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 5 min ischemia, more pERK1/2 immunolocalization was detected in epithelial cells of the crypt part. Up to 60 min, the pERK1/2 immunoreactivity was remarkably increased in wide areas of epithelial cells. In the 20 and 60 min ischemia groups, phosphorylated CREB was also immunostained in the nuclei of the same epithelial cell areas of pERK1/2. In 20 min ischemia with 60 min reperfusion 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.

Immunohistochemical and Morphofunctional Studies of Skeletal Muscle Tissues with Electric Nerve Stimulation by <i>In Vivo</i> Cryotechnique

“…The present application of IVCT to morphological analyses of skeletal muscle tissues has clearly demonstrated the congestion of erythrocytes usually flowing in blood vessels along with the decrease of sarcomere length of contracted muscle fibers. Such erythrocyte congestion has already been observed in other organs of living mice under abnormal conditions, where blood circulation was considered to be impaired, because of heart-arrest conditions [ 17 , 28 ], ischemic model by blood vessel ligation [ 3 ] or thrombus formation by tumor cell metastasis [ 26 ]. It has been suggested that the blood flow into contracting muscle tissues is usually perturbed upon intensive muscle contraction, presumably due to the temporarily increased intramuscular pressure [ 1 , 10 ].…”

The Histochem Cell Biol conspectus: the year 2013 in review

“…Ischemia/reperfusion damage also occurs in other tissues, such as intestine. Chen et al (2013) have used their well-documented in vivo cryotechnique (IvCT), known to preserve soluble cellular components (such as signal transduction molecules) to investigate the involvement of the eRK1/2 signal transduction pathway after mouse intestinal ischemia/reperfusion by immunostaining for peRK1/2 and pCReB on frozen, freeze-substituted tissue sections. In control tissue sections, eRK immunoreactivity was widely observed in the cytoplasm of intestinal epithelial cells, whereas peRK1/2 was much more restricted in distribution to some epithelial cells present in the crypt region and at the tops of villi.…”