Developmental milestones in neonatal and juvenile C57Bl/6 mouse - Indications for the design of juvenile toxicity studies.

There is a growing demand for wild type mice and mouse models of disease that may be more representative of human conditions but there is little information on neonatal and juvenile mouse anatomy. This project produces sound and comprehensive histology background data on the developing neonatal mouse at different time points from Day 0 until Day 28. The work describes optimal methods for tissue harvesting, fixation and processing from the neonatal and juvenile mice which can be used in routine toxicology studies. A review of the available literature revealed inconsistencies in the developmental milestones reported in the mouse. Although it is true that the sequence of events during the development is virtually the same in mice and rats, important developmental milestones in the mouse often happen earlier than in the rat, and these species should not be used interchangeably.

The large intestine from fetal period to adulthood and its impact on the course of colonoscopy.

The human large intestine in the living adult has a total length of about 1300 mm, ranging from 1100 to 2108 mm. The development of the gut continues after birth, up to the age 4-5. The large intestine ascends at the beginning in the right abdominal quadrant, then it traverses the abdominal cavity, and finally it descends to the anus. The left and right colic flexures are the basic flexions between the transverse, ascending and descending colon, respectively. Additionally, there are secondary bendings between intestinal segments. The angles between the neighbouring parts can vary between examined subjects. Most of the angulations can be found in the transverse (range 2-9) and sigmoid colon (range 1-9), making them the most troublesome parts to pass with a colonoscope. Colonoscopy (usually performed in the left lateral or supine position) is one of the most important examination of the large intestine mucus membrane. During this procedure the endoscope is passed through the colon into the cecum or terminal ilium. The individual anatomical features (tortuosity, supernumerary loops and elongation) may slow down or interfere with the progress of the scope. We summarize current knowledge on the human large intestine from the fetal period to adulthood and carve out some aspects that are currently less known to colonoscopists.

Expression and Localization of Aquaporin 4 and Aquaporin 5 along the Large Intestine of Colostrum-Suckling Buffalo Calves.

Aquaporins (AQPs) are membrane channel proteins that play a role in regulating water permeability in many tissues. To date, seven isoforms of AQPs have been reported in the gastrointestinal tract in different mammalian species. In contrast, both tissue distribution and expression of AQPs are unknown in the buffalo. The purpose of this study was to investigate the expression of both AQP4 and AQP5 mRNAs and their relative proteins in the large intestinal tracts of buffalo calves after colostrum suckling using reverse transcriptase polymerase chain reaction (RT-PCR), Western blotting and immunohistochemistry. Our results revealed a diversified tissue AQP4 and AQP5 immunolocalization accompanied by their highest expression in the tissues of colostrum-suckling buffalo calves confirmed by Western blotting. In particular, AQP4 was distributed along the endothelium and enterocytes while AQP5 in the endocrine cells. These findings provide direct evidence for AQP4 and AQP5 expression in the large intestine, suggesting that different AQPs collaborate functionally and distinctively in water handling during intestinal development, especially during the first period after delivery.

Pattern selection in growing tubular tissues.

Tubular organs display a wide variety of surface morphologies including circumferential and longitudinal folds, square and hexagonal undulations, and finger-type protrusions. Surface morphology is closely correlated to tissue function and serves as a clinical indicator for physiological and pathological conditions, but the regulators of surface morphology remain poorly understood. Here, we explore the role of geometry and elasticity on the formation of surface patterns. We establish morphological phase diagrams for patterns selection and show that increasing the thickness or stiffness ratio between the outer and inner tubular layers induces a gradual transition from circumferential to longitudinal folding. Our results suggest that physical forces act as regulators during organogenesis and give rise to the characteristic circular folds in the esophagus, the longitudinal folds in the valves of Kerckring, the surface networks in villi, and the crypts in the large intestine.

Galacto-oligosaccharides derived from lactulose exert a selective stimulation on the growth of Bifidobacterium animalis in the large intestine of growing rats.

There is increasing interest in identifying novel dietary nondigestible carbohydrates capable of modulating the composition and/or metabolic activities of the gut microbiota. This work assessed the differential modulatory influence of novel galacto-oligosaccharides derived from lactulose (GOS-Lu) in comparison with commercial galacto-oligosaccharides derived from lactose (GOS-La) in gut microbiota of growing rats (5 weeks old). Rats were fed either a control diet or diets containing 1% (w/w) of GOS-Lu or GOS-La, and cecal and colonic contents were collected after 14 days of treatment. Compared to controls, GOS-Lu had significantly more bifidobacteria within the large intestine, showing a significant and selective increase of Bifidobacterium animalis in the cecum and colon; however, no significant differences in the number of bifidobacteria among GOS-Lu and GOS-La groups were observed. Both types of GOS significantly increased the number of the Eubacterium rectale / Clostridium coccoides group. These findings support a prebiotic role of galactosyl-fructoses in functional foods.

The role of Hes genes in intestinal development, homeostasis and tumor formation.

Notch signaling regulates intestinal development, homeostasis and tumorigenesis, but its precise downstream mechanism remains largely unknown. Here we found that inactivation of the Notch effectors Hes1, Hes3 and Hes5, but not Hes1 alone, led to reduced cell proliferation, increased secretory cell formation and altered intestinal structures in adult mice. However, in Apc mutation-induced intestinal tumors, inactivation of Hes1 alone was sufficient for reducing tumor cell proliferation and inducing differentiation of tumor cells into all types of intestinal epithelial cells, but without affecting the homeostasis of normal crypts owing to genetic redundancy. These results indicated that Hes genes cooperatively regulate intestinal development and homeostasis and raised the possibility that Hes1 is a promising target to induce the differentiation of tumor cells.

Morphometric characteristics of the small and large intestines of Mus musculus during postnatal development.

The objective of this study was to investigate the size of the small and large intestine in postnatal development of Mus musculus mice. The gut was obtained from 2-, 4-, 6-, and 12-week-old animals. The morphometric analysis was performed at microscopic level. Measurements and calculations included dimensions of villi (height, diameter) and their number per 1 mm(2) surface area in the proximal, middle, and distal section of the small intestine, as well as the length and surface area (external and internal) of the small and large intestines. To find the allometric relationship between the size of the small and large intestines and body mass, reduced major axis regression was applied. The length and surface area of both intestinal segments gradually increased with age. The increase in the internal surface area of the small intestine was the result of lengthening of the intestine and increasing diameter of the villi in its proximal and middle sections. No increase in villus height during the studied period was detected. A marked increase in the size of the intestinal segments was observed between the 2(nd) and 4(th) weeks of life, when the length doubled and the surface area tripled in size. Allometric analysis revealed that the increase in length and internal surface area of the small and large intestines was more rapid than the body mass increase during the weaning period, while it was not different from isometry after the weaning. In conclusion, the greatest changes in the structure and size of the small and large intestines of mice occurred in the weaning period. During this period these two segments of intestine grew faster than the rest of the body and reached adult proportions.

Wnt signaling specifies and patterns intestinal endoderm.

Wnt signaling has been implicated in many developmental processes, but its role in early endoderm development is not well understood. Wnt signaling is active in posterior endoderm as early as E7.5. Genetic and chemical activation show that the Wnt pathway acts directly on endoderm to induce the intestinal master regulator Cdx2, shifting global gene away from anterior endoderm and toward a posterior, intestinal program. In a mouse embryonic stem cell differentiation platform that yields pure populations of definitive endoderm, Wnt signaling induces intestinal gene expression in all cells. We have identified a set of genes specific to the anterior small intestine, posterior small intestine, and large intestine during early development, and show that Wnt, through Cdx2, activates large intestinal gene expression at high doses and small intestinal gene expression at lower doses. These findings shed light on the mechanism of embryonic intestinal induction and provide a method to manipulate intestinal development from embryonic stem cells.

Regulation of Dab2 expression in intestinal and renal epithelia by development.

Disabled-2 (Dab2) is an intracellular adaptor protein proposed to function in endocytosis. Here, we investigate the intestinal and renal Dab2 expression versus maturation. Dab2 mRNA levels measured by RT-PCR are greater in the small than in the large intestine. Immunological studies localize Dab2 to the terminal web domain of the enterocytes and reveal the presence of a 96-kDa Dab2 isoform in the apical membrane of the jejunum, ileum, and renal cortex of the suckling and adult rat. A 69-kDa Dab2 isoform is only observed in the apical membranes of the suckling ileum. During the suckling period, the Dab2 mRNA levels measured in the enterocytes and crypts and those of the 96-kDa Dab2 isoform are greater in the ileum than in the jejunum. No segmental differences are observed in the adult intestine. In the intestine, the levels of Dab2 mRNA and those of the 96-kDa Dab2 isoform decrease to adult values at weaning, whereas in the kidney they increase with development. Weaning the pups on a commercial milk diet slows the periweaning decline in the levels of Dab2 mRNA in the crypts and of those of the 96-kDa isoform. This is the first report showing that the 96-kDa Dab2 isoform is expressed at the apical domain of rat small intestine, that ontogeny regulates Dab2 gene expression in intestine and kidney and that retarding weaning affects intestinal Dab2 gene expression.

Ontogeny regulates creatine metabolism in rat small and large intestine.

The ontogeny of intestinal CRT, AGAT and GAMT was investigated in foetuses, newborn, suckling, weaning and adult rats. In the colon, CRT mediates creatine transport because it was Na(+)- and Cl(-) dependent and inhibited by creatine and GPA. In addition, Northern assays showed two CRT transcripts (2.7-kb and 4.2-kb) and the in situ hybridisation revealed that CRT mRNA is restricted to the colon epithelial cells. The immunohistochemistry revealed that CRT protein was at the apical membrane of colon epithelia. Maturation decreased colonic CRT activity to undetectable levels and increased CRT mRNA abundance. Western assays revealed 57-, 65-, 80- and 116-kDa polypeptides at the intestinal apical membrane. The abundance of the 65-, 80- and 116-kDa polypeptides decreased with age, and that of 57-kDa was only observed in adult rats. The small and large intestine express AGAT and GAMT mRNAs. Maturation decreased AGAT mRNA abundance without affecting that of GAMT. For comparison, renal AGAT mRNA levels were measured and they were increased with age. The study reports for the first time that: i) the apical membrane of rat colon have an active CRT, ii) development down-regulates CRT activity via post-transcriptional mechanism(s), iii) the intestine might synthesize creatine and iv) intestinal and renal creatine synthesis is ontogenically regulated at the level of AGAT gene expression.

[Digestive enzyme activities in rats kept on standard or surplus breast-feeding and on low-protein diet directly after weaning].

Activities of digestive enzymes (maltase, alkaline phosphatase, amino peptidase M, and glycyl-L-leucine dipeptidase) in small and large intestine, liver, and kidney were studied in rats of different ages kept in normal (8) and low (3) amounts of pups per litter. The low-protein diet for 10 days at once after weaning was found to change the mass of the organs and their digestive enzyme activities in all studied rat groups. The revealed changes were more prominent in rats kept under conditions of breast-overfeeding. In adult animals of this group, distribution of the alkaline phosphatase activity along the small intestine differed from that in control rats. The obtained results seem to confirm the fact that any disturbance of the nutrition quality in early ontogenesis leads to disturbance of the "metabolic programming of enzyme systems" of digestive and non-digestive organs.

Ontogeny of FOXP3(+) regulatory T cells in the postnatal human small intestinal and large intestinal lamina propria.

FOXP3(+) regulatory T cells (Treg) suppress innate and adaptive immune responses and are critical for intestinal immune homeostasis. Our objective was to define the postnatal developmental regulation of Treg in relationship to other T cells in the human intestinal tract. We analyzed 41 small and 18 large intestinal paraffin-embedded tissue samples from preterm and term infants with and without necrotizing enterocolitis (NEC) for the presence of CD3(+), CD4(+), CD8(+), and FOXP3(+) cells by immunohistochemistry. We compared labeled cells against age, gestational age (GA), or (corrected) postmenstrual age (PMA). The GA ranged from 23 to 40 weeks, with a mean of 32 (standard deviation, 4.7) weeks. Independent of age, GA, or PMA, the numbers of CD4(+) cells were higher in the small intestine compared to the large intestine (P = 0.046), except in patients with NEC. FOXP3(+) cells could be detected as early as 23 weeks in GA in both large and small bowel, and similar quantities were detected at the highest GA examined (40 weeks). We saw no statistically significant effect of GA, age, or PMA on total number of FOXP3(+) cells or by comparing FOXP3(+) to CD4(+) or FOXP3(+) to CD8(+) ratios, indicating intact ontogeny of Treg in intestinal tissue early in gestation. Human infants exhibit presence of mucosal FOXP3(+) cells in the small and large intestinal mucosa at birth and as early as 23 weeks GA. The frequency of FOXP3(+) cells and the ratios of FOXP3(+) to CD4(+) or CD8(+) cells do not change with increasing intrauterine development or postnatal age.

Weaned piglets display low gastrointestinal digestion of pea (Pisum sativum L.) lectin and pea albumin 2.

A study was conducted to investigate the biochemistry of digestion of field pea (Pisum sativum L.) albumins and globulins in the stomach and along the small intestine of weaned piglets with a particular emphasis on the respective roles of these compartments in pea protein digestion. Twenty-four piglets were weaned at 28 d of age. They were allocated to 2 diets (control and pea) and 3 slaughter times (3, 6, or 9 h after the last meal) in a 2 x3 factorial arrangement of treatments in a randomized complete block design. Pea flour provided 30% of total dietary protein in the pea diet. The diets were fed for 2 wk after weaning. After slaughter, gastrointestinal tract (GIT) compartments were weighed, digesta were collected, and pH was measured. Digesta from the stomach and cranial, middle, and caudal small intestine (SI) were extracted for soluble proteins and analyzed for specific pea proteins using SDS-PAGE, immunoblotting, and mass spectrometry. Tissue weight of the whole GIT (P = 0.015), cecum (P <0.001), and colon (P <0.001) was greater in the pea diet. Digesta pH in the stomach and caudal SI was lower (P = 0.02) in the pea diet than the control diet. In the stomach, vicilin, lectin, and pea albumin 2 were not digested, whereas legumin was only partly digested. Legumin and vicilin were totally digested in the SI in less than 3 h. A resistant peptide of 15 kDa located at the N-terminus of pea albumin 2 was transiently detected at 3 h. A protein band at 20 kDa was consistently identified as lectin. It was present in high intensity in intestinal digesta of pea-fed piglets at all times after the meal compared with those fed the control diet (P <0.001). Various proteins of, presumably, endogenous origin displayed differential digestion patterns between the control and the pea-fed piglets (P<0.05). In conclusion, differences in digestion between specific pea proteins were observed along the GIT of piglets. They could be partly explained by differences in protein digestion in the stomach.

Postnatal development of the mucosal plexus in the porcine small and large intestine.

Knowledge regarding the foetal and postnatal development of the enteric nervous system is crucial for the understanding of congenital disorders. While lot of information exists regarding the myenteric and submucosal plexuses, the development of the mucosal plexus has not been previously studied. The mucosal innervation seems to play an important role in the local reflex activity of the gut. In this study, we examined the development of enteric mucosal innervation in the pig at various ages of life. Small and large bowel paraffin-embedded specimens were stained with PGP 9.5 and neurofilament protein in three piglets from six age groups (60 and 90 days gestation, newborn, 4 and 12 weeks old, and adult pigs). Small and large bowel demonstrated identical innervation patterns. Myenteric and submucosal plexuses were stained with PGP 9.5 at 60 days gestation. However, the mucosal staining was first noted clearly at the newborn period. By 4 weeks, PGP 9.5 staining was noted in small amounts within the mucosa. Inner proprial and villous fibres were seen ahead in time to the subepithelial fibres. Both inner proprial and villous staining became quiet prominent by 12 weeks of age and remained unchanged into adulthood. However, the subepithelial fibres appear to increase in adulthood. This study demonstrates for the first time that enteric mucosal innervation first appears only at birth. The immaturity of the mucosa generated reflex activity, and secretory functions may have implication in the management of functional intestinal obstruction in the premature infant.

Maternal adaptive immunity influences the intestinal microflora of suckling mice.

The microflorae in the intestine of breast-fed infants are distinct from those that typically populate the intestine of formula-fed infants. Although the acquisition of passive immunity through breast-feeding may play a critical role in influencing the pattern of bacterial colonization of the gut, the precise mechanisms underlying the differences in the commensal microflorae of breast and formula-fed children have not been established. We hypothesized that the assemblage of commensal microflorae in suckling and weaned mice may be influenced by the maternal adaptive immune system. To test this hypothesis, we analyzed the intestinal microflorae of mice reared in the presence (wild-type) or absence of an intact maternal immune system (T- and B-cell deficient). Several types of bacteria (Lactobacillus, Enterococcus, Clostridium perfringens, Bifidobacterium, and Bacteroides) were isolated and enumerated from both the small and large intestine of 10-, 18-, 25- and 40- to 60-d old mice using selective media. The densities of bacteria were significantly lower in the small intestine of weaned mice that were reared by wild-type (WT) compared with immunodeficient (ID) dams. However, the microflorae were generally more abundant in the large intestine of suckling pups reared by WT compared with ID dams. Our results indicate that intestinal microflorae change throughout the suckling phase of development and that the maternal adaptive immune system influences the pattern and abundance of bacteria within the gut in an age- and site-specific manner.

Development of the digestive tract in the ostrich (Struthio camelus).

A study was conducted to investigate changes in the development of the gastrointestinal tract (GIT) in relation to body growth of growing ostriches. There was an 11-fold increase (P < 0.001) in body weight between 3 and 72 days of age. The relative (to body weight) weight of the proventriculus/gizzard, caeca and colon also increased (P < 0.001) with age. The relative weight of the small intestine peaked at 41 days of age and then tended to decline (P < 0.05) subsequently. The relative weight of the pancreas peaked at 27 days of age and remained fairly stable thereafter. The activities of chymotrypsin and lipase declined (P < 0.001) with age between 3 and 72 days. At 3 days of age, the protein content of the duodenal mucosal homogenate was higher (P < 0.001) than that of the jejunum or ileum, but at all subsequent periods the jejunal protein content was the highest. The protein content of the intestinal brush-border membrane was higher (P < 0.001) at the jejunum than at the duodenum or ileum. The specific activity of maltase declined (P < 0.001) with age in all three regions, most especially between 3 and 27 days of age. The activity of alkaline phosphatase (AP) at 41 and 55 days of age was higher (P < 0.001) in the duodenum than in the jejunum or ileum. The activity of AP fluctuated with age in the duodenum but there was a more defined decline (P < 0.001) with age in the jejunum and ileum. The relative protein content of the liver increased (P < 0.001) with age, with two peaks at 27 and 55 days of age. Arginase activity was not detected in the liver of 3-day old chicks and was not significantly affected by age between 27 and 72 days of age. The pattern of development observed is similar to that in growing poultry. There is, however, a need for evaluation at closer intervals in early life as well as an in-depth assessment of the morphometry of the intestinal mucosa.

Acquisition of neuronal and glial markers by neural crest-derived cells in the mouse intestine.

Enteric neurons and glia arise from the neural crest. The phenotype of crest-derived cells was examined as they differentiated into neurons or glia in the mouse small and large intestine. Previous studies have shown that undifferentiated enteric crest-derived cells are Phox2b(+)/Ret(+)/p75(+)/Sox10(+), and at embryonic day (E) 10.5, about 10-15% of the crest-derived cells in the small intestine have started to differentiate into neurons. In the current study, by E12.5 and E14.5, about 25% and 47%, respectively, of Phox2b(+) cells in the small intestine were immunoreactive to the pan-neuronal protein, ubitquitin hydrolase (PGP9.5), and the percentage did not change dramatically from E14.5 onward. The differentiation of crest-derived cells into neurons in the colon lagged behind that in the small intestine by several days. Differentiating enteric neurons showed high Ret, low p75, and undetectable Sox10 immunostaining. Glial precursors were identified by the presence of brain-specific fatty acid binding protein (B-FABP) and detected first in the fore- and rostral midgut at E11.5. Glial precursors appeared to be B-FABP(+)/Sox10(+)/p75(+) but showed low Ret immunostaining. S100b was not detected until E14.5. Adult glial cells were B-FABP(+)/Sox10(+)/p75(+)/S100b(+). A nucleic acid stain (to identify all ganglion cells) was combined with immunostaining for PGP9.5 and S100b to detect neurons and glial cells, respectively, in the postnatal intestine. At postnatal day 0, fewer than 5% and 10% of cells in myenteric ganglia of the small and large intestine, respectively, were neither PGP9.5(+) nor S100b(+). Because some classes of neurons are not present in significant numbers until after birth, the expression of PGP9.5 by developing enteric neurons appeared to precede the expression of neuron type-specific markers.

Effects of pre- and postnatal protein deprivation and postnatal refeeding on myenteric neurons of the rat large intestine: a quantitative morphological study.

We investigated the effect of protein deprivation and refeeding on weight gain, the size of the colon, and the numbers and sizes of enteric neurons. Neurons were located by reduced nicotinamide adenine dinucleotide (NADH) diaphorase staining. Protein deprivation of the mother throughout pregnancy, and the mother and unweaned rat pups in the first 21 postnatal days, reduced the weights of pups to about 50% of control. The size of the colon was also reduced, by about 40%. Despite this, total numbers of neurons in the colon were not reduced. However, there was a small, but significant, 15% reduction in the areas of neuron profiles. After 21 days the remaining pups were removed from the mothers, and either maintained on the control diet, maintained on the protein-deprived diet, or changed from the protein-deprived diet to a normal diet (refed group). These rats were examined after a further 21 days. Refeeding restored body weight to 20% below control, restored colon size, and restored nerve cell size. After a total of 42 days of protein deprivation, nerve cell numbers were not significantly different from control. In undernourished rats at 21 and 42 days, neurons were less well stained than control for NADH diaphorase. Refeeding between 21 and 42 days restored the normal appearance of the neurons. It is concluded that enteric neurons are protected from loss even when there is a substantial reduction in body weight and organ size caused by protein deprivation. The neurons become smaller, but recover size after refeeding.

The truncated metabolite GLP-2 (3-33) interacts with the GLP-2 receptor as a partial agonist.

The therapeutic potential of the intestinotrophic mediator glucagon-like peptide-2 (1-33) [GLP-2 (1-33)] has increased interest in the pharmacokinetics of the peptide. This study was undertaken to investigate whether the primary degradation product GLP-2 (3-33) interacts with the GLP-2 receptor. Functional (cAMP) and binding in vitro studies were carried out in cells expressing the transfected human GLP-2 receptor. Furthermore, a biologic response of GLP-2 (3-33) was tested in vivo. Mice were allocated to groups treated for 10 days (twice daily) with: (1) 5 microg GLP-2 (1-33), (2) 25 microg GLP-2 (3-33), (3) 5 microg GLP-2 (1-33)+100 microg GLP-2 (3-33), or (4) 5 microg GLP-2 (1-33)+500 microg GLP-2 (3-33). The intestine was investigated for growth changes. GLP-2 (3-33) bound to the GLP-2 receptor with a binding affinity of 7.5% of that of GLP-2 (1-33). cAMP accumulation was stimulated with an efficacy of 15% and a potency more than two orders of magnitude lower than that of GLP-2 (1-33). Increasing doses of GLP-2 (3-33) (10(-7)-10(-5) M) caused a shift to the right in the dose-response curve of GLP-2 (1-33). Treatment of mice with either GLP-2 (1-33) or (3-33) induced significant growth responses in both the small and large intestines, but the response induced by GLP-2 (3-33) was much smaller. Co-administration of 500 microg of GLP-2 (3-33) and 5 microg GLP-2 (1-33) resulted in a growth response that was smaller than that of 5 microg GLP-2 (1-33) alone. Consistent with the observed in vivo activities, our functional studies and binding data indicate that GLP-2 (3-33) acts as a partial agonist with potential competitive antagonistic properties on the GLP-2 receptor.