Midazolam

The Göttingen minipigs were given pre-medication by intramuscular injection (Azaperone, 4 mg/kg BW; Stresnil, Janssen, Beerse, Belgium. Ketamine, 10 mg/kg BW; Ketavet 10%, Vet Way, Elvington, Great Britain. Midazolam, 1 mg/kg BW; Midazolam-ratiopharm, Ratiopharm, Ulm, Germany). A venous cannula was inserted into the veins of the left and right ears. Propofol (Propofol-Lipuro, Braun, Melsungen, Germany) was then administered to induce anesthesia, enabling endotracheal intubation. The further preparations and the operation were carried out with balanced anesthesia with isoflurane, propofol and fentanyl.
The procedure set out above was initially carried out for anesthesia during MRI and the subsequent killing. In addition to this, a central venous catheter was inserted into the jugular vein and a transurethral bladder catheter was also inserted [29 ]. Purely intravenous anesthesia with pentobarbital was administered throughout the entire examination period. During the examination in the MRI machine, the animal was ventilated using a mechanical ventilator (Servo 300A, Maquet, Rastatt, Germany).
The pigs were killed under deep anesthetic by means of an intravenous injection of 20 ml saturated potassium chloride solution. Hypodynamic circulatory arrest documented in the electrocardiogram and by auscultation.

Before the surgery, a combination of anesthetics consisting of midazolam (5.0 mg/kg, Ratiopharm GmbH, Ulm, Germany), fentanyl (0.05 mg/kg, CuraMED Pharma, Karlsruhe, Germany) and medetomidine (0.5 mg/kg, Pfister Pharma, Berlin, Germany) were injected subcutaneously. After anesthetizing, unilateral ligation of the right femoral artery was performed, while the same operation was done on the left side without closing the surgical thread to obtain an internal sham control [5 (link)]. The operation led to unilateral initiation of angiogenesis in the gastrocnemius muscle, which could be investigated by further tissue processing. Tissue collection was performed on day 3 or day 7 after ligation of the femoral artery. For this purpose, after another anesthesia, the hindlimb was perfused first with adenosine buffer (5% bovine serum albumin (BSA, Sigma-Aldrich) and 1% adenosine (Sigma-Aldrich, dissolved in PBS)) and for fixation of the muscle tissue with 3% paraformaldehyde (PFA, Merck, Darmstadt, Germany; dissolved in PBS, pH 7.4). Finally, gastrocnemius muscle from both legs was harvested and stored at −80 °C after being embedded in Tissue-Tek compound (Sakura Finetek Germany GmbH, Staufen, Germany).

To induce angiogenesis, 8–10 weeks old mice were initially anesthetized with a combination of fentanyl (0.05 mg/kg, CuraMED Pharma, Karlsruhe, Germany), midazolam (5.0 mg/kg, Ratiopharm GmbH, Ulm, Germany), and medetomidine (0.5 mg/kg, Pfister Pharma, Berlin, Germany). Once anesthetized, the mice were submitted to an unilateral FAL of the right hindlimb, while the left hindlimb was sham operated as previously described (Limbourg et al., 2009 (link); Lasch et al., 2019b (link)). For tissue sampling 2 or 7 days after the surgical procedure, mice were anesthetized as described above and were perfused with adenosine buffer [1% adenosine (Sigma-Aldrich, St. Louis, MO, United States), 5% bovine serum albumin (BSA, Sigma-Aldrich), dissolved in phosphate buffered saline (PBS, PAN Biotech, Aidenbach, Germany, pH 7.4)] followed by a perfusion with 3% paraformaldehyde (PFA, Merck, Darmstadt, Germany; for cryopreservation), or 4% PFA (for paraffin embedding) in PBS, pH 7.4. After the perfusion, the gastrocnemius muscles were carefully isolated and stored for further processing.