Animals
Twenty male German landrace pigs (12-16 weeks, 28-35 kg) were acquired from a local private farm and were treated as described previously(10, 11).
Intervention
Following the baseline measurements, flexible bronchoscopy was performed using a single-use fiberoptic bronchoscope (Ambu aScope Regular, Ambu GmbH, Bad Nauheim, Germany). The right and left caudal main bronchi were identified and inspected, and after confirmed insertion of the endoscope into the respective bronchus, the animals were randomized into three groups.
Group 1 (“Intrabronchial Histones”), n=7: 50 ml of a saline solution containing 100 mg of mixed calf thymus histones (LS002548, Worthington Biochemical Corp, Lakewood, NJ, USA) was instilled through the bronchoscope into each caudal main bronchus separately, adding up to a total of 100 ml and 200 mg of histones.
Group 2 (“Sham”), n=7: 50 ml of a saline solution without any additives was instilled through the bronchoscope into each caudal main bronchus separately, adding up to a total of 100 ml.
Group 3 (“Intravenous histones, positive control”), n=6: Intravenous injection of 200 mg of the histone solution.
Monitoring
After the intervention, the animals were monitored for 8 hours, and sample collection was performed as described below. During the monitoring period, the mean arterial blood pressure was kept over 60 mmHg using a norepinephrine drip if necessary and glucose was substituted to maintain the levels above 80 mg/dl. The ventilation parameters were adjusted according to the ARDS network guidelines(12) once oxygen saturation decreased below 93%.
Measurements/sample collection:
Cardiopulmonary data were constantly measured and collected during the duration of the experiment using a Datex Ohmeda S5 monitor (GE Healthcare, Munich, Germany). These variables included respiratory rate, ventilation pressures, oxygen fractions, oxygen saturation, intra-arterial blood pressure, pulmonary artery pressure, heart rate and core temperature. Additionally, blood gas analyses and cardiac output (CO) measurements were taken at baseline and every hour after the intervention as described before(11).
After termination, both lungs were harvested, and samples from the cranial and caudal left lung lobes (central dorsal and ventral) were either snap frozen for biomolecular analyses or preserved in 2% formaldehyde solution for histologic fixation. Histopathologic scoring and interleukin 6 (IL-6) and tumor necrosis factor alpha (TNFα) expression analyses were performed via ELISA and RT-PCR as described previously (13, 14).
As the primary outcome parameter, pulmonary function represented by the Horovitz ratio (PaO2/FiO2) was determined. Secondary outcomes were the histological organ damages and the proinflammatory cytokine expressions.
The experiment was terminated with the animals being euthanized using high doses of propofol (200 mg) and potassium chloride (40 mmol).
Statistical analysis
Since this was a pilot study with no previous data to draw from, no adequate animal number calculation could be performed and animal numbers were chosen empirically. Statistical analyses were performed using 2-way ANOVA intergroup tests with a post hoc Bonferroni correction for repeated measurements as well as Mann-Whitney U test for single measurements via GraphPad Prism 8 software (GraphPad Software, Inc., La Jolla, CA, USA). Data in the text are presented as the mean (standard deviation). P-values < 0.05 were considered significant.