Segmental Difference of Mucosal Damage along the Length of a Mouse Small Intestine in an Ussing Chamber

Abstract

SummaryThe Ussing chamber is often used for in vitro investigations into the transport physiology of epithelia including the small intestine. However, the morphological and func tional integrity of the small intestine incubated in an Ussing chamber has been largely ignored. The present study attempted to compare the mucosal injury among different seg ments of the mouse small intestine when incubated in an Ussing chamber. To assess the functional damage, we measured changes in the transmural potential difference (PD) evoked by adding glucose to the mucosal solution and forskolin to the mucosal and serosal solutions. Morphological deterioration was assessed histologically. The villi in the duode num and proximal jejunum were morphologically damaged by 2h of incubation in an Ussing chamber and almost completely destroyed within 4h, while crypts remained intact. The villi were moderately damaged in the distal jejunum. In contrast, the integrity of the villi and crypts was maintained in the ileum for 4h. The basal PD and forskolin-induced PD were maintained up to 4h of incubation in all segments. On the other hand, the glucose induced PD was not apparent in the duodenum at 0h, and was gradually suppressed to zero in the proximal jejunum by 4h, although the glucose-induced PD was maintained for 4h in the ileum. The loss of villous epithelial integrity was correlated with the disappearance of the glucose-induced PD, while both the basal and forskolin-induced PD were retained, even in the tissue with disrupted villi. It is concluded that the mucosa of the proximal small intes tine was rapidly injured in the Ussing chamber, particularly in the villi, while the integrity of the mucosa of the distal small intestine was maintained for 4h. The glucose-induced PD, but not the basal or forskolin-induced PD, can be used as a marker of the villous integrity.

In Vitro Diffusion Barriers of the Mouse Jejunum in Ussing Chambers

“…1A and B), as reported previously ( 8 ). The lucifer yellow flux ( J LY ) from the mucosal to serosal side rapidly increased during the initial 1 h period and then slowly increased thereafter until 4 h after the start of incubation ( Fig.…”

“…The ileal whole-thickness preparations had previously been shown to maintain epithelial cells on the villus region, as well as the PD induced by glucose and forskolin during 4 h in the Ussing chambers (8). As is shown in Fig.…”

“…All procedures used in this study were performed in accordance with the "Guiding Principles for the Care and Use of Animals in the Field of E-mail: e-ina@nuas.ac.jp Physiological Sciences" published by the Physiological Society of Japan. The tissue preparation, Ussing chambers, and electrical measurements were essentially similar to those described previously ( 8 ). Briefly, male mice (30-40 g, 7-8 wk old, Std:ddY; Japan SLC, Hamamatsu, Japan) had free access to a conventional diet (MF; Oriental Yeast, Tokyo, Japan) until the time of the experiment.…”

“…A histological examination was performed on the tissue that had been incubated for 4 h with haematoxylin/eosin staining according to the routine method ( 8 ). Each tissue sample was fixed with 10% formalin in PBS at pH 7.1.…”

“…We have previously reported that mouse proximal jejunum, when incubated in Ussing chambers for 4 h, exhibited morphological deterioration of the villi with denudation of the epithelial cells, although the crypt structure remained intact ( 8 ). We have also demonstrated that, while the transmural potential difference (PD) induced by glucose was abolished under these conditions, the PD level resulting from the cAMP-induced activation of Cl Ϫ secretion was maintained.…”