The experimental procedures were approved by the ethics committee for animal experiments at Rakuno Gakuen University (Protocol Number: VH19B14). Care and handling of the animals were performed in accordance with the guidelines of the National Institutes of Health.
Animal preparation
All studies were performed by a qualified and experienced research team using female pigs (Landrace × Large White × Duroc [LWD], weighing 29–34 kg). Food was withheld from the animals for 12 h before the start of the experiment; however, water was allowed 6 h prior. After getting used to the environment and following physical examination by a veterinarian, the animals were sedated by an intramuscular injection of medetomidine hydrochloride (40.0 μg/kg), midazolam (0.2 mg/kg), and butorphanol tartrate (0.2 mg/kg). Tracheal intubation was performed after induction of anesthesia using propofol, and general anesthesia was maintained with inhaled 2.0% sevoflurane (Sevoflo®, Dainippon-Sumitomo Pharma, Osaka, Japan). Anesthetics were adapted if the depth of anesthesia was insufficient. Following anesthesia induction, the pigs were placed in a supine position and administered a fluid infusion of lactated Ringer’s solution (LR, Terumo Co., Tokyo, Japan) at 10 mL/kg/h, through a 22-gauge catheter (Supercath, Medikit Co., Tokyo, Japan) placed in the right marginal ear vein. All animals were mechanically ventilated in volume-control mode (Flow-i, Maquet, Sonia, Sweden) after intravenous administration of 2 mg/kg vecuronium (Musculate®, Fuji Pharma Co., Tokyo, Japan), followed by a constant-rate infusion at 0.1 mg/kg/h, administered through the left marginal ear vein. The ventilation settings were 8–10 mL/kg tidal volume with a respirator, with positive end-expiratory pressure set at 5 cm H2O. The fraction of inspired oxygen (FIO2) was set at 0.5, with an inspiration to expiration ratio of 1:2; the respiratory rate was adjusted to 16–20/min to maintain artPCO2 50 ± 5 mmHg. Body temperature was maintained at 37.0 °C ± 0.5 °C using a heating pad.
Experimental protocol
After each catheterization was completed, all animals were allowed to stabilize for 30 min, and each initial value was regarded as the baseline value. To induce a progressive hemorrhagic shock status, blood was withdrawn from the central venous catheter and stored in sterile bags with a volume of citrate–phosphate-dextrose-adenine (CPDA-1) appropriate for 30 mL/kg of blood (14 mL CPDA-1 per 100 mL of blood). Blood withdrawal (BW) was performed three times by each 10 mL/kg, and a total of 30 mL/kg was collected. The measurement points where the loss of circulating blood volume reached 10, 20, and 30 mL/kg were defined as BW10, BW20, and BW30, respectively. Next, the animals were resuscitated by transfusing them with the stored blood. Blood transfusion (BT) was performed at intervals of 10 mL/kg, transfusing 30 mL/kg in total. The points where the gain of circulation blood volume reached 10, 20, and 30 mL/kg were defined as BT10, BT20, and BT 30, respectively (Fig. 1). Before each measurement, the animals were allowed to stabilize for 10 min.
Instrumentations
A 7-Fr PAC (Edward Lifesciences, Irvine, CA, USA) was inserted into the left internal jugular vein and advanced into the pulmonary artery. Assessment of pulmonary artery pressure and wedge waveforms confirmed the correct position of the PAC. Simultaneously, the right femoral artery was catheterized with a thermistor-tipped 4-Fr PiCCO catheter (PV2014L16, Pulsion Medical Systems AG, Munich, Germany) connected to the PulsioFlex platform (Pulsion Medical System, Munich, Germany). Arterial blood pressure was measured via the femoral artery using a PiCCO catheter. A 6-Fr double-lumen central venous catheter (UK catheter kit UB-0610-W, 21G, 10 cm, Unitika Medical, Osaka, Japan) was inserted into the right jugular vein and positioned at the cranial end of the superior vena cava for blood withdrawal and transfusion. The distal ports of the PAC were connected to the PulsioFlex monitor sensor for the same time as for the measurement of CO via an injection of ice-cold physiological saline 0.9% (Isotonic Sodium Chloride Solution®). All transducers were zeroed and positioned at the level of the right atrium.
Transcutaneous CO2 and O2 measurements
The transcutaneous monitoring system (TCM4; Radiometer, Copenhagen, Denmark) provides non-invasive and continuous measurement of tcPO2 and tcPCO2. Prior to the fixation of transcutaneous sensor (tc Sensor 84; Radiometer, Copenhagen, Denmark), the skin was cleaned, and an adhesive ring with two drops of contact gel was applied, according to the manufacturer’s instructions. Measurements were performed with the animals in the supine position with a skin probe positioned on the right ear. Calibration was conducted automatically, with the temperature of the skin probe set at 44 °C. The tcPCO2 and tcPO2 values were obtained and recorded before CO measurement, during each hemodynamic stage, by an investigator blinded to the experiments.
Measurements of hemodynamic parameters
CO was measured by pulmonary artery thermodilution (COPATD: using PAC) and transpulmonary thermodilution (COTPTD: using PulsioFlex); 10 mL of ice-cold physiological saline 0.9% (Isotonic Sodium Chloride Solution®) was used as an indicator and was injected through the left distal port of PAC. PATD and TPTD were measured simultaneously at each time point by the same operator (YE), who was blinded to the transcutaneous CO2 and O2 measurements and laboratory data. To unify the COTPTD measurement method and eliminate the nominative effects, COTPTD was measured by the following methods: (1) each measurement was triplicated, and the averaged values of measurements were used for the analyses, (2) the indicator was injected by the same operator (YE) throughout the overall study period, (3) ΔT in TPTD (the change in blood temperature after indicator injection) was recorded; optimal = ΔT > 0.3, good = ΔT > 0.2, and bad = ΔT < 0.2, to verify the reliability of the measurement method, and (4) the indicator was injected exactly for 2–3 s (to avoid faster or slower injection); and (5) the temperature of the indicator was accurately controlled at 0 °C using an ice and cooler box. In this study, ΔT > 0.3 occurred 38 times, ΔT > 0.2 occurred 4 times, and ΔT < 0.2 did not occur at all, indicating that TPTD at all measurement points was quite accurate according to the TPTD instruction.
Hemodynamic parameters measured by PAC (pulmonary artery thermodilution [PATD]) included the central venous pressure (CVP), pulmonary artery wedge pressure (PAWP), stroke volume (SVPATD), and CO (COPATD). Transpulmonary thermodilution [TPTD] parameters obtained by PiCCO technology (Pulsion Medical System, Munich, Germany) included the mean arterial pressure (MAP), SV (SVTPTD), CO (COTPTD), global end-diastolic blood volume (GEDV), stroke volume variation (SVV), pulse pressure variation (PPV), cardiac function index (CFI, CO/GEDV), cardiac power output (CPO, mean arterial pressure [mmHg] × CO [L/min] × Κ, where Κ = 0.0022 [a conversion factor]). The shock index (SI), defined as heart rate divided by systolic blood pressure, at each measurement point was calculated. SI of > 1.0 was indicative of worsening hemodynamic status and shock [22]. PATD and TPTD could be measured at each time point. SVV was not observed at two time points in one pig for an unknown reason; all other hemodynamic variables displayed each value.
Additionally, a perfusion index (PI), which was derived from pulse oximetry readings (Masimo SET Radical-7TM, Masimo Inc, Irvine, CA, USA), was monitored simultaneously. The Masimo Radical-7 uses transcutaneous, multi-wavelength analysis for non-invasive measurement of arterial oxygen saturation, PI, and pleth variability index (PVI), which are measures of local blood flow. A sensor (LNOP Neo-L, Masimo Inc., Irvine, CA, USA) was attached to the tail after clipping the hair. Esophageal temperature and heart rate were also recorded (BSM-6000; Nihon Kohden Inc., Tokyo, Japan).
Arterial blood gas analysis
Arterial blood samples were withdrawn anaerobically from the PiCCO catheter and collected in a plastic syringe heparinized with 1000 U/mL of sodium heparin (Novo-heparin for injection, Mochida Pharmaceutical Co., Tokyo, Japan), using an evacuation technique to minimize sample dilution. The blood samples were analyzed by an investigator blinded to the experiments immediately after collection (< 5 min) to measure artPO2, artPCO2, hemoglobin (Hb), and lactate, using a blood gas analyzer (ABL-90 FLEX; Radiometer, Copenhagen, Denmark).
Systemic oxygen delivery
An inappropriate level of systemic oxygen delivery (DO2) capacity fails to satisfy the metabolic oxygen need in the tissue. It is well established that tissues exhibit a level of systemic DO2, known as the critical DO2 (DO2 crit), below which the extraction cannot increase sufficiently to sustain O2 uptake; O2 consumption then becomes supply-dependent [23,24,25,26]. DO2 was calculated using the following equation: DO2 (mL/kg/min) = CO × hemoglobin [Hb] × 1.36 × arterial oxygen saturation (SaO2) + (partial pressure of oxygen [PaO2] × 0.0031) [27]. In this study, tcPO2 was used to define DO2crit since tcPO2 reflects tissue oxygenation under experimental conditions. Given that the lowest baseline tcPO2 associated with survival was 28 mmHg [28], the lowest threshold for tcPO2 was set as 30 mmHg. This is consistent with previous studies, which demonstrated that tcPO2 values < 25 mmHg reflects a 50% decrease in oxygen consumption, preceding cardiac arrest [29], and that a tcPO2 value of 40 mmHg is a critical artPO2 in the subcutaneous tissue [30, 31].
Statistical analysis
Values are expressed as mean ± standard error of the mean. An unpaired two-tailed Student’s t-test or Mann–Whitney U test was performed to compare two independent groups, as appropriate. Linear regression and Bland–Altman analyses were performed to determine the correlation between tcPCO2 and artPCO2. Repeated one-way analysis of variance (ANOVA) followed by Dunnett's correction and Friedman test followed by Dunn's correction were performed for post-hoc comparisons of normally and non-normally distributed data, respectively. Spearman's correlation coefficients (r) were calculated to evaluate the correlation between any two parameters. To examine the accuracy of tc-artPCO2, SI, and arterial lactate for predicting DO2crit, receiver operating characteristic (ROC) curve analyses were performed. The area under the curve (AUC) between two pairs of potential predictors was compared using a nonparametric test. Statistical significance was defined as a two-sided p-value of < 0.05. All statistical analyses were performed using GraphPad Prism version 8.3.0 (GraphPad Software, San Diego, CA, USA) and Microsoft Excel (Microsoft 365, Microsoft Corporation, Redmond, WA, USA).