*2.2. Resveratrol Administration*

Dogs were randomly allocated to receive either no resveratrol supplementation (C-control group; *n* = 6) or seven days of 1000 mg (>10 mg/kg) per day of micronized trans-resveratrol (Micronized Resveratrol Micro500, Harmoni-T, Las Vegas, NV 89119, USA) orally (R-resveratrol group; *n* = 6), via a computer-generated random number sequence (Excel, Microsoft Corporation, Redmond, WA, USA). The final dose of resveratrol was administered in the morning of anesthesia. Serum and urine supernatant collected during the enrolment screening process for all dogs was divided into aliquots and stored at −80 ◦C as "baseline" samples for subsequent analysis of creatinine, protein, gamma-glutamyl transferase (GGT), and AKI biomarker concentrations. Animals in both groups were cared for in the same premises, under the same conditions, for seven days prior to anesthesia. Throughout this period, water was provided ad lib, and dogs were fed an intestinal health diet (Hills I/D) twice daily. All dogs received a deworming prophylactic medication (Popantel®, Jurox Animal Health, Rutherford, NSW, Australia) at the time of enrolment into the study. Dogs in both groups were examined daily by a veterinarian to ensure maintenance of good health, and any abnormalities detected on physical examination recorded.

#### *2.3. Anesthesia*

For all dogs, food was removed at least eight hours prior to the procedure but access to water provided until premedication. Dog were anesthetized by a veterinary anesthesiologist (AR) using a standardized protocol. Premedication with methadone 0.3 mg/kg (Ilium Methadone 10 mg/mL, Troy Laboratories, Glendenning, NSW, Australia) IM was performed 30 min prior to induction of general anesthesia with alfaxalone (Alfaxan injection 10 mg/mL, Jurox, Glendinning, NSW, Australia) 2.2–3.2 mg/kg intramuscularly. Endotracheal intubation was used to maintain anesthesia with isoflurane (I.S.O., Veterinary Companies of Australia Pty Ltd, Kings Park, NSW, Australia) in 30% oxygen through a circle rebreathing system. End tidal isoflurane was maintained at 1.3–1.4%. Infusion of a balanced isotonic crystalloid solution (Compound Sodium Lactate, Baxter Healthcare, Toongabbie, NSW, Australia) was provided at 10 mL/kg/hour IV throughout anesthesia, along with IV fentanyl (Fentanyl injection 50 µg/mL, AstraZeneca, Macquarie Park, NSW, Australia) 2 µg/kg/hour. Arterial carbon dioxide tension was maintained between 35 and 45 mmHg by provision of mechanical ventilation by an anesthesia workstation (Datex-Ohmeda S/5 Aespire Anaesthesia Machine, GE Healthcare, Chicago, IL, USA). Active warming maintained esophageal temperature between 36.0 and 38.0 ◦C.

#### *2.4. Instrumentation Procedures*

Instrumentation procedures were performed within the first 60 min of anesthesia. Dogs were positioned in dorsal recumbent position during this period. The cranial vena cava was cannulated via the right jugular vein, using a 14-gauge 13 cm cannula, to facilitate collection of venous blood and injection of lithium chloride. To allow measurement of arterial blood pressure and cardiac output (Qt) via the lithium dilution technique, and to facilitate controlled hemorrhage, a 14-gauge 9 cm cannula was placed in the left femoral artery. Prior to surgical exposure of the femoral artery and cannula placement, a femoral nerve block was performed using 0.1 mL/kg bupivacaine (Bupivacaine hydrochloride 5.0 mg/mL, Pfizer, Sydney, NSW, Australia). Non-distensible tubing filled with heparinized saline connected the arterial cannula to an electronic pressure transducer (DTX Plus, Argon Critical Care Systems, Singapore) for measurement of arterial blood pressure using a multi-parameter monitor (Surgivet V9203; Smiths Medical, Minneapolis, MN, USA). Before anesthesia of each animal, the transducer was calibrated using a mercury manometer (at 40, 80 and 120 mmHg). Once connected, the transducer was placed level with the manubrium of the sternum (i.e., approximate level of the right atrium) and zeroed to atmospheric pressure. Hourly during the study (just prior to each data collection interval), damping factor was subjectively assessed via a rapid flush test. Lack of baseline drift was confirmed at the end of each anesthetic by ensuring pressure read zero upon re-opening the transducer to the atmosphere. An 8 Fr 55 cm Foley urinary catheter was inserted into the bladder for collection of urine samples and measurement of urine output (UOP).

#### *2.5. Experimental Design*

This study utilized a predefined blood pressure (mean arterial pressure-MAP of ≤40 mmHg) target to assess whether the volume of blood loss needed to achieve such target was different between the two groups (see Figure 1). Following instrumentation and collection of baseline samples (T0), bleeding was commenced by removing blood from the femoral artery cannula until MAP fell to ≤40 mmHg and remained at that level for 60 mins. After the 60-min hypotensive period was completed, intravenous fluid resuscitation was initiated using 20 mL/kg of succinylated gelatine solution 4% (Gelofusine®, B. Braun, Bella Vista, NSW, Australia) at 1200 mL/h (without crystalloid boluses) until MAP was ≥60 mmHg. The dogs then underwent a further three hours of anesthesia with their MAP maintained at <sup>≥</sup>60 mmHg, by infusing more Gelofusine® if

required. Approximately about five hours following induction of anesthesia, the animals were euthanized using pentobarbitone (Lethabarb Euthanasia Injection 300 mg/mL, Virbac, Milperra, NSW, Australia) 150 mg/kg IV. The total amount of blood volume needed to achieve and maintain the 60 min hypotensive period and the amount of colloid fluid needed to achieve normotension subsequently were recorded.

**Figure 1.** Timeline of the pressure-targeted hemorrhage model performed in 12 adult male Greyhounds. MAP = mean arterial pressure. T0–T4 were time points at which cardiac output was measured, and blood (arterial and venous) and urine samples were collected.

#### *2.6. Data Collection*

Data collection was performed at T0 (baseline: 60 min after induction of anesthesia, following instrumentation, prior to ischemia), T1 (60 min after MAP first reached ≤40 mmHg, prior to reperfusion), T2 (60 min after commencing reperfusion), T3 (120 min after reperfusion commenced), and T4 (180 min after reperfusion commenced).

#### 2.6.1. Cardiovascular Parameters

At each time point, the mean of five consecutive MAP measurements was calculated. A blood gas analyzer and co-oximeter (ABL 725, Radiometer, Copenhagen) was used to measure blood gas variables (oxygen tension, carbon dioxide tension, bicarbonate concentration, calculated base excess, oxygen saturation) on arterial and central venous blood collected into heparinized syringes (PICO syringe, Radiometer, Copenhagen). These samples were analyzed immediately following collection. The same analyzer was used to measure hemoglobin and sodium concentration in central venous blood, necessary for the Qt measurement using lithium dilution. The lithium dilution technique, previously validated for Qt measurement in dogs, was performed at each time interval [22]. The mean of two consecutive Qt measurements that were not different by >10% was used for analysis. The calculation of the oxygen extraction ratio (OER) is described in Table 1. OER reflects the balance between systemic oxygen delivery and demand, and an OER greater than 0.5 in anesthetized dogs implied the presence of anaerobic metabolism according to previous studies [23–25].



BSA = body surface area; CaO<sup>2</sup> = arterial oxygen content; CcvO<sup>2</sup> = central venous oxygen content; DO2I = oxygen delivery index; Hb = hemoglobin concentration; PaO<sup>2</sup> = arterial oxygen tension; PcvO<sup>2</sup> = central venous oxygen tension; Qt = cardiac output; SaO<sup>2</sup> = arterial oxygen saturation; ScvO<sup>2</sup> = central venous oxygen saturation; VO2I = oxygen consumption index.
