3D Cranial Morphometrics in Mouse Pups

μCT images were obtained on 15, 20 and 25 day post-natal mouse pup skulls with a SkyScan 1174 (Kontich, Belgium) at a 22.57 μm voxel resolution. Scans were obtained on 181 animals (Male = 42.8%; Female = 43.3%; Undetermined = 13.9%). Mouse skulls were reconstructed with NRecon v1.6.4.8 (BrukermicroCT, Kontich, Belgium) as previously described and imported into Amira v5.0 where it was exposed to a Gaussian Smoothing image filter (r50.3 in X, Y, and Z dimensions; isometric kernel size53) to reduce extraneous noise in the images [47 ]. Threshold settings were then set to only visualize bone volume within the skull. Measurements of the length and width of the cranial vault were collected by a single experienced rater (TEP) from each reconstructed mouse skull. Cranial vault length was defined as the linear distance between landmarks opisthion and nasion. Cranial vault width was defined as the linear distance between the left and right sqzy landmarks, which are defined as the point of junction between the posterior zygomatic arch and squama of the temporal bone. The above landmarks can be visualized at the following website: http://getahead.psu.edu/viewer.html?id=Adult_Mouse_Skull. Cranial vault width and length measurements were used to define the cranial vault index (width x 100 / length) to further analyze 3D morphometric alterations due to treatment. Additionally, the widths of the coronal and sagittal sutures were measured at 25, 50, and 75 percent of their length as defined by the distance from the bregma to the pterion and from the bregma to the lambda, respectively. The width of the suture was defined as the distance between bony fronts at each of these points. Measures were compared at each time point by split-plot ANOVA or Kruskal-Wallis where appropriate by postnatal time point or suture for effects by dose where applicable; p≤0.05 was considered significant for post-hoc Bonferonni analyses. Sex of the pups was recorded for future post-hoc investigation but was not considered a factor in current analyses. All statistical analyses were completed using SPSS 23.0 (IBM, Armonk, NY, USA). All measurements are presented as mean ±SEM.