Ringerfundin

The experimental protocol is delineated in Fig 1. After instrumentation, animals were allowed to rest for 30 minutes, after which baseline (t_bsl) hemodynamic measurements, blood gas analyses and laboratory testing were performed. After baseline data were recorded, blood was drained from the arterial catheter placed in the left external carotid artery by direct suctioning with a 50 mL syringe, until the SV index (SVI) dropped by 50% of its initial value (t₀), then measurements were repeated. After reaching the target reduction of SVI, values of MAP were taken as targets of fluid replacement during the rest of the study. The difference between MAP_tbsl−MAP_t0 was divided equally into four target values, which were aimed to be achieved in four steps during fluid resuscitation (t_1–4) to reach the baseline MAP by t₄. Fluid loading was carried out with boluses of balanced crystalloid Ringer Fundin (B. Braun AG., Melsungen, Germany). For practical reasons the total amount of crystalloid to be used for resuscitation was limited to 4.5 times the volume of the drained blood. After reaching each step, 10 minutes were allowed for equilibrium, then hemodynamic and blood gas parameters were measured. At the end of resuscitation, the animals were euthanized with Na-pentobarbital (120 mg/kg) while still under anesthesia (dx.doi.org/10.17504/protocols.io.mrac52e)

The general condition of the animals was evaluated at 6 h after the ip. injections and every 12 h thereafter using a modified 0–9 point rat-specific RSS scoring system^{33 (link)}, where a cumulative value above 6 was considered a humane endpoint for euthanasia (Supplementary Table S3). At time points of sickness assessment, the animals received 10 mL/kg crystalloid solution sc. (Ringerfundin, B. Braun, Hungary) to avoid dehydration and 15 µg/kg buprenorphine sc. (Bupaq, Merck, USA) to maintain analgesia^{2 (link)}.

After induction of anesthesia, a concomitant norepinephrine infusion will be started at 2 μg/kg/h until the end of surgery, and the randomized crystalloid solution (Ringerfundin®; B Braun Medical AG, Sempach, Switzerland) for group 1 or the G5-K solution for group 2 (Bichsel, Interlaken, Switzerland) will be infused at a rate of 1 ml/kg/h until the bladder has been removed, followed by 3 ml/kg/h until the end of surgery. If hypotension is observed (mean arterial pressure (MAP) <60 mmHg), norepinephrine will be titrated to a maximum of 8 μg/kg/h after an initial bolus of 10 μg. If hypotension persists, a bolus of 250 ml of balanced Ringer’s solution (Ringerfundin®) in both groups will be given.
In both groups, blood loss of >500 ml will be substituted with an equal amount of Ringerfundin®. Packed red blood cells (PRBC) will be transfused if hemoglobin values are less than 80 g/l (<100 g/l in patients with coronary artery disease). Additional boluses of Ringerfundin® (250 ml) will be infused as a rescue medication if MAP persists at less than 60 mmHg after the aforementioned correction, and in cases of severe intraoperative metabolic acidosis (base excess < −5, pH <7.25) attributable to severe hypovolemia.

For the immunofluorescence-based biodistribution study, the tumor-bearing mice were intravenously injected once with 60 µg/mouse of IL2-7NP2-TNF^mut, IL2-F8-TNF^mut, and IL2-KSF-TNF^mut diluted in Ringerfundin (B. Braun, Bethlehem, PA, USA). At 24 h post-administration, the mice were sacrificed and the tumors and organs were collected in a cryo-embedding medium (NEG-50 Thermo Fisher) for the further analysis. The cryostat sections were stained and detected, as previously described.

The HES product used in this study was a potato-derived HES with a molecular weight of 130 kDa and a molar substitution of 0.42 in a concentration of 60 g/L (6 % solution) in an electrolyte-balanced and buffered carrier solution (Tetraspan®, BBraun Melsungen AG, Melsungen, Germany). The crystalloid was a modified Ringer’s acetate (RA) solution (Ringerfundin®, BBraun Melsungen AG, Melsungen, Germany).

Severe sepsis was induced either by continuous intravenous infusion of Pseudomonas aeruginosa (1 × 10⁹ colony-forming units/mL, n = 3 in non-AKI group, n = 2 in AKI group) or fecal peritonitis (n = 2 in non-AKI group, n = 3 in AKI group) as described previously [3 ]. The infusion rate of Pseudomonas aeruginosa was titrated to clinical goal of moderate pulmonary hypertension (MPAP 35-40 mmHg). In the peritonitis group, sepsis was induced by inoculating 0.5 g/kg of autologous feces incubated in 200 ml saline for 8 h at 37 °C through the drains into the abdomen. Continuous infusion of balanced crystalloids (Plasmalyte, Baxter Healthcare, Deerfield, IL, United States or Ringerfundin, BBraun Melsungen Ag, Melsungen, Germany) were used as a fluid replacement in dose 15 ml/kg/h during the surgery and reduced to 7 ml/kg/h thereafter. In addition to crystalloid solution, 6% hydroxyethyl starch 130 kD/0.4 (Voluven 6%, Fresenius Kabi Deutschland GmbH, Bad Homburg, Germany) was infused to maintain normovolemia in a goal-directed fashion guided by filling pressures response and ITBV measurement. Continuous i.v. noradrenaline was administered if mean arterial pressure (MAP) fell below 65 mmHg and titrated to maintain MAP above 70 mmHg. When the last set of data had been obtained, the animals were euthanized by potassium chloride injection under deep anesthesia and section was performed.