**2. Effect of perinatal infection and toxicants on the developing brain**

In a continuing quest to understand the nature of gene-environment interactions in ASD, we have recently completed two animal studies examining the effect of perinatal exposure of thimerosal (TM) and lipopolysaccharides (LPS) on the developing rat central nervous system (CNS). Both TM and infections (modeled by LPS exposure) have been implicated in autistic pathology.

Organic mercury compounds are powerful toxicants with a range of harmful neurological effects in humans and animals. TM, which is metabolized to ethyl mercury, has been discon‐ tinued as a preservative from infant vaccines but continues to be used in several vaccines including a flu vaccine administered to pregnant and lactating mothers (Sulkowski et al., 2012). Perinatal maternal exposure of two strains of rats, Sprague Dawley (SD) and spontane‐ ously hypertensive (SHR) rats, to thimerosal (200 ug/kg body weight) resulted in both sex- and strain-specific abnormalities in the neonatal rats (Sulkowski et al., 2012). Behavioral abnor‐ malities included delayed startle response and decreased motor learning, with the effects being both sex- and strain-specific. TM exposure also resulted in a significant increase in cerebellar levels of an oxidative stress marker (3-nitrotyrosine) and a decrease in cerebellar type 2 deiodinase, responsible for local intrabrain conversion of thyroxine to the active hormone, 3', 3,5,-triiodothyronine (T3). These effects were associated with an increased expression of several genes negatively regulated by T3 (Sulkowski et al., 2012); Khan et al., 2012) suggesting that perinatal exposure to TM impacts the developing brain at the genetic level. As TM exposure during the postnatal phase coincided with lactation, some of the TM was delivered through the milk to the GIT and may have had an effect on the developing gut microbiome known to be sensitive to heavy metal exposure (Lapanje et al., 2007). This effect may be in part due to competition with zinc resulting in a disturbance in metallothionein function and general chelating capacity for other metals. Thus, at least part of the neonatal impact of TM/mercury could be mediated via its action on the gut microbiome.

In a related study (Xu et al., submitted) we examined the effect of E.coli lipopolysaccharides (LPS) exposure during critical developmental periods on the developing brain employing the animal model of infection. Clinical and epidemiological data suggest that maternal infection during pregnancy and nursing increases the probability of neonatal brain injury and may have a long-lasting impact on brain functions. Maternal infection during pregnancy has been linked to neurological and neurobehavioral disorders in humans such as cerebral palsy (Schendel, 2001; Schendel et al., 2002), neonatal strokes (Ferrieo, 2004), schizophrenia (Watson et al., 1999; Pearce, 2001) and affective disorders (Watson et al., 1999). Animal studies implicate bacterial infection in the pathology of Parkinson's disease (Carvey et al., 2003), and notably, schizophrenia and autism (Patterson, 2002). The triggering signals for cytokine production are endotoxins, major components of the outer membrane of Gram-negative bacteria.

LPS exposure is one of the most acceptable models of infection; LPS is a sufficient trigger for cytokine production. LPS administered to the pregnant mother are transferred to the fetus through the placenta (Kohmura et al., 2000), and result in increased cytokines levels in the amniotic fluid (Urakabo et al., 2001; Gayle et al., 2004) and the fetal brain (Urakabo et al., 2001). Bacterial infection of lactating mothers also results in an increased level of cytokines in milk (Bannerman et al., 2004). Pretreatment of suckling rats with LPS (10 mg/kg-day x 5 days – the dose which produces weak, transient signs of endotoxemia) results in reduced pancreatic secretion and attenuates acute pancreatitis at adult age due to an increased concentration of the antioxidative enzyme SO in the pancreatic tissue, and to the modulation of cytokines production (Jaworek at al., 2007a, b). This late-effect of LPS is accompanied by dose-dependent reduction of mRNA signal for CCK1 receptor on pancreatic acini as well as modified expres‐ sion of acinar pro-apoptotic heat shock protein-60 (HSP60) and Bax proteins (Jaworek et al., 2007b, 2008). Early postnatal LPS exposure results in inceased expression of toll-like receptor 4 (TLR4) and caspase-3 and 9- proteins in the pancreatic tissue of adult rats (Bonior et al., 2012). These studies clearly indicate that perinatal exposure to LPS may have long lasting consequences on the GIT function, and as expected, though not studied in detail, on the braingut axis.

system(s) in autism as a consequence of altered gut microbiome. It is possible that aberrant regulation of the reward system(s) underlines behavioral abnormalities in ASD that could be

In a continuing quest to understand the nature of gene-environment interactions in ASD, we have recently completed two animal studies examining the effect of perinatal exposure of thimerosal (TM) and lipopolysaccharides (LPS) on the developing rat central nervous system (CNS). Both TM and infections (modeled by LPS exposure) have been implicated in autistic

Organic mercury compounds are powerful toxicants with a range of harmful neurological effects in humans and animals. TM, which is metabolized to ethyl mercury, has been discon‐ tinued as a preservative from infant vaccines but continues to be used in several vaccines including a flu vaccine administered to pregnant and lactating mothers (Sulkowski et al., 2012). Perinatal maternal exposure of two strains of rats, Sprague Dawley (SD) and spontane‐ ously hypertensive (SHR) rats, to thimerosal (200 ug/kg body weight) resulted in both sex- and strain-specific abnormalities in the neonatal rats (Sulkowski et al., 2012). Behavioral abnor‐ malities included delayed startle response and decreased motor learning, with the effects being both sex- and strain-specific. TM exposure also resulted in a significant increase in cerebellar levels of an oxidative stress marker (3-nitrotyrosine) and a decrease in cerebellar type 2 deiodinase, responsible for local intrabrain conversion of thyroxine to the active hormone, 3', 3,5,-triiodothyronine (T3). These effects were associated with an increased expression of several genes negatively regulated by T3 (Sulkowski et al., 2012); Khan et al., 2012) suggesting that perinatal exposure to TM impacts the developing brain at the genetic level. As TM exposure during the postnatal phase coincided with lactation, some of the TM was delivered through the milk to the GIT and may have had an effect on the developing gut microbiome known to be sensitive to heavy metal exposure (Lapanje et al., 2007). This effect may be in part due to competition with zinc resulting in a disturbance in metallothionein function and general chelating capacity for other metals. Thus, at least part of the neonatal impact of TM/mercury

In a related study (Xu et al., submitted) we examined the effect of E.coli lipopolysaccharides (LPS) exposure during critical developmental periods on the developing brain employing the animal model of infection. Clinical and epidemiological data suggest that maternal infection during pregnancy and nursing increases the probability of neonatal brain injury and may have a long-lasting impact on brain functions. Maternal infection during pregnancy has been linked to neurological and neurobehavioral disorders in humans such as cerebral palsy (Schendel, 2001; Schendel et al., 2002), neonatal strokes (Ferrieo, 2004), schizophrenia (Watson et al., 1999; Pearce, 2001) and affective disorders (Watson et al., 1999). Animal studies implicate bacterial infection in the pathology of Parkinson's disease (Carvey et al., 2003), and notably, schizophrenia and autism (Patterson, 2002). The triggering signals for cytokine production are

endotoxins, major components of the outer membrane of Gram-negative bacteria.

**2. Effect of perinatal infection and toxicants on the developing brain**

targeted by future microbiome-targeting therapies.

62 Recent Advances in Autism Spectrum Disorders - Volume I

could be mediated via its action on the gut microbiome.

pathology.

Perinatal maternal exposure of two strains of rats, SHR or SD rat dams to LPS (200 µg/kg body weight) resulted in increased rollover time, delayed startle, and decreased motor learning, with the effects being both strain- and sex-specific. LPS challenge also resulted in a trend towards an increase in cerebellar levels of 3-NT and a decrease in D2 activities in LPS-exposed pups (Xu et al., submitted). Several genes were affected by LPS. Notably Type 2 deiodinase 2 (DIO2) and brain derived neurotrophic factor (BDNF) expression was significantly elevated, while transthyretin (TTR) expression was decreased following LPS exposure. *In vitro*, acute exposure of cerebellar cultures to LPS resulted in a decreased size of the dendritic area of Purkinje cells. Our data thus demonstrate that perinatal infection impacts the developing cerebellum in a sex- and strain-dependent manner via mechanisms involving oxidative stress, enzymes involved in maintaining local TH homeostasis, and downstream gene expression. Interestingly, gene changes observed in the brains of LPS-exposed rats were distinct from TMassociated gene effect suggesting that the underlying macromolecular mechanism may be trigger-specific.

Perinatal LPS exposure could have a profound effect on the gut microbiome similar to the effect of repeated treatment with antibiotics. Experiments in healthy mice have shown that disrupting the normal balance of the gut microbiome with antibiotics caused changes in mice behavior and was accompanied by changes in BDNF which has been linked to depression and anxiety (Bercik et al., 2011; Neufeld et al., 2011). Perinatal LPS exposure most likely affects gut motility as suggested by studies of irritable bowel syndrome (IBS), where mild bacterial overgrowth-associated motility disorder can be reversed by antimicrobials (Scarpignato and Pelosini, 1999). Animal studies have also shown that stress can change the composition of the microbiome, where the changes are associated with increased vulnerability to inflammatory stimuli in the GIT. Could gut dysbiosis be induced by recurrent infections? We have observed an increase in neurotrophin levels in the cerebella of rats exposed to LPS (Sajdel-Sulkowska et al, unpublished observation) and brain region-specific changes in neurotrophin levels in ASD (Sajdel-Sulkowska et al., 2011). Together these observations suggest that a bacterial infection could trigger the gut microbiome to induce cytokine overproduction leading to an imbalance of brain neurotrophins and contribute to developmental abnormalities.

form large size mobile vacuoles in the upper part of the cell enabling the transfer of intact colostral molecules into the blood (Baintner 2002). Approximately two days after birth, following substantial intake of colostral bioactive substances, the permeability of the gut epithelium is dramatically reduced to macromolecules due to the rapid replacement of fetal type enterocytes by adult type enterocytes, a phenomenon known as gut closure; the cell replacement is made by a receptor-mediated apoptosis involving TGF-β1 and TNF-α as mediators (Godlewski et al., 2005, Strzałkowski et al., 2007). Consequently, adult type enterocytes do not contain ACS and large vacuoles. Interestingly, in the gut of neonatal pigs the cells undergoing apoptosis, which is followed by unzipping-zipping events markedly disrupting epithelial cell continuity, are located on the entire length of the villi (Godlewski et al., 2005; see also Fig. 1). In contrast, in adult animals the apoptotic cells are observed only on the villi top, forming a so called extrusion zone. Therefore, in neonates there is a much wider absorptive surface that is potentially subject to environmental stimuli as compared to adults. Though, one population of fetal type enterocytes disappears within the first few days after birth, there is still another population of fetal type enterocytes existing in the lower small intestine, in piglets observed until approximately three weeks after birth. These enterocytes are important for the intracellular digestion of nutrients by lysosomal en‐ zymes, and form digestive vacuoles as a result of non-selective macromolecule uptake. Their massive loss in piglets is observed 2-3 weeks after birth. Nevertheless the protection by intestinal mucus and colostral biologically active peptides and proteins, extensive apopto‐ sis and unzipping-zipping of a great number of epithelial cells at the same time may potentially open epithelial gates for any xenobiotics and harmful bacteria, and thereby

Gut Microbiome and Brain-Gut Axis in Autism — Aberrant Development of Gut-Brain Communication…

http://dx.doi.org/10.5772/55425

65

Studies of preterm piglets and intrauterine growth retarded (IUGR) piglets demonstrated that the gut barrier in both groups of animals is open for a longer time than in full-term-appropriate weight piglets. Namely, the lower part of the small intestine of 28 day-old IUGR piglets still contained fetal type enterocytes expressing digestive vacuoles indicating marked delay in gut mucosa development (Mickiewicz et al. 2012 JPP). The gut epithelium continuity in IUGR and preterm neonates is not as finely controlled as in control rats; abnormalities of the gut epithe‐ lium may facilitate exposure of the gut and in turn the whole organism to external factors or xenobiotics. It is possible that gut permeability is altered in critically ill children and predispose them to bacterial translocation via a mechanism that creates a hostile environment in the gut and alters the gut microbiome favoring the growth of pathogens that promote bacterial

Recent studies indicate that the vagus nerve is involved in immunomodulation as suggested by its ability to attenuate the production of proinflammatory cytokines in experimental models of inflammation (de Jonge and Ullola, 2007). Furthermore, functional development of the vagus nerve occurs at two stages with the neuronal population in the dorsal motor nucleus of the vagus (DMNV) maturing ahead of the sensory neuron population of the vagal sensory nucleus NTS (Islami et al., 2008). There appears to be an important link between the vagus nerve and memory recall in infancy suggesting that social learning, modulated by autonomic

nervous system, may be jeopardized in preterm infants (Haley et al., 2010)

facilitate their transfer into blood circulation.

translocation (Papoff et al., 2012).
