A minimally invasive technique for serial intraosseous perfusion measurements in the murine tibia using laser Doppler flowmetry.

In biomedical and preclinical research, the current standard method for measuring blood perfusion inside murine bone, radiolabeled microspheres, is a terminal procedure that cannot be used to monitor longitudinal perfusion changes. Laser Doppler flowmetry (LDF) can assess perfusion within the proximal tibial metaphysis of mice in vivo but requires a surgical procedure to place the measurement probe directly onto the bone surface. Sustained inflammation for over a month following this technique was previously reported, and previous studies have used LDF as an endpoint-only procedure. We developed a modified, minimally invasive LDF procedure to measure intraosseous perfusion in the murine tibia without stimulating local or systemic inflammation or inducing gait abnormalities. This modified technique can be used to measure perfusion weekly for up to at least a month in the murine tibia.•Unlike previous endpoint-only techniques, this modified LDF procedure can be performed weekly to monitor serial changes to intraosseous perfusion in the murine tibia•The modified LDF technique utilizes a smaller, more localized incision to minimize invasiveness and speed recovery.

Minimally invasive laser Doppler flowmetry is suitable for serial bone perfusion measurements in mice.

In vivo laser Doppler flowmetry (LDF) has previously been used to quantify blood perfusion accurately at a single timepoint in the murine tibial metaphysis. However, this procedure entailed substantial disruption to soft tissues overlying the bone and caused notable localized inflammation for several weeks after the procedure, impeding serial measurements in the same mouse. In this study, we tested a less invasive technique to measure perfusion in the tibia with LDF and determined that it can be used serially in the same mouse without causing signs of inflammation or gait perturbations. Twenty 14-week-old C57Bl/6J mice were evenly divided into groups that either had daily treadmill exercise or remained sedentary. Within these activity groups, mice were evenly subdivided into groups that received LDF measurements either weekly or only once at the study endpoint. Bone perfusion was measured with LDF in the anteromedial region of the right tibial metaphysis. Serum concentrations of interleukin 6, incision site wound area, and interlimb coordination during gait were measured weekly for four weeks. Tibial perfusion did not differ significantly between exercise and sedentary groups within the weekly or endpoint-only LDF groups at any timepoint. Perfusion was significantly increased in the third week in the weekly LDF group relative to measurements in the second and fourth weeks. Ligation of the femoral artery caused consistent, rapid reductions in tibial perfusion, validating that LDF is sensitive to changes in tibial blood supply. Weekly LDF procedures did not adversely affect gait, as interlimb coordination during treadmill locomotion was similar between weekly and endpoint-only LDF groups at every timepoint. Images of the incision site show wound closure within one week, and serum concentrations of interleukin 6 were not significantly different between weekly and endpoint-only groups. Together, these findings demonstrate that our minimally invasive LDF technique is suitable for serial in vivo measurements of intraosseous blood perfusion without inducing localized inflammation or negatively affecting gait patterns in mice.

Stroke Prevents Exercise-Induced Gains in Bone Microstructure But Not Composition in Mice.

Ischemic stroke induces rapid loss in bone mineral density that is up to 13 times greater than during normal aging, leading to a markedly increased risk of fracture. Little is known about skeletal changes following stroke beyond density loss. In this study, we use a mild-moderate middle cerebral artery occlusion model to determine the effects of ischemic stroke without bedrest on bone microstructure, dynamic bone formation, and tissue composition. Twenty-seven 12-week-old male C57Bl/6J mice received either a stroke or sham surgery and then either received daily treadmill exercise or remained sedentary for 4 weeks. All mice were ambulatory immediately following stroke, and limb coordination during treadmill exercise was unaffected by stroke, indicating similar mechanical loading across limbs for both stroke and sham groups. Stroke did not directly detriment microstructure, but exercise only stimulated adaptation in the sham group, not the stroke group, with increased bone volume fraction and trabecular thickness in the sham distal femoral metaphysis. Stroke differentially decreased cortical area in the distal femoral metaphysis for the affected limb relative to the unaffected limb, as well as endosteal bone formation rate in the affected tibial diaphysis. Although exercise failed to improve bone microstructure following stroke, exercise increased mineral-to-matrix content in stroke but not sham. Together, these results show that stroke inhibits exercise-induced changes to femoral microstructure but not tibial composition, even without changes to gait. Similarly, affected-unaffected limb differences in cortical bone structure and bone formation rate in ambulatory mice show that stroke affects bone health even without bedrest.

Mass Spectrometry Imaging (MSI) of Fresh Bones using Infrared Matrix-Assisted Laser Desorption Electrospray Ionization (IR-MALDESI).

We report an effective strategy for direct analysis and two-dimensional (2D) matrix-assisted laser desorption electrospray ionization (IR-MALDESI) mass spectrometry imaging (MSI) of mouse bones that underwent no chemical treatments prior to analysis. To unravel the chemistry in bones under near-physiological conditions, we cut a flash-frozen bone in half longitudinally, placed it in a mold facing flat side down, and poured Plaster of Paris on top of and around the bone. After Plaster of Paris had set, the bone with embedding material was removed from the mold, and placed on the IR-MALDESI imaging stage. Plaster of Paris acted as a fixture to keep every spot on the sample surface the same distance away from the laser focus. To demonstrate the feasibility of IR-MALDESI MSI for analyses of unmodified bones, we imaged bones derived from healthy and stroke-affected mice and generated ion heatmaps showing the spatial distribution of putatively annotated features.

Thrombin-Responsive Transcutaneous Patch for Auto-Anticoagulant Regulation.

A thrombin-responsive closed-loop patch is developed for prolonged heparin delivery in a feedback-controlled manner. This microneedle-based patch can sense activated thrombin and subsequently releases heparin to prevent coagulation in the blood flow. This "smart" heparin patch can be transcutaneously inserted into skin without drug leakage and can sustainably regulate blood coagulation in response to thrombin.