Chartpro

In a subset of mice, freshly isolated jejunum rings were prepared for measures of isometric tension. Each mouse yielded 2 rings of jejunum tissue. All segments of the jejunum were subjected to the same procedures, and data were averaged and considered representative for that animal, considered n = 1. Isometric tension was measured using standard isolated tissue procedures. Briefly, jejunum rings were mounted between 2 stainless steel wires that were passed through the lumen of each jejunum ring, with wires connected to a force transducer (159901A, Radnoti, Monrovia, CA). Jejunum rings were submerged in a 15 ml bath containing a Krebs bicarbonate ringer, in mM: 115 NaCl, 25 NaHCO₃, 5 KCl, 1.2 MgCl₂, and 1.2 CaCl₂ (pH 7.4), and equilibrated at 37°C with a 95% oxygen/5% carbon dioxide gas mixture. Jejunum rings were stretched and maintained at ~0.275 g resting tension and equilibrated for ~40 min with frequent bath changes. Dose-response curves of tension generated by cumulative addition of increasing concentrations of potassium chloride were obtained (with steady-state contractile responses achieved at each dose, 0–100 mM). Tension development was continuously recorded and collected by PowerLab with ChartPro computerized data acquisition system software (AD Instruments Inc., Colorado Springs, CO).

At the end of the hypoxic or normoxic protocols, animals were re-anesthetized. A stainless-steel guide cannula (13 mm long, 0.6 mm o.d., 0.4 mm i.d.) was implanted into the lateral cerebral ventricle (−0.6 mm to Bregma, 1.5 mm lateral to the midline and −3.6 mm ventral to dura mater) and a catheter (PE-10 connected to PE-50; Clay Adams, Parsippany, USA) inserted into the abdominal aorta, through the femoral artery, for pulsatile arterial pressure measurements. MAP and HR were processed from PAP by a data acquisition system. Post-surgery, rats were treated again with analgesic and antipyretic and veterinary antibiotic. Two days later, the arterial catheter was connected to a pressure transducer (MLT0380; ADInstruments, Sidney, Australia), and in turn, to an amplifier (ML221; ADInstruments). PAP was recorded in conscious, freely moving rats under normoxic conditions (1 kHz; Chart Pro, PowerLab 4/25, ML845; ADInstruments). Alst [2 mM, 10 µl; Phoenix Pharmaceuticals, Inc., Burlingame, USA^{33 (link)}] was administered intracerebroventricullarly [(25 µl syringe; Hamilton Company, Reno, USA) (needle 33-gauge; Small Parts, Miami Lakes, USA)]. The correct placement of the guide cannula was confirmed at the end of the experiment by injection of Evans Blue (2% in 10 μl; Sigma-Aldrich, St. Louis, USA) and its visible presence in the intracerebroventricular system.

Cardiac functions were analyzed by transthoracic echocardiography with a 13-MHz probe and a VIVID i ultrasound machine (GE Health). The mice were anesthetized with 3% isoflurane. Two-dimensional targeted M-mode imaging was obtained from the short axis immediately below the level of the mitral valve. M-mode tracings were recorded, and measurements were made with the operator blinded to the mouse groups.
Mice were anesthetized with pentobarbital by intraperitoneal injection of 70 mg/kg and catheterized with a 1.4-French catheter (SPR-839, Millar Instruments). Hemodynamics were recorded by PowerLab 8/35 with Chart Pro (AD Instruments) similar to methods described.⁴² (link) Data for each animal were calculated from at least 30 cardiac cycles of chart recording.