Experimental Protocol
Food was withheld for at least 4 hours and water was provided ad libitum prior to anaesthesia. A ten-gauge intravenous (IV) catheter (Angiocath, Becton, Dickinson and Company) was aseptically placed in the jugular vein. Horses were sedated with IV xylazine (Rompun, Dechra Pharmaceuticals, 0.6 mg/kg bwt) and anaesthesia was induced with IV ketamine (Zetamine, VetOne®, 2.5 mg/kg bwt) and diazepam (Dash Pharmaceuticals, 0.05 mg/kg bwt). Upon recumbency, horses were orotracheally intubated with a size 26 mm internal diameter silicone, cuffed tracheal tube and positioned in dorsal recumbency. Anaesthesia was maintained with isoflurane (Isospire, Dechra Pharmaceuticals) in 100% oxygen on a large animal rebreathing circuit and anaesthesia machine (SurgiVet LDS 3000, Surgivet Inc.). Isoflurane vaporizer settings were adjusted to achieve an end-tidal isoflurane of 1.3*Minimum Alveolar Concentration (MAC) to mimic a surgical plane of anaesthesia (absence of brisk palpebral or nystagmus). Mechanical ventilation was performed in all horses with a tidal volume of 12 mL/kg bwt, inspiratory:expiratory ratio of 1:2-3, and respiratory rate of 5-7 breaths per minute adjusted to achieve an arterial partial pressure of carbon dioxide less than 55 mmHg determined via arterial blood gas analyses (i-STAT, Abbott Laboratories). Fluid therapy during anaesthesia was provided with lactated Ringer’s solution (LRS) at 3 mL/kg bwt/h IV (Vetivex®, Dechra Pharmaceuticals). Atracurium besylate (Meitheal® Pharmaceuticals, loading dose: 0.1 mg/kg bwt IV; constant rate infusion: 0.2 mg/kg bwt/hr IV) was used for neuromuscular blockade to prevent ventilator asynchrony. In addition to the arterial blood pressure measurements (see below), monitoring included an ECG, pulse oximetry, and end-tidal carbon dioxide. Two additional 10-gauge jugular venous catheters were placed for controlled haemorrhage and return of autologous blood. Prior to haemorrhage (baseline), a constant rate infusion of dobutamine (Hospira Inc., rate 0.5 - 3.0 mcg/kg bwt/min IV) was titrated as needed to maintain normotension from invasive blood pressure measured in the facial artery (MAP ≥ 70 mmHg) to mimic clinical conditions. If dobutamine was required to maintain normotension at baseline, it was continued at that same infusion rate throughout the experiment. Atracurium, LRS, and dobutamine were all administered through a peripheral 14-gauge IV catheter placed in the medial saphenous vein (Angiocath, Becton, Dickinson and Company).