METHOD DETAILS
Pulmonary Function Tests
Lung tissue physiology was evaluated by derivation of the Constant Phase Model parameters from the lung input impedance measurements(1). Animals were anesthetized with an intraperitoneal injection of 90 mg/kg ketamine (Imalgène ®, Merial, Lyon, France) and 10 mg/kg xylacine (Rompun®, Bayer AG, Leverkusen, Germany). Endotracheal intubation was performed using the BioLite system (Biotex, Houston, Texas), to illuminate the trachea with a fiber optic stylet. After intubation, animals were connected to a Flexivent rodent ventilator (Scireq, Montreal, Canada), at a rate of 200 breaths/min and a tidal volume of 10 ml/kg. Animals were kept breathing isoflurane at 2% concentration until complete relaxation was achieved. They were kept breathing 0.5% isoflurane during the lung function tests and micro-CT data acquisition.
Measurements were performed three times to reduce variability, using a single-frequency forced oscillation and fitting the measured data to a first order single compartment model, which consists of a conduit (with resistance R) serving a reservoir (with compliance C).
Histological analysis
After micro-CT imaging, mice were anesthetized with ketamin and xylacin and then sacrificed by exsanguination. A 22G cannula was inserted in the trachea and secured with a thread. The lungs and the heart were removed en bloc and then fixed for one hour with 4% formaldehyde introduced in the lungs through the cannula at a constant pressure of 20 cm H2O. The lungs were immersed in fresh 4% formaldehyde for 24 hours and then in 70% ethanol for 24 hours, before being included in paraffin. Three different paraffin blocks were built from each lung, dividing the lobes in the following way: block 1 contained right lobes 1 and 2; block 2 contained right lobe 3 divided in two; and block 3 contained the left lobe divided in two. The right lobe 4 was reserved for RNA extraction. Three non-consecutivesections per paraffin block werestained with hematoxylin and eosin, resulting in 9 slides per mouse, each containing two lobe pieces.
Images of the lobe sections were acquired with a Zeiss Axioplan 2iemicroscope (Carl Zeiss, Jena, Germany) with a 10x magnification.Lobular walls were automatically segmented and analyzed in order to detect andremove vessels and alveolar walls. This step was necessary becausethese structures would otherwise affect the quantification ofemphysema. Finally the Lm and D2 parameters were automaticallycomputed(2). D2 represents the mean area of the alveoli sections corrected with the standard deviation and the skew to account for variability in alveolar sizes. In order to reduce computation time, theanalysis was executed in parallel on 5 machines. The algorithms wereprogrammed in hybrid Python/C++ code and made use of the ITKlibrary.
RNA cytokine expression
The right accessory lobe of every animalwas dissected and frozen in isopentane previously cooled in liquid nitrogen. Tissue was then stored at -80ºC for further analysis.Lysate was obtained by physic homogenization of the frozen tissue with a steel mortar and a mixer mill (Retsch MM301).
Total RNA isolation of the lysate was performed using standard QUIAGEN RNeasy Micro KIT (including the DNase step) following manufacturer's instruction. RNA concentrations and the A260/A280 ratio were measured with a a NanoDrop® ND-1000 (NanoDrop Technologies, Montchanin, DE, USA). Absence of DNA contamination was checked by running samples on 2% agarose gels.
1 µg purified RNA was reverse transcribed. Before transcription, RNA was denatured for 5 min at 65°C followed by cooling on ice. First strand cDNA synthesis was carried out with SuperScript™ III Reverse Transcriptase (Invitrogen) and random primers (Invitrogen) in a total volume of 20 μl. Reverse transcription was performed at 50°C for 50 minutes followed by 70°C for 15 min. Finally, RNase H was added to the reaction mixture followed by incubation for 20 min at 37°C. cDNA was stored at -80°C until RT-PCR analysis. Each RNA sample was controlled for genomic DNA contamination by a reaction mix without reverse transcriptase addition. All cDNAs were diluted 1:10 before being used as PCR template.qRT-PCR was performed with an Applied Biosystems 7900HT Fast Real-time PCR System.
A set of seven inflammatory cytokines was selected based on a literature search on cytokines present in chronic obstructive pulmonary disease (COPD). The selected cytokines were: interleukin 1-β (IL1-β), interleukin 6 (IL6), inmune protein 10 (IP10), keratinocyte chemoattractant (KC), monocyte chemoattractant protein 1 (MCP1), macrophage inflammatory protein 1 α (MIP1-α), and tumor necrosis factor α (TNF-α). In addition, Beta-2 microglobulin (B2M) was used as an endogenous control for each sample, because it provided homogeneous results at different melting temperatures. The primers for each gene are specified in Table E1.
GENE / Annealling Temperature / Sense5'3' / Antisense
5'3' / Product
size (bp)
IL1-β / 59 ºC / GGATGAGGACATGAGCACCT / TAATGGGAACGTCACACACC / 108
IL6 / 60 ºC / TCTCTGGGAAATCGTGGAAA / TTCTGCAAGTGCATCATCGT / 83
IP10 / 60 ºC / AATCATCCCTGCGAGCCTAT / TTTTTGGCTAAACGCTTTCATT / 131
KC / 58 ºC / TGGGATTCACCTCAAGAACA / TTTCTGAACCAAGGGAGCTT / 137
MCP1 / 60 ºC / AGGTCCCTGTCATGCTTCTG / GGGATCATCTTGCTGGTGAA / 128
MIP1-α / 60 ºC / CTGCCTGCTGCTTCTCCTAC / CCCAGGTCTCTTTGGAGTCA / 148
TNF-α / 58 ºC / GCCTCTTCTCATTCCTGCTT / AGGGTCTGGGCCATAGAACT / 134
B2M / 60 ºC / ACCCTGGTCTTTCTGGTGCT / ATGTTCGGCTTCCCATTCTC / 111
Table E1 Annealling temperature, primers and product size for the genes used in the qRT-PCR process for measurement of inflammatory cytokine RNA expression.
The 2-∆∆CT method was used to quantify relative changes in gene expression between control and treated animals, where
∆∆CT= (CT,Target gene – CT, B2M)Treated – (CT,Target gene – CT, B2M)Control
Protein cytokine expression
The concentration of cytokines and chemokines in plasma was determined simultaneously using Luminex ® Xmap technology. Briefly, blood samples were extracted from mice by facial vein puncture before sacrifice for histological analysis. Blood was added in EDTA tubes, plasma was removed after double centrifugation (1500 g for 10 min) and frozen at -80ºCfor storage. A mouse cytokine MILLIPLEX™ MAP kit (Millipore, Billerica, MA, USA) was used for measurement of 7 immune-modulatory cytokines and chemokines including IL-1β, IL-6, IP-10, KC, MCP-1, MIP-1α and TNF-α. The procedure for quantification was performed according to the manufacturer’s protocol. A Luminex 100 IS System (Luminex, Austin, TX, USA) was used to read the samples, compute standard curves and estimate cytokine concentrations.
REFERENCES
1.Hantos Z, Daroczy B, Suki B, Nagy S, Fredberg JJ (1992) Input impedance and peripheral inhomogeneity of dog lungs. J Appl Physiol72:168-78
2.Parameswaran H, Majumdar A, Ito S, Alencar AM, Suki B (2006) Quantitative characterization of airspace enlargement in emphysema. J Appl Physiol100:186-93