Validated Laser Doppler protocol for measurement of mouse bone blood perfusion - response to age or ovariectomy...

The physiological role of bone vascularization in bone metabolism begins to be understood; however, its involvement in pathological situations remains poorly explored. Bone blood supply depends on both vascular density and blood flow. However, in mice, the specific evaluation of perfusion in bone suffers from a lack of easy-handling measurement tools. In the present study, we first developed a Laser Doppler Perfusion Measurement (LDPM) protocol in mouse tibia, which we validated with ex vivo and in vivo experiments. Then we carried out a study associating both structural (vascular quantitative histomorphometry) and functional (LDPM) approaches. We studied the effects of aging in 4, 7 and 17 month-old male mice and the early effects of ovariectomy in 4 month-old females. Both studies were carried out in inbred mice (C57BL/6) and in mice of mixed background (129sv/CD1). The significant differences we observed between strains in unchallenged 4 month-old animals concerned both perfusion and vascular density and depended on gender. Additionally, the age-related bone loss observed in male mice was not temporally associated with vascular changes in either strain. Between 7 and 17 months, we did not find any decrease in bone vascular density or perfusion. In contrast, ovariectomy triggered early vascular structural and functional adaptations which differed between genetic backgrounds. We observed that bone vessel density did not generally account for bone perfusion levels. In conclusion, we describe here a LDPM-based experimental protocol which provides a reproducible quantitative evaluation of bone perfusion in mouse tibia, hence allowing intergroup comparisons. This integrative structural and functional approach of bone vascularization showed that bone vascular adaptation occurs during aging or after ovariectomy and is affected by the genetic background.