General information

This study was approved by the Animal Care and Use Committee of the Amsterdam University Medical Centers, location AMC at the University of Amsterdam, the Netherlands. The procedures were performed in accordance with the European Parliament directive (2010/63/EU) and the national law of the Experiments on Animals Act (Wod, 2014). 72 male Sprague Dawley rats (Envigo, Indianapolis, IN, USA), 12–13 weeks of age and weighing around 400 g, were group housed in standard cages under normal conditions of a 12:12-h light:dark cycle receiving food and water ad libitum for at least 7 days before the experiment. Studies were conducted and reported following the ARRIVE guidelines (Additional file 2: Appendix S2) [23].

Pneumosepsis model

Streptococcus pneumoniae (serotype 3, ATCC 6303; Rockville, MD, USA) were cultured on Colombian Agar with 5% sheep blood plates (COS) (Biomerieux Benelux B.V, Zaltbommel, the Netherlands). After 12 h of growth, 5–6 separate colonies were isolated and transferred to 20 mL brain heart infusion (BHI) broth. After overnight incubation at 37 °C, the culture was centrifuged for 10 min at 2700 g, 20 °C (5804 R, Eppendorf AG, Hamburg, Germany). Afterwards, medium was removed and bacteria were washed with sterile 0.9% saline and centrifuged a second time for 10 min at 2700 g, 20 °C (5804 R, Eppendorf AG, Hamburg, Germany). Using a spectrophotometer (Secoman S250), optical density (OD) was measured. Assuming that an OD620 of 0.350 = 1 × 108 colony forming units (CFU), inoculum was made using sterile 0.9% NaCl. Prepared inocula were plated on COS plates in serial tenfold dilutions and grown overnight to quantify.

Following anesthesia using 5% isoflurane/60% FiO2 mix, rats underwent intratracheal inoculation with 3–5 × 108 CFU S. Pneumoniae (ATCC6303) (BSL-2) in 100 µl sterile 0.9% NaCl. In pilot experiments, this dose range induced pneumosepsis, defined as lactatemia with growth of S. pneumonia in lung and blood cultures, in all animals. Then, animals were transferred to their cages and allowed to wake up (Additional file 1: Fig. S1). During the experiments the following exclusion criteria were used: animals receiving too low (< 3 × 108 CFU) or high (> 5 × 108 CFU) amounts of S. pneumoniae. Animals without growth of S. pneumonia in either lung or blood cultures. Animals that died before start of intervention. Exclusion criteria were established before start of experiments.

Twenty-four hours after inoculation (T = 0), animals were randomized to 5 intervention and 2 control groups (N = 11 per group) with opaque envelopes using a 1:1 allocation. Intervention groups received resuscitation with either Ringer’s lactate (RL), 5% human albumin, rat-derived fresh frozen plasma (rFFP), human-derived fresh frozen plasma (hFFP) or human-derived solvent detergent plasma (SDP). Positive controls (n = 11) underwent all procedures while not receiving any resuscitation. Healthy controls (n = 6) underwent all procedures but were sham inoculated with 100 µL sterile NaCl 0.9%. Researchers were not blinded to group allocation during conduct of the experiment and data analyses. A 21G venflon was inserted in the tail vein under anaesthesia with 1–3% isoflurane/60% FiO2 while breathing spontaneously. After obtaining a baseline blood sample (T = 0). Fluids were administered at 8 ml/kg in 1 h (perfusor® fm; B Braun, West Bloomfield Township, MI, USA). Animals received no additional fluids between inoculation and start of resuscitation (T = 0).

After resuscitation, the catheter was removed and anesthesia was stopped. After 5 h, rats were exsanguinated through heart puncture (T = 5). Fifteen min before sacrifice, 0.5 mL saline containing 12.5 mg/ml fluorescein isothiocyanate (FITC)-labelled 70 kDa dextran (Sigma Aldrich, St. Louis, MO, USA) was administered through a newly placed tail vein catheter. Immediately after exsanguination, 2 mL of heparinized saline (1250 iE/mL, Leo Pharma BV, Amsterdam) was administered to prevent clotting. Thereafter, laparotomy and thoracotomy were performed, urine was collected by bladder puncture, and the inferior vena cava was cut. The circulation was flushed using 100 ml 0.9% NaCl, administered into the right ventricle, to remove excess intravascular FITC-dextran. Before flushing, the left pulmonary and renal hilum were ligated for wet-to-dry (W/D) ratios. After flushing, both lungs and kidneys were taken out for later assessment.

Animal welfare

Animals were monitored during the whole study period using a modified M-CASS scoring sheet (Additional file 1: Results S2) [17, 24]. Scoring 4 points in any of the categories was considered a humane endpoint and would result in early termination by heart puncture under anesthesia. No analgesics were administered during the study period. Animals were monitored during predefined moments by the responsible researcher (After inoculation, T12, T24, T26.5 and T29) or animal caretaker (T8, T20). Monitoring frequency was increased when unusual progression of symptoms was witnessed. Autopsy was performed on all animals not reaching the end of the study period to assess cause of death.

Intervention products

Rat fresh frozen plasma (rFFP) transfusion product was prepared from donor rats according to national blood bank standards. In short; rats were anesthetized using 5% isoflurane and whole blood was obtained through cardiac puncture using a 19G syringe containing 10% citrate–phosphate–dextrose (Sigma, USA). Blood was centrifuged at 2000G, 18 °C for 10 min after which plasma was separated centrifuged again at 2000G, 4 C° for 15 min (5804 R, Eppendorf AG, Hamburg, Germany). Thereafter, upper 2/3rd of plasma was taken, snap-frozen in liquid nitrogen and stored at − 80 °C. Human albumin (Albuman 200®, Prothya Biosolutions Netherlands, the Netherlands) was diluted 1:3 using sterile ringer’s lactate to a concentration of 5% and stored at – 80 °C. Both FFP (Sanquin, the Netherlands) and SDP (Omniplasma®, Octapharma, Switzerland) products were AB- and retrieved from Sanquin, the national blood bank. Prior to the experiment, all plasma products were aliquoted, snap frozen and stored at – 80 °C. At the day of the experiment, required infusion products were quickly thawed in a water bath at 37 °C, after which they were kept at room temperature until infusion.

Measurements

Blood was drawn before resuscitation (T = 0) and before exsanguination (T = 5) (Additional file 1: Fig. S1). Blood gas analyses (RAPIDPoint 500, Siemens, Germany) and total blood counts (cell Coulter AC•T diff2 Hematology analyser, Beckmann, Germany) were performed at both timepoints. All other biochemical assessments were measured only at termination (T = 5). Blood was centrifuged twice (2000g, 15 min, acceleration 9, brake 0, 18 C˚; 5804 R, Eppendorf AG, Hamburg, Germany), aliquoted, snap frozen in liquid nitrogen and stored at – 80 °C till further evaluation.

Pulmonary vascular leakage

To quantify the amount of pulmonary FITC-dextran leakage, the superior lobe of the right lung was thawed and homogenized using a radio immunoprecipitation assay (RIPA) buffer and tissue homogenizer (TH-115, Omni international, Kennesaw, Georgia, USA). 100 µL of homogenates were measured for fluorescence using a spectrophotometer (spectramax M2, molecular devices, San Jose, California, USA).

Pulmonary histopathology

The left inferior lobe of the right lung was fixed in 10% formaldehyde and embedded in paraffin, after which 4-µm-thick sections were cut and stained with haematoxylin and eosin (H&E). A pathologist blinded for treatment allocation evaluated the lung tissue for the presence of oedema, bronchitis, endothelialitis, interstitial inflammation and thrombi formation on a scale of 0–4 (0 = absent, 1 = mild, 2 = moderate, 3 = severe, 4 = very severe), as described previously [25, 26]. For the full scoring list, see the supplement (Additional file 1: Table S1). No other organs were assessed for histopathology.

Bacterial growth

The right inferior lobe of the lung was homogenized in sterile 0.9% NaCl. Lung homogenates were plated on COS plates in serial tenfold dilutions. Whole blood retrieved during termination (T = 5) was plated on COS plates, growth of S. pneumonia in whole blood was defined as bacteremia.

Parameters of systemic and organ injury

As a reflection of systemic endothelial injury and glycocalyx degradation, soluble levels of syndecan-1 (Elabscience, USA), thrombomodulin (TM) (Lifespan Biosciences, Inc. USA), von Willebrand factor (vWF) (Elabscience, USA) and intercellular adhesion molecule-1 (ICAM-1) (Elabscience, USA) were measured using commercially available ELISA kits according to manufacturer guidelines. ELISA values that were lower than the reference value were set at the lowest detection range. To assess liver and kidney injury, aspartate transaminase (AST), alanine transaminase (ALT), albumin and creatinine were measured by standard enzymatic reactions using spectrophotometric, colorimetric or turbidimetric measurement methods.

Sample size and statistical analyses

The primary outcome was pulmonary leakage of FITC-dextran. In a previous sepsis model, resuscitation with plasma compared to normal saline reduced syndecan-1 levels (21.8 vs 31.0 ng/mL) [17]. These data were extrapolated to a sample size with 7 groups. Using a one-way ANOVA analysis of variance (V = 17.5) and a common standard deviation of 9, the use of 10 rats had a power of 80% to reach a statistically significant difference in vascular leakage. With an expected mortality of 5% in our model, 11 rats were included per group.

Statistical analysis was done using SPSS Statistics V.26 (IBM). Graphs were made using GraphPad Prism 8 (GraphPad Software). All data were regarded as non-parametric and are shown as median with interquartile range (IQR). Data were tested for differences using the Kruskal Wallis test with post-hoc multiple comparisons. A p value < 0.05 was considered statistically significant. No correction was used for multiple testing. All data collected till the moment of death was used in analyses.