Spatiotemporal mapping of the motility of the isolated chicken caecum

We studied the caecal contractile activity of the chicken (Gallus gallus) using single caeca that had been cannulated at their proximal and distal ends, and in paired caeca, maintained in situ on excised segments of gut that were cannulated at the colonic and small intestinal ends. Longitudinal and circular contractile patterns were characterised using high-definition spatiotemporal mapping. Low amplitude longitudinal contraction waves of frequency 14.1 cycles/min occurred in the absence of major contractile events. These were termed fast phasic and appeared to be mediated by slow waves. The nature of major spontaneous contractions occurring in the single caecum varied with the level of caecal distension. Type A contractions occurred when the caecum was not distended, originated from variable sites and propagated in both directions. Type B or C contractile events occurred when the caecum was moderately or fully distended, originated from a predominantly distal site and propagated proximally. On diameter maps, each type B event comprised a succession of contractions which had similar propagation speeds, frequency and direction to fast phasic contractions. Type C events were comprised of a succession of higher amplitude contractions with no appreciable propagation. Perfusion of saline via the colon resulted in fluid entering both caeca and the onset of aborad contractions in their proximal canals. Saline was also seen to flow between caeca during contractile events however no saline was seen to enter the small intestine as has been postulated by other workers.     

Spatiotemporal mapping of ex vivo motility in the caecum of the rabbit

We used high definition radial, strain rate and intensity spatiotemporal mapping to quantify contractile movements of the body and associated structures of the rabbit caecum when the terminal ileum was being perfused with saline at a constant rate. This perfusion caused gradual distension of the caecum as a result of relative restriction of outflow from the ampulla caecalis. The body of the caecum exhibited two patterns of motility that appeared autonomous, i.e. occurred independently of any contractile activity at the inlet or outlet. Firstly, the pattern that we termed ladder activity consisted of an orderly sequential contraction of bundles of axially oriented circular muscle between the spiral turns of longitudinal muscle and proceeded either from base to tip or from tip to base at a similar frequency and velocity. Secondly, less-localised, rapidly propagating synchronous contractions of both circular and longitudinal muscle, which were more common when the caecum was distended, that were termed mass peristalsis. Movements of the ileum and sacculus rotundus occurred at the same frequency and were broadly coordinated. Distension of the distal sacculus occurred synchronously with contraction of the ileum and did not propagate in an orderly manner across the structure, i.e. was instantaneous. This pattern was consistent with hydrostatic distension. Contractions propagated through the ampulla caecalis in either an orad or an aborad direction at a similar frequency to, and broadly correlated with, those in the ileum. The frequencies of distension of the sacculus and of contraction in the ileum and ampulla were momentarily augmented during mass peristalsis. The authors conclude that there was some coordination between the contractile activity of the terminal ileum and the caecal ampulla during periods of ongoing inflow from the ileum and between these structures and the caecum during mass peristalsis.     

Myeloelectric complex of the small intestine and variations in the intestinal motility in sheep]

The motor pattern of the small intestine of the sheep fed ad libitum is characterized by the regular occurrence of myoelectric complexes comprising a phase of regular and irregular spiking activities. Each complex is propagated along the ovine small intestine at a mean velocity of 17 cm/min and originated on the duodenal bulb at 70-100 mn intervals. Reduction and increase in duration of the phase of irregular spiking activity of the complex occurred during fasting and overfeeding respectively. Reduction in spiking activity is paralleled by an increased velocity of propagation whilst a lower migration and a reduced number of the complexes are characteristic of overfeeding. It is concluded that duration, number and velocity of propagation of the myoelectric complexes are adaptative factors in changes of the intestinal flow rate.     

Intraepithelial lymphocytes express junctional molecules in murine small intestine

Intestinal intraepithelial lymphocytes (IEL) that reside at basolateral site regulate the proliferation and differentiation of epithelial cells (EC) for providing a first line of host defense in intestine. However, it remains unknown how IEL interact and communicate with EC. Here, we show that IEL express junctional molecules like EC. We identified mRNA expression of the junctional molecules in IEL such as zonula occludens (ZO)-1, occludin and junctional adhesion molecule (JAM) (tight junction), beta-catenin and E-cadherin (adherens junction), and connexin26 (gap junction). IEL constitutively expressed occludin and E-cadherin at protein level, while other T cells in the thymus, spleen, liver, mesenteric lymph node, and Peyer's patches did not. Gammadelta IEL showed higher level of these expressions than alphabeta IEL. The expression of occludin was augmented by anti-CD3 Ab stimulation. These results suggest the possibility of a novel role of IEL concerning epithelial barrier and communication between IEL and EC.     

Ghrelin stimulates motility in the small intestine of rats through intrinsic cholinergic neurons

BACKGROUND AND PURPOSE: Ghrelin is a peptide discovered in endocrine cells of the stomach. Since ghrelin mRNA expression and plasma levels are elevated in the fasting state, we investigated the effects of ghrelin on the interdigestive migrating myoelectric complex (MMC) in the small intestine in vivo and compared with motor effects of ghrelin in vitro. Methods: Sprague-Dawley rats were supplied with a venous catheter and bipolar electrodes in the duodenum and jejunum for electromyography of small intestine in awake rats. In organ baths, isometric contractions of segments of rat jejunum were studied. RESULTS: Ghrelin dose-dependently shortened the MMC cycle length at all three recording points. At the duodenal site, the interval shortened from 17.2+/-2.0 to 9.9+/-0.8 min during infusion of ghrelin (1000 pmol kg(-1) min(-1)) and at the jejunal site from 17.5+/-2.2 to 10.5+/-0.8 min. Ghrelin contracted the muscle strips with a pD2 of 7.97+/-0.47. Atropine (10(-6) M) in vitro and (1 mg kg(-1)) in vivo blocked the effect of ghrelin. CONCLUSION: Ghrelin stimulates interdigestive motility through cholinergic neurons. Ghrelin also stimulates motility, in vitro, suggesting that ghrelin receptors are present in the intestinal neuromuscular tissue and mediate its effects via cholinergic mechanisms.     

Anticarcinogenesis pathways activated by bovine lactoferrin in the murine small intestine

Oral administration of bovine lactoferrin (bLF) inhibits carcinogenesis in the colon and other organs in rats, and lung metastasis in mice. A likely mechanism by which bLF mediates its anticarcinogenesis effects is by enhanced expression of cytokines and subsequent activation of immune cells. Oral administration of bLF enhances expression of interleukin-18 (IL-18) mRNA in the mucosa of the small intestine of mice. Importantly, the pepsin hydrolysate of bLF (bLFH) also induced expression of IL-18 mRNA in the mouse small intestine and a peptide produced by pepsin digestion of bLF, bovine lactoferricin (bLFcin), induced expression of mature IL-18 in organ culture. In addition to IL-18, bLF and bLFcin both induced significant increases in caspase-1 activity in peritoneal macrophages and in organ cultures. The increase of mature IL-18 by macrophages was inhibited by caspase-1 inhibitor: caspase-1 is known to cleave the proform of IL-18 to produce active mature IL-18. Finally, bLF also induced expression of IFNgamma by peritoneal macrophages. Importantly, in IFNgamma knockout (GKO) mice, bLF administration resulted in increased expression of caspase-1 protein, but induction of IL-18 mRNA, caspase-1 activity, and mature IL-18 was not observed. These results indicate that orally administered bLF can induce expression of IFNgamma and caspase-1 in the small intestine. IFNgamma in turn increases expression of target genes, including IL-18. Active caspase-1 then cleaves pro-IL-18 to generate mature IL-18. Thus, bLF activates an effector pathway mediated by IFNgamma, caspase-1, and IL-18. We also show that ingested bLF is able to activate more than a single effector pathway. For example, in GKO mice while bLF administration could not activate the IFNgamma/caspase-1/IL-18 effector pathway, it was able to inhibit tumor growth and metastasis by activation of an IFNalpha/IL-7 effector pathway.     

The effect of extragastric vagotomy on gastric emptying and motility of the small intestine

Selective parasympathetic denervation of small and some large intestine has been performed in dogs. Chronic experiment on these dogs has revealed that this operation: has no effect on frequency and amplitude of intestine contractions during the first phase of the digestive process but it is accompanied by significant relaxation of the motor intestine activity in the second phase, causes a retardation of the rate of evacuation from stomach by 56.0% in dogs subjected to extragastric vagotomy as well as pH of chyme in the duodenum by 1-1.5 units above the norm.     

Expression of the chloride channel ClC-2 in the murine small intestine epithelium

The chloride channel ClC-2 has been implicated inneonatal airway chloride secretion. To assess its role in secretion by the small intestine, we assessed its subcellular expression in ilealsegments obtained from mice and studied the chloride transport properties of this tissue. Chloride secretion across the mucosa ofmurine ileal segments was assessed in Ussing chambers as negative short-circuit current (I_sc). If ClC-2contributed to chloride secretion, we predicted on the basis ofprevious studies that negative I_sc would bestimulated by dilution of the mucosal bath and that this response woulddepend on chloride ion and would be blocked by the chloride channelblocker 5-nitro-2-(3-phenylpropylamino) benzoic acid but not by DIDS.In fact, mucosal hypotonicity did stimulate a chloride-dependent changein I_sc that exhibited pharmacological propertiesconsistent with those of ClC-2. This secretory response is unlikely tobe mediated by the cystic fibrosis transmembrane conductance regulator(CFTR) channel because it was also observed in CFTR knockout animals.Assessment of the native expression pattern of ClC-2 protein in themurine intestinal epithelium by confocal and electron microscopy showedthat ClC-2 exhibits a novel distribution, a distribution patternsomewhat unexpected for a channel involved in chloride secretion.Immunolabeled ClC-2 was detected predominantly at the tight junctioncomplex between adjacent intestinal epithelial cells.

     

Differential expression of glutathione S-transferase isoenzymes in murine small intestine and colon

Glutathione (GSH) S-transferase (GST) isoenzymes of the small intestine and colon of female A/J mice have been purified and characterized to determine their interrelationships with other murine GSTs. Cytosolic GST activity in the small intestine was at least due to six isoenzymes with isoelectric points (pI) of 9.5, 9.3, 9.1, 8.5, 6.2 and 5.5. Small intestine isoenzymes with pI values of 9.5, 9.3, 8.5, and 6.2 were identical to the mGSTA1-1 (Alpha class), mGSTP1-1 (Pi class), mGSTM1-1 (Mu class) and mGSTA4-4 (Alpha class), respectively, of other A/J mouse tissues on the basis of their reverse-phase HPLC elution profile, immunological cross-reactivity and/or N-terminal region amino acid sequence. Even though GST9.1 of the small intestine cross-reacted with the antibodies raised against Pi class GST, reverse-phase HPLC and N-terminal amino acid sequence analyses suggested that this isoenzyme may be structurally different from mGSTP1-1 as well as mGSTP2-2. Likewise, despite immunological similarity with the Mu class GSTs, small intestine GST5.5 appeared to be different from other Mu class murine GSTs characterized previously. Cytosolic GST activity in the colon was mainly due to four isoenzymes with pI values of 9.8, 9.4, 6.6 and 5.8. While the identity of colon GST6.6 could not be established due to its low abundance, GST9.8, GST9.4 and GST5.8 were identical to mGSTP1-1, mGSTM1-1 and mGSTA4-4, respectively, of other A/J mouse tissues including the small intestine. Isoenzymes corresponding to small intestine GST9.1 and GST5.5 could not be detected in the colon. The results of the present study indicate that the small intestine of female A/J mice is better equipped for protection against toxic effects of electrophiles than colon.     

Epithelial apoptosis in mechanistically distinct methods of injury in the murine small intestine

Gut epithelial apoptosis is involved in the pathophysiology of multiple diseases. This study characterized intestinal apoptosis in three mechanistically distinct injuries with different kinetics of cell death. FVB/N mice were subjected to gamma radiation, Pseudomonas aeruginosa pneumonia or injection of monoclonal anti-CD3 antibody and sacrificed 4, 12, or 24 hours post-injury (n=10/time point). Apoptosis was quantified in the jejunum by hematoxylin and eosin (H&E), active caspase-3, terminal deoxynucleotidyl transferase dUTP-mediated nick end labeling (TUNEL), in situ oligoligation reaction (ISOL,) cytokeratin 18, and annexin V staining. Reproducible results were obtained only for H&E, active caspase-3, TUNEL and ISOL, which were quantified and compared against each other for each injury at each time point. Kinetics of injury were different with early apoptosis highest following radiation, late apoptosis highest following anti CD3, and more consistent levels following pneumonia. ISOL was the most consistent stain and was always statistically indistinguishable from at least 2 stains. In contrast, active caspase-3 demonstrated lower levels of apoptosis, while the TUNEL assay had higher levels of apoptosis in the most severely injured intestine regardless of mechanism of injury. H&E was a statistical outlier more commonly than any other stain. This suggests that regardless of mechanism or kinetics of injury, ISOL correlates to other quantification methods of detecting gut epithelial apoptosis more than any other method studied and compares favorably to other commonly accepted techniques of quantifying apoptosis in a large intestinal cross sectional by balancing sensitivity and specificity across a range of times and levels of death.     

Estimates of the number of clonogenic cells in crypts of murine small intestine

There is a proliferative cell hierarchy in the mouse intestinal crypt with ancestral stem cells which can regenerate all components of the lineage after injury (clonogenic cells). The number of these clonogenic or regenerative cells per crypt can be estimated from radiobiological experiments where doses of radiation are used to kill cells and ablate crypts. Various approaches can be adopted which provide different estimates of this number of cells. One of the conventional approaches used in the past provided estimates of about 70-80 clonogenic cells per crypt (i.e. about 50% of the proliferative or 30% of all crypt cells). A technically simpler approach has recently been suggested. This has been used here to provide many independent estimates of the number of crypt clonogenic cells. These suggest about 32 clonogenic cells exist per crypt i.e. about half the previous estimate and about twice the number of putative "functional" stem cells (those which operate as stem cells in the normal steady-state crypt). The reasons for the differences are discussed. The new estimates are compatible with the hypothesis that the crypt contains a ring of about 16 functional stem cells which are expected to be clonogenic, besides which there is a second ring of 16 clonogenic cells which represent early transit cells (the immediate daughters of the stem cells) which can act as clonogenic cells if required after radiation injury.