Smooth Muscle Organization and Nerves in the Rat Vagina: A First Look Using Tissue Clearing and Immunolabeling

Abstract

Smooth muscle fibers within the vagina, as well as the nerve fibers that contribute to their control mechanisms, are important for the maintenance and alteration of vaginal length and tone. Vaginal smooth muscle (VaSM) is typically described as being arranged into two distinct concentric layers: an inner circular muscular layer and an outer longitudinal muscular layer. However, the distribution of VaSM oriented in the longitudinal direction (LD) and circumferential direction (CD) has never been quantified. In this study, tissue clearing and immunohistochemistry were performed so that the VaSM, and surrounding nerves, within whole rat vaginas ([Formula: see text]) could be imaged without tissue sectioning, preserving the three-dimensional architecture of the organs. Using these methods, the vagina was viewed through the full thickness of the muscularis layer, from the distal to the proximal regions. The VaSM orientation in the proximal and distal regions and the VaSM content along the LD and CD were quantified. Additionally, a qualitative assessment of vaginal nerves was performed. When compared using a permuted version of the Watson [Formula: see text] test, the orientation of VaSM in the proximal and distal regions were found to be significantly different in 4 of the 6 imaged rat vaginas ([Formula: see text]). While the distal vagina contained a similar amount of VaSM oriented within [Formula: see text] of the LD and within [Formula: see text] of the CD, the proximal vagina contained significantly more VaSM oriented towards the LD than towards the CD. Nerve fibers were found to be wavy, running both parallel and perpendicular to vascular and non-vascular smooth muscle within the vagina. Micro-structural analyses, like the one conducted here, are necessary to understand the physiological function and pathological changes of the vagina.

Keywords: Confocal microscopy; Immunohistochemistry; Innervation; Smooth muscle; Tissue clearing; Vagina.