Repeated Laser Doppler Flowmetry Procedures in Bone Do Not Alter Perfusion, Localized Inflammation, or Gait Patterns

Bone is a highly vascularized tissue that requires adequate blood perfusion to remodel in response to loading and heal after fracturing. Laser Doppler flowmetry (LDF) is a technique that has been validated to measure blood flow in mouse hindlimb bones in vivo, but to date it has only been used for measurements at a single timepoint, because the invasive surgery required to place the probe on the bone surface induced prolonged inflammation. The goal of this study was to determine if a modified, minimally invasive LDF technique could be performed repeatedly at the same bone site without altering gait patterns or causing substantial inflammation at the wound site, which would confound bone measurements in studies using exercise therapy. Twenty 14-week-old C57Bl/6J mice were divided into two activity groups and two LDF groups for 4 weeks prior to sacrifice. The exercise group performed daily treadmill exercise (30 min/day, 5 days/week), while the sedentary group spent a matched time on a stationary treadmill. Mice were further divided into either a repeated LDF group, which received an LDF procedure weekly (n=5 per activity group), or an endpoint LDF group, which underwent the LDF procedure only once (n=5 per activity group). LDF measurements were taken on the anteromedial surface of the right proximal tibial metaphysis. The effect of the LDF procedure was assessed with measures of blood perfusion, wound healing, and gait kinematics. Repeated LDF procedures did not alter blood perfusion measurements across weeks, either in the exercise or sedentary group. Wound area was measured from weekly photographs to determine the extent of inflammation. All wounds were fully closed within one week by the time of the next procedure. Changes in mouse gait kinematics were analyzed using weekly high-speed video of treadmill running. Duty cycle and phase dispersion remained constant from week to week with repeated LDF procedures. These results validated that the modified, minimally invasive LDF technique can be used for serial measurements of intraosseous blood perfusion without inducing potentially confounding effects of inflammation or altered gait or undesired changes in the perfusion itself. These findings will enable future longitudinal studies of bone perfusion with various pathologies, such as diabetes and stroke.