Angiotensin 1–7 in an experimental septic shock model

Study setting

The study followed the EU Directive (2010/63/EU) for animal experiments and was approved by the local animal ethics committee (Protocol number 772N, Comité Ethique du Bien-Être Animal, from the Université Libre de Bruxelles (ULB) in Brussels, Belgium). Experiments were performed in the Experimental Laboratory of Intensive Care of the ULB (LA1230406). The ARRIVE guidelines and MQTiPSS recommendations for translational research in sepsis were followed [20, 21].

An ovine model of fecal peritonitis, adapted from previous experiments [22,23,24], was used, with 14 domestic female adult (6–8 months) Suffolk sheep included. Only females were used to facilitate access to bladder catheterization and increase homogeneity.

Experimental procedure

On the day of the experiment, the animals were weighed, premedicated with an intramuscular mixture of 0.25 mg/kg midazolam and 20 mg/kg ketamine, and placed in the supine position. An 18G peripheral cannula was inserted into the cephalic vein to ensure vascular access.

After administration of an intravenous bolus of 30 μg/kg of fentanyl citrate, 1 mg/kg of propofol, and 0.1 mg/kg of rocuronium bromide, an 8 mm endotracheal tube was introduced. All the animals were sedated with 1.8–2.4% alveolar concentration of sevoflurane, and a continuous intravenous infusion of morphine at a rate of 0.2–0.4 mg/kg/h. The optimal dose was determined through repeated pain tests, and in the absence of other explanatory factors, additional boluses of 0.1 mg/kg were given. Rocuronium bromide was administered at 0.1 mg/kg/h for muscle paralysis. Hypoglycemia was avoided by giving a continuous infusion of a 20% glucose solution. A 60 cm long plastic tube (inner diameter 1.8 cm) was inserted via the esophagus into the rumen to drain its content and to prevent rumen distension.

Mechanical ventilation was started in a volume-controlled mode (Primus, Dräger, Lübeck, Germany) using a tidal volume of 8 mL/kg, positive end-expiratory pressure of 5 cmH2O, a fraction of inspired oxygen of 30%, ratio of inspiratory time to expiratory time of 1:2 and a square-wave pattern. Respiratory rate was adjusted to maintain end-tidal carbon dioxide pressure (PetCO2) between 35 and 45 mmHg. Animals were under mechanical ventilation until the end of the experiment.

A 4.5 G arterial catheter was introduced into the left femoral artery under ultrasound guidance (Vivid E90, GE Machines, USA) connected to a pressure transducer and zeroed at the mid-thorax level. Pulse pressure variation (PPV) was automatically calculated from the arterial femoral signal using the formula [PPV = PPmax − PPmin/(PPmax + PPmin)/2], with PP being the pulse pressure (i.e., the difference between systolic and diastolic arterial pressures), continuously displayed (SC9000, Siemens, Munich, Germany), and exported to a recording station (Notocord-Hem 4.4, Notocord, France). In addition, an 8 Fr introducer was inserted into the left jugular vein, to introduce a 7.5F Swan-Ganz catheter (CCO, Edwards LifeSciences, Irvine, California, USA) into the pulmonary artery. A three-lumen central line catheter was inserted in the right jugular vein to provide fluids and drug infusion. A 14 Fr Foley catheter was inserted into the bladder and connected to a manometer to monitor intra-vesical pressure and to a urine collection bag for monitoring of urine output.

A midline laparotomy was performed. After cecotomy, 1.5 g/kg body weight of feces was collected and stored. The cecum was then closed and repositioned in the abdominal cavity. Two plastic tubes were left behind for later introduction of the feces and peritoneal lavage. After abdominal surgery, the animals were placed prone. Baseline measurements were taken, and feces were then injected into the abdominal cavity.

Immediately thereafter, seven of the animals received a continuous infusion of 10 μg/kg/h of Ang-(1–7) (Chemcube, Bochum, Germany—Ang-(1–7) group), and seven received a corresponding volume of saline solution (placebo) until the end of the experiment (Fig. 1). The selected dose of Ang-(1–7) was derived from a study using a rat model of ARDS [16], in which a low dose of 0.27 μg/kg/h Ang-(1–7) improved oxygenation and a high dose of 60 μg/kg/h reduced inflammation; an intermediate dose was therefore selected in this experiment.

Fig. 1figure 1

During the first 4 h, fluids were maintained at 2 mL/kg/h. Then, fluid resuscitation was then started with equal amounts of crystalloid (Plasmalyte, Baxter, USA) and colloid (Geloplasma, Fresenius Kabi, France) solutions, targeting a PPV < 13% in case of MAP ≤ 65 mmHg. Peritoneal lavage was performed using four liters of warm (38° Celsius) saline through the abdominal drain tubes. Intravenous norepinephrine was started if the mean arterial pressure (MAP) was ≤ 65 mmHg despite fluid administration, and titrated to a maximum dose of 5 μg/kg/min.

Four hours after injection of the feces, meropenem was administered as an intravenous bolus of 20 mg/kg, followed by a continuous infusion of 2.5 mg/kg/h until the end of the experiment. Experiments were continued until spontaneous death or for 24 h at which point the animals were euthanatized under deep anesthesia with a bolus injection of 40 mL of potassium chloride solution.

Data collection and blood sampling

Variables, including MAP (mmHg), pulmonary artery (PA) pressure (mmHg), right atrial pressure (mmHg) and PA wedge pressure (mmHg), were continuously displayed (SC9000, Siemens, Munich, Germany) and exported to an A/D recording station (Notocord-Hem 4.4, Notocord, France). Variables were referenced to the mid-chest level and obtained at end expiration. Core temperature (°C) and cardiac output (L/min) (Vigilance II; Edwards Lifesciences, California, USA), as well as minute volume (mL), plateau pressure (mmHg), expiratory tidal volume (mL), and end-tidal carbon dioxide pressure (mmHg) were continuously monitored. Cardiac index (L/min/m2), stroke volume index (mL/m2), systemic vascular resistance (dynes/s/cm5), and pulmonary vascular resistance (dynes/s/cm5) were calculated using standard formulas.

Urine output (UO) was monitored hourly. Arterial and mixed central venous blood gas samples were obtained every hour. Additional arterial samples were obtained at baseline and T4, T8, T12, T16, T20 and T24 hours after sepsis induction for later determination of blood creatinine and interleukin (IL)-6 levels. They were sampled in EDTA-syringes and centrifuged at 3000 rounds per minute for 15 min, then immediately frozen at − 20 °C until analysis.

Multiplex cytokine magnetic bead panel assay

Protein levels of IL-6 and IL-10 in systemic arterial plasma were determined using a cytokine magnetic bead panel assay (MILLIPLEX® Ovine Cytokine Multiplex Assay, Merck, Germany), according to manufacturer’s instructions. Cytokine concentrations were obtained by referring to a standard curve realized in parallel. Results represented the mean value of two separate measurements performed in duplicate at each time point.

Statistical analysis

The number of animals was selected based on our previous experience with this animal model [22, 24]. Statistical analysis was performed using Prism 9 (Version 9.1.2 (225). San Diego, CA, USA). Continuous variables are presented as means ± standard deviation (SD) or median [25, 75% interquartile range (IQR)]. To estimate the effect of Ang-(1–7) administration during the observational period, a mixed-effects model with Greenhouse–Geisser correction was used. The effects of time and group, as well as the interaction between group and time, were tested as fixed effects, and animals were introduced as random effects. If there were significant differences, the two-stage linear procedure of Benjamini, Krieger, and Yekutieli, with individual variances, was used for comparison of the means of these variables between the groups at each time point. Differences in time to develop several predefined organ failure parameters and survival time between groups were tested using a log-rank test. A p value of < 0.05 was considered statistically significant.

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