Ileal VIP submucous neurons: confocal study of the area enlargement induced by myenteric denervation in weanling rats

Vasoactive Intestinal Peptide (VIP) neurons are maturing during suckling and weaning periods and the neuropeptide VIP is thought to be neurotrophic during ontogenesis. We have previously demonstrated that suckling rats with myenteric ablation have significantly higher mitotic index and an increase on villus height and crypt depth 15 days after treatment. In the current study, we measured the area of VIP neurons of submucous plexus in the ileum of weanling rats, in which myenteric neurons were ablated by serosal application of benzalkonium chloride (BAC). The area of VIP immunoreactive cell bodies, reconstructed under confocal microscope, was significantly increased in response to denervation. This result suggests that the myenteric plexus may have an inhibitory role over submucous plexus in the normal intestine. The enhanced production of VIP may be correlated with the increased epithelial proliferation induced by denervation in a critical period of life, from suckling to weaning time.     

Effect of hydrocortisone on sialyltransferase activity in the rat small intestine during maturation. Changes along the villus-crypt axis and in fetal organ culture

Sialyltransferase activity was assayed in rat intestinal cells isolated as fractions reflecting the villus-crypt axis of differentiation. In 13-day-old rats both endo- and exogenous sialyltransferase activity reached their maximum in undifferentiated crypt cells and their peaks overlapped. In contrast, sialyltransferase of the adult intestine was 4-fold lower than that of sucklings in the crypts, with slight tendency to be transferred to the villus cells. Hydrocortisone applied to 10-day-old rats caused three days later a precocious drop of sialyltransferase activity in the crypt cells. Unlike in vivo, glucocorticoid responsiveness was accompanied by increased sialyltransferase activity in fetal small intestine cultivated for 17 days.     

Mucosal IgA increase in rats by continuous CLA feeding during suckling and early infancy

The aim of this work was to establish the effect of the cis9,trans11 conjugated linoleic acid (CLA) isomer on mucosal immunity during early life in rats, a period when mucosal immunoglobulin production is poorly developed, as is also the case in humans. CLA supplementation was performed during three life periods: gestation, suckling, and early infancy. The immune status of supplemented animals was evaluated at two time points: at the end of the suckling period (21-day-old rats) and 1 week after weaning (28-day-old rats). Secretory IgA was quantified in intestinal washes from 28-day-old rats by ELISA technique. IgA, TGFbeta, and PPARgamma mRNA expression was measured in small intestine and colon by real time PCR, using Taqman specific probes and primers. IgA mucosal production was enhanced in animals supplemented with CLA during suckling and early infancy: in 28-day-old rats, IgA mRNA expression was increased in small intestine and colon by approximately 6- and 4-fold, respectively, and intestinal IgA protein by approximately 2-fold. TGFbeta gene expression was independent of age and type of tissue considered, and was not modified by dietary CLA. Gene expression of PPARgamma, a possible mediator of CLA's effects was also upregulated in animals receiving CLA during early life. In conclusion, dietary supplementation with CLA during suckling and extended to early infancy enhances development of the intestinal immune response in rats.     

Ontogeny of mitochondrial calcium transport in spontaneously hypertensive (SHR) and WKY rats

The current studies were designed to investigate calcium uptake by intestinal jejunal sacs as well as in intestinal mitochondria of spontaneously hypertensive rats and their genetically matched WKY control rats. Kinetics of jejunal calcium uptake by jejunal sacs of adult SHR and WKY rats showed a significant decrease in Vmax of calcium uptake in SHR (227 +/- 24 versus 423 +/- 22 nmol.g tissue-1.3 min-1) compared to WKY rats P less than 0.001. To explore the intracellular handling of calcium by the intestinal mitochondria, calcium uptake was characterized by intestinal mitochondria before (suckling and weanling periods) and after (adult period) development of hypertension. Calcium uptake by intestinal mitochondria was driven by ATP in the presence of succinate as a respiratory substrate. Calcium uptake was stimulated several fold by the presence of ATP compared to no ATP conditions. Maximal calcium uptake occurred between 15-30 min and was significantly greater in adult SHR and WKY rats compared to corresponding values in weanling and suckling rats. Maximal ATP dependent calcium uptake in adult, weanling and suckling WKY rats was significantly greater compared to corresponding mean values in each age group in SHR (P less than 0.001). Oligomycin (10 micrograms/mg protein) inhibited calcium uptake partially. Ruthenium red (0.25 microM), 1 mM sodium azide and 0.5 mM dinitrophenol inhibited calcium uptake by more than 80% in both SHR and WKY rats. Kinetic parameters for ATP stimulated calcium uptake at 10 s revealed a Vmax of 0.56 +/- 0.6, 3.46 +/- 0.23 and 3.95 +/- 0.52 nmol/mg protein/10 s in suckling, weanling and adult WKY rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pre- and postnatal development of differentiated functions in rat intestinal epithelial cells

A panel of monoclonal antibodies to intestinal cell surface components has been used to compare the expression of differentiation-specific antigens in the epithelial cells of fetal, suckling, and adult rat small intestine. Indirect immunofluorescence staining, and immunopurification of detergent-solubilized membrane proteins, followed by single- and two-dimensional slab gel electrophoretic analysis, have demonstrated that fetal intestinal cells (at day 21 of gestation) express most differentiation-specific markers typical of adult absorptive villus cells. A marked heterogeneity in antigen expression was observed among different villus cell populations in suckling rat intestine, and three cell surface components were identified which are exclusively present during this period of intestinal development. Striking changes in the patterns of antigen expression in crypt and villus cells, and variations in the apparent isoelectric points for most luminal membrane components, were associated with the maturation of the intestinal mucosa at weaning. These changes could not be prematurely induced by cortisone injection in newborn rats, suggesting that factors other than glucocorticoids are responsible for the postnatal development of the intestinal epithelium. These results suggest that basic differences in biological properties and regulatory mechanisms exist among intestinal epithelial cells at different stages of pre- and postnatal maturation.     

Fetal gut mesenchyme induces differentiation of cultured intestinal endodermal and crypt cells

An experimental model was designed to analyze the effect of fetal gut mesenchyme on the cytodifferentiation of crypt cells and of embryonic progenitor cells. The cells used were the rat intestinal crypt cell line, IEC-17, and primary cell cultures prepared form isolated 14-day-old fetal intestinal endoderm (EC). Both cultures prepared from isolated 14-day-old fetal rat intestinal endoderm (EC). Both types of cells were associated with 14-day-old fetal rat gut mesenchyme (Rm) and grafted under the kidney capsule of adult rats. Seventy percent of the Rm/EC and ten percent of the Rm/IEC recombinants, recovered after 9 days, exhibited well-vascularized structures in which the mesenchyme had induced morphogenesis of the cells into a villus epithelium. The four main intestinal epithelial cell types, absorptive, goblet, endocrine, and Paneth cells, were identified using electron microscopy. Biochemical determinations of enzyme activities associated with brush border membranes revealed that alkaline phosphatase, lactase, sucrase, and maltase were expressed in both types of associations. These results were confirmed by immunofluorescence staining using monoclonal antibodies to brush border enzymes. Both enzyme assays and immunocytochemistry showed that the amount of enzymes present in the brush border membrane of Rm/IEC grafts was in general lower than that of the Rm/EC recombinants. The results indicate that fetal rat gut mesenchyme enables morphogenesis and cytodifferentiation of both crypt and embryonic progenitor cells.     

Crypt cell development in newborn rat small intestine

Three monoclonal antibodies were prepared against luminal membranes from small intestinal cells of 3-d-old rats (YBB 1/27, YBB 3/10) and crypt cell membranes from adult rats (CC 4/80). The antibodies were shown to define specific stages of development of the intestinal crypt cells. The YBB 1/27 antigen was first detected at the luminal membrane of the epithelial cells in fetal intestine at day 20 of gestation; it was confined to the crypt cells and lower villus cells between 1 and 20-22 d after birth, and could not be detected in any region of the intestine in older animals. The YBB 3/10 antigen, identified as a set of high Mr proteins, was localized over the entire surface membrane of fetal intestinal cells and of crypt and villus cells after birth; after weaning (20-22 d after birth) it gradually disappeared from the villus cells and became confined to the region of the crypts. The CC 4/80 antigen, identified as a protein (or a set of related proteins) of molecular mass 28-34 kD, was shown to appear in the crypt cells 10-14 d after birth. Its distribution changed after weaning, when it disappeared from the crypts, and was localized in the absorptive lower villus cells. This change in pattern could, in part, be prematurely elicited by cortisone injection in younger animals. These results have demonstrated the presence of specific surface membrane components on the intestinal crypt cells, and suggested that fetal antigens may be retained in these cells after birth.     

EFFECT OF VILLUS LENGTH ON CELL PROLIFERATION AND MIGRATION IN SMALL INTESTINAL EPITHELIUM

For the interpretation of data supporting the hypothesis of a feedback regulation of proliferative activity in intestinal crypts by the functional villus cell compartment the life span and migration rate of epithelial cells on villi of experimentally reduced length should be known. Autoradiographic studies and scintillation counting of isolated villi at different time intervals after ³H-thymidine labelling were carried out 36, 48 and 60 hr intervals after X-irradiation. The results showed that the life span of epithelial cells in rat small intestine (36–48 hr) is independent of the villus length. In villi of reduced length the migration rate of the epithelial cells was found to be decreased compared with controls. Changes in the migration rate in turn seem to be dependent on the production of epithelial cells in the crypt. Comparative studies on the recovery of crypt and villus epithelium after various doses (300 and 700 R) of X-radiation support the hypothesis that increased proliferative activity in the crypt cell compartment is related to a reduction of the number of functional villus cells below a critical villus length. The importance of these findings in the interpretation of data on (micro) biochemical analyses of certain cell differentiation characteristics during increased proliferative activity is discussed.     

Rapid expansion of intestinal secretory lineages following a massive small bowel resection in mice

Following massive small bowel resection (SBR) in mice, there are sustained increases in crypt depth and villus height, resulting in enhanced mucosal surface area. The early mechanisms responsible for resetting and sustaining this increase are presently not understood. We hypothesized that expansion of secretory lineages is an early and sustained component of the adaptive response. This was assessed in the ileum by quantitative morphometry at 12 h, 36 h, 7 days, and 28 days and by quantitative RT-PCR of marker mRNAs for proliferation and differentiated goblet, Paneth cell, and enterocyte genes at 12 h after 50% SBR or sham operation. As predicted, SBR elicited increases of both crypt and villus epithelial cells, which were sustained though the 28 days of the experiment. Significant increases in the overall number and percentage of both Paneth and goblet cells within intestinal epithelium occurred by 12 h and were sustained up to 28 days after SBR. The increases of goblet cells after SBR were initially observed within villi at 12 h, with marked increases occurring in crypts at 36 h and 7 days. Consistent with this finding, qRT-PCR demonstrated significant increases in the expression of mRNAs associated with proliferation (c-myc) and differentiated goblet cells (Tff3, Muc2) and Paneth cells (lysozyme), whereas mRNA associated with differentiated enterocytes (sucrase-isomaltase) remained unchanged. From these data, we speculate that early expansion of intestinal secretory lineages within the epithelium of the ileum occurs following SBR, possibly serving to amplify the signal responsible for initiating and sustaining intestinal adaptation.     

Regulation of liver corticoid metabolism during early postnatal life: effects of surgical stress

Corticosterone metabolism by liver slices was investigated in suckling (10-day-old), weanling (21-day-old) and adult male rats. During the suckling period adrenalectomy as well as sham adrenalectomy increase the rate of steroid A ring reduction and also the rate of steroid side chain degradation by 20-40%. In older animals such changes were not detected. The results support an earlier assumption that liver steroid metabolism is regulated in an age specific manner.     

Calpain from rat intestinal epithelial cells: Age-dependent dynamics during cell differentiation

Micromolar and millimolar Ca²⁺-requiring neutral protease (calpain I and calpain II) along with their endogenous inhibitor calpastatin were isolated and partially purified from the same preparation of rat intestinal epithelial cells. Calpain I and II were partially purified by 1300 and 900-fold with 57 and 53 per cent yield, respectively. The optimum assay conditions revealed pH 7.5, 20 min incubation at 25° C and 0.24% casein substrate for both calpains. The optimum calcium concentration obtained for calpain I and II were 25 M and 4 mM, respectively. Distribution of rat intestinal epithelial cells calpain I and II along with calpastatin during cell differentiation stages in weanling to senescence age were studied. Calpain I in weanling rats was in an increasing order from villus to crypt regions. Adult rats indicated well expressed consistent calpain I throughout the differentiation stages. Whereas, significant lowering towards crypt region cells were evident in old rats. Calpain II in weanling and adult rats was found to be consistent throughout the differentiation stages. Old animals revealed an increasing trend from villus to crypt region with insignificant activity present in upper villus cells. Concomitantly, different concentrations of calpastatin were observed throughout the differentiation stages in all the age groups. Moreover, the levels of calpains exceeded that of calpastatin in most of the epithelial cell populations during developmental stages. In addition to casein, intestinal epithelial cell membranes were found to be equally good substrates for calpains. Proteolytic susceptibility of weanling, adult and old rat membrane proteins varied significantly all along the ageing process in rats. Simultaneous age-dependent calpastatin response were also evident. Taken together the results obtained provided strong evidence that calpain plays significant role in rat intestinal cell differentiation and ageing process with calpastatin as its specific regulatory protein.Abbreviations DEAE-cellulose O-(Diethylaminoethyl)-cellulose - EDTA Ethylene Diamine Tetra Acetic Acid - Tris Tris (hydroxymethyl) amino methane - KH₂PO₄ potassium dihydrogen orthophosphate - Na₂HPO₄ disodium hydrogen phosphate - CaCl₂ Calcium Chloride - TCA Trichloroacetic Acid - PMSF Phenylmethylsulfonyl Fluoride     

Effect of neonatal hypoxia on the development of hepatic lipase in the rat

Increases in plasma lipids occur during hypoxia in suckling but not in weaned rats and may result from altered hepatic enzyme activity. We exposed rats to 7 days of hypoxia from birth to 7 days of age (suckling) or from 28 to 35 days of age (weaned at day 21). Hypoxia led to an increase in hepatic lipid content in the suckling rat only. Hepatic lipase was decreased to approximately 45% of control in 7-day-old rats exposed to hypoxia but not in hypoxic 35-day-old rats. Hypoxic suckling rats also had a 50% reduction in lactate dehydrogenase activity, whereas transaminase activity and CYP1A and CYP3A protein content were not different between hypoxic and normoxic groups. Additional rats were studied 7 and 14 days after recovery from hypoxic exposure from birth to 7 days of age; hepatic lipase activity had recovered to 85% by 7 days and to 100% by 14 days in the rats previously exposed to hypoxia. Administration of dexamethasone to neonatal rats to simulate the hyperglucocorticoid state found in hypoxic 7-day-old rats led to a moderate decrease ( approximately 75% of control) in hepatic lipases. Developmentally, in the normoxic state, hepatic lipases increased rapidly after birth and reached levels more than twofold that of the newborn by 7 days of age. Hypoxia delays the maturation of hepatic lipases. We suggest that the decrease in hepatic lipase activity contributes to hyperlipemia in the hypoxic newborn rats.     

Intestinal enzymes activities in isolated villus and crypt cells during postnatal development of the rat

Summary A modification of Weiser's (1973) cell isolation method was used in order to study the developmental pattern of various intestinal enzyme activities in villus and crypt cells of normal rats from 5 days after birth until 8 weeks. Alkaline phosphatase and enterokinase activities were always located in the upper villus zone during postnatal development. Enterokinase activity was higher in the upper villus cells during the third week of life than after this period. Aminopeptidase activity was located in the crypt cells during the first week, its maximum activity remained in this area until the third week. At this time, sucrase activity appeared in the crypt cells, then aminopeptidase and sucrase activities rose to the villus zone during the fourth week. Amylase activity was detected along the entire crypt-villus axis 5 days after birth, reaching maximum activity in crypt cells at the end of the first week and in the upper villus cells after the fourth week. In contrast with the other enzymes studied almost all amylase activity was soluble in the youngest animals whereas at weaning most of the activity appeared in a particulate form in the villus cells. But in the crypt cells the ratio between particulate and soluble form remained unchanged until the adult stage. Various hypotheses are advanced to explain the patterns of evolution of the different enzymes.     

Neonatal hypoxic hyperlipidemia in the rat: effects on aldosterone and corticosterone synthesis in vitro

Neonatal hypoxia increases aldosterone production and plasma lipids. Because fatty acids can inhibit aldosterone synthesis, we hypothesized that increases in plasma lipids restrain aldosteronogenesis in the hypoxic neonate. We exposed rats to 7 days of hypoxia from birth to 7 days of age (suckling) or from 28 to 35 days of age (weaned at day 21). Plasma was analyzed for lipid content, and steroidogenesis was studied in dispersed whole adrenal glands untreated and treated to wash away lipids. Hypoxia increased plasma cholesterol, triglycerides, and nonesterified fatty acids in the suckling neonatal rat only. Washing away lipids increased aldosterone production in cells from 7-day-old rats exposed to hypoxia, but not in cells from normoxic 7-day-old rats or from normoxic or hypoxic 35-day-old rats. Addition of oleic or linolenic acid to washed cells inhibited both aldosterone and corticosterone production, although cells from hypoxic 7-day-old rats were less sensitive. We conclude that hypoxia induces hyperlipidemia in the suckling neonate and that elevated nonesterified fatty acids inhibit aldosteronogenesis.     

The effect of continuous irradiation on cell proliferation and maturation in small intestinal epithelium.

Autoradiographic studies and scintillation counting of crypt material after pulse labelling with 3H-thymidine showed that during continuous irradiation with 290 rads/day a reduced proliferative activity is present in the crypts of rat small intestine after 1 day of irradiation and of normal activity during the remaining period (5 days) irradiation. After cessation of irradiation an increase in proliferative activity can be observed after 1 day of recovery. From the time (36-48 hr after starting of the irradiation) that the number of villus cells is reduced an expansion of the proliferation zone in the crypt was observed. Both effects last until 1 day of recovery after cessation of irradiation. The process of crypt cell maturation and of villus cell function has also been studied during and after continuous irradiation by micro-chemical enzyme analyses in isolated crypts and villi. It was found that the expansion of the proliferation zone in the crypt is accompanied by a decrease in activity of only those enzymes (i.e. non-specific esterases) which normally become active during crypt cell maturation. The activity of enzymes normally present mainly in the functional villus cells remained relatively unaffected by changes in crypt cell kinetics. A hypothesis of different regulation mechanisms of the proliferative activity in the intestinal crypt and a possible explanation of the different behaviour of various enzyme activities as a result of changes in crypt cell proliferation is discussed.     

ALTERATION OF CELLULAR PROLIFERATION IN THE ILEAL EPITHELIUM OF SUCKLING AND WEANED RATS: THE EFFECTS OF ISOPROTERENOL

The purpose of this study was to analyse cell proliferation in the ileum before and after neonatal closure to macromolecules and to determine the effects of the sympathomimetic amine, isoproterenol (IPR), on cell cycle parameters. 8- and 28-day-old rats were employed in this study representing the neonatal periods of pre- and post-closure ileum respectively. The duration of the cell cycle phases was determined by the percentage of labeled metaphases technique (PLM) with computerized analysis of the curves. The generation cycle time was longer in 8-day-old suckling rats (18.63 hr) as compared to the older weaned rats (11.85 hr), and most of this 6.78 hr difference was in the G₁-period (4.5 hr). Other proliferative indices were also lower in the suckling rats—mitotic index (1.9 ± 0.4% as compared to 6.7 ± 0.9%), labelling index (26.8 ± 2.5% versus 44.2 ± 2.6%) and migration rate measured as per cent labeled villus cells (8.9 ± 3.1% versus 45.2 ± 3.4%). IPR was found to inhibit cellular proliferation in the ileal epithelium of both age groups. The cell cycle of the ileal epithelium of 8-day-old rats was lengthened from 18.63 to 21.07 hr and 28-day-old rats from 11.85 to 13.98 hr. IPR produced a decrease in mitotic index from 1.9 ± 0.4% to 1.6 ± 0.4% in pre-closure ileum and from 6.7 ± 0.9% to 5.1 ± 0.6% in post-closure ileum. Labeling index decreased from 26.8 ± 2.5% to 20.0 ± 2.0% in 8-day-old rats and from 44.2 ± 2.6% to 31.1 ± 3.0% in 28-day-old rats after IPR administration. There were also significant differences in growth fraction between age groups and a significant decrease in growth fraction after IPR-treatment. From the results of this study it appears that β-adrenergic stimulation has an inhibitory effect on neonatal ileal epithelium.     

THE EFFECT OF TRANSPOSITION TO JEJUNUM ON EPITHELIAL CELL KINETICS IN AN ILEAL SEGMENT

Epithelial cell kinetics were studied in an ileal segment after transposition to proximal jejunum. The number of cells per villus column in the transposed ileum increased after 4-7 days to reach values normal for jejunum after 14-30 days. This increase was accompanied by a simultaneous increase in the number of cells per crypt column up to 130% of values in jejunum and ileum in situ. The percentage of labelled crypt cells, after labelling with ³H-thymidine, and the relative size of the proliferative cell compartment in the crypt in the transposed ileum did not differ from values in the ileum in situ at any time interval after surgery. The total proliferative activity per crypt, which was determined by scintillation counting of isolated crypts after ³H-thymidine labelling, increased two-fold from 7 days after surgery. Cell migration studies showed that the increase in the number of villus cells was probably not caused by a change in the life span of the epithelial cells. It seems that the increase in the number of villus cells in ileal epithelium after transposition to proximal jejunum is brought about by an enlargement of the crypt, while the relative size of the proliferative cell compartment in the crypt remains unchanged.     

Alanyl-glutamine promotes intestinal epithelial cell homeostasis in vitro and in a murine model of weanling undernutrition

Alanyl-glutamine (Ala-Gln) has recently been shown to enhance catch-up growth and gut integrity in undernourished children from Northeast Brazil. We hypothesized that the intestinal epithelial effects of Ala-Gln in malnourished weanling mice and mouse small intestinal epithelial (MSIE) cells would include modulation of barrier function, proliferation, and apoptosis. Dams of 10-day-old suckling C57BL/6 pups were randomized to a standard diet or an isocaloric Northeast Brazil "regional basic diet," moderately deficient in protein, fat, and minerals. Upon weaning to their dam's diet on day of life 21, pups were randomized to Ala-Gln solution or water. At 6 wk of age, mice were killed, and jejunal tissue was collected for morphology, immunohistochemistry, and Ussing chamber analysis of transmucosal resistance and permeability. Proliferation of MSIE cells in the presence or absence of Ala-Gln was measured by MTS and bromodeoxyuridine assays. MSIE apoptosis was assessed by annexin and 7-amino-actinomycin D staining. Pups of regional basic diet-fed dams exhibited failure to thrive. Jejunal specimens from undernourished weanlings showed decreased villous height and crypt depth, decreased transmucosal resistance, increased permeability to FITC-dextran, increased claudin-3 expression, and decreased epithelial proliferation and increased epithelial apoptosis (as measured by bromodeoxyuridine and cleaved caspase-3 staining, respectively). Undernourished weanlings supplemented with Ala-Gln showed improvements in weight velocity, villous height, crypt depth, transmucosal resistance, and epithelial proliferation/apoptosis compared with unsupplemented controls. Similarly, Ala-Gln increased proliferation and reduced apoptosis in MSIE cells. In summary, Ala-Gln promotes intestinal epithelial homeostasis in a mouse model of malnutrition-associated enteropathy, mimicking key features of the human disease.