Acknowledge software

At the end of the 12-week training protocol, rats were anesthetized intraperitoneally with ketamine hydrochloride (50 mg/kg ip) and xylazine hydrochloride (10 mg/kg ip) (Dopalen, Sespo Indústria e Comércio Ltda., Vetbrands Division) and submitted to catheterization surgery. Hemodynamic data were obtained by a micromanometer (MikroTip^TM SPR 320, Millar Instruments, USA) inserted from the right carotid artery and positioned immediately above the aortic valves to monitor the aortic and LV pressure records. SBP and DBP (mmHg), HR (bpm), LV systolic and end-diastolic pressures (LVSP and LVEDP; mmHg), maximum positive (+dP/dtmax; mmHg/s) and negative (-dP/dtmax; mmHg/s) derivatives of LV pressures, and LV relaxation time constant (TAU; s) were acquired and analyzed using a computer program (Acknowledge Software, Biopac System, USA).

Ankle joint torque produced during voluntary plantar flexion was measured using a calibrated isokinetic dynamometer (Biodex S4, Shirley, NY) while subjects were seated with the hip and knee joints both flexed, at 80° (0° = anatomical position) and 60° (0° = full extension) respectively. Data was obtained only from the right foot, which was tightly attached to the dynamometer’s footplate accessory. The ankle joint was aligned with the dynamometer’s motor axis. Subjects were fastened to the chair by two shoulder harnesses and one abdominal harness to minimize the body’s movement and to limit the contribution of other muscle groups aside from ankle plantar- and dorsi-flexors in net torque production. Particular care was taken in avoiding head movements, and a neck brace was used to reduce the influence of neck muscle electrical activity on the EEG signal. Net torque and angular position were recorded at 2 kHz using a Biopac MP150 system and Acknowledge software (Biopac Systems, Santa Barbara, CA, USA). The “baseline” torque produced by the limb weight plus accessories was systematically subtracted from all active torque signals.

The EMG activities of the right side SOL, MG and tibialis anterior (TA) muscles were recorded by pairs of 8 mm diameter silver-silver chloride surface electrodes (inter-electrode distance: 2 cm) and sampled at 5 kHz using a Biopac MP150 system and Acknowledge software (Biopac Systems, Santa Barbara, CA, USA; common mode rejection ratio CMRR > 110 dB, gain: 1000, bandwidth: 10–500 Hz). After preparing the skin to reduce impedance below 5 kΩ, SOL, MG and TA electrodes were placed in accordance with the SENIAM recommendations^{44 (link)}. The SOL and MG electrodes were thus fixed lengthwise over the middle of their respective muscle bellies. If needed, the placements were adjusted with the aim of obtaining the greatest M-wave and H-reflex amplitudes in response to tibial nerve stimulation. Because spinal excitability can be affected by antagonist muscle activity, the EMG activity of the TA was also recorded, in order to ensure that no difference in coactivation that could influence the spinal excitability would occur between contraction types^{45 (link)}. The TA electrodes were positioned one-third of the way between the proximal fibula tip and medial malleolus. The reference electrode was placed on the patella on the left leg.

Immediately after echocardiography, baseline hemodynamic evaluation was performed under adjusted anesthesia (K-X mixture) and oxygen-enriched ventilation with a closed chest. The left femoral vein was accessed for drug administration, and a 2-F gauge Millar catheter-tip micromanometer (model SPR-320, Millar Instruments, Houston, TX, USA) was inserted into the right carotid artery into the LV cavity. Moreover, an ultrasound flow probe (Transonic System Inc., Ithaca, NY, USA) was positioned in the ascending aorta. The following data were analyzed (Acknowledge software, Biopac System, Santa Barbara, CA, USA): LV systolic (SP) and end-diastolic pressures (EDP), rate of change of LV pressure (+dP/dt and −dP/dt), heart rate, and cardiac output (CO), and stroke volume (SV). Stroke work index (SWI) was stated as previously described (dos Santos et al., 2010 (link)). Thereafter, sudden LV afterload increases were achieved using a single phenylephrine in bolus injection (15–25 μg/kg, i.v.) (dos Santos et al., 2010 (link)).

Twenty-eight days after the 2K1C or sham operations, mice were anesthetized with a combination of ketamine/xylazine (91/9.1 mg/kg, i.p.), and a catheter (0.040 mm outer × 0.025 mm inner diameters, MicroRenathane, Braintree Science, Massachusetts, USA) was inserted into the right carotid artery to take mean arterial pressure (MAP) and heart rate (HR) recordings. The free end of the catheter was tunneled under the back skin to the level of the shoulder blades. Two days after the catheter placement, hemodynamic measurements were performed in conscious, freely moving mice in their cages, as already validated by others [30 (link)] and standardized in our laboratory [13 (link)-17 (link)] as a sufficient period for complete recovery from surgery. For the MAP and HR recordings, the arterial catheter was plugged into a disposable blood pressure transducer (Cobe Laboratories, Colorado, USA) connected to a pressure processor amplifier and data-acquisition system (MP100, Biopac Systems, California, USA). At the beginning of the experimental session, a period of approximately 30 min was allowed for stabilization of cardiovascular parameters before the measurement of basal MAP and HR values in conscious mice (Acknowledge software, Biopac Systems, California, USA).