The Animal Research Ethical Committee of Uppsala University approved the study (nr C 145/14). The study was performed according to Arrive 2.0 Guidelines [11].
Thirteen piglets, between two and three months old and with a weight between 22.5 and 25.7 kg, were studied.
All animals were given an endotoxin infusion to induce a septic-like state. They were randomized into two groups. In one group—the Edema group—the abdominal tract of the thoracic duct was ligated a couple of centimeters from its passage into the diaphragm, while the other group—the Control group—received a similar surgery to identify the thoracic duct, which was maintained intact.
A 1:1 randomization was performed using the random numbers generator application on GraphPad software (version 8.4.2.)
Preparation
Animals were pre-medicated with Zoletil Forte™ (tiletamine and zolazepam) 6 mg kg−1 and Rompun Vet™ (xylazine) 2.2 mg kg−1 i.m. After around 5 min, the animals were placed supine on an operating table, a bolus of fentanyl 10–20 µg kg−1 was given through a cannulated ear vein and a tracheotomy was performed inserting a 7 mm ID endotracheal tube (Mallinckrodt Medical, Ireland).
Ventilation was started in volume-control mode by a Servo-I ventilator (Maquet, Sweden) with a tidal volume (VT) of 6 mL kg−1, inspiratory:expiratory ratio (I:E) 1:2, FiO2 0.5, respiratory rate (RR) 25 cycles/min and PEEP 5 cmH2O.
Anesthesia was then maintained with a continuous iv infusion of ketamine 30 mg kg−1 h−1, midazolam 0.1 mg kg−1 h−1 and fentanyl 3.75 µg kg−1 h−1. After checking that anesthesia was sufficient to prevent responses to painful stimulation, muscle relaxation was added as a continuous i.v. infusion of rocuronium 3 mg kg−1 h−1. Thirty ml kg−1 Ringer’s Acetate were administered during the preparation to both groups.
In all animals, a triple-lumen thermistor-tipped balloon catheter (Swan–Ganz catheter, 7 Fr) was placed in the pulmonary artery from the right external jugular vein. Through the same access, a central venous catheter was inserted. A neck artery was cannulated and a second triple-lumen thermistor-tipped balloon catheter was placed in the hepatic vein from the left internal jugular vein (positioning was ascertained by fluoroscopy). A 20 cm laparotomy was performed at 3–4 cm left from the median abdominal line, then spleen and intestine were displaced to reach the aorta, on the left side of which the thoracic duct is normally positioned. In the Edema group, the duct was identified and ligated with a silk suture thread. The intestine was put back in place and the spleen was exposed for the cannulation of the splenic vein through a Seldinger technique, a 4 Fr catheter was positioned into the portal vein via the splenic vein. The spleen was carefully repositioned in the abdomen and peritoneum, muscle layers, and skin were sutured.
In the Control group we performed similar surgery, but once the thoracic duct was identified, we proceed directly with the cannulation of the portal vein. A schematic drawing of the animals’ preparation setting is shown in Fig. 1.
The catheters were used for blood sampling and pressure measurements when appropriate.
Cardiac output was measured by thermo-dilution.
A bladder catheter was inserted to collect urine and to measure intra-abdominal pressure (IAP). Abdominal perfusion pressure (APP) was calculated as:
MAP (mean arterial pressure) – IAP (intra-abdominal pressure) [12, 13].
Protocol
After baseline measurements, an endotoxin infusion was started (Sigma, Lipopolysaccharide from Escherichia Coli, 0111:B4, L2630-100 mg, 129K4025). The infusion rate was 15 μg kg−1 h−1 for 2 h, then decreased to 5 μg kg−1 h−1.
A noradrenaline infusion was titrated to maintain MAP equal to or above 65 mmHg as recommended by the guidelines for resuscitation in the Third International Consensus Definition for Sepsis and Septic Shock [14].
As soon as a stable condition was reached (most of the time during the first hour of observation), PEEP was gradually increased to 15 cmH2O in both groups.
A higher amount of fluids (around 500 ml in total) was administered to the Edema group, in order to magnify the effect of the thoracic duct ligation.
After 6 h of endotoxin infusion, four animals per groups were taken to the magnetic resonance imaging (MRI) Research unit for image acquisition, while the rest of the animals were euthanized by the injection of a KCl bolus. The animals examined with MRI, were killed once back in the laboratory, using the same method.
General anesthesia was maintained during the imaging acquisition, while endotoxin was discontinued.
During autopsy, samples from the lungs (upper and lower lobes of both), the duodenum, the colon, the liver, the spleen and both kidneys were taken and used to measure wet–dry weight, immunohistological analysis (cytokines concentration measurement), and histopathological assessment.
DW-MRI
Diffusion-weighted MRI was performed on a Philips 1.5 T scanner. Settings were: FOV 300 mm, 34 slices, thickness 8 mm, repetition (TR) and echo time (TE) of 1500 ms and 82 ms. Respiratory triggering was used as well as three diffusion sensitizing directions, i.e., 9 b-values 0, 20, 50, 75, 100, 200, 300, 600, 900 s/mm2.
The apparent diffusion coefficient (ADC) was measured; it is a measure of the magnitude of diffusion (random movement of water molecules), and was consider as a proxy for edema, (unit µm2/ms).
It is obtained by applying a mono-exponential model [15].
Using the intravoxel incoherent motion method (bi-exponential model), the f value, defined as the contribution of perfusion to the global movement of water molecules in a voxel [16], was calculated.
Regions of interest (ROIs) were drawn for edema and perfusion measurements in four target organs: liver, kidneys, intestine and spleen. For each organ four to six ROIs were drawn and the mean value of the ADC and f value was calculated. A goodness of fit was calculated to assess the quality of the measurements and ROIs with a goodness of fit lower than 95% were discarded.
Data collected
General data
Hemodynamic and respiratory data were collected at the baseline and every hour during the entire observation time (6 h) (see Tables 1 and 2 for a comprehensive list).
Arterial pressure, pulmonary arterial pressure, central venous pressure and oxygen saturation were monitored continuously.
Intra-abdominal pressure was measured following the instruction of the World Society of the Abdominal Compartment Syndrome [17], through the bladder catheter and injecting a volume of 10 ml in the bladder (instead of 25 ml volume, recommended for human adults) at baseline and at every hour.
At baseline and every hour during the observation time, blood gas analyses were performed on samples taken from 4 different vascular beds: artery, pulmonary artery, portal vein and hepatic vein.
Edema
Edema was assessed by comparing wet and dry weight in the lungs (samples were taken from the upper and lower lobes in both lungs), the duodenum, the colon, the liver, the spleen, and both kidneys.
ADC was also used as an edema assessment of the small intestine, the liver, the spleen, and the kidneys. Hemoglobin concentration was used as a surrogate of the hemoconcentration produced by the extravasation of plasma.
The abdominal circumference of the animals was measured at the baseline and after 6 h. The difference between the circumference measured at the end of the experiment and the baseline was used as an approximative assessment of edema and ascites amount in the abdomen.
An edema score (from 0 to 4) was given to lung (lower and upper lobes), small intestine and colon tissue samples by the veterinary pathologist on histopathological samples.
Perfusion
Perfusion was assessed using DW-MRI data [16]. f value was compared between the groups as a measurement of perfusion in the small intestine, the liver, the spleen, and the kidneys.
Inflammation
Samples from the duodenum, colon, kidneys, liver, and spleen were used to perform histopathological analyses of inflammation. The main features analyzed to produce the histopathological score in intestinal samples were: number and type of leukocytes, site of leukocytes localization (surface of the villi, side of the villi, or the crypts), type, intensity and extension of damage (necrosis, exfoliation, degeneration, apoptosis or erosion) [18]. In parenchymatous organs the score included the evaluation of leukocytes’ infiltrates, the presence of hemorrhage and general damages to the organ structure. A veterinary pathologist, blinded to the protocol, analyzed the samples.
Inflammation was also assessed by measurement of interleukin-6 (IL6), tumor necrosis factor α (TNFα) and interleukin 1 (IL1) concentration (by ELISA test) in blood from different vascular beds (artery, pulmonary artery, portal vein, and hepatic vein) at baseline and at the end of the observation time, and ascites and organs samples (the lungs—upper and lower lobes, the duodenum, the colon, the liver, the spleen and both kidneys).
The tissue samples were taken, as close as possible, in the same anatomical region as had previously been studied by DW-MRI.
Since the observation time in the study was short, 6 h, IL-6, TNFα and IL1 were selected for analysis, being among the earliest pro-inflammatory cytokines produced in sepsis [19]. A biologist, blinded to the protocol, performed the biochemical tests.
Statistical analysis
Data are presented as median and range. Based on distribution (Shapiro–Wilk test), comparisons between the two groups were performed using Student’s t-test or Mann–Whitney test and, when an analysis of multiple time points was performed, using ANOVA and multiple t-tests (correction for multiple comparison using Bonferroni–Dunn method). Pearson’s or Spearman’s coefficient were calculated to correlate different variables.
Cytokines and lactate concentrations as log-normally distributed data were log-transformed before analysis.
p < 0.05 will be defined as statistically significant.
Statistical analysis was performed using Prism version 8.4.2 (GraphPad software); and R (open source software, version 3.6.3).
