Neonatal Rat Colon Research Articles

Cotransplantation of neuroepithelial stem cells with interstitial cells of Cajal improves neuronal differentiation in a rat aganglionic model

BackgroundRecent advances have raised the possibility of treating Hirschsprung's disease (HSCR) with transplanted neural stem cells. Although these cells have been shown to migrate and differentiate after transplantation, the restoration of intestinal motility by grafted cells is also mediated via the interstitial cells of Cajal (ICCs). We therefore aimed to evaluate the effect of cografting with ICC on neural stem cells after transplanted into aganglionic bowel. MethodsNeuroepithelial stem cells (NESCs) and ICCs were isolated from neural tube of embryonic rat (embryonic day 11.5) and colon of neonatal rats respectively. After cotransplantation into the benzalkonium chloride-induced rat aganglionic colon, survival and differentiation of the implanted cells were assessed 8weeks posttransplantation using immunofluorescence and Western blotting. Colonic motility was assessed by measuring the changes of intraluminal pressure responding to inflating stimulation and the response of muscle strips to electrical field stimulation. ResultsCotransplantation with ICC accelerated neuronal differentiation compared with transplantation of NESCs alone. Moreover, the contractile response to inflation and EFS-induced reaction of the colon after coimplantation were successfully evoked. ConclusionsThese data indicate that reconstitution of elements in the intestinal environment such as ICC enhances efficacy of NESCs therapy. This may aid development of a stem cell-based treatment for HSCR.

Effect of bowel transection and fecal passage deprivation on the enteric nervous system in neonatal rats

Introduction A temporary stoma is a relatively common procedure at the neonatal age for a number of disorders. At birth, the enteric nervous system (ENS) is not fully developed; temporary colostomy causes morphological changes in the ENS. We studied whether transection of bowel or deprivation of stool is responsible for these changes. Moreover, reversibility of the ENS changes after stoma closure was investigated. Methods Male Lewis rats aged 3 weeks were randomly divided into 3 groups. Group 1 (stoma group) received a stoma (t = 0), stoma closure 2 weeks later (t = 1), and were killed 2 weeks after stoma closure; group 2 (transection group) had a colon transection and immediate anastomosis (t = 0) and were killed 2 weeks afterward. Group 3 (sham group) had no operation. In all groups, the nerve/muscle fiber ratio (NMR) in the proximal and distal colon was assessed with full thickness biopsy specimens at operation or after killing. The NMR in the stoma, transection, and sham group at t = 1 were used to assess the effect of deprivation of passage of feces and transection itself on the development of the ENS of the colon. Results of the stoma group at t = 1 and t = 2 were compared to assess possible reversibility of ENS changes after stoma closure. Results There was a significantly higher NMR in the distal colon compared to the proximal colon in both the stoma and transection groups at t = 1 (2.0 ± 0.38, P < .001 and 2.8 ± 0.97, P < .001, respectively), whereas there was no significant difference in NMR ratio at t = 1 (1.1 ± 0.18, P = .34) in group 3. In group 1, the NMR ratio increased further after stoma closure to 3.1 ± 0.37, P < .001, at t = 2. Conclusion Transection of the bowel rather than deprivation of fecal passage causes nerve fiber hypertrophy in the distal colon of neonatal rats. Restoration of bowel continuity does not result in recovery of these ENS changes.

The Vanilloid Receptor Initiates and Maintains Colonic Hypersensitivity Induced by Neonatal Colon Irritation in Rats

Robust chemical or mechanical irritation of the colon of neonatal rats leads to chronic visceral hypersensitivity. The clinical and physiologic relevance of such noxious stimulation in the context of human irritable bowel syndrome is questionable. The aims of this study were to determine whether mild chemical irritation of the colon of neonatal rats produced persistent changes in visceral sensitivity and to evaluate the role of transient receptor potential vanilloid 1 (TRPV1) in the initiation and maintenance of visceral hypersensitivity. Ten-day-old rat pups received an intracolonic infusion of 0.5% acetic acid in saline. TRPV1 inhibitors were administered 30 minutes before acetic acid sensitization. Sensitivity of the colon to balloon distention (CRD) in adults was measured by grading their abdominal withdrawal reflex and electromyographic responses. In adult rats, TRPV1 antagonist was injected intraperitoneally 30 minutes before CRD. Neonatal acetic acid treatment resulted in higher sensitivity to CRD in adult rats compared with controls in the absence of histopathologic signs of inflammation. Treatment of colons of adult rats with acetic acid did not produce persistent sensitization. Antagonism of the TRPV1 before neonatal administration of acetic acid and after established visceral hypersensitivity attenuated sensitivity to CRD. TRPV1 expression was increased in dorsal root ganglia-containing colon afferent neurons. We have described a new model for persistent colonic sensory dysfunction following a transient noxious stimulus in the neonatal period and a potentially important role for TRPV1 in initiation and maintenance of persistent visceral hypersensitivity.

The Role of PKCδ and PKCε in the Neonatal Rat Colon in Response to Hypoxia Challenge

Previous studies have determined that, in response to bacterial endotoxin, the colonic mucosa of the 10-d-old neonatal rat was more susceptible to injury than was the colon of the 25-d-old mature animal. Furthermore, it is known that certain isoforms of protein kinase C (PKC), specifically PKCdelta and PKCepsilon, mediate intestinal inflammatory responses to specific challenges. Therefore, in the present study, we have examined the association between the activation of these PKC isoforms and the enhanced susceptibility to hypoxia-induced challenge. In response to exposure to a hypoxic environment (14% O2/86% N2, 30 min), the degree of inflammation and tissue damage was significantly greater in 10- than in 25-d-old rats. The injury in 10-d-old rats was associated with activation of PKCdelta and PKCepsilon as estimated by translocation of the isoform from cytosolic to membrane fraction of the tissue lysate. There was no activation of either isoform in colons from 25-d-old rats after hypoxia. Pretreatment of 10-d-old rats with epidermal growth factor (EGF) (10 microg/kg) but not 16,16 dimethyl prostaglandin E2 (2 microg/kg) significantly reduced the extent of colonic injury, whereas neither agent was able to exert significant protection of the colonic mucosa of 25-d-old rats. PKC activation associated with hypoxia was not evident after EGF treatment in 10-d-old rats. In 25-d-old rats, prostaglandin E2 treatment was linked with an activation of PKCepsilon only. In conclusion, these data suggest that activation of PKCdelta and PKCepsilon is associated with the enhanced susceptibility to injury evident in suckling neonatal rat colon. EGF-mediated protection of the colon in these animals results in a removal of this PKC activation.

Enhanced susceptibility to LPS-induced inflammation in the neonatal rat colon: Role of neutrophils and IL-6

Enhanced susceptibility to LPS-induced inflammation in the neonatal rat colon: Role of neutrophils and IL-6

Enhanced susceptibility to LPS-induced inflammation in the neonatal rat colon: Role of neutrophils and IL-6

Enhanced susceptibility to LPS-induced inflammation in the neonatal rat colon: Role of neutrophils and IL-6

Cellular differentiation of absorptive cells in the neonatal rat colon: an electron microscopic study.

Cellular differentiation of the absorptive cells in the neonatal-suckling rat colon was investigated using horseradish peroxidase as a macromolecular tracer with a scanning or transmission electron microscope. Numerous villi were lined on oblique folds in an orderly fashion and smaller villi were lined between the folds in the proximal colon in 0- to 12-day-old rats. Three different types of absorptive cells were observed in the epithelium covering their villi: type I cells, type II cells, and type III cells. The type I cells were most numerous, and similar to typical absorptive cells of the adult rat large intestine. The type II cells endocytosed macromolecules into an apical endocytic membrane system without giant lysosome from the large intestinal lumen. The type III cells were characteristic of the well-developed endocytic membrane system, including a large supranuclear giant lysosome specialized for endocytosis, storage, and intracellular digestion as an ileal absorptive cell of the suckling rat. At no time was macromolecular tracer detected in the Golgi apparatus of the absorptive cells or in the intercellular space of the epithelium. These results suggest that the villial absorptive cells of the proximal colon have differentiated to three types at specific neonatal-suckling times. The type II and type III cells were not observed in the epithelium after the disappearance of the villi.

Role of mast cells, neutrophils and nitric oxide in endotoxin-induced damage to the neonatal rat colon.

1. The mechanisms involved in mediating bacterial endotoxin lipopolysaccharide (LPS)-induced injury in the colon of neonatal rat pups aged 10-12 days was examined. 2. Administration of LPS (3 mg kg(-1), i.p.) caused a time-related increase in the plasma concentration of rat mast cell protease-II (RMCP-II) which was attenuated dose-dependently, by the non-selective mast cell stabilizer doxantrazole (0.05-5 mg kg(-1), i.p.). The selective connective tissue mast cell stabilizer ketotifen (5-25 mg kg(-1), i.p.) was without effect at the lower dose and had only a limited inhibitory effect at the higher dose. 3. In addition, doxantrazole (5 mg kg(-1), i.p.) inhibited mast cell degranulation in response to LPS in sections of neonatal rat colon, but ketotifen (5 mg kg(-1), i.p.) was without effect. 4. The increase in plasma RMCP-II concentration in response to LPS treatment preceded increases in tissue myeloperoxidase (MPO) activity, inducible nitric oxide synthase (iNOS) activity and tissue lipid peroxidation. These events were all attenuated by pretreatment with doxantrazole (5 mg kg(-1), i.p.), antineutrophil serum (100 microl kg(-1), i.p.), dexamethasone (2 mg kg(-1), i.p.) and the selective iNOS inhibitor, aminoguanidine (25 mg kg(-1), i.p.). 5. In addition, lipid peroxidation was inhibited by pre-administration of the antioxidant enzymes superoxide dismutase (2000 u kg(-1), i.p.) and catalase (2000 u kg(-1), i.p.), the xanthine oxidase inhibitor allopurinol (100 mg kg(-1), i.p.) and the peroxyl scavenger deferoxamine (10 mg kg(-1), i.p.), suggesting the involvement of reactive oxygen metabolites in the colonic injury. 6. These findings suggest that the sequence of events resulting in colonic damage in the neonatal rat following administration of LPS include mast cell degranulation, neutrophil infiltration, elevation in iNOS activity and subsequent lipid peroxidation.

Ontogeny of nitric oxide synthase activity and endotoxin-mediated damage in the neonatal rat colon.

Nitric oxide (NO) is synthesized by most regions of the gastrointestinal tract and is an important regulator of mucosal function and integrity. In this study we examined the ontogenic appearance of constitutively expressed Ca2+-dependent NO synthase (cNOS) and inducible Ca2+-independent NO synthase (iNOS) activity, in the colon of neonatal rats. Furthermore, the susceptibility of the colon to damage after induction of iNOS activity following bacterial endotoxin treatment was also examined. Segments of distal colon were removed from either control rat pups (aged between 10 and 25 d) or from animals pretreated with the following agents: 1) Escherichia coli lipopolysaccharide [LPS; 3 mg/kg, intraperitoneally (i.p.), 4 h before sacrifice], 2) dexamethasone (2 mg/kg, i.p., 1 h before administration of LPS) or aminoguanidine (25 mg/kg, i.p., at the same time as LPS). NOS activity was measured via the conversion of L-[14C]arginine to L-[14C]citrulline. Samples of colon were assessed for damage by either light microscopy or by measurement of the malondialdehyde content to estimate lipid peroxidation. In untreated animals cNOS activity increased during the first 20 postnatal days and fell postweaning at 25 d. LPS treatment resulted in a significant increase in iNOS activity in all age groups examined, with maximal activity occurring between 10 and 15 d of age. This coincided with the greatest histologic damage score and lipid peroxidation. Dexamethasone or aminoguanidine attenuated the effects of LPS suggesting the involvement of iNOS in these responses. These data suggest that colonic cNOS activity in the neonatal rat may be important during development and maturation of that tissue. Furthermore, the colon of the preweaned rat is more susceptible to the detrimental effects of LPS-induced NO production than the colon of postweaned animals.

The expression of soluble and cell-bound α2,6 sialyltransferase in human colonic carcinoma CaCo-2 cells correlates with the degree of enterocytic differentiation

alpha 2,6 sialyltransferase towards the N-acetyllactosaminyl sequence (alpha 2,6 ST, E.C. 2.4.99.1) is one of the major sialyltransferases in human colonic cells; it strongly increases in human colorectal tumors and is largely expressed in fetal and neonatal rat colon. In this study we demonstrate that human colon carcinoma CaCo-2 cells, which differentiate spontaneously into enterocytes when maintained confluent for several days, exhibit a very high expression of alpha 2,6 ST both in the cell-bound and soluble form. When the CaCo-2 cells were cultured on porous membranes the soluble alpha 2,6 ST was mainly detected in the medium collected from the chamber corresponding to the basolateral face of the monolayer. The soluble alpha 2,6 ST could be concentrated and purified from the alpha 2,3 sialyltransferase by affinity chromatography on Blue Sepharose.

Potassium secretion by neonatal rat distal colon.

Electrogenic K+ secretion across the distal colon of young rats was investigated by measuring the sensitivity of the short-circuit current to Ba2+ added to the mucosal side of the tissue. Ba2+-sensitive short-circuit current (IBasc) was high during the suckling and weaning periods but very low in adult animals. Increasing the mucosal K+ concentration was accompanied by the inhibition of the serosa-to-mucosa IBasc and the induction of the mucosa-to-serosa IBasc. The IBasc was decreased by serosal omission of either Na+ or Cl- as well as by serosal addition of furosemide or ouabain. Mucosal omission of Na+ did not change IBasc. By increasing the plasma level of aldosterone (low-sodium diet) IBasc rose by 95% whereas treatment decreasing this level (high-sodium diet) reduced IBasc by 76%. Bilateral adrenalectomy lowered IBasc by 59% and treatment of adrenalectomized rats with deoxycorticosterone acetate prevented the reduction of IBasc. Tetraethylammonium and quinidine had similar effects on Isc as Ba2+. These data are consistent with the presence of a high level of K+ secretion in the distal colon of neonatal rats. This secretory pathway is electrogenic and independent of Na+ absorption. It appears to be mediated by the Na-K-ATPase as well as a furosemide-sensitive Na-Cl or Na-Cl-K cotransport on the basolateral side and by Ba2+-sensitive K+ conductive pathways on the mucosal side. The results suggest that this K+ secretion can be regulated by mineralocorticoids. The mineralocorticoids are necessary for "stimulated" K+ secretion but they are not essential for maintaining "basal" K+ secretion.

Amiloride-sensitive sodium transport of the rat distal colon during early postnatal development.

To evaluate developmental changes in colonic sodium transport, the sensitivity of the transepithelial potential and short-circuit current to amiloride was investigated. The amiloride-sensitive short-circuit current (IscNa), which represents the electrogenic sodium transport through Na+ channels, rose significantly from day 5, reached a peak on day 10, and entirely disappeared after weaning. The maximum rate of electrogenic, amiloride-sensitive sodium transport was 12.0 microEq/cm2 X h. The IscNa was suppressed by adrenalectomy and/or premature weaning but not by a mineralocorticoid antagonist, spironolactone. On the contrary, treatments which increase aldosterone levels in vivo (low-sodium diet, furosemide-induced natriuresis, high dietary intake of potassium) stimulated the IscNa. The effect of adrenalectomy increased during postnatal development. The sensitivity of IscNa to aldosterone was highest at the end of the weaning period. High-sodium diet, which causes a decrease in circulating aldosterone, was associated with a partial inhibition of IscNa (P less than 0.016). These data suggest that the distal colon of neonatal rats can transport sodium via an electrogenic, amiloride-sensitive mechanism and that adrenocortical hormones exert the main regulatory control of this pathway.