Akihiro Nakama

Disturbed intestinal movement, bile reflux to the stomach, and deficiency of c-kit-expressing cells in Ws Ws mutant rats

BACKGROUND & AIMS Interstitial cells of Cajal (ICCs) are believed to initiate the basic contractile activity of the gastrointestinal tract. Because ICCs in the intestine of mice express c-kit receptor tyrosine kinase and because rats are more commonly used than mice for pathophysiological investigations of the gastrointestinal tract, the number of the c-kit messenger RNA-expressing cells was compared with gastrointestinal movement in rats. METHODS The c-kit messenger RNA-expressing cells were detected by in situ hybridization. The autonomous contraction of excised segments of the ileum was recorded. The function of the pyloric sphincter was evaluated by measuring the content of bile acids in the stomach. RESULTS The c-kit messenger RNA-expressing cells were not detectable in the stomach of Ws/Ws mutant rats with a small deletion at the tyrosine kinase domain of c-kit, and the number of c-kit messenger RNA-expressing cells decreased to 7% that of normal control rats in the ileum of Ws/Ws rats. The contractile activity of the ileum was apparently impaired, and the content of bile acids in the stomach was significantly increased in Ws/Ws rats. CONCLUSIONS The abnormalities in the ileal movement and pyloric sphincter function in Ws/Ws rats were attributable to the deficiency of c-kit messenger RNA-expressing cells.

RGM1, A CELL LINE DERIVED FROM NORMAL GASTRIC MUCOSA OF RAT

The epithelium of gastric mucosa undergoes constant renewal. However, the mechanisms of regulation of epithelial cell proliferation and differentiation in the gastric mucosa are poorly understood. Many investigators have used primary cultures of gastric mucosal cells to investigate cell physiology of gastric mucosal epithelium (5,8,14). Because this technique resulted in mixed cell populations of the stomach (5,15), the results may be influenced by complex cell-tocell interactions and are difficult to interpret. For this reason, a cell line made of a single type of cells is more ideal for precise investigation of proliferation or differentiation of gastric mucosal cells. However, cell lines from normal gastric mucosa have not been established. Recently, Matsui and Ohno (in preparation) established a new cell line, RGM1 (rat gastric mucosal cell first), from a rat glandular stomach by a method modified from Matsuoka et ah (7). In brief, the stomach was harvested from an anesthetized Wistar rat aged 4 wk and inverted. After washing the mucosa with phosphate-buffered saline (PBS) at 4 ~ C, the inverted pouch was immersed in a 0.2% pronase E solution at 37 ~ C. The solution was changed every 15 rain and centrifuged to collect the exfoliated gastric cells. Thereafter, the c.ells were washed twice with PBS, and cultivated in a 1:1 mixture of Dulbeccos modified Eagles medium (DMEM) and Hams F-12 medium supplemented with 20% fetal calf serum (FCS). When the cells were passaged to the 10th generation, this cell line was named RGM1. We examined character and usefulness of RGM1 cells using 3 0 4 0 times passaged cells. RGM1 cells are homogeneous epitheliallike cells with large oval nuclei and a polygonal shape (Fig. 1 A). They grow as monolayers of closely opposed cells, and many granules are visible in the cytoplasm. These cells were stained with periodic acid-Schiff (PAS) reagent and were negative for Bowie staining for chief cells (3) and succinic dehydrogenase activity for parietal cells (9). The cells were positive for cytokeratin in cytoplasm by immunohistochemistry. Transmission electron microscopy demonstrated that RGM1 cells had some microvilli on the surface. In the cytoplasm, a few homogeneous, dense cytoplasmic granules, presumably secretory granules, were observed. Junctional complexes were seen at the cell boundaries (Fig. 1 B and C). These data indicate that RGM 1 cells are epithelial in origin and like gastric mucous epithelial cells or mucous neck cells. RGM1 cells grew in an exponential manner with a population doubling time of 15.7 h, with saturation density of 1.97 + 0.38 X

Endogenous Nitric Oxide Modulates Ethanol-Induced Gastric Mucosal Injury in Rats

BACKGROUND/AIMS Endothelium-derived relaxing factor regulates vascular tone via vasodilation. The relative contribution of endogenous nitric oxide to the pathophysiology of ethanol-induced gastric mucosal microcirculatory disturbances was investigated in anesthetized rats. METHODS Macroscopic and microscopic gastric mucosal damage and gastric mucosal hemodynamics including blood flow and hemoglobin oxygen saturation (ISO2) were assessed by pretreatment with a specific NO synthase inhibitor, N omega-nitro-L-arginine (L-NNA), before and after intragastric administration of ethanol. RESULTS Pretreatment with L-NNA significantly increased macroscopic (7.7-fold) and microscopic damage caused by 30% ethanol. Concurrent administration of L-arginine, but not D-arginine, significantly reduced the increase in mucosal damage. Similar results were obtained with 60% ethanol. Pretreatment with L-NNA decreased both mucosal blood flow and ISO2 in the basal period and enhanced decreases in both mucosal blood flow (2.7-fold) and ISO2 (4.3-fold) induced by 30% ethanol compared with controls. Concurrent administration of L-arginine, but not D-arginine, significantly inhibited the effect of L-NNA on blood flow and ISO2 in the basal period as well as after intragastric administration of 30% ethanol. CONCLUSIONS Endogenous NO modulates ethanol-induced gastric mucosal injury through the regulation of gastric mucosal microcirculation.

Expression of the cyclooxygenase-2 gene in gastric epithelium.

This study examined the mRNA level of cyclooxygenase-2 (cox-2) in a rat gastric mucosal cell line after growth stimulation in vitro and in rat gastric mucosa before and after acid-induced injury in vivo. RGMI cells were stimulated with fetal calf serum in the in vitro study. Thereafter, the cox-2 mRNA level was examined by Northern analysis. Effects of NS-398, a specific inhibitor of cox-2, and indomethacin on prostaglandin production and cell proliferation were examined in RGM1 cells. In the in vivo study, rats were given 1 ml of 0.6 N hydrochloric acid into the stomach. The level of cox-2 mRNA was examined in rat gastric mucosa after acid administration. Cox-2 mRNA increased 20-60 min after growth stimulation in RGM1 cells. Both NS-398 and indomethacin suppressed prostaglandin production and cell proliferation after growth stimulation. Expression of cox-2 mRNA was observed 40 and 60 min after administration of 0.6 N HCl. Gastric lesions developed within 60 min after HCl administration and healed significantly within 48 h. The present study shows the expression of cox-2 in gastric epithelial cells in vitro and in gastric mucosa after injury in vivo. The results also suggest that cox-2 is involved in de novo synthesis of prostaglandins and cell proliferation in gastric epithelium.

Disturbed pyloric motility in Ws/Ws mutant rats due to deficiency of c-kit-expressing interstitial cells of Cajal.

Interstitial cells of Cajal (ICC) are believed to lnitlate the basic contractile activity of the gastrointestlnal tract. Interstitial cells of Cajal express c‐kit receptor tyroslne kinase and are deficient in Ws/Ws mutant rats with a small deletion of the c‐kit gene. As Ws/Ws rats show remarkable bile reflux to the stomach, the contraction pressure of the pylorus was compared between Ws/Ws and control +/+ rats. The contraction pressure of the pylorus was measured using a mlcrotransducer, which was Inserted through a pln‐hole in the anterlor wall of the stomach under anesthesla. The magnitude of bile reflux was estimated by measurlng the content of bile acids In the stomach. The c‐kit messenger RNA‐expressing cells were detected by in sltu hybrldlzatlon. Frequency and the maxlmum pressure of the contractlon were comparable between Ws/Ws and +/+ rats, but the duration of the contractlon was significantly shorter In Ws/Ws rats than In +/+ rats. The number of c‐kit messenger RNA‐expresslng ICC in the pylorus of Ws/Ws rats was 1.7% that of +/+ rats. The bile reflux observed in Ws/Ws rats was attributed to the decrease in the duration of the pyloric contraction, which appeared to result from the deficlency of c‐kit messenger RNA‐expressing ICC.

Roles of double-balloon endoscopy in the diagnosis and treatment of Crohn’s disease: a multicenter experience

BackgroundDouble-balloon endoscopy (DBE) examinations are not yet widely accepted as routine procedures for examining the small bowel of patients with Crohn’s disease (CD).AimTo evaluate the feasibility and usefulness of DBE for CD in tertiary-care hospitals.MethodsBetween July 2004 and September 2008, 1444 DBE procedures were performed for 704 patients in 6 tertiary-care hospitals. Patient profile, indication, diagnosis and treatment of DBE were evaluated using a multicenter database.ResultsDBE examinations were most frequently performed in 75 patients with CD, corresponding to 10.5% of all the patients examined by DBE. Fifty patients were diagnosed with CD before DBE, while DBE was performed for the diagnosis of 25 new CD patients. Small bowel lesions were often detected even when the terminal ileum was not involved. In the 75 patients, 21 patients were asymptomatic at the time of DBE examinations. Active inflammatory lesions were detected in 51.2% of the CD patients, and were even detected in 33.3% of the asymptomatic CD patients. The treatment was altered in 53.3% of the CD patients after the DBE evaluation. No severe complications were experienced.ConclusionsDBE procedures can be safely performed in patients with CD and should be considered for the precise evaluation of and to determine the treatment strategy for CD.

Flow cytometric analysis of cell proliferation kinetics during duodenal ulcer healing

Cell proliferation in the gastroduodenal mucosa of patients with duodenal ulcers was evaluated using flow cytometry. Forty patients with duodenal ulcers and 12 normal subjects were investigated. Biopsy samples were obtained during endoscopic examination and subjected to DNA analysis by flow cytometry. Thirty patients with duodenal ulcers were healed within 3 months with H2 blockers (tractable or responsive ulcers), whereas 10 patients did not respond to treatment (intractable ulcers). The percentage of cells at the DNA‐synthetic phase, an index of cell proliferation, was constant in the adjacent duodenal mucosa 2cm from ulcer margin and antral mucosa during duodenal ulcer healing. The index at the margin of tractable ulcers was elevated during the active stage (12.9 ± 1.3), peaked during the healing stage (15.4 ± 2.8) and returned to the same level at the scarring stage (10.9 ± 2.0) as normal controls (10.3 ± 1.7). However, the index was not elevated in intractable ulcers (10.3 ± 1.7 in the healing stage) and was smaller than in tractable ulcers. These data indicate that augmented mucosal cell proliferation at the ulcer margin plays an important role in duodenal ulcer healing and intractable ulcers are characterized by an abnormal failure to accelerate DNA synthesis to achieve ulcer repair.