Labchart 5

On the day of the experiment (24 h after artery catheterization), the rats were placed in Plexiglas chambers (5 l) for respiratory measurements. The femoral artery catheter was then flushed with heparinized saline to prevent clotting and connected to a pressure transducer (BRPL2, WPI, Sarasota, FL, USA) coupled to an amplifier and an acquisition system (PowerLab, ADInstruments, Bella Vista, NSW, Australia) running LabChart 5.0 (ADInstruments, Bella Vista, NSW, Australia). MAP (mmHg) and HR (bpm) were derived from the pulsatile arterial pressure (PAP, mmHg). Baseline BP and HR were recorded for 60 min. After establishing baseline cardiovascular parameters, ventilatory activity was measured using the whole-body plethysmographic method according to (Malan, 1973 (link)). After each icv microinjection, the plexiglas chamber was closed and air flow was suspended for short periods (3 min). Before breath recordings, we performed a volume calibration via injecting 1 ml of air into the chamber and subsequently captured air displacement by the respiratory cycles via a pressure differential transducer connected to a signal amplifier (ML141 Spirometer, 196 PowerLab; ADInstruments). Tidal volume (V_T, mL) and minute volume (V_M, mL) were derived from the breath according to the method reported by Bartlett and Tenney (1970 (link)).

Six weeks after silver clip implantation in the renal artery or sham surgery, the rats were anesthetized with ketamine and xylazine (75 and 10 mg/kg, i.p., respectively) to insert polyethylene catheters in the femoral vein and artery for drug injection and arterial pressure recordings, respectively. BP and heart rate (HR) measurements were recorded 24h after catheter implantation in conscious rats using a pressure transducer coupled to an acquisition system (PowerLab; ADInstruments, CastleHill, NSW, Australia) connected to a computer running LabChart 5.0 software (ADInstruments).

Mice were anaesthetized by intraperitoneally injecting pentobarbital sodium (80 mg/kg) and fixed on a heating pad to maintain body temperature. Extensor digitorum longus (EDL) muscle was exposed, and the distal tendon was tied with a surgical suture, and the tendon was dissected at the distal end of the knot. Next, the muscle was separated up to the proximal muscle attachment site, and the other end of the suture was tied to a force transducer and maintained at a low base tension. The muscle was connected to an isolated pulse stimulator (A‐M SYSTEMS MODEL 2100, USA) by a platinum wire and stimulated at 20 V/cm. Analogue signals were converted using an A/D converter and recorded using LabChart 5.0 (ADInstruments). Stimulation with a 5 ms square wave was used while gradually increasing the base tension until the optimal base tension was discovered. With the optimal base tension being used, 5 ms pulses at 0.2 Hz were applied thrice to determine the maximal isometric twitch force. Maximal isometric tetanic force was detected thrice by stimulating the muscle with 5 ms pulses at 100 Hz for 300 ms with a 60 s interval between stimulations; subsequently, the muscle was isolated and transected, and the maximal cross‐sectional area (CSA) was determined using a stereoscope (Nikon SMZ25) and Image‐Pro Plus.