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).