Abstract
BACKGROUND: The aim of this study was to define the level of peak inspiratory pressure (PIP) and mean airway pressure (
) at which a pneumothorax is produced in an in vivo ARDS neonate model. In addition, we analyzed the hemodynamic response and cerebral parameters during the progressive increase of intrathoracic pressure.
METHODS: We designed a prospective, experimental study with 11 Landrace × Large White pigs < 48 h from their birth. With the pigs under general anesthesia, tracheal intubation, invasive hemodynamic monitoring with a pediatric arterial thermodilution catheter, intracranial pressure, cerebral oximetry through near-infrared spectroscopy, and bilateral chest tube catheterization were performed. The ARDS model was developed with bronchoalveolar lavages. The rise in inspiratory pressure was performed achieved by increasing PEEP in stepwise increments at a constant driving pressure. PEEP was increased 5 cm H2O every 2 min until a pneumothorax was observed. A descriptive analysis, a Kaplan-Meier curve, and a regression analysis by using a generalized estimation equation were performed.
RESULTS: A pneumothorax was observed in a median (interquartile range [IQR]) 
of 54 (46–56) cm H2O and median (IQR) PIP of 65 (58–73) cm H2O; asystole at median (IQR) 
of 49 (36–54) cm H2O and median (IQR) PIP of 60 (48–65) cm H2O. Hemodynamic changes in the median artery pressure, cardiac output, and myocardial contractility were observed above the range of 
of 14 cm H2O (PIP 25 and PEEP 10 cm H2O). Disturbances in intracranial pressure and cerebral oximetry through near-infrared spectroscopy appeared when deep hypotension and asystole occurred.
CONCLUSIONS: A progressive increase of PEEP at a constant driving pressure did not increase severe adverse events at the range of pressures that we routinely use in neonates with ARDS. Asystole, pneumothorax, and cerebral compromise appeared at high intrathoracic ranges of pressure. Hemodynamics must be strictly monitored in all patients during the performance of lung recruitment maneuvers because hemodynamic deflections emerge early, at a range of pressures commonly used in ventilated neonates with ARDS.
- ARDS
- neonates
- lung recruitment maneuver (LRM)
- mechanical ventilation
- pneumothorax
- hemodynamic
- intracranial pressure (ICP) and cerebral oximetry through near-infrared spectroscopy
Introduction
ARDS is a non-homogenous inflammation of the lung that alters the alveolo-capillary barrier and produce diffuse alveolar damage.1 These mechanisms are responsible for the ventilation-perfusion disruption and for the loss of pulmonary compliance, both characteristics of ARDS. Consequently, they generate a pathological pulmonary shunt (which manifests with hypoxemia) and an increase in physiologic dead space (which favors hypercapnia) in a variable degree.2 Lung recruitment maneuvers consist of a transitory and controlled increase in transpulmonary pressure to reverse and treat the alveolar collapse, almost constantly present in ARDS. To reverse this collapse, the inspiratory pressure should surpass the critical opening pressure.3 Lung recruitment maneu-vers are indicated to treat atelectasis, which improves gas exchange in patients on mechanical ventilation.4,5
Currently, randomized controlled trials on lung recruitment maneuvers in preterm, neonates, and infants are limited.6 Although some of these studies demonstrate that lung recruitment maneuvers are beneficial for these patients,7-9 their use is not widespread.10,11 The recent study by Vento et al8 shows that the efficacy of surfactant treatment was improved after the lung recruitment maneuver (intubate-recruitment-surfactant-extubate) compared with the standard technique (intubate-surfactant-extubate) in extremely preterm neonates. However, there is no agreement on indication nor in the technique to apply lung recruitment maneuvers to reverse this collapse (high-frequency oscillatory ventilation vs progressive increases in pressure on conventional mechanical ventilation).12,13
Performance of lung recruitment maneuvers might have adverse events. Although these events are typically mild and self-limited (such as hypotension, a decrease in 
, or bradycardia),14 there is the possibility to induce more-severe consequences, such as barotrauma or even cardiac arrest.15 Previous studies in neonate models with no lung pathology demonstrated that lung recruitment maneuvers carried out by stepwise increases in PEEP at constant driving pressures are safe for both pulmonary and hemodynamic considerations, given that the investigators found no significant incidence of hypotension, bradycardia, or barotrauma until 30 cm H2O of peak inspiratory pressure (PIP).5,16,17
However, in neonates with ARDS, to our knowledge, there are no studies on the critical pressure of lung opening or about the inspiratory pressures that might involve barotrauma. In addition, hemodynamic repercussion during lung recruitment maneuvers in these patients has not been widely studied neither have their implications on certain cerebral parameters, such as cerebral oximetry and intracranial pressure (ICP). The main objective of the present study was to determine the level of PIP and mean airway pressure (
) at which a pneumothorax is produced in an in vivo ARDS neonate model through the stepwise increasing PEEP method at a constant driving pressure. In addition, we analyzed the hemodynamic response and cerebral parameters during the progressive increase of intrathoracic pressure so as to determine what level of pressure implies consequences that are non-compatible with life.
Quick Look
Current Knowledge
ARDS is a non-homogenous inflammation of the lung that causes loss of pulmonary compliance. Lung recruitment maneuvers, through a transitory and controlled increase of the transpulmonary pressure, reverse alveolar collapse; however, in neonates with ARDS, their use is not widespread. In addition, there is no agreement on their indication or in the technique to apply them.
What This Paper Contributes to Our Knowledge
In an in vivo ARDS neonatal model, through the stepwise increasing PEEP method at a constant driving pressure, we found (a) the appearance of asystole, pneumothorax, and cerebral compromise at high ranges of pressures (
of 37 cm H2O, peak inspiratory pressure 50 and PEEP 35 cm H2O); and (b) the appearance of hemodynamic changes in mean arterial pressure, cardiac output, and myocardial contractility at a range of pressures commonly used in mechanical ventilation in ARDS neonates (
of 14 cm H2O, peak inspiratory pressure 25 and PEEP 10 cm H2O).
Methods
Animals
Eleven Landrace × Large White pigs < 48 h old were used. The mean ± SD weight was 2.54 ± 0.22 kg. All of the pigs were examined by the same veterinarian (M.S) from our unit before the study to eliminate any animal with a health problem. The animals were handled according to the European and national regulations for protection of experimental animals (2010/63/UE and RD 53/2013). Ethics approval for this study (Ethical Committee CEEA 008/2017 Ref. PROEX 234/17) was provided by the Institutional Animal Care and Use Committee, Puerta de Hierro-Segovia de Arana Health Research Institute, Madrid, Spain.
Experimental Design
On each study day, one unmedicated animal was transported to an operating room close to the housing room and anesthesia was induced by using sevoflurane 8% vaporized in an 
of 0.4. Self-adhering patches were applied to the skin for electrocardiogram and heart rate recording, and hemoglobin 
was continuously recorded by placing a pulse oximeter (Infinity Delta; Dräger, Lübeck, Germany) on the hoof. A peripheral 24-gauge polyethylene catheter was then introduced in the medial auricular vein in the posterior side of the ear to get peripheral venous access. Then, 0.2 mg/kg of morphine chlorhydrate and 0.15 mg/kg of cisatracurium were administrated. Only after the obtention of an adequate anesthetic depth, endotracheal intubation was performed with a 3.5-mm internal diameter endotracheal tube without cuff. By using silk thread, we sealed the trachea around the endotracheal tube to guarantee the absence of leaks.
After endotracheal intubation, mechanical ventilation was initiated with an anesthesia machine, Flow-i C20 (Getinge, Solna, Sweden), with the following baseline ventilatory parameters: volume-controlled ventilation with a tidal volume of 6–8 mL/kg, breathing frequency of 30–50 breaths/min, 
of 0.30, PEEP of 3 cm H2O, and inspiratory (TI) to expiratory ratio (TE) of 1:1. These parameters were then adjusted to obtain an end-tidal CO2 between 40 and 50 mm Hg, with 
> 90%, and when maintaining a correct hemodynamic stability. We also controlled the temperature with a rectal probe to maintain normothermia (37–39°C) with a hot air blanket (EQUATOR Convective Warmer; Smiths Medical ASD, Minneapolis, Minnesota). Then, for the maintenance of anesthesia, sevoflurane at 2.5% (1.0 MAC) was used,18 with a cisatracurium infusion at 0.12 mg/kg/h, and intra-operative analgesia was achieved with intravenous morphine chlorhydrate (0.2 mg/kg/h). The fluid used during the procedure was a Ringer’s lactate solution at 4 mL/kg/h. No volume load was realized, nor administration of other drugs before the experiments, to preserve the hemodynamical parameters in the same conditions. However, we administrated 10 mL/kg of saline solution (SS 0.9%) if mean arterial pressure was < 45 mm Hg and an extra bolus of morphine chlorhydrate of 0.1 mg/kg was administrated if heart rate increased more than 20% from baseline.
To monitor hemodynamic parameters (systolic arterial pressure, diastolic arterial pressure, and mean arterial pressure, cardiac output [Q̇T] through the pulse wave, changes in arterial pressure per unit of time as an indirect parameter of left ventricle contractility (dP max)) and to obtain arterial blood samples (to analyze pH, 
, and 
), we inserted a femoral right artery with a thermodilution pediatric catheter of 3 French and 7 cm of length (Pulsiocath PV2013L07A, Pulsion Medical Systems AG, Munich, Germany) and was then connected to a PiCCO monitor (PULSION). Then, to complete monitoring and for the administration of medication, a central venous access was obtained through the insertion of a central venous catheter of 5.5 French and 8 cm of length in the right internal jugular vein (Seldiflex 66714J18, Promided, Cedex, France).
For monitoring ICP, the animals were positioned in prone. Then, we used a manual craniotome with a number 26 drill to insert an ICP catheter (Camino 1104B; Natus, Pleasaton, California) into the right hemisphere parenchyma and connected it to the Camino ICP Monitor (Natus, Pleasaton, California). In addition, to measure cerebral oximetry through near-infrared spectroscopy, the skin was shaved and cleaned with a 90% alcohol solution, and an oximetry probe was positioned in the left hemisphere (INVOS Cerebral/Somatic Oximetry Infant-Neonatal Sensors 12-PM-0012a, Medtronic, Minneapolis, Minnesota) and connected to an INVOS 510°C Cerebral/Somatic Oximeter monitor (Medtronic). In this sense, it was not possible to place the oximetry probe in the right hemisphere because there was not enough space, given the volume of the ICP catheter. Also, the animal was positioned in a supine position.
After checking the correct monitoring, 5 bronchoalveolar lavages were performed with 10 mL/kg of 0.9% saline solution at 38°C, which was instilled intra-tracheally.19 During the lavages, we maintained the baseline ventilation parameters (tidal volume of 6–8 mL/kg, breathing frequency of 30–50 breaths/min, 
: 
of 1:1, PEEP of 3 cm H2O), in which 
was increased to 1.0. None of the animals were manually ventilated to avoid excessive peak pressures that could lead to an unnoticed barotrauma. We maintained the baseline ventilation parameters 5 min after the lavages (tidal volume of 6–8 mL/kg, breathing frequency of 30–50 breaths/min, 
: 
of 1:1). PEEP was increased to 5 cm H2O to accomplish the diagnostic criteria of ARDS,2,10 with a 
of 1.0. This experimental model was then checked by 2 measures: (a) in arterial blood gas, we checked that 
/
was < 200 mm Hg,2 and (b) we verified that there was a decrease of at least 40% of the dynamic compliance. If those 2 criteria were not met, then we repeated the bronchoalveolar lavages as many times as needed to accomplish them.
Also, we placed a 10 French chest tube (Kendall Argyle, Covidien, Minneapolis, Minnesota) in each hemithorax, at the anterior extremities middle line, with a careful surgical dissection. Before the insertion of each tube through the last plane of the muscular fascia, the animal was disconnected from the ventilation through the endotracheal tube to provide a pulmonary collapse, avoiding any pulmonary damage. Then, each chest tube was fixed to the epidermic plane of the chest wall by using silk sutures to avoid peri-tube leaks. Both chest tubes were then connected to a trap (1 cm H2O) on their distal side to surveil pulmonary damages through the observation of bubbling.
Before the initiation of the experiments, the animals were maintained with the baseline ventilation parameters previously described (tidal volume of 6 mL/kg, breathing frequency of 30–50 breaths/min, 
: 
of 1:1, PEEP of 5 cm H2O, and 
of 1.0) during 20 additional min. After this, a constant driving pressure of 15 cm H2O, was programmed, with a breathing frequency of 35 breaths/min, 
: 
of 1:1 and 
up to 1.0. On each increase of PIP) we checked for the absence of air trapping and auto-PEEP with the surveillance of the flow-time curve. This rise in inspiratory pressure was performed through increases in PEEP of 5 cm H2O every 2 min until reaching a PEEP up to 50 cm H2O, which corresponded to a PIP of 65 cm H2O. If no pneumothorax appeared at this level of PIP, the PEEP was maintained and the driving pressure was increased to rise PIP (because there is no possibility to program PEEP above 50 cm H2O on anesthesia machines) in steps of 5 cm H2O until a pneumothorax was observed.
The variables that we studied were the presence of pneumothorax, heart rate, mean arterial pressure, Q̇T, changes in arterial pressure per unit of time as an indirect parameter of left ventricle contractility (dP max), central venous pressure, ICP, and cerebral oximetry through near-infrared spectroscopy. 
was registered from the anesthesia machine. The data obtained from monitoring were recorded in the last 15 s of each step of PIP/PEEP. We considered the presence of a pneumothorax if (a) we observed a continued bubbling (in inspiration and expiration) on the water traps, or (b) we visualized sudden changes on the ventilator curves (flow − time, pressure − time). Hence, we defined the pneumothorax triggering pressure as that level of PIP/PEEP, in which one of these phenomena appeared and we considered the asystole triggering pressure as the level of PIP/PEEP where electromechanical dissociation appeared.
Statistical Analysis
Sample size and power calculations were conducted before data collection. They were based on previous data from similar studies and determined by using the Granmo 7.12 software program (Institut Municipal d’Investigació Mèdica, Barcelona, Spain). Accepting an α risk of 0.05 and β risk of 0.05 in a 2-sided test, 7 animals per group were deemed adequate to obtain a statistically significant difference of ≥10 cm H2O in mean arterial pressure (a 20% drop in mean arterial pressure from baseline). The SD was assumed to be ±7. We carried out a descriptive analysis in which we presented the step of PIP and 
, in which a pneumothorax, asystole, bradycardia, hypotension, and a drop in Q̇T occurred.
Bradycardia, hypotension, and a drop in Q̇T are defined as a 20% decrease from baseline.3 The results are presented as median (interquartile range [IQR]). In addition, we performed a Kaplan-Meier curve to determine the PIP that may involve barotrauma. To analyze the hemodynamic and cerebral changes during the 
increment, a regression analysis by using a generalized estimation equation was performed. The average estimate, together with the 95% CI was calculated, and the effect size was expressed as the effect-size coefficient and its 95% CI. Statistical significance was set at P < .05. All of the statistical analyses were performed by using STATA 15 2017. (Stata Statistical Software: Release 15, StataCorp LLC, College Station, Texas).
Results
Eleven male pigs were included in the present study. However, 2 of them were not included in the analysis due to the following issues: in one of them, hemodynamic values were not obtained due to a defect in the arterial catheter; in the other one, the chest tube was not correctly positioned. All of the pigs met ARDS criteria after 5 bronchoalveolar lavages.
Pneumothorax Pressures
The median (IQR) pneumothorax triggering PIP was 65 (58−73) cm H2O, and the median (IQR) 
was 54 (46–56) cm H2O (Table 1, Fig. 1). The first pneumothorax event occurred at PIP of 50 cm H2O; above this level, a pneumothorax was observed in each step of increment of PIP/PEEP in the Kaplan-Meier curve (Fig. 2). We observed right lung rupture in 56% of the animals (n = 5), whereas the rupture in the left lung was in 33% of the animals (n = 3). Bilateral rupture occurred in 11% of the animals (n = 1).
Box plots that compare mean airway pressures (
) in cm H2O, causing the main experimental outcomes: pneumothorax, asystole, bradycardia, hypotension, drop in cardiac output (CO). Bradycardia, hypotension, and a drop in CO are defined by a 20% decrease from baseline. The box plots show the first and third quartiles, with the center line denoting the median, and the 2 whiskers showing the minimum and maximum.
A Kaplan-Meier survival estimate curve that represents the appearance of a pneumothorax during increasing steps of peak inspiratory pressure (PIP)/PEEP. Abscissa axis represents the PIP in cm H2O. Ordinate axis represents the appearance of a pneumothorax: first event appears at PIP of 50 cm H2O. Above this level, a pneumothorax was observed in each step of increment of PIP/PEEP at the Kaplan-Meier curve.
PIP and 
that Correspond to the Appearance of Pneumothorax, Asystole, Bradycardia, Hypotension, and a Drop in the Cardiac Output
Hemodynamics
Our regression model showed that Q̇T during the experiment was inversely proportional to the increase of 
. This decrease turns significant in the step 
of 14 cm H2O (PIP 25 and PEEP 10 cm H2O), in which the mean Q̇T was 0.93 (95% CI 0.73–1.12) L/min, whereas the mean baseline Q̇T was 1.15 (95% CI 0.97-1.34) L/min, which involves a mean drop in Q̇T of 0.22 (95% CI−0.44 to −0.01) (P = .04) (Fig. 3). We observed a 20% decrease in Q̇T at a median (IQR) 
of 19 (14–21) cm H2O and at a median (IQR) PIP of 30 (25–33) cm H2O (Table 1, Fig. 1).
Hemodynamic and cerebral variables at each step of PIP/PEEP. Abscissa axis represents the mean airway pressure (
) generated in cm H2O. Data that correspond to 
of 6 cm H2O are the baseline average values. (A) HR = heart rate (beats/min); (B) MAP = mean arterial blood pressure (mm Hg); (C) CO = cardiac output (L/min); (D) 
= ventricular contractility (mm Hg/s); (E) CVP = central venous pressure (mm Hg); (F) ICP = intracranial pressure (mm Hg); and (G) SrO2 = cerebral oximetry (%). Results are shown as average estimated, together with the 95% CI. Statistical significance from baseline value, *P < .05, **P < .01.
The dP max (which indirectly estimates ventricular contractility) is inversely proportional to the increase of 
. At the step of 
= 14 cm H2O (PIP 25 and PEEP 10 cm H2O), it significantly dropped to mean 748 (95% CI 581−916) mmHg/sec, in which the mean baseline dP max was 1,045 (95% CI 878−1,212) mm Hg/sec, which meant a mean decrease of 297 (95% CI −503 to −90) mm Hg/sg (P = .005) (Fig. 3). In our regression model, we observed that the mean arterial pressure was also inversely proportional to the increase of 
. It was at the step 
= 14 cm H2O (PIP 25 and PEEP 10 cm H2O) in which the mean arterial pressure significantly diminished, to 42 (95% CI 34−50) mm Hg, whereas the baseline mean arterial pressure was 51 (95% CI 43−60) mm Hg, which represented a mean reduction of 10 (95% CI −18 to −1) mm Hg (P = .01) (Fig. 3). Hypotension (defined as a 20% decrease from the mean arterial pressure baseline) was registered at a median (IQR) 
of 14 (14–39) cm H2O and at a median (IQR) PIP of 25 (25–50) cm H2O (Table 1, Fig. 1).
The heart rate in our regression model was inversely proportional to the increase of 
. It was at the step 
= 34 cm H2O (PIP 45 and PEEP 30 cm H2O) in which the mean heart rate was 134 (95% CI 103−166) beats/min, whereas the mean baseline heart rate was 185 (95% CI 153−216) beats/min, which indicated a mean decrease of 50 (95% CI −92 to −9) beats/min (P = .02) (Fig. 3). Bradycardia (defined as a 20% decrease from the heart rate baseline) was registered at a median (IQR) 
= 39 (31–49) cm H2O and at a median (IQR) PIP = 50 (43–60) cm H2O (Table 1, Fig. 1). In our regression model, the mean baseline central venous pressure was 4 (95% CI 2−7) mm Hg, and it remained unchanged until 
= 44 cm H2O (PIP 55 and PEEP 40 cm H2O), in this step central venous pressure increases to 11 (95% CI 7–15) mm Hg (P = .01). From this step, central venous pressure continued to increase proportional to the increase in 
(Fig. 3).
Asystole
Asystole was observed at a median (IQR) 
of 49 (36–54) cm H2O and at a median (IQR) PIP of 60 (48–65) cm H2O (Table 1, Fig. 1).
Neurologic Monitoring
The mean ICP was 9 (95% CI 7−12) mm Hg, and it remained unchanged until 
= 39 cm H2O (PIP 50 and PEEP 35 cm H2O), in which ICP started to decrease proportionally to the increase of 
. From the step 
= 49 cm H2O (PIP 60 and PEEP 45 cm H2O), the mean ICP diminished 4 (95% CI −7 to −0.16) mm Hg when compared with the baseline ICP (P = .04) (Fig. 3). Also, we did not find significant variations with cerebral oximetry through near-infrared spectroscopy until 
= 34 cm H2O (PIP 45 and PEEP 30 cm H2O), in which it decreases to mean cerebral oximetry through near-infrared spectroscopy of 31% (95% CI 19%–42%), whereas the mean baseline value was 46% (95% CI 35%–58%). This represents a reduction of 20% from the baseline value (P = .03) (Fig. 3).
Discussion
We observed that severe adverse events (pneumothorax, asystole) during the lung recruitment maneuver in our neonatal ARDS model occurred at a range of pressures higher than the range of pressures used in daily clinical practice. Indeed, even the minimum PIP at which those events occurred was over the limit routinely used (50 cm H2O). However, we observed hemodynamic deflections (a decrease in mean arterial pressure, Q̇T, and ventricular contractility) when we applied the initial increments of PIP/PEEP, over the range of 
= 14 cm H2O (PIP 25 and PEEP 10 cm H2O). Thus, bradycardia occurred later, at 
= 34 cm H2O (PIP 45 and PEEP 30 cm H2O). Central venous pressure remained unchanged until a high 
level (>44 cm H2O). Also, disturbances in ICP and cerebral oximetry through near-infrared spectroscopy were clinically important when hypotension and asystole occurred.
García-Fernández et al17 studied which pressures were involved in pneumothorax development. They compared 2 groups: in one group, PEEP was fixed at 20 cm H2O and the driving pressure was increased progressively (constant PEEP and variable driving pressure), and, in the other group, the driving pressure was fixed at 15 cm H2O and the PEEP was increased progressively (constant driving pressure and variable PEEP). A pneumothorax was developed sooner in the group with the constant PEEP and variable driving pressure. These results supported that the pressure most related to pneumothorax development is PIP, and the second one is driving pressure. This supported the concept that cyclic mechanical stress (driving pressure) is worse than static mechanical stress (PEEP) for developing a pneumothorax.
One of our previous investigations, in which we studied what level of pressures may involve barotrauma in a neonatal healthy lung model showed that pneumothorax only occurred at a very high range of pressures (PIP 92 ± 14.8 cm H2O; a minimum PIP value of 70 cm H2O).5 When comparing the pneumothorax triggering pressure between the healthy neonatal model and our ARDS neonatal model, we appreciate that the last one was more prone to a pneumothorax because this triggering pressure was lower median (IQR) PIP 65 (60–70) cm H2O than in the healthy model. Hence, lung heterogeneity in ARDS might be one of the sources of this phenomena, given that those alveoli that are ventilated might present cyclic overdistention and atelectrauma, even with ventilatory parameters that are “protective.”20 Moreover, positron emission tomographic studies have shown that inflammation is not only confined to areas whose architecture were affected but also to the ventilated areas (being these areas traditionally considered as “healthy”).21 Thus, it is possible that the rupture occurs in ventilated areas, which are also inflamed and subject to cyclic overdistention.
There are no related safety studies in humans with which we can compare our results, for ethical reasons. A recent clinical study in which a high PIP level (close to our study) was used is the ART trial,22 in which the investigators observed a higher risk of a pneumothorax when lung recruitment maneuvers were applied in adult subjects with ARDS. In this study, they performed lung recruitment maneuver with a maximum PIP of 60 cm H2O and PEEP of 45 cm H2O for 1 min, and, after PEEP titration, a new lung recruitment maneuver was conducted in one step by using the same maximum PIP/PEEP (60/45 cm H2O) for 2 min. In our study, the minimum pneumothorax triggering pressure was 50 cm H2O. Although there are several differences between an adult and a neonatal chest wall, analysis of the ART trial22 data demonstrate that ARDS is an independent risk factor for pneumothorax.20 In the case of neonatal and pediatric populations, we believe that the PIP limit should be ≤ 50 cm H2O because of the hemodynamic consequences, as we presented.
It is well known that the increase in intrathoracic pressure during the performance of lung recruitment maneuvers involve some degree of hemodynamic compromise.13,23 Hemodynamic disruptions are closely related to 
level, which is determined by PEEP, inspiratory time, expiratory time, and plateau pressure. 24,25 Previous studies have shown that an elevated 
leads to decreased Q̇T in the infant population during both conventional mechanical ventilation and high-frequency oscillatory ventilation.26,27
The results obtained confirmed that all of our variables (heart rate, mean arterial pressure, Q̇T, and ventricle contractility) were inversely proportional to the increase of 
. There are studies that present hemodynamic changes due to lung recruitment maneuver as transient,3,25,28 being the most common cardiac rhythm alterations.29 However, in our neonatal ARDS model, bradycardia was observed at higher levels of 
= 34 cm H2O (PIP 45 and PEEP 30 cm H2O), compared with Q̇T, mean arterial pressure, and myocardial contractility decrease, which occurred at 
= 14 cm H2O (PIP 25 and PEEP 10 cm H2O). This could be explained by the following mechanism: the reduction in the right-ventricular preload, and afterload are the responsible for Q̇T and mean arterial pressure reduction.23 This causes a decrease in coronary perfusion that leads to bradycardia, which can end in asystole if 
is not reduced. It is possible that, at higher levels of intrathoracic pressure, extrapulmonary shunts are opened with right-to-left shunting, worsening systemic oxygenation. All of these pathophysiologic changes produced by extreme levels of PIP are those that are likely to trigger potentially fatal hemodynamic events.
The fact that the central venous pressure remained unchanged during the first steps of pressure showed that the venous return was maintained in our subjects. A previous study found that subjects who were euvolemic were less prone to hemodynamic disturbances during the lung recruitment maneuver.30 Although venous return was maintained, the increment in central venous pressure observed at a high 
is probably due to increased lung vascular resistance, thus increased right-ventricular afterload.31 This behavior is described as transitory during the performance of the lung recruitment maneuver16,25,30; however, in our study, because there was no release of pressure, this increase was progressive as the 
increased.
The behavior of Q̇T, mean arterial pressure, heart rate, and central venous pressure are widely studied. But there are a few studies that analyze myocardial contractility during the lung recruitment maneuver. Gernoth et al31 revealed a transient decrease in the cardiac power index due to increased right-ventricular stress and strain, a consecutive acute leftward shift of the interventricular septum, which resulted in a decrease left-ventricular end-diastolic area, although venous return was maintained. We did not find studies in neonates performed with advanced hemodynamic monitoring that examined the association between myocardial contractility and the increase of intrathoracic pressure.
In daily clinical practice in neonates, it is unusual to have advanced invasive monitoring that records Q̇T or myocardial contractility, hence these consequences can go unnoticed. On the contrary, the mean arterial pressure is an accessible parameter with no basic invasive monitoring and could be the variable with which we detect early the effects of increasing intrathoracic pressure. From our results, we believe that new studies have to be designed to evaluate the efficacy of the lung recruitment maneuver with open critical pressures lower than those commonly used in neonatal ARDS (35–40 cm H2O)3 to minimize the hemodynamic impact.
When it comes to cerebral monitoring, the mean baseline ICP in our study was 9 (95% CI 7–12) mm Hg. During the first steps of the increase of PIP/PEEP, ICP remained constant. However, over a 
of 39 cm H2O (PIP 50 and PEEP 35 cm H2O) ICP started to decrease. This reduction was significant over a 
of 49 cm H2O (PIP 60 and PEEP 45 cm H2O), in which we find a mean reduction of 4 mm Hg (95% CI −7 to −0.16). This drop in ICP was in line with the maximum hemodynamic compromise and asystole. However, the mean baseline cerebral oximetry through near-infrared spectroscopy, (which reflects the balance between oxygen supply and consumption) was 46% (95% CI 35%–58%). We did not find significant variations until a 
of 34 cm H2O (PIP 45 and PEEP 30 cm H2O), when the mean value was 31% (95% CI 19%–42%), which meant a drop of 20% from the baseline value. This reduction was in line with the maximum hemodynamic compromise and asystole.
Muench et al32 analyzed the effect of increasing PEEP on ICP and cerebral blood flow in male pigs without intracranial pathology but found no effect on ICP or cerebral blood flow. In a later approach, in which they analyzed the same variables in subjects with acute subarachnoid hemorrhage, they concluded that the negative effects of PEEP on ICP and cerebral blood flow depend on mean arterial pressure changes.32 Hence, this finding was explained by the cerebral autoregulation, which can maintain a constant cerebral blood flow between a range of mean arterial pressures.33 Other studies in humans with intracranial pathology from different origins showed that subjects with preserved cerebral autoregulation can tolerate a decrease in mean arterial pressure with no generation of ischemia.32,34,35 However, in patients with cerebral damage and this mechanism altered, low levels of PEEP (4–8 cm H2O) may increase ICP.36 Our results support that the increase in intrathoracic pressure, when cerebral autoregulation is preserved, might not affect ICP or cerebral blood flow (as far as macro-hemodynamic is preserved).
We emphasize that the hemodynamic results noted in our study may not be comparable with those obtained by using the typical lung recruitment maneuver because these changes are mostly related to the duration of PIP/PEEP and higher pressurization steps. In addition, we are subjecting the models to progressive pressurization with no pressure relief at any time, which is different from conventional lung recruitment maneuvers in which, after the higher step of pressurization, a release in pressure that facilitates hemodynamic recovery normally occurs.3,25,37 Pressure relief also plays a role in ICP and cerebral oximetry through near-infrared spectroscopy values as well. Hence, hemodynamic and intracranial changes described in this study might be interpreted to understand the pathophysiology of the extreme increase of intrathoracic pressures, and further studies that analyze cerebral and hemodynamic compromise during the performance of conventional lung recruitment maneuvers in neonates are required.
Our study had some limitations. First, we studied piglets, and our results require a clinical context, so it is not possible to apply these results literally to humans. However, our piglet neonatal model is widely validated to study pulmonary and hemodynamic effects due to its good correlation with human patients, being a model superior to those proposed in other animals. In addition, for ethical reasons, it is not possible to perform this study in humans. In addition, bronchoalveolar lavage with saline solution is one of the first models used in animal experimentation.19 However, lung damage produced by bronchoalveolar lavages is more similar to ARDS secondary to hyaline membrane disease secondary to a lack of surfactant and different than ARDS due to pneumonia or other pathologies,25 being similar to ARDS due to hyaline membranes because of the lack of pulmonary surfactant. Nonetheless, to increase the validity of our study, during the lung-injury generation phase of generation, we repeated the instillation of saline solution at least 5 times until obtaining 
/
< 200 mmHg that was stable. Another limitation was our sample size, which was reduced according to the limitations of the ethics and animal wellness committee, who insisted on the use of the least number of animals. However, it is also true that experimental studies normally present limited inter-individual variation, which allows the use of smaller sample sizes. In this study, we previously calculated the sample size with the Granmo software. In addition, this sample size was similar to previous experimental studies published.
Conclusions
Progressive stepwise increases in PEEP at a constant driving pressure did not increase severe adverse events (pneumothorax, asystole, brain perfusion) at a range of pressure of 
< 37 cm H2O (PIP 50 and PEEP 35 cm H2O) in a neonatal ARDS model. However, we observed hemodynamic disturbances (mean arterial pressure, Q̇T, and myocardial contractility) at a range of pressures of 
> 14 cm H2O (PIP 25 and PEEP 10 cm H2O). Hemodynamics must be strictly monitored in all patients during the performance of lung recruitment maneuvers because hemodynamic deflections emerge early, at a range of pressures commonly used in ventilated neonates with ARDS.
Acknowledgments
The authors thank Ana Royuela PhD, Biostatistics Unit, Instituto de Investigación Sanitaria Puerta de Hierro-Segovia de Arana, CIBER Epidemiology and Public Health, for her valuable assistance in the study.
Footnotes
- Correspondence: Alberto Gutiérrez Martínez MD, Department of Anesthesiology, Intensive Care and Pain Hospital Universitario Puerta de Hierro-Majadahonda. C/Joaquín Rodrigo 1, 28222 Majadahonda, Spain. E-mail: gutylon{at}hotmail.com
The study was performed at Instituto de Investigación Sanitaria Puerta de Hierro-Segovia de Arana. Hospital Universitario Puerta de Hierro-Majadahonda. C/Joaquín Rodrigo 1, 28222 Majadahonda, Spain.
Dr González-Pizzaro has disclosed a relationship with Mindray Biomedical. The other authors have disclosed no conflicts of interest.
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