A. Weyns

Diet-dependent Mucosal Colonization and Interleukin-1β Responses in Preterm Pigs Susceptible to Necrotizing Enterocolitis

Objectives: Intestinal colonization challenges the neonatal innate immune system, especially in newborns with an immature immune response lacking the supportive bioactive components from mothers milk. Accordingly, formula-fed preterm pigs frequently show bacterial overgrowth, mucosal atrophy, and gut lesions reflecting necrotizing enterocolitis (NEC) within the first days after birth. We hypothesized that NEC development is related to a diet-dependent bacterial adherence and a subsequent proinflammatory cytokine response in the gut mucosa immediately after introduction of enteral food. Materials and Methods: Premature piglets (92% gestation) received 2 to 3 days of total parenteral nutrition followed by 0, 8, or 17 hours of enteral formula or sows colostrum feeding. Results: Following 8 hours, but not 17 hours, of colostrum feeding, a reduced number of intestinal samples with adherent bacteria (both Gram-negative and Gram-positive bacteria) was counted compared with 0 or 8 hours of formula feeding. Besides a more dense colonization, formula feeding leads to higher intestinal interleukin-1β (IL-1β) levels and more NEC-like lesions from 8 hours onward. The load of adherent bacteria was especially high in NEC lesions. Toll-like receptor 4 was detected in enteroendocrine, neuronal, and smooth muscle cells, potentially mediating the increase in IL-1β levels by Gram-negative bacteria. Conclusions: 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.

NITRIC OXIDE SYNTHASE EXPRESSION IN ENTERIC NEURONS DURING DEVELOPMENT IN THE PIG DUODENUM

Abstract 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.

Stereologic Characteristics of Pig Small Intestine During Normal Development

Stereologic methods were used to study the behavior of the pigs intestinal wall during periods that are characterized by a high incidence of gastrointestinal disorders. For this purpose conventionally stained transverse and vertical paraffin sections were made of the small intestine (duodenum, jejunum, and ileum) of fetal, neonatal, and weaned pigs. The volumes of the intestinal walls were estimated using Cavalieris method. Subsequently, the surface density (Sv) of the tunica mucosa and the volume densities (Vv) of the different small intestinal elements were estimated. Finally, the surface and volumes per serosal surface area (Ss and Vs) were calculated. The decrease of Sv can be attributed to the finding that the mucosal surface increases to a lesser extent compared with the volume of the intestinal wall. The Vs of the various layers increased postnatally, illustrating that the intestinal wall thickens. Despite an increasing total mucosal surface, this postnatal thickening causes Ss to decline. Each of these changes is temporally related to dietary changes, an increased antigen load, and an increased need for protection. Additionally, the regional differences of the various parameters match the qualitative descriptions of the small intestine of the pig and relate to region-specific functions.

Formula induces intestinal apoptosis in preterm pigs within a few hours of feeding.

BACKGROUND Nutrition regimens influence postnatal small intestinal development, which shows prominent changes after 6 hours of suckling. Such influences are particularly important in preterm neonates as inappropriate feeding responses may predispose to gastrointestinal disorders such as necrotizing enterocolitis (NEC). The authors investigated the early morphological responses to enteral feeding, prior to the time period when a large proportion of preterm pigs normally develop clinical NEC symptoms. METHODS Preterm piglets (106-107 days of gestation) were fed parenteral nutrition (PN) for 2 days with or without a subsequent 8-hour or 17-hour period of enteral nutrition (EN) with sows colostrum or formula. Another group of piglets was delivered at 108-109 days of gestation and used for comparison to PN pigs before enteral feeding. Stereological measurements of the mucosal surface density and the volume densities of the tunica mucosa, tunica muscularis, proliferative, and apoptotic cells were made and related to microscopical NEC-lesion score. In addition, villus length and crypt depth were measured. RESULTS PN-fed piglets showed minimal PN-induced mucosal atrophy, although their crypts were deeper, together with lower cell proliferation and higher apoptotic indices, than newborn (NB) unfed piglets. After PN, enteral feeding with colostrum, for just 8 hours, induced a rapid increase in the mucosal volume density while formula feeding was associated with an elevated number of both proliferating and apoptotic cells and a higher NEC lesion score than PN- or colostrum-fed pigs. CONCLUSION 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.

Postnatal and diet-dependent increases in enteric glial cells and VIP-containing neurones in preterm pigs

Abstract  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 βIII‐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.

Parenteral and enteral feeding in preterm piglets differently affects extracellular matrix proteins, enterocyte proliferation and apoptosis in the small intestine

The preterm intestine is immature and responds differently to total parenteral nutrition (TPN) and enteral nutrition, compared with the term intestine. We hypothesised that in preterms, diet composition and feeding route affect mucosal morphology, enterocyte mitosis and apoptosis, and the distribution of laminin-1, fibronectin and collagen IV (extracellular matrix proteins (ECMP)). Preterm piglets (93.5 % of gestation) were delivered via caesarean section and birth weight-matched allocated to one of the four experimental groups: the piglets were either euthanised immediately after delivery, after 3 d of TPN or after 2 d enteral feeding with colostrum or milk formula, following 3 d of TPN. We combined immunohistochemistry, image analysis and stereological measurements to describe the intestinal mucosal layer. No significant changes occurred after 3 d of TPN. Feeding colostrum or milk replacer for 2 d after TPN was associated with an increased crypt depth. Only enteral feeding with colostrum resulted in an increased villus height and mitotic index. Neither TPN nor enteral feeding changed the distribution pattern of ECMP or the occurrence of bifid crypts. The immature distribution pattern of ECMP in TPN-fed piglets, coupled with unchanged enterocyte mitosis and apoptosis indices, illustrates that feeding preterm pigs 3 d TPN does not lead to mucosal atrophy. Despite the invariable distribution of ECMP, colostrum was associated with crypt hyperplasia resulting in an increased villus height. These data illustrate that some mechanisms regulating cell turnover are immature in preterms and may in part explain the abnormal gut responses to TPN and enteral feeding in prematurely born pigs.

Enteral Feeding Reduces Endothelial Nitric Oxide Synthase in the Caudal Intestinal Microvasculature of Preterm Piglets

The initiation of enteral feeding represents a challenge to the neonatal intestinal microcirculation, especially in preterms where it predisposes to necrotizing enterocolitis (NEC). We hypothesized that a structural microvascular deficiency may occur when enteral feeding is initiated in preterm piglets susceptible to NEC. Stereologic volume densities of a pan-endothelial marker (vWF), and the main vasodilator endothelial nitric oxide synthase (eNOS), were determined along the small intestine of 1) unfed preterm piglets, 2) piglets receiving total parenteral nutrition (TPN) for 2–3 d, and 3) piglets fed 2 d sows colostrum (TPN+SOW) or milk formula (TPN+FOR) following TPN. In the mucosa, vWF-density decreased in a cranio-caudal direction. A corresponding mucosal eNOS gradient appeared only after initiating enteral feeding. In TPN+SOW, eNOS induction may lag behind the mucosal growth of the caudal region. In TPN+FOR, formula-related factors (i.e. bacteria, cytokines) may suppress mucosal eNOS, indicated by increased stress-sensitive nuclear HIF1α staining. The low mucosal endothelial eNOS density was related to the presence of NEC lesions, maybe via increased hypoxia-sensitivity, especially in the caudal region as indicated by nuclear HIF1α-staining. Our results suggest an insufficient structural adaptation of the microvasculature to enteral feeding, especially of mucosal eNOS, which may lead to NEC.

Stereologic description of the changing expression of constitutive nitric oxide synthase and heme oxygenase in the enteric plexuses of the pig small intestine during development.

The similarities between heme oxygenase‐2 (HO‐2) and nitric oxide synthase (nNOS) and the transient expression of nNOS during development led us to investigate whether both systems are similarly affected by changes that occur during development and by regional differences along the small intestine. By combining NADPH diaphorase histochemistry and HO‐2 immunohistochemistry on whole‐mount preparations and by using stereologic methods, a qualitative and quantitative description of HO‐2 and nNOS expression was obtained. Examinations were carried out on the small intestine of fetal, 1–2‐day and 5–6‐week‐old pigs. In all age groups, three enteric plexuses were distinguished. The presence of HO‐2‐immunoreactive (HO‐2‐IR) and NADPH diaphorase‐positive neurons corresponded to earlier morphological and physiological reports. Nevertheless, the total number of nitrergic neurons remained constant or decreased in the enteric plexuses, whereas the total number of HO‐2‐IR neurons displayed an overall increase. Changing concentrations of glucocorticoids, target‐derived signals, presynaptic input, and an effect of HO‐2 activity on nNOS synthesis are likely to play roles in the observed developmental changes. The numerical density of HO‐2‐IR neurons remained relatively constant along the intestinal tract; in contrast, the nitrergic neurons were most numerous in the inner submucous and myenteric plexus in the duodenum and ileum, respectively. It is believed that the duodenal nitrergic neurons in the inner submucous plexus could be involved in the regulation of duodenal secretion processes, whereas the region‐dependent density in the myenteric plexus possibly forms the morphological basis for a regionally different participation of NO in the relaxation of the small intestine. J. Comp. Neurol. 437:118–128, 2001.

Localization of neurokinin A and chromogranin A immunoreactivity in the developing porcine adrenal medulla.

SummaryThe presence of neurokinin A immunoreactivity was studied in the chromaffin cells of the porcine adrenal medulla and in the nerve fibres innervating the adrenal gland during ontogenic development. For comparison, chromogranin A immunoreactivity was used as a marker for chromaffin cells.Whereas chromogranin A was found in chromaffin cells through all steps in embryonic development, three developmental stages of neurokinin A immunoreactivity could be distinguished. In the first and second trimester of gestation, neurokinin A was observed in some groups of chromaffin cells, but no neurokinin-immunoreactive nerve fibres could be detected. In the last trimester of gestation, neurokinin A-reactive chromaffin cells and nerve fibres were both found in adrenal glands. However, in adrenal glands of neonatal piglets, neurokinin A was found only in nerve fibres and not in chromaffin cells. From these results a hypothesis is proposed that neurokinin A might act as a neurotrophic factor in the early stages of the developing porcine chromaffin cells. Biochemical studies are being performed in order to confirm these morphological results and to study the possible role of neurokinin A as a neurotrophic factor in the adrenal gland.

A stereological evaluation of secretin and gastric inhibitory peptide-containing mucosal cells of the perinatal small intestine of the pig

Stereological methods were used to quantify secretin and gastric inhibitory peptide (GIP)‐immunoreactivity (GIP‐IR) in paraffin sections of the duodenum, jejunum and ileum of fetal and neonatal piglets. In addition, sections were processed for GLP‐1‐immunohistochemistry. The volume density of the tunica mucosa increased after birth, giving rise to a decreased volume density of the tela submucosa and tunica muscularis. Generally known region‐specific morphological distinctions were reflected in differing volume densities of the various layers. The highest volume density of GIP‐IR epithelial cells was observed in the jejunum of the neonate. In contrast, the volume density of secretin‐IR epithelial cells was highest in the duodenum of both fetal and neonatal piglets. The volume occupied by GIP‐IR and secretin‐IR epithelial cells increased in the jejunum after birth. Additionally, ileal secretin‐IR epithelial cells were more numerous in the neonatal piglet. In conclusion, the quantitative and qualitative presence of GIP‐IR and secretin‐IR epithelial cells agree with earlier reports of their presence and co‐localization between GIP‐IR and GLP‐1‐IR, in the pig small intestine. Furthermore, the differences suggest that age‐ and region‐related functional demands are temporally and probably causally related with the morphological diversification of the intestine and its endocrine cells.