Validating Minimally Invasive Laser Doppler Flowmetry 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 validated that it can be used serially in the same mouse without causing 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 can be used for serial in vivo measurements of intraosseous blood perfusion without inducing localized inflammation or negatively affecting gait patterns in mice. Highlights Modified, minimally invasive laser Doppler flowmetry (LDF) technique was validated for serial measures of tibial perfusion in mice. Weekly LDF procedures did not induce inflammation or alter gait patterns that could confound metrics of interest in bone studies. Ligation of the femoral artery confirmed the LDF technique measures functional perfusion within the bone.


Introduction
changes in bone perfusion for individual subjects, thereby improving understanding of disease 23 progression and intervention effectiveness. 24 First proposed as a tool to measure intraosseous perfusion by Nilsson et al. in 1980 [22], 25 laser Doppler flowmetry (LDF) directs a monochromatic light source over a perfused tissue and 26 measures backscattered light from fluid movement with a photodetector to provide a relative 27 measure of blood perfusion. Perfusion is a functional measure of blood flow that is affected not 28 only by the amount and velocity of red blood cells but also capillary density, vascular permeability, 29 and flow direction [23,24]. LDF was first used to measure blood perfusion in the cancellous bone 30 of pig mandibles by Hellem et al. in 1983 [25] and thereafter was rapidly adopted in orthopaedic 31 clinics as an intraoperative tool to aid surgeons in identifying non-viable bone for debridement in 32 patients with osteomyelitis, osteonecrosis of the femoral head, and lower limb traumatic injury 33 [23]. LDF has also been used as an endpoint measure to compare relative intraosseous perfusion 34 between groups in murine research studies [26,27]. Recently, it was validated as a tool to quantify 35 perfusion in the mouse tibia, but the technique used in that study involved a relatively large incision 36 that resulted in inflammation at the incision site up to three months after the procedure [28]. To 37 monitor longitudinal changes in murine bone perfusion, a less invasive LDF procedure is needed 38 that will not induce significant localized inflammation or limping during gait, which could have 39 both biological and mechanical confounding effects on bone. 40 We developed a minimally invasive LDF procedure and have used it to measure changes in 41 tibial perfusion in mice in response to diet-induced obesity, ischemic stroke, and treadmill exercise end of the study (endpoint groups). For the weekly LDF groups, starting in Week 2, images of the 69 incision site were taken during the LDF procedure prior to making an incision to assess the wound 70 from the previous week. Blood samples were collected from the submandibular vein of all mice 71 under anesthesia (at the end of the LDF procedure for the weekly groups). Blood samples were 72 centrifuged at 2,000 x g for 10 min, and the isolated serum was stored at -80°C until analysis with 73 an enzyme-linked immunosorbent assay (ELISA). At five days after each of the first three LDF 74 procedures, gait patterns were assessed using high-speed video. Immediately following the last 75 LDF procedure and serum collection, mice were euthanized using CO2 asphyxiation followed by 76 cervical dislocation.  All mice were fasted for 6-8 hours before each LDF procedure. Anesthesia was induced and 81 maintained with isoflurane (2%) in pure oxygen throughout the procedure (about 15 minutes).

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After anesthesia induction, the fur over the right knee was shaved, mice were placed supine on a  heated pad, and the right leg was taped to the surgical platform. A 2-5-mm long incision was made 84 over the anteromedial surface of the right proximal tibial metaphysis, the bone was exposed, and 85 a small region of the periosteum was scraped away. LDF measurements were recorded using an 86 LDF monitor with a 785-nm light source (MoorVMS-LDF, Moor Instruments Ltd, Axminster, 87 UK), and a 3 kHz lowpass filter selected. A VP4 Needle Probe (0.8 mm outer diameter, 0.25 mm 88 fiber separation) was placed directly on the exposed bone surface (Fig. 2)  At the end of the study, just prior to euthanasia, additional LDF measurements were  Criterion. Effect differences were calculated based on LSM with Tukey-Kramer adjustments for 166 multiple comparisons. All data are presented as mean ± standard deviation, except LDF and gait 167 data, which are presented as LSM ± 95% confidence interval.    Figure 6. Example images of the incision wound site (arrows) from the weekly LDF group taken one week following the procedure. Incisions were either A) fully closed or B) closed with small, dry granulation tissue. C) Only one incision did not fully heal, but it was healed by the following week. Scale bars are 1 mm.
Circulating levels of proinflammatory marker IL-6 were below the detectable limit for all animals 200 at each timepoint, except one mouse in the endpoint sedentary group at Week 4 (52.1 pg/mL).

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Since the lower threshold of the test is 7.8 pg/mL, and serum samples were diluted up to four times 202 to allow for duplicate measurements, serum levels of IL-6 were below 31.2 pg/mL. These levels  shown that changes to gait kinematics and limb patterning affect bone strain [44,48], we were 254 concerned the LDF procedure could affect locomotion patterns and confound exercise effects by 255 altering functional strain. We found no differences in duty cycle or interlimb coordination between 256 weekly and endpoint-only groups at any timepoint, indicating that weekly LDF measurements do 257 not alter gait patterns (and thus functional strain) during treadmill exercise.

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Although not the main focus of this study, aerobic treadmill exercise was anticipated to cause 259 increased tibial perfusion over time due to vascular growth and adaptation, as we have previously

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The increased perfusion lasted only one week and was present in both exercise and sedentary mice.

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This study had several limitations that warrant attention in future studies. A primary 280 concern of this technique is the removal of a small area of the periosteum, a highly vascularized 281 tissue that contains osteoblast precursor cells [52].   insulin secretion, signal transduction, nitric oxide release, and redox status in a clonal