Ethical Statement
The study was approved by the Committee on Animal Experimentation at the National Institute of Cardiology - Ignacio Chavez (INCICH) in Mexico City (reference number 09F12). Anaesthesia was used in all surgical interventions. Experiments described in this study were performed in adherence to Good Laboratory Practices (GLP) [19][20], National regulations NOM-062-ZOO-1999 [21], General Considerations for Animal Studies for Cardiovascular Devices [22], and Animal Research: Reporting of In Vivo Experiments (ARRIVE guidelines) [23] (Additional file 1: Table S1). The study was monitored by an independent quality assurance unit.
Sample Size
Previous experimental protocols in an ischaemia-reperfusion swine model have determined a standard deviation for XL of 0.20 -jW [24, 25], considering a significance with a p-value <0.05, a confidence interval of 0.95 and a margin of error of 10%, the sample size for the shock group is at least 15 subjects, and 6 subjects for the control group.
Surgical Preparation
After subjects were prepped and draped in a sterile fashion, a left carotid artery catheter was placed for blood chemistry, blood gas measurements (Radiometer, ABL 835) and systemic arterial pressure monitoring (Matron, BPM-1000). A double lumen catheter was inserted in the right internal jugular vein for fluid and drugs administration. A left external jugular pulmonary artery catheter (PAC) (7.5 French, model 774F75, Edwards Lifesciences) using Medex transducers (Pressure Transducer MX9504, Medex) was placed for measuring hemodynamic variables and sampling of mixed venous blood gases, and continuous cardiac output (Edwards, Monitor Vigilance II), and a Foley catheter was placed.
Measurements
Vital signs and Hemodynamic
Vital signs such as electrocardiogram, pulse oximetry, temperature, mean arterial pressure (MAP), heart rate (HR), and central venous pressure (CVP) were measured every thirty minutes. Hemodynamic variables from PAC are SvO2, pulmonary artery systolic pressure (PASP), pulmonary artery diastolic pressure (PADP), pulmonary artery wedge pressure (PAWP), cardiac output (CO), systemic vascular resistance (SVR), pulmonary vascular resistance (PVR), and stroke volume (SV) among others, were obtained only for SG. Calculated variables such as oxygen delivery (DO2), oxygen consumption (VO2), arterial oxygen content (CaO2), venous oxygen content (CvO2), oxygen extraction ratio (REO2), and mean pulmonary artery pressure (MPAP) were determined through the PAC software, that applies the equations described in [26–28]. At the same time, additional parameters were taken from blood gas readings.
Gastric Impedance Spectroscopy & Gastric reactance (XL)
XL is a quantity derived from the electrical impedance of the gastric wall, a feature that provides a distinctive fingerprint of tissue structure; pathologies like ischemia, infarct, or necrosis cause cellular alterations that are reflected by changes in impedance. The biological and mathematical basis for the calculation of XL has been published in peer-reviewed scientific literature by independent research groups [5, 6, 8, 29]. The GIS device determines the magnitude of XL through proprietary algorithms applied to impedance readings which are acquired through a feeding tube fitted with an impedance sensor.
During the experiment, correct positioning of the feeding tube and the impedance sensor was confirmed endoscopically (Fujinon 200), after this, the tube was connected to the bedside monitor and XL measurements were taken every minute.
Histological and Endoscopic
All biopsies were stored in a 3.7 M phosphate buffer (formol), pH 7.2 solution. Endoscopic assessments were performed during every biopsy. For the CG, two biopsies (after tracheostomy, at the end of the procedure) were taken and, for the SG, four biopsies (after tracheostomy, two during haemorrhage, and one at the end of the procedure) were taken. Biopsies were embedded in paraffin and cut with a microtome to a thickness of 4 microns and stained with hematoxylin-eosin. Microscopic description and a photomicrography were documented for each cut. A qualitative assessment was made for each biopsy identifying superficial inflammation, oedema and superficial detachment by a pathologist following an empirical scale: 0%, 12.5%, 25%, 50%, 75%, 100%. On the other hand, the macroscopic appearance of the gastric mucosa was classified by a gastroenterologist into five levels: normal mucosa (level 0), stippling or epithelial haemorrhage (level 1), pale mucosa (level 2), violet mucosa (level 3), and marmoreal mucosa (level 4).
Laboratory Tests
Venous and arterial blood gases were measured in both groups every thirty minutes (ABL 835 Radiometer Blood gas analyser). Blood gas samples include pH, partial pressure of carbon dioxide in arterial blood (PaCO2), partial pressure of oxygen in arterial blood (PaO2), haemoglobin (Hb), sodium (Na+), potassium (K+), chloride (Cl-), ionized calcium (Ca2+), glucose, base excess (ECF), partial pressure of carbon dioxide in venous blood (PvCO2), partial pressure of oxygen in mixed venous blood (PvO2), carbon dioxide (CO2), arterial oxygen saturation (SaO2), and lactate.
On the other hand, the following laboratory tests were performed at baseline conditions in both groups (tests were also performed at the end of the experiment in the SG): blood creatinine, urea, alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin, lactate dehydrogenase (LDH), alkaline phosphatase (ALP), fibrinogen (Daytona RX, Randox), prothrombin time (PT), activated partial thromboplastin time (PTT) (Thrombotimer, Behnk Elektronic), complete blood count (CBC) with cell differentials, white blood cell count (WBC), platelet count (BC-2800Vet, Mindray), and complete blood chemistry.
All laboratory tests were carried out at the Clinical Pathology Department of the Faculty of Veterinary Medicine at the National Autonomous University of Mexico following manufacturer’s guidelines.
Necropsy
Necropsies were performed in twenty-one subjects through a thoracoabdominal incision, all organs in the thoracic and abdominal cavities were collected. Macroscopic photographs of the outer and mucosal surfaces of stomach were taken. All organs were fixed for 48h in 10% buffered formalin. Samples from different organs were preserved: fundus, body, and antrum of the stomach, duodenum, ileum, large intestine, lung, liver, spleen, and kidney. Tissue samples were embedded in paraffin and cut into a microtome with a thickness of 4 microns and stained with haematoxylin-eosin.
Experimental protocol
The study screened twenty-five York-Landrace swine subjects sourced from a licensed vendor (Centre for Education, Research and Extension in Pig Production, Mexico). Four subjects were excluded for not meeting inclusion criteria (health certificate, 27-35 kg, castrated, fasting ≥ 16h) and only twenty-one subjects were included (Fig. 1). The study was divided in two stages, in the first stage, baseline measurements of gastric reactance, vital signs, laboratory tests and histopathological samples were taken from CG of 5 subjects. In the second stage, the same measurements plus haemodynamic variables were taken from SG of 16 subjects; in this case, baseline conditions were followed by an induced hypovolemic shock with a controlled haemorrhage at a rate of 15-20 mL/min until a MAP of 40-30 mmHg was achieved (Additional file 2: Figure S1).
Housing and husbandry
All swine were transported safely with proper ventilation, the target temperature ranges were defined as 10°C to 29°C. A veterinarian performed the clinical examination, codification and placed each subject in a barnyard. The swine were acclimatized to the environment under a regular diurnal cycle (12:12 light: dark) with ab libitum access to water and a controlled food intake, chopped corn cob was used as bedding, and vital signs were recorded daily until the procedure day. In addition, all animals underwent fasting at least 16h before surgery but were allowed water ab libitum.
Anaesthesia
All subjects were pre-treated with atropine (0.1 mg/kg, intramuscular), Zoletil® (tiletamine hydrochloride and zolazepam hydrochloride [7-10mg/kg, intramuscular]), and azaperone (4mg/kg, intramuscular). Continuous inhaled isoflurane (1-2%) was used to maintain anaesthesia during the experiment, the rate was adjusted as needed to provide adequate anaesthesia; all variations from to this rate were recorded (Vaporizer, Datex Ohmeda Tec 4).
Tracheostomy and Mechanical Ventilation
A midline cervical incision was made, and a tracheostomy was performed to warrant adequate ventilation (Modulus II plus, Datex Ohmeda). Initial settings were tidal volume 12 Vt (mL/kg), respiratory rate (RR) of 12-14/min titrated to maintain PaCO2 within the normal range (35-45 mmHg), fraction of inspired oxygen (FiO2) of 75%, Inspiration/Exhalation ratio 1:2, and positive end-expiratory pressure (PEEP) of 3 cmH2O.
Injury
Haemorrhage in the SG was initiated after a period of 20-40 min baseline stabilization. As mentioned before, exsanguination of the animal is performed at a rate of 15-20 mL/min until reaching a mean arterial pressure (MAP) of 30-40mmHg and 40% of the circulating blood volume was withdrawn. Volume was continuously recorded, and blood was refrigerated for the duration of the experiment. Hypovolemia and hypotension conditions were maintained for about 4 hours by infusing, and titrating crystalloids, according to the requirements of the subject.
Shock criterion
The shock criterion was established as MAP ≤ 48 mmHg, which is two standard deviations below the mean of the CG. Considering this, the relevant timepoints to be evaluated are T-2: two hours before shock; T-1:one hour before shock; T0: shock; T1: one after hypovolemic shock; T2: two hours after hypovolemic shock for systemic and hemodynamic variables.
Euthanization
Once anaesthetized and all study procedures completed, an intravenous injection of Potassium Chloride solution 1-2mmol/kg was given to induce euthanasia.
Statistical analysis
Continuous variables were tested for normality using the Shapiro-Wilk test using the residuals method. A non‐parametric one-way ANOVA (Kruskal‐Wallis test) was performed to detect significative differences between CG and SG at the relevant timepoints defined by the shock criterion (all subjects were considered); the main effects were evaluated through post-hoc Dunn's multiple‐comparison test to control type I error. One-way repeated measures ANOVA by ranks (Friedman test) were made to detect significative differences between each relevant timepoint using SG paired data (same subjects per timepoint group); the main effects were evaluated with post-hoc Durbin's multiple‐comparison test to control type I error. Holm adjustment method for p-values for multiple comparisons was used [30]. In all cases, differences were considered significative at a two-tailed p-value of <0.05.
Receiver operating characteristic (ROC) curves were calculated to identify optimal cut-off values and their individual ability to predict hypovolemic shock for lactate and XL at the relevant timepoints defined by the shock criterion (MAP ≤ 48 mmHg). ROC curves with 95% confidence interval (CI) were computed using the bootstrap method. The curves were generated using maximum lactate values per subject, per event (Lac_Max) and minimum XL value per subject, per event (XL_Min) between CG and SG as the binary classifier considering the shock criterion described above.
Confounders were considered in the design of statistical analysis. All analyses were conducted using the R Statistical language (version 4.0.5) and RStudio (version 1.4.1106) on Windows 10 using the packages pROC (version1.17.0.1) [31], gtsummary (version 1.4.1) [32] and ggstatsplot (version 0.8.0) [33].