Tissue Clearing and 3D Imaging of Gap Junctions in Rat Uterine Myometrium

The propagation patterns of electrical activity in the uterus are not well understood. However, the number and size of gap junctions between uterine smooth muscle cells towards the end of pregnancy increases, which may be a catalyst for the onset of the coordinated contractions required for successful labor. In non-pregnancy, there is some evidence that the number and distribution of gap junctions varies throughout the reproductive hormonal cycle. Gap junctions are hemichannel proteins in the cell membrane which allow fast electrical and ionic communication between cells. In this study, we outline the use of state-of-the-art tissue clearing, labelling, and imaging techniques in order to image connexin-43 gap junction in rat uterine tissue. We used stage-scanning line confocal microscopy to image a 3D volume of uterine tissue, and quantified the change in fluorescence of connexin-43 along the longitudinal length of the uterine horn. We observed an increased fluorescence signal at the cervical end of the uterine horn, compared with the ovarian end. This pilot study shows the efficacy of this approach for analyzing distributions of gap junctions in rat uterine smooth muscle.