**3. Effect of environmental perturbations on the developing components of the brain-gut axis: Intestinal permeability, inflammation and gut microbiome**

As indicated above the perinatal development of the CNS structure and function greatly depends on the gastrointestinal GIT function. Little is known about the regulation of embry‐ onic gut epithelium or the effect of prenatal infection. The two key developmental time-points in the regulation of the GIT both occur postnatally, the first few days after birth when all gut digestive functions are launched by first colostrum ingestion and the second at weaning when the digestive system has to modify its function following a switch from mother's milk to solid food. The first time-point is particularly relevant for all mammalian species since it is associated with a complex of dynamic changes in the GIT structure and function leading to a temporary drop in the gut permeability barrier. The secretion of digestive juices (e.g., gastric and pancreatic juice secretion) is obviously close to null before birth. Our studies indicate that in neonatal calves the exocrine pancreas secreted low but measurable amounts of pancreatic juice from the first postnatal day. The secretion responded to colostrum feeding showing a clearcut cephalic phase associated with plasma pancreatic polypeptide (PP) elevation but no gastric or intestinal phase. Further studies involving vagal blockades and pharmacological cholecys‐ tokinin receptor antagonists indicated that in neonatal calves pancreatic response to first colostrum feeding is already controlled by a neuro-hormonal mechanism involving CCK and long vago-vagal reflex (Zabielski et al., 2002). Thus, the brain-gut axis control of the exocrine pancreas observed in one- and two-month old milk-fed calves is already present at birth, only the magnitude of the response increases with age reaching its peak at four weeks. The other GIT function in neonatal calves closely associated with the brain-gut axis control include periodic activity of GIT motility (migrating motor complex, MMC) and secretion (pancreatic juice periodic secretion) observed already two-three days after birth along with plasma PP oscillations (Zabielski et al., 2002). Plasma PP, a marker of efferent vagal activity, increased with age indicating that brain-gut axis further develops after birth (and may be potentially sensitive to any environmental modifications).

Intestinal functions in neonates are far more complex than in adults due to intensive developmental processes. The small intestine is one of the fastest body organs to grow in size postnatally as well as the fastest organ in rebuilding its structure. Relatively little is known regarding the development of the large intestine, a major organ inhabited by gut bacteria. At birth, the small intestinal mucosa is lined by enterocytes ready for rapid uptake of colostral macromolecules (open gut). These enterocytes, so called fetal type enterocytes, are equipped with a system of vesicles and cisterns (apical canalicular system, ACS) which

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 facilitate their transfer into blood circulation.

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

**3. Effect of environmental perturbations on the developing components of**

As indicated above the perinatal development of the CNS structure and function greatly depends on the gastrointestinal GIT function. Little is known about the regulation of embry‐ onic gut epithelium or the effect of prenatal infection. The two key developmental time-points in the regulation of the GIT both occur postnatally, the first few days after birth when all gut digestive functions are launched by first colostrum ingestion and the second at weaning when the digestive system has to modify its function following a switch from mother's milk to solid food. The first time-point is particularly relevant for all mammalian species since it is associated with a complex of dynamic changes in the GIT structure and function leading to a temporary drop in the gut permeability barrier. The secretion of digestive juices (e.g., gastric and pancreatic juice secretion) is obviously close to null before birth. Our studies indicate that in neonatal calves the exocrine pancreas secreted low but measurable amounts of pancreatic juice from the first postnatal day. The secretion responded to colostrum feeding showing a clearcut cephalic phase associated with plasma pancreatic polypeptide (PP) elevation but no gastric or intestinal phase. Further studies involving vagal blockades and pharmacological cholecys‐ tokinin receptor antagonists indicated that in neonatal calves pancreatic response to first colostrum feeding is already controlled by a neuro-hormonal mechanism involving CCK and long vago-vagal reflex (Zabielski et al., 2002). Thus, the brain-gut axis control of the exocrine pancreas observed in one- and two-month old milk-fed calves is already present at birth, only the magnitude of the response increases with age reaching its peak at four weeks. The other GIT function in neonatal calves closely associated with the brain-gut axis control include periodic activity of GIT motility (migrating motor complex, MMC) and secretion (pancreatic juice periodic secretion) observed already two-three days after birth along with plasma PP oscillations (Zabielski et al., 2002). Plasma PP, a marker of efferent vagal activity, increased with age indicating that brain-gut axis further develops after birth (and may be potentially

Intestinal functions in neonates are far more complex than in adults due to intensive developmental processes. The small intestine is one of the fastest body organs to grow in size postnatally as well as the fastest organ in rebuilding its structure. Relatively little is known regarding the development of the large intestine, a major organ inhabited by gut bacteria. At birth, the small intestinal mucosa is lined by enterocytes ready for rapid uptake of colostral macromolecules (open gut). These enterocytes, so called fetal type enterocytes, are equipped with a system of vesicles and cisterns (apical canalicular system, ACS) which

**the brain-gut axis: Intestinal permeability, inflammation and gut**

of brain neurotrophins and contribute to developmental abnormalities.

64 Recent Advances in Autism Spectrum Disorders - Volume I

**microbiome**

sensitive to any environmental modifications).

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 translocation (Papoff et al., 2012).

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)

66 Recent Advances in Autism Spectrum Disorders - Volume I

Fig. 1.

While much of this biodiversity remains unexplained, extrinsic factors such as diet, environ‐ ment, and early microbial exposure, and the intrinsic factors such as host genetics have been implicated (Human Microbiome Project Consortium, 2012); our own studies (Sulkowski et al., 2012) suggest that sex may play an important role. Diet-derived carbohydrates that are not fully digested in the upper gut are metabolized by bacteria in the human large intestine. These nondigestable carbohydrates influence microbial fermentation and total bacterial number in the colon. Human milk, unlike milk of other mammalian species, contains high amounts of oligosaccharides of yet unknown function, but one can speculate that dietary oligosaccharides may play an important function in the development of the microbiome in human neonates. Evidence exists that the amount and type of nondigestable carbohydrates influence the species composition of the intestinal microbiome. Individual variation in the gut microbiome may, in part reflect differences in dietary intake, but the response of the gut microbiome to dietary

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

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

67

Furthermore, an outcome of the exposure to infectious microbes or their toxins is also influ‐ enced by both microbial and host genes. Some host genes encode defense mechanisms, whereas others assist pathogen function. Extensive human diversity in cell lethality dependent on toxin binding and uptake has been observed (Martchenko et al., 2012). Furthermore, there is evidence that individuals may evolve their own specific microbiome (Clayton, 2012).

Results of our recent animal studies (Sulkowski et al, 2012; Khan et al, 2012, Xu et al, submitted; see also Fig. 2) indicate that the sensitivity of the developing CNS to both environmental toxins and infection, are both sex- and rat strain-dependent. It can be extrapolated that the sensitivity of the human microbiome is also sex-dependent. Because of this individual variability in host response it is not surprising that the results of human postmortem studies of ASD brains are

The human GIT harbors a large number (1000 to 1150) of bacterial species and is involved in maintaining homeostasis and well-being. Functions of this microbiome include the regulation of the mucosal immune system, GIT motility, epithelial barrier regulation, gut secretion, digestion and metabolism (Grenham et al., 2011). One of the main functions of gut microbes is to extract nutrients from otherwise indigestible fibers (Tremaroli and Backhed, 2012). The microbiome, absent at birth, is gradually colonized by facultative bacteria and anaerobic

Several lines of evidence point to both brain-gut axis and gut microbiome abnormalities in autism which are summarized in Fig 3. Children with ASD frequently present a variety of gastrointestinal (GI) symptoms, although some claim that the data supporting increased GI symptomology in autistic children not to be rigorous enough (Erickson et al., 2005). The socalled "bacterial theory" of autism proposes the GIT symptoms are associated with changes in microbial composition and that these changes could be involved in the pathogenesis or

progression of several childhood diseases including autism (Somma et al., 2010).

change can also differ among individuals (Flint, 2012)

difficult to interpret.

**5. Microbiome**

bacteria (Grenham et al., 2011).

Fig. 1. **Figure 1. Packetts of apoptotic cells in the epithelium of neonatal piglets.** Massive apoptosis is evidenced by scanning electron microscope (SEM) image by shortened microvilli, several yet unzipped spaces between cells are present. **Figure 1. Packetts of apoptotic cells in the epithelium of neonatal piglets.** Massive apoptosis is evidenced by scan‐ ning electron microscope (SEM) image by shortened microvilli, several yet unzipped spaces between cells are present. (SEM images generously supplied by dr. Tomasz Skrzypek, Catholic University of Lublin, Poland)

In conclusion, maturation of the autonomic nervous system may be delayed in preterm and IUGR animals. Furthermore, delayed development of the GIT in preterm and IUGR animals, including longer gut permeability, facilitates the toxic effect of external factors including bacterial translocation. Furthermore, the immature gut seemingly fails to stimulate the development of the vagus nerve. Importantly, there is some evidence pointing to altered gut permeability (leaky gut) in autism and possibly genetic predisposition to abnormalities in tight junctions in ASD (White, 2003; de Magistris et al, 2010). (SEM images generously supplied by dr. Tomasz Skrzypek, Catholic University of Lublin, Poland)
