Length measurement of cholinergic innervation in the tunica albuginea of rat testis

Abstract The testicular albuginea is not a simple receptacle that contains the testis parenchyma. It is a biologically active structure involved, among other things, in the progression of sperm from the seminiferous tubules to the rete testis. This function, attributed to the albuginea myofibroblasts’ contractile properties, is mediated by its vegetative innervation. Therefore, the quantification of innervation seems relevant to deepen the knowledge of the testicular envelope’s physiology. The albuginea contains cholinergic nerve fibers that are not very evident and with a marked directional arrangement (anisotropy). These structural characteristics require that the quantification of the fibers’ length be carried out using stereological measurements, which ensure the absence of bias in the process. The authors have developed stereological tools usually carried out on vertical VUR (vertical uniform random) sections in the present work. This approach’s novelty is that the albuginea is treated "in toto" as if it were a thick section (slice) on which a cycloid grid is applied through different optical planes. Thus, on a pilot study of three specimens, cholinergic nerve fibers’ length density was estimated, as well as the estimates of the coefficient of error of measurements and the biological variability of the sample. Furthermore, after estimating the albuginea volume, the mean of the fibers’ absolute length can be obtained. This stereological approach can be used after visualization of the immunostained nerve fibers for various markers, as long as the reagents’ penetration into the tissue is ensured. Moreover, it can be used in other biological structures, such as the intestine of small animals, which can also be processed as whole mounts and treated as thick VUR sections.