Supplementary Materials and Methods
Animals and Treatment Groups
The mice were anesthetized with intraperitoneal ketamine hydrochloride (60 mg/kg) and
atropine sulfate (0.025mg/kg), and given either intranasal porcine pancreatic elastase
(100 U/kg) (Sigma-Aldrich) in 20 µl of sterile saline solution (elastase-induced
emphysema) or intranasal saline alone (control). Three weeks after elastase
administration, 5 animals were sacrificed from each of these groups.
Additional treatments were administered to four subgroups from the elastaseinduced emphysema or control animals (N  5 animals per each subgroup). In a first
subgroup, after the initial elastase treatment, intranasal recombinant HGF (12.5 µg/dose
in 20 µl solution) was given twice/week for 1, 2 or 4 weeks. In a second subgroup, also
initially elastase-treated, intranasal saline was given twice/week for 1, 2 or 4 weeks. In a
third subgroup, which had initially received saline, intranasal recombinant HGF was
given twice/week for 1, 2 or 4 weeks. In a fourth subgroup, also initially saline-treated,
intranasal saline was given twice/week for 1, 2 or 4 weeks. Animals in these subgroups
were sacrificed after these secondary treatments.
The elastase/HGF treatment protocol was repeated three times with wild type C57BL/6J
mice: once for histopathology, once for lung mechanics measurements and once for
protein and serum collections. An additional series was done with mice transplanted with
bone marrow from GFP-transgenic mice for identification of the role of bone marrowderived cells (7 animals/group C, D and E and 5 animals/all other groups in each time).
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In a preliminary study using blue dye, we confirmed that a liquid volume of 20 µl
achieves near homogenous distribution to all lobes of both lungs when given by both the
intranasal and intra-tracheal instillation techniques. As the intranasal route is less invasive
and more physiological, we used it for both elastase and HGF administrations.
Lung histopathology and quantitation of emphysema.
Mean linear intercept (Lm) was determined by counting alveolar wall intersections with
an array of test grid lines drawn horizontally on a printout of the digital image of each
section and calculated according to the equation: Lm=2LT/Iw, where Iw is the number of
times the test line intersected with an alveolar wall and LT is the length of the test line.
Lung mechanics measurements
Mice were terminally anesthetized with Ketamine (250mg/kg i.p.) then tracheostomized
and the trachea were cannulated with a 20-gauge cannula connected to a computercontrolled small animal ventilator (flexiVent; Scireq, Montreal, PQ, Canada) [1].
Diaphragmatic paralysis was obtained by xylazine hydrochloride (12 mg/kg, i.p.) and
mice were mechanically ventilated at 150 breaths/min with 6 ml/kg tidal volume. A
positive end-expiratory pressure of 3 cmH2O was applied by submerging the expiratory
line in water. Six-second sigh maneuvers to 3 times the tidal volume were performed
three times to ensure similar volume history and establish a stable baseline respiratory
system resistance and elastance. The compliance was determined by recording the
relaxation pressures during inflation and deflation in 0.1-ml steps between 0 and 20 cm
H2O. Pressures from the normalized compliance curves were extrapolated at 0.1-ml
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increments and used to establish the mean static lung compliance and elastance in each
group. Pressure volume curves were generated and automatically extrapolated and lung
mechanics values calculated by the FelxiVent software.
Lethal irradiation and reconstitution of bone marrow
Adoptive transfer of bone marrow cells was performed as follow. Recipient C57BL/6
male mice were irradiated using two doses of 5 Gy, separated by 3 hours. Within 3 hours
after irradiation, the recipient C57BL/6 mice were injected intravenously with donor bone
marrow cells from GFP-transgenic mice (4 x 106 cells/200 µl media). Three weeks after
transplantation, over 95% of the circulating white blood cells were GFP-positive,
indicating that the recipient C57BL/6 mice had completely reconstituted with bone
marrow cells of GFP mouse origin.
FACS analysis
Single cell suspensions were prepared from the right lungs of GFP-chimeric mice as
follow. To remove most of the blood from lungs, animals were bled by transecting the
abdominal aorta. The pulmonary vasculature was perfused with 10 ml of PBS at
25cmH2O through the right ventricle. One ml dispase I (2 unites/ml, Roche Diagnostics,
Indianapolis, IN) was injected through the trachea, then lungs were incubated in a 37°C
shaking incubator for 45 minutes in 10 ml of dispase, 1 ml of 0.001% DNAse (Sigma)
and 1 ml of 2 μg/mL collagenase/dispase (Roche). The trachea and bronchi were
removed, and lung minced and re-incubated for 10 minutes. This suspension was filtered
through 100 μm mesh (BD Falcon, San Jose, CA), and centrifuged at 1000 rpm, 5 min at
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4°C. Red blood cells were depleted by RBC lysis buffer (Sigma). Cells were resuspended in PBS at 1 x 106 cells/100 μL, and stained with anti-mouse Sca-1-APC,
CD34-FITC, c-Kit-PE or CD45-PE and CD31-PE antibodies. Then, cells were analyzed
by the FACSCalibur flow cytometer (Becton Dickinson).
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Supplementary References:
1.
Schuessler, TF, Bates, JH (1995) A computer-controlled research ventilator for
small animals: design and evaluation. IEEE Trans Biomed Eng 42: 860-866.
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