Detection of MAPK Signal Transduction Proteins in an Ischemia/Reperfusion Model of Small Intestines

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 in conventional tissue sections. For immunohistochemistry, we have proposed that soluble molecules including cell-signaling proteins are well retained by our in vivo cryotechnique (IVCT). 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 IVCT. Under normal conditions, although ERK1/2 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 20 min ischemia, more pERK1/2 immunolocalization was detected in epithelial cells of the crypt part. In 60 min ischemia, however, its immunoreactivity was remarkably increased in wide areas of epithelial cells. In these 20 min and 60 min ischemia groups, another phosphorylated CREB was also immunostained in the nuclei of the same epithelial cell areas of pERK1/2. By ischemia 20 min reperfusion 60 min experiments, pERK1/2 immunointensity was reduced in the crypt areas. In 60 min ischemia with 60 min 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.