Validation of prenatal versus postnatal valproic acid rat models of autism: A behavioral and neurobiological study

https://doi.org/10.1016/j.pnpbp.2020.110185Get rights and content

Highlights

  • Prenatal and postnatal VPA exposure protocols induce autistic-like behaviors in rats.

  • Prenatal and postnatal VPA elevate brain inflammatory cytokines and oxidative stress.

  • Prenatal and postnatal VPA induce brain histological neurodegeneration and apoptosis.

  • Prenatal VPA causes more autistic-like behaviors & less mortality than postnatal VPA.

Abstract

Despite the increasing prevalence of autism spectrum disorder (ASD), there is still a deficiency in understanding its exact pathophysiology and treatment, therefore validation of translational ASD animal model is warranted. Although strong evidences support the valproic acid (VPA) model of autism, yet a controversy exists regarding the best timing of exposure whether prenatal or postnatal. Accordingly, this study was designed to compare the time dependent effects of VPA exposure as regard its ability to induce autistic like changes in male Wistar rats. In this study, two different protocols of VPA exposure (prenatal and postnatal) were compared at different levels (behavioral, neurochemical and histopathological). Results of this study revealed that both prenatal and postnatal VPA exposures induced autistic-like behaviors manifested by reduced social interaction, increased repetitive stereotyped behavior and anxiety, cognitive dysfunction, lowered sensitivity to pain, and neurodevelopmental delay. Furthermore, inflammatory cytokines and oxidative/nitrosative stress markers were elevated in prefrontal cortex and hippocampal homogenates. Likewise, histopathological and immunohistochemical assessment confirmed the neurodegenerative and the apoptotic changes in prefrontal cortex, hippocampus and cerebellum exhibited by decreased viable cells number and Nissl's granules optical density, and increased caspase-3 immunoreactivity respectively. Interestingly, ASD core symptoms and histopathological changes were significantly (P < 0.05) altered in prenatal VPA model compared to postnatal VPA model. Additionally, postnatal mortality in prenatal model (4.3%) was much lower compared to the postnatal model (22.7%). In conclusion, our study overweighs the ability of prenatal VPA model over postnatal VPA model to induce behavioral and neuropathological alterations that simulate those 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

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.

References (70)

  • D.M. Kerr et al.

    Alterations in the endocannabinoid system in the rat valproic acid model of autism

    Behav. Brain Res.

    (2013)
  • K.C. Kim et al.

    The critical period of valproate exposure to induce autistic symptoms in Sprague-Dawley rats

    Toxicol. Lett.

    (2011)
  • H. Kumar et al.

    Memantine ameliorates autistic behavior, biochemistry & blood brain barrier impairments in rats

    Brain Res. Bull.

    (2016)
  • X. Li et al.

    Elevated immune response in the brain of autistic patients

    J. Neuroimmunol.

    (2009)
  • A.M. Mohamed et al.

    Amisulpride alleviates chronic mild stress-induced cognitive deficits: Role of prefrontal cortex microglia and Wnt/β-catenin pathway

    Eur. J. Pharmacol.

    (2020)
  • M. Narita et al.

    Nonexploratory movement and behavioral alterations in a thalidomide or valproic acid-induced autism model rat

    Neurosci. Res.

    (2010)
  • C. Nicolini et al.

    The valproic acid-induced rodent model of autism

    Exp. Neurol.

    (2018)
  • F. Pohl-Guimaraes et al.

    Early valproic acid exposure alters functional organization in the primary visual cortex

    Exp. Neurol.

    (2011)
  • F.I. Roullet et al.

    In utero exposure to valproic acid and autism--A current review of clinical and animal studies

    Neurotoxicol. Teratol.

    (2013)
  • T. Schneider et al.

    Gender-specific behavioral and immunological alterations in an animal model of autism induced by prenatal exposure to valproic acid

    Psychoneuroendocrinology

    (2008)
  • H. Wu et al.

    Fingolimod (FTY720) attenuates social deficits, learning and memory impairments, neuronal loss and neuroinflammation in the rat model of autism

    Life Sci.

    (2017)
  • O. Abdel-Salam et al.

    Nuclear factor-kappa B and other oxidative stress biomarkers in serum of autistic children

    Open J. Mol. Integr. Physiol.

    (2015)
  • S. Abuelezz et al.

    Targeting oxidative stress, cytokines and serotonin interactions via indoleamine 2, 3 dioxygenase by coenzyme Q10: Role in suppressing depressive like behavior in rats

    J. NeuroImmune Pharmacol.

    (2016)
  • T. Al Sagheer et al.

    Motor impairments correlate with social deficits and restricted neuronal loss in an environmental model of autism

    Int. J. Neuropsychopharmacol.

    (2018)
  • A.A. Ali

    Pomegranate and folate ameliorate isolation-induced autistic like behavior in experimental rat model: Impact on oxidative, inflammatory, and apoptotic machineries

    EC Pharmacol. Toxicol.

    (2020)
  • G. Anderson et al.

    Redox regulation and the autistic spectrum: Role of tryptophan catabolites, immuno-inflammation, autoimmunity and the amygdala

    Curr. Neuropharmacol.

    (2014)
  • K. Anshu et al.

    Altered attentional processing in male and female rats in a prenatal valproic acid exposure model of autism spectrum disorder

    Autism Res.

    (2017)
  • J. Baio et al.

    Prevalence of autism spectrum disorder among children aged 8 years - Autism and developmental disabilities monitoring network, 11 sites, United States, 2014

    MMWR Surveill. Summ.

    (2018)
  • C.D. Barnhart et al.

    Using the Morris water maze to assess spatial learning and memory in weanling mice

    PLoS One

    (2015)
  • G. Bjørklund et al.

    Oxidative stress in autism spectrum disorder

    Mol. Neurobiol.

    (2020)
  • R.L. Bromley et al.

    The prevalence of neurodevelopmental disorders in children prenatally exposed to antiepileptic drugs

    J. Neurol. Neurosurg. Psychiatry

    (2013)
  • M.R. Bronzuoli et al.

    Neuroglia in the autistic brain: Evidence from a preclinical model

    Mol. Autism

    (2018)
  • K.K. Chadman

    Animal models for autism in 2017 and the consequential implications to drug discovery

    Expert Opin. Drug Discov.

    (2017)
  • A. Chauhan et al.

    Increased DNA oxidation in the cerebellum, frontal and temporal cortex of brain in autism

  • R.M. Deacon

    Digging and marble burying in mice: Simple methods for in vivo identification of biological impacts

    Nat. Protoc.

    (2006)
  • Cited by (0)

    View full text