Surfactant replacement therapy in combination with different non-invasive ventilation techniques in spontaneously-breathing, surfactant-depleted adult rabbits
Surfactant replacement therapy in combination with different non-invasive ventilation techniques in spontaneously- breathing, surfactant-depleted adult rabbits
Francesca Ricci 0 1
Costanza Casiraghi 0 1
Matteo Storti 0 1
Francesco D'Alò 0 1
Chiara Catozzi 0 1
Roberta Ciccimarra 1
Francesca Ravanetti 1
Antonio Cacchioli 1
Gino Villetti 0 1
Maurizio Civelli 0 1
Xabi Murgia 1
Virgilio Carnielli 1
Fabrizio Salomone 0 1
0 Chiesi Farmaceutici, R&D Department , Parma , Italy , 2 Department of Veterinary Science, University of Parma , Parma , Italy , 3 Department of Drug Delivery, Helmholtz Institute for Pharmaceutical Research Saarland, SaarbruÈcken, Germany, 4 Division of Neonatology, Polytechnic University of Marche and Salesi Children's Hospital , Ancona , Italy
1 Editor: Francesco Staffieri, University of Bari , ITALY
Nasal intermittent positive pressure ventilation (NIPPV) holds great potential as a primary ventilation support method for Respiratory Distress Syndrome (RDS). The use of NIPPV may also be of great value combined with minimally invasive surfactant delivery. Our aim was to implement an in vivo model of RDS, which can be managed with different non-invasive ventilation (NIV) strategies, including non-synchronized NIPPV, synchronized NIPPV (SNIPPV), and nasal continuous positive airway pressure (NCPAP). Forty-two surfactantdepleted adult rabbits were allocated in six different groups: three groups of animals were treated with only NIV for three hours (NIPPV, SNIPPV, and NCPAP groups), while three other groups were treated with surfactant (SF) followed by NIV (NIPPV+SF, SNIPPV+SF, and NCPAP+SF groups). Arterial gas exchange, ventilation indices, and dynamic compliance were assessed. Post-mortem the lungs were sampled for histological evaluation. Surfactant depletion was successfully achieved by repeated broncho-alveolar lavages (BALs). After BALs, all animals developed a moderate respiratory distress, which could not be reverted by merely applying NIV. Conversely, surfactant administration followed by NIV induced a rapid improvement of arterial oxygenation in all surfactant-treated groups. Breath synchronization was associated with a significantly better response in terms of gas exchange and dynamic compliance compared to non-synchronized NIPPV, showing also the lowest injury scores after histological assessment. The proposed in vivo model of surfactant deficiency was successfully managed with NCPAP, NIPPV, or SNIPPV; this model resembles a moderate respiratory distress and it is suitable for the preclinical testing of less invasive surfactant administration techniques.
Data Availability Statement: All relevant data are
within the paper.
Funding: Chiesi Farmaceutici has funded this study
and provided support in the form of salaries for
authors [FR, CC, MS, FDA, CC, GV, MC, FS], but
did not have any additional role in the study design,
data collection and analysis, decision to publish, or
preparation of the manuscript. The specific roles of
these authors are articulated in the `author
Preterm birth is characterized by a marked immaturity of all organ systems. In very preterm
infants, the signs of lung immaturity manifest shortly after birth. Alveolar instability, arising
from the incapacity of the underdeveloped alveolar epithelium to synthesize and secrete
adequate quantity of surfactant [
], leads to alveolar collapse, which in turn compromises the
vital systemic oxygen delivery and, ultimately, the life of infants who suffer the so called
respiratory distress syndrome (RDS).
RDS infants routinely require artificial respiratory support and surfactant replacement therapy
]. The classic treatment against RDS entailed early tracheal intubation of the infants followed
by the intratracheal instillation of exogenous surfactant and a variable period of intensive
mechanical ventilation [
]. Although this approach has proved to be a life-saving therapy, the association
between mechanical ventilation through an endotracheal tube and the incidence of chronic lung
disease [7±9] led to the search of alternative, non-invasive ventilation (NIV) strategies.
Nasal continuous positive airway pressure (NCPAP) has been the earliest form of
non-invasive respiratory support [
]. Its use as a first line treatment against RDS (without surfactant
treatment) has become nowadays standard clinical practice [
]. However, despite its
efficacy, this technique cannot always avoid tracheal intubation, particularly for very
low-gestational-age preterm infants [
]. Nasal intermittent positive pressure ventilation (NIPPV) is
an enhanced form of NCPAP, which superimposes a pre-set number of mandatory ªbreathsº
to the background NCPAP . Moreover, it is possible to synchronize the mandatory
ªbreathsº provided by the ventilator with the spontaneous breathing efforts of the infant, as in
synchronized NIPPV (SNIPPV) [
]. Breath synchronization further improves pulmonary
mechanics, reduces the work of breathing, and enhances gas exchange [
A limitation regarding the universal application of NIV to all preterm infants is that
newborns with moderate to severe RDS will be inadequately supported by NIV, ultimately
requiring intubation and surfactant therapy at a later stage [
]. The solution to avoid the failure of
NIV is the timely administration of exogenous surfactant. Recently, successful approaches of
less invasive surfactant administration have been described, consisting of delivering an
intratracheal bolus of surfactant through a thin tube, while infants are managed with NCPAP
]. Surfactant aerosol delivery in combination with NCPAP has been investigated as the
ideal, non-invasive alternative which holds the potential to couple NIV with surfactant
replacement [21±26]. Nevertheless, the technical challenge of delivering medical aerosols to neonates
is high  and needs further preclinical development, including appropriate in vivo
experiments with models that can be managed with NIV.
In a previous study, we have implemented and fully characterized the
spontaneouslybreathing, lung-lavaged adult rabbit as an in vivo model of respiratory distress which can be
managed with NCPAP [
]. Interestingly, this model showed a significant improvement of the
pulmonary status if surfactant therapy was applied in combination with NCPAP compared to
NCPAP only. In the present study, our primary aim was to address the feasibility of
implementing this model with alternative techniques of NIV such as NIPPV and SNIPPV. In
addition, we hypothesized that the use of NIPPV or SNIPPV could improve the pulmonary
outcomes after surfactant treatment compared to NCPAP.
Materials and methods
Animal handling and surfactant delivery protocol
The experiments were carried out in 6- to 7-week-old rabbits. The experimental procedure
was approved by the intramural Animal Welfare Body and the Italian Ministry of Health
2 / 15
(Prot.nÊ 1300-2015-PR) and complied with the European and Italian regulations for animal
Rabbits (body weight of 1.5±2.5 kg) were initially sedated with intramuscular (i.m.)
medetomidine (Domitor1, 2 mg/kg) and handled as previously described [
]. Briefly, the throat of
the animals was first shaved and local anaesthesia was applied in the anterior neck with
lidocaine gel (Luan1 2.5%). Thirty minutes later the animals received 50 mg/kg of ketamine
(Imalgene 10001, Merial-Boehringer Ingelheim, France) and 5 mg/kg of xylazine (Rompun1,
Bayer, Germany) i.m. Rabbits, in supine position, were intubated and stabilized on positive
pressure ventilation (Fabian HFO, Acutronic, Zug, Switzerland) with the following settings:
FiO2 = 100%, Flow = 10 L/min, respiratory rate (RR) = 40 breaths/min, positive end-expiratory
pressure (PEEP) = 3 cmH2O, tidal volume (VT) targeted to 7 ml/kg (with the peak inspiratory
pressure, PIP, not exceeding 15 cmH2O) and inspiratory time of 0.5 sec. Airway flow, mean
airway pressure (MAP) and VT were monitored with a flow sensor connected to the
endotracheal tube, as long as the animals were intubated. Body temperature was monitored with a
rectal probe and it was maintained by placing a heating pad underneath the animal.
After endotracheal intubation, a catheter was inserted into the right jugular vein for
continuous infusion of 1 mg/ml of ketamine and 0.1 mg/ml of xylazine (100 μl/min). Trometamol
(tris-hydroxymethyl aminomethane, THAM, 1M, Sigma-Aldrich, USA) was also infused
during the surfactant depletion procedure. A second catheter was inserted into the right carotid
artery for blood sampling. After instrumentation, the baseline blood gases were measured with
an emogas analyzer (Radiometer Medical, Denmark).
If the initial inclusion criteria of arterial oxygen partial pressure (PaO2) value > 400 mmHg at
PIP < 15 cmH2O were met, the animal was included in the study. Repeated broncho-alveolar
lavages (BALs) were performed by flushing the airways with 20 ml/kg of pre-warmed (37ÊC) 0.9%
NaCl solution, followed by a short recovery period in-between, until a PaO2 value < 100 mmHg
was reached. Then, if after 15 min of stabilization on mechanical ventilation the respiratory failure
was confirmed (PaO2 < 100 mmHg, with PIP not exceeding 23 cmH2O), the animal was allocated
in one of the study groups. NIV support was applied using nasal prongs (Fisher & Paykel,
Auckland, New Zealand). Just before extubation, the animals assigned to the surfactant + NIV groups
received a bolus of 200 mg/kg of surfactant (SF, Curosurf, Chiesi Farmaceutici, Parma, Italy).
Animals included in the NCPAP (n = 6) and NCPAP+SF (n = 6) groups were maintained
in NCPAP (5 cmH2O, Fabian HFO, Acutronic, Zug, Switzerland) for 180 minutes, as
previously described [
The animals included in the NIPPV (n = 8) and NIPPV+SF (n = 7) groups were managed
with non-synchronized NIPPV (Fabian HFO, Acutronic, Zug, Switzerland) for 180 min. The
initial settings of NIPPV support were: PIP = 21±23 cmH2O, PEEP 5 cmH2O, RR (guaranteed) = 60
breaths/min, inspiratory time = 0.5.
The SNIPPV (n = 7) and SNIPPV+SF (n = 8) groups were managed with SNIPPV using
the Sophie ventilator (Fritz Stephan GmbH, Gackenbach, Germany) with the Graseby
abdominal capsule-triggering device (Fritz Stephan GmbH, Gackenbach, Germany) for breath
synchronization. Briefly, this system detects the diaphragmatic mechanical perturbations induced
by the animals' respiratory effort, which are then converted into a stable, fast-reacting (<30
ms) trigger signal. The initial settings of SNIPPV support were: PIP = 20 cmH2O, PEEP 5
cmH2O, RR (guaranteed) = 60 breaths/min, inspiratory time = 0.5.
Arterial carbon dioxide partial pressure (PaCO2) and PaO2 were measured right after the
induction of the anaesthesia (baseline), after inducing the respiratory failure by repeated BALs,
3 / 15
and after the stabilization period following the insult to confirm the respiratory failure
(Radiometer Medical, Denmark). Arterial blood gases were also measured right after placing the
animals on NIV support, 15 and 30 min after the start of NIV support, and then every 30 min
until the end of the experiment.
The ventilation efficacy index (VEI) and the Oxygenation index (OI) were calculated at
baseline, after surfactant depletion by repeated BALs, and at the end of the observational period.
Once the observational period was completed, animals were shifted from NIV to invasive
mechanical ventilation for a brief period of time, with the same settings used at baseline
(before the BALs: FiO2 100%, Flow = 10 L/min; RR = 40 bpm, PEEP = 3 cmH2O, VT targeted
to 7 ml/kg, and inspiratory time of 0.5).
VEI was calculated to evaluate the overall ventilation efficiency of mechanically ventilated
animals independently from the ventilation settings, as follows:
Dynamic compliance (Cdyn) was determined in all animals at baseline, after BAL-induced
surfactant depletion, and again at the end of the follow-up period, after re-intubation. Cdyn was
calculated by dividing the changes in lung volume (ΔV) by the changes in pressure (ΔP)
multiplied by the weight of the animal in Kg. ΔV and ΔP were obtained by spirometry (Acutronic).
The right lung was sampled in the cranial, middle and caudal lobes to represent
non-dependent, intermediate and dependent lung regions. The samples were fixed in 10% neutral
buffered formalin, then dehydrated in graded alcohol solutions, xylene clarified, paraffin
infiltrated by means of an automatic processor (ATP 700 Tissue Processor, Histo-line
laboratories, Italy), and embedded with the dorsal surface of the slice down (EG 1160, Leica
Biosystems, Mannheim, Germany). Sections, 5 μm thick, were obtained using a rotary microtome
(Slee Cut 6062, Slee Medical, Mainz, Germany). Three slides for each lung region were then
further deparaffinized, rehydrated in descending grades of ethanol, and finally stained with
hematoxylin and eosin (Sigma). Images of the samples were acquired at 40X magnification by
a digital slide scanner (NanoZoomer, Hamamatsu, Hamamatsu City, Japan). Lung injury was
scored by an investigator blinded to the experimental design using a semi-quantitative scoring
]. All parenchyma within a histological slide was scored considering multiple and
not overlapping ROIs at 10X magnification. According to internal standard procedure, the
ROIs occupied for more than 30% in area by conductive airways or blood vessels were
excluded. Alveolar and interstitial inflammation, alveolar and interstitial hemorrhage, edema,
and atelectasis were each scored on a 0 to 4 point scale. Each ROI was scored as 0 if the item
was absent or normal, as 1 if the item was present in 25% of the field, as 2 if it was present in
4 / 15
50% of the field, as 3 if it was present in 75% of the field and as 4 if the item was apparent
throughout the whole field. The total injury score was calculated as a sum of these scores and
the average of the ROIs was reported for each section.
Physiological parameters are presented as mean ± SEM. Raw data were analyzed and
compared by repeated measures two-way analysis of variance (ANOVA) as a function of group
and time or one-way ANOVA, followed by Tukey's post-hoc test. The data of the histological
score are presented as mean ± SEM and analysed by the non-parametric Kruskal-Wallis test.
Statistical analysis was performed using GraphPad software, version 7.0.
The body weight of the animals, the PaO2 and the Cdyn at baseline and the number of BALs
needed to achieve respiratory failure were not significantly different between the groups
(Table 1). BALs led to a marked (P<0.0001) decrease of PaO2 and Cdyn values, indicative of a
dramatic reduction of the alveolar surfactant pool [
After surfactant depletion, all groups showed mean PaO2 values below 80 mmHg, even though
the FiO2 was set to 100%. As expected, PaO2 values increased in all SF-treated groups
immediately after SF instillation (Fig 1). In particular, PaO2 values were back to the basal levels (before
BALs) after 60 minutes in the SNIPPV+SF group and after 120 minutes in the NCPAP+SF
group. Arterial oxygenation was clearly superior in SF-treated animals throughout the follow
up period. Among SF-treated groups, the highest mean PaO2 values were observed for the
SNIPPV+SF group, which were significantly higher compared to NIPPV+SF at any
timepoint. The mean PaO2 values of the NCPAP+SF group were higher than the values observed
for NIPPV, reaching statistical significance at 60 and 120 minutes.
The mean PaCO2 values increased in all groups due to the respiratory failure induced by
the BALs. Even though a slight downward trend of the mean PaCO2 values could be observed
within the first 30 min in the SF-treated groups, the arterial carbon dioxide levels remained
rather high in all groups, without significant differences in the first two hours of the follow up
period. The worst PaCO2 values were observed in the NCPAP group; in the last hour of the
follow up the mean PaCO2 of the NCPAP group was over 100 mmHg, significantly higher
0.33 ± 0.05
0.31 ± 0.06
0.37 ± 0.07
0.29 ± 0.03
0.42 ± 0.07
0.32 ± 0.04
1.85 ± 0.06
1.75 ± 0.08
1.90 ± 0.06
1.91 ± 0.05
1.86 ± 0.06
1.70 ± 0.06
Nasal continuous positive airway pressure, NCPAP; surfactant, SF; nasal intermittent positive pressure ventilation, NIPPV; synchronized NIPPV, SNIPPV; arterial
oxygen partial pressure, PaO2; dynamic compliance, Cdyn.
P vs. baseline values < 0.0001.
5 / 15
Fig 1. Mean PaO2 values over time of surfactant-depleted rabbits managed with non-invasive ventilation.
Surfactant-depleted adult rabbits were treated with nasal continuous positive pressure ventilation (NCPAP, white
diamonds), with NCPAP + intratracheal surfactant (NCPAP+SF, black diamonds), with nasal non-synchronized
intermittent positive ventilation (NIPPV, white squares), with NIPPV + intratracheal surfactant (NIPPV+SF, black
squares), with Synchronized Intermittent Positive Pressure Ventilation (SNIPPV, white circles), or with SNIPPV
+ intratracheal surfactant (SNIPPV+SF, black circles). Surfactant-treated animals (black symbols) received a bolus of
surfactant before extubation, immediately after the 0 time-point. Values are shown as the mean ± SEM. P vs. NIPPV
+SF group < 0.01; # P vs. SNIPPV group < 0.01; § P vs. NCPAP group < 0.01; & P vs. NIPPV group < 0.01.
compared to NIPPV and NIPPV+SF groups at 150 minutes and to all the other groups at 180
minutes (Fig 2).
To directly compare the pulmonary status of the animals before and after treatment, at the end
of NIV follow-up period, all animals were re-intubated and mechanically ventilated with the
same ventilation settings as at baseline (before the BALs). The OI and the VEI were therefore
calculated for the time-points in which the animals were managed with invasive mechanical
ventilation: 1) at baseline, 2) after surfactant depletions by repeated BALs, and 3) at the end of
the experiment (180 minutes).
The mean OI at baseline was below 1.4 in all groups, indicative of an optimal pulmonary
oxygen exchange. However, surfactant depletion by repeated BALs induced a significant
respiratory failure which increased the OI in all animals, yielding a mean value of 19.2 ± 7.6,
considering all the animals featured in the study. The tested NIV techniques without SF treatment
could not restore the OI to baseline values; moreover, the pulmonary response to the different
NIV modalities without surfactant treatment was not uniform within the groups showing a
great intra-group variability (Fig 3A). At the end of the experimental period, the mean OI
values of the NIPPV, SNIPPV and NCPAP groups were 12.98 ± 4.27 (range 2.46±35.9),
15.17 ± 3.7 (1.76±33.22), and 16.61 ± 2.88 (3.37±22.59), respectively. Surfactant therapy in
combination with NIV, however, was associated with a significantly better OI score compared
with NIV alone. The combined use of surfactant and SNIPPV or NCPAP could revert the high
mean OI values observed after the BALs to baseline values; in addition, it is noteworthy that
6 / 15
Fig 2. Mean PaCO2 values over time of surfactant-depleted rabbits managed with non-invasive ventilation.
Surfactant-depleted adult rabbits were treated with NCPAP only (white diamonds), with NCPAP+SF (black
diamonds), with NIPPV only (white squares), with NIPPV+SF (black squares), with SNIPPV only (white circles), or
with SNIPPV+SF (black circles). Surfactant-treated animals (black symbols) received a bolus of surfactant before
extubation, immediately after the 0 time-point. Values are shown as the mean ± SEM P vs. NIPPV group < 0.01; # P
vs. NIPPV+SF group < 0.01; § P vs. all other groups < 0.01.
the pulmonary response to the combined treatment in these two groups was remarkably
homogeneous: the mean OI values for the SNIPPV+SF and NCPAP+SF groups were
1.35 ± 0.04 (range 1.15±1.46) and 1.91 ± 0.22 (1.24±2.84), respectively. Surfactant therapy
followed by non-synchronized NIPPV also improved the OI significantly compared to animal
groups treated with NIV only. Nevertheless, the mean OI of the NIPPV+SF group was slightly
higher (4.29 ± 1.18, range 1.57±10.42) than in the SNIPPV+SF and NCPAP+SF groups.
The VEI was markedly influenced by the elevated PaCO2 values observed in all groups (Fig
3B). The mean VEI for all the animals at baseline, before the induction of the respiratory failure,
was 0.25 ± 0.001, indicative of normal ventilation. The VEI dropped dramatically after the BALs
to 0.01 ± 0.001 (P<0.001), as expected. At the end of the experiment the highest mean VEI values
were achieved by the SNIPPV+SF group (0.17 ± 0.03), which were in turn significantly higher
than those achieved by the SNIPPV (0.06 ± 0.01), NCPAP (0.06 ± 0.01), NIPPV+SF (0.08 ± 0.02),
and NCPAP+SF (0.10 ± 0.02) groups (P<0.01).
Cdyn values were assessed at those time intervals in which the animals were ventilated with
invasive positive pressure ventilation. Cdyn was equivalent for all groups at baseline (NIPPV,
1.11 ± 0.1 mL/cmH2O/kg; NIPPV+SF, 1.05 ± 0.06 mL/cmH2O/kg, SNIPPV, 0.82 ± 0.02 mL/
cmH2O/kg; SNIPPV+SF, 0.90 ± 0.1 mL/cmH2O/kg; NCPAP, 0.94 ± 0.06 mL/cmH2O/kg;
NCPAP+SF, 0.88 ± 0.07 1.11 ± 0.1 mL/cmH2O/kg). However, Cdyn dropped dramatically after
surfactant depletion to a mean value of 0.36 ± 0.02 mL/cmH2O/kg. After three hours of
follow-up, mean Cdyn values increased in all surfactant-treated groups (Fig 4). The highest mean
Cdyn value was observed for the SNIPPV+SF group (0.80 ± 0.17 mL/cmH2O/kg), which was
7 / 15
Fig 3. The oxygenation index (OI) and the ventilation efficacy index (VEI) of surfactant-depleted rabbits
managed with non-invasive ventilation. To the left of the dashed line, the baseline and post-surfactant depletion OI
(A) and VEI (B) values of all the animals featured in the study are given. To the right of the dashed line, the OI and VEI
values of the different groups after 180 minutes of management with NIV are shown. Surfactant-treated groups are
represented by the box-plots with a grey filling, whereas the groups treated with NIV only are represented by the white
box-plots. The small squares within each box-plot indicate the mean of the group. The whiskers indicate the maximum
and minimum values observed for each group. P < 0.01 vs. any group not treated with surfactant; # P < 0.01 vs.
SNIPPV+SF group. NCPAP: nasal continuous positive airway pressure, SF: surfactant, NIPPV: nasal
(nonsynchronized) intermittent positive pressure ventilation, and SNIPPV: synchronized NIPPV.
significantly higher compared to NIPPV+SF, SNIPPV and NCPAP groups (P<0.01). In the
animal groups treated with NIV only, the mean Cdyn values remained low in the SNIPPV and
NCPAP groups (0.36 ± 0.08 and 0.37 ± 0.07 mL/cmH2O/kg, respectively) and slightly
increased in the NIPPV group (0.53 ± 0.07 mL/cmH2O/kg).
8 / 15
Fig 4. Dynamic compliance (Cdyn) of surfactant-depleted rabbits managed with non-invasive ventilation. To the
left of the dashed line, the baseline and post-surfactant depletion Cdyn values of all the animals featured in the study are
given. To the right of the dashed line, the Cdyn values of the different groups after 180 minutes of management with
NIV are shown. Surfactant-treated groups are represented by the box-plots with a grey filling, whereas the groups
merely treated with NIV are represented by the white box-plots. The small squares within each box-plot indicate the
mean of the group. The whiskers indicate the maximum and minimum values observed for each group. # P < 0.01 vs.
SNIPPV+SF group. NCPAP: nasal continuous positive airway pressure, SF: surfactant, NIPPV: nasal
(nonsynchronized) intermittent positive pressure ventilation, and SNIPPV: synchronized NIPPV.
The histological examination of the lungs showed a marked inflammatory interstitial and
alveolar neutrophilic infiltration, alveolar and interstitial haemorrhage, thickening of the alveolar
wall, and accumulation of proteinaceous oedema and atelectasis: these findings are typical
features of an acute inflammation (examples in S1 Fig). From the overall view of the samples,
surfactant-treated groups showed a moderate decrease of the neutrophil infiltrates and of the
haemorrhagic areas both in the alveolar space and interstitium compared to their reference
groups treated with NIV only (Fig 5 and Table 2).
The sum score for SNIPPV+ SF and NCPAP+SF groups was significantly lower compared
to the SNIPPV and NCPAP groups, respectively. Among surfactant-treated groups, SNIPPV
+SF and NCPAP+SF performed significantly better than NIPPV+SF. Indeed, the SNIPPV+SF
group had the lowest scores among all groups.
Irrespective of whether surfactant was administered or not, synchronization of the animal's
breathing pattern and the ventilator's drive during nasal intermittent ventilation was
associated with significantly better histological outcomes.
In the present study, we have successfully implemented an in vivo model of surfactant
depletion induced by repeated BALs, which can be managed with different NIV techniques,
9 / 15
Fig 5. Histological microphotographs of surfactant-depleted rabbits managed with non-invasive ventilation.
Histological overview of lung parenchyma presenting representative inflammatory infiltrate, hemorrhage, edema and
atelectasis for all groups. SF: surfactant; NCPAP: nasal continuous positive airway pressure (A,B); NIPPV: nasal
intermittent positive pressure ventilation (C,D); SNIPPV: synchronized NIPPV (E,F). Haematoxylin & Eosin staining,
scale bar 250 μm.
Table 2. Histological scores of surfactant-depleted adult rabbits.
NCPAP 3.5 ± 0.2#
NCPAP+SF 2.8 ± 0.1
NIPPV 3.9 ± 0.2²
NIPPV+SF 3.2 ± 0.2
SNIPPV 3.1 ± 0.2
SNIPPV+SF 2.3 ± 0.2
0.9 ± 0.1³
0.9 ± 0.1
2.3 ± 0.2²§
1.6 ± 0.2 #
1.3 ± 0.2
0.8 ± 0.1
PLOS ONE | https://doi.org/10.1371/journal.pone.0200542
including NCPAP, NIPPV, and SNIPPV. As evidenced by the marked drop of arterial
oxygenation and Cdyn, the respiratory distress induced to the animals after lung lavage was severe
enough to preclude the full pulmonary recovery by merely applying NIV. However, early
surfactant instillation followed by any of the tested NIV techniques was associated with a
significant improvement of the arterial oxygenation. It is worth mentioning that the combined
treatment of early surfactant followed by SNIPPV was found to maximize the initial response
to surfactant therapy, achieving a rapid and uniform recovery of the baseline PaO2 values, a
significant improvement of the VEI, a marked increase of the Cdyn, and the best histological
outcomes among all groups.
The use of NCPAP without surfactant treatment has become a standard clinical practice for
the treatment of mild-to-moderate RDS [
]. Moreover, nasal ventilation has
demonstrated great clinical value not only in preventing extubation failures [
] and as a treatment
of apnea of prematurity , but also as a first line treatment against RDS [
Unfortunately, in many cases the application of NIV alone is not enough to adequately support RDS
infants, particularly those born before the 28th week of gestation, in which the NCPAP failure
fraction stands at approximately 40±50% . In the present study, neither NCPAP, NIPPV
nor SNIPPV alone could revert the respiratory failure induced after BALs, which indicates a
marked depletion of the surfactant pool with the applied lung lavage protocol [
of the ventilation modality, arterial oxygenation remained low in all groups treated with NIV
only. The histological scores were as well significantly worse in the NIV only treated groups
compared the ones observed in their equivalent SF-treated groups. Of note, the mean PaCO2
of the NCPAP group, at the end of the follow-up period, was significantly higher compared to
the groups ventilated with intermittent positive pressure. We attribute this difference to the
higher capacity of NIPPV and SNIPPV to flush the CO2 from the airways.
The results from this study suggest that, in the setting of a more severe RDS, early surfactant
followed by NIV, avoiding intratracheal intubation, might represent the most effective
therapeutic approach. Several attempts have been made to deliver a dose of surfactant without
intubation. Surfactant aerosol delivery was developed with the aim to administer surfactant in
combination with NIV. The safety and the efficacy of this approach were investigated in small
clinical studies [23±26]. Although the therapy was proven to be safe, the clinical efficacy of this
technique is not yet convincing [
]. Surfactant administration using the laryngeal mask
airway has also been investigated as a less invasive surfactant administration method [
Recently, a minimally invasive way of surfactant administration has been successfully
implemented in preterm infants: this technique consists of delivering an intratracheal bolus of
surfactant through a thin tube, while the infants are supported with NCPAP [
approach reduces the need for mechanical ventilation, supplemental oxygen, and NCPAP.
Most of the aforementioned less invasive surfactant administration techniques used NCPAP
immediately after surfactant delivery. The use of NCPAP following surfactant instillation was
also associated with a rapid and uniform improvement of oxygenation in our in vivo model.
However, the results obtained in this study bring up the following scientific question: should
SNIPPV rather than NCPAP be used as a primary non-invasive respiratory support
immediately after surfactant administration? The available clinical evidence remains controversial.
Chen at al. did not find any statistical differences in the rate of endotracheal ventilation in
twin pairs receiving surfactant and supported with either NCPAP or NIPPV .
Nevertheless, they found a marginal statistically significant difference favouring the use of NIPPV in
infants with a gestational age of 32±33 weeks. Of note, the study by Chen et al. did not use
breath-synchronization. Gizzi et al. compared the chart data of 31 infants undergoing the
InSurE (Intubate-Surfactant-Extubate) approach supported with NCPAP with the data of 33
infants undergoing InSurE but supported with SNIPPV [
]. In this single-centre study, the
11 / 15
use of SNIPPV significantly reduced the InSurE failure (2/33 in the SNIPPV group vs. 11/31 in
the NCPAP group, P < 0.004) and the need for mechanical ventilation. These clinical studies
suggest that the use of nasal ventilation might be of advantage compared to NCPAP after SF
administration. In particular breath-synchronization has the potential to maximize SF-therapy
and the overall pulmonary ventilation. For instance, during non-synchronized NIPPV,
effective patient-ventilator synchronization has been described to occur only in 25% of the cycles
]. The remaining asynchronous cycles are delivered by the ventilator at all phases of the
infant's respiratory cycle, including the early-, mid- and late-expiration [
]. This mismatch
alters the spontaneous respiratory rhythm, induces glottal narrowing, increases the work of
breathing (WOB), and can ultimately cause volutrauma [
]. Conversely, during SNIPPV, the
positive pressure is delivered by the ventilator with the opened glottis and is therefore
effectively transmitted to the lungs, reducing the thoraco-abdominal asynchrony, the inspiratory
effort, and the WOB, yielding an improved pulmonary ventilation [
In the present study, the most prominent pulmonary improvement among all SF-treated
groups, as well as the lowest lung injury scores, were also observed when SNIPPV was applied
right after surfactant instillation. Therefore, this study is in line with the clinical reports that
found a benefit in synchronizing the ventilator's drive with the patient's spontaneous
breathing efforts. To achieve an efficient synchronization, we used the Graseby abdominal pneumatic
capsule; this device has shown to correctly detect inspiration 88% of the times [
to non-synchronized NIPPV, the use of SNIPPV immediately after surfactant instillation was
associated with significantly better PaO2, VEI, and Cdyn values. In addition, the SNIPPV+SF
group had a significantly lower injury score compared to the NIPPV+SF group and showed
the best outcomes for all the studied items (inflammation, hemorrhage, edema, atelectasis)
among all the study groups. We speculate that the coordinated ªbreathsº delivered by the
ventilator during SNIPPV might have helped spreading the surfactant immediately after
instillation, accounting for a better initial pulmonary distribution of surfactant that led to a rapid
increase of the PaO2, a significant improvement of the VEI, and a better histological
We acknowledge that the present in vivo study has several limitations: on the one hand, the
surfactant-depleted adult rabbit does not completely resemble the complex pathogenesis of
RDS, which takes places in a significantly larger time-scale compared to the relatively short
follow-up of our protocol. On the other hand, PaCO2 values remained above physiological levels
in all groups, irrespective of the applied treatment. We assume that the high PaCO2 levels are
due to the increased resistance to air flow of the customized nasal cannulas, which have been
originally designed for humans and not for rabbits. Nevertheless, we have implemented an in
vivo model of surfactant deficiency that can be alternatively managed with NCPAP, NIPPV, or
SNIPPV; this model resembles a moderate respiratory distress, which cannot be reverted by
merely applying NIV support. Conversely, surfactant administration followed by NIV was
associated with a rapid improvement of arterial oxygenation, making this model suitable for
the preclinical testing of alternative surfactant delivery methods, e.g. surfactant nebulization
and laryngeal mask surfactant administration [
]. The present study also suggests that
SNIPPV applied immediately after surfactant instillation may maximize the effects of
surfactant therapy compared to other NIV modalities.
S1 Fig. Examples of histological findings in surfactant-depleted rabbits managed with
non-invasive ventilation. Histological examples of lung parenchyma presenting inflammatory
12 / 15
infiltrate, hemorrhage, edema and atelectasis. Haematoxylin & Eosin staining.
Conceptualization: Francesca Ricci, Costanza Casiraghi, Matteo Storti, Francesco D'Alò,
Chiara Catozzi, Francesca Ravanetti, Gino Villetti, Maurizio Civelli, Xabi Murgia, Virgilio
Carnielli, Fabrizio Salomone.
Data curation: Francesca Ricci, Costanza Casiraghi, Matteo Storti, Francesco D'Alò, Chiara
Catozzi, Francesca Ravanetti, Antonio Cacchioli, Xabi Murgia, Fabrizio Salomone.
Formal analysis: Francesca Ricci, Costanza Casiraghi, Matteo Storti, Francesco D'Alò, Chiara
Catozzi, Roberta Ciccimarra, Francesca Ravanetti, Antonio Cacchioli, Xabi Murgia,
FabriInvestigation: Francesca Ricci, Costanza Casiraghi, Matteo Storti, Francesco D'Alò, Chiara
Catozzi, Roberta Ciccimarra, Francesca Ravanetti, Xabi Murgia, Fabrizio Salomone.
Methodology: Francesca Ricci, Costanza Casiraghi, Matteo Storti, Francesco D'Alò, Chiara
Catozzi, Roberta Ciccimarra, Francesca Ravanetti, Antonio Cacchioli, Gino Villetti,
Maurizio Civelli, Xabi Murgia, Fabrizio Salomone.
Project administration: Francesca Ricci, Costanza Casiraghi, Gino Villetti, Fabrizio
Murgia, Fabrizio Salomone.
Supervision: Francesca Ricci, Costanza Casiraghi, Francesca Ravanetti, Antonio Cacchioli,
Gino Villetti, Maurizio Civelli, Virgilio Carnielli, Fabrizio Salomone.
Validation: Francesca Ricci, Costanza Casiraghi, Matteo Storti, Francesco D'Alò, Chiara
Catozzi, Gino Villetti, Maurizio Civelli, Virgilio Carnielli, Fabrizio Salomone.
Visualization: Francesca Ricci, Francesco D'Alò, Chiara Catozzi, Francesca Ravanetti, Xabi
Writing ± original draft: Francesca Ricci, Costanza Casiraghi, Matteo Storti, Francesca
Ravanetti, Antonio Cacchioli, Gino Villetti, Maurizio Civelli, Xabi Murgia, Virgilio Carnielli,
Writing ± review & editing: Francesca Ricci, Costanza Casiraghi, Francesca Ravanetti,
Antonio Cacchioli, Gino Villetti, Xabi Murgia, Virgilio Carnielli, Fabrizio Salomone.
13 / 15
14 / 15
1. Nkadi PO , Merritt TA , Pillers D- AM . An Overview of Pulmonary Surfactant in the Neonate: Genetics, Metabolism, and the Role of Surfactant in Health and Disease . Mol Genet Metab . 2009 ; 97 : 95 ± 101 . https://doi.org/10.1016/j.ymgme. 2009 . 01 .015 PMID: 19299177
2. Zimmermann LJI , Janssen DJMT , Tibboel D , Hamvas A , Carnielli VP . Surfactant Metabolism in the Neonate . Neonatology . 2005 ; 87 : 296 ± 307 .
3. Sweet DG , Carnielli V , Greisen G , Hallman M , Ozek E , Plavka R , et al. European Consensus Guidelines on the Management of Respiratory Distress SyndromeÐ2016 Update. Neonatology . 2016 ; 107 ± 125 . https://doi.org/10.1159/000448985 PMID: 27649091
4. Walsh BK , Daigle B , DiBlasi RM , Restrepo RD . AARC Clinical Practice Guideline. Surfactant Replacement Therapy: 2013. Respir Care . 2013 ; 58 : 367 LP± 375 .
5. Speer CP , Robertson B , Curstedt T , Halliday HL , Compagnone D , Gefeller O , et al. Randomized European multicenter trial of surfactant replacement therapy for severe neonatal respiratory distress syndrome: single versus multiple doses of Curosurf . Pediatrics. 1992 ; 89 : 13 ± 20 . PMID: 1727997
6. Morley CJ , Greenough A , Miller NG , Bangham AD , Pool J , Wood S , et al. Randomized trial of artificial surfactant (ALEC) given at birth to babies from 23 to 34 weeks gestation . Early Hum Dev . 1988 ; 17 : 41 ± 54 . PMID: 3061771
7. Hayes D , Feola DJ , Murphy BS , Shook LA , Ballard HO . Pathogenesis of bronchopulmonary dysplasia . Respiration . 2010 . pp. 425 ± 436 . https://doi.org/10.1159/000242497 PMID: 19786727
8. Northway WHJ , Rosan RC , Porter DY . Pulmonary disease following respirator therapy of hyaline-membrane disease: Bronchopulmonary dysplasia . N Engl J Med . 1967 ; 276 : 357 ± 368 . https://doi.org/10. 1056/NEJM196702162760701 PMID: 5334613
9. Jobe AH , Bancalari E. Bronchopulmonary Dysplasia . Am J Respir Crit Care Med . American Thoracic SocietyÐAJRCCM; 2001 ; 163 : 1723 ± 1729 . https://doi.org/10.1164/ajrccm.163.7.2011060 PMID: 11401896
10. Kattwinkel J , Nearman HS , Fanaroff AA , Katona PG , Klaus MH . Apnea of prematurity: Comparative therapeutic effects of cutaneous stimulation and nasal continuous positive airway pressure . J Pediatr . 1975 ; 86 : 588 ± 592 . PMID: 1092821
11. Saunders R a , Milner a D , Hopkin IE . The effects of continuous positive airway pressure on lung mechanics and lung volumes in the neonate . Biol Neonate . 1976 ; 29 : 178 ± 186 . https://doi.org/10.1159/ 000240862 PMID: 782570
12. Rojas-Reyes MX , Morley CJ , Soll R . Prophylactic versus selective use of surfactant in preventing morbidity and mortality in preterm infants . Cochrane database Syst Rev . 2012 ; 3 : CD000510 .
13. Stoll BJ , Hansen NI , Bell EF , Shankaran S , Laptook AR , Walsh MC , et al. Neonatal outcomes of extremely preterm infants from the NICHD Neonatal Research Network . Pediatrics. 2010 ; 126 : 443 ± 56 . https://doi.org/10.1542/peds.2009-2959 PMID: 20732945
14. Dunn MS , Kaempf J , de Klerk A , de Klerk R , Reilly M , Howard D , et al. Randomized Trial Comparing 3 Approaches to the Initial Respiratory Management of Preterm Neonates . Pediatrics . 2011 ; 128 : e1069 LP ± e1076 .
15. Davis Peter G; Morley Colin J; Manley BJ . Noninvasive Respiratory Support: An Alternative to Mechanical Ventilation in Preterm Infants . In: Polin Richard A; Bancalari E , editor. The Newborn Lung: Neonatology Questions and Controversies . 2nd ed. Elsevier; 2012 . pp. 265 ± 283 .
16. Owen LS , Manley BJ . Nasal intermittent positive pressure ventilation in preterm infants: Equipment, evidence, and synchronization . Semin Fetal Neonatal Med . Elsevier; 2017 ; 21 : 146 ± 153 .
17. Moretti C , Gizzi C , Montecchia F , Barbàra CS , Midulla F , Sanchez-Luna M , et al. Synchronized Nasal Intermittent Positive Pressure Ventilation of the Newborn: Technical Issues and Clinical Results. Neonatology . 2016 . pp. 359 ± 365 . https://doi.org/10.1159/000444898 PMID: 27251453
18. Dargaville PA , Gerber A , Johansson S , De Paoli AG , Kamlin COF , Orsini F , et al. Incidence and Outcome of CPAP Failure in Preterm Infants . Pediatrics . 2016 ; 138 : e20153985± e20153985 . https://doi. org/10.1542/peds.2015-3985 PMID: 27365307
19. GoÈpel W , Kribs A , Ziegler A , Laux R , Hoehn T , Wieg C , et al. Avoidance of mechanical ventilation by surfactant treatment of spontaneously breathing preterm infants (AMV): An open-label, randomised, controlled trial . Lancet . 2011 ; 378 : 1627 ± 1634 . https://doi.org/10.1016/S0140- 6736 ( 11 ) 60986 - 0 PMID: 21963186
20. Dargaville PA , Aiyappan A , Cornelius A , Williams C , De Paoli AG. Preliminary evaluation of a new technique of minimally invasive surfactant therapy . Arch Dis ChildÐFetal Neonatal Ed. 2011 ; 96 : F243 LP ± F248 .
21. Walther FJ , HernaÂndez-Juviel JM , Waring AJ . Aerosol delivery of synthetic lung surfactant. Longo M, editor . PeerJ . 2014 ; 2: e403 . https://doi.org/10.7717/peerj.403 PMID: 24918030
22. Dijk PH , Heikamp A , Oetomo SB . Surfactant nebulisation prevents the adverse effects of surfactant therapy on blood pressure and cerebral blood flow in rabbits with severe respiratory failure . Intensive Care Med . 1997 ; 23 : 1077 ± 1081 . PMID: 9407244
23. Arrøe M , Pedersen-Bjergaard L , Albertsen P , BodeÂ S , Greisen G , Jonsbo F , et al. Inhalation of aerosolized surfactant (Exosurf®) to neonates treated with nasal continuous positive airway pressure . 1998 .
24. Jorch G , Hartl H , Roth B , Kribs A , Gortner L , Schaible T , et al. To the editor: Surfactant aerosol treatment of respiratory distress syndrome in spontaneously breathing premature infants . Pediatric Pulmonology . 1997 . pp. 222 ± 224 .
25. Berggren E , Liljedahl M , Winbladh B , Andreasson B , Curstedt T , Robertson B , et al. Pilot study of nebulized surfactant therapy for neonatal respiratory distress syndrome . Acta Paediatr . 2000 ; 89 : 460 ± 4 . PMID: 10830460
26. Finer NN , Merritt TA , Bernstein G , Job L , Mazela J , Segal R. An open label, pilot study of Aerosurf® combined with nCPAP to prevent RDS in preterm neonates . J Aerosol Med Pulm Drug Deliv . 2010 ; 23 : 303 ± 309 . https://doi.org/10.1089/jamp. 2009 .0758 PMID: 20455772
27. Mazela J , Polin RA . Aerosol delivery to ventilated newborn infants: Historical challenges and new directions . European Journal of Pediatrics . 2011 . pp. 433 ± 444 .
28. Ricci F , Catozzi C , Murgia X , Rosa B , Amidani D , Lorenzini L , et al. Physiological, Biochemical, and Biophysical Characterization of the Lung-Lavaged Spontaneously-Breathing Rabbit as a Model for Respiratory Distress Syndrome . PLoS One . 2017 ; 12 : e0169190. https://doi.org/10.1371/journal.pone. 0169190 PMID: 28060859
29. Mrozek JP , Smith KM , Bing DR , Meyers PA , Simonton SC , Connet JE , et al. Exogenous surfactant and partial liquid ventilation: Physiologic and pathologic effects . Am J Respir Crit Care Med . 1997 ; 156 : 1058 ± 1065 . https://doi.org/10.1164/ajrccm.156.4.9610104 PMID: 9351603
30. Zimmermann AM , Roberts KD , Lampland AL , Meyers PA , Worwa CT , Plumm B , et al. Improved gas exchange and survival after KL-4 surfactant in newborn pigs with severe acute lung injury . Pediatr Pulmonol . 2010 ; 45 : 782 ± 788 . https://doi.org/10.1002/ppul.21252 PMID: 20597076
31. Barrington KJ , Bull D , Finer NN , Objective A . Randomized trial of nasal synchronized intermittent mandatory ventilation compared with continuous positive airway pressure after extubation of very low birth weight infants . Pediatrics . 2001 ; 107 : 638 ± 641 . PMID: 11335736
32. Khalaf MN , Brodsky N , Hurley J , Bhandari V. A prospective randomized, controlled trial comparing synchronized nasal intermittent positive pressure ventilation versus nasal continuous positive airway pressure as modes of extubation . Pediatrics . 2001 ; 108 : 13 ± 17 . PMID: 11433048
33. Lin CH , Wang ST , Lin YJ , Yeh TF . Efficacy of nasal intermittent positive pressure ventilation in treating apnea of prematurity . Pediatr Pulmonol . 1998 ; 26 : 349 ± 353 . PMID: 9859905
34. Bhandari V , Gavino RG , Nedrelow JH , Pallela P , Salvador a , Ehrenkranz R a et al. A randomized controlled trial of synchronized nasal intermittent positive pressure ventilation in RDS . J Perinatol . 2007 ; 27 : 697 ± 703 . https://doi.org/10.1038/sj.jp. 7211805 PMID: 17703184
35. Sai Sunil Kishore M , Dutta S , Kumar P . Early nasal intermittent positive pressure ventilation versus continuous positive airway pressure for respiratory distress syndrome . Acta Paediatr . 2009 ; 98 : 1412 ±5. https://doi.org/10.1111/j.1651- 2227 . 2009 . 01348 . x PMID : 19523049
36. Mazela J , Merritt TA , Finer NN . Aerosolized surfactants . Curr Opin Pediatr . 2007 ; 19 : 155 ± 162 . https:// doi.org/10.1097/MOP.0b013e32807fb013 PMID: 17496758
37. Roberts KD , Lampland AL , Meyers PA , Worwa CT , Plumm BJ , Mammel MC . Laryngeal mask airway for surfactant administration in a newborn animal model . Pediatr Res . 2010 ; 68 : 414 ± 418 . https://doi. org/10.1203/PDR.0b013e3181ef7619 PMID: 20613684
38. Chen L , Wang L , Li J , Wang N , Shi Y. Noninvasive Ventilation for Preterm Twin Neonates with Respiratory Distress Syndrome: A Randomized Controlled Trial . Sci Rep . Nature Publishing Group; 2015 ; 5 : 14483 . https://doi.org/10.1038/srep14483 PMID: 26399752
39. Gizzi C , Papoff P , Giordano I , Massenzi L , Barbàra CS , Campelli M , et al. Flow-Synchronized Nasal Intermittent Positive Pressure Ventilation for Infants <32 Weeks' Gestation with Respiratory Distress Syndrome . Crit Care Res Pract . Hindawi Publishing Corporation; 2012 ; 2012 : 301818. https://doi.org/ 10.1155/ 2012 /301818 PMID: 23227317
40. Owen LS , Morley CJ , Dawson JA , Davis PG . Effects of non-synchronised nasal intermittent positive pressure ventilation on spontaneous breathing in preterm infants . Arch Dis ChildÐFetal Neonatal Ed. 2011 ; 96 : F422 LP ± F428 .
41. Gizzi C , Montecchia F , Panetta V , Castellano C , Mariani C , Campelli M , et al. Is synchronised NIPPV more effective than NIPPV and NCPAP in treating apnoea of prematurity (AOP)? A randomised crossover trial . Arch Dis ChildÐFetal Neonatal Ed. 2015 ; 100 : F17 LP ± F23 .
42. Chang HY , Claure N , D'Ugard C , Torres J , Nwajei P , Bancalari E. Effects of synchronization during nasal ventilation in clinically stable preterm infants . Pediatr Res . 2011 ; 69 : 84 ± 89 . https://doi.org/10. 1203/PDR.0b013e3181ff6770 PMID: 20924313
43. HuÈtten MC , Kuypers E , Ophelders DR , Nikiforou M , Jellema RK , Niemarkt HJ , et al. Nebulization of Poractant alfa via a vibrating membrane nebulizer in spontaneously breathing preterm lambs with binasal continuous positive pressure ventilation . Pediatr Res . 2015 ; 78 : 664 ± 669 . https://doi.org/10.1038/pr. 2015 .165 PMID: 26322413