Visualization of interstitial cells of Cajal in the mouse colon by vital staining

Abstract

Interstitial cells of Cajal (ICCs) are believed to be a major element in generating the spontaneous rhythm of the gastrointestinal tract. A prominent problem in the study of these cells has been the difficulty in observing them in intact tissues. We used the lipophilic dye DiI to stain ICCs in the submucosal-circular muscle border of freshly dissected mouse colon. The placement of small DiI crystals in this area resulted in the labeling of ICC-like cells. Two main morphological cell types, viz., bipolar and multipolar, were noted. Bipolar cells had two primary processes emerging from the poles of an elongated soma. The mean length of these processes was 78.7 microm. These cells constituted 42.3% of the sample (n=105). Multipolar cells (54.3% of total) had a less elongated soma and extended 3-6 main processes whose mean length was 56.3 microm. These processes showed no preferred direction. The length of the primary processes of bipolar cells was 40% greater than that of multipolar cells (P<0.02). Three cells (2.9%) had only one primary process. The DiI stain could be converted into a stable electron-opaque product. Electron-microscopic observations showed that these cells had the typical appearance of ICCs reported in previous studies. This staining method should be useful for physiological investigations of ICCs in gastrointestinal tissues.

Guide to the identification of interstitial cells of Cajal

“…One such dye of low cell toxicity may be the lipophilic DiI (see Hanani et al, 1999). Recently, it was demonstrated in the mouse colon that DiI under appropriate conditions labeled cells that under the TEM were identified as ICC (Hanani et al, 1998). The ultrastructure of the labeled cells was preserved, together with contacts with nerves and smooth muscle cells.…”

In situ recording from gut pacemaker cells

“…Hanani et al observed ICC in living tissue and showed its network properties using Nomarski optics and Lucifer yellow injection [5]. The present method expands on this technical achievement by the development of a preparation that allows patch clamp recording from ICC, which can now also be applied to other auxiliary cells such as glial cells [6].…”

Visualization of interstitial cells of Cajal in living, intact tissues

“…The cells were found along the submucosal border of the circular muscle layer in apposition to smooth muscle cells and nerve endings. The cells had all the structural characteristics of ICC: they were rich with caveolae and mitochondria and had discontinuous basal lamina (see Hanani et al, 1998).…”

“…Staining with DiI was performed as described previously (Hanani et al, 1998). Briefly, DiI (Molecular Probes) was dissolved at 3% in DMSO; this solution was diluted to 0.15% DiI in 96% ethanol and was further diluted to 0.003% in Krebs solution, causing precipitation of the dye as small crystals.…”

“…For long-term storage, the tissues were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.3) for 30 minutes, and were subsequently mounted on a glass slide and coverslipped in 50% glycerol in PBS. The DiI stain was converted to an insoluble product by photoconversion as described by Hanani et al (1998). Briefly, the tissue was fixed in glutaraldehyde (1.6%) in sodium cacodylate buffer (0.05 M, pH 7.4) for 4 hours, and it was then washed in cacodylate buffer (0.1 M, pH 7.4) containing 0.22 M sucrose.…”