Virtual Dissection of Complex Masticatory Muscles with DiceCT

Purpose: Weaning is a critical stage in the life history of altricial mammals, with far-reaching impacts on growth trajectories and survival. Post-weaning diet(s) are known to affect craniofacial skeletal morphology as well as masticatory muscle volumes, physiological cross-sectional areas (PCSA), and fiber type ratios. These muscles and their subparts can be functionally grouped into vertical elevators, protractors, and retractors. However, the extent to which these functional groups differ in their responses to dietary changes is currently underappreciated, particularly where they are difficult to isolate through traditional dissection methods. Here we use diffusible iodine-based contrast-enhanced computed tomography (diceCT) to perform digital dissections of small, complex masticatory muscles to assess the effects of longitudinal variation in diet on the growth of functional groups of these muscles. Methods: Sprague-Dawley rats were raised from weaning (21 days) to adulthood (16 weeks), and randomly sorted into hard and/or soft dietary treatment groups. Post-sacrifice, cranial tissues were fixed in 4% PFM for 36 hours and stored in 70% EA at 4℃. Specimens were stained in 11.25% Lugol’s solution (I2KI) for 48 hours before microCT scanning. In 3D Slicer, muscles were manually segmented every 10 slices, the “Fill Between Slices” function was applied, and volumes were quantified. Volumetric measurements were compared using Kruskall-Wallis tests and pairwise Mann-Whitney U-tests (α = 0.05). Results: Contrary to our expectations, preliminary results suggest a trend for animals raised on softer diets to have larger temporalis and superficial masseter muscles than those raised on hard diets. However, no statistically significant differences were observed among or between treatments likely due to small sample size (n Conclusions: DiceCT is a promising method for soft tissue analysis that complements CT analyses of bone. Volumetric data can be obtained for small and/or complex musculature where limitations exist for traditional dissection methods.