Straight spring wire

Wire-induced injury of the femoral artery was performed at day 7 of empagliflozin treatment as previously described (19 (link)). In brief, mice were anesthetized as described earlier. A straight spring wire (0.38 mm in diameter, Cook Medical, Bloomington, IN, United States) was then inserted through the profunda femoris artery up to 1 cm into the femoral artery and left in place for 1 min to achieve an adequate wire-induced vessel injury. After removal of the wire, the profunda femoris artery was ligated (7-0 Prolene, Ethicon, Johnson & Johnson, Norderstedt, Germany) and perfusion of the dilated femoral artery was re-established. Mice were sacrificed at 10 or 21 days by cervical dislocation. The femoral artery was carefully harvested and embedded in Tissue-Tek OCT compound medium (Sakura Finetek Europe B.V., Staufen im Breisgau, Germany). Then, the arteries were snap-frozen and stored at –80°C until sectioning.

C57BL/6 strain (WT) mice and Wistar rats were purchased from CLEA Japan Inc. Elovl6^−/− mice were a kind gift from Dr H. Shimano (Tsukuba University). These mice were intercrossed into Elovl6^−/− or Elovl6^−/+ mice. Littermates were genotyped by PCR. The femoral artery of 10‐ to 12‐week‐old male WT mice (n=24, 24‐28 g) and Elovl6^−/− mice (n=23, 22‐26 g) was injured with a wire, as previously described,14 with some modifications.15 Briefly, a straight spring wire (0.38 mm in diameter, Cook Medical, Bloomington, IN) was inserted into the right femoral artery and pushed forward toward the iliac artery. The wire was left in place for 1 minute to denude and dilate the artery and then was removed. Blood flow was reconstituted after ligation of the profunda femoris branch. Mice were sacrificed 14 days after this injury, and the femoral arteries were isolated. Uninjured femoral arteries were isolated from sham controls. The carotid artery of rats was injured with a balloon as described previously.16 See Data S1 for further details. Animal experiments using these mice were approved by and performed according to the guidelines of the Committee of Experimental Animal Research of Gunma University (Permit Number: 10‐019).