LAUSR

Bone is a highly vascular tissue and angiogenesis, the formation of new capillaries from pre-existing vessels, plays a pivotal role in skeletal growth. Therapies that increase angiogenesis have been shown to improve bone repair (e.g., [1]). The coupling of blood vessel and bone formation has received wide attention in mammalian models of bone healing (e.g., [2]), but little is known of the interrelationship between angiogenesis in osteogenesis within profoundly regenerative environments. In particular, these data are lacking for the pre-eminent model of vertebrate regeneration, the axolotl (Ambystoma mexicanum). This salamander, unlike the more widely employed zebrafish model, is capable of extensive, repeated, endochondral bone regrowth throughout its lifespan. Genetic tools facilitating imaging of these animals, however, are as yet extremely limited compared to those available for zebrafish. This necessitates the development of other tissue labeling techniques. To develop a methodological foundation for investigating the role of angiogenesis in osteogenesis within the context of endochondral bone regeneration, we have combined and adapted tissue preparation methods more widely used in other vertebrate models to permit simultaneous whole mount imaging of the regenerating vasculature and bone in axolotls. We have previously [3] developed perfusion-based methods for labeling the vasculature within regenerating axolotl tissue. In the present work, we sought to determine if the specimen processing required for bone labeling was compatible with this vascular marking technique. Vascular perfusion combined with whole mount labeling allows us to examine the structures of interest in the regenerating appendage within a three dimensional context. The methods provide an inexpensive, straightforward means for simultaneous imaging of the microvasculature and bone using light microscopy.