SUPPLEMENTARY MATERIALS AND METHODS
This study was approved by the Ethics Committee of the Health Sciences Center,
Federal University of Rio de Janeiro. All animals received humane care in compliance
with the “Principles of Laboratory Animal Care” formulated by the National Society for
Medical Research and the “Guide for the Care and Use of Laboratory Animals” prepared
by the National Academy of Sciences, USA.
Animal Preparation and Experimental Protocol
A total of 186 C57BL/6 mice: 144 females and 42 males (20-25 g) and 6 male
green fluorescent protein (GFP) transgenic mice (20-22 g) were used. In 84 female mice,
static lung elastance, histology, and molecular biology were analyzed. The remaining 60
mice were used to evaluate total and differential cell count as well as cytokines in
bronchoalveolar lavage fluid (BALF). All females were randomly assigned to two
groups. In control (C) groups, sterile saline solution (0.9% NaCl) was intratracheally
instilled (0.05 mL, Cp) or intraperitoneally injected (0.5 mL, Cexp). In ALI groups, mice
received E. coli lipopolysaccharide (LPS) (serotype O55:B5, Sigma Chemical Co., St
Louis, MO, USA) either intratracheally (40 g diluted in 0.05 mL of saline/mouse, to
induce ALIp) or intraperitoneally (400 g diluted in 0.5 mL of saline/mouse, ALIexp)
(Fig.1). For intratracheal instillation, mice were anesthetized with sevoflurane. A 1-cm
midline cervical incision was made to expose the trachea, and LPS or saline were
instilled using a bent 27-gauge tuberculin needle. The cervical incision was closed with
5.0 silk suture and the mice were returned to their cage. At six hours, C and ALI groups
were further randomized into subgroups receiving saline (0.05 mL, SAL) or BMDMC
(2x106 cells/0.05 mL of saline) intravenously (iv).
Extraction of bone marrow-derived mononuclear cells
Bone marrow cells from all male C57BL/6 and GFP mice were extracted and
administered on the day of collection. Briefly, under anesthesia with ketamine (25 mg/kg)
and xylazine (2 mg/kg) iv, bone marrow cells were aspirated from the femur and tibia by
flushing the bone marrow cavity with Dulbecco’s modified Eagle’s medium (DMEM)
(Life Technologies, Grand Island, NY, USA). After a homogeneous cell suspension
was achieved, the cells were centrifuged (400×g for 10 min), re-suspended in DMEM and
added to Ficoll-Hypaque (Histopaque 1083, Sigma Chemical Co., St. Louis, MO, USA),
and again centrifuged and supplemented with sterile phosphate-buffered saline (PBS).
Cells were counted in a Neubauer chamber with Trypan Blue for evaluation of viability.
For the administration of saline or BMDMC, mice were anesthetized with sevoflurane.
The jugular vein of each mouse was dissected, and cells were slowly injected using a bent
27-gauge tuberculin needle.
Lung Mechanics
One and 7 days after saline or LPS administration, animals were sedated (diazepam
1 mg intraperitoneally), anesthetized (thiopental sodium 20 mg/kg intraperitoneally),
tracheotomized, paralyzed (vecuronium bromide, 0.005 mg.kg iv), and ventilated with a
constant flow ventilator (Samay VR15; Universidad de la Republica, Montevideo,
Uruguay) with the following parameters: frequency of 100 breaths/min, tidal volume
(VT) of 0.2 mL, and fraction of inspired oxygen of 0.21. The anterior chest wall was
surgically removed and a positive end-expiratory pressure (PEEP) of 2 cmH2O was
applied. After a 10-min ventilation period, lung mechanics were computed and, at the end
of the experiment (approximately 30 min), the lungs were prepared for histology and
molecular biology.
Airflow, volume and tracheal pressure (Ptr) were measured (1). In an open chest
preparation, Ptr reflects transpulmonary pressure (PL). Static lung elastance (Est,L) was
computed as end-inspiratory transpulmonary plateau pressure – PEEP divided by VT (23). All data were analyzed using ANADAT data analysis software (RHT-InfoData, Inc.,
Montreal, Quebec, Canada).
Histology
A laparotomy was done immediately after the determination of lung mechanics, and
heparin (1,000 IU) was intravenously injected in the vena cava. The trachea was clamped
at end-expiration, and the abdominal aorta and vena cava were sectioned, yielding a
massive hemorrhage that quickly killed the animals. Right lung, kidney, and liver were
then removed, fixed in 3% buffered formaldehyde and paraffin-embedded. Four-µmthick slices were cut and stained with hematoxylin-eosin.
Lung morphometric analysis was performed with an integrating eyepiece with a
coherent system consisting of a grid with 100 points and 50 lines (known length) coupled
to a conventional light microscope (Olympus BX51, Olympus Latin America-Inc.,
Brazil). The volume fraction of the lung occupied by collapsed alveoli or normal
pulmonary areas were determined by the point-counting technique (4) at a magnification
of X200 across 10 random, non-coincident microscopic fields.
Collagen (picrosirius-polarization method) (5) and elastic fibers (Weigert’s resorcin
fuchsin method modified with oxidation) (6) were quantified in the alveolar septa. The
area occupied by fibers was determined by digital densitometric recognition and divided
by the length of each studied septum (7).
Electron microscopy
Three slices measuring 2x2x2 mm were cut from three different segments of the left
lung and fixed [2.5% glutaraldehyde and phosphate buffer 0.1 M (pH = 7.4)] for electron
microscopy (JEOL
1010 Transmission Electron Microscope, Tokyo, Japan) analysis.
For each
electron microscopy image (15/animal), the following structural damages were analyzed:
a) alveolar capillary membrane, b) type II epithelial cells, and c) endothelial cells.
Pathologic findings were graded according to a 5-point semi-quantitative severity-based
scoring system as: 0=normal lung parenchyma, 1=changes in 1 to 25%, 2=changes in 26
to 50%, 3=changes in 51 to 75%, and 4=changes in 76 to 100% of examined tissue (2-3).
Confocal microscopy
For recipients of GFP marrow transplants, frozen sections were treated with 4’,6diamidino-2-phenylindole dihydrochloride (DAPI)-supplemented mounting medium
(Vectashield, Vector Labs, Burlingame, CA), cover slipped and examined for GFP
expression by confocal microscopy. Background autofluorescence was determined
through examination of 10 simultaneously prepared negative control sections that were
stained with DAPI alone. All sections were examined using a Zeiss LSM-410 laserscanning confocal microscope equipped with GFP (green) and DAPI (blue) filter sets.
Apoptosis assay of lung and distal organs
Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL)
staining was used in a blinded fashion by two pathologists to assay cellular apoptosis.
Apoptotic cells were detected using In Situ Cell Death Detection Kit, Fluorescin
(Boehringer, Mannheim, Germany). The nuclei without DNA fragmentation stained blue
as a result of counterstaining with hematoxylin (8). Ten fields per section from the
regions with apoptotic cells were examined at a magnification of X400. A 5-point semiquantitative severity-based scoring system was used to assess the degree of apoptosis,
graded as: 0=normal lung parenchyma; 1=1-25%; 2=26-50%; 3=51-75%; and 4=76100% of examined tissue.
Evaluation of BALF
Animals for BALF evaluation were killed in a CO2 chamber and exsanguinated.
The lungs were flushed four times with 0.5 mL sterile saline preheated to 37oC (0.9%
NaCl) via the tracheal cannula. Total cell counts in the BALF were performed in
Neubauer chambers under light microscopy after dilution of the samples in Türk solution
(2% acetic acid). Differential neutrophil counts were performed in cytospin smears
stained by the May-Grünwald-Giemsa method. Levels of IL-6, KC (murine IL-8
homolog), IL-10 and vascular endothelial growth factor (VEGF) were evaluated by
enzyme-linked immunosorbent assay on cell-free BALF according to manufacturer’s
protocol (Duo set, R & D Systems, Minneapolis, MN).
Expression of growth factors and caspase-3 mRNA
Quantitative real-time reverse transcription (RT) polymerase chain reaction
(PCR) was performed to measure the relative levels of expression of VEGF, transforming
growth factor (TGF)-, platelet derived growth actor (PDGF), insulin growth factor
(IGF) and caspase-3 genes. Central slices of left lung were cut, collected in cryotubes,
quick-frozen by immersion in liquid nitrogen and stored at -80ºC. Total RNA was
extracted from the frozen tissues using the Trizol reagent (Invitrogen, Carlsbad, CA)
according to manufacturer's recommendations. RNA concentration was measured by
spectrophotometry in Nanodrop® ND-1000. First-strand cDNA was synthesized from
total RNA using M-MLV Reverse Transcriptase Kit (Invitrogen, Carlsbad, CA). The
following PCR primers for target gene (Invitrogen, Carlsbad, CA) were used: VEGF:
forward
[5’-CCACGACAGAAGGAGAGCA-3’]
AATCGGACGGCAGTAGCTT-3’],
and
TGF-:
reverse
forward
[5’[5’-
ATACGCCTGAGTGGCTGTC-3’ and reverse [5’-GCCCTGTATTCCGTCTCCT-3’],
PDGF:
forward
[5’-TCGAAGTCAGATCCACAGCA-3’]
CTTGTCTCCAAGGCATCCTC-3’],
IGF:
and
reverse
forward
[5’[5’-
CTGGTGGATGCTCTTCAGTTC-3’] and reverse [5’-CAGTCTCCTCAGATCACAGC3’], and caspase 3: forward [5’-TACCGGTGGAGGCTGACT-3’ and reverse [5’GCTGCAAAGGGACTGGAT-3’]. Relative mRNA levels were measured with a SYBR
green detection system using Applied Biosystems 7500 Real-Time PCR (Applied
Biosystems, Foster City, CA) (95°C, 10 min; 40 cycles 95°C, 15 s; 60°C, 1 min). All
samples were measured in triplicate. The relative amount of expression of each gene was
calculated as a ratio of studied gene and control gene [glyceraldehyde-3-phosphate
dehydrogenase – GAPDH: forward [5’-ACCACGGTGCACGCCATCAC-3’] and reverse
[5’-TCCACCACCCGTTTGCTGTA-3’] and expressed as fold change relative to Cp.
Statistical analysis
The normality of the data (Kolmogorov-Smirnov test with Lilliefors’ correction)
and the homogeneity of variances (Levene median test) were determined. If both
conditions were satisfied, differences between the groups at day 1 were assessed by twoway ANOVA followed by Tukey’s test. At day 7, the effects of BMDMC therapy were
analyzed using three-way ANOVA followed by Tukey’s post-hoc test. To compare nonparametric data, three-way ANOVA on ranks was selected. Survival curves were derived
by the Kaplan-Meier method and compared by log-rank test. A p value < 0.05 was
considered significant. Parametric data were expressed as mean ±SD, while nonparametric data were expressed as median (interquartile range). SigmaStat 3.1 statistical
software package (Jandel Corporation, San Raphael, CA, USA) was used.
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