Structural brain maturation differs between preterm and term piglets, whereas brain activity does not.

AIM: The aim of the study was to investigate whether amplitude-integrated electroencephalography (aEEG) and cerebral magnetic resonance imaging (MRI) in preterm piglets would provide measures of cerebral functional, microstructural and anatomical maturation, which might reflect the signs of functional brain immaturity, documented in preterm piglets. METHODS: During July-October 2013 at the NEOMUNE Centre, Copenhagen University, Denmark, 31 preterm (90% gestation) and 10 term piglets underwent aEEG on days 1, 2, 4 and 11, and MRI on day 25. Physical activity levels were recorded. RESULTS: Preterm showed delayed neonatal arousal and physical activity, relative to term piglets. Preterm piglets had lower growth rates and brain volume than term piglets, but aEEG patterns were similar. MRI mean diffusivity was also similar, but fractional anisotropy (FA) was lower in preterm piglets (p < 0.001). CONCLUSION: Functional brain maturation, as assessed by aEEG, was relatively advanced in preterm piglets. Conversely, the low FA in the preterm piglets suggests that the white matter microstructure remains less mature in preterm compared to term piglets at postnatal day 25. The results might be utilised to define whether and how preterm piglets may contribute to preclinical models for brain development in preterm infants.

Intrauterine growth-restricted piglets have similar gastric emptying rates but lower rectal temperatures and altered blood values when compared with normal-weight piglets at birth.

Intrauterine growth-restricted (IUGR) piglets have lower survival rates and are more likely to have empty stomachs 24 h after birth than normal piglets. Although hypoglycemia may result from low colostrum intake per se, it is not known if slow gastric emptying may be an additional risk factor for poor immunization and glucose absorption in IUGR piglets. It is estimated that IUGR piglets consume less colostrum per kilogram BW than normal-weight piglets within the first 24 h, which could be due to a slower gastric emptying rate and a compromised energy metabolism. Therefore, we hypothesized that the gastric emptying rate and blood glucose would be lower in IUGR piglets. We investigated gastric emptying rates in normal and IUGR piglets and blood glucose and rectal temperatures at birth and after 15, 30, 60, and 120 min. In addition, blood parameters relevant for metabolism were studied. Forty-eight piglets (24 normal and 24 IUGR) were classified at birth as either normal or IUGR on the basis of head morphology. Piglets were removed from the sow at birth before suckling, and birth weight was recorded. Pooled porcine colostrum was tube-fed to all piglets at 12 mL/kg BW as soon as possible after birth (t = 0 min). The piglets were randomly allocated to be euthanized at 15, 30, 60, and 120 min (all groups, = 6) after bolus feeding, and the weights of the stomach and its residuals were recorded. There was no difference in gastric emptying rates between normal and IUGR piglets ( = 0.129); however, gastric DM residuals tended to by greater in IUGR piglets than normal piglets ( = 0.085). Overall, IUGR piglets had lower rectal temperatures (36.2°C ± 0.2°C vs. 37.5°C ± 0.2°C; < 0.001) and plasma glucose levels (2.8 ± 0.2 vs. 4.1 ± 0.2 mmol; < 0.001) than normal piglets. Interactions between piglet classification and time were observed in plasma values for NEFA, -3-hydroxybutyrate, albumin, aspartate, and alanine amino transferase, with greater levels in normal piglets at 15 min ( < 0.05) and 30 min for bile acid ( < 0.05) compared to IUGR piglets. In conclusion, the gastric emptying rates between normal and IUGR piglets were similar, but gastric DM residuals tended to be greater in IUGR piglets. Differences were observed in blood values and rectal temperatures, with lower values in IUGR piglets. Therefore, it is likely that factors like hypothermia and possibly reduced metabolic function are more important during the first hours after birth than gastric retention per se.

Endocrine regulation of gut maturation in early life in pigs.

After birth, the newborn must adapt to the acute challenges of circulatory changes, active respiration, thermoregulation, microbial colonization, and enteral nutrition. Whereas these processes normally occur without clinical complications in neonates born at term, birth at a preterm state of gestation is associated with high morbidity and mortality. In commercial pig production, perinatal mortality is higher than in any other mammalian species. Asphyxia, hypothermia, hypoglycemia, sepsis, and gut dysmotility, represent some of the most common findings. The intestine is a particularly sensitive organ after birth, as it must adapt acutely to enteral nutrition and microbial colonization. Likewise, during the weaning phase, the intestine must adapt to new diet types. Both critical phases are associated with high morbidity. This review focuses on the endocrine changes occurring around birth and weaning. There are a number of endocrine adaptations in late gestation and early postnatal life that are under influence of development stage and environmental factors such as diet. The review discusses general endocrine changes in perinatal life but specifically focuses on the role of glucagon-like peptide-2. This gut-derived hormone plays a key role in development and function of the intestine in early life.

Glucagon-like peptide 2 treatment may improve intestinal adaptation during weaning.

Transition from sow's milk to solid feed is associated with intestinal atrophy and diarrhea. We hypothesized that the intestinotrophic hormone glucagon-like peptide 2 (GLP-2) would induce a dose- and health status-dependent effect on gut adaptation. In Exp. 1, weaned pigs (average BW at weaning 4.98 ± 0.18 kg) were kept in a high-sanitary environment and injected with saline or short-acting GLP-2 (80 µg/(kg BW·12 h); n = 8). Under these conditions, there was no diarrhea and GLP-2 did not improve gastrointestinal structure or function. In Exp. 2, weaned pigs (average BW at weaning 6.68 ± 0.27 kg) were kept in a low-sanitary environment, leading to weaning diarrhea, and injected with saline or short-acting GLP-2 (200 µg/(kg BW·12 h); n = 11). Treatment with GLP-2 increased goblet cell density (P < 0.05) and reduced short chain fatty acid concentration in the colon (P < 0.01) but had limited effects on diarrhea. In Exp. 3, weaned pigs (average BW at weaning 6.90 ± 0.32 kg) were kept in a low-sanitary environment and injected with saline or a long-acting acylated GLP-2 analogue (25 µg/(kg BW·12 h); n = 8). In this experiment, GLP-2 increased intestinal weight (+22%; P < 0.01) and activity of brush border enzymes (+50-100%; P < 0.05). Circulating GLP-2 levels were in the pharmacological range in Exp. 3 (constant levels >20,000 pmol/L) and Exp. 2 (increases to 20,000 pmol/L for a few hours each day) while they were in the supraphysiological range in Exp. 1 (50-200 pmol/L). In conclusion, GLP-2 may improve gut structure and function in weanling pigs. However, the effects may be significant only under conditions of diarrhea and if GLP-2 exposure time is extended using long-acting analogues.

Invited review: the preterm pig as a model in pediatric gastroenterology.

At birth, the newborn mammal undergoes a transition from a sterile uterine environment with a constant nutrient supply, to a microbe-rich environment with intermittent oral intake of complex milk nutrients via the gastrointestinal tract (GIT). These functional challenges partly explain the relatively high morbidity and mortality of neonates. Preterm birth interrupts prenatal organ maturation, including that of the GIT, and increases disease risk. Exemplary is necrotizing enterocolitis (NEC), which is associated closely with GIT immaturity, enteral feeding, and bacterial colonization. Infants with NEC may require resection of the necrotic parts of the intestine, leading to short bowel syndrome (SBS), characterized by reduced digestive capacity, fluid loss, and dependency on parenteral nutrition. This review presents the preterm pig as a translational model in pediatric gastroenterology that has provided new insights into important pediatric diseases such as NEC and SBS. We describe protocols for delivery, care, and handling of preterm pigs, and show how the immature GIT responds to delivery method and different nutritional and therapeutic interventions. The preterm pig may also provide a sensitive model for postnatal adaptation of weak term piglets showing high mortality. Attributes of the preterm pig model include close similarities with preterm infants in body size, organ development, and many clinical features, thereby providing a translational advantage relative to rodent models of GIT immaturity. On the other hand, the need for a sow surgical facility, a piglet intensive care unit, and clinically trained personnel may limit widespread use of preterm pigs. Studies on organ adaptation in preterm pigs help to identify the physiological basis of neonatal survival for hypersensitive newborns and aid in defining the optimal diet and rearing conditions during the critical neonatal period.

Casein addition to a whey-based formula has limited effects on gut function in preterm pigs.

Preterm infants are susceptible to necrotizing enterocolitis (NEC). Using preterm pigs, we determined whether a whey-casein-based formula would be superior to a formula based on whey protein alone. Twenty cesarean-derived preterm pigs (92% gestation) were given total parenteral nutrition for 36 h followed by 30 h of enteral feeding with whey [protein fraction of milk formula based on whey (WHEY); n = 11] or casein and/or whey [protein fraction of milk formula based on a combination of casein and whey (CASEIN); n = 9]-based formulas. Sugar absorptive function was investigated at 6 and 30 h after initiation of enteral feeding using bolus feedings with galactose and mannitol. Pigs were killed after the last in vivo sugar absorption test and evaluated for NEC and the mid intestine was used for ex vivo measurements of hexose absorption. Microbiota profile and short chain fatty acid (SCFA) levels were studied in gut contents. Severity of NEC lesions was similar between diet groups but galactose absorption was markedly higher in CASEIN than in WHEY (P < 0.01) although only 6 h after the start of the enteral feeding period. There were no differences in ex vivo (14)C-D-glucose uptake, digestive enzymes, microbiota profile, or SCFA concentration. Casein may transiently stimulate intestinal sugar absorption but has limited effects on gut structure, microbiota, and NEC in preterm pigs.

Dietary long-chain n-3 PUFA, gut microbiota and fat mass in early postnatal piglet development--exploring a potential interplay.

Dietary n-3PUFA and gut bacteria, particularly Bacteroidetes, have been suggested to be related to adiposity. We investigated if n-3PUFA affected fat storage and cecal bacteria in piglets. Twenty-four 4-day-old piglets were allocated to formula rich in n-3PUFA (∼3E%) from fish oil (FO) or n-6PUFA from sunflower oil (SO) for 14 days. We assessed body weight, fat accumulation by dual-energy X-ray absorptiometry and microbial molecular fingerprints. Dietary PUFA-composition was reflected in higher erythrocyte n-3PUFA in the FO- than the SO-group (P<0.001). Principal component analysis revealed group differences in the overall microbiotic composition, which involved a larger Bacteroides community in the SO-group (P=0.02). There was no significant difference in body fat percentage and no relationship between fat accumulation and gut Bacteroides. Hence, this study does not support an impact of n-3PUFA or microbiota on fat accumulation during the postnatal maturation period. The impact of dietary PUFA on the gut Bacteroides warrants further investigation.

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

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.

Elective cesarean delivery affects gut maturation and delays microbial colonization but does not increase necrotizing enterocolitis in preterm pigs.

Although preterm birth and formula feeding increase the risk of necrotizing enterocolitis (NEC), the influences of cesarean section (CS) and vaginal delivery (VD) are unknown. Therefore, gut characteristics and NEC incidence and severity were evaluated in preterm pigs (92% gestation) delivered by CS or VD. An initial study showed that newborn CS pigs (n = 6) had decreased gastric acid secretion, absorption of intact proteins, activity of brush-border enzymes and pancreatic hydrolases, plasma cortisol, rectal temperature, and changes in blood chemistry, indicating impaired respiratory function, compared with VD littermates (n = 6). In a second experiment, preterm CS (n = 16) and VD (n = 16) pigs were given total parenteral nutrition (36 h) then fed porcine colostrum (VD-COL, n = 6; CS-COL, n = 6) or infant milk formula (VD-FORM, n = 10; CS-FORM, n = 10) for 2 days. Across delivery, FORM pigs showed significantly higher NEC incidence, tissue proinflammatory cytokines (IFN-gamma and IL-6), Clostridium colonization, and impaired intestinal function, compared with COL pigs. NEC incidence was equal for CS (6/16) and VD (6/16) pigs, CS pigs had decreased bacterial diversity and density, higher villus heights, and increased brush-border enzyme activities (lactase, aminopeptidases) compared with VD pigs. In particular, VD-FORM pigs showed reduced mucosal proportions, reduced lactase and aminopeptidases, and increased proinflammatory cytokine IL-6 compared with CS-FORM (P < 0.06). Despite the initial improvement of intestinal and metabolic functions following VD, gut function, and inflammation were similar, or more negatively affected in VD neonates than CS neonates. Both delivery modes exhibited positive and negative influences on the preterm gut, which may explain the similar NEC incidence.

The intestinal trophic response to enteral food is reduced in parenterally fed preterm pigs and is associated with more nitrergic neurons.

In term neonates, total parenteral nutrition (TPN) induces mucosal atrophy, whereas the first intake of milk is followed by intestinal growth. This may be explained in part by an NO-mediated increased blood flow. We hypothesized that the immature gut has an altered response to TPN and enteral nutrition. In Expt. 1, preterm caesarean-delivered pigs were administered elemental nutrients for 3 d, infused parenterally (TPN, n = 7) or enterally (TENT, n = 7). In Expt. 2, preterm pigs were fed sow's colostrum, cow's colostrum, or infant formula for 2 d after a 3-d TPN period (TPN-SOW, TPN-COW, TPN-FORM, n = 8-11). Intestinal morphology and the number of enteric neurons containing nitric oxide synthase-1 (NOS-1) were quantified. Both the TPN and TENT groups had increases in intestinal mass, circumference, and mucosal mass, volume, and surface density, relative to values at birth (+30-50%, P < 0.05). In Expt. 2, the magnitudes of the intestinal trophic responses to feeding were similar to those in Expt. 1, but were also associated with an increased number of nitrergic myenteric neurons and some mucosal damage, most frequently observed for the formula group. We conclude that 1) a short period of TPN does not induce mucosal atrophy in preterm pigs, whereas elemental nutrients infused luminally do not mimic the trophic response seen with milk diets, 2) enteral feeding of preterm pigs after a short period of TPN is associated with a modest, diet-dependent trophic response that may be related in part to the actions of an increased population of enteric NOS-1 neurons.