L8 18i rs

Animals are anesthetized with isoflurane (1–2%), placed in the supine position, and a 1–2 cm ventral midline cervical skin incision is made between the mandible and sternum. The left vagus nerve is isolated from the carotid sheath and placed on a bipolar electrode and stimulated (1 V, 30 Hz, 2 ms pulse width, 5 min). Following stimulation or sham surgery, the right carotid artery is dissected free from surrounding tissue for a distance of 5 mm, and the artery is bathed in 5 μL of a 10% ferric chloride (FeCl₃) solution for 3 min (F8KO mice) or a 5% FeCl₃ solution for 1 min (C57BL6/J and α7KO mice). The carotid artery is then rinsed with 0.9% normal saline. A doppler ultrasound probe (L8–18i-RS, GE) attached to a GE Logiq e ultrasound system is placed over both carotid arteries to monitor blood flow for a total of 25 min. Experimental endpoints include cessation of blood flow for >1 min as determined by the absence of arterial waveform on motion mode (m-mode) and doppler signal as compared to the contralateral, unaffected carotid artery. If the occlusion is not observed after 25 min, the time is recorded as 25 min for statistical comparisons.

Body height, body weight and circumferences of waist and hip were measured in accordance with the International Society for the Advancement of Kinanthropometry (ISAK) standards (18 ). The BMI was calculated according to the formula BMI = body weight (kg)/body height (m)² (19 ).
For the assessment of body fat, subcutaneous adipose tissue (SAT) thicknesses were measured by an ultrasound (US) technique (20 (link)). This method has been chosen since it was shown to accurately measure SAT in people ranging from extremely low fat layers to obese (21 (link)) whereas the application of other methods is limited in people with extreme body composition (22 (link)). SAT layers were measured at eight standardized body sites (upper abdomen, lower abdomen, erector spinae, distal triceps, brachioradialis, external oblique, front thigh, and medial calf). Ultrasound measurements were performed with a conventional US system (GE Logiq-e, General Electric) using a linear probe (L8-18i RS) operated at 8–16 MHz. The semiautomatic evaluation software (Rotosport, Stattegg, Austria) was applied for the US images evaluation. For further analysis, the value D_INCL was used. D_INCL is the calculated sum of the eight SAT thicknesses.