The overall goal of this procedure is to properly position and restraint a rat hind limb for the acquisition of focused high resolution images of the bone micro-architecture of the proximal tibia and distal femur using In Vivo micro-computed tomography. This method can help answer key questions about the acquisition of In Vivo images by micro CT and provides guidance for the proper positioning and restraint of the hind limb. The main advantage of this procedure is that highly consistent images can be acquired and then used for quantification of bone micro-architecture changes that occurs during longitudinal studies.

Place the rat in the supine position on a carbon fiber scanner bed. After confirming the appropriate level of sedation by lack of response to toe pinch, apply ophthalmic lubrication to the animal's eyes. Restraint the foot in a malleable foam tube with the toes extending out of one end, and apply dental wax to hold the foot tightly within the foam, taking care that the foam does not extend over the region of interest.

Tightly seal the tube with tape and insert the foam into the tube. Confirm a tight fit between the foam and the tube and slide the tube into the x-ray scanner bed. Extend the hind limb until the leg is taut.

Confirming a lack of overextension by palpating the knee joint position and pull the opposite hind limb and the tail towards the torso. Extending the hind limb away from the torso until the leg is taut ensure that the x-ray will pass through all of the regions of the bone in as similar a pattern as possible. Next, blot the adhesive side of several pieces of masking tape and use the modified tape to secure the non-extended hind limb and tail, so that they remain out of the scanning field of view.

Then, secure the hips, shoulders, head and nose cone to the scanning bed. Wrap the animal in vet wrap to limit heat loss and image the animal by micro computed tomography using the appropriate experimental parameters according to standard protocols. When all of the images have been acquired, allow the animal to recover in an individual partially heated cage with monitoring until full recumbency.

Proper positioning of the rat hind limb involves the leg being fully extended and the entire foot and ankle being restrained in foam, resulting in an acquired image of sufficient quality for analysis of the trabecular and cortical micro architecture. Insufficient placement and restraint of the hind limb can result in images with movement artifacts, while a tail that is not fully removed from the scanning field of view will interfere with x-ray attenuation by the scan samples and alter the bone marrow and tissue mineral density measurements. Once mastered, this procedure can be properly performed in 5-10 minutes.

While performing this procedure, it's important to always be cognizant of the breathing rate, sedation level, and amount of radiation exposure of the rat. This procedure can be altered and used to assess other skeletal sites, such as the distal femur and lumber vertebrae. Moreover, this procedure can be modified and used for other rodent species such as mice.

After watching this video, you should have a good understanding of how to properly position and restrain the hind limb of the rat, for the focus and consistent high resolution imaging of the bone micro-architecture of the proximal tibia.