Long-Term Neuropsychiatric Developmental Defects after Neonatal Organophosphate Exposure: Mitigation by Synthetic Neurosteroids.

Discussion

In this study, we show for the first time that neurosteroid treatment mitigates long-term neurologic effects from acute DFP exposure in pediatric rats. The salient findings of this study include: 1) acute exposure to DFP in pediatric rats resulted in the progressive deterioration of cognitive deficits and behavioral functions for up to 10 months; 2) the behavioral and cognitive dysfunctions were associated with widespread neurodegeneration, neuroinflammation, abnormal neurogenesis, and mossy fiber sprouting in the brain; 3) GX treatment mitigated the DFP-induced chronic behavioral and cognitive impairments; 4) GX treatment showed robust neuroprotection by reducing neurodegeneration and neuroinflammation; and 5) GX treatment showed decreased mossy fiber sprouting and an increase in neurogenesis in the hippocampus. Although the underlying mechanisms are unclear, the outcomes from neuropathological defects are consistent with neurologic functional deficits. These findings highlight the potential of neurosteroids to counteract the progressive neuropsychiatric sequelae following acute pediatric OP exposure.

Nerve agent exposure is associated with serious and long-lasting neurologic effects (Abou-Donia et al., 2016). In this study, DFP exposure in pediatric rats led to marked behavioral impairments that included increased aggression, which effectively simulated both the presentation and progression of aggressive-like symptoms in children or pediatric rats after acute OP exposure (Ricceri et al., 2006; Venerosi et al., 2008; Langston et al., 2012; Muñoz-Quezada et al., 2013; Talabani et al., 2018). Limbic areas, such as the amygdala and hippocampus, are linked to mediate the expression of aggressive behaviors and impulse control (Price and Drevets, 2010). Rodents with hippocampal and amygdala lesions have been shown to significantly alter the occurrence of aggressive behaviors (Blanchard et al., 1970, 1978; Davis et al., 1994). As consistent with our histopathological findings, it is possible that the neurodegeneration experienced in these limbic regions may underlie the emergence of aggressive behavior.

DFP is a widely used chemical threat agent for OP intoxication research. It is considered a surrogate for nerve agents. It produces consistent seizures, neuronal damage, and long-term neurologic effects reminiscent of humans exposed to nerve agents (Deshpande et al., 2010; Pouliot et al., 2016; Kuruba et al., 2018; Wu et al., 2018; Reddy et al., 2019, 2021). In this study, the DPF protocol used is widely regarded as the most commonly used and accepted model of OP exposure (Flannery et al., 2016; Rojas et al., 2016; Wu et al., 2018; Reddy et al., 2020). This protocol involves pretreatment with pyridostigmine, a reversible acetylcholinesterase inhibitor, and immediate post-treatment with 2-PAM and atropine. These standard antidote regimens are administered to prevent immediate mortality and create a more accurate representation of the clinical scenario. The pretreatment with pyridostigmine serves a crucial purpose, allowing a fraction of the enzyme to be spared for reactivation with the antidotes and reducing the overall toxicity of DFP. However, it should be noted that although this pretreatment is essential for the simulation of OP exposure in military personnel, it would not be practical or feasible for children. Despite the use of pretreatment in the pediatric OP model, it was observed that this did not have any significant impact on the long-term neurologic effects of OP exposure in children as demonstrated in this study.

In this study, DFP exposure in pediatric rats leads to the chronic presentation of behaviors indicative of anxiety and depression, consistent with the previous studies in adult animals and children (Eskenazi et al., 1999; Chen, 2012). Neuronal damage in several key regions, including the frontal lobe and cingulate cortex, as well as the limbic region (amygdala and hippocampus), may possibly account for the observed heightened anxiety in DFP-exposed animals (Ohman, 2005; Kennedy et al., 2009; Adolphs, 2010). Rats use their olfactory and visual systems to perceive information in their surroundings; in particular, their visual system consists of neural circuits that are related to emotional memory associated with the limbic system, which in turn relays sensory information to the cingulate and frontal cortical areas. Chronic neurodegenerative disorders in children that affect the sensory and limbic systems are commonly associated with an increased incidence of anxiety (Brandt et al., 2003; LaGrant et al., 2020) Therefore, the neurodegeneration occurring in these specific areas could provide a plausible explanation for the emergence of anxiety-like behavior in DFP-intoxicated animals.

DFP exposure causes chronic cognitive deficits in adult models (Delgado et al., 2004; Brewer et al., 2013; Rojas et al., 2016; Lumley et al., 2019). Our study in a pediatric model confirms and extends these observations with persistent learning and memory deficits. Developmental limitations due to widespread neuronal damage, inflammation, and reduced neurogenesis can be linked to these outcomes. Neuroinflammation, characterized by microgliosis and astrogliosis, has been suggested as a contributing factor to cognitive dysfunction (Kofman et al., 2006; Flannery et al., 2016). Neurodegeneration, specifically the loss of hippocampal principal neurons in the CA1 and CA3, has also been linked to cognitive deficits associated with an array of neurodegenerative disorders (Kotloski et al., 2002; Padurariu et al., 2012). We also observed decreased neurogenesis and the development of mossy fiber sprouting in the hippocampus, which could further explain the development of cognitive impairment (Ikegaya et al., 2000; Bonde et al., 2006; Ma et al., 2012). Surprisingly, SE has been found to enhance aberrant neurogenesis; however, it subsequently declines below normal levels during later stages (Shapiro et al., 2007; D’Alessio et al., 2010). In humans as well, research investigating the long-term consequences of seizures, particularly studies involving human tissue, has indicated a subsequent reduction in neurogenesis (Mathern et al., 2002; Fahrner et al., 2007). Decreased neurogenesis following early neuronal injury or SE could reduce the number of normal functional granule cells generated within the DG. This paucity of normal granule cells in the hippocampus could magnify the disruptive potential of abnormal cells that are generated after the insult. Therefore, decreased neurogenesis as observed in our study associated with abnormal mossy fiber sprouting and aberrant integration in networks possibly explained the emergence of cognitive impairment following DFP intoxication (Murphy et al., 2011).

Neurosteroids have been suggested as an option for mitigating OP neurotoxicity (Reddy, 2016). Mechanistically, they are unique compounds for protecting seizures and promoting neuroprotection due to their dual actions at synaptic and extrasynaptic GABA-A receptors (Reddy, 2010; Carver and Reddy, 2016; Reddy, 2018; Chuang and Reddy, 2018). This is a superior feature of neurosteroids as compared with benzodiazepines that lack activity at extrasynaptic GABA-A receptors (Reddy et al., 2015). In this study, GX treatment provided protection against behavioral and cognitive impairments in DFP-exposed animals. Like other neurosteroids, GX is a powerful anticonvulsant and neuroprotectant (Reddy and Rogawski, 2010; Chuang and Reddy, 2018, 2020). GX potentiates inhibitory currents through a novel mechanism of action that activates all isoforms of synaptic and extrasynaptic receptors. GX targets preferentially extrasynaptic GABA-A receptors, which play a key role in the effective suppression of persistent seizures and SE (Scimemi et al., 2005; Reddy, 2019b). Due to its broad impact on various types of GABA-A receptors, GX has demonstrated greater efficacy compared with benzodiazepines, even when given after 30 minutes (Zolkowska et al., 2018; Saporito et al., 2019). This is attributed to its ability to generate potent inhibitory tonic currents, which remain unaffected even when synaptic GABA-A receptors undergo internalization during prolonged SE (Naylor et al., 2005; Goodkin et al., 2008). Besides this mechanism, neurosteroids can directly promote neuroprotection and restore the loss of neurons by promoting neurogenesis (Charalampopoulos et al., 2008). Thus, neurosteroid intervention for pediatric OP exposure would be a powerful alternative to benzodiazepine treatment that offers mechanistic advantages for targeted neuroprotection in mitigating the long-term impact of OP neurotoxicity.

Neurosteroid treatment prevented neuronal loss following DFP-induced SE. It is likely that neurosteroid-mediated tonic inhibition and attenuation of excitotoxicity and neuroinflammatory response contribute to such protection (Reddy, 2019b; Deshpande and DeLorenzo, 2020; Pulkrabkova et al., 2023). This is evident from decreased neurodegeneration, rescue of neurogenesis, and reduced the neuroinflammatory response. These effects likely led to the improvements observed in behavioral and cognitive outcomes. Improvement in neurobehavioral deficits is observed with agents that dampen excitotoxicity and inflammation in OP models (Sharma et al., 2011; de Araujo Furtado et al., 2012; Ciarlone et al., 2017; Lumley et al., 2019; Dhir et al., 2020). GX can directly modulate the immune response, potentially leading to a more direct relationship in the reduction of neuroinflammation associated with OP exposure (Müller and Kerschbaum, 2006). Overall, due to their multimodal actions at inhibitory networks and neuroprotection properties, neurosteroids may offer an option for mitigating the long-term deficits of acute OP exposure in a pediatric population.

In conclusion, these results show that neurosteroid treatment with GX mitigates the enduring behavioral and cognitive impairments associated with acute DFP exposure in pediatric animals. Neurosteroid treatment attenuates DFP-induced chronic neurodegeneration, neuroinflammation, and mossy fiber sprouting in the hippocampus, along with the facilitation of neurogenesis, which possibly explains its neuroprotectant features. In addition, there is a strong correlation between the neuropathological outcomes with behavioral deficits, indicating a potential mechanistic basis of neurosteroid protection. Future studies are warranted to advance the therapeutic potential of neurosteroids in mitigating the long-term neuropsychiatric effects following DFP exposure.