A Novel Three-Dimensional Analysis Method for Accurate Evaluation of Cutaneous Small Sensory Nerve Fibers in Mice

The evaluation of intraepidermal nerve fiber (IENF) density is commonly used to diagnose peripheral neuropathy. However, conventional two-dimensional (2D) analysis methods using rodent models show high interstudy variability. Three-dimensional (3D) IENF analysis has been proposed for human skin biopsies because the spatial location of each nerve is easily determined. However, to date, no studies have compared 2D and 3D analysis of mouse cutaneous nerve fibers under the same conditions. This study aimed to establish a more accurate analysis method for mouse cutaneous nerve fibers, addressing the limitations of 2D analysis. Using tissue transparency, nerve fiber-specific antibodies, confocal microscopy, and 3D analysis software (IMARIS), we analyzed the 3D structure of IENFs in mice. In the 2D analysis, the IENF density was not accurately determined due to the inability to ascertain the continuity of the nerve from dermis to epidermis. In contrast, the 3D analysis clearly defined branching points and continuity, allowing for the accurate measurement of IENF density. As a result, the actual IENF density from the 3D analysis was significantly less than that indicated by the 2D analysis. This 3D approach enables the precise capture of IENF trajectories with various parameters, establishing a standard method for evaluating peripheral neuropathy models.