Am J Physiol Lung Cell Mol Physiol 312: L32–L41, 2017.

First published November 23, 2016; doi:10.1152/ajplung.00416.2016.

RESEARCH ARTICLE Real-Time Visualization of Lung Function: From Micro to

Macro

Effectiveness of individualized lung recruitment strategies at birth: an

experimental study in preterm lambs
X David G. Tingay,1,2,3,4 Anushi Rajapaksa,1,4 Emanuela Zannin,5 Prue M. Pereira-Fantini,1,4
Raffaele L. Dellaca,5 Elizabeth J. Perkins,1,2 Cornelis E. E. Zonneveld,1 Andy Adler,6 Don Black,1
Inéz Frerichs,7 Anna Lavizzari,1,8 Magdy Sourial,1 Bartłomiej Grychtol,9 Fabio Mosca,8
and Peter G. Davis1,3,10

Downloaded from http://ajplung.physiology.org/ by 10.220.33.3 on January 22, 2017

1
Neonatal Research, Murdoch Children’s Research Institute, Parkville, Australia; 2Neonatology, The Royal Children’s
Hospital, Parkville, Australia; 3Neonatal Research, The Royal Women’s Hospital, Parkville, Australia; 4Department
of Paediatrics, University of Melbourne, Melbourne, Australia; 5TBM Laboratory, Dipartimento di Elettronica, Informazione
e Ingegneria Biomedica-DEIB, Politecnico di Milano University, Milano, Italy; 6Systems and Computer Engineering,
Carleton University, Ottawa, Canada; 7Department of Anaesthesiology and Intensive Care Medicine, University Medical
Centre Schleswig-Holstein, Campus Kiel, Kiel, Germany; 8NICU, Fondazione IRCCS Ca’ Granda, Ospedale Maggiore
Policlinico-Università degli Studi di Milano, Milano, Italy; 9Fraunhofer Project Group for Automation in Medicine
and Biotechnology, Mannheim, Germany; and 10Department of Obstetrics and Gynaecology, University of Melbourne,
Melbourne, Australia
Submitted 16 September 2016; accepted in final form 20 November 2016

Tingay DG, Rajapaksa A, Zannin E, Pereira-Fantini PM,
Dellaca RL, Perkins EJ, Zonneveld CE, Adler A, Black D, Fre-
richs I, Lavizzari A, Sourial M, Grychtol B, Mosca F, Davis PG.
Effectiveness of individualized lung recruitment strategies at birth: an
experimental study in preterm lambs. Am J Physiol Lung Cell Mol
Physiol 312: L32–L41, 2017. First published November 23, 2016;
doi:10.1152/ajplung.00416.2016.—Respiratory transition at birth in-
volves rapidly clearing fetal lung liquid and preventing efflux back
into the lung while aeration is established. We have developed a
sustained inflation (SIOPT) individualized to volume response and a
dynamic tidal positive end-expiratory pressure (PEEP) (open lung
volume, OLV) strategy that both enhance this process. We aimed to
compare the effect of each with a group managed with PEEP of 8
cmH2O and no recruitment maneuver (No-RM), on gas exchange,
lung mechanics, spatiotemporal aeration, and lung injury in 127 ⫾ 1
day preterm lambs. Forty-eight fetal-instrumented lambs exposed to
antenatal steroids were ventilated for 60 min after application of the
allocated strategy. Spatiotemporal aeration and lung mechanics were
measured with electrical impedance tomography and forced-oscilla-
tion, respectively. At study completion, molecular and histological
markers of lung injury were analyzed. Mean (SD) aeration at the end
of the SIOPT and OLV groups was 32 (22) and 38 (15) ml/kg,
compared with 17 (10) ml/kg (180 s) in the No-RM (P ⫽ 0.024,
1-way ANOVA). This translated into better oxygenation at 60 min
(P ⫽ 0.047; 2-way ANOVA) resulting from better distal lung tissue
aeration in SIOPT and OLV. There was no difference in lung injury.
Neither SIOPT nor OLV achieved homogeneous aeration. Histological
injury and mRNA biomarker upregulation were more likely in the
regions with better initial aeration, suggesting volutrauma. Tidal
ventilation or an SI achieves similar aeration if optimized, suggesting
that preventing fluid efflux after lung liquid clearance is at least as
important as fluid clearance during the initial inflation at birth.
Address for reprint requests and other correspondence: D. Tingay,
Neonatal Research, Murdoch Children’s Research Institute, Royal Chil-
dren’s Hospital, Flemington Rd., Parkville 3052 Victoria, Australia (e-mail:
david.tingay@rch.org.au).
L32 1040-0605/17 Copyright © 2017 the American Physiological Society
preterm; infant; open lung ventilation; sustained inflation; lung me-
chanics; regional lung injury
THE INITIAL TRANSITION from in utero life poses many problems
for the preterm infant (14). Effective tidal ventilation requires
the underdeveloped lung to rapidly transition from a fluid-filled
to aerated state (12, 14). This complex process requires rapid
clearance of fetal lung fluid from the airway and alveoli,
establishing a functional residual capacity and then prevention
of fluid efflux back into the alveoli spaces during expiration
(13). Inability to successfully complete this process exposes
the preterm lung to heterogeneous states of aeration and ven-
tilation, both increasing the risk of injury (14, 40).
Both positive end-expiratory pressure (PEEP) and an initial
sustained lung inflation (SI) have been suggested as methods of
optimizing respiratory transition (13, 18). In rabbit pups, an
initial SI has been shown to assist clearance of lung fluid, with
PEEP then preventing fluid efflux back into alveolar spaces
during subsequent tidal ventilation (31). Subsequent clinical
and preclinical data regarding an SI have been conflicting (16,
18, 28, 31), with some studies showing no substantive benefit
over tidal ventilation with sufficient PEEP (11, 39). The inter-
pretation of these studies has been hampered by inconsistencies
in both the SI and PEEP strategies used and the use of proven
lung protective therapies, such as antenatal corticosteroids and
exogenous surfactant (38). We have previously shown that
tidal ventilation at birth with a dynamic PEEP, or open lung
ventilation (OLV), approach that exploited hysteresis resulted
in more homogenous spatiotemporal aeration and better out-
comes than ventilation with a fixed PEEP of 8 cmH2O or a 30-s
SI (33, 39). The advantage of an OLV approach is that it uses
real-time feedback on the mechanical properties of an individ-
ual’s lung (3, 15). In contrast, SI strategies have traditionally
http://www.ajplung.org
 INDIVIDUALIZED LUNG RECRUITMENT STRATEGIES AT BIRTH
used a predetermined pressure, duration, or delivered volume
(24, 30, 31, 33, 37–39).
To be effective, an SI must overcome the long time con-
stants of the fluid-filled respiratory system (30), which are
highly variable even in standardized preterm animal models
(34, 40). Recently, we demonstrated that a “volumetric” SI, in
which the SI duration was individualized to achieving stable
aeration optimized outcomes compared with a predefined 30-s
SI (34). It is possible that the SI strategy used in our previous
OLV studies, and by many other groups, was inappropriate
given the variable mechanical characteristics of the preterm
lung. Notwithstanding this, the observation that an OLV strat-
egy achieved lung aeration without the need for an initial SI to
rapidly clear lung fluid is intriguing. To date, optimal SI and
OLV approaches have not been compared in the preterm lung
exposed to antenatal steroids.
We hypothesized that optimizing lung recruitment at birth
using an SI or OLV would create different pathways of
spatiotemporal aeration during respiratory transition. The spe-
cific aims of the study were to compare the effects of an
individualized SI, dynamic PEEP OLV, and a static PEEP
approach on gas exchange, lung mechanics, spatiotemporal
aeration, and lung injury in preterm lambs exposed to antenatal
steroids.
METHODS
All techniques and procedures were approved by the Animal Ethics
Committee of the Murdoch Children’s Research Institute, Melbourne,
Australia in accordance with National Health and Medical Research
Committee guidelines.
Experimental instrumentation. Surfactant-deficient Border-Leices-
ter/Suffolk 127 ⫾ 1 day preterm lambs (term ⬃145 days; n ⫽ 12–13
per group) born to date-mated ewes who received 11.7 mg of beta-
methasone 24 and 48 h before delivery were delivered via Caesarean
section under general anesthesia and instrumented as described in
detail previously (33, 34, 38, 40). Twin pregnancies were preferred to
minimize maternal variability. After we exposed the fetal head and
neck, the carotid artery and external jugular vein were cannulated, an
ultrasonic flow probe placed around the contralateral carotid artery,
airway intubated with a 4.0 cuffed endotracheal tube (ETT), and lung
fluid passively drained. Electrical impedance tomography (EIT)
(Goe-MF II EIT system; CareFusion, Hoechberg, Germany) needles
were then positioned around the chest (20, 33, 34, 37, 39, 40). At
delivery, the lambs were weighed, placed supine, and commenced on
intravenous infusions of ketamine and midazolam.
Measurements. Peripheral oxygen saturation (SpO2), heart rate
(HR), arterial blood pressure (HP48S monitor, Hewlett Packard,
Andover, MA), carotid blood flow (TS420 Perivascular Flow Module;
Transonic Systems, Ithaca, NY), airway pressure (PAO), gas flow, and
tidal volume (VT) at the airway opening (Florian; Acutronic Medical
Systems, Hirzel, Switzerland) were measured continuously from birth.
Global and regional lung volume changes were acquired by EIT at 25
scans/s (24, 33, 34, 39), and the unfiltered global lung volume change
was displayed in real-time using the Thorascan software package
(CareFusion). Arterial blood gases were measured at 5 min of life and
every 15 min from birth. The respiratory system resistance (RRS) and
reactance (XRS) were computed using the forced oscillation technique
(FOT) (4) from the pressure and flow signals measured at the inlet of
the tracheal tube during a small-amplitude (5 cmH2O) 5-Hz oscilla-
tory pressure superimposed onto the ventilation waveform for 10 s.
These were conducted on completion of the ventilation strategy at
birth and immediately after every arterial blood gas sample (5, 6, 41).
Ventilation strategies. Lambs were randomly assigned before de-
livery to receive one of the following strategies at birth using a
AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00416.2016 • www.ajplung.org
L33
computer-generated system that ensured equal matching of group
permutations and no repetition of the same strategy within twined
ewes. 1) No intentional recruitment maneuver (No-RM) group: pos-
itive-pressure ventilation (PPV; SLE5000; SLE, South Croydon, UK)
in volume-targeted ventilation (VTV) mode at PEEP 8 cmH2O,
inspiratory time 0.4 s, rate 60 inflations/min and set VT of 7 ml/kg
[inspiratory pressure (PIP) 40 cmH2O] (39). 2) Optimized SI (SIOPT)
group: an optimal aeration SI at 40 cmH2O individualized to each
lamb, defined as 10 s after aeration plateau visually determined by two
investigators (CEZ, DT) in the global EIT volume signal on the
Thorascan display (34). The SI was administered with a Neopuff
Infant T-Piece Resuscitator (Fisher & Paykel Healthcare, Auckland,
New Zealand) at 8 l/min flow. On completion of the SI, the lung was
held at a PEEP of 8 cmH2O for 5 s before the clamping of the ETT
and transferring the lamb to the SLE5000 ventilator and PPV ⫹ VTV
per No-RM (34, 39). 3) OLV group: step-wise PEEP strategy (OLV)
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using PPV ⫹ VTV (VT 7 ml/kg) (33, 39). PEEP was increased by 2
cmH2O every 10 inflations from an initial PEEP of 6 cmH2O until 20
cmH2O (PEEPMAX) and then similarly decreased to 6 cmH2O. PPV ⫹
VTV was then commenced at PEEP 8 cmH2O after a transient
10-inflation rerecruitment at PEEP 20 cmH2O (170 –180-s total du-
ration) followed by PPV ⫹ VTV per No-RM.
Ventilation strategy and general management after birth. Addi-
tional 10-s FOT measurements were made at PEEPMAX (OLV), on
completion of the SIOPT and OLV, and 90 s and 180 s (No-RM and
SIOPT). All lambs were initially supported in 0.21 fraction-inspired
oxygen (FIO2). PPV ⫹ VTV was applied for 60 min using a standard-
ized strategy, including titration of FIO2 and VTV to maintain SpO2
88 –94% and PaCO2 40 – 60 mmHg after the first arterial gas. At the
end of the study period, all lambs received a lethal dose of pentobar-
bitone. The ETT was then disconnected to atmosphere until lung
collapse. A static in vivo super-syringe pressure-volume curve was
generated (maximum pressure 40 cmH2O) to determine the static
mechanical properties of the respiratory system and calibrate the EIT
signal. An additional (1, 34) 10 age-matched fetuses (unventilated
controls, UVC group) were euthanized at delivery for injury analysis
comparison.
Data acquisition and analysis. Physiological parameters were re-
corded at 1,000 Hz (LabChart V7; AD Instruments, Sydney, Austra-
lia) and analyzed at key time points (34). Together with these data,
EIT data were monitored in real-time and recorded (Thorascan,
Carefusion) continuously for the first 15 min, and subsequently for 2
min with each arterial blood gas. EIT signals were also recorded
during the static in vivo PV curve. PIP, PEEP, and inspiratory change
in pressure (⌬P) were determined from the PAO data, and dynamic
compliance (Cdyn) was calculated from the ⌬P and VT data. XRS
(indicator of lung recruitment) and RRS were computed from FOT
recordings (34, 42). Static respiratory system compliance (CRS) was
determined from the PV curve. The alveolar-arterial difference in
oxygen (AaDO2) was calculated from the arterial blood gases.
Time-course EIT image data were reconstructed using an anatom-
ically correct custom-built GREIT algorithm (2) with non-lung re-
gions excluded during postprocessing and filtering to the respiratory
domain, as we have described previously (39). The trough values of
the EIT signal were used to determine global and regional change in
end-expiratory volume (aeration; ⌬EEV) and the trough to peak
amplitudes VT. The global ⌬EEV was calibrated (ml/kg) to the
change in impedance during the static PV curve (34, 39). Functional
EIT images of aeration throughout the imaged cross-sectional slice
were also computed to determine the spatiotemporal patterns of
aeration (39). The fraction of VT within each gravity-dependent third
of the lung weighted to equal lung pixel area was determined (39) and
calibrated to ml/kg from the corresponding Florian VT value.
Lung injury analysis. Immediately after euthanasia, the lung was
removed. The right upper lobe was fixed at 20 cmH2O with 4%
paraformaldehyde and five hematoxylin and eosin-stained 5-␮m sec-
tions from each of the upper, middle, and lower gravity-dependent
L34 INDIVIDUALIZED LUNG RECRUITMENT STRATEGIES AT BIRTH
sections scored for lung injury (n ⫽ 15 total/lamb) on the following
criteria: 1) alveolar wall thickness, 2) detached epithelial cells, 3)
hyaline membranes, and 4) alveolar collapse/atelectasis by an inves-
tigator blinded to treatment allocation (34, 39). Bronchoalveolar
lavage with saline was performed on the left lung and total protein
concentration determined using the Lowry method (17). Lung tissue
samples were collected from the gravity-dependent (lower) and non-
dependent (upper) zones of the right lower lobe and immediately snap
frozen in liquid nitrogen. RNA was extracted from lung tissue using
TRIzol, and 0.1 kg RNA was reverse-transcribed into complementary
DNA. Primers of early biomarkers of lung injury [connective tissue
growth factor (CTGF), cysteine-rich 61 (CYR61), early growth re-
sponse protein 1 (EGR1), and the interleukins-1B, ⫺6, and ⫺8] were
designed using the Roche Universal ProbeLibrary Assay Design
Center. All reactions were performed in triplicate on the Light-Cycler
480 System (Roche, Mannheim, Germany). The 2⫺⌬⌬Ct method was
used to calculate relative changes in gene expression, determined from
qRT-PCR experiments using GAPDH as a housekeeping gene and
relative to the UVC group (22).
Statistical analysis. On the basis of our previous studies with this
model (34), a sample size of 12 lambs per group would detect a
clinically meaningful difference (SD) in Cdyn of 0.08 (0.067)
ml·kg⫺1·cmH2O⫺1 at 60 min (power of 0.8 and ␣ error 0.05). Data
were first tested for normality and analyzed with t-tests, one-way
ANOVA, Kruskal-Wallis test, or two-way repeated-measures
ANOVA (using time and ventilation strategy as factors) and Tukey’s,
Dunn’s, or Dunnett’s (against UVC) posttests as appropriate. Statis-
tical analysis was performed with GraphPad PRISM 6 (GraphPad
Software, San Diego, CA), and P ⬍ 0.05 was considered significant.
RESULTS
Forty-eight lambs were studied. The groups were well
matched for weight, gestation, drained lung fluid, and fetal
wellbeing (Table 1). Four lambs were excluded because of
severe fetal hypoxia, two in the fetal UVC group, one attrib-
utable to unrecognized esophageal intubation (No-RM), and
one severe intrauterine growth restriction (OLV). Four lambs
developed pneumothoraces after inflation to 40 cmH2O during
the static PV curve, three in the No-RM and one in the SIOPT
groups.
Initial aeration during respiratory transition. The time
needed to achieve plateau aeration during the SI was a median
(range) 72 (36 –132) s and resulted in a mean (SD) 32 (22)
ml/kg ⌬EEV from birth (Fig. 1A). This was comparable to the
⌬EEV of 38 (15) ml/kg at PEEPMAX (90 s, OLV), mean (95%
CI) difference 6 (⫺13, 24) ml/kg, Tukey’s posttest (2-way
repeated-measure ANOVA). The OLV maneuver resulted in
hysteresis with a similar EEV at completion (PEEP 8 cmH2O)
Table 1. Study group characteristics
No-RM
Number 12
Gestational age, days 127.0 (1.0)
Female, n (%) 5 (42%)
Singleton, n (%) 1 (8%)
First born, n (%) 7 (64%)
Birth weight, g 3,169 (519)
Fetal lung fluid, m/kg 15.9 (8.6)
Arterial cord pH 7.33 (0.04)
Arterial cord Pao2, mmHg 22.0 (5.1)
Static CRS, ml·kg⫺1·cmH2O⫺1 0.94 (0.26)
All applicable data are means (SD). No differences between groups (1-way ANOVA or ␹2 test as appropriate). No-RM, no recruitment maneuver; SIOPT,
optimized sustained inflation; OLV, open lung ventilation; UVC, unventilated controls; CRS, respiratory system compliance.
AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00416.2016 • www.ajplung.org
and PEEPMAX, and both greater than the EEV of 19 (13) ml/kg
immediately before the OLV (10-s life) (P ⫽ 0.0008, 1-way
ANOVA). The ⌬EEV at the end of the OLV maneuver was
similar to the SIOPT group at 180 s. In contrast, the No-RM
group demonstrated less time-based recruitment although this
was still significant from birth, increasing from 10 (6) ml/kg at
10 s to 17 (10) ml/kg by 180 s (P ⫽ 0.004, 1-way ANOVA).
⌬EEV was significantly higher in the OLV compared with
No-RM group from 90 s onward (P ⫽ 0.033, 1-way ANOVA).
SIOPT and OLV had similar regional aeration patterns on
completion of the recruitment maneuvers (Fig. 1, B and C),
with lower relative aeration in the most gravity-dependent third
of the chest compared with the least gravity-dependent and
middle regions (P ⫽ 0.0007 and P ⫽ 0.0008, respectively,
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1-way ANOVA). The No-RM group had more heterogeneous
aeration with both the most and least gravity-dependent third of
the chest having lower relative aeration at 90 s and 180 s (both
P ⬍ 0.0001). Within each region, the OLV group had rela-
tively greater aeration in the least dependent third at 90
s/PEEPMAX and 180 s compared with No-RM group (P ⫽
0.0228 and P ⫽ 0.0478, respectively). Although not statisti-
cally significant, the SIOPT group had the most homogenous
aeration at 180 s of life. There were no differences within each
region of interest over time for all strategies.
Lung mechanics. No-RM and OLV groups showed time-
dependent improvement in Cdyn immediately after birth (Fig.
2A). Cdyn was higher in the No-RM group at 90 s (P ⬍ 0.0001)
compared with 10 s. At the end of the SI, Cdyn was similar to
that of the OLV group at the same time point (PEEPMAX) and
greater than No-RM group (P ⫽ 0.16, Tukey’s posttest). At
180 s, Cdyn was lower in the SIOPT and No-RM groups than the
OLV group (end of RM) (P ⫽ 0.036 and P ⫽ 0.0002,
respectively). Thereafter, SIOPT and OLV groups behaved
similarly and with better Cdyn than No-RM although this was
only significant at 5 min of life (P ⫽ 0.0087, OLV vs. No-RM;
Tukey’s posttest). This translated to better PV curves at the end
of the study (Fig. 2B).
No-RM and OLV groups showed improvement in XRS from
10 s and PEEPMAX (OLV) (both P ⬍ 0.0001, 1-way ANOVA)
(Fig. 2C). XRS did not change over time in the SIOPT group, but
the post-SI XRS was higher than the XRS at 10 s for No-RM and
OLV groups. XRS was higher at the end of the OLV RM
compared with No-RM group at 180 s (P ⬍ 0.025, 2-way
repeated-measures ANOVA, Tukey’s posttest) but not SIOPT.
By 5 min of life, there was no difference among the groups.
SIOPT OLV Fetal UVC
13 13 10
127.0 (0.8) 127.0 (0.8) 127.0 (0.7)
7 (58%) 7 (54%) 6 (60%)
0 (0%) 0 (0%) 1 (10%)
5 (42%) 5 (38%) 3 (30%)
3,042 (420) 3,120 (724) 2,765 (432)
16.5 (10.0) 20.1 (5.4) N/A
7.33 (0.06) 7.36 (0.06) 7.20 (0.19)
22.7 (3.7) 22.3 (6.2) 21.4 (14.7)
1.20 (0.35) 1.16 (0.39) N/A
 INDIVIDUALIZED LUNG RECRUITMENT STRATEGIES AT BIRTH
Fig. 1. Change in global end-expiratory volume (⌬EEV) from birth (ml/kg) (A)
following the no recruitment maneuver (No-RM) (black circles), open lung
ventilation (OLV) (gray circles), and optimized sustained inflation (SIOPT)
(open diamonds) strategy. *OLV vs. No-RM; P ⬍ 0.05 2-way repeated-
measures ANOVA with Tukey’s posttest. †OLV vs. 10-s life EEV, ‡No-RM
and OLV vs. 10-s life EEV; P ⬍ 0.05 1-way ANOVA with Tukey’s posttest.
B: relative regional aeration at 90 s, end of SI or maximum positive end-
expiratory pressure (PEEPMAX) in the least gravity-dependent (black), middle
(gray), and most gravity-dependent (white) third of the cross section of the
thorax, expressed as a percentage of total aeration. C: relative regional aeration
at 180 s or end of OLV maneuver. *P ⬍ 0.05, **P ⬍ 0.01, ***P ⬍ 0.001;
1-way ANOVA. All data are means ⫾ SD.
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L35
Overall, RRS behaved similarly between all groups (Fig. 2D).
RRS was significantly lower at PEEPMAX and at the end of
OLV compared with 10 s (P ⫽ 0005, repeated-measures 1-way
ANOVA).
Ventilator parameters. All groups demonstrated a reduction
in ⌬P requirements with time (all groups P ⬍ 0.0001, RM
1-way ANOVA), with OLV having the greatest decrease in ⌬P
during early respiratory transition (Fig. 3A). ⌬P was higher in
the No-RM group throughout the study, reaching significance
against OLV, but not SIOPT, from 180 s (P ⫽ 0.001, Tukey’s
posttest) to 10 min of life (P ⫽ 0.049). No strategy was within
target VT range before 180 s, with No-RM and OLV groups
being below range and SIOPT group above range (Fig. 3B).
Measured VT was lower in the No-RM group at 10 s compared
with OLV group (P ⫽ 0.0149). Both No-RM and OLV groups
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had a lower VT at 90 s compared with the SIOPT post-SI (P ⬍
0.0001). Thereafter, VT was the same in all groups.
Figure 4 shows VT within the middle, least, and most
gravity-dependent thirds of the lung. At 5 min, the OLV and
SIOPT groups had higher VT in the least dependent regions
compared with middle (both) and most dependent (SIOPT) (P ⫽
0.029 and P ⫽ 0.011, respectively, 1-way ANOVA). By 60
min, all groups had significantly higher VT in the least depen-
dent third of the lung compared with all other regions [P ⫽
0.020 (No-RM), P ⫽ 0.001 (SIOPT), P ⫽ 0.004 (OLV)]. Only
the No-RM groups demonstrated increased spatiotemporal re-
distribution between 5 and 60 min (Fig. 4C), with significant
changes in VT toward the least dependent lung (P ⫽ 0.0007).
Oxygenation. OLV and SIOPT attained target SpO2 (in FIO2
0.21) by 180 s and 5 min, respectively (Fig. 3C), compared
with 15 min for the No-RM group [P ⬍ 0.0001 against OLV
(90 s to 5 min) and SIOPT (3 and 5 min), Tukey’s posttest].
Required FIO2 was similar for SIOPT and OLV (Fig. 3D) and
lower than No-RM. By 60 min, FIO2 was a mean (95% CI) 0.26
(0.03, 0.42) higher than OLV in the No-RM group (Tukey’s
posttest). AaDO2 was higher in the No-RM group (Fig. 3E)
from 30 min (P ⫽ 0.014, OLV) and 45 min (P ⫽ 0.036, SIOPT).
All groups could be ventilated within the target PaCO2 range
(Fig. 3F) although the initial 5-min PaCO2 was higher in the
No-RM group (P ⫽ 0.0005, OLV and P ⫽ 0.0077, SIOPT).
EEV. OLV and SIOPT had similar ⌬EEV beyond the initial
period of respiratory transition (Fig. 1A). The No-RM group
had a lower ⌬EEV at all time points of the study, and this was
significant compared with OLV [with mean (95% CI) differ-
ences ranging between 19 (1, 38) ml/kg (30 min) and 20 (1, 39)
ml/kg (10 min), Tukey’s posttest]. All groups showed redistri-
bution in aeration from the most dependent lung regions to the
middle regions over the duration of the study (Fig. 5). This was
most apparent in the No-RM group. Both RM groups behaved
similarly.
Lung histology. All strategies had similar total lung injury
scores on histology, and all were greater than UVC overall
(P ⬍ 0.0001, 1-way ANOVA). Regionally there was no
difference in lung injury between all groups in the middle
regions (Fig. 6A). Only SIOPT had higher injury in the most
gravity-dependent lung regions compared with UVC (P ⫽
0.0156, Tukey’s posttest), and all strategies demonstrated sig-
nificantly more injury in the least gravity-dependent (upper)
region compared with UVC (P ⫽ 0.0002, No-RM and SIOPT;
P ⫽ 0.07, OLV). Within strategies, regional injury distribution
differed. The No-RM and SIOPT strategies had higher scores in
L36 INDIVIDUALIZED LUNG RECRUITMENT STRATEGIES AT BIRTH
Fig. 2. Dynamic compliance (Cdyn) (A),
static pressure-volume curves (B), respira-
tory system reactance (XRS) (C) and respi-
ratory system resistance (RRS) (D). Sym-
bols per Fig. 1A. P ⬍ 0.05 *SIOPT vs.
No-RM (90 s), †OLV vs. No-RM and
SIOPT, ‡OLV vs. No-RM; 2-way repeated-
measures ANOVA and **OLV and No-
RM, ††OLV vs. 10 s or ‡‡SIOPT post-SI
vs. OLV and No-RM at 10 s; 1-way
ANOVA. All data are means ⫾ SD.
the least gravity-dependent regions compared with the most
(No-RM, P ⫽ 0.030) and middle (SIOPT, P ⫽ 0.011). Injury
scores were higher in the middle compared with least depen-
dent regions in the OLV strategy (P ⫽ 0.001). There was no
difference in total lung protein (Fig. 6B).
Molecular evidence of injury. Gene expression of markers of
lung injury (Fig. 6C) was increased in all interventional groups
when compared with UVC in the nondependent lung (all
markers P ⱕ 0.004, Kruskal-Wallis test) although SIOPT
(CTGF) did not differ on subgroup analysis (P ⫽ 0.076
respectively, Dunnett’s posttest). In the dependent lung, all
strategies exhibited increased IL1␤, IL6, IL8, CYR61, and
EGR1 (P ⱕ 0.0017) gene expression compared with the UVC
group but not CTGF (P ⫽ 0.90).
DISCUSSION
This is the first study to compare the mechanical and injury
responses after two different active lung recruitment maneu-
vers individualized to the mechanical response of the lung and
designed to optimize aeration at birth. One strategy focused on
rapid lung liquid clearance (SIOPT) and the other on preventing
fluid efflux via PEEP and gradual tidal aeration (OLV). Both
strategies produced similar benefits over a control group using
tidal ventilation with no active recruitment maneuver in ste-
roid-exposed preterm lambs that would represent clinically
meaningful short-term differences. This has implications for
clinical practice, as optimal approaches to PEEP and SI have
yet to be determined in human infants; our study suggests that
both could be equally effective.
Birth involves the rapid transition from a fluid-filled to
aerated lung state, a process that is essential for successful
respiratory function (14). Moving lung fluid from the airways
and into the interstitium can only happen when a driving
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pressure gradient is applied to the lung (12). Thereafter, suffi-
cient end-expiratory pressure is needed to prevent fluid efflux
back into the alveolar spaces during tidal ventilation (12, 13).
This mechanical explanation of aeration at birth emphasizes
the need for sufficient applied pressure. The SIOPT and OLV
strategies we employed both aimed to optimize aeration at birth
but focused on pressure during different components of the
respiratory transition, the initial inflation pressure (SIOPT) and
dynamic PEEP during tidal inflations. The median SIOPT du-
ration was similar to the time to OLV PEEPMAX, and both
achieved similar absolute lung volumes that were greater than
No-RM. The improved aeration was associated with beneficial
mechanical changes in the lung. Thus it is not unexpected that
the subsequent clinical outcomes were similar. How best to
support the preterm lung at birth remains unknown (18). SIs
have been extensively investigated (11, 16, 24, 28, 33, 34,
37– 40), with large clinical trials ongoing (7), but investigation
of the role of PEEP has been limited (32). Our study reiterates
the need for active maneuvers to facilitate lung aeration at
birth, but, unlike previous studies, ours emphasizes that more
than one effective option exists. Whichever maneuver is cho-
sen, it is critical that it is applied optimally.
A striking finding of our study was the high variability in the
aeration duration and subsequent volumes achieved irrespec-
tive of strategy; between 36 and 132 s was needed to optimize
the SI response. We have previously demonstrated that lung
aeration at birth depends on both the strategy employed and the
mechanical properties of the recipient’s lung. For this reason,
our sample size was considerably greater than previous phys-
iological studies (24, 28, 30, 31, 33, 40). Most of these studies
have used predefined SI durations of ⬍60 s (11, 24, 28, 33,
38 – 40) and/or static PEEP (27, 31). Our study reinforces that
such an approach to both SI and PEEP is very unlikely to create
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Fig. 3. Change in pressure (⌬P) (A), tidal
volume (VT) (B), SpO2 (C), FIO2 (D), alve-
olar-arterial difference in oxygen (AaDO2)
(E) and PaCO2 (F). Symbols per Fig. 1A. P ⬍
0.05 *SIOPT vs. No-RM, †OLV vs. No-RM,
‡No-RM vs. OLV and SIOPT (2-way repeat-
ed-measures ANOVA), ‡‡SIOPT post SI vs.
OLV and No-RM 90 s (1-way ANOVA). All
data are means ⫾ SD.

uniform outcomes across an intervention group. This may
explain the conflicting and inconclusive outcomes of previous
human (26) and animal studies (11, 24, 28, 30, 31, 38, 39). Our
dynamic stepwise PEEP strategy is based on the OLV concept
(33). This patient-defined approach aims to place ventilation on
the deflation limb of the pressure-volume relationship, the
region known to optimize lung mechanics in the already
aerated lung (9, 35, 36). Our results suggest that future clinical
studies should also focus on providing meaningful physiolog-
ical feedback mechanisms at the bedside. In this context, EIT
offers promise as a direct measure of aeration that is robust in
the presence of poor face mask leak and can demonstrate upper
airway obstruction (8).
The EIT data showed that the benefits seen with both SIOPT
and OLV were due to improved distal lung aeration, particu-
larly within the least gravity-dependent lung. In contrast, aer-
ation was mainly limited to regions associated with the major
airways and adjacent alveoli (middle third of chest) in the

Fig. 4. Regional VT (ml/kg) in the least-gravity dependent (black), middle (gray), and most-gravity dependent (white) third of the cross section of the
thorax at 5 min (A) and 60 min (B). C: change in VT in each region between 5 and 60 min. *P ⬍ 0.05, **P ⬍ 0.01, ***P ⬍ 0.001; 1-way ANOVA. All
data are means ⫾ SD.

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Fig. 5. Functional electrical impedance tomography (fEIT) graphs of the regional gravity-dependent distribution of relative aeration (end-expiratory volume,
EEV) within 22 nondependent (top) to dependent (bottom) slices of the thorax immediately after the recruitment maneuver (SIOPT and OLV) or at 180 s of life
(No-RM) and 60 min (right) following No-RM, SIOPT, and OLV strategies. fEIT images divided into the least-gravity dependent (dotted), middle (nonfilled) and
most-gravity dependent (dashed) thirds shown in Fig. 1B and C. All bars are means ⫾ SD. There was no spatial difference between 2 time points within each
group.

No-RM group. Despite this, both SIOPT and OLV failed to
achieve truly uniform aeration in our surfactant-deficient
lambs, mainly attributable to poorer recruitment of the most
gravity-dependent lung. This is not unexpected, as these lung
regions are the hardest to recruit in poorly compliant diseased
lungs (3, 10, 21). EIT measures relative aeration and ventila-
tion differences, so it cannot determine whether these lung
regions were anatomically atelectatic. Interesting, across all
groups, lung injury markers were greater in those regions
easiest to engage in ventilation and aeration. The two recruit-
ment strategies resulted in the greatest heterogeneity of VT by
5 min. This suggests that volutrauma is an important compo-
nent of preterm lung injury in early life, potentially more so
than atelectasis, and that there may be a risk from excessive
recruitment.
That both recruitment approaches already had evidence of
significant lung injury by 60 min and failed to demonstrate any
meaningful benefit in injury over No-RM despite more uniform
aeration is intriguing and not simply explained by the potential
effects of antenatal steroids. This finding challenges the hy-
pothesis that fluid/aeration inhomogeneity in the lung at birth
increases injury (13). It is possible that protecting lung regions
that are very poorly compliant, and thus hardest to aerate, from
the initial high driving pressures placed on the lung during
aeration may be beneficial (37). The use of VTV with a
threshold maximum PIP allows this to be achieved, as the
volume exposure is primarily defined by lung mechanics. The
VT during the first 3 min of life in the two PPV groups was
much less than the intended 7 ml/kg, and changes then mir-
rored the temporal increases in Cdyn, RRS, and XRS as more

Fig. 6. Hematoxylin and eosin lung injury score in the most dependent (open), middle (shaded), and least (solid) dependent regions of lung (A). P ⬍ 0.05,
*between regions, †against all other strategies, ‡against SIOPT (1-way ANOVA). B: total bronchoalveolar fluid protein by strategy. C: IL1B, IL6, IL8, CTGF,
CYR61, and EGR1 gene expression in the most and least gravity-dependent lung regions. P ⬍ 0.05, *between regions, †against all other strategies
(Kruskal-Wallis test with Dunn’s posttest), ‡against unventilated controls (UVC) (Kruskal-Wallis test with Dunnett’s posttest). All bars are means ⫾ SD.

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 INDIVIDUALIZED LUNG RECRUITMENT STRATEGIES AT BIRTH
lung units engaged in tidal ventilation. The simultaneous as-
sessment of breath-to-breath regional aeration, ventilation, and
FOT-derived mechanics, unique to our study, allows the lung
behavior at birth to be defined both spatially and temporally.
This approach clearly demonstrates that focusing on immediate
and uniform aeration is too simplistic a model of lung protec-
tion in the preterm lung at birth.
A lack of consistency in animal models, SI strategies, and
PEEP levels has hampered interpretation of preclinical studies
(including our own) of respiratory transition. We have previ-
ously found that a PEEP of 8 cmH2O optimized aeration in our
lambs, a finding supported by data from other groups (25).
However, we acknowledge that this is higher than in other
studies (11, 28). We contend that the focus should be on the
intent of the strategy. The SIOPT focused on achieving aeration
through a prolonged but rapid inspiratory driving pressure
upon the highly resistive fluid-filled lung. In contrast, the OLV
uses a slower approach with a higher transient PEEP applied to
overcome the viscoelastic forces, allowing fluid efflux and
alveoli collapse during expiration between ongoing tidal fluid
clearance and recruitment via exploiting hysteresis. This is
based on the observation that recruitment is an ongoing process
in the diseased lung and differentiates the OLV from both the
No-RM and SIOPT strategies (15). OLV strategies are used in
atelectatic lung conditions (19) but outside our group have not
been reported at birth. On the basis of observations in ⬍40-g
rabbit pups (30), it was recently proposed that the flow dynam-
ics and tissue mechanics of the lung make it difficult to
ventilate at birth using high pressures and short inspiratory
times (tidal ventilation) without overinflating and injuring
aerated lung regions (13). Lung injury was not reported in
these studies (30, 31). Our study, using a larger sample size and
lambs with different chest mechanics, refutes this hypothesis,
as similar regional aeration and injury characteristics were
reported using both tidal ventilation and an SI, which all
resulted in similar reductions in RRS. This suggests that pre-
venting fluid efflux over time and understanding of the dy-
namic mechanical responses of the lung may be more impor-
tant than how the airways are cleared of fluid.
In our steroid-exposed lambs without exogenous surfactant
therapy, the initial benefits of EEV were universally lost by 60
min, associated with a gradual redistribution of aeration toward
the least dependent lung (39) and were not prevented using
antenatal steroids. This complicates the interpretation of the
regional injury results. It is possible that the complex hetero-
geneous injury pattern at study completion was a response to
one, or both, of either the initial aeration event or the subse-
quent spatiotemporal changes. Spatiotemporal changes are
most evident in the regional VT pattern of the No-RM group.
We have now observed this in both the steroid- and nonsteroid-
exposed surfactant-deficient preterm lamb lung by 60 min of
life (39). The spatiotemporal redistribution of aeration after
birth is likely to be a major contributor to early development of
lung injury but is poorly understood. Clearly long-term eval-
uation is needed, as the spatiotemporal behavior beyond 60
min of life may differ again, perpetuating new injury patterns.
We postulate that the beneficial effects of surfactant relate to
stabilizing spatiotemporal injury pathways (29, 40), and our
study suggests that there may be an important clinical interac-
tion between recruitment strategies at birth and surfactant
efficacy. The breath-to-breath imaging by EIT, coupled with
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L39
newer methods of injury evaluation with proteomics (23),
offers potential to detail injury mechanisms at a high temporal
resolution.
This study has some other limitations. Similar to other
translational studies in this field (11, 12, 24, 25, 27, 28), the
lambs were anaesthetized, intubated, and managed with a
cuffed ETT, inconsistent with clinical practice and likely to
result in less variable aeration than in humans managed with a
face mask, but necessary to ensure the accuracy of the me-
chanical measures. In spontaneously breathing human infants,
the variability we observed is likely to be greater still, and
future work should consider developing spontaneously breath-
ing animal models or using our EIT methods in the delivery
room (8). Most previous animal studies of preterm ventilation
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at birth (18, 24, 25, 28, 30, 31, 33, 37, 39, 40) have not exposed
fetal lambs to corticosteroids, which significantly alters the
mechanical benefits of the ventilation strategies (38). It is for
this reason that we chose to expose lambs to steroids before
birth, but we did not use surfactant after birth, despite also
having proven benefit (29). Our sample size was almost double
that of most similar studies, but injury outcomes were still
underpowered. Future studies should be larger still if lung
injury is a primary outcome. We focused on alveolar injury
only, but Hillman and coworkers (11) have recently shown that
ventilation strategy at birth also influences airway injury.
Furthermore, lung inflammation was limited to mRNA expres-
sion in fetuses without overt infection. Immunohistochemistry
and examination of other pathways, such as surfactant catab-
olism and fibrosis, may have yielded different patterns (11) and
may not translate to fetuses exposed to chorioamnionitis.
Finally, the limitations of EIT are well described elsewhere (8).
Specifically, EIT is only able to measure regional volume
characteristics through a single cross-sectional plane, and as-
sumptions regarding whole lung patterns should not be made.
In the preterm lamb, similar outcomes can be achieved using
an SI and dynamic PEEP recruitment maneuver (OLV) as long
as both are applied using a strategy that accounts for the
individual variability of the mechanical state of the preterm
lung at birth. We found that similar aeration and injury out-
comes can be achieved using tidal ventilation alone as long as
a dynamic PEEP approach was used, suggesting that prevent-
ing fluid efflux after lung liquid clearance may be the critical
step in successful respiratory transition at birth.
ACKNOWLEDGMENTS
The authors acknowledge Georgina Huan for preparing and scoring the
hematoxylin and eosin data, Dan Pavlic for assisting in PCR analysis, and
Sarah White and Rebecca Sutton for assistance in preparation of the ewes.
GRANTS
This study is supported by a National Health and Medical Research Council
Project Grant (Grant ID 1009287) and the Victorian Government Operational
Infrastructure Support Program (Melbourne, Australia). D. Tingay is supported
by a National Health and Medical Research Council Clinical Career Develop-
ment Fellowship (Grant ID 1053889). P. Davis is supported by a National
Health and Medical Research Council Program Grant (Grant ID 606789). P.
Davis is supported by a National Health and Medical Research Council
Practitioner Fellowship (Grant ID 556600).
DISCLOSURES
Politecnico di Milano University, the institution of Emanuela Zannin and
Raffaele L. Dellaca, owns a patent on the use of forced oscillation technique
L40 INDIVIDUALIZED LUNG RECRUITMENT STRATEGIES AT BIRTH
for the detection of lung volume recruitment/derecruitment. The other authors
have no competing interests to declare.
AUTHOR CONTRIBUTIONS
D.G.T., A.E.R., E.Z., P.M.P.-F., E.P., C.E.E.Z., D.B., A.L., and M.S.
performed experiments; D.G.T., A.E.R., E.Z., P.M.P.-F., R.D., E.P., C.E.E.Z.,
D.B., and B.G. analyzed data; D.G.T., A.E.R., E.Z., P.M.P.-F., R.D., C.E.E.Z.,
A.A., I.F., and P.G.D. interpreted results of experiments; D.G.T., A.E.R., and
P.M.P.-F. prepared figures; D.G.T. and A.E.R. drafted manuscript; D.G.T.,
E.Z., R.D., E.P., C.E.E.Z., A.A., I.F., and P.G.D. edited and revised manu-
script; D.G.T., A.E.R., E.Z., P.M.P.-F., R.D., E.P., C.E.E.Z., A.A., I.F., A.L.,
M.S., B.G., F.M., and P.G.D. approved final version of manuscript.
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