Unilateral hind limb ischemia was performed after femoral artery excision as previously described [32 (link)]. LASER Doppler perfusion imaging (Perimed, Craponne, France) was performed to quantify hind limb perfusion on a 37 °C heated bed under isoflurane anesthesia (induction at 5%, maintenance at 1.5% in air, Iso-vet, Piramal). Each LASER Doppler acquisition lasted 120 s and was repeated 3 times for each animal. On day 0, LASER Doppler was used to check and quantify the induced perfusion defect in the right hind limb, allowing the constitution of 2 homogeneous groups of 10 mice accordingly, for subsequent experiments. Hind limb perfusion was then quantified on days 1, 3, 7, 14, 21, and 28 post-ischemia. Results were expressed as a mean ± SD ratio of ischemic-to-contralateral (i/c) hind limb blood flow, and graphically represented as a mean ± SD reperfusion ratio to day 0. A motility impairment score, inspired by Suffee et al., and as previously published [32 (link),33 (link)], was calculated for each mouse on day 28, as follows: 1—unrestricted active movement; 2—restricted active foot; 3—use of the other leg only; 4—leg necrosis; 5—self-amputation.
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