Diversity of the intestinal microbiota differently affects non-neuronal and atropine-sensitive ileal contractile responses to short-chain fatty acids in mice.

Non-neuronal and atropine-sensitive ileal contractile responses to short chain fatty acids (SCFAs) are detected in the neonatal stage, and change with age or inflammatory conditions. However, the roles of luminal SCFAs in developmental changes have not yet been elucidated. We examined ileal contractile responses to SCFAs in mice colonized with different SCFA-producing intestinal microbiota under normal and inflammatory conditions. Using conventional (Conv), germ-free (GF), and gnotobiotic mice infected with Bifidobacterium (GB-bif), Propionibacterium (GB-prop), or Lactobacillus (GB-lact), ileal contractions were measured in 1-day-old neonates and 7-week-old mice using an isotonic transducer. Contractions occurred in all 1-day-old neonates, and were significantly desensitized in the adult stage in the Conv, GB-bif, and GB-prop groups, but not in the GF and GB-lact groups. An injection of lipopolysaccharide frequently restored desensitized contractions; however, the contraction rate did not change in the GF and GB-lact groups. The relative mRNA expression of a SCFA receptor (GPR43) or nicotinic acetylcholine receptor α7 was weaker in the GF group (0.3-fold or 0.4-fold expression level, respectively) than in the Conv group. In conclusion, the luminal inhabitation of SCFA-producing bacteria may potentiate the regulation of non-neuronal and atropine-sensitive ileal contractile responses to SCFAs under healthy and inflammatory conditions.

Non-neuronal, but atropine-sensitive ileal contractile responses to short-chain fatty acids: age-dependent desensitization and restoration under inflammatory conditions in mice.

Intestinal epithelial cells sense short-chain fatty acids (SCFAs) to secrete non-neuronal acetylcholine (ACh). However, the roles of luminalSCFAs and epithelialACh under normal and pathological conditions remain unknown. We examined ileal contractile responses toSCFAs at different ages and their mucosal cholinergic alterations under inflammatory conditions. Ileal contractile responses toSCFAs in 1-day-old pups to 7-week-old mice were compared using an isotonic transducer, and responses to an intraperitoneal injection of lipopolysaccharide (LPS) were analyzed in 7-week-old mice. ThemRNAexpression levels of aSCFAactivate free fatty acid receptor, acetylcholinesterase (AChE), choline acetyltransferase (Chat), and choline transporter-like protein 4 (CTL4) were measured using real-time quantitativeRT-PCRAChE was analyzed by histochemical and optical enzymatic assays. Atropine-sensitive ileal contractile responses toSCFAs occurred in all 1-day-old pups, but were frequently desensitized after the weaning period. These contractile responses were not inhibited by tetrodotoxin and did not appear when the mucosal layer had been scraped off. Contractile desensitization in 7-week-old mice was abolished in the presence of theAChE inhibitor, eserine, which was consistent with increasedAChE activity after weaning. Ileal contractions toSCFAs in adult mice were restored byLPS, which significantly increased the epithelialmRNAexpression of Chat andCTL4. Atropine-sensitive ileal contractile responses toSCFAs constitutively occur in the newborn period, and are desensitized during developmental stages following the up-regulated expression ofAChE in the villous mucosa, but are restored under inflammatory conditions possibly via the release of epithelialACh.

Long-term oral feeding of lutein-fortified milk increases voluntary running distance in rats.

To evaluate the effects of lutein-fortified milk administration on running exercise, a voluntary wheel-running model was performed in rats. Four-week-old F344 rats were administered test milk (10 mL/kg) daily following a 4-h fasting period, and their running distances were measured each day for a 9-week period. Total weekly running distance significantly increased from the sixth week until the end of the test period in lutein-supplemented rats (lutein-fortified milk administered) compared with control rats (vehicle administered). This increase was not apparent in rats administered lutein alone. In the lutein-fortified-milk exercise group compared with the sedentary control group, carnitine palitroyltransferase 1 (CPT-1), total AMP-activated protein kinase (tAMPK), and phosphorylated AMP-activated protein kinase (pAMPK) contents were significantly increased in the gastrocnemius muscle, with a concomitant decrease in triglyceride and total cholesterol levels in the blood and liver. Furthermore, the lutein level in blood of lutein-administered rats significantly decreased with exercise. These results suggest that lutein-fortified milk may enhance the effect of exercise by effective utilization of lipids when combined with voluntary running.

Effects of constant light during perinatal periods on the behavioral and neuronal development of mice with or without dietary lutein.

Constant light conditions (LL) carry a risk of disrupting the biological clock of developing animals. Our purpose in this study was to investigate what disorders occur in animals receiving an LL stress during the late embryonic and suckling periods as compared with animals housed in dark-light (14 h-10 h) conditions (DL). In addition, we examined ameliorating effects against the disorder by the oral administration of lutein as an antioxidant. LL caused hypertrophy of the spleen and induced a higher expression of serotonin transporter (5HTT) in the corpus striatum and hippocampus in 15-day-old pups. In 9-week-old offspring, LL caused abnormal behavior in the elevated plus-maze test. The expression levels of 5HTT in the brain of the LL group changed to lower than those in DL group. The oral administration of lutein lessened the abnormality in behavior and 5HTT expression in the hippocampus to a certain degree although the expression levels of 5HTT in the corpus striatum were not altered by lutein diet. LL also induced disorders in the maternal brain with lower expression levels of 5HTT and neuregulin 1. These results indicate that LL during the perinatal periods may induce some neuronal abnormalities in both offspring and mothers that may be partially ameliorated by dietary lutein as an antioxidant.

Expression and distribution of acyl-CoA thioesterases in the white adipose tissue of rats.

Acyl-CoA thioesterases (Acots) are enzymes that catalyze the hydrolysis of fatty acyl-CoAs to free fatty acids and coenzyme A, and have the potential to regulate the intracellular levels of these molecules. In this study, we show that a cytosolic isoform, Acot1, is expressed and distributed in immature adipocytes located in the perivascular region of the white adipose tissue (WAT) of rats. Immunoblot analyses detected Acot1 in all of the WATs examined, while immunohistochemistry revealed positively stained layered structures surrounding the adventitia of blood vessels in the subcutaneous WAT. When the subcutaneous WAT was digested with collagenase and centrifuged, Acot1 was recovered in the stromal vascular fraction (SVF), and not in the large mature adipocytes. In the SVF, undigested cells attached to short tubular fragments of blood vessels showed positive immunostaining, as well as a proportion of the dispersed cells. These fibroblast-like cells contained fine particulate lipid droplets, stained by oil-red O dye, in their cytoplasm, or expressed fatty acid-binding protein 4, an adipocyte marker. After induction of adipocyte differentiation following a 15-day preculture without insulin, the dedifferentiated cells showed increased Acot1 expression with a diffuse distribution throughout the cytosol. These findings suggest that Acot1 expression is transiently upregulated at an early stage of adipocyte maturation, possibly to maintain cytosolic acyl-CoAs below a certain level until the cells acquire their full capability for fat storage.

Non-neuronal release of ACh plays a key role in secretory response to luminal propionate in rat colon.

Colonic chloride secretion is induced by chemical stimuli via the enteric nervous reflex. We have previously demonstrated that propionate stimulates chloride secretion via sensory and cholinergic systems of the mucosa in rat distal colon. In this study, we demonstrate non-neuronal release of ACh in the secretory response to propionate using an Ussing chamber. Mucosa preparations from the colon, not including the myenteric and submucosal plexuses, were used. Luminal addition of propionate and serosal addition of ACh caused biphasic changes in short-circuit current (Isc). TTX (1 µm) had no effects, while atropine (10 µm) significantly inhibited the Isc response to propionate and abolished that to ACh. In response to luminal propionate stimulation, ACh was released into the serosal fluid. A linear relationship was observed between the maximal increase in Isc and the amounts of ACh released 5 min after propionate stimulation. This ACh release induced by propionate was not affected by atropine and bumetanide, although both drugs significantly reduced the Isc responses to propionate. Luminal addition of 3-chloropropionate, an inactive analogue of propionate, abolished both ACh release and Isc response produced by propionate. RT-PCR analysis indicated that isolated crypt cells from the distal colon expressed an enzyme of ACh synthesis (ChAT) and transporters of organic cation (OCTs), but not neuronal CHT1 and VAChT. The isolated crypt cells contained comparable amounts of ACh to the residual muscle tissues including nerve plexuses. In conclusion, the non-neuronal release of ACh from colonocytes coupled with propionate stimulation plays a key role in chloride secretion, via the paracrine action of ACh on muscarinic receptors of colonocytes.

Bifidobacterium bifidum improves intestinal integrity in a rat model of necrotizing enterocolitis.

Neonatal necrotizing enterocolitis (NEC) is a major cause of morbidity and mortality in premature infants. Oral administration of probiotics has been suggested as a promising strategy for prevention of NEC. However, little is known about the mechanism(s) of probiotic-mediated protection against NEC. The aim of this study was to evaluate the effects of Bifidobacterium bifidum treatment on development of NEC, cytokine regulation, and intestinal integrity in a rat model of NEC. Premature rats were divided into three groups: dam fed (DF), hand fed with formula (NEC), or hand fed with formula supplemented with 5 x 10(6) CFU B. bifidum per day (B. bifidum). All groups were exposed to asphyxia and cold stress to develop NEC. Intestinal injury, mucin and trefoil factor 3 (Tff3) production, cytokine levels, and composition of tight junction (TJ) and adherens junction (AJ) proteins were evaluated in the terminal ileum. B. bifidum decreased the incidence of NEC from 57 to 17%. Increased levels of IL-6, mucin-3, and Tff3 in the ileum of NEC rats was normalized in B. bifidum treated rats. Reduced mucin-2 production in the NEC rats was not affected by B. bifidum. Administration of B. bifidum normalized the expression and localization of TJ and AJ proteins in the ileum compared with animals with NEC. In conclusion, administration of B. bifidum protects against NEC in the neonatal rat model. This protective effect is associated with reduction of inflammatory reaction in the ileum, regulation of main components of mucus layer, and improvement of intestinal integrity.

Decreased development of necrotizing enterocolitis in IL-18-deficient mice.

Necrotizing enterocolitis (NEC) is a devastating gastrointestinal disease predominantly of prematurely born infants, characterized in its severest from by extensive hemorrhagic inflammatory necrosis of the distal ileum and proximal colon. Proinflammatory cytokines have been implicated in the development of NEC, and we have previously shown that IL-18 is significantly elevated in the well-established neonatal rat model of NEC. To determine whether IL-18 contributes to intestinal pathology in NEC, we subjected IL-18 knockout mice to the protocol used to develop experimental NEC in newborn rats. Newborn B6.129P2-Il18(tm1Aki)/J (NEC IL-18(-/-)) and wild-type (NEC WT) mice were hand fed every 3 h with cow's milk-based formula and exposed to asphyxia and cold stress twice daily. After 72 h, animals were killed and distal ileum and liver were removed. Disease development was determined via histological changes in the ileum as scored by a blinded evaluator. The number of TNF-alpha-, IL-12-, and IL-1beta-positive cells and macrophages were determined in both ileum and liver via immunohistology. IkappaB-alpha and IkappaB-beta were determined from protein extracts from both ileum and liver using Western blot analysis. The incidence and severity of NEC was significantly reduced in NEC IL-18(-/-) mice compared with NEC WT. Furthermore, mean ileal macrophages and hepatic IL-1beta were significantly reduced in IL-18(-/-) mice subjected to the NEC protocol. There were no statistically significant changes in Kupffer cells, hepatic TNF-alpha, ileal IL-1beta, or IL-12. IkappaB-alpha and IkappaB-beta were significantly increased in NEC IL-18(-/-) mice ileum and liver, respectively. These results confirm that IL-18 plays a crucial role in experimental NEC pathogenesis.

Reduced thymic size and numbers of splenic CD4+ and CD8+ cells in artificially reared mouse pups.

The effect of early nutrition on the development of the immune tissue and T cells of mouse pups was examined. Newborn mice were divided into three experimental groups: mother-reared (MR) pups, pups that were fed on a milk substitute from the first day (AR-0), and the third day (AR-2), using a hand-feeding system. The average thymic size of the AR-2 pups was respectively significantly larger and smaller than that of the AR-0 and MR pups. In contrast, the splenic sizes of the AR-0 and AR-2 pups were greater than that of the MR pups. The numbers of CD4+CD8- and CD4-CD8+ cells in the spleen of the MR pups were significantly higher than those in the AR-0 pups. These results indicate that early nutrition affected the sizes of the thymus and spleen and the composition of CD4+CD8- or CD4-CD8+ T cells in the spleen.

A chemically derived milk substitute that is compatible with mouse milk for artificial rearing of mouse pups.

The object of this study was to prepare a chemically derived milk substitute that is compatible with mouse-milk. Milk was independently collected from ICR, BALB/c, and FVB/N mice, and analyzed for the protein, fat, and mineral contents to formulate a milk substitute. Thereafter, ICR mouse pups were artificially reared on the milk substitute to evaluate the rate of increase of their body weights. A gastric cannula tube was placed through the esophageal way into 8-day-old ICR pups, and the mice were fed with the milk substitute by computer-regulated infusion pumping by the pup-in-a-cup method. The analytical mean values of total protein and total fat in milk from ICR, BALB/c, and FVB/N mice were 10.23 +/- 0.49% and 21.34 +/- 1.31%, respectively. The milk substitute was constituted from purified bovine casein and whey proteins, five edible oils, including MCT oil, minerals, and vitamins. After 8 days of artificial rearing with the new milk substitute, 36 of the 42 pups had survived, and the growth rate of these mice was not significantly different from that of maternally reared littermate pups. In conclusion, we have succeeded in the preparation of a chemically derived milk substitute for mice pups which is available for clarifying the roles of dietary components such as milk-bone substance during the suckling period in mice pups including those of knockout and transgenic mice.

Intraperitoneal injection of lactoferrin ameliorates severe albumin extravasation and neutrophilia in LPS-induced inflammation in neonatal rats.

Lactoferrin (LF) plays various anti-inflammatory roles in inflammation experimentally induced by lipopolysaccharides (LPS). But the effects of LF on albumin extravasation and neutrophilia have not been elucidated. We aimed to study the effects of LF on albumin extravasation, neutrophilia and/or on other symptoms in inflammation caused by LPS in rats. Human lactoferrin (hLF) was injected (10 mg/100 mL in PBS) 18 h, or 15 min prior to, or 60 min after intraperitoneal injection of LPS in 13 days old Sprague Dawley rats. Prophylactic injection of hLF significantly ameliorated albumin extravasation in ascitic fluid at 5 h and neutrophilia in the blood at 24 h after LPS injection, but the after-injection of hLF did not. Interestingly, an injection of rat anti-TNFalpha IgG 15 min prior to LPS injection did not ameliorate albumin extravasation. Prophylactic injection of hLF significantly ameliorated other symptoms like mortality, and the decrease of phagocytotic activity of peritoneal polymorpho-nuclear leukocytes (PMNL), but did not ameliorate the decrease of platelets in the plasma. These findings suggest that hLF may be available as a medical treatment prior to surgery for prophylaxis of side effects like albumin extravasation or neutrophilia.

Intestinal adherent bacteria and bacterial translocation in breast-fed and formula-fed rats in relation to susceptibility to infection.

The barrier function of the intestinal mucosa is immature in the newborn mammal, and is strengthened by breast milk. We investigated this effect of breast milk by comparing the susceptibility to infection assessed in terms of adherent bacterial colonization of the intestinal tissue (AdC) and bacterial translocation (BT) between breast-fed and formula-fed newborn rats. Three-day-old rat pups were assigned to one of three groups: mother-reared (MR), pseudo-cannulated (sham), and artificially reared (AR). AR rats were infused with formula through an intragastric cannula, under the control of a computer-regulated pumping machine. MR and sham rat pups were reared with their respective dams and received breast milk until weaning in a specially designed cage. In 10-d-old rats, there was no significant difference in the fecal or cecal flora between the AR and MR groups, whereas the AdC and the BT to the liver were greater in the AR than MR group. Enterobacteriaceae, Streptococcus and/or Enterococcus, and Staphylococcus were dominantly detected as microorganisms in AdC flora and BT. The AdC flora did not directly reflect the bacterial colonization flora. These findings suggest that AR rat pups mature normally, although there is a greater colonization of Enterobacteriaceae and BT in AR than MR pups. Consequently, the intestinal barrier function of the pups reared by artificial feeding may become susceptible to BT, and AdC may be more indicative than bacterial colonization of the susceptibility to BT.

Reactivity of secretory IgA antibodies in breast milk from 107 Japanese mothers to 20 environmental antigens.

The secretory IgA (sIgA) antibody response to 20 environmental antigens, including microorganisms, toxins, food, and inhaled allergens, was evaluated in the breast milk from 107 Japanese mothers 1-10 days after delivery. Specific sIgA antibody responses were detected in most milk samples against almost all of the antigens tested, although there was a wide variation in the specific sIgA antibody profiles of each individual's milk. With regard to twelve bacterial antigens, highly specific sIgA antibody responses were detected against Escherichia coli, Yersinia enterocolitica, and Pseudomonas aeruginosa. With regard to eight nonbacterial antigens, highly specific sIgA antibody responses were detected against rotavirus, cholera, and pertussis toxins. Similar sIgA antibody profiles were obtained when the 107 milk specimens were divided into colostrum (milk 1-5 days after delivery, n = 36) and transitional milk (milk 6-10 days after delivery, n = 71). This study provides information on the possible protective role of human milk sIgA antibodies and will serve as a baseline for future studies.