Detection of MAPK signal transduction proteins in an ischemia/reperfusion model of mouse intestine using in vivo cryotechnique

Abstract

Intestinal ischemia and ischemia-reperfusion rapidly progress to tissue destruction and reconstruction of functional organs. To date, precise immunolocalizations and the timing of appearance of cell signaling components under such conditions have not been well visualized. Mitogen-activated protein kinase (MAPK) signal transduction pathways have been reported to be activated under various types of cell damage, and cyclic AMP response element-binding protein (CREB) was directly phosphorylated with various cellular stimuli. In this study, both the expression and the immunolocalization of ERK1/2, a member of the MAPK family, were examined in mouse intestinal tissues by in vivo cryotechnique, which is useful to retain soluble molecules including cell signaling molecules. Under normal conditions, although ERK was widely immunolocalized in the cytoplasm of epithelial cells, phosphorylated (p) ERK1/2 was slightly detected in a small amount of epithelial cells in crypt and top parts of the villi. In 5min ischemia, more pERK1/2 immunolocalization was detected in epithelial cells of the crypt part. Up to 60min, the pERK1/2 immunoreactivity was remarkably increased in wide areas of epithelial cells. In the 20 and 60min ischemia groups, phosphorylated CREB was also immunostained in the nuclei of the same epithelial cell areas of pERK1/2. In 20min ischemia with 60min reperfusion experiments, pERK1/2 immunointensity was reduced in the crypt areas. In 60min ischemia with 60min reperfusion, however, it was still strongly immunolocalized in epithelial cells of the crypts. Thus, rapidly changing ERK1/2 phosphorylation was visualized in the intestinal epithelial stem cells of mouse small intestine.