3.2.1 Mice
TAp73 mutant mice with targeted deletion of exons 2 and 3 of the Trp73 gene were a generous gift from Dr. Tak Mak (Princess Margaret Cancer Centre, Toronto, Canada) (Tomasini et al., 2008). miR449 mutants were previously described (Song et al., 2014). Both strains were maintained in C57Bl/6 background (n8) at the animal facility of the European Neuroscience Institute Göttingen, Germany in full compliance with institutional guidelines.
The study was approved by the Animal Care Committee of the University Medical Centre Goettingen and the authorities of Lower-Saxony under the number 16/2069 (TAp73xmiR449 KO mice) and G12/963 (naphthalene). As described in (Wildung et al., 2018).
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WT (C57BL/6) mice used for DEP experiments were obtained from Harlan Laboratories (Horst, The Netherlands) and maintained in the animal facility of the Ghent University. The Animal Ethical Committee of the Faculty of Medicine and Health Sciences of Ghent University approved the in vivo manipulations using DEP.
Experiments including the exposure to CS and nontypeable Haemophilus influenzae (NTHi) were approved by the “Landesamt für Soziales, Gesundheit und Verbraucherschutz” of the State of Saarland following the national guidelines for animal treatment. During the experiment WT and miR449-/- mice were maintained under specific pathogen-free conditions in the animal facility of the Institute for Experimental Surgery of the Saarland University Homburg.
3.2.2 Naphthalene-induced injury model
Naphthalene (Merck) was dissolved in sunflower oil to a concentration of 30 mg/ml in a sterile environment on the day of use.12-14 weeks old male WT and miR449-/- mice were injected with naphthalene (200 mg/kg) or with oil (vehicle control) i.p. in the morning on d0.
Changes in body weight were monitored every day during the first week and then again at the endpoint (d14). Lung analysis was performed at d1, d3, d7, and d14.
3.2.3 DEP-induced acute inflammation model
DEP instillation was performed as described previously (Provoost et al., 2016). Briefly, DEP (standard reference material (SRM) 2975) was obtained from the National Institute for Standards and Technology (Gaithersburg, MD, USA) and suspended in saline containing 0.05% Tween 80 to a concentration of 2 mg/ml. 6-8 weeks old anesthetized mice (i.p.
ketamine (70 mg/kg; Ketamine 1,000 CEVA, Ceva Sante Animale, Libourne, France) - xylazine (7 mg/kg; Rompun2%, Bayer, Leverkusen, Germany) were held vertically and 50 µl saline or DEP solution (100 µg) was pipetted just above their vocal cords. By grasping the tongue, the mice were prevented from swallowing. Mice were instilled at d1, d4, and d7 and sacrificed at d9 by a lethal dose of i.p. pentobarbital (Ceva Sante Animale). Experiment was performed by S. Provoost.
3.2.4 NTHi-induced chronic inflammation model
A clinical isolate of NTHi was grown on selective chocolate agar supplemented with 300 µl IsoVitaleX per 10 cm plate (Becton Dickinson, Heidelberg, Germany) for 24 h at 37°C in 5%
CO2. After harvesting, bacteria were incubated for 24 h in brain-heart infusion (700 ml) broth (Becton Dickinson) supplemented with 3.5 mg/ml β-Nicotinamide adenine dinucleotide (Merck) and 5% Fildes enrichment (Becton Dickinson). After a centrifugation step at 2,500 g for 15 min at 4°C, the culture was washed, resuspended in PBS (20 ml), and heat inactivated at 70°C on a shaker for 45 min. Following sonication (three times for 30 seconds), the protein concentration was determined and adjusted to 2.5 mg/ml using the
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Pierce BCA-protein assay (Thermo Fisher Scientific). For bacterial challenge, 4 months old WT and miR449-/- mice were transferred to a plexiglass box connected to a Pari MASTER®
nebulizer (Pari GmbH, Starnberg, Germany) and treated with the inactivated NTHi lysate for 1 h per day and 5 times per week for 3 months. Experiment was performed by C. Herr.
3.2.5 Cigarette smoke-induced COPD model
4 months old WT and miR449-/- mice were exposed to CS (3R4F cigarettes, College of Agriculture, Reference Cigarette Program, University of Kentucky, Lexington, Kentucky, USA) using a TE-10 smoking machine (Teague Enterprises, Woodland, California, USA).
The concentration of CS was 120 mg/m3 total suspended particles. Mice were subjected to CS for 5 h per day and 5 days per week over a period of 6 months. The daily smoking protocol consisted of three smoking phases each with 87 min, which were interrupted by room-air exposures for 40 min. Experiment was performed by C. Herr.
3.2.5.1 Pulmonary function
Pulmonary function analysis was carried out using the FlexiVent system (Scireq, Montreal, Canada). Mice were anesthetized by a mixture of xylazine (Rompun, 7 mg/kg, Bayer) and ketamine (Ursotamin, 100 mg/ml, Serumwerk Bernburg, Bernburg, Germany).
Subsequently, the trachea was exposed and cannulated using the 18 G cannula supplied with the FlexiVent system. Data acquisition was performed with the flexiWare 7.1 software (Scireq, Montreal, Canada). Experiment was performed by C. Herr.
3.2.5.2 Bronchoalveolar lavage
Prior to bronchoalveolar lavage (BAL) the animals were deeply anesthetized with a mixture of ketamine (Ursotamin, 105 mg/ml, Serumwerk Bernburg) and xylazine (Rompun, 7 mg/kg, Bayer). After reaching anesthesia the trachea was exposed and cannulated. The BAL was performed by rinsing the lungs 3 times repeatedly with 1 ml sterile PBS containing protease inhibitors (cOmplete ULTRA Tablets, Mini, Roche Diagnostic, Mannheim, Germany). The collected BAL fluid (BALF) was centrifuged for 5 min at 300 g. The number of viable cells was determined by resuspending the cell pellet in 1 ml of sterile PBS and counting on an improved Neubauer chamber after trypan blue staining. For the cellular composition of the BALF a cytospin was stained with DiffQuik (Medion Diagnostic, Gräfelfing, Germany) and at least 200 cells were counted and differentiated based on their morphology. Experiment was performed by C. Herr.
3.2.5.3 Stereology
For stereological analysis, the lung was prepared for uniform random sampling as described previously (Knust et al., 2009). Briefly, fixation of the lung was performed by instillation of freshly prepared PBS-buffered 4% formalin under a constant hydrostatic pressure of 20 cm
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for 20 min. After that, the trachea was ligated to preserve intrapulmonary pressure. For additional fixation, the lung was incubated in PBS-buffered 4% formalin for 24 h. The lung volume used as reference for stereological measurements was determined by fluid displacement. After embedding in agar, the lung was cut into regular slices prior to paraffin embedding. Stereological analysis was performed on hematoxylin and eosin (H&E) stained lung sections. The mean chord length (mean linear intercept, Lm) was determined using the Visiopharm Integrator System package (Visiopharm,Hoersholm, Denmark) on an Olympus BX51 microscope equipped with an 8-position slide loader (Olymus, Tokio, Japan).
Experiment was performed by C. Herr.