2.7 Animal studies
Animal studies adhered strictly to the Italian Ministry of Health guidelines for the use and care of experimental animals (protocol #549 and 733). Research with P. aeruginosa RP73 isolate from CF individuals has been approved by the Ethics Commission of Hannover Medical School, Germany. The patient and parent gave informed consent before the sample collection. Approval for storing of biological materials was obtained by the Ethics Commission of Hannover Medical School, Germany. C57Bl/ 6NCrlBR male mice (8 to 10 weeks of age) from Charles River were challenged with 1x106 CFUs of MDR-RP73 embedded in agar beads for chronic infection by intratracheal administration, as previously described (Bragonzi, 2010; Paroni et al., 2013; Facchini et al., 2014).
First, mice were treated by gavage with Roflumilast (5 mg/Kg) or vehicle (4,4%DMSO in saline) daily, starting two hours before infection. Health and body weight was monitored daily. Mice were sacrificed five days after infection, two hours after the last treatment. Lungs were excised and analysed for bacterial load, by measuring Colony Forming Units (CFU). Bronchoalveolar lavage fluid (BALF) was analysed for total and differential cell count, amount of free DNA and cytokine content. Free DNA was measured by Quant-iTTM dsDNA high-sensitivity assay kit as for in vitro experiments. Cytokines were measured using a competitive ELISA method or a Luminex multi-analyte assay (ProcartaPlex, Thermo Fisher Scientific, Monza, Italy).
Next, mice were treated per aerosol with roflumilast (5 mg/kg) or vehicle (4,4% DMSO in saline) using Penn Century as previously described (Cutone et al., 2019). The drug or vehicle were administered once a day, starting from 4 hours after infection. Each group of treatment was divided in two: one group of animals was sacrificed 28 hours after infection (2 hours after treatment), to analyse the effect of treatments on the acute phase of the infection, whereas the other group was sacrificed five days after infection (2 hours after the last treatment), to analyse the effect of treatments in chronic infection.
Body weight was determined, and aerosol administration was carried out under anaesthesia (5% isoflurane–oxygen, running at 4 l/min) according to established procedures once a day. At the end of the experiment, BALF was collected and analysed for cell, free-DNA and cytokine content. A fraction of BALF containing a fixed number of neutrophils (1 x 105) was centrifuged, and the pelleted cells were immediately frozen and stored for western-blot analysis of citrullinated Histone H3. Lungs were excised, homogenized and CFU counts performed as reported (Bragonzi, 2010; Paroni et al., 2013; Facchini et al., 2014; Cutone et al., 2019).