The expression of the constitutive neural isoform of nitric oxide synthase (bNOS) is dynamic and thus forms an ideal parameter to evaluate whether development and region affect the enteric nervous system. By applying NADPH-diaphorase histochemistry on whole-mount preparations of the myenteric and submucosal plexuses and by using the 'unbiased counting frame', a qualitative and quantitative description of bNOS-expression in enteric neurons in the pig duodenum in various developmental stage and region was obtained. Examinations were carried out on the oral and aboral duodenum of fetal pigs from the second half of gestation, of 1-2-day-old pigs and of 6-8-week-old pigs. In the pig duodenum, three enteric plexuses were readily distinguished: the inner submucous, the outer submucous and the myenteric plexuses. All three plexuses already harboured, to different degrees, bNOS-expressing neurons at midgestation. Although the enteric nervous system was present at midgestation, the enteric neurons had not yet reached their adult phenotype and morphology. During gestation, the number of inner submucous bNOS-expressing neurons increased approximately 50-fold, whereas after birth that number fell to about 10% of the prenatal value. During further postnatal development it returned to prenatal values. In addition, the number of bNOS-expressing myenteric neurons doubled postnatally. These changes favour a role for NO in mediating the development of enteric neurons and point to a greater necessity for inhibitory innervation in the adult pig as compared with the fetal pig. Furthermore, the number of bNOS-expressing outer submucosal and myenteric neurons was significantly higher in the oral duodenal segment compared with the aboral duodenal segment. This regional difference suggests that the oral duodenal segment is more prominently involved in the regulation of NO-mediated gastrointestinal processes than the aboral one. The developmentally and regionally dependent bNOS-expression can be explained by shifts and differences in the balanced system of hormones, presynaptic input and target-derived signals that affects neurotransmitter expression.

Formula feeding facilitates bacterial adherence and the development of a proinflammatory state of the intestine, which may be among the key factors that predispose formula-fed preterm neonates to NEC.

A mature enteric nervous system (ENS) is required to ensure a normal pattern of intestinal motility in order to regulate digestion after birth. We hypothesized that neuronal and glial components of the ENS would mature during the first postnatal days in preterm pigs that are a sensitive animal model of food intolerance and necrotizing enterocolitis (NEC). Stereological volume densities of the general neuronal population [assessed by betaIII-tubulin immunoreactivity (IR)] and subsets of neuronal (VIP-IR and nitrergic IR) and glial cells (GFAP-IR and S100-IR) were determined in the small intestine of newborn preterm piglets (93% gestation), after 3 days of receiving total parenteral nutrition (TPN) and after 3 days of TPN plus 2 days of enteral feeding with sow's colostrum or milk formula. Following TPN, VIP in the myenteric and inner submucous plexus and GFAP in the inner submucous plexus increased, while the relative volume of the total neuronal population remained constant. Introduction of enteral food induced variable degrees of food intolerance and NEC, especially after formula feeding, a diet that gave rise to a higher myenteric VIP and GFAP content in the inner submucous plexus than colostrum feeding. However, the ENS seemed unaffected by the presence of NEC-like intestinal lesions. Nevertheless, this study shows that the ENS is highly plastic during the first days after premature birth and adapts in an age- and diet-dependent manner. The observed postnatal adaptation in enteric VIP and GFAP may help to maintain intestinal homeostasis during suboptimal feeding regimens in preterm neonates.

Enteral feeding of formula, for only a few hours, induces rapid enterocyte turnover and mucosal structural changes that may predispose to later development of NEC.