Intestinal inflammation in a murine model of autism spectrum disorders Caroline G.M. de Theije a,⇑ , Pim J. Koelink a , Gerdien A.H. Korte-Bouws a , Sofia Lopes da Silva a,b , S. Mechiel Korte a , Berend Olivier a , Johan Garssen a,b , Aletta D. Kraneveld a a Division of Pharmacology, Utrecht Institute for Pharmaceutical Sciences, Faculty of Science, Utrecht University, Universiteitsweg 99, 3584 CG Utrecht, The Netherlands b Nutricia Research, Uppsalalaan 12, 3584 CT Utrecht, The Netherlands article info Article history: Received 13 September 2013 Received in revised form 25 November 2013 Accepted 2 December 2013 Available online 7 December 2013 Keywords: Autism spectrum disorders Prenatal Valproic acid Neurodevelopment Gut-brain axis Serotonin Intestinal tract abstract Autism spectrum disorder (ASD) is a cluster of neurodevelopmental disorders characterized by impair- ments in communication, social interest and stereotypical behaviour. Dysfunction of the intestinal tract is reported in patients with ASD and implicated in the development and severity of ASD symptoms. How- ever, more research is required to investigate the association of intestinal problems with ASD and the potential underlying mechanisms. The purpose of this study was to investigate comorbid symptoms of intestinal inflammation in a murine model of ASD induced by prenatal exposure to valproic acid (VPA). Pregnant BALB/c females were treated subcutaneously with 600 mg/kg VPA or phosphate buffered saline on gestational day 11. Offspring were housed with their mother until weaning on postnatal day 21 (P21). All pups were exposed to a social behaviour test on P28. Inflammatory correlates and activity of the serotonergic system were measured in brain and intestinal tissue. Here we demonstrate, in addition to reduced social behaviour and increased expression of neuroinflammatory markers in the brain, that VPA in utero- exposed male offspring showed epithelial cell loss and neutrophil infiltration in the intes- tinal tract. Furthermore, reduced levels of serotonin were not only observed the prefrontal cortex and amygdala of VPA in utero- exposed males, but also in the small intestine. Overall, we demonstrate that gender-specific inflammatory conditions are present in the small intestines of VPA in utero- exposed mice and are accompanied by a disturbed serotonergic system in the brain as well as in the intestinal tract. Ó 2013 Elsevier Inc. All rights reserved. 1. Introduction Autism spectrum disorder (ASD) is a heterogeneous cluster of severe neurodevelopmental disorders. It is characterized by impairments in social interaction and communication and the presence of stereotyped behaviours (American-Psychiatric-Associ- ation, 2000). Although the aetiology of ASD is unknown, it is thought that ASD is a multifactorial disorder with a strong genetic component (Bailey et al., 1995; Folstein and Rosen-Sheidley, 2001). A variety of environmental factors is suggested to contribute to ASD development. For example, prenatal exposure to teratogens has been shown to be a significant risk factor for ASD (Dufour- Rainfray et al., 2011). Indeed, maternal use of the anticonvulsant valproic acid (VPA) is associated with the development of ASD in the offspring (Christensen et al., 2013; Moore et al., 2000; Rasalam et al., 2005). The mechanism for VPA-induced symptoms of ASD is still unclear, but several pathways have been proposed. These include attenuation of folic acid metabolism, inhibition of histone deacetylases and increased oxidative stress (Ornoy, 2009). In search of underlying mechanisms, animal models of VPA-induced autism-like behaviours have been established in rats and mice. In a well-characterized murine model, VPA in utero- exposed mice exhibit developmental and behavioural deficits comparable to those observed in ASD patients, including deficits in social behav- iour (Kataoka et al., 2013; Roullet et al., 2010), stereotyped behav- iour (Wagner et al., 2006), anxiety and impairments in cognition (Kataoka et al., 2013). Furthermore, observations were more prom- inent in male offspring compared to female offspring (Kataoka et al., 2013; Kim et al., 2013), which reflects the human situation where a marked male preponderance is observed in ASD patients (Fombonne, 2005; Lord et al., 1982). Disturbances in the immune system are repeatedly reported in various organs of ASD patients. Since ASD is primarily a disorder of the central nervous system, the brain is a major target for immuno- logical research. In post-mortem brains of patients with ASD, marked activation of astroglia and microglia is observed when compared to controls (Fatemi et al., 2008; Laurence and Fatemi, 2005; Morgan et al., 2010; Tetreault et al., 2012; Vargas et al., 2005), indicative of neuroinflammation. Enhanced activation of neuroglia was also observed in various murine models of autism (Derecki et al., 2012; Ratnayake et al., 2012; Yuskaitis et al., 2010). In addition, enhanced levels of a wide range of cytokines and chemokines were found in the brain (Li et al., 2009) and in 0889-1591/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bbi.2013.12.004 ⇑ Corresponding author. Tel.: +31 30 253 7353; fax: +31 30 253 7900. E-mail address: c.g.m.detheije@uu.nl (C.G.M. de Theije). Brain, Behavior, and Immunity 37 (2014) 240–247 Contents lists available at ScienceDirect Brain, Behavior, and Immunity journal homepage: www.elsevier.com/locate/ybrbi