Diet- and Colonization-Dependent Intestinal Dysfunction Predisposes to Necrotizing Enterocolitis in Preterm Pigs
2006; Elsevier BV; Volume: 130; Issue: 6 Linguagem: Inglês
10.1053/j.gastro.2006.02.026
ISSN1528-0012
AutoresPer Torp Sangild, Richard H. Siggers, M. Schmidt, Jan Elnif, Charlotte Reinhard Bjørnvad, Thomas Thymann, Marie Louise Grøndahl, Axel Kornerup Hansen, Soeren K. Jensen, Mette Boyé, Lars Moelbak, Randal K. Buddington, Björn Weström, Jens J. Holst, Douglas G. Burrin,
Tópico(s)Clinical Nutrition and Gastroenterology
ResumoBackground & Aims: Preterm birth and formula feeding are key risk factors associated with necrotizing enterocolitis (NEC) in infants, but little is known about intestinal conditions that predispose to disease. Thus, structural, functional, and microbiologic indices were used to investigate the etiology of spontaneous NEC development in preterm pigs. Methods: Piglets were delivered by cesarean section at 92% gestation, reared in infant incubators, and fed infant formula or colostrum every 3 hours (n = 120) until tissue collection at 1–2 days of age. Results: Clinical and histopathologic signs of NEC were observed in 57% of pigs fed FORMULA (26/46) and in 5% of pigs fed COLOSTRUM (2/38) (P < .05). Relative to COLOSTRUM, both healthy and sick FORMULA pigs had reduced intestinal villous heights, enzyme activities, nutrient absorption, and antioxidant levels and higher inducible nitric oxide synthetase activity (P < .05). In healthy pigs, mucosal microbial diversity remained low and diet independent. NEC pigs showed bacterial overgrowth, and a high mucosal density of Clostridium perfringens was detected in some but not all pigs. Germ-free conditions and antiserum against Clostridium perfringens toxin prevented intestinal dysfunction and NEC in formula-fed pigs, whereas the gut trophic factors, epidermal growth factor, and glucagon-like peptide 2 had limited effects. Conclusions: A subclinical, formula-induced mucosal atrophy and dysfunction predispose to NEC and bacterial overgrowth. The adverse feeding effects are colonization dependent and may be reduced by factors in colostrum that include antibodies against aggressive toxins such as those of Clostridium perfringens. Background & Aims: Preterm birth and formula feeding are key risk factors associated with necrotizing enterocolitis (NEC) in infants, but little is known about intestinal conditions that predispose to disease. Thus, structural, functional, and microbiologic indices were used to investigate the etiology of spontaneous NEC development in preterm pigs. Methods: Piglets were delivered by cesarean section at 92% gestation, reared in infant incubators, and fed infant formula or colostrum every 3 hours (n = 120) until tissue collection at 1–2 days of age. Results: Clinical and histopathologic signs of NEC were observed in 57% of pigs fed FORMULA (26/46) and in 5% of pigs fed COLOSTRUM (2/38) (P < .05). Relative to COLOSTRUM, both healthy and sick FORMULA pigs had reduced intestinal villous heights, enzyme activities, nutrient absorption, and antioxidant levels and higher inducible nitric oxide synthetase activity (P < .05). In healthy pigs, mucosal microbial diversity remained low and diet independent. NEC pigs showed bacterial overgrowth, and a high mucosal density of Clostridium perfringens was detected in some but not all pigs. Germ-free conditions and antiserum against Clostridium perfringens toxin prevented intestinal dysfunction and NEC in formula-fed pigs, whereas the gut trophic factors, epidermal growth factor, and glucagon-like peptide 2 had limited effects. Conclusions: A subclinical, formula-induced mucosal atrophy and dysfunction predispose to NEC and bacterial overgrowth. The adverse feeding effects are colonization dependent and may be reduced by factors in colostrum that include antibodies against aggressive toxins such as those of Clostridium perfringens. Infants delivered preterm have an immature gastrointestinal tract (GIT) with underdeveloped capacity to digest and absorb carbohydrates and other nutrients. When the nutrient load exceeds the digestive capacity, bacterial overgrowth, and fermentation may occur and predispose to the most serious GIT disease in neonatal wards, necrotizing enterocolitis (NEC).1Shulman R.J. Schandler R.J. Lau C. Heitkemper M. Ou C.N. Smith E.O. Early feeding, feeding tolerance and lactase activity in preterm infants.J Pediatr. 1998; 133: 645-649Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 2Berseth C.L. Bisquera J.A. Paje V.U. Prolonging small feeding volumes early in life decreases the incidence of necrotizing enterocolitis in very low birth weight infants.Pediatrics. 2003; 111: 529-534Crossref PubMed Scopus (286) Google Scholar, 3Caicedo R.A. Schanler R.J. Li N. Neu J. The developing intestinal ecosystem implications for the neonate.Pediatr Res. 2005; 58: 625-628Crossref PubMed Scopus (109) Google Scholar Feeding formula is associated with a higher incidence of NEC than mother’s milk, but it is not known how the absence of natural milk, combined with the consequences of preterm birth (hypoxia, hyperthermia, GIT immaturity), may predispose to NEC. The symptoms can be induced in newborn rodents and pigs by installation of acidified diets into the GIT lumen, coupled with hypothermia, and/or hypoxia exposure.4Caplan M.S. Jilling T. The role of polyunsaturated fatty acid supplementation in intestinal inflammation and neonatal necrotizing enterocolitis.Lipids. 2001; 36: 1053-1057Crossref PubMed Scopus (41) Google Scholar, 5Di Lorenzo M. Krantis A. Altered nitric oxide production in the premature gut may increase susceptibility to intestinal damage in necrotizing enterocolitis.J Pediatr Surg. 2001; 36: 700-705Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar Such acute disease models have provided important information about some pathologic mechanisms, but they do not provide insights into intestinal physiology prior to spontaneous disease outbreak. NEC is a disease that typically starts with feeding intolerance, reduced bowel motility, and moderate fecal blood loss. As NEC progresses, there is intestinal dilation and immotility and gas accumulation in the intestinal lymph vessels and portal vein (pneumatosis intestinalis). Finally, peritonitis and transmural necrosis may develop, and the affected bowel segment becomes perforated (Bell stages I, II, and III).6Walsh M.C. Kliegman R.M. Necrotizing enterocolitis treatment based on staging criteria.Pediatr Clin North Am. 1986; 33: 179-201Crossref PubMed Scopus (1306) Google Scholar, 7Bell M.J. Ternberg J.L. Feigin R.D. Keating J.P. Marshall R. Barton L. Brotherton T. Neonatal necrotizing enterocolitis. Therapeutic decisions based upon clinical staging.Ann Surg. 1978; 187: 1-7Crossref PubMed Scopus (2611) Google Scholar Although the ischemic affliction is usually restricted to the ileum and proximal colon, necrosis is sometimes found throughout the GIT, including the stomach.6Walsh M.C. Kliegman R.M. Necrotizing enterocolitis treatment based on staging criteria.Pediatr Clin North Am. 1986; 33: 179-201Crossref PubMed Scopus (1306) Google Scholar, 8Travadi J.N. Patole S.K. Simmer K. Gastric pneumatosis in neonates revisited.Paediatr Child Health. 2003; 39: 560-562Crossref PubMed Scopus (33) Google Scholar The incidence and severity of NEC are highly variable, and subclinical intestinal dysfunctions may be present in children who appear clinically healthy. Inappropriate immunologic responses of premature enterocytes to bacterial colonization have been implicated in the development of NEC.9Nanthakumar N.N. Fusunyan R.D. Sanderson I. Walker W.A. Inflammation in the developing human intestine a possible pathophysiologic contribution to necrotizing enterocolitis.Proc Natl Acad Sci U S A. 2000; 97: 6043-6048Crossref PubMed Scopus (382) Google Scholar, 10Claud E.C. Walker W.A. Hypothesis inappropriate colonization of the premature intestine can cause neonatal necrotizing enterocolitis.FASEB J. 2001; 15: 1398-1403Crossref PubMed Scopus (340) Google Scholar Although the Clostridium species have been frequently associated with NEC,11De La Cochetiere M.F. Piloquet H. Des Robert C. Darmaun D. Galmiche J.P. Roze J.C. Early intestinal bacterial colonization and necrotizing enterocolitis in premature infants the putative role of clostridium.Pediatr Res. 2004; 56: 366-370Crossref PubMed Scopus (185) Google Scholar, 12Waligora-Dupriet A.J. Dugay A. Auzeil N. Huerre M. Butel M.J. Evidence for clostridial implication in necrotizing enterocolitis through bacterial fermentation in a gnotobiotic quail model.Pediatr Res. 2005; 58: 629-635Crossref PubMed Scopus (75) Google Scholar no single pathogen is causative, and the ability of the microflora to colonize the epithelium and to ferment unabsorbed nutrients may be more important than the strain itself. Typically, NEC patients have higher circulating levels of proinflammatory cytokines (eg, tumor necrosis factor α [TNF-α], interleukin [IL]-1β, IL-6) but not until the inflammatory responses have resulted in severe necrosis.13Morecroft J.A. Spitz L. Hamilton P.A. Holmes S.J. Plasma interleukin-6 and tumour necrosis factor levels as predictors of disease severity and outcome in necrotizing enterocolitis.J Pediatr Surg. 1994; 29: 798-800Abstract Full Text PDF PubMed Scopus (36) Google Scholar, 14Edelson M.B. Bagwell C.E. Rozycki H.J. Circulating pro- and counterinflammatory cytokine levels and severity in necrotizing enterocolitis.Pediatrics. 1999; 103: 766-771Crossref PubMed Scopus (209) Google Scholar Thus, the surge in plasma inflammatory mediators appears to be a relatively late phenomenon during disease progression. Poorly coordinated production of nitric oxide (NO) and NO synthetase (NOS) isoforms during the early phases of disease could result in the altered intestinal blood flow and ischemic damage typically seen in NEC.5Di Lorenzo M. Krantis A. Altered nitric oxide production in the premature gut may increase susceptibility to intestinal damage in necrotizing enterocolitis.J Pediatr Surg. 2001; 36: 700-705Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar Collectively, these findings suggest that the early phases of NEC include a combination of inadequate digestion and nutrient fermentation, localized ischemia, inappropriate immune responses induced by resident bacteria, and decreased mucosal protection (eg, antioxidants15Finer N.N. Peters K.L. Hayek Z. Merkel C.L. Vitamin E and necrotizing enterocolitis.Pediatrics. 1984; 73: 387-393PubMed Google Scholar). However, neither of the proposed functional GIT deficits has been demonstrated in a mammalian animal model that incorporates prematurity, diet, bacterial colonization, and spontaneous outbreak of disease. We hypothesized that NEC would develop spontaneously in a significant proportion of preterm, FORMULA pigs and that FORMULA-induced deficits in GIT function would be colonization-dependent and present in clinically healthy tissue prior to pathologic manifestations of NEC. Hence, the first objective (experiment 1) was to document spontaneous development of NEC in FORMULA pigs and to identify functional deficits in nonnecrotic tissues from FORMULA pigs. The second objective was to test whether a FORMULA-induced mucosal atrophy and dysfunction in preterm pigs would be colonization-dependent, using gnotobiotic experimental conditions (experiment 2). Finally, a third objective was to test the potential protective effects of providing antiserum against some common pathogens in neonatal piglets (Clostridium perfringens, Escherichia coli)16Feng J. El-Assal O.N. Besner G.E. Heparin-binding EGF-like growth factor (HB-EGF) and necrotizing enterocolitis.Semin Pediatr Surg. 2005; 14: 167-174Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar or administration of 2 gut trophic factors, epidermal growth factor (EGF) or glucagon-like peptide 2 (GLP-2) (experiment 3). EGF was chosen because of its presence in maternal milk, the expression of EGF receptors by the preterm GIT, and the reported potential for EGF to reduce NEC incidence.17Dvorak B. Epidermal growth factor and necrotizing enterocolitis.Clin Perinatol. 2004; 31: 183-192Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar The choice of GLP-2 was based on its trophic and functional effects on the immature mucosa18Petersen Y.M. Schmidt M. Elnif J. Sangild P.T. Glucagon-like peptide 2 enhances maltase-glucoamylase and sucrase-isomaltase gene expression and activity in parenterally-fed premature neonatal pigs.Pediatr Res. 2002; 52: 498-503Crossref PubMed Scopus (63) Google Scholar and the stimulation of intestinal blood flow and metabolism in newborn pigs.19Guan X. Stoll B. Lu X. Tappenden K.A. Holst J.J. Hartmann B. Burrin D.G. GLP-2-mediated up-regulation of intestinal blood flow and glucose uptake is nitric oxide-dependent in TPN-fed piglets.Gastroenterology. 2003; 125: 136-147Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar Forty-five pigs (Danish Landrace × Large White) from 6 sows were delivered by cesarean section at 105–108 days gestation (term = 115 days), as described in detail previously.20Sangild P.T. Petersen Y.M. Schmidt M. Elnif J. Petersen T.K. Buddington R.K. Michaelsen K.F. Greisen G. Burrin D.G. Preterm birth affects the gastrointestinal responses to parenteral and enteral nutrition in the newborn pig.J Nutr. 2002; 132: 2673-2681PubMed Scopus (66) Google Scholar Pigs from each litter were randomly allocated to receive either milk replacer (FORMULA, n = 14), sow’s colostrum (COLOSTRUM, n = 13), or were killed (sodium pentobarbitone, 60 mg kg-1, intravenously, [IV]) immediately after birth (NEWBORN, n = 18). Pigs assigned to the feeding groups were transferred to heated infant incubators within 5 minutes of birth and supplied with extra oxygen over the first 12 hours of life until arterial blood oxygenation exceeded 95%. Within 4 hours of delivery, each pig was fitted with a vascular catheter (infant feeding tube, 4F; Portex, Kent, United Kingdom) inserted into the dorsal aorta via the transected umbilical cord and prepared with an orogastric feeding tube (6F, Portex).20Sangild P.T. Petersen Y.M. Schmidt M. Elnif J. Petersen T.K. Buddington R.K. Michaelsen K.F. Greisen G. Burrin D.G. Preterm birth affects the gastrointestinal responses to parenteral and enteral nutrition in the newborn pig.J Nutr. 2002; 132: 2673-2681PubMed Scopus (66) Google Scholar The pigs were fed COLOSTRUM or FORMULA (15 mL/kg) every 3 hours after placement of the catheters. The COLOSTRUM group received passive immunologic protection via absorption of colostral immunoglobulins. The FORMULA pigs were immunized systemically by arterial administration of serum taken from their own mothers that had received no vaccination regime (15 mL/kg over the first 12 hours). This provides adequate immunologic protection of preterm, COLOSTRUM-deprived newborn pigs.20Sangild P.T. Petersen Y.M. Schmidt M. Elnif J. Petersen T.K. Buddington R.K. Michaelsen K.F. Greisen G. Burrin D.G. Preterm birth affects the gastrointestinal responses to parenteral and enteral nutrition in the newborn pig.J Nutr. 2002; 132: 2673-2681PubMed Scopus (66) Google Scholar Before tissue collection at 20–30 hours after birth (mean age, 24 ± 2 hours), an arterial blood sample was taken, and the pigs were killed with an arterial injection of pentobarbitone (60 mg/kg, IV). Blood was analyzed immediately (blood chemistry values), or the plasma was separated and stored (−20°C) for later biochemical measurements. Tissue samples were collected (see below, Tissue Collection and Blood Chemistry section) and stored fixed in formaldehyde or frozen in liquid nitrogen for later structural, biochemical, and microbiologic analyses. A total of 35 cesarean-delivered pigs from 3 litters (Danish Landrace × Large White, 106 days gestation) were randomly allocated to 3 treatment groups: COLOSTRUM (COLOSTRUM, n = 11), FORMULA (FORMULA, n = 12), and FORMULA pigs delivered and reared sterile (FORMULA/GNOTO, n = 12). Sterile delivery and transfer via an iodine bath into a gnotobiotic incubator followed standard procedures. Feeding regimen and tissue collection procedures at 24–40 hours were as described for experiment 1, except that samples of nasal fluid and colon contents were taken prior to tissue collection, for anaerobic and aerobic microbiology. Analyses of tissues from experiment 2 focused on the clinical and histopathologic signs of NEC in the 3 treatment groups combined with some selected structural and functional analyses (villous morphology, enzyme activities). A total of 58 cesarean-delivered pigs from 5 litters (Danish Landrace × Large White, 105–108 days gestation) were allocated to 5 treatment groups. Four of the groups received either sow’s COLOSTRUM (COLOSTRUM, n = 14), formula (FORMULA, n = 20), FORMULA + EGF (human EGF20–31, Bachem H-6175, 20 μg/kg, given intraperitoneally [IP] every 6 hours, n = 9, FORMULA/EGF), or FORMULA + GLP-2 (human GLP-21–33, NOVO Nordisk, Bagsværd, Denmark, 50 μg/kg, given IP every 6 hours, n = 9, FORMULA/GLP-2). The dose of EGF was chosen based on a dose that has previously been shown to induce marked mucosal maturation in newborn pigs.21James P.S. Smith M.W. Tivey D.R. Wilson T.J. Epidermal growth factor selectively increases maltase and sucrase activities in neonatal piglet intestine.J Physiol. 1987; 393: 583-594PubMed Google Scholar The GLP-2 dose was chosen based on the observation that a similar dose administered over several days to preterm parenterally fed pigs induced marked intestinal mucosal growth.22Petersen Y.M. Hartmann B. Holst J.J. Le Huerou-Luron I. Bjornvad C.R. Sangild P.T. Introduction of enteral food increases plasma GLP-2 and decreases GLP-2 receptor mRNA abundance during pig development.J Nutr. 2003; 133: 1781-1786PubMed Scopus (61) Google Scholar While the FORMULA, FORMULA/EGF, and FORMULA/GLP-2 groups received normal sow’s plasma for passive immunization, the remaining FORMULA/ANTI group received plasma from a sow immunized with a commercially available vaccine (Toxicol; Schering-Plough, Farum, Denmark) directed against the α, β, and ϵ toxins of Clostridium perfringens (CP) and Escherichia coli F4ab, F4ac, F5, and F6 antigens (15 mL/kg, IV, within 12 hours postpartum, n = 6). Vaccination of pregnant sows with this product normally provides full protection of newborn piglets against necrotizing enteritis related to CP α, β, and ϵ toxins via their uptake of antibodies from colostrum ingestion.23Springer S. Selbitz H.J. The control of necrotic enteritis in sucking piglets by means of a Clostridium perfringens toxoid vaccine.FEMS Immunol Med Microbiol. 1999; 24: 333-336Crossref PubMed Google Scholar The circulating antibody titer for CP β toxin (international units [IU] per mL) was measured in plasma by standard ELISA techniques using purified CP β toxin obtained from pure cultures of CP type C.23Springer S. Selbitz H.J. The control of necrotic enteritis in sucking piglets by means of a Clostridium perfringens toxoid vaccine.FEMS Immunol Med Microbiol. 1999; 24: 333-336Crossref PubMed Google Scholar The sow’s serum was provided to FORMULA piglets systemically because FORMULA is known to facilitate poor uptake of intestinal immunoglobulins.24Jensen A.R. Elnif J. Burrin D.G. Sangild P.T. Development of intestinal immunoglobulin absorption and enzyme activity in neonatal pigs is diet-dependent.J Nutr. 2001; 131: 3259-3265PubMed Scopus (129) Google Scholar Delivery of pigs, feeding, and sample collection procedures were identical to those in experiment 1, except that all the pigs were reared for a slightly longer period (40 ± 4 hours). Analyses of tissues from experiment 3 focused on the clinical and histopathologic signs of NEC in the 5 treatment groups combined with some selected structural and functional analyses (villous morphology, enzyme activities). Finally, distal small intestine samples were used for molecular microbiology analyses (terminal restriction fragment length polymorphism [T-RFLP] and fluorescent in situ hybridization). Colostrum was pooled from different sows that were milked within 6 hours of parturition and was kept frozen until use and contained 6800 kJ/L, 146 g protein per L, and other nutrients as described previously.24Jensen A.R. Elnif J. Burrin D.G. Sangild P.T. Development of intestinal immunoglobulin absorption and enzyme activity in neonatal pigs is diet-dependent.J Nutr. 2001; 131: 3259-3265PubMed Scopus (129) Google Scholar All COLOSTRUM was collected from sows that had been vaccinated prior to delivery (90 days gestation) with the product mentioned above (Toxicol). The milk formula was prepared by combining 3 commercial products (SHS International, Liverpool, UK) used for feeding hospitalized infants at 0–2 years of age (Table 1). The principal source of carbohydrate was hydrolyzed corn syrup (maltodextrins, Pepdite), the fat was largely obtained from coconut oil (MCT Liquigen), and the protein was provided by a mixture of low molecular proteins from cow’s milk whey and vegetables (Pepdite + Maxipro). The formula was mixed to have energy and protein levels comparable with sow’s milk.24Jensen A.R. Elnif J. Burrin D.G. Sangild P.T. Development of intestinal immunoglobulin absorption and enzyme activity in neonatal pigs is diet-dependent.J Nutr. 2001; 131: 3259-3265PubMed Scopus (129) Google Scholar, 25Sangild P.T. Xu R.J. Colostrum.in: Pond W.G. Bell A.W. Encyclopedia of animal science. Marcel Dekker, New York2004: 229-231Google Scholar The osmolality of the formula diet was 182 mOsm/L, compared with 344 mOsm/L for colostrum.Table 1The Nutrient Composition of Formula (per L formula)Macronutrients and vitaminsMinerals (mg)Amino acids (g)Energy (kJ)4151Na345Ala3.1Protein (g)63.9K728Arg1.9Carbohydrate (g)46.9Cl245Asp6.0Total sugars (g)9.0Ca513Cys1.9Lactose (g)5.2P392Glu1.4Fat (g)61.3Mg66Gln8.5Saturated fat (g)44.3Fe5.6Gly2.1Monounsaturated fat (g)9.7Cu304His1.3Polyunsaturated fat (g)3.7Zn4.0Ile3.9Vitamin A (μg)422Mn0.30Leu6.2Vitamin D (μg)6.8I0.038Lys5.2Vitamin E (mg)2.6Mb0.011Met1.7Vitamin C (mg)32Se0.009Phe3.0Vitamin K (μg)17Cr0.008Pro3.8Vitamin B1 (mg)0.3Ser2.8Vitamin B2 (mg)0.5Thr4.0Vitamin B6 (mg)0.4Trp1.5Vitamin B12 (μg)1.0Tyr2.7Folacin (μg)30Val4.1Biotin (μg)21Tau0.03NOTE. Data are calculated from prescribed contents in the commercially available products (80 g Peptide, 70 g Super Soluble Maxipro, 75 mL MCT Liquigen per 1000 mL formula, all products kindly donated by SHS International, Liverpool, UK). Open table in a new tab NOTE. Data are calculated from prescribed contents in the commercially available products (80 g Peptide, 70 g Super Soluble Maxipro, 75 mL MCT Liquigen per 1000 mL formula, all products kindly donated by SHS International, Liverpool, UK). The pigs were observed at least every 3 hours, with the onset of NEC based on lethargy, abdominal distention, and bloody diarrhea. Each animal was subsequently evaluated using a combination of the clinical symptoms just before death, and the magnitude of GIT lesions from macroscopic and histologic examination of tissues from 4 regions: stomach, proximal and distal small intestine, and proximal colon. Pigs without clinical signs of discomfort and feeding intolerance, and lacking abdominal distention or macroscopic hemorrhage, edema, or bleeding of the gastrointestinal mucosa, and with no mucosal abnormality in any of the 4 regions, were assigned a grade 1. Grades 2–6 were assigned to pigs exhibiting feeding intolerance, runny stool with or without blood, abdominal distention with or without intestinal pneumatosis. The specific score was determined by the average degree of mucosal alteration and severity of lesions in the 4 GIT regions. Specifically, when 20% or more of the mucosa in a particular region was hyperemic with clearly stunted villi, it was assigned a grade of 2. Regions graded as 3 and 4 were characterized by more widespread hemorrhagic and partly necrotic mucosa, and grades 5 and 6 were assigned when the damage was extensive and widespread, with evidence of transmural necrosis. Pigs were considered to have clinical signs of NEC when the average score for the 4 regions exceeded 1.5. Thus, the minimum required for NEC diagnosis was extensive mucosal swelling and hyperemia in at least 1 of the 4 GIT regions. Digital photos were taken of the GIT harvested from all pigs for later confirmation of the data collected at necropsy. During the first 1–2 postnatal days, enterocytes of the term pig intestine absorb intact protein macromolecules, including colostral immunoglobulins.24Jensen A.R. Elnif J. Burrin D.G. Sangild P.T. Development of intestinal immunoglobulin absorption and enzyme activity in neonatal pigs is diet-dependent.J Nutr. 2001; 131: 3259-3265PubMed Scopus (129) Google Scholar Transcytosis was evaluated in vivo before clinical signs of NEC and in vitro after tissue harvest. Bovine serum albumin and human serum albumin (BSA; A-4503, or HSA; A-1653, respectively; Sigma Chemical Co, St. Louis, MO) were added to the food (colostrum or formula, 20 mg/mL) at the first feeding (0 hours, BSA) or at 12 hours (HSA). Arterial blood samples were taken at intervals (0, 3, 6, 9, and 12 hours postfeeding) for measurement of circulating BSA and HSA levels. Plasma concentrations of BSA, HSA, and porcine immunoglobulin G (IgG) were measured by quantitative immunoelectrophoresis.24Jensen A.R. Elnif J. Burrin D.G. Sangild P.T. Development of intestinal immunoglobulin absorption and enzyme activity in neonatal pigs is diet-dependent.J Nutr. 2001; 131: 3259-3265PubMed Scopus (129) Google Scholar The absorption of galactose following an oral load of lactose was investigated at 18–24 hours after birth and before any pigs exhibited clinical symptoms of NEC. Lactose was dissolved in sterile water (10%) and was administered via the orogastric tube at a dose of 1.5 g/kg body weight 3 hours after the last feeding. Arterial blood samples were collected before and at 10, 20, 30, 40, and 70 minutes after the lactose bolus. Plasma galactose was measured by spectrophotometry using galactose-dehydrogenase (Boehringer Mannheim, Darmstadt, Germany). An increase in plasma galactose levels following an oral load of lactose reflects the combined activity of brush border lactase-phloridzin hydrolase and the sodium-glucose cotransporter 1 (SGLT-1), which transports galactose and glucose. When the clinical symptoms of NEC were clearly observed in 1 or more pigs, the entire litter was killed within 8–10 hours (sodium pentobarbitone, 60 mg/kg, IV). This approach allowed us to compare piglets that were clinically healthy (but at risk of developing clinical symptoms of NEC) with those that were clearly pathologic. Before tissue collection, an arterial blood sample was taken and analyzed immediately for pH, partial carbon dioxide pressure, O2, O2 saturation, glucose, hematocrit, hemoglobin, and electrolytes using an automated blood gas and electrolyte analyzer (NOVA Biomedical, Waltham, MA) and a hemoximeter (Radiometer, Copenhagen, Denmark). Blood samples taken from pigs with terminal NEC symptoms were excluded. Plasma was separated and stored (−20°C) for later measurements of cortisol by ELISA (Biomar Diagnostics, Marburg, Germany) and for radioimmunoassays to quantify plasma haptoglobin26Petersen H.H. Nielsen J.P. Jensen A.L. Heegaard P.M. Evaluation of an enzyme-linked immunosorbent assay for determination of porcine haptoglobin.J Vet Med A Physiol Pathol Clin Med. 2001; 48: 513-523Crossref PubMed Scopus (20) Google Scholar and GLP-2.22Petersen Y.M. Hartmann B. Holst J.J. Le Huerou-Luron I. Bjornvad C.R. Sangild P.T. Introduction of enteral food increases plasma GLP-2 and decreases GLP-2 receptor mRNA abundance during pig development.J Nutr. 2003; 133: 1781-1786PubMed Scopus (61) Google Scholar Following death, the entire GIT was rapidly removed, and the small intestine, from the pyloric sphincter to the ileo-colonic junction, was isolated by cutting along the mesenteric border and weighed without contents. Intestinal length was measured in a relaxed state on a tabletop and divided into 3 segments of equal length, which were designated proximal, middle, and distal small intestine. The lungs, liver, spleen, heart, adrenals, kidneys, stomach, and pancreas were removed, and the wet masses were recorded. Tissue samples from the pancreas, the stomach (fundus region), the 3 regions of small intestine, and the proximal colon were frozen in liquid nitrogen and stored at −70°C for later biochemical and microbiologic analyses. Three intestinal sections were collected from the middle of each intestinal region. A 10–15-cm section was used for measuring rates of macromolecule and nutrient absorption. Another 10-cm adjacent section was opened along its length for measurements of intestinal circumference, wet mass, and percentage of mucosa that could be removed by gentle scraping with a plastic slide. The proportion of mucosa was determined on a dry matter basis after drying both the mucosa and the underlying tissues (50°C for 72 hours). Additional sections from each region were frozen for biochemical and microbiologic studies. Finally, a 1–2-cm section was fixed in 4% buffered formaldehyde for 2–3 days and then stored in 70% alcohol for later histologic evaluation and for in situ hybridization using specific bacterial probes. For villous height and crypt depth measurements, the samples were embedded in paraffin, sectioned (5 μm), and stained with eosin and hematoxylin.24Jensen A.R. Elnif J. Burrin D.G. Sangild P.T. Development of intestinal immunoglobulin absorption and enzyme activity in neonatal pigs is diet-dependent.J Nutr. 2001; 131: 3259-3265PubMed Scopus (129) Google Scholar Intestinal morphometry data were collected only from tissue sections that had intact villi without extensive atrophy or necrosis. The intestine and colon samples were homogenized in 1.0% Triton X-100 (6 mL per g tissue) and the homogenates assayed for disaccharidase (lactase, maltase, sucrase) and peptidase (aminopeptidases N, aminopeptidase A, dipeptidylpeptidase IV) activities using specific substrates, as described previously.18Petersen Y.M. Schmidt M. Elnif J. Sangild P.T. Glucagon-like peptide 2 enhances maltase-glucoamylase and sucrase-isomaltase gene expression and act
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