Validation of prenatal versus postnatal valproic acid rat models of autism: A behavioral and neurobiological study
Introduction
Autism spectrum disorder (ASD) is a childhood neurobehavioral disorder presenting with impairments in social interaction and repetitive stereotyped patterns of behavior (DSM-5, 2013). It is a worldwide problem where 1 child in every 59 has an ASD and it is four times more common in males than in females (Baio et al., 2018).
Although the underlying pathophysiology of ASD is not well understood, yet ample evidence points to a close link between neuro-inflammation, oxidative stress, and ASD. Postmortem neuroimaging studies and animal models of ASD have unveiled neuroanatomical changes in different brain regions especially the frontal cortex, hippocampus and cerebellum which were related to the behavioral abnormalities observed in ASD (Roullet et al., 2013; Varghese et al., 2017). Furthermore, body of evidence revealed increased inflammatory cytokines such as interlukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) with the activation of nuclear factor kappa-of B-cells (NF-κB) linked to autistic-like behavioral impairments (Li et al., 2009; Rodriguez and Kern, 2011; Young et al., 2016).
Additionally, oxidative and nitrosative stress (O&NS) pathways have been found to play a significant role in driving the alterations exists in early developmental etiology and course of ASD (Anderson and Maes, 2014; Bjørklund et al., 2020). Clinical and experimental studies illustrated altered O&NS markers in autistic individuals where reactive oxygen species (ROS), nitric oxide (NO) and lipid peroxidation were elevated while the endogenous antioxidant defenses, specifically reduced glutathione (GSH) and catalase (CAT) were depleted (Rodriguez and Kern, 2011; Sandhya et al., 2012; Abdel-Salam et al., 2015; Kumar and Sharma, 2016).
A pivotal tool in understanding the etiology of human diseases and determining effective therapies is the presence of a valid animal model. Ideally, a valid autism model is required to have both “face validity” (the phenotypes similar to human ASD clinical presentation) and “construct validity” (the molecular defects that mimic what is seen in human ASD) (Chadman, 2017). Despite different animal models used to elicit autistic like behaviors that resemble autistic changes in humans and provide the opportunity to understand the ASD and developing new treatments, valproic acid (VPA) has been the most extensively studied (Schneider and Przewlocki, 2005; Markram et al., 2008; Kim et al., 2014; Nicolini and Fahnestock, 2018).
VPA is an antiepileptic drug that is also used in migraine headaches and in bipolar disorder as a mood stabilizer. Clinical studies have demonstrated that VPA consumption during pregnancy was associated with increased rates of ASD in the offspring besides increased incidence of neural tube defects, developmental delay and cognitive impairments (Bromley et al., 2013; Roullet et al., 2013). The deleterious effects of the VPA animal model of autism were confirmed by different experimental studies in which similarities between the behavioral changes in VPA exposed rats and the disturbed behavior in autistic patients were observed including: marked decrease in social interaction and repetitive stereotyped behavior, which were further supported by numerous biochemical and histopathological studies (Schneider and Przewlocki, 2005; Wagner et al., 2006; Kim et al., 2014; Kumar and Sharma, 2016; Wang et al., 2016; Wu et al., 2017; Cezar et al., 2018). The exposure, to a neurotoxicant agent like VPA during critical periods was found to disrupt neurobehavioral development by altering neural migration, circuitry, and/or synaptogenesis of brain areas required for expression of these behaviors, resulting in behavioral retardation, regression, and/or intrusions (Wagner et al., 2006). According to the literature, prenatal and postnatal VPA exposures in rodents are the most commonly used models to induce autistic-like neurobehavioral defects which are analogous to the deficits observed in humans with autism (Schneider and Przewlocki, 2005; Wagner et al., 2006; Mony et al., 2016; Gedzun et al., 2017).
The prenatal VPA model was introduced in 1996, when a single large dose of VPA administered to rats on gestational day 12.5 to elicit autistic-like features (Rodier et al., 1996). This period corresponds to 1st human trimester following neural tube closure (Kim et al., 2011). As regard the postnatal VPA model, VPA is administered to rodents during the early postnatal period (0–14 days; which roughly corresponds to the human third trimester where the brain development occur) to disrupt brain development and to elicit autistic like behaviors (Wagner et al., 2006; Reynolds et al., 2012).
Remarkably, literature controversy exists regarding the best timing and dosing of VPA exposure either in prenatal or postnatal ASD models (Rodier et al., 1996; Schneider and Przewlocki, 2005; Markram et al., 2008; Kim et al., 2011; Reynolds et al., 2012; Morakotsriwan et al., 2016; Wang et al., 2016; Gedzun et al., 2017). Yet, to the authors' knowledge, experimental studies comparing the validity of these two models in rats have not been conducted hitherto. Accordingly, this study was designed to compare the validity of prenatal VPA exposure versus the postnatal VPA exposure to induce autistic like changes at different levels (behavioral, neurobiological and histopathological) in Wistar rats with assessment of postnatal mortality % as well.
Section snippets
Animals
Twenty-six adult Wistar rats (males and females) weighing 150–250 g were purchased from the Nile for Pharmaceuticals and Chemical Industries, El Sawah, Cairo, Egypt. Animals were acclimated for 7 days before experimentation after that they were allowed to mate together. Female rats were used only once for breeding and then were excluded from the experiment. Throughout the experiment, animals were maintained under controlled conditions of temperature (24 °C) and a 12 h. light/dark cycle and were
Three-chamber sociability test (3-CST)
Fig. 2 shows that prenatal VPA exposure displayed more impairment in social interaction than postnatal VPA exposure. Only prenatal VPA model significantly (P < 0.0001) reduced sociability index compared to its respective control. Regarding social novelty preference phase, both prenatal and postnatal VPA protocol exposures exhibited a significant decrease in social novelty preference index (P < 0.001, P < 0.05 respectively) compared to their respective controls. Additionally, prenatal VPA model
Discussion
To the best of our knowledge, this is the first study to compare prenatal versus postnatal VPA induced ASD models at the behavioral, neurochemical, histopathological levels and postnatal mortality % in Wistar rats. Both prenatal and early postnatal VPA exposure protocols were able to induce autistic-like behaviors manifested by reduced social interaction (in 3CST), increased repetitive stereotyped behavior and anxiety (in MBT & OFT), cognitive impairment (in MWM) and lowered sensitivity to pain
Conclusion
In conclusion, the present work overweighs the ability of prenatal over postnatal VPA exposure to simulate behavioral and neuropathological alterations observed in autistic individuals with a lower postnatal animal mortality, highlighting the privilege of prenatal over postnatal VPA exposure as a translational model for understanding pathophysiology and developing novel targets for management of ASD. However, it explicitly raises the question when, where and how alterations/deficits in neuronal
Funding
This research received no specific Grant from any funding agency in the public, commercial or not-for-profit sectors.
Authors' statement
All authors certify that they had participated sufficiently in the work and took public responsibility for the content, including participation in the concept, design, analysis, writing, and revision of the manuscript. Furthermore, all authors certify that this material is not under publication or published in any other publication.
Ethical statement
The present study was conducted in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) and was approved by the ethics committee of faculty of medicine, Ain Shams University (FMASU REC). All efforts were made to minimize animal suffering as well as to reduce the number of animals used.
This Study has not been published previously, and it is not under consideration for publication elsewhere. Its publication is approved by all authors. If accepted, it will
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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