Innervation of the anal canal revisited through micro-CT imaging

Studies in anatomy are frequently performed through dissection and histology as investigative methods. These techniques are, however, destructive and struggle to capture the peripheral nerves over larger areas, such as the pelvis, and their trajectories thus remain elusive. This study aimed to explore the capabilities of non-destructive micro-CT in identifying small nerves in the periphery of the pelvic area and map them in human fetal development. Six human fetal pelvic samples were derived from the Dutch Fetal Biobank; samples were contrast enhanced with B-Lugol and scanned through micro-CT imaging. Scans were segmented using Avizo Amira software to quantify nerves entering the rectum by crossing the longitudinal muscle layer and visualize their trajectory in relationship to pelvic organs across development age range and sex. Results were verified with haematoxylin and eosin stained histological slices. The following conclusions were found: (1) Micro-CT is able to resolve nerves with a diameter larger than 60 μm in the periphery of the pelvic area in full fetal pelvises of 4 cm in size. (2) Both above and below the level of the levator ani, small lateral nerves enter the anal canal by penetrating the longitudinal muscle layer. (3) Males appear to have more crossing nerves than females. (4) A developmental shift was observed with more nerve crossings below the levator ani muscle in older fetuses. Micro-CT was successfully applied to scan human fetal pelvic samples to study nerves and provides a new way to analyse topographical nerve development. Microscale nerves cross to the anal canal at all levels, which should be taken into account when operating in this area.