1
Electronic supplementary material
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3
Neurally adjusted ventilatory assist decreases ventilator induced lung injury and
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non-pulmonary organ dysfunction in rabbits with acute lung injury
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7
Running head: NAVA in rabbits with lung injury
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9
1,7
1,6
, François Lecomte 1, Howard Leong-Poi
10
Lukas Brander
11
Jennifer Beck
12
Parker 2,6, Jesús Villar 6,8, Haibo Zhang 1,6, Arthur S. Slutsky 1,6
4,6
, Christer Sinderby
2,6
, James N. Tsoporis 2, Rosanna Vaschetto 1, Marcus J. Schultz
, David Bell 3,
1,5
, Thomas G.
13
14
15
1
16
Critical Care Medicine, St. Michael’s Hospital, Toronto, Canada
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2
18
3
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Hospital, Toronto, Canada
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4
Department of Pediatrics, University of Toronto, Canada
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5
Department of Intensive Care Medicine and Laboratory of Experimental Intensive Care and
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Anesthesiology, University of Amsterdam, The Netherlands
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Toronto, Canada
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7
Department of Intensive Care Medicine, University Hospital - Inselspital, Bern, Switzerland
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8
CIBER de Enfermedades Respiratorias, Instituto de Salud Carlos III, Madrid, Spain; Translational
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Research on Organ Dysfunction, Research Unit, Hospital Universitario Dr. Negrin, Las Palmas de
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Gran Canaria, Spain
Interdepartmental Division of Critical Care Medicine, University of Toronto, Department of
Division of Cardiology, University of Toronto, St. Michael’s Hospital, Toronto, Canada
Department of Laboratory Medicine and Pathobiology, University of Toronto, St. Michael's
Keenan Research Center at the Li Ka Shing Knowledge Institute of St. Michael's Hospital,
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30
The work was performed at St. Michael’s Hospital and the University of Toronto, Toronto, Canada
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This article is discussed in the editorial available at: doi: 10.1007/s00134-009-1632-z
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1
Materials and methods
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3
The study was performed according to the National Institutes of Health guidelines for the use of
4
experimental animals. The protocol was approved by the Animal Care Committee, St. Michael’s
5
Hospital, Canada.
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7
Instrumentation, anaesthesia, and monitoring
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30 adult male New Zealand white rabbits weighing 3.6–4.6 kg (Charles River Laboratories, St
9
Constant, QC, Canada) were intramuscularly premedicated with ketamine hydrochloride 35 mg/kg
10
(de Wyeth-Ayerst Inc., Guelph, ON, Canada) and xylacin 10 mg/kg (Bayer Inc., Toronto, ON,
11
Canada), followed by bilateral cannulation of the ear veins and arteries (22GA-Angiocath, Becton
12
Dickinson Inc., Sandy, UT, USA), and by installation of a tracheal cannula (4.0 ID, Mallinckrodt
13
Inc., St. Louis, MO, USA). All procedures were performed under sterile conditions. Anaesthesia
14
was maintained with continuous intravenous infusion of ketamine hydrochloride 40 mg/kgh-1 and
15
xylazine 4 mg/kgh-1. Ringer’s lactated solution (Baxter Corporation, Toronto, ON, Canada) 5
16
ml/kgh-1 was infused intravenously via an infusion pump (ColleagueTM, Baxter Healthcare
17
Corporation, Deerfield, IL, USA) throughout the experiment. The volume of withdrawn blood was
18
replaced by Ringer’s lactate solution.
19
20
No other fluids, including bicarbonates, and no vasoactive drugs were administered during the
21
study.
22
23
Transcutaneous oxygen saturation was continuously measured by pulse oxymetry (NONIN 8600
24
VTM, Nonin Medical Inc., Plymouth, MN, USA) at the tail and the heart rate was calculated from the
25
signal. Blood pressure was measured using quartz pressure transducers (TruWave PX12N, Edwards
26
Lifesciences, Irvine, CA, USA) referenced to the midcardiac plane and displayed continuously on a
27
multimodular monitor (S/5 Critical Care MonitorTM, Datex-Ohmeda, Helsinki, Finland). Rectal
28
temperature was monitored continuously and body temperature was maintained at 39 +/- 0.5 °C
29
using a heating lamp.
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31
NAVA technology
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NAVA was used as previously described [1-3]. The electrical activity of the crural part of the
33
diaphragm (EAdi) was derived from an array of electrodes mounted on an 8F esophageal catheter.
2
1
Proper positioning of the catheter in relation to the diaphragm was verified by online display of the
2
electrocardiogram detected by the electrodes, and by correllograms of EAdi signals along the
3
electrode array [4].
4
5
The EAdi signal was amplified, digitized, and processed with algorithms previously described [5,6],
6
and was translated into a voltage every 16 ms which was then multiplied by a proportionality factor
7
(NAVA level; cmH2O/unit of EAdi) throughout inspiration, and was used to control the pressure
8
generated by the ventilator (Servo300, Maquet Critical Care, Solna, Sweden). Hence, with NAVA
9
airway pressure (Paw) during inspiration is linearly proportional to EAdi (Paw = EAdi x NAVA
10
level). Since with NAVA the command signal to the pressure generator of the ventilator is updated
11
essentially in real time (every 16ms = 62.5 times per second), the pressure delivered to the animal’s
12
airways is virtually synchronous and instantaneously proportional to respiratory demand as reflected
13
by the EAdi (i.e. the intra-breath assist profile closely mirrors the profile of the EAdi).
14
15
The EAdi is influenced by facilitatory and inhibitory feedback loops that integrate information from
16
mechano- and chemo-receptors as well as voluntary and behavioral inputs [1]. If the delivered assist
17
exceeds the subject’s respiratory demand, EAdi will reflexively be down-regulated resulting in less
18
assist for the same NAVA level. If the delivered assist falls short of the subject’s respiratory
19
demand, EAdi will be up-regulated, resulting in delivery of more assist for the same NAVA level.
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Thus, the Paw delivered by the ventilator in response to a changed NAVA level always depends on
21
the concurrent change in the EAdi level.
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23
In the present study, NAVA was only applied during inspiration and therefore EAdi based trigger-on
24
and cycling-off algorithms were implemented. EAdi based trigger-on was set to initiate ventilatory
25
assist when the EAdi exceeded the random noise-variability. Given that the variability of the noise
26
level was low, the trigger threshold was set to a fixed level that permitted early detection of an EAdi
27
increase without causing auto triggering when the diaphragm is inactive. EAdi based cycling-off
28
was set to terminate ventilatory assist when the EAdi fell below 80% of peak inspiratory EAdi.
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Experimental protocol (Figure E1)
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A commercially available ventilator (Servo 300TM, Maquet Critical Care, Solna, Sweden) modified
32
for NAVA was used. The animals were ventilated with a time cycled, volume targeted mode using a
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Vt of 6-ml/kg (VC 6-ml/kg), a fraction of inspired oxygen (FiO2) of 0.5, and a PEEP of 3 cm H2O
3
1
during instrumentation and during the subsequent 30 minute stabilisation period. Stable mean
2
arterial blood pressure (>60 mmHg), arterial oxygen tension (PaO2) to FiO2 ratio >400 mmHg,
3
PaCO2 4.7-6.0 kPa (35-45 mmHg), and pH 7.30-7.50 were required at baseline before induction of
4
lung injury to proceed with induction of lung injury.
5
6
Induction of lung injury
7
After induction of neuromuscular paralysis with pancuronium bromide 0.02 mg/kg (Sabex Inc.,
8
Boucherville, QC, Canada) and after EAdi was no longer detectible, hydrochloric acid (pH 1.5) was
9
instilled intratracheally with the rabbit in the lateral position (0.75 ml/kg each side), followed by a
10
ventilation pause at a positive airway pressure (CPAP) of 25 cm H2O for four seconds. The HCl
11
instillation and the recruitment maneuver were repeated after five minutes.
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13
After instillation of HCl, all animals were ventilated for another thirty minutes with VC (Vt 6 ml/kg;
14
FiO2 0.5, PEEP 3 cm H2O). A PaO2/FiO2 ratio between 80 and 200 at 30 minutes after induction of
15
ALI was required to proceed to randomization.
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17
Randomization to NAVA, VC - ml/kg, or VC 1- ml/kg
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Three out of 30 animals were sacrificed before randomization because their PaO2/FiO2 ratio was
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below the lower limit of 80. 27 animals were equally randomized (n=9 per group; concealed
20
allocation) to 5.5 hours ventilation with one of the following 3 strategies:
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- NAVA: No neuromuscular paralysis. NAVA was initiated as soon as the EAdi was sufficient to
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control the ventilator. We used a NAVA level of 0.5 cmH2O per unit of EAdi throughout the
24
experiment in all animals based on our experience from previous studies using the same animal
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model where we demonstrated that the animals keep Vt constant at higher NAVA levels due to
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down regulation of their EAdi [2]. Note that employing a uniform NAVA level does not necessarily
27
result in a uniform level of assist since the animals up- or down regulate their EAdi (and hence the
28
pressure delivered) based on information from the respiratory system (e.g. from receptors sensitive
29
to lung distension or to oxygen tension) on a breath by breath basis. With NAVA the ventilatory
30
pattern including the Vt, the respiratory rate, and the intra-breath assist profile is entirely defined by
31
the EAdi.
32
4
1
- VC 6-ml/kg or VC 15-ml/kg: In order to prevent lung recruitment with inspiratory muscle activity
2
or with tonic muscle activity during expiration [1], muscle paralysis was continued after
3
randomization in both volume controlled (VC) groups with an infusion of pancuronium bromide
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(0.25 mg/kgh-1) and additional boluses of 0.02 mg/kg if needed to suppress any activity of the
5
continuously monitored EAdi. The pressure rise time was set at 5% and the end-inspiratory pause
6
time was set at 10% of the breath cycle in both VC groups. An additional dead space of 25 ml was
7
used in VC 15-ml/kg so that the ventilatory rate of both VC groups would be roughly the same. The
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ventilatory rate was adjusted to maintain PaCO2 levels between 4.7-6.0 kPa (35-45 mmHg).
9
10
Positive end-expiratory pressure (PEEP)
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PEEP was adjusted similarly throughout the experiment in NAVA and VC 6-ml/kg aiming at the
12
highest PEEP level possible while maintaining mean arterial pressure ≥60 mmHg. The PEEP was
13
lowered in steps of 1 cmH2O if mean arterial pressure decreased below 60 mmHg. This approach
14
was chosen in order to control for the effect of PEEP on cardiac function similarly in both groups.
15
To prevent disparities between the groups (e.g. hemodilution, tissue edema, fluid overload,
16
metabolic derangements) we did not administer additional fluid to maintain mean arterial pressure
17
above 60 mmHg. In VC 15-ml/kg animals PEEP was maintained at 1 cmH2O throughout the
18
protocol.
19
20
Fraction of inspired oxygen (FiO2)
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FiO2 was kept at 0.5 and increased up to 1.0 if needed to maintain oxyhemoglobin saturation in
22
arterial blood (SaO2) above 90% if possible. Recruitment manoeuvres and suctioning of secretions
23
were not performed throughout the study.
24
25
At the end of the protocol, the animals were sacrificed with an overdose of pentobarbital
26
(intravenous bolus of at least 20mg/kg).
27
28
29
Measurements and sample collection
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Respiratory, hemodynamic, and temperature measurements
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Vt, ventilatory rate, Paw, and PEEP were measured using the ventilator’s pneumotachograph and
32
pressure transducers. Dynamic respiratory system compliance CRSdyn = Vt in ml / (peak airway
33
pressure in cmH2O – PEEP in cmH2O) was assessed during neuromuscular paralysis in all animals
5
1
before induction of ALI, immediately before randomization, and after completion of the protocol
2
(NAVA animals were paralyzed for measuring CRSdyn after completion of the protocol) using
3
uniform ventilator settings (Vt 6-ml/kg, PEEP 3 cmH2O) in all animals. Mean arterial pressure, heart
4
rate, and rectal temperature were recorded immediately before induction of ALI and every 30
5
minutes thereafter.
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7
Analyses in blood and urine samples
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Arterial blood samples were drawn before induction of ALI and hourly thereafter at a FiO 2 of 0.5
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and processed immediately. Arterial blood gases (corrected for actual rectal temperature), and
10
arterial lactate concentration was measured using standard electrodes (ABL 520, Radiometer,
11
Copenhagen, Denmark). SaO2 and arterial hemoglobin concentration were analyzed using a
12
spectrophotometry method (OSM 3, Radiometer, Copenhagen, Denmark) designed for rabbit blood.
13
4 ml arterial blood was withdrawn before as well as 3 and 6 hours after induction of ALI,
14
immediately centrifuged and the supernatant was stored in aliquots at -80°C until further analyzed.
15
The urinary bladder was emptied before induction of ALI by gently compressing the lower
16
abdomen. Urin was collected after 3 hours by transcutaneous puncture and at the end of the protocol
17
by direct puncture of the bladder (after median laparatomy) using a 23G needle to collect the entire
18
urine produced during the experimental period.
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Echocardiography and oxygen delivery
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Echocardiography was performed immediately before induction of ALI and hourly thereafter using a
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Sonos 5500 system (Philips Ultrasound, Bothell, WA) equipped with a 8-12-MHz broad-band
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phased-array s12 transducer in 7 animals in each group (the equipment was not available in the other
24
animals). Digital image frames and image loops were saved to magnetic-optical disk for off-line
25
analysis (Medarchive Viewer, version 2.1). From a short-axis cross-sectional view of the left
26
ventricle (LV) at the midpapillary muscle level, maximal LV end-diastolic dimension (EDD) and
27
end-systolic dimension (ESD) were measured and LV fractional shortening (FS) was calculated as
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FS = [(EDD-ESD)/EDD]. LV ejection fraction (LVEF) was calculated as LVEF=[(EDD3-
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ESD3)/EDD3]. With the ultrasound beam parallel to the long axis of the pulmonary artery (PA), the
30
Doppler sample volume was placed above the pulmonary valve in the mid-lumen of the main PA, to
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record spectral profiles of blood flow velocity. From pulsed Doppler spectral recordings at
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maximum sweep speed, velocity time integral (VTI), peak velocity (Vmax), acceleration time (AcT),
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deceleration time (DcT), and flow period (FP) were measured. Mean acceleration (Acmean) was
6
1
calculated as Vmax /AcT. PA diameter (d) was measured using the same view and PA cross section
2
area was calculated as (d/2)2. Right ventricular (RV) stroke volume = (PA cross section area 
3
VTI); and cardiac output (CO) = (RV stroke volume  heart rate) were calculated. Heart rate was
4
calculated from the PA Doppler tracings for this equation.
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6
Arterial oxygen content (CaO2 in [ml/L]) was calculated as: (1.36 [mlO2/g] x haemoglobin
7
concentration [g/L] x SaO2 [%] / 100) + (0.03 [ml/L·mmHg-1] x PaO2 [mmHg]). Oxygen delivery
8
(DO2 in [ml/min·kg-1]) was calculated as: CO [L/min·kg-1] x CaO2 [ml/L].
9
10
Broncho-alveolar lavage (BAL) fluid
11
After sacrificing the animals, the heart-lung block was removed with the lungs inflated at a CPAP of
12
20 cmH2O. The left main bronchus and the right main lower lobe bronchus were both tightly
13
occluded before 20 ml of normal saline was instilled intratracheally. After applying a CPAP of 20
14
cmH2O for five seconds, the BAL fluid was aspirated, immediately centrifuged at 4 °C with 2000
15
rounds per minute (rpm), and the supernatant was stored in aliquots at -80°C until further analyzed.
16
17
Lung wet to dry ratio
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Lung wet to dry ratio was assessed in all animals as well as in 6 healthy, non-ventilated controls.
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The right lower lung lobe was divided from ventral to dorsal into a caudal and a cranial portion.
20
Both portions were further cut into a dependent (dorsal) and a non-dependent (ventral) portion. The
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caudal two portions were weighed before and after exposure to 40°C for 72 hours and lung wet-to-
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dry ratio = [(lung wet weight – lung dry weight) / lung dry weight] was calculated for dependent and
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non-dependent lung portions separately.
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Lung and extra-pulmonary organ tissue
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The cranial two portions of the right lower lung lobe and fragments of the heart (apex), right kidney
27
(caudal third), small intestine, liver (middle lobe), and spleen were removed after exsanguination,
28
stored in TritonX 0.2% (Sigma-Aldrich Corp. St. Louis, MO, USA) on ice, homogenized
29
immediately, centrifuged at 4°C for 10 minutes, and the supernatant was stored in aliquots at -80°C
30
until analyzed. The left lung was filled with paraformaldhyde 4% at a hydrostatic pressure of 15
31
cmH2O, submerged with the other organs in paraformaldhyde 4% for 24 hours, rinsed in phosphate
32
buffered saline 1% (Sigma-Aldrich Inc, St. Louis, MO) for another 24h, and then stored in alcohol
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70% at 4°C until embedded in paraffin.
7
1
2
Measurements after the experiments
3
Complete sets of material for the assessment of lung histology; of inflammation and coagulation
4
parameters; and of apoptosis in non-pulmonary organs were available in 5 VC 15-ml/kg animals, in
5
7 VC 6-ml/kg animals, and in 7 NAVA animals. For comparison, inflammation and coagulation
6
parameters were also measured in 4 healthy, anesthetised, non-ventilated control animals that were
7
sacrificed immediately after tracheostomy. Biochemical markers of organ dysfunction were
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measured in all ventilated animals.
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Lung and non-pulmonary organ histology
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A pathologist blinded to the study groups assessed eight high power fields (x 400) in paraffin
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embedded, hematoxylin and eosin (HE) stained sections of the dependent (dorsal) and non-
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dependent (ventral) portions of the left lower and upper lung separately (4 sections per animal x 8
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fields each) and scored histological lung injury qualities per section (see Table 2) as follows:
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none=0, mild=1, moderate=2, severe=3 (for perivascular / peribronchial hemorrhage and edema, and
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for bronchial epithelial lesions); none=0, only few=1, in <50% lung volume=2, and in >50% lung
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volume=3 (for vascular congestion / distension, and for alveolar membranes and alveolar edema);
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none=0, <20%=1, >20%=2, >30%=3 (for alveolar collapse); none=0, up to 6 red blood cells per
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alveolus=1, 7-30 red blood cells per alveolus=2, >30 red blood cells per alveolus=3 (for
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intraalveolar hemorrhage), and none=0, only one found=1, more than one found=2, four or more
21
found=3 (for intravascular thrombi). Alveolar polymorph-nuclear neutrophils and macrophages
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were calculated as average number per alveolus.
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Sections of the heart, kidney, liver, and small intestines of all animals were examined by the same
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pathologist for histological organ injury, specifically for intravascular thromboemboli and for micro-
26
infarctions.
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Inflammatory, coagulation and fibrinolysis mediators
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We measured IL-8 concentrations as a surrogate marker of a pro-inflammatory response because IL-
30
8 was increased in animal [7-9] and human studies on VILI [10], and because plasma IL-8 levels
31
were associated with both, mortality as well as ventilator and organ failure free days in patients with
32
established ALI/ARDS [11]. In patients at risk for ARDS, Donelly et al. demonstrated that BAL IL-
33
8 concentration was higher in those patients that subsequently progressed to ARDS suggesting that
8
1
IL-8 might be an early marker of ARDS. Measurement of inflammatory mediators in rabbit models
2
is generally limited by the availability of rabbit specific assays.
3
4
Plasma, BAL fluid, and tissue levels of interleukin 8 (IL-8) were measured using a human IL-8
5
ELISA kit (BioSource International Inc, Camarillo, CA, USA). It has been shown that the antibody
6
against human IL-8 is cross-reactive with rabbit IL-8 [13]. Tissue IL-8 levels were normalized to
7
lung tissue total protein concentrations (BIO-RAD Protein Assay, Hercules, CA, USA).
8
9
There is extensive cross–talk between inflammatory mediators and coagulation products, causing
10
reciprocal modulation. We measured tissue factor (TF) and plasminogen activator inhibitor type 1
11
(PAI-1) because levels of these proteins reflect the extent of coagulopathy in various forms of lung
12
injury, including VILI [16]. TF levels are low in the normal lung and elevated in disease [17,18]. In
13
patients with ARDS an increase in soluble TF in BAL fluid has been demonstrated [19]. In
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association with enhanced fibrin production, fibrinolytic activity is depressed in BALF of patients
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with ALI/ARDS [20], related to high pulmonary concentrations of PAI–1. PAI–1 is increased in
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ALI/ARDS [21,22]. Alveolar PAI–1 levels have also been found to be associated with higher
17
mortality in patients with ALI/ARDS [23].
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19
PAI-1 was measured using a commercial kit (IMUBIND® Tissue PAI-1 Kit, American Diagnostica
20
Inc., Stamford, CT, USA; lower detection limit 50 pg/mL), and TF using a kit (IMUBIND® Tissue
21
Factor Kit, American Diagnostica Inc.; lower detection limit 10 pg/ml).
22
23
These kits have previously been used in rabbits [14,15].
24
25
Biochemical markers of non-pulmonary organ function
26
Measured parameters: Concentrations of creatinine, urea nitrogen, potassium, and sodium were
27
measured in plasma withdrawn immediately before induction of ALI, and before sacrificing the
28
animals. Concentrations of creatinine, urea nitrogen, sodium, and potassium, as well as osmolarity
29
were measured in urine collected at the end of the experimental period.
30
31
Calculated variables: Plasma creatinine clearance (Ccreat) in ml/min = [(creatinineUrine in mol/L
32
x volumeUrine in ml) / (creatininePlasma in mol/L x sampling time in minutes)]; Urine sodium to
33
potassium ratio = [sodiumUrine in mmol/L / potassiumUrine in mmol/L]; Transtubular potassium
9
1
gradient (TTKG) = [(potassiumUrine in mmol/L x osmolarityPlasma in mOsmol/kg) / (potassiumplasma in
2
mmol/L x osmolarityUrine in mOsmol/kg)]; Fractional excretion of sodium (FENa) = [(sodiumurine in
3
mmol/L x creatinineplasma in mol/L / sodiumplasma in mmol/L x creatinineurine in mol/L)].
4
5
Apoptosis rate in non-pulmonary organs
6
Apoptosis staining: Nuclei with damaged DNA were detected using the in situ terminal
7
deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) assay for in-situ
8
end labeling, adapted to an automated in-situ hybridization instrument (Discovery Ventana
9
Medical Systems Inc., Tuscon, AZ, USA). As per Discovery protocol, the instrument used 4m
10
thick deparaffinized tissue sections mounted on positive charged glass slides, with subsequent
11
digestion with Protease I (Ventana Medical Systems Inc, Cat# 250-2018, Tuscon, AZ, USA) for 12
12
minutes. The assay uses recombinant terminal deoxynucleotidyl transferase (Invitrogen, Cat#
13
10533-065, Carlsbad, CA, USA) for adding homo-polymer tails to the 3’ ends of cleaved DNA,
14
characteristic in cells undergoing programmed cell death. Biotin-16,2'-deoxyuridine-5'-triphosphate
15
(Roche Diagnostics, cat# 1-093-070, Laval, QC, Canada) was the label used for this reaction.
16
Colorimetric visualization using avidin-horse radish peroxidase and 3,3’-diaminobenzidine detection
17
method was performed. The counterstain of choice was hematoxylin.
18
19
Counting of apoptotic cells: Counting of the apoptotic cells was carried out by a single investigator
20
who was blinded to the group assignment of the animals. First, images were taken and digitized
21
from randomly chosen light microscopic fields of all TUNEL stained sections following a
22
standardized procedure in order to assess high power fields evenly distributed over the entire cross-
23
section of the organs. Twelve pictures were taken of the heart (transversal, mid-ventricular cross-
24
section; left and right ventricles separately), of the liver, and of the kidney cortex (mid-portion
25
cross-section, only tubular epithelial cells were counted) with an original magnification x200, and
26
15 pictures were taken randomly of the small intestine (well-oriented villi and crypts separately;
27
original magnification x400). Second, all nuclei were hand marked and counted using imaging
28
software. Apoptotic nuclei were then defined as those nuclei exhibiting a clear brown to dark brown
29
color as compared to the surrounding blue nuclei. Because nuclei change shape and density during
30
the process of apoptosis, shape was not used as a defining characteristic for apoptosis. An apoptotic
31
index (TUNEL positive nuclei per total nuclei per high power field) was calculated for each section
32
(expressed in % total nuclei), and the apoptotic indices of all images of each organ were averaged to
33
ultimately provide a single, average apoptotic index for each organ of each animal.
10
1
2
3
Statistical analysis
4
Statistical analysis was performed with SigmaStatTM (version 3.10, Systat Software Inc., San Jose,
5
CA). Kolmogorov-Smirnov test was used to assess normal distribution of data. Differences among
6
groups were analyzed using one way analysis of variance (ANOVA) or ANOVA on ranks as
7
appropriate. Repeated measurements were analyzed using either ANOVA on ranks (Friedman test)
8
or two-way ANOVA with the ventilatory mode as the between-group factor and time after
9
randomization as the repeated-measures factor. Holm-Sidak method was used for post test, pair wise
10
multiple comparison procedures. Groups with paired data were compared with t-test or Wilcoxon
11
Signed Rank as appropriate. Level of significance was p<0.05.
11
1
Legend to Figure in the ESM
2
3
Figure E1
4
Schematic study protocol. § PEEP was continuously adjusted in VC 6-ml/kg and NAVA, aiming at
5
the highest PEEP possible while maintaining mean arterial pressure >60 mmHg. Surgical and
6
anesthesia procedures as well as total amount of fluids infused was equal in all animals, except for
7
administration of paralytic drugs that were withhold in NAVA after induction of acute lung injury
8
(ALI).
12
1
Reference List ESM
2
1. Allo JC, Beck JC, Brander L, Brunet F, Slutsky AS, Sinderby CA (2006) Influence of neurally
3
adjusted ventilatory assist and positive end-expiratory pressure on breathing pattern in rabbits
4
with acute lung injury. Crit Care Med 34:2997-3004
5
2. Beck J, Campoccia F, Allo JC, Brander L, Brunet F, Slutsky AS, Sinderby C (2007) Improved
6
synchrony and respiratory unloading by neurally adjusted ventilatory assist (NAVA) in lung-
7
injured rabbits. Pediatr Res 61:289-294
8
3. Spahija J, Beck J, de Marchie M, Comtois A, Sinderby C (2005) Closed-loop control of
9
respiratory drive using pressure-support ventilation: target drive ventilation. Am J Respir Crit
10
Care Med 171:1009-1014
11
4. Beck J, Sinderby C, Lindstrom L, Grassino A (1996) Influence of bipolar esophageal electrode
12
positioning on measurements of human crural diaphragm electromyogram. J Appl Physiol
13
81:1434-1449
14
15
5. Beck J, Tucci M, Emeriaud G, Lacroix J, Sinderby C (2004) Prolonged neural expiratory time
induced by mechanical ventilation in infants. Pediatr Res 55:747-754
16
6. Sinderby C, Spahija J, Beck J (2001) Changes in respiratory effort sensation over time are
17
linked to the frequency content of diaphragm electrical activity. Am J Respir Crit Care Med
18
163:905-910
19
7. Imai Y, Parodo J, Kajikawa O, de Perrot M, Fischer S, Edwards V, Cutz E, Liu M, Keshavjee
20
S, Martin TR, Marshall JC, Ranieri VM, Slutsky AS (2003) Injurious mechanical ventilation
21
and end-organ epithelial cell apoptosis and organ dysfunction in an experimental model of
22
acute respiratory distress syndrome. JAMA 289:2104-2112
23
8. Kotani M, Kotani T, Ishizaka A, Fujishima S, Koh H, Tasaka S, Sawafuji M, Ikeda E,
24
Moriyama K, Kotake Y, Morisaki H, Aikawa N, Ohashi A, Matsushima K, Huang YC, Takeda
25
J (2004) Neutrophil depletion attenuates interleukin-8 production in mild-overstretch
26
ventilated normal rabbit lung. Crit Care Med 32:514-519
27
9. Tremblay L, Valenza F, Ribeiro SP, Li J, Slutsky AS (1997) Injurious ventilatory strategies
28
increase cytokines and c-fos m-RNA expression in an isolated rat lung model. J Clin Invest
29
99:944-952
30
10. Ranieri VM, Suter PM, Tortorella C, De Tullio R, Dayer JM, Brienza A, Bruno F, Slutsky AS
31
(1999) Effect of mechanical ventilation on inflammatory mediators in patients with acute
32
respiratory distress syndrome: a randomized controlled trial. JAMA 282:54-61
13
1
11. Parsons PE, Eisner MD, Thompson BT, Matthay MA, Ancukiewicz M, Bernard GR, Wheeler
2
AP (2005) Lower tidal volume ventilation and plasma cytokine markers of inflammation in
3
patients with acute lung injury. Crit Care Med 33:1-6
4
12. Donnelly SC, Strieter RM, Kunkel SL, Walz A, Robertson CR, Carter DC, Grant IS, Pollok
5
AJ, Haslett C (1993) Interleukin-8 and development of adult respiratory distress syndrome in
6
at-risk patient groups. Lancet 341:643-647
7
8
13. Ito A, Imada K, Sato T, Kubo T, Matsushima K, Mori Y (1994) Suppression of interleukin 8
production by progesterone in rabbit uterine cervix. Biochem J 301 ( Pt 1):183-186
9
14. Chi L, Gibson G, Peng YW, Bousley R, Brammer D, Rekhter M, Chen J, Leadley R (2004)
10
Characterization of a tissue factor/factor VIIa-dependent model of thrombosis in
11
hypercholesterolemic rabbits. J Thromb Haemost 2:85-92
12
15. Watanabe A, Kurabayashi M, Arai M, Sekiguchi K, Nagai R (2001) Combined effect of
13
retinoic acid and basic FGF on PAI-1 gene expression in vascular smooth muscle cells.
14
Cardiovasc Res 51:151-159
15
16
16. Schultz MJ, Haitsma JJ, Zhang H, Slutsky AS (2006) Pulmonary coagulopathy as a new target
in therapeutic studies of acute lung injury or pneumonia--a review. Crit Care Med 34:871-877
17
17. Jesmin S, Gando S, Matsuda N, Sakuma I, Kobayashi S, Sakuraya F, Hattori Y (2004)
18
Temporal changes in pulmonary expression of key procoagulant molecules in rabbits with
19
endotoxin-induced acute lung injury: elevated expression levels of protease-activated
20
receptors. Thromb Haemost 92:966-979
21
18. Maris NA, de Vos AF, Bresser P, van der Zee JS, Meijers JC, Lijnen HR, Levi M, Jansen HM,
22
van der PT (2005) Activation of coagulation and inhibition of fibrinolysis in the lung after
23
inhalation of lipopolysaccharide by healthy volunteers. Thromb Haemost 93:1036-1040
24
19. Miller DL, Welty-Wolf K, Carraway MS, Ezban M, Ghio A, Suliman H, Piantadosi CA (2002)
25
Extrinsic coagulation blockade attenuates lung injury and proinflammatory cytokine release
26
after intratracheal lipopolysaccharide. Am J Respir Cell Mol Biol 26:650-658
27
20. Gunther A, Mosavi P, Heinemann S, Ruppert C, Muth H, Markart P, Grimminger F, Walmrath
28
D, Temmesfeld-Wollbruck B, Seeger W (2000) Alveolar fibrin formation caused by enhanced
29
procoagulant and depressed fibrinolytic capacities in severe pneumonia. Comparison with the
30
acute respiratory distress syndrome. Am J Respir Crit Care Med 161:454-462
31
32
21. Idell S (2002) Endothelium and disordered fibrin turnover in the injured lung: newly
recognized pathways. Crit Care Med 30:S274-S280
14
1
2
22. Marshall BC, Sageser DS, Rao NV, Emi M, Hoidal JR (1990) Alveolar epithelial cell
plasminogen activator. Characterization and regulation. J Biol Chem 265:8198-8204
3
23. Prabhakaran P, Ware LB, White KE, Cross MT, Matthay MA, Olman MA (2003) Elevated
4
levels of plasminogen activator inhibitor-1 in pulmonary edema fluid are associated with
5
mortality in acute lung injury. Am J Physiol Lung Cell Mol Physiol 285:L20-L28
6
15
1
Figure E1
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