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).