Fe221

Animals, from both groups, control (n = 4) and DL treated (n = 4), were heparinized (200 I.U., i.p.) and after 15 min the heart was removed and mounted in an aortic perfusion system of the Langendorff type, on a constant flow (10 mL/min).¹⁴ The hearts were continuously perfused with Krebs-Henseleit solution (in mM: 120 NaCl, 5.4 KCl, 1.2 MgCl₂, 1.25 CaCl₂, 2 NaH₂PO₄, 27 NaHCO₃, 11 glucose), previously filtered through a cellulose acetate membrane (0.45 µm), pH was adjusted to 7.4 and oxygenated (95% O₂ + 5% CO₂) and maintained at 37 ± 0.1ºC (Haake F3, Berlin, Germany). Electrocardiographic (ECG) heart signals were captured using three electrodes (Ag/AgCl/NaCl, 1 M) that were placed inside the chamber close to the heart. The signals were amplified, digitalized (PowerLab 4/35 ADInstruments, USA) and stored in a computer. Left ventricular development pressure (LVDP, mmHg) and HR (bpm) were measured using a water-filled balloon introduced into the cavity of the left ventricle. This device was coupled to a pressure transducer (FE221, Bridge Amp, ADInstruments, USA) and an amplifier (PowerLab 4/35, ADInstruments). The system was calibrated using a column of mercury. Time of peak (ms), which is defined as the time necessary to achieve the peak of maximal ventricular contraction, and relaxation time, were also analysed.

Left intraventricular pressure was measured using a water-filled balloon introduced into the cavity of the left ventricle with a constant diastolic pressure of 15 mmHg by adjusting the volume of the balloon, connected to a pressure transducer (FE221, Bridge Amp, ADInstruments, Australia) coupled to an amplifier (PowerLab 8/35, ADInstruments). Ventricular pressures were processed using a dedicated software (LabChart 8 Pro, ADInstruments).

A 1.5–2 cm incision was made in the inner part of the left leg to expose the femoral artery, where a heparinized (20 IU/ml) cannula (0.6 mm outer diameter) was inserted and secured using 4.0 silk sutures. The cannula was connected to a calibrated pressure transducer (AD-Instruments, MLT1199) and coupled to a bridge amplifier and power supply (AD-Instruments, FE221 and ML826, respectively). BP measurements were obtained continuously during the acute studies and exported at 1,000 and 100 bits per second, respectively. A PowerLab data acquisition system and LabChart Pro software (both from AD Instruments, Colorado Springs, CO, United States) were used to digitalize and visualize the data.

The mean arterial pressure (MAP) was measured with a catheter in the femoral artery, and the drug delivery was administrated in the femoral vein. n = 6 rats were used for this study, 1–3 physiological tests per rat. A 1.0–1.5 cm incision in the medial aspect of the leg was made to expose both vessels, and a cannula (0.6 mm outer diameter) previously filled with heparinized saline (20 IU/mL) was inserted and secured to the muscle using 4.0 silk sutures. The arterial cannula was connected to a previously calibrated pressure transducer (AD-Instruments, MLT1199) coupled to a bridge amplifier and power supply modulus (AD-Instruments, FE221 and ML826, respectively) for continuously MAP evaluation. The venous cannula was coupled to an infusion system to administrate the vasoactive drug, nitroprusside (5 mg/mL, a bolus of 2.5 µg/g weight; 71778 Sigma Aldrich). Simultaneous nerve activity was recorded with the sutrodes placed on the cVN and SNVP-1 to 4. PowerLab data acquisition system (AD Instruments, Colorado Springs, CO) and LabChart Pro V8 (AD Instruments, Colorado Springs, CO) software were used to process and analyze the mean arterial pressure data.

Colonic tissue samples were dissected, weighed and placed in an oxygenated Krebs solution. Tissue strips were suture-mounted in tissue baths (10 ml, Panlab Two Chamber Compact Organ Bath, ML0126/10-220; ADInstruments Ltd, UK) connected to force transducers (MLT0420, ADInstruments Ltd, UK) and bridge amplifiers (FE221, ADInstruments Ltd, UK). Tissues were equilibrated in oxygenated Krebs solution for 60 min at 37 °C and an initial tension of 0.5 g was maintained. During equilibration, the tissues were perfused with three washes of oxygenated Krebs solution. Basal activity was recorded and collected using PowerLab 2/26 data acquisition system (PL2602/P, ADInstruments Ltd, UK), and analysed using LabChartPro v8.