reflecting a marked reduction in pathological HFO activity. Similarly, GD-exposed animals showed a significant increase in average fast ripple rate (~160 fast ripples/day) compared to sham, consistent with an epileptic or diseased state (Fig. 4A and 4C). GX-treated animals exhibited a pronounced reduction in mean fast ripple frequency (~26 fast ripples/day), suggesting effective disease modification. The algorithm also computed the average daily counts of ripples and fast ripples across the entire recording period, which are presented in Figures 4D and 4E. The MDZ + GX treatment group exhibited a significant reduction in both daily ripple and fast ripple occurrences. Importantly, this study represents the first continuous computational quantification of HFOs per minute, offering a novel approach for longitudinal characterization of pathological oscillatory activity. These findings collectively demonstrate that GX treatment effectively attenuates pathological high-frequency oscillations associated with epileptogenesis, highlighting its potential as a therapeutic intervention.
Fig. 4. Effect of ganaxolone (GX) on ripples, fast ripples, and interictal spikes in soman (GD)-exposed pediatric rats.
(A) Representative EEG traces illustrating the appearance of ripples, fast ripples, and interictal spikes in EEG. (B) Average rate of ripples per day in each cohort. (C) Number of ripples per day over the entire recording period. (D) Average fast ripple rate per day in each cohort. (E) Number of fast ripples per day over the entire recording period. (F) Representative EEG traces illustrating interictal spikes in various cohorts. (G) Average interictal spike rate in each cohort. (H) Number of interictal spikes per hour over the entire recording period. Data represents mean ± SEM (n=6-7 per group). Panels B, C and G were analyzed using the Kruskal-Wallis test followed by Dunn’s post hoc test. *p < 0.05 versus age-matched control group; #p < 0.05 versus GD-exposed animals; &p < 0.05 versus GD + MDZ animals.
3.4. GX decreases the frequency of interictal spiking events in GX-treated rats
To assess the effects of GX on interictal spiking activity, we quantified the average interictal spike frequency for 60 days (Fig. 4). Across all groups, animals displayed normal interictal spike morphology without qualitative abnormalities. Representative EEG traces of electrographic interictal spikes from GD, MDZ-treated, and GX-treated rats are shown in Figure 4F. Quantitative analysis revealed that GD-exposed animals exhibited a marked increase in interictal spike frequency (~450 spikes/hour) compared with sham controls (Fig. 4F). Both MDZ alone and GX alone significantly reduced interictal spike counts relative to the GD-only group (Fig. 4G). Notably, animals receiving the GX + MDZ combination demonstrated the greatest reduction, with an ~83% decrease in spike frequency, indicating enhanced suppression of aberrant neuronal activity (Figs. 4F–H). These results highlight the efficacy of GX in mitigating hyperexcitable neural network activity that underlies electrographic interictal events.
3.5. Reduced aggressive-like behavioral reactivity in GX-treated rats
To assess GX’s long-term effect on aggression in soman exposed rats, we evaluated the behavior reactivity and startle responses of the rats. Soman exposure resulted in a significant increase in the aggression score at each timepoint in the vehicle treated group compared to controls (p < 0.05; Fig. 5A). MDZ (1 mg/kg) administration at 40 min did not reduce aggression scores; however, there was a significant decrease in aggression score in the group treated with combination of MDZ and GX (10 mg/kg) compared to the group treated with MDZ alone (p < 0.05). Moreover, GX (10 mg/kg or 20 mg/kg) also significantly reduced aggression scores compared to the GD control group and MDZ treated group (p < 0.05). These findings indicate that GX either alone or in combination with MDZ effectively reduces aggressive-like behavior in pediatric rats exposed to soman.
Fig. 5. Effect of ganaxolone (GX) on aggressive reactivity, depression, and anxiety-like behaviors in soman (GD)-exposed pediatric rats.
(A) Aggressive reactivity in GX-treated groups was scored at 1-, 2-, and 3 months after GD exposure. The mean aggression score was scored using a scale of 1-5. (B) The social interaction test assessed social anxiety/depressive-like behavior as total time in contact with another control rat. (C) Number of contacts with another rat in social interaction test. Anxiety-like behavior in the elevated plus-maze test. (D) Time spent in open arms. (E) Time spent in closed arms. The aggression data in Panel 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 ± SEM (n = 8 to 10 per group). *p < 0.05 versus age-matched control group; #p < 0.05 versus GD-exposed animals; &p < 0.05 versus GD + MDZ animals.
3.6. Reduced anxiety/depressive-like behavior in GX-treated rats
To assess the long-term effect of GX on social anxiety/depressive-like behavior in soman-exposed p21 rats, we analyzed social contact times in the SIT. Vehicle-treated animals showed reduced contact time and number of contacts at all time points compared with controls—approximately 35% at 1 month (p < 0.05), 50% at 2 months (p < 0.05), and 45% at 3 months (p < 0.05) (Fig. 5B, 5C). MDZ treatment alone resulted in contact numbers comparable to those of GD controls (Fig. 5C). In contrast, rats receiving MDZ + GX (10 mg/kg) displayed significantly fewer anxiety/depressive-like behaviors than those given MDZ alone (p < 0.05). GX monotherapy at either 10 mg/kg or 20 mg/kg also significantly increased both contact time and contact number compared with vehicle (Fig. 5B, 5C). Collectively, these results demonstrate that GX, whether administered alone or in combination with MDZ, effectively alleviates social anxiety/depressive-like behavior in pediatric rats following soman exposure.
3.7. Reduced anxiety in GX-treated rats
To examine the long-term effect of GX on anxiety-like behavior in soman-exposed p21 rats, the EPM test was used. In the vehicle-treated group, soman exposure significantly reduced time spent in the open arms compared with controls—by ~55% at 1 month (p < 0.05), ~50% at 2 months (p < 0.05), and ~47% at 3 months (p < 0.05) (Fig. 5D). Conversely, time spent in the closed arms was markedly increased—by ~210% at 1 month (p < 0.05), ~270% at 2 months (p < 0.05), and ~200% at 3 months (p < 0.05) (Fig. 5E). MDZ treatment alone produced behavior similar to that of vehicle-treated rats. In contrast, co-administration of GX (10 mg/kg) with MDZ significantly reduced anxiety-like behavior compared with MDZ alone, as indicated by decreased closed-arm time across all time points (Fig. 5E). GX monotherapy (10 mg/kg or 20 mg/kg) also significantly increased open- and closed-arm times toward control levels compared with soman vehicle-treat rats (Figs. 5D, 5E). These results indicate that GX, alone or combined with MDZ, can provide sustained mitigation of soman -induced anxiety-like behavior in pediatric rats.
3.8. GX Reduces exploratory deficits and anxiety
To assess the long-term effects of GX on exploratory and anxiety-like behaviors in soman-exposed p21 rats, we conducted the OFT (Figs. 6A, 6B). In the vehicle-treated group, soman exposure significantly reduced dwell time in the center zone compared with controls—by ~75% at 1 month (p < 0.05), ~72% at 2 months (p < 0.05), and ~41% at 3 months (p < 0.05) (Fig. 6A). MDZ alone produced outcomes similar to vehicle treatment. In contrast, all GX-treated groups—either dose alone (10 mg/kg or 20 mg/kg) or in combination with MDZ—showed significantly increased center-zone dwell time compared with vehicle (p < 0.05), indicating reduced anxiety-like behavior (Fig. 6A). The number of center entries did not differ significantly among groups (Fig. 6B), suggesting that GX-treated animals spent more time per entry in the center rather than making more frequent entries. These results demonstrate that GX, whether alone or combined with MDZ, effectively attenuates soman-induced exploratory deficits anxiety-like behavior in pediatric rats.
Fig. 6. Effect of GX on exploratory and anxiety-like behavior, object recognition memory, and spatial learning in soman (GD)-exposed rats.
(A) Total time in the center as observed in the Open Field Test (OFT). (B) Number of entries in the center as observed in the OFT. (C) Total time spent with the novel object as observed in the novel object recognition task (NORT). (D) Discrimination index as observed in the NORT. (E) Mean latency to reach the platform during the learning trials as observed in the Morris water maze (MWM) test. (F) Short-term memory (probe test 1) and memory retention (probe test 2) were evaluated on days 1 and 8, respectively, after the training session, in the MWM. Data in all panels were analyzed by one-way ANOVA followed by Tukey’s test. Panels A, B, and D are presented as box-and-whisker plots showing the interquartile range (25–75%) and SD. Data in all other panels are presented as mean ± SEM (n = 8–10 per group). *p < 0.05 versus age-matched control group; #p < 0.05 versus GD-exposed animals; &p < 0.05 versus GD + MDZ animals.
3.9. GX Reduces object recognition memory deficits
To evaluate the long-term effects of GX on exploratory behavior and interest in novel objects in GD-exposed p21 rats, we performed the NORT (Figs. 6C, 6D). The NORT assessed short-term memory recognition and cognitive function. Soman exposure significantly decreased both time spent with the novel object and the discrimination index in vehicle-treated rats compared to controls—by approximately 39% at 1 month, 42% at 2 months, and 47% at 3 months (p < 0.05). Rats treated with MDZ alone showed similar impairments as the vehicle group. However, co-administration of GX (10 mg/kg) with MDZ significantly increased time spent with the novel object compared to soman vehicle-treated rats. GX (10 mg/kg or 20 mg/kg) administered alone at both doses also significantly improved novel object exploration relative to vehicle treatment (p < 0.05). These results suggest that GX may alleviate soman-induced memory and cognitive impairments in pediatric rats.
3.10. GX Reduces long-term deficits in spatial learning and memory
To assess the long-term effects of GX on hippocampus-dependent spatial learning and memory in soman-exposed p21 rats, the MWM was examined. During the 7-day learning phase, soman-exposed animals consistently showed significantly higher latency to reach the platform compared to healthy controls, indicating impaired learning ability (Fig. 6E). Significant differences were observed between healthy controls and soman vehicle-treated rats throughout the learning phase. Co-administration of GX (10 mg/kg) with MDZ reduced latency times by over 60% on day 7 compared to vehicle-treated rats (p < 0.05). GX alone (10 mg/kg or 20 mg/kg) similarly decreased latency, with reductions exceeding 70% on day 7 (Fig. 6E). During the probe trials on day 8 (24 hours after the last training session. Probe 1) and day 15 (Probe 2), vehicle- and MDZ-treated soman-exposed rats spent approximately 30% and 50% less time, respectively, in the target quadrant than controls, indicating impaired memory retention (Fig. 6F). Treatment with GX (10 mg/kg) combined with MDZ significantly increased time spent in the target quadrant compared to vehicle and MDZ alone groups. Notably, rats treated with GX alone (10 mg/kg or 20 mg/kg) spent significantly more time in the target quadrant than both vehicle- and MDZ-treated soman-exposed rats during both probe trials (Fig. 6F). These results suggest that GX, either alone or combined with MDZ, effectively mitigates spatial learning and memory deficits induced by early-life soman exposure.
3.11. MRI Analysis of soman induced long-term neuropathology and protection by GX treatment.
To investigate whether exposure to soman during the pediatric stage affects brain volume development at 3 months, sagittal MRI scans were used to assess long-term, broad-scale brain volume changes. The sagittal plane, being the shortest axis of the rat brain, required fewer images to encompass the entire brain. Representative sagittal slices for control and soman-exposed animals are shown in Fig. 7A. Overall, the sagittal morphometric images reveal normal global brain volumes in animals exposed to soman, regardless of the presence or absence of GX (10 mg/kg) treatment (Fig. 7B). Soman exposure appears to have minimal impact on overall brain volume, with the soman group showing comparable brain volume to the control group. This indicates that soman alone does not significantly alter brain volume. Furthermore, the addition of GX treatment, whether administered alone or in combination with MDZ, does not appear to further influence brain volume. These findings suggest that soman does not induce brain volume alterations.
Fig. 7. MRI T2-weighted images of full sagittal sections taken 3 months after soman exposure in P21 rats, with or without ganaxolone (GX) treatment.
(A) Representative sagittal sections of randomly selected age-matched controls, and GD-exposed animals with and without GX treatment, are shown. The rat brain atlas highlights regions including the thalamus (THA), hypothalamus (HYPO), hippocampus (HPC), corpus callosum (CC), and lateral ventricle (LV). The T2-weighted 1.5-mm thick sagittal slices have a TE/TR of 37/2600 milliseconds. No major anatomical differences were observed among the three groups. (B) Total brain volumes were comparable across age-matched, and GD-exposed animals with and without midazolam (MDZ) and GX treatment groups. Data are presented as mean ± SEM (n = 6-8 per group). Scale bar = 10 mm.
3.11.1. GX prevents soman-induced chronic morphological alterations in the hippocampus.
Although overall brain volume remained unchanged, regional differences were possible, as the hippocampus is particularly vulnerable to soman -induced neurotoxic damage (Apland et al., 2017; de Araujo Furtado et al., 2012; Gullapalli et al., 2010; Reddy et al., 2021). To investigate potential neuroprotective effects of GX, coronal MRI of the hippocampus was performed at 3 months post-exposure in P21 rats (Fig. 8A). Hippocampal volume and T2 relaxation times were quantified from coronal slices 8–10 (~2.2 mm, 3.7 mm, and 5.2 mm posterior to bregma). Representative images from age-matched controls, GD alone, GD + MDZ, and GD + GX (10 mg) treatment groups are shown in Fig. 8A. Volumetric analysis revealed significant hippocampal atrophy in soman-exposed animals (Fig. 8B), while T2 relaxation times remained similar across all groups (Fig. 8C).
Fig. 8. MRI T2-weighted images revealed comparative changes in the hippocampus and ventricles 3 months after soman (GD) exposure in P21 rats, with or without ganaxolone (GX) treatment.
(A) Representative MR images are shown for age-matched controls and GD-exposed animals with and without MDZ or GX (10 mg/kg) treatment. Anatomical diagrams from the Paxinos and Watson’s Rat Brain Atlas (reference) are compared with respective coronal sections of a randomly selected control and GD-exposed (bottom three panels) rats. Yellow arrows indicate areas of pathological abnormalities. Scale bar = 10 mm. (B) GX treatment significantly prevented the decrease in hippocampus volumes. (C) T2 relaxation times for the hippocampus were similar across groups. (D) GX treatment significantly prevented the increase in right lateral ventricle (LV) volume. (E) T2 relaxation times for the ventricles were significantly higher in GD-exposed animals. Data are presented as mean ± SEM (n = 6-8 per group). *p < 0.05 versus age-matched control group; #p < 0.05 versus GD-exposed animals; &p < 0.05 versus GD + MDZ animals.
Regional analysis demonstrated marked volume loss in the left anterior, left medial, and posterior hippocampal subregions following soman exposure (Fig. 9A). MDZ treatment alone failed to improve posterior hippocampal volume relative to soman-exposed rats and remained significantly reduced compared with GX-treated groups. In contrast, both GX (10mg/kg) alone and MDZ + GX (10 mg/kg) treatments effectively prevented hippocampal volume loss in all affected regions (Figs. 8B, 9A). These findings highlight the capacity of GX to protect against soman -induced hippocampal atrophy and its potential to preserve related cognitive functions.
Fig. 9. Comparative average volumes and T2 relaxation times of various brain regions 3 months after soman (GD) exposure in P21 rats, with or without ganaxolone (GX) treatment.
(A) Brain tissue volumes significantly decreased in the posterior hippocampus (HPC), left medial and anterior hippocampus, and right anterior thalamus in GD-exposed groups. The volume of the right lateral ventricle (LV) significantly increased in GD-exposed groups. GX (10 mg/kg) treatment significantly prevented the decrease in HPC and the increase in LV volumes in GD-exposed rats. LP and RP: left and right posterior; LM and RM: left and right medial; LA and RA: left and right anterior; L and R: left and right. (B) T2 relaxation times were similar between groups in all brain regions except the right lateral ventricles, which exhibited heightened T2 signals due to massive fluid expansion in GD-exposed groups. GX treatment significantly prevented the increase of T2 signals in LV in GD-exposed rats. All data represent mean ± SEM (n = 6-8 per group). *p < 0.05 versus age-matched control group; #p < 0.05 versus GD-exposed animals; &p < 0.05 versus GD + MDZ animals.
3.11.2. GX prevents soman-induced long-term alterations in the lateral ventricles
To determine whether soman exposure produces lasting structural changes in the lateral ventricles, we assessed cerebrospinal fluid expansion 3 months post-exposure in P21 rats. Analyses followed the same imaging protocol used for the hippocampus, targeting coronal slices at 0.7, 2.2, 3.7, and 5.2 mm posterior to the bregma (Fig. 8). Volume measurements revealed a significant enlargement of the right lateral ventricle in soman-exposed animals (Figs. 8D, 9A). T2 relaxation times were also markedly elevated in this group, consistent with persistent T2 hyperintensity (Figs. 8E, 9B). MDZ treatment alone failed to improve right lateral ventricle relaxation times relative to soman exposure and remained significantly different from GX- and MDZ + GX (10 mg)-treated groups. In contrast, GX alone or in combination with MDZ effectively prevented both volume increases and T2 relaxation time prolongation. These results indicate that soman causes chronic morphological alterations in the lateral ventricles, which can be mitigated by GX treatment.
3.11.3. GX prevents soman-induced long-term morphological changes in extrahippocampal regions
Permanent thalamic alterations were evaluated using the same imaging protocol applied to the hippocampus, focusing on three corresponding coronal slices (Fig. 9). Among all regions analyzed, only the right anterior thalamus showed a significant volume reduction relative to controls 3 months after soman exposure. Treatment with MDZ, GX, or MDZ + GX effectively prevented this reduction compared to soman-exposed animals (Fig. 9A). T2 relaxation times remained stable across all groups (Fig. 9B). These results indicate that soman produces only limited and region-specific morphological changes in the thalamus.
3.12. GX Preserves PV(+) interneurons in the hippocampus of soman-exposed rats
To determine whether GX mitigates the loss of GABAergic inhibitory interneurons following soman exposure, hippocampal sections were immunostained for parvalbumin-positive (PV(+)) cells. Representative images (Fig. 10A) show PV(+) interneuron distribution in vehicle controls, GD-exposed animals, and groups treated with MDZ (1 mg/kg) or GX (10 mg/kg, 20 mg/kg). Soman exposure caused a marked reduction of PV(+) cells in CA1, CA3, and DG compared with controls. GX-treated animals showed attenuated PV(+) cell loss in these regions, indicating a neuroprotective effect. Stereological quantification (Fig. 10B) revealed that soman exposure reduced PV(+) interneurons to ~31% of control levels across the hippocampus, with losses of ~33% in CA1, ~38% in CA3, and ~31% in DG, while CA2 was largely unaffected. MDZ alone conferred modest protection (~22% reduction in loss). GX treatment at 10 mg/kg or 20 mg/kg significantly preserved interneuron numbers across all affected subfields. The MDZ + GX (10 mg/kg) combination provided the strongest protection, with PV+ cell numbers approaching control values: CA1 (~100% protection), CA3 (~78%), and DG (~93%). Soman exposure also produced significant hippocampal atrophy, with volume loss in CA1, CA3, and DG (Fig. 10E). MDZ alone did not prevent this shrinkage. In contrast, GX alone or combined with MDZ preserved hippocampal volume, consistent with MRI findings (Fig. 9) and persistent behavioral deficits at 3 months post-exposure. Overall, these results demonstrate that GX, particularly in combination with MDZ, confers strong long-term neuroprotection against soman -induced inhibitory interneuron loss and hippocampal atrophy.
Fig. 10. Analysis of PV(+) inhibitory interneuron loss in the hippocampus (HPC) 4 months after soman (GD) exposure in pediatric P21 rats.
(A) Representative sections of PV(+) staining in the hippocampus subfields (CA1, CA3, and DG). (B) Stereological quantification of the absolute PV(+) cells in hippocampal subfields. (C) Percent protection of PV(+) cells in each hippocampal subfield. (D) Cell density by stereology in hippocampal subfields. (E) Tissue volume measurements in various brain regions. The top panel (1.25× objective) depicts the entire hippocampus, while the bottom panels (20× objective) illustrate the CA1, CA3, and DG regions.CA1, cornus ammonis 1; CA2, cornus ammonis 2; CA3, cornus ammonis 3; DG, dentate gyrus. Data represent mean ± S.E.M. (n = 6-8 per group). *p < 0.05 versus age-matched control group; #p < 0.05 versus GD-exposed animals; &p < 0.05 versus GD + MDZ animals.
3.13. GX preserves PV(+) interneurons in extra-hippocampal brain regions of soman exposed rats
The distribution of surviving PV(+) interneurons after soman challenge was examined in extra-hippocampal regions, including the thalamus, hypothalamus, amygdala, piriform cortex, somatosensory cortex, and entorhinal cortex (Fig. 11A). Neuropathology-based scoring revealed that soman exposure markedly reduced PV(+) interneuron density in the thalamus, amygdala, piriform cortex, somatosensory cortex, and entorhinal cortex compared with controls (Fig. 11B and 11C). MDZ treatment alone conferred minimal protection, whereas GX, either alone (10 mg/kg or 20 mg/kg) or combined with MDZ, significantly preserved PV(+) interneuron populations in all assessed extra-hippocampal regions. These results indicate that GX affords substantial neuroprotection against soman -induced interneuron loss beyond the hippocampus.
Fig. 11. Analysis of PV(+) inhibitory interneuron loss in the extrahippocampal regions 4 months after soman (GD) exposure in pediatric P21 rats.
(A) Representative sections of PV(+) staining in the thalamus (Thal), hypothalamus (Hypo), amygdala (AMY), piriform cortex (Pir), somatosensory cortex (SS), and entorhinal cortex (Ent). (B) Neuropathological scores of PV(+) cells in the extrahippocampal regions. (C) Percent protection of PV(+) cells in the extrahippocampal regions. Data represent mean ± S.E.M. (n = 6-8 per group). *p < 0.05 versus age-matched control group; #p < 0.05 versus GD-exposed animals; &p < 0.05 versus GD + MDZ animals.
3.14. GX prevents NeuN(+) principal neurons loss in the hippocampus of soman-exposed rats
To assess the neuroprotective effects of GX against soman -induced loss of hippocampal principal neurons, NeuN(+) immunostaining was performed. Representative images (Fig. 12A) show NeuN(+) principal neuron distribution across hippocampal subfields in control, GD-vehicle, and MDZ- or GX-treated rats. Soman exposure produced a marked reduction in NeuN(+) neurons in CA1, CA2, CA3, and DG compared to control, indicating substantial neuronal loss. GX treatment markedly attenuated this loss across all subfields. Stereological quantification (Fig. 12A, 12B) revealed an overall hippocampal neuronal loss of ~42% following soman exposure, with reductions of ~34% in CA1, ~44% in CA2, ~29% in CA3, ~43% in DG and ~60% in DH. MDZ (1 mg/kg) provided limited protection, reducing loss by ~26% overall. In contrast, GX at 10 mg/kg or 20 mg/kg significantly preserved neuronal populations in all regions. Notably, MDZ + GX (10 mg/kg) yielded near-complete protection, with NeuN(+) counts in CA1 (~95%), CA2 (~75%), CA3 (~98%), DG (~89%) and DH (~66%) approaching control values (Fig. 12 C). These results demonstrate that delayed GX treatment confers substantial, long-term protection against soman -induced hippocampal neuronal loss.
Fig. 12. Analysis of NeuN(+) principal neuron degeneration in the hippocampus (HPC) 4 months after soman (GD) exposure in pediatric P21 rats.
(A) Representative sections of NeuN(+) staining in the hippocampus subfields (CA1, CA3, and DG). (B) Stereological quantification of the absolute NeuN(+) cells in hippocampal subfields. (C) Percent protection of NeuN(+) cells in each hippocampal subfield. Data represent mean ± S.E.M. (n = 6-8 per group). *p < 0.05 versus age-matched control group; #p < 0.05 versus GD-exposed animals; &p < 0.05 versus GD + MDZ animals.
3.15. GX reduces cytokines and other inflammatory markers in soman-exposed rats
To evaluate the effects of GX on soman-induced inflammatory responses, multiplex cytokine analysis was performed to quantify circulating levels of pro-inflammatory markers 3 months post-exposure. Cytokine data for IL-1α, IL-6, IL-17A, IL-18, MCP-1, TNF-α, and IL-1β are presented across control, GD-exposed, and treatment groups (Table 2). Soman exposure produced a robust increase in these cytokines, indicating sustained systemic inflammation. Compared to control, soman-exposed animals showed significant elevations in IL-1α, IL-6, MCP-1, TNF-α, IL-17A, IL-18, and IL-1β (*p < 0.05), with the largest increases observed in IL-6 and MCP-1. MDZ treatment partially reduced cytokine levels relative to GD control (#p < 0.05), but values remained elevated compared to control. GX treatment at 10 mg/kg produced similar reductions to MDZ across most analytes, indicating comparable efficacy. In contrast, GX at 20 mg/kg markedly suppressed cytokine levels (#p < 0.05 vs GD), with several analytes approaching control levels. MDZ and GX (10 mg/kg) combination produced the greatest reduction, with cytokine levels significantly lower than those in both the GD and GD+MDZ groups (#p < 0.05 vs. GD; &p < 0.05 vs. GD+MDZ). IL-1α, MCP-1, TNF-α, and IL-1β were reduced to levels not significantly different from control, while IL-6 and IL-18 remained modestly elevated (Table 2). These findings demonstrate that GX effectively attenuates soman-induced systemic inflammation, with greater efficacy observed at higher doses.
Table 2.
Multiplex cytokine assay for analysis of plasma cytokines.
| Control | GD | GD+MDZ | GD+ GX-10 | GD+MDZ+GX10 | GD+GX20 | |
|---|---|---|---|---|---|---|
| IL-1α | 83.82 ± 20.79 | 724.88 ± 64.88* | 410.32 ± 66.15*# | 295.74 ± 24.28*# | 108.65±12.76#& | 185.48 ± 8.92*#& |
| IL-6 | 1820±210.3 | 5125.16±420.7* | 3480.22±185.44*# | 2955.67±160.28*#& | 1988.54±145.12#& | 1425.33 ± 120.65#& |
| IL-17A | 68 ± 9 | 332 ± 61* | 228 ± 31*# | 214 ± 27*# | 96 ± 11#& | 84 ± 10#& |
| IL-18 | 148 ± 17 | 672 ± 102* | 418 ± 49*# | 391 ± 46*# | 231 ± 29#& | 201 ± 24#& |
| MCP-1 | 1025.44±120.32 | 2450.03±310.4* | 1850.6 ± 210.94*# | 1455.8 ± 180.26*#& | 1105.31±130.12#& | 900.89 ± 110.27#& |
| TNF-α | 9.8 ± 2.1 | 61.4 ± 8.7* | 39.1 ± 5.0*# | 36.8 ± 4.6*# | 13.7 ± 2.4#& | 12.1 ± 1.8#& |
| IL-1β | 2.4 ± 0.9 | 41.7 ± 6.8* | 19.6 ± 3.5*# | 17.8 ± 3.0*# | 6.1 ± 1.3#& | 5.4 ± 1.1#& |
Values are expressed as mean ± SEM (n=8-10 per group). One-way ANOVA followed by Tukey’s multiple comparisons test.
p < 0.05 versus age-matched control group;
p < 0.05 versus GD-exposed animals;
p < 0.05 versus GD + MDZ animals.
3.16. GX mitigates inflammatory microgliosis in the hippocampus and amygdala of soman-exposed rats
To evaluate the effect of GX on soman-induced microglial activation and inflammation for 3 months post-exposure, IBA1(+) immunostaining was performed. Representative images in Figure 13A illustrate microglial distribution across hippocampal subfields and the amygdala (AMY) for control, soman-exposed, MDZ-treated, and GX-treated groups. Soman exposure elicited widespread microgliosis, evident in all hippocampal subfields and the AMY, with morphological hallmarks such as enlarged soma and ramified processes indicating neuroinflammation. In contrast, GX-treated brains exhibited markedly reduced microgliosis, suggesting anti-inflammatory activity. Quantitative densitometric analysis confirmed significant increases in IBA1(+) expression throughout the hippocampus and AMY 3 months following soman exposure, with region-specific elevations in CA1 (~178%), CA3 (~209%), DG (~125%), and AMY (~162% of control) (Fig. 13B and 13C). MDZ treatment (1 mg/kg) provided minimal protection, whereas GX at 10 or 20 mg/kg substantially attenuated microglial activation. Notably, combined MDZ and GX (10 mg/kg) treatment produced robust neuroprotection, with substantial reductions in IBA1(+) cell densities in CA1 (~52%), CA3 (~47%), DG (~89%), and AMY (~91% of protection) (Fig. 13C). These results indicate that GX attenuates soman-induced long-term neuroinflammation by suppressing microglial activation in pediatric rats.
Fig. 13. Analysis of IBA1(+) microgliosis and neuroinflammation in the hippocampus (HPC) and amygdala (AMY) 4 months after soman (GD) exposure in pediatric P21 rats.
(A) Representative sections of IBA1(+) immunostaining in the hippocampus subfields (CA1, CA3, and DG) and amygdala. (B) Area Fractional (AF) densitometry quantification of IBA1(+) expression in each hippocampal subfield and amygdala. (C) Percent protection from IBA1(+) expression in each hippocampal subfield and amygdala. Data represent mean ± S.E.M. (n = 6-8 per group). *p < 0.05 versus age-matched control group; #p < 0.05 versus GD-exposed animals; &p < 0.05 versus GD + MDZ animals.
3.17. GX enhances neurogenesis in soman-exposed rats
To assess the long-term effects of acute pediatric soman exposure on neurogenesis, DCX(+) newborn neurons in the dentate gyrus (DG) were quantified. GD-exposed rats showed a significant reduction (~40%) in DCX(+) neurons compared to controls (Fig. 14), indicating impaired neurogenesis. MDZ treatment (1 mg/kg) offered minimal benefit, with DCX(+) counts similar to the GD-only group. In contrast, GX at 10 or 20 mg/kg, alone or combined with MDZ, preserved DCX(+) neuron numbers at levels comparable to controls. GX-treated animals demonstrated significant protection of neurogenesis relative to both GD and GD+MDZ groups (Fig. 14). These results indicate that acute soman exposure in P21 rats produces lasting deficits in neurogenesis, whereas GX effectively preserves and restores hippocampal neurogenesis.
Fig. 14. Quantification of DCX(+) newborn neurons in the dentate gyrus 4 months after acute soman (GD) exposure in pediatric P21 rats.
(A) Representative sections of DCX(+) staining in the hippocampus DG regions. (B) Stereological quantification of the absolute DCX(+) cells in the DG regions. (C) Percent protection of DCX(+) cells in the hippocampus. Data represent mean ± S.E.M. (n = 6-8 per group). *p < 0.05 versus age-matched control group; #p < 0.05 versus GD-exposed animals; &p < 0.05 versus GD + MDZ animals.
Discussion
In this study, we demonstrate for the first time that GX therapy exerts long-term protective effects following acute soman exposure in pediatric rats, effectively counteracting hippocampal atrophy, neuroinflammation, neurodegeneration, and associated cognitive deficits and epileptic seizures. Key findings from this study include (Table 3): (1) GX exhibited anti-epileptogenic effects leading to decline in the incidence of SRS and overall reduction in seizure burden; (2) GX effectively halted the development of epileptiform discharges and interictal spiking events; (3) GX also suppressed the number of HFOs and interictal spikes, which are electrographic biomarkers of epileptogenesis; (4) GX exhibited a significant efficacy in reducing depression and exploratory anxiety-like behavior; (5) GX mitigated immediate, long-term, and spatial memory impairments; (6) GX effectively prevented widespread neurodegeneration in principal neurons and interneurons in the hippocampus; (7) GX drastically reduced cytokines and peripheral pro-inflammatory markers; (8) GX reduced inflammatory microgliosis in the hippocampus and amygdala; (9) GX facilitated neurogenesis, which possibly explains its neuroprotectant features; (10) GX provided dose-related protection with greater protection in the high dose group. Overall, these findings demonstrate GX as an effective therapy for preventing epileptogenesis, dampening inflammation, reducing neurodegeneration, and alleviating the long-term behavioral impairments associated with acute nerve agent exposure to pediatric rats.
Table 3.
Summary of GX long-term neuroprotection and antiepileptogenic efficacy in the pediatric soman model.
| Thematic Study | Experimental Endpoint | GD / Vehicle Group | Midazolam (MDZ) Alone | Ganaxolone (GX) Alone | GX + MDZ Combination | Overall Interpretation |
|---|---|---|---|---|---|---|
| EEG | Acute seizures | Persistent seizures and SE | Moderate seizure suppression | Strong seizure suppression | Robust seizure suppression | Antiseizure effect |
| Spontaneous recurrent seizures (SRS) | High chronic seizure burden | Moderate reduction in SRS | Significant reduction in SRS | Greatest reduction in SRS | Antiepileptogenic effect | |
| Epileptiform discharges | Persistent epileptiform activity | Partial improvement | Significant suppression | Strongest suppression | Reduced network stability | |
| Interictal spikes | Frequent interictal spikes | Mild reduction | Marked reduction | Greatest reduction in spike burden | Decreased network hyperexcitability | |
| High-frequency oscillations (HFOs) | Elevated pathological HFOs | Moderate reduction | Significant reduction | Maximal suppression of HFOs | Reduced epileptic network remodeling | |
| MRI | Hippocampal atrophy | Severe hippocampal volume loss | Partial preservation | Significant preservation | Near-complete preservation | Structural neuroprotection |
| Ventricular enlargement | Marked ventricular dilation | Mild attenuation | Significant reduction | Greatest normalization | Reduced neurodegeneration | |
| Structural brain abnormalities | Extensive neuropathology | Partial improvement | Major reduction in pathology | Most pronounced protection | Improved long-term brain integrity | |
| Behavior | Spatial memory (MWM) | Severe learning deficits | Partial improvement | Significant recovery | Robust performance | Improved hippocampal-dependent cognition |
| Recognition memory (NORT) | Impaired recognition memory | Mild benefit | Significant improvement | Strongest improvement | Enhanced cognitive restoration | |
| Anxiety-like behavior | Increased anxiety phenotype | Limited improvement | Reduced anxiety behavior | Greatest anxiolytic effect | Improved emotional outcomes | |
| Exploratory behavior | Reduced exploratory activity | Mild improvement | Significant restoration | Near-normal exploration | Improved behavioral recovery | |
| Social interaction | Impaired social engagement | Minimal improvement | Improved interaction | Strongest recovery | Better neurobehavioral outcome | |
| Histology | PV+ interneurons | Significant interneuron loss | Partial preservation | Significant protection | Highest preservation | Maintenance of inhibitory circuitry |
| NeuN+ neurons | Extensive neuronal degeneration | Mild neuroprotection | Significant neuronal survival | Greatest neuronal survival | Reduced neurotoxicity | |
| DCX+ neurogenesis | Reduced neurogenesis | Minimal restoration | Enhanced neurogenesis | Highest neurogenic recovery | Improved neuronal repair mechanisms | |
| IBA1+ microgliosis | Marked microglial activation | Partial reduction | Significant reduction | Strongest suppression | Potent anti-inflammatory effect | |
| Bio-chemistry | Cytokines: IL-1β, TNF-α, IL-6, MCP-1 | Elevated pro-inflammatory cytokines | Mild cytokine reduction | Significant cytokine suppression | Greatest reduction in inflammatory mediators | Strong attenuation of neuroinflammation |
Organophosphate intoxication produces lasting neuronal injury, neurobehavioral impairments, and cognitive dysfunction (Andrew and Lein, 2021; Delgado et al., 2004; McDonough Jr and Shih, 1997; Ross et al., 2010; van den Dries et al., 2020). Rodent studies and clinical observations both indicate significant hippocampal and cortical vulnerability, paralleling long-term neurological sequelae in humans (Ramakrishnan et al., 2025; Singh et al., 2024b; Voorhees et al., 2017). Consistent with adult organophosphate models (Delgado et al., 2004; Lumley et al., 2019; Reddy, 2024; Singh et al., 2024a; Wu et al., 2018), our pediatric model reveals persistent deficits in learning and memory, reflecting high developmental vulnerability (Neff and Reddy, 2024; Ramakrishnan et al., 2025; Singh et al., 2024b). These outcomes likely arise from combined neuronal injury, impaired neurogenesis, and sustained neuroinflammation, the latter involving microgliosis and astrogliosis (Flannery et al., 2016; Kofman et al., 2006; Neff and Reddy, 2024; Ramakrishnan et al., 2025). Recent soman studies further show that microglial and astrocytic phenotypes remain persistently altered across multiple brain regions, reinforcing inflammation as a therapeutic target (Andrew et al., 2024; Reddy, 2024).
Soman exposure in pediatric rats also produced anxiety- and depression-like behaviors in our studies, consistent with human and animal reports of organophosphate-induced affective disturbances (Chen, 2012; Eskenazi et al., 2007; Ramakrishnan et al., 2025). Neuronal injury within the prefrontal, cingulate, and limbic circuits likely underlies these effects (Adolphs, 2010; Kennedy et al., 2009; Öhman, 2005), consistent with evidence that disruption of these networks in children increases anxiety risk (Brandt et al., 2006, 2003; LaGrant et al., 2020).
Hippocampal neurodegeneration, including loss of interneurons, and granule cells, appears central to impairments in memory, memory coding, and spatial and contextual information (Kotloski et al., 2002; Kuruba et al., 2018; Padurariu et al., 2012). Our findings also reveal suppressed neurogenesis, which is critical for encoding new memories. These pathological changes have been previously linked to long-term cognitive dysfunction in epilepsy and pesticide models, as reductions in immature granule cells impair learning and memory (Kempermann et al., 2015; Neff and Reddy, 2024; Reddy, 2024; Scorza et al., 2024; Shapiro and Ribak, 2006; Zhao et al., 2008). Human studies similarly show reduced neurogenesis in epileptic and organophosphate-exposed tissue, supporting the translational relevance of preclinical models (Ammothumkandy et al., 2025; Fahrner et al., 2007; Gonçalves et al., 2016; Kempermann et al., 2015; Zhao et al., 2008). Our data, including MRI analyses, confirm that impaired neurogenesis persists beyond the acute seizure phase, highlighting the need for therapies that extend past seizure termination.
Despite being the standard of care, benzodiazepines such as midazolam, which selectively act on GABA-A receptors, show limited efficacy once seizures progress to refractory status epilepticus. Classical benzodiazepines, such as midazolam, selectively enhance phasic inhibition by binding at the α/γ2 interface, effective only on receptors containing α1, α2, α3, or α5 subunits, while α4- and α6-containing receptors remain benzodiazepine-insensitive (Deeb et al., 2012; Lumley et al., 2019; Möhler et al., 2001; Reddy et al., 2018, 2015; Reddy and Reddy, 2015; Sieghart and Sperk, 2002). This loss of efficacy is primarily attributed to GABA-A receptor internalization and reduced synaptic inhibition (Aroniadou-Anderjaska et al., 2024; McDonough Jr et al., 1999; RamaRao et al., 2014; Reddy, 2024; Reddy et al., 2024b, 2015; Reddy and Reddy, 2015; Shih et al., 1999; Singh et al., 2024a; Wu et al., 2018). Consistent with these reports, our results in this study show that MDZ provided minimal neuroprotection, as principal cell and interneuron counts remained significantly lower. Similarly, other studies indicate that benzodiazepine-only regimens fail to halt ongoing neuropathology after organophosphate exposure, leaving survivors susceptible to long-term hippocampal injury and cognitive deficits (Kundrick et al., 2020; Marrero-Rosado et al., 2018; McDonough Jr et al., 1999; Shih et al., 1999; Spampanato et al., 2019; Wu et al., 2018). These limitations underscore the urgent need for adjunctive therapies capable of addressing benzodiazepine-refractory seizures and their downstream neurodegenerative consequences.
Neurosteroids have emerged as a promising therapeutic strategy for mitigating both acute and long-term consequences of nerve agent exposure (Reddy, 2024). Notably, extrasynaptic GABA-A receptors containing α4, α6, and δ subunits mediate tonic inhibition and display high sensitivity to neurosteroids (Carver et al., 2014; Carver and Reddy, 2016; Chuang and Reddy, 2018; Gangisetty and Reddy, 2010; Maguire and Mennerick, 2024; Ramakrishnan et al., 2025; Reddy and Woodward, 2004; Wu et al., 2013). This dual activity makes neurosteroids particularly effective in conditions where benzodiazepines are insufficient, such as refractory SEor organophosphate-induced seizures, by acting on benzodiazepine-insensitive extrasynaptic receptor subtypes (Maguire and Mennerick, 2024; Reddy, 2024; Vaitkevicius et al., 2022). Unlike benzodiazepines, which act on receptor subtypes susceptible to internalization and desensitization, neurosteroids such as allopregnanolone and its synthetic analogs maintain tonic inhibition and effectively terminate benzodiazepine-refractory seizures (Carver et al., 2014; Carver and Reddy, 2013; Chuang and Reddy, 2018; D. Reddy, 2019; Reddy, 2024; Wu et al., 2013). Neurosteroids also mitigate neuroinflammation, attenuate excitotoxicity, and may provide mitochondrial protection following OP exposure (Ramakrishnan et al., 2025; Reddy, 2024; Reddy et al., 2024b, 2023). Our findings show this therapeutic rationale, suggesting that neurosteroid-based treatments may protect hippocampal neurodegeneration, reverse behavioral abnormalities, and improve long-term outcomes in OP-exposed pediatric populations.
GX treatment conferred robust neuroprotection against soman-induced behavioral and cognitive impairments. Neurodegeneration in the frontal lobe, cingulate cortex, amygdala, and hippocampus is known to underlie mood, anxiety, and memory deficits (Adolphs, 2010; Kennedy et al., 2009; LaGrant et al., 2020; Öhman, 2005). Targeting these regions, GX reduced anxiety- and depression-like behaviors and improved recognition, short- and long-term memory after soman exposure. Beyond symptoms, neurosteroids including GX modulate neuroinflammation—a major contributor to cognitive decline. Current studies show GX and similar neurosteroids dampen microglial activation and proinflammatory cytokine production (Barker et al., 2020; Batchelor et al., 1999; Frade and Barde, 1998; Maguire and Mennerick, 2024; Neff and Reddy, 2024; Ramakrishnan et al., 2025, 2024; Reddy, 2024; Troncoso-Escudero et al., 2018). Consistent with these reports, our multiplex cytokine analysis confirmed significant elevations in IL-1α, IL-6, MCP-1, TNF-α, IL-17A, IL-18, and IL-1β following soman exposure, with GX treatment reducing these levels in a dose-dependent manner. These findings align with recent reports of sustained cytokine upregulation after soman exposure (Massey et al., 2025). Persistent inflammation, mediated by activated microglia, astrocytic reactivity, and cytokines such as IL-1β and TNF-α, can hinder neuronal regeneration and exacerbate long-term cognitive impairment (Andrew and Lein, 2021; Ramakrishnan et al., 2025; Singh et al., 2024b). Importantly, neuroinflammation and mitochondrial stress may persist long after the initial cholinergic crisis resolves (Karami-Mohajeri and Abdollahi, 2013). Our studies shows neurosteroids dampen microglial activation, consistent with GX’s protective potential in mitigating both neuroinflammation and its behavioral consequences (Aryanpour et al., 2017; Cutler et al., 2007; Hua et al., 2011; Lei et al., 2014; Neff and Reddy, 2024; Ramakrishnan et al., 2025).
This study demonstrates that soman exposure profoundly impairs hippocampal neurogenesis, an effect not rescued by standard benzodiazepine treatment. These results are consistent with the broader literature indicating that conventional benzodiazepine antiseizure drugs fail to prevent long-term neurotoxicity after nerve agent exposure. By contrast, neurosteroid-based interventions show promise in overcoming benzodiazepine-refractory seizures and protecting hippocampal function. Targeting both seizure activity and secondary neuropathological mechanisms may therefore represent the most effective therapeutic strategy.
In addition to neurosteroids, several emerging alternatives have been investigated for the management of benzodiazepine-refractory seizures after nerve agent exposure, where glutamatergic hyperexcitation plays a central role in seizure propagation and neuronal injury. AMPA receptor antagonists such as perampanel have shown significant promise as adjunctive therapies; for example, perampanel administered after midazolam reduces seizure duration, spontaneous seizures, and neurodegeneration in soman-exposed models, supporting its utility as a second-line agent in benzodiazepine-refractory SE (Steier et al., 2025). Similarly, glutamatergic antagonists such as LY293558 (tezampanel) demonstrate robust antiseizure and neuroprotective efficacy, even when administered with delayed timing, and combination strategies targeting AMPA, NMDA, and muscarinic pathways have shown near-complete neuroprotection (Apland et al., 2018a; Aroniadou-Anderjaska et al., 2020). Perampanel, in combination with allopregnanolone, is a potential adjunct to midazolam for benzodiazepine-refractory organophosphate-induced SE (Dhir et al., 2020). Recent studies also highlight novel glutamate receptor antagonists (e.g., IEM-1925) that outperform benzodiazepines in reducing seizure severity, neuronal damage, and cognitive deficits (Lin et al., 2026). Clinically, levetiracetam and perampanel are increasingly used as second- or third-line agents in refractory SE due to their favorable safety profiles and distinct mechanisms, although their efficacy may depend on timing and combination with other therapies. Despite these advances, benzodiazepines remain limited by reduced efficacy with delayed treatment, likely due to GABA-A receptor internalization and diminished inhibitory tone during prolonged seizures. In contrast, neurosteroids offer a complementary and potentially superior approach by enhancing both synaptic and extrasynaptic GABA-A receptor–mediated inhibition, including benzodiazepine-insensitive receptor populations, while also exerting anti-inflammatory and neuroprotective effects. Thus, compared to glutamatergic antagonists and conventional antiseizure drugs, neurosteroids provide a multimodal mechanism that addresses both seizure termination and underlying epileptogenic processes, supporting their strong translational potential either as monotherapy or in combination regimens for refractory SE.
Levetiracetam (LEV), a widely used antiseizure medication with a unique mechanism involving synaptic vesicle protein 2A modulation, has also been explored as a comparator and adjunctive therapy in benzodiazepine-refractory seizures. Although LEV exhibits favorable safety and pharmacokinetic profiles, its efficacy in terminating established SE, particularly in OP-induced models, appears limited when used as monotherapy. Preclinical studies indicate that LEV does not effectively terminate ongoing SE but may enhance the anticonvulsant efficacy of other agents when used in combination (Myhrer et al., 2011). Importantly, LEV has demonstrated neuroprotective effects by attenuating sustained intracellular calcium dysregulation (“Ca2+ plateau”), a key mechanism underlying long-term neuronal injury and epileptogenesis following OP-induced SE (Deshpande and DeLorenzo, 2020). Furthermore, LEV-containing combination regimens have been shown to improve seizure control and survival outcomes in severe nerve agent exposure models (Myhrer et al., 2018). Clinically, LEV is frequently used as a second-line therapy due to its tolerability; however, its limited efficacy in refractory SE highlights the need for more robust agents. Compared to glutamatergic antagonists such as perampanel and multimodal therapies like neurosteroids, LEV primarily serves as an adjunctive strategy that may mitigate downstream neurotoxicity rather than providing strong standalone antiseizure or disease-modifying effects.
There are several gaps in translating preclinical findings to clinical application, particularly in pediatric populations exposed to nerve agents. From a translational perspective, the current study models a clinically relevant delayed-treatment paradigm (~40 min post-exposure), reflecting real-world scenarios where immediate intervention is often not feasible, thereby enhancing its applicability to mass-exposure settings. In this study, pediatric rats were exposed at P21, a developmental stage that corresponds broadly to the early childhood in humans, approximately 2–3 years of age, based on comparative neurodevelopmental milestones. This age is highly relevant because the developing brain is especially vulnerable to nerve agent–induced seizures, neuroinflammation, impaired neurogenesis, and long-term cognitive and behavioral deficits. Thus, the protective effects of GX in P21 rats support its potential clinical relevance for pediatric nerve agent exposure, particularly in young children who may experience delayed treatment during mass-casualty or accidental exposure scenarios. Mechanistically, GX offers a distinct advantage over benzodiazepines by targeting both synaptic and extrasynaptic GABA-A receptors, including δ-subunit–containing receptors that remain functional during SE, thereby restoring inhibitory tone even in benzodiazepine-refractory states (Maguire and Mennerick, 2024; Reddy, 2024). In addition, GX exerts multimodal neuroprotective effects by attenuating glutamate-driven excitotoxicity, suppressing neuroinflammation, preserving interneuron populations, and promoting neurogenesis—key processes underlying epileptogenesis and long-term neurological dysfunction. Importantly, GX is already FDA-approved for pediatric epilepsy, providing a well-established safety and pharmacokinetic profile that significantly accelerates its translational potential for repurposing in nerve agent exposure. From a clinical application standpoint, our findings support GX as both a monotherapy and adjunct to benzodiazepines, particularly in delayed-treatment or refractory seizure scenarios, where current standard-of-care therapies are insufficient. Furthermore, its compatibility with combination regimens (e.g., with midazolam or glutamatergic modulators) aligns with evolving treatment paradigms for nerve agents. Nevertheless, future studies are needed to address key translational gaps, including sex- and age-related variability, optimal dosing windows, long-term safety, and clinical trials. Collectively, these data position neurosteroid-based therapy as a highly promising, mechanistically grounded, and clinically relevant strategy for mitigating both seizures and long-term neurological consequences of nerve agent exposure.
A limitation of the present study is that the effects of GX, either as monotherapy or in combination with MDZ, were primarily evaluated in the chronic phase following soman exposure in pediatric male rats, without direct continuous EEG quantification of acute SE duration or early intergroup differences in seizure suppression. While assessment of acute SE dynamics would further refine the distinction between initial injury modification and true disease-modifying effects, several lines of evidence strongly support the rigor and validity of the current findings. Notably, comparable acute seizure severity across treatment groups, the short pharmacokinetic profiles of both GX and MDZ, and the lack of dose-dependent differences in early seizure outcomes argue against acute SE termination as the sole driver of long-term protection. Importantly, GX demonstrated robust and consistent efficacy across multiple independent and mechanistically distinct endpoints—including reduction in spontaneous seizures, suppression of epileptiform discharges and HFOs, preservation of interneurons and principal neurons, attenuation of neuroinflammation, restoration of neurogenesis, and improvement in cognitive and behavioral outcomes—thereby supporting a true disease-modifying effect rather than a transient antiseizure action. Furthermore, the use of a clinically relevant delayed-treatment paradigm (~40 min post-exposure), a pediatric paradigm (modelling children 2-3 years of age), and comprehensive longitudinal assessments (EEG, MRI, behavior, and histopathology) collectively strengthen the translational relevance and scientific rigor of the study. Nevertheless, we acknowledge this limitation and have highlighted future studies aimed at integrating acute EEG monitoring and early injury biomarkers to further delineate GX’s effects on SE dynamics and initial neurotoxic injury.
In conclusion, this study provides compelling and comprehensive evidence that neurosteroid therapy with GX is a highly effective strategy for mitigating both the acute and long-term neurological consequences of nerve agent exposure in the developing brain. In a field relevant pediatric model of soman-induced neurotoxicity, GX demonstrated robust disease-modifying effects, significantly reducing epileptogenesis, seizure burden, and pathological electrographic biomarkers while improving cognitive, emotional, and behavioral outcomes (Table 3). Unlike standard benzodiazepine therapy, which showed limited long-term benefit, GX—administered alone or in combination with midazolam—conferred sustained neuroprotection, preserving hippocampal structure, preventing loss of inhibitory interneurons and principal neurons, enhancing neurogenesis, and markedly attenuating neuroinflammation. The suppression of interictal spikes and HFOs further underscores its potential to modify the underlying epileptogenic networks rather than merely suppressing symptoms, thereby reinforcing its translational relevance as an optimized therapeutic strategy for pediatric nerve agent exposure.
Acknowledgements
This work was supported by the National Institute of Neurological Disorders and Stroke of the National Institutes of Health (grants U01NS117278, U01NS117209 and UG3NS138911 to D.S.R.). This work was partly supported by the Office of the Assistant Secretary of Defense for Health Affairs through the Epilepsy Research Program (grants W81XWH2210275, HT94252510237 and HT94252410174) and the Texas A&M Presidential X-Grant award (to D.S.R.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health and the Department of Defense. This research utilized the Texas A&M Preclinical Phenotyping Core (TPPC; RRID: SCR_023263), a facility within the Institute for Genome Sciences and Society at Texas A&M University, and acknowledge them for providing technical assistance with the multiplex assay. We acknowledge the technical assistance of Rithi Kowtha with MRI studies. The technical assistance of Elle Holder, Trisha Challa, Preet Patel and Hetvi Desai with stereology and densitometry analysis is greatly appreciated.
Abbreviations:
- ACh
acetylcholine
- AChE
acetylcholinesterase
- AMY
amygdala
- CA1
cornu ammonis 1
- CA2
cornu ammonis 2
- CA3
cornu ammonis 3
- CNS
central nervous system
- DFP
diisopropyl-fluorophosphate
- DG
dentate gyrus
- DH
dentate hilus
- DI
discrimination index
- EEG
electroencephalography
- EPM
elevated plus maze
- GABA-A
γ-aminobutyric acid
- GD
soman
- GX
ganaxolone
- HFO
high-frequency oscillation
- IBA1
Ionized calcium binding adaptor molecule 1
- MRI
magnetic resonance imaging
- MWM
Morris water maze
- NeuN
neuronal nuclei antigen
- NORT
novel object recognition test
- OFT
open field test
- OP
organophosphate
- PV
parvalbumin
- SE
status epilepticus
- SRS
spontaneous recurrent seizures
- SIT
social interaction test
Footnotes
Conflict of Interest
The authors declare no competing financial interests.
Data Availability Statement
The authors declare that all the data supporting the findings of this study are contained within the paper.
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Data Availability Statement
The authors declare that all the data supporting the findings of this study are contained within the paper.