Forene

Surgical procedures were divided into two steps, which were 2–4 days apart. In the first surgery, after fixing the mouse head in a stable position in a stereotaxic frame, an incision was made along the mid-line to expose the skull. After cleaning the bone, bonding reagent (ONE COAT 7 Universal, Coltene) was applied, and then a head cap was created using layers of light-cured dental cement (SYNERGEY D6 Flow, Coltene). Finally, a custom-made aluminum head post was attached to the head cap. These procedures were carried out under isoflurane (4% for induction and 1.5–2% for maintenance, Forene, AbbVie). The second surgery involved a craniotomy, cortical viral injection and chronic window implantation. With a dental drill, a small piece of skull was removed above the sensory cortex to expose the barrel cortex. A glass pipette and hydraulic pump were used to inject 150 nL of virus (injecting speed 50–70 nL per minute) at a depth of 350 µm beneath the brain surface into the whisker areas identified by Intrinsic optical imaging (see below). Immediately after the injections, a 3 × 3 mm coverslip was fixed right above the exposed brain and secured with dental cement to the head cap. Buprenorphine (Temgesic 0.1 mg/kg) was given before and after surgical procedures for 3 days.

Ultrasound imaging was performed in rats under anesthesia 2% (v/v) isoflurane (Forene; Abbvie, Belgium) using a high-frequency ultrasound system (Vevo2100, VisualSonics). Body temperature was maintained at 36–37°C and heart rate was kept at 400 ± 50 beats/min. Abdominal aorta PWV (aPWV) was determined according to the method developed by Di Lascio et al. with a 24-MHz transducer [43 ]. Briefly, pulse-wave Doppler was used to measure abdominal aortic flow velocity (V). Aortic diameter (D) was measured using B-mode images of the abdominal aorta in ECG-gated Kilohertz Visualization (EKV) imaging mode. The ln(D)-V loop method was then applied to calculate aPWV, using MathLab v2014 software (MathWorks). Abdominal aortic distensibility was calculated based on EKV images using the VevoVasc software (VisualSonics).

All surgical procedures were performed in spontaneously breathing animals anaesthetized with isoflurane (Forene, AbbVie Germany) and nitrous oxide (Westfalen AG, Muenster, Germany). Perioperative analgesia was achieved via subcutaneous injection of 2μl Meloxicam per 10g body weight (Metacam, Boehringer Ingelheim Pharma GmbH & Co. KG, Germany). Median laparotomy was performed in supine position. The small bowel was moved to the side and a distal colonic segment at the recto-sigmoid junction was dissected but not resected preserving the vascular arcades of the large bowel. A standardized end-to-end anastomosis was performed using 8 single stitches of 7–0 absorbable suture material (PDS II, Ethicon, Johnson & Johnson Medical GmbH, Norderstedt, Germany). The technique was consistent in all animals, each with 4 full thickness single stitches of the anterior and posterior wall leaving small spaces between the stitches to provoke leakage formation (Fig 1). After recovery from the procedure the animals were removed to their cages where they had free access to regular drinking water and food ad libitum.