Powerlab 4 25t

The descending thoracic aorta was isolated from sacrificed rats of both groups and placed in ice-cold Krebs solution (in mmol/L: 118.3 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25 NaHCO3, 0.026 EDTA, 5.5 glucose; pH 7.45). The aorta was cleaned of perivascular fat and connective tissue and cut into aortic rings of 3 mm in length. Aortic rings were horizontally mounted between two steel hooks, one of which was fixed and the other connected with an isometric force transducer (MLT 050/A, AD Instruments) and a data acquisition system (PowerLab 4/25 T, AD Instruments). Aortic rings were placed in individual tissue baths containing Krebs solution, maintained at 37 °C and continuously oxygenated. Passive tension was applied, and rings were equilibrated for 1 h. During this period, aortic rings were washed three times for 20 min with fresh Krebs solution and were then precontracted with 10⁻⁷ mol/L phenylephrine (PE, Sigma-Aldrich, Diegem, Belgium). After reaching a stable plateau phase, 10⁻⁶ mol/L acetylcholine (ACh, Sigma-Aldrich) was added to the tissue baths to check vessel viability and endothelial integrity. Aortic rings that failed to react upon PE or ACh application were excluded from further experiments. After washing aortic rings three times for 10 min with fresh Krebs solution, vasocontraction and vasorelaxation responses were assessed.

Fitness level and VO₂ max were assessed using the Cooper 12-Minute Run Test [25 (link)]. This test was performed according to the protocol developed by Dr. Kenneth H. Cooper, where participants were instructed to run as quickly as possible for 12 min at a steady pace between two points of a certain distance, and the total distance covered was measured. The estimated VO₂ max was calculated based on the distance covered, following the procedure outlined in the original protocol [25 (link)].
HRV was recorded and analyzed according to the published standard guidelines using the Powerlab data acquisition system (PowerLab 4/25 T and Chart v5.4 Pro, AD Instruments, NZ) [26 (link)]. HRV was recorded with the participant lying in a supine position in a quiet room for 12 min. Subsequently, an artefact-free segment of 300 s of recording was selected for analysis. HRV data were analyzed for the time, frequency, and Poincare Plot.

Lead II Electrocardiograms (ECG) was recorded for 20 minutes in conscious animals using a signal transducer (PowerLab 4/25T, ADInstrument) and operated through Chart 5.0 program. ECG data was then analyzed using the frequency-domain method by the MATLAB program. High-frequency (HF) component was considered as a marker of parasympathetic tone while low-frequency (LF) component was considered as a marker of parasympathetic tone and sympathetic tone. The cardiac sympathetic/parasympathetic balance was reported as the LF/HF ratio. Increased LF/HF ratio indicates cardiac sympathovagal imbalance37 (link)38 (link). HRV was recorded at week 0 (baseline), 12, 16, 20 and 24.