Animals and housing
The study was approved by the ethical committee for animal studies in Bavaria, Germany. Housing and all medical and surgical procedures were performed in the Center for Preclinical Research (ZPF) of the Klinikum rechts der Isar (Munich) in accordance with the national animal protection act (Tierschutzgesetz). German landrace female pigs (~60 kg) were kept in animal housing for about 4–7 days to allow for acclimatization before the surgical procedure. The timeline of the experiments is schematically described in Fig. 1. Every step was performed following carefully prepared standard operating procedures (SOPs) as the study was designed and procedures were put in place to comply with good laboratory practice (GLP) and assure data quality and integrity. The institution has its own policies and procedure in compliance with the local laws and guidelines, but was not GLP certified.
Surgical procedure
The sepsis-like swine model was developed in two steps: induction of liver injury and further development to multiple organ dysfunction through superimposed endotoxemia. Before the experiments, pigs remained fasting for 12 h with free access to water. In order to prevent gastric and duodenal ulceration, pantoprazol (80 mg) was daily administered from the day of admission of animals in ZPF [20].
On day 0, two cannulas were inserted into the ear veins to establish intravenous access. Intramuscular premedication consisted of ketamine (15 mg/kg), azaperone (2 mg/kg), and atropine (0.5–1 mg/kg). Anesthesia was induced with ketamine (1–2 mg/kg) and 2% propofol (1–2 mg/kg) and was maintained with the latter (60–100 mg, i.v.). The pigs were mechanically ventilated through endotracheal intubation following the recommendations of the Acute Respiratory Distress Syndrome Network [21]. The aim was to keep the arterial oxygen pressure (PaO2) around 80 mmHg by adequately adjusting the tidal volume (8 ml/kg). We adjusted the respiratory rate (up to 35) for a better control of the acid-base status. The inspiratory plateau was set at a pressure ≤30 cm H2O, which might be exceeded, if necessary, to treat respiratory acidosis (i.e., pH < 7.3). The fraction of inspiratory oxygen (FiO2) and positive end-expiratory pressure (PEEP) combinations employed can be found in the supplement (Additional file 1: Table S1). Adequacy of anesthesia was clinically assessed ensuring that animals had sufficient relaxation and analgesia and was adjusted accordingly [22]. Animals received buprenorphine (0.6–1.2 mg/24 h) on day 0 and metamizol (p.o. or i.m. 40 mg/kg) were administered before surgery on day 0 and on days 1 and 2. Intravenous infusions of propofol 2% and remifentanil were used to maintain anesthesia on day 3.
Induction of liver injury
Induction of liver injury was performed based on the surgical procedure described by Awad and colleagues [23], with minor modifications [24]. Briefly, on day 0, laparotomy was performed and the bile ducts and portal vein in the hepatoduodenal ligament were exposed in order to ligate the cystic, common hepatic, and the common bile duct (Vicryl® 2/0, Ethicon Inc., Norderstedt, Germany). The latter was ligated twice to ensure complete obstruction of bile flow. Afterwards, the portal vein and inferior (caudal) vena cava were partially clamped before a functional end-to-side portosystemic anastomosis was established. Arterial supply of the liver was not interrupted, and the development of splanchnic congestion was avoided by ensuring an adequate portal flow during partial clamping [24]. Cefuroxime (i.v. 500 mg) was infused during surgery. The animals were returned to their pens where they were clinically observed.
Superimposed endotoxemia
On day 3 after induction of liver injury, animals were re-admitted to the operation room and were anesthetized and further challenged with E. coli lipopolysaccharide (serotype: B0111:B4, VWR International GmbH, Darmstadt, Germany), starting with a dose of 4 μg/kg/h and continuing with twofold stepwise increments every hour for 7½ h, up to a total dose of 764 μg/kg. The endotoxin was dissolved in saline and administered through an auricular vein. Paracetamol (i.v. 1–2 g) over 15 min was given to all animals participating in the study when endotoxin infusion started. Endotoxins can lead to variable elevation of hypothalamic set point for body temperature with resultant violent shivering and fever of the animals. Paracetamol as antipyretic was given to control these symptoms and avoid differences between groups, which could have led to bias in the final results.
The procedures carried out in this study have been validated in two previous publications where 7 [24] and 14 pigs [10] in each case were employed. In addition, 32 additional animals were necessary in order to set an adequate endotoxin dosing protocol (unpublished observations). Consequently, we have developed a stable swine sepsis-like model that allowed us to evaluate the safety and efficacy of a three-circuit albumin dialysis-based extracorporeal organ support system (ADVOS).
ADVOS procedure
A laboratory prototype (Hepa Wash GmbH, München, Germany) was employed to conduct the ADVOS procedure as already described in [10]. The treatment consists of an albumin dialysis performed through a three-circuit system (i.e. blood, dialysate, and ADVOS multi). The dialysate circuit allows to eliminate the excess of protein-bound and water-soluble toxins from patients’ body (Fig. 2). In the ADVOS multi circuit, toxin-loaded albumin dialysate is divided into two. Before reaching the filters, acid (HCl) or base (NaOH) is added and each part is subjected to a pH and temperature change that favors toxin removal from albumin. The resulting dialysates containing toxin-free albumin join each other in order to reach the desired pH before entering the hemodialyzers.
The treatment was started 2½ h after induction of endotoxemia and continued for 7½ h. Blood circulated between 225 and 250 ml/min through two 1.8 m2 surface hemodialyzers (Fresenius Medical Care, Bad Homburg, Germany). Dialysate containing Na+, Cl−, K+, Mg2+, HPO4
2−, CO3
2−, glucose, and 3% albumin flowed at 1200 ml/min co-currently to blood. Postdilution (2 l/h) was performed with PrismaSol2® (Gambro Hospal GmbH, Gröbenzell, Germany). Thanks to the recycling circuit, albumin was supplied only at the beginning of the treatment.
The same anticoagulation protocol (with heparin) described previously was employed [10]. However, in order to enable a change to citrate anticoagulation if necessary, the dialysate solution did not contain any calcium. Therefore, external calcium infusions were needed to be administered so that calcium loss was corrected. None of the animals receive citrate anticoagulation throughout the study.
Experimental design
Randomization
Ten pigs were randomly allocated to either control (n = 5) or ADVOS group (n = 5) following a block randomization with SPSS® for Windows (Additional file 1: Table S2). We performed randomization on day 1 of the experiments.
End points of the study
The primary end point of the study was to evaluate the potential survival benefit of the ADVOS procedure in a swine model with a sepsis-like syndrome. Animals were considered dead if cerebral perfusion pressure (CPP) was lower than 5 mmHg for 5 min. Surviving animals were sacrificed with an intravenous lethal dose of pentobarbitone and KCl 10 h after start of endotoxemia (T10). Additionally, the effects of the ADVOS procedure in the course of an endotoxin-induced sepsis-like syndrome were evaluated, paying special attention of those systems involved in the estimation of the SOFA score (coagulation, cardiovascular, cerebral, renal, respiratory, and hepatic systems).
Monitoring and sample analysis
Fluid balance
On day 3, cannulation and adjustment of fluid therapy by the PiCCO system (Pulsion Medical Systems AG, Munich, Germany) were performed, as described previously [10, 24]. A dialysis catheter (13 F high flow two-lumen 20 cm, Achim Schulz-Lauterbach VMP, Iserlohn, Germany) was inserted, placing the tip in the right atrium of the heart. PiCCO parameters were recorded each hour between T0 and T10. The administered fluids were adjusted according to the electrolyte status and included crystalloids like normal saline, dextrose 5–20% with or without KCl and/or bicarbonate. Target for fluid therapy was to keep extravascular lung water index (ELWI) <12 ml/kg and global end-diastolic volume index (GEDI) between 500 and 800 ml/m2.
In the case of metabolic acidosis (pH < 7.3), sodium bicarbonate (8.4%) was administered to increase bicarbonate levels (aim 28–30 mmol/l). Infusion fluids were supplemented with potassium (KCl, 20–80 ml, 1 M) or calcium (calcium gluconate 10%, 10–100 ml/h) to prevent hypokalemia (<3 mmol/l), or hypocalcemia (<1.2 mmol/l), respectively. Additionally, potassium levels above 4.8 mmol/l were treated by insulin injections (5–25 IU) in boli with simultaneous adjustment of glucose infusions (5 or 20%, to maintain levels between 110 and 150 mg/dl). Ninety minutes were allowed for hemodynamic parameters to stabilize after completing all surgical procedures.
Intracranial pressure measurement
Intracranial pressure (ICP) and temperature were monitored every 15 min between T0 and T10 using an intraparenchymal transducer combined with Datalogger MPR2 logO (Raumedic AG, Münchberg, Germany), as previously described [24].
Cardiovascular monitoring
Cardiac rhythm was monitored via a standard lead II electrocardiogram. Hemodynamic and respiratory parameters such as oxygen saturation, arterial blood pressure, end-tidal volume, or heart rate were monitored every 15 min between T0 and T10 using the Compact Critical Care Monitor (Datex-Ohmeda, Helsinki, Finland).
Blood sample analysis
Blood samples for biochemical analyses (among others, liver enzymes, creatinine, lactate, BUN, and ammonia) were collected on day 3 just after anesthesia and intubation (day 3 pre-endotoxemia), immediately before induction of liver injury (day 0) and endotoxemia, i.e., after completing minor surgical procedures and stabilization period (T0), and every 2 h after endotoxemia (T2, T4, T6, T8, and T10 or prior to death). Samples were sent to the in-house laboratory. Blood gas analysis (including glucose and electrolyte measurement) was performed more frequently (Rapidpoint® 405, Siemens Health Care Diagnostics Inc., Eschborn, Germany) to ensure quick adjustment of glucose and PaO2.
Statistics
The log-rank test was employed to evaluate survival, whereas Student’s t test for paired samples was used to compare the pre-endotoxemia parameters between day 0 and day 3. A repeated measures ANOVA was used to evaluate the effects of the ADVOS procedure on the course of endotoxemia and for intergroup comparison (T6). A two-tailed p value lower than 0.05 was considered to indicate statistical significance. Data were documented and analyzed using IBM SPSS 19.0 for Windows®. If any data, especially at the end of the experiments, were missing due to death of the animal, they were assumed to be equal to the latest measured value in accordance to the last observation carried forward (LOCF) method [25].