2. Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, ON N1G2W1, Canada.
Background: Mechanoreceptors at the terminal ends of afferent nerves in the foot sole are critical for gait and balance. While previous studies have mapped afferent receptive fields and innervation densities across the foot sole using microneurography, the location and distribution of mechanoreceptors in the foot sole skin is unknown. In humans, histological investigations are limited to specimens obtained during amputation or embalmed tissues. These can be difficult to obtain and the latter poorly preserved at the extremity of the foot sole. Mice serve as an appropriate animal model to study mechanoreceptors whose function is conserved across mammalian species, however serial sectioning of intact mouse hind paws is challenging due to the dense bones and variety of soft tissues present. Consequently, digits and footpads are typically isolated for analysis and inferences made regarding mechanoreceptor density across the whole hind paw. There are no published protocols for serial histological sectioning of mouse hind paws and attempts at serially sectioning intact rat hind paws have been unsuccessful.
Methods: We conducted eight experiments optimizing tissue preparation, chemical processing and sectioning techniques to achieve serial sectioning and staining of intact mouse hind paws. Two additional approaches (isolated hind paw skin and footpad biopsies) were compared to the intact hind paw approach to determine the optimal method for mechanoreceptor visualization and localization.
Results: The optimized protocol for serial sectioning of intact hind paws included five days of fixation in 4% paraformaldehyde, ten days of decalcification in Cal-Ex™️ II, a 9-hour automated tissue processing protocol, embedding in a special formulated paraffin, and sectioning with specific techniques. Of the three approaches, intact hind paws provided the most context to structures visualized in the hind paw skin and thus, was the recommended protocol for future studies. Meissner-like corpuscles were located in the footpads and most abundantly in the footpads adjacent to digits II to V of the hind paw.
Conclusion: The method for serial sectioning of the intact hind paws outlined in this study will allow future analysis of mechanoreceptor distribution and density in transgenic or disease mouse models.
Keywords: Histology, serial sectioning, mouse, hind paws, decalcification, Meissner’s corpuscles, mechanoreceptors, fixation, tissue processing