Debra L. McWilliam

Up-regulation of IL-18 and IL-12 in the ileum of neonatal rats with necrotizing enterocolitis

Necrotizing enterocolitis (NEC) is a common and devastating gastrointestinal disease of premature infants. Because the proinflammatory cytokines IL-18, IL-12, and interferon (IFN)-γ have been implicated in other diseases of the small intestine, we hypothesized that these cytokines would play an important role in NEC pathogenesis. NEC was induced in newborn rats via enteral feeding with rat milk substitute and asphyxia and cold stress (RMS). Dam-fed, asphyxia- and cold-stressed littermates were used as controls (DF). After 96 h, the distal ileum was removed from all animals and processed to determine expression and localization of IL-18, IL-12, and IFN-γ using real-time reverse transcriptase PCR and immunohistology. IL-18 and IL-12 mRNA from the RMS group were increased (p ≤ 0.05) compared with DF controls, and there was a correlation between increasing IL-18 and IL-12 mRNA levels and progression of tissue damage (r = 0.629 and 0.588, respectively;p ≤ 0.05). Immunohistology revealed IL-18 in the cytoplasm of villi and crypt enterocytes and IL-12–positive monocytes/macrophages were increased with disease progression (r = 0.503, p ≤ 0.05). No differences in the number of IFN-γ–positive cells were observed between groups. These data demonstrate up-regulation of IL-18 and IL-12 in experimental NEC and a correlation between production of these proinflammatory cytokines and progression of tissue damage.

Artificial formula induces precocious maturation of the small intestine of artificially reared suckling rats.

BackgroundThe artificially reared rat model was used successfully to study the effect of nutrition during the early postnatal period on growth and development of the neonate. Overgrowth and morphologic changes of the gastrointestinal tract are known consequences of artificial rearing. The major goal of our study was to elucidate whether artificial rearing–enhanced gut development is caused by artificial diet or by gastrostomy and the artificial rearing technique itself. MethodsSuckling rats at day 8 of age underwent intragastric cannulation and were machine fed either a cows milk–based artificial rats milk substitute or pooled rats milk for 4 days. Dam-fed littermates served as a control. ResultsBody growth did not differ in the three experimental groups. In rats receiving rats milk substitute, small intestinal wet weight was approximately 60% greater than in rats fed rats milk or control rats. Additionally, the entire small intestine was approximately 20% longer in the rats milk substitute group. Morphologically, rats milk substitute–fed pups demonstrated significantly greater intestinal villus length and crypt depth compared with rats milk–fed or control rats. Jejunum and midjejunum of the rats milk and control groups did not differ in these parameters. Intestinal sucrase activity of rats milk substitute–fed rats was significantly elevated compared with rats milk–fed rats or control animals. ConclusionsThese results indicate that cows milk–based formula, not gastrostomy or artificial feeding technique, is a principal cause of the small intestine overgrowth and precocious maturation of some intestinal functions observed in artificially reared sucklings.

Milk-borne epidermal growth factor modulates intestinal transforming growth factor-α levels in neonatal rats

Epidermal growth factor (EGF) is present in milk from various mammalian species, but its physiologic function in neonatal development remains unclear. Transforming growth factor-α (TGF-α) is a peptide structurally related to EGF, and its presence is detected in the developing small intestine of rats. The purpose of the present study was to examine the effect of milk-borne EGF on endogenous production of EGF and TGF-α in the small intestine of suckling rats. Neonatal rats were fed via gastrostomy either growth factor-free rat milk substitute (RMS) or RMS supplemented with EGF (100 ng/mL of RMS) from 8 to 12 d of age. Artificially reared rats were then compared with their dam-fed littermates. Animals fed the EGF-deficient diet RMS had markedly increased EGF and TGF-α mRNA levels in duodenum and ileum compared with dam-fed controls and significantly elevated total intestinal content of TGF-α peptide. Intestinal EGF content and EGF serum levels were significantly decreased in the RMS group compared with controls. The addition of EGF to the RMS diet normalized TGF-α mRNA levels in the duodenum and ileum, EGF mRNA levels in the ileum, and total intestinal TGF-α content and EGF serum levels to the levels measured in dam-fed littermates. Motility studies showed that enteral administration of EGF did not affect stomach emptying and intestinal transit. These studies indicate that exogenous milk-borne EGF modulates endogenous production of TGF-α in developing small intestine. It is likely that neither TGF-α nor EGF are solely responsible for small intestinal overgrowth of artificially reared neonatal rats.

The expression of epidermal growth factor and transforming growth factor-α mRNA in the small intestine of suckling rats: organ culture study

Epidermal growth factor (EGF) and transforming growth factor‐α (TGF‐α) are associated with regulation of various gastrointestinal functions. In order to better understand their role in developing small intestine EGF, TGF‐α and EGF‐R steady‐state mRNA levels and transcript stability were determined. Reverse transcription (RT) competitive‐polymerase chain reaction (PCR) revealed that intestinal TGF‐α mRNA levels were 10‐fold higher in comparison with EGF mRNA. The primary intestinal culture technique was used to evaluate mRNA stability. The stability of TGF‐α mRNA was remarkably lower than the stability of EGF mRNA. High levels of TGF‐α mRNA accompanied by high degradation rate of this mRNA suggested a rapid turnover of intestinal TGF‐α mRNA.

Transforming growth factor-alpha delays gastric emptying and small intestinal transit in suckling rats.

Transforming growth factor-alpha (TGF-alpha) is a biologically potent polypeptide detected in the gastrointestinal tract in suckling rats. The major goal of the present study was to test the hypothesis that the administration of TGF-alpha affects gastric emptying and small intestinal transit in suckling rats. Suckling (12-day-old) rats fasted 16 h received rat TGF-alpha subcutaneously (s.c.) or orogastrically in varying doses (0, 0.5, 1.0 microg/rat in 0.1% BSA). Control animals received 0.1% BSA only. Poly R-478 dye was used as a motility marker. Rats were decapitated 45 min after marker administration and the amount of dye in the stomach and the small intestine was measured by spectrophotometry. Subcutaneous administration of TGF-alpha significantly delayed stomach evacuation. In controls, the stomach contained 21.4 +/- 1.4% (mean +/- s(x)) of the Poly R-478 marker, whereas in TGF-alpha treated rats the stomach contained 37.2 +/- 2.8% of the total Poly R-478 dye given to animals. The delaying effect of TGF-alpha was time- and dose-dependent. Small intestinal transit was also significantly delayed. The proximal jejunum of TGF-alpha treated rats contained a 1.4-fold higher amount of marker in comparison with control rats. Orogastrically administered rTGF-alpha did not affect gastric emptying or intestinal transit. In conclusion, s.c. administration of rat TGF-alpha significantly delayed the gastrointestinal motility in vivo in suckling rats.

Milk-Borne EGF and Necrotizing Enterocolitis in Neonatal Rat Model

Neonatal necrotizing enterocolitis (NEC) is the most common gastrointestinal (GI) disease of premature infants with excessive morbidity and mortality that afflicts 3,000 to 4,000 babies in the United States each year.1 Many factors contribute to the development of NEC, mainly prematurity, enteral feeding, infectious agents and/or intestinal hypoxia-ischemia. Enteral feeding is nearly always a prerequisite for the development of NEC, but the exact mechanism of NEC pathogenesis is poorly understood. The protective role of maternal milk in NEC pathogenesis has been reported.2 Various components of milk have been tested to protect the gut against NEC.3 Epidermal growth factor (EGF) is a promising candidate for the treatment of NEC. Mammalian milk of many species contains high concentrations of EGF. Moreover, maternal milk is the major source of EGF for neonates during the suckling period.4 In contrast, EGF is absent in all commercial infant formulas. Strong effects of exogenous EGF on healing of damaged gastrointestinal mucosa or on intestinal adaptation after injury are reported in a number of studies.5 The aim of this study was to examine the effects of milk-borne EGF on the development of NEC in a neonatal rat model.