Evaluation of hydroxyurea-induced fetal skeletal changes in Dutch belted rabbits by micro-computed tomography and alizarin red staining

This laboratory has been investigating the utility of X-ray micro-computed tomography (micro-CT) to produce high-resolution, 3D images of skeletal structures in common laboratory species. The present investigation uses micro-CT evaluation of skeletons from rabbit fetuses exposed to the known teratogen, hydroxyurea.Groups of 4-6 mated Dutch Belted female rabbits each were administered vehicle or hydroxyurea (62.5 to 500 mg/kg) once on GD 12. On GD 28, all live fetuses were weighed, euthanized, and viscera removed. Up to 7 fetuses per litter were placed into a custom-made polystyrene holder and scanned in the micro-CT imaging system. Raw projection data were acquired in approximately 15 seconds, and reconstructed images at 100-micron cubic voxel dimension could be viewed as early as 20 minutes later. Fetuses were subsequently stained with alizarin red, and findings recorded separately for each method without knowledge of treatment group.Except for a few isolated cases, micro-CT evaluation detected the same skeletal malformations, variations, and incomplete ossifications as seen by the staining method. Skeletal elements that are very small (e.g., caudal-most vertebrae, metacarpal no. 1) or those with a minimal degree of ossification were occasionally not observed with micro-CT. However, this difference did not impact the overall study conclusions. Femur length was easily measured by micro-CT.These results indicate that micro-CT imaging can effectively assess rabbit fetal skeletal structures, and for those laboratories with this resource, may be used to significantly reduce time prior to skeletal evaluation and hazardous wastes associated with staining.