Effects of positive end-expiratory pressure and spontaneous breathing activity on regional lung inflammation in experimental acute respiratory distress syndrome

Abstract

Objective:

To determine the impact of positive end-expiratory pressure (PEEP) during mechanical ventilation with and without spontaneous breathing (SB) activity on regional lung inflammation in experimental non-severe ARDS.

Design:

Laboratory investigation.

Setting:

University-hospital research facility.

Subjects:

Twenty-four pigs (28.1 to 58.2 kg).

Interventions:

In anesthetized animals, intrapleural pressure sensors were placed thoracoscopically in ventral, dorsal and caudal regions of the left hemithorax. Lung injury was induced with saline lung lavage followed by injurious ventilation in supine position. During airway pressure release ventilation (APRV) with low tidal volumes, PEEP was set 4 cmH₂O above the level to reach a positive transpulmonary pressure in caudal regions at end-expiration (best-PEEP). Animals were randomly assigned to one of four groups (n=6/group; 12 hours): 1) no SB activity and PEEP= best-PEEP-4 cmH₂O; 2) no SB activity and PEEP= best-PEEP+4 cmH₂O; 3) SB activity and PEEP= best-PEEP+4 cmH₂O; 4) SB activity and PEEP= best-PEEP-4 cmH₂O.

Measurements and Main Results:

Global lung inflammation assessed by specific [¹⁸F]fluorodeoxyglucose uptake rate [median (25%−75% percentiles), min⁻¹] was decreased with higher compared to lower PEEP both without SB activity [0.029(0.027–0.030) vs. 0.044(0.041–0.065), P=0.004] and with SB activity [0.032(0.028–0.043) vs. 0.057(0.042–0.075, P=0.016). SB activity did not increase global lung inflammation. Lung inflammation in dorsal regions correlated with transpulmonary driving pressure from SB at lower (r=0.850, P=0.032) but not higher PEEP (r=0.018, P=0.972). Higher PEEP resulted in a more homogeneous distribution of aeration and regional transpulmonary pressures at end-expiration along the ventral-dorsal gradient, as well as a shift of the perfusion center towards dependent zones in the presence of SB activity.

Conclusions:

In experimental mild to moderate ARDS, PEEP levels that stabilize dependent lung regions reduce global lung inflammation during mechanical ventilation, independent from SB activity.

Introduction

In the acute respiratory distress syndrome (ARDS), lung protection is more easily achieved during mechanical ventilation without spontaneous breathing (SB) activity (1). However, the suppression of SB activity may require higher levels of sedation and muscle paralysis, which have been associated with respiratory muscle atrophy and proteolysis, possibly delaying weaning from mechanical ventilation (2). Whereas low levels of SB activity can preserve respiratory muscle integrity in ARDS (3), SB may promote further damage in injured lungs.

The potential of SB activity to damage the lungs depends on the severity of the underlying injury (4), but stability of alveolar units at end-expiration could play an important role too (5). Positive end-expiratory pressure (PEEP) that keeps the lungs open reduces lung damage due to SB activity in experimental severe ARDS (6, 7). However, it remains unclear if SB activity is injurious in mild to moderate ARDS, where SB activity is more frequently observed (8).

We aimed at determining the impact of SB activity on the degree and distribution of lung inflammation at different levels of PEEP in experimental mild to moderate ARDS. Given that lung inflammation is usually accompanied by infiltration and activation of neutrophils (9, 10), and that the rate of glucose utilization of neutrophils is higher than other inflammatory or lung cells (11, 12), we used the regional uptake of the glucose analogue [¹⁸F]-fluorodeoxyglucose ([¹⁸F]FDG) measured with positron emission tomography (PET) as surrogate of inflammation. We hypothesized that in mild to moderate ARDS, SB activity associated with inappropriate low levels of PEEP increases lung inflammation, especially in dependent lung regions.

Materials and Methods

Following approval of the investigation protocol by the Internal Review Board and the Government of the State of Saxony, Germany, 24 pigs (German landrace) weighing 28.1 to 58.2 kg were intravenously anesthetized as described in detail in Supplemental Data File 1. Animals were oro-tracheally intubated and mechanically ventilated in supine position. Lungs were ventilated in volume controlled mode using fraction of inspired oxygen (FIO₂) of 1.0, V_T of 6 mL/kg, positive end-expiratory pressure (PEEP) of 5 cmH₂0, inspiratory:expiratory (I:E) ratio of 1:1 and respiratory rate (RR) to achieve an arterial pH (pHa) between 7.38 and 7.42. FIO₂ was kept at 1.0. Arterial and pulmonary artery pressures were monitored, and urine collected.

Under one-lung ventilation, pressure sensors (n=3) were attached to ventral, dorsal-cranial and dorsal-caudal regions of the left hemi-thorax (Supplemental Figure 1 and Figure 2). Following lung re-expansion, two-lung ventilation was resumed (Pre-Injury, Figure 1).

Figure 2 – Pleural and transpulmonary pressures at end-expiration.

End-expiratory pleural and transpulmonary (airway minus pleural) pressures determined in ventral, dorsal-cranial and dorsal-caudal regions of the left hemi-thorax at start of therapy (T0). Symbols and vertical lines represent means and standard deviations, respectively. CV-PEEP_high, controlled mechanical ventilation with positive end-expiratory pressure (PEEP) titrated to positive transpulmonary pressure in the dorsal-caudal region (best-PEEP) plus 4 cmH₂O; CV-PEEP_low, controlled mechanical ventilation with best-PEEP minus 4 cmH₂O; SB-PEEP_high, mechanical ventilation with spontaneous breathing activity and best-PEEP plus 4 cmH₂O; SB-PEEP_low, mechanical ventilation with spontaneous breathing activity and best-PEEP minus 4 cmH₂O. #, P <0.05.

Figure 1 – Time course of interventions and measurements.

VATS, video-assisted thoracoscopy; VILI, ventilator-induced lung injury; PEEP, positive end expiratory pressure; PET, positron emission tomography; CT, computed tomography; T1, T2 and T3, Time 1, Time 2 and Time 3, respectively; VCV, volume controlled ventilation; APRV, airway pressure release ventilation; V_T, tidal volume; FIO₂, inspiratory oxygen fraction; RR, respiratory rate; pHa, arterial pH.

Experimental ARDS was induced with saline lung lavage and ventilator-induced lung injury (VILI) with a fixed driving pressure of 30 cmH₂O at PEEP of 0 cmH₂O (see Supplemental Digital File 1) until the PaO₂ was lower than 100 mmHg for at least 30 minutes (Injury).

Respiratory signals, gas exchange and hemodynamics

Airflow, airway pressure and pleural pressure signals were obtained. Transpulmonary pressure was calculated by subtracting pleural from airway pressure. The pressure time product (PTP) of SB activity was computed regionally.

Cardiac output was determined with a pulmonary artery catheter (thermodilution method). The intrapulmonary shunt fraction and oxygen consumption were calculated.

Protocol for measurements

The time course of experiments is presented in Figure 1. Following lung recruitment, the PEEP was set at 26 cmH₂O and decreased in steps of 2 cmH₂O (PEEP trial). The “best-PEEP” level was defined as the lowest PEEP yielding positive end-expiratory transpulmonary pressure in the dorsal-caudal region. After further lung recruitment, Post-Injury data were obtained under airway pressure release ventilation (APRV) with plateau pressure to V_T of 6 mL/kg, but limited to a maximum driving pressure of 15 cmH₂O, PEEP according to best-PEEP + 4 cmH₂O, I:E ratio of 1:1, and RR to achieve pHa > 7.30.

Animals were randomly assigned to 4 groups of mechanical ventilation (n=6/group): 1) without SB activity and best PEEP + 4 cmH₂O (CV-PEEP_high); 2) without SB activity and best PEEP – 4 cmH₂O (CV-PEEP_low); 3) with SB activity and best PEEP + 4 cmH₂O (SB-PEEP_high); 4) with SB activity and best PEEP – 4 cmH₂O (SB-PEEP_low). In SB-PEEP_high and SB-PEEP_low groups, the infusion of the neuromuscular block agent was stopped and RR set at the ventilator titrated to allow 40 to 60% of total minute ventilation from SB activity, whereas CV-PEEP_high and CV-PEEP_low were sham ventilated over a time period of 60 minutes.

Respiratory system mechanics, gas exchange and hemodynamics and were assessed at start of therapy and every 4 hours during 12 hours (T0, T1, T2 and T3, respectively), and lung imaging was obtained at the end.

Computed tomography

Detailed CT methods are presented in Supplemental Data File 1. Briefly, a CT scan of the thorax was obtained during ventilation for attenuation correction of PET images (CTAC). CTAC scans were used to compute gas fraction from Hounsfield units (HU) (F_GAS = HU/–1.000). Masks obtained from CTAC were applied to PET images.

Before the end of experiments, a static high-resolution helical CT of the thorax was performed under respiratory hold at mean airway pressure under muscle paralysis. The center of aeration along the ventral-dorsal axis was determined as the median of the respective distribution.

Dynamic CT recordings were obtained caudal to the heart. Images were divided in 5 isogravimetric regions of interest (ROIs) along the ventral-dorsal gradient (ventral, mid-ventral, central, mid-dorsal and dorsal).

Gas and tissue contents were calculated (13), and hyper-aerated, normally aerated, poorly-aerated, and non-aerated compartments computed (14). In dynamic CT images, tidal reaeration and hyperaeration (15), as well as tidal changes in the poorly-aerated compartment alone, were calculated. Pendelluft, a surrogate of inhomogeneity of ventilation, was determined as described elsewhere (16). The center of ventilation along the ventral-dorsal axis, which is a surrogate for was determined as the median of the respective distribution.

Positron emission tomography

[¹⁸F]FDG was injected as bolus (200 MBq), and sequential PET frames were obtained. Tracer plasma activity was measured in a γ-counter cross-calibrated with the PET scanner. The field of view (15 cm) was set above the diaphragmatic dome. Images were reconstructed iteratively with correction for scatter and attenuation. PET images were also divided into ROIs. The [¹⁸F]FDG uptake (K_i) in each ROI was calculated using an adapted Sokoloff three-compartment model (17). To account for differences in F_GAS and blood volume fraction (F_BLOOD), K_i was normalized to lung tissue fraction (F_TISSUE=1-F_GAS-F_BLOOD), yielding the specific K_i (K_iS = K_i/F_TISSUE).

The distribution of specific regional perfusion was determined with PET using a 68-Gallium(⁶⁸Ga)-labeled tracer, as previously described (18) (Supplemental Data File 1). The center of perfusion along the ventral-dorsal axis was determined as the median of the respective distribution.

Statistics

The sample size calculation was based on effect estimates obtained from previous studies (19) and our previous experience (20, 21). We expected that a sample size of 6 animals per group would provide the appropriate power (1-β = 0.8) to identify significant (α = 0.05) differences in K_iS between SB-PEEP_high and SB-PEEP_low using the Mann-Whitney test and taking an effect size d = 2.5, equal number of animals per group, two-sided test and multiple comparisons (n = 4) into account (α* = 0.0125, α* Bonferroni adjusted).

Variables were tested with Kruskall-Wallis, Mann-Whitney, unpaired t-tests and repeated measures 2-way ANOVA, as appropriate. Associations between variables were assessed with Pearson’s correlation coefficients. The global significance level for all performed tests was α = 0.05 and Bonferroni-Holm adjustments were used for multiple comparisons. The statistical analysis was conducted with R (R, http://www.r-project.org, 2014), SPSS (IBM SPSS Statistics for Windows, Version 23, Armonk, NY, IBM Corp.) and GraphPad Prism (GraphPad Prism version 6.00 for Windows, GraphPad Software, La Jolla California USA, www.graphpad.com).

Results

Basic characteristics of animals are given in Supplemental Table 1. After randomization, pHa was lower in groups with higher PEEP (Supplemental Table 2). In all animals, PaO₂/FIO₂ values were higher than 100 mmHg.

Total V_T, RR and minute ventilation did not differ among groups (Supplemental Table 3). RR and MV due to SB activity were higher in SB-PEEP_low than SB-PEEP_high.

At comparable PEEP levels, peak and plateau airway pressures were lower with SB activity than controlled ventilation.

Regional transpulmonary pressures at the different time points are presented in Supplemental Table 4. The end-expiratory pleural and transpulmonary pressures at T0 were higher and more homogenously distributed along the ventral-dorsal axis with higher compared to lower PEEP (Figure 2).

PTP per minute, but not per breath, was higher during SBV-PEEP_low than SB-PEEP_high, mainly in dorsal-cranial and dorsal-caudal regions (Table 1).

Table 1 -.

Pressure-time-product (PTP) from pleural pressure sensors

Variable	Group	T0	T1	T2	T3	ANOVA
						Group	Time	Group x Time
PTPvent (cmH2O·s)	SB-PEEPhigh	1.7 ± 1.0	1.8 ± 1.3	1.4 ± 0.8	1.7 ± 1.0	0.674	0.457	0.538
	SB-PEEPlow	1.6 ± 0.6	1.7 ± 0.5	1.7 ± 0.7	1.8 ± 0.7
PTPdors-cran (cmH2O·s)	SB-PEEPhigh	2.1 ± 1.2	2.8 ± 3.0	1.5 ± 0.3	2.1 ± 0.4	0.464	0.163	0.104
	SB-PEEPlow	2.0 ± 0.4	2.2 ± 0.6	2.4 ± 0.5	2.4 ± 0.9
PTPdors-caud (cmH2O·s)	SB-PEEPhigh	2.6 ± 1.0	2.9 ± 2.5	1.7 ± 0.6	2.3 ± 0.7	0.135	0.23	0.231
	SB-PEEPlow	2.9 ± 0.9	3.0 ± 0.6	3.2 ± 1.1	3.2 ± 1.0
PTPvent (cmH2O·s·min−1)	SB-PEEPhigh	99 ± 88	73 ± 51	65 ± 49	81 ± 66	0.175	0.629	0.425
	SB-PEEPlow	115 ± 52	103 ± 28	124 ± 65	117 ± 53
PTPdors-cran (cmH2O·s·min−1)	SB-PEEPhigh	122 ± 105	97 ± 66	69 ± 32	96 ± 41	0.005	0.279	0.075
	SB-PEEPlow	146 ± 56	136 ± 32	171 ± 43###	157 ± 63
PTPdors-caud (cmH2O·s·min−1)	SB-PEEPhigh	150 ± 109	109 ± 46	84 ± 43	109 ± 53	<0.001	0.448	0.193
	SB-PEEPlow	211 ± 101	187 ± 20##	218 ± 73##	202 ± 73#

Legend: All values are given as mean ± standard deviation. Sensors were placed in ventral (vent), dorsal-cranial (dors-cran) and dorsal-caudal (dors-caud) regions of the left hemi-thorax. SB-PEEP_high, mechanical ventilation with spontaneous breathing activity and positive end-expiratory pressure (PEEP) titrated to positive transpulmonary pressure in the dorsal-caudal region (best-PEEP) plus 4 cmH₂O; SB-PEEP_low, mechanical ventilation with spontaneous breathing activity and best-PEEP minus 4 cmH₂O. Statistical analysis was performed using two-factorial repeated measures ANOVA with with-in factor therapy time and between factor therapy group and covariate the respective values at T0, T1, T2 and T3, respectively (therapy period). Significance was accepted at P≤0.05. For therapy time points T0 difference were assessed by ANOVA followed by t-test (P-values above time point values). Differences in time points during therapy were tested in the same manner but only, if group effects were significant according to ANOVA; pressure time product (PTP) at ventral, dorsal and caudal regions.

Figure 3 shows the maps of distribution of lung inflammation, aeration compartments and perfusion in one representative animal of each group.

Figure 3 – Single-slice images of specific [18F]-fluorodeoxyglucose uptake rate, aeration compartments and perfusion in representative animals.

Left column: slice images of specific [18F]-fluorodeoxyglucose uptake rate (K_iS) calculated voxel by voxel using the Patlak method from positron emission tomography (PET), and normalized to tissue fraction, as obtained with computed tomography (CT). R, right; L, left. Right column: aeration compartments obtained with CT; hyper, hyperaerated compartment; normally, normally aerated compartment; poorly, poorly aerated compartment, non, non-aerated compartment. Right column: distribution of perfusion obtained with ⁶⁸Ga microspheres and PET. CV-PEEP_high, controlled mechanical ventilation with positive end-expiratory pressure (PEEP) titrated to positive transpulmonary pressure in the dorsal-caudal region (best-PEEP) plus 4 cmH₂O; CV-PEEP_low, controlled mechanical ventilation with best-PEEP minus 4 cmH₂O; SB-PEEP_high, mechanical ventilation with spontaneous breathing activity and best-PEEP plus 4 cmH₂O; SB-PEEP_low, mechanical ventilation with spontaneous breathing activity and best-PEEP minus 4 cmH₂O.

As depicted in Figure 4, K_iS was lower with higher PEEP, both with and without SB activity. With higher PEEP, K_iS decreased in all lung regions during controlled ventilation, but only in ventral zones during SB activity (Supplemental Figure 3). The same global and regional behavior was observed when inflammation was assessed by non-normalized K_i measurements (Supplemental Figures 4 and 5, respectively). In dorsal lung zones, K_iS correlated with the regional transpulmonary pressure gradient of SB cycles at lower, but not higher PEEP (Supplemental Figure 6).

Figure 4 – Specific [18F]-fluorodeoxyglucose uptake rate in groups.

Specific [18F]- fluorodeoxyglucose uptake rate measured with positron emission tomography and modeling of tracer kinetic according to Sokoloff followed by normalization to tissue fraction (K_iS). Horizontal lines represent median values. CV-PEEP_high, controlled mechanical ventilation with positive end-expiratory pressure (PEEP) titrated to positive transpulmonary pressure in the dorsal-caudal region (best-PEEP) plus 4 cmH₂O; CV-PEEP_low, controlled mechanical ventilation with best-PEEP minus 4 cmH₂O; SB-PEEP_high, mechanical ventilation with spontaneous breathing activity and best-PEEP plus 4 cmH₂O; SB-PEEP_low, mechanical ventilation with spontaneous breathing activity and best-PEEP minus 4 cmH₂O. NS, not significant.

The normally aerated lung tissue increased, while poorly aerated lung tissue decreased with higher PEEP, independently from SB activity (Supplemental Figure 7). SB-PEEP_low showed more non-aerated lung tissue than SB-PEEP_high. Dynamic CT videos of a representative animal of each group are shown in Supplemental Video Files 1 to 4. In ventral lung regions, tidal hyperaeration was higher with SB-PEEP_high than SB-PEEP_low (Supplemental Figure 8). In mid-ventral regions, tidal reaeration, as well as changes in poorly-aerated tissue only, were more pronounced with CV-PEEP_low compared to CV-PEEP_high (Supplemental Figures 9 and 10, respectively). Pendelluft did not differ among groups (Supplemental Figure 11).

In SB-PEEP_high compared to SB-PEEP_low, the centers of aeration and perfusion were shifted towards dependent lung zones (Supplemental Figure 12). The centers of ventilation along the ventral-dorsal gradient did not differ significantly among groups (Supplemental Figure 13).

Discussion

We found that in experimental mild to moderate ARDS: 1) a PEEP level able to stabilize the most dependent lung region reduced lung inflammation during mechanical ventilation with and without SB activity; 2) SB activity did not increase global lung inflammation neither with lower, nor higher PEEP; 3) in dorsal lung zones, the transpulmonary pressure gradient during SB cycles correlated with regional lung inflammation.

To our knowledge, this is the first study determining the effects of PEEP and SB activity on the regional distribution of lung inflammation in experimental mild to moderate ARDS. We induced ARDS with a double-hit since it better reproduces typical features of human ARDS (22), while keeping PaO₂/FIO₂ > 100 mmHg. We opted for the use of PET with [18F]FDG since this technique represents a well-established method to assess pulmonary inflammation in vivo (23). [18F]FDG accumulates especially in neutrophils, as shown in pigs (24) and patients with pneumonia (25). We also individualized PEEP through titration to a positive transpulmonary pressure in the dorsal-cranial (most dependent) lung region aiming at stabilizing lungs at end-expiration. Furthermore, we achieved pressure time products in dorsal-cranial and dorsal-caudal regions that were situated within the expected range during spontaneous breathing, increasing the possible clinical relevance of the findings.

The observation that lung inflammation was decreased with higher compared to lower PEEP, mainly in ventral lung zones, is in line with previous data from our group in small animals (26), and can be explained by different factors. First, higher PEEP yielded increased normally aerated with decreased poorly aerated lung tissue. Second, the distribution of transpulmonary pressures along the ventral-dorsal gradient at end-expiration was more even with higher PEEP, leading to reduced stress mainly in ventral lung regions. The finding that higher PEEP results in more homogeneous distribution of transpulmonary pressures is in agreement with previous data (7, 27). Also, the reduction in inflammation with higher PEEP is in agreement with the concept that during SB activity for a given V_T the regional impact of mechanical stress on inflammation is reduced when the end-expiratory lung volume is increased (20, 21). Third, higher PEEP led to a shift of perfusion, but not ventilation, towards dorsal lung regions, mainly during SB activity, possibly contributing to less injury in ventral lung areas. In fact, it has been shown that higher blood flow is associated with more pronounced cyclic changes in perivascular pressures surrounding extra-alveolar vessels, possibly contributing to VILI (28), which is likely increased by strain. It is worth noting that the amount of atelectrauma was relatively low in our study when compared to experimental severe ARDS (6). Thus, we cannot completely rule out that higher amounts of atelectrauma might have had a greater effect on lung inflammation. Also, the fact that SB did not result in tidal changes in poorly-aerated tissue, suggest that bronchiolotrauma (29) did not play a major role in our animals.

It is worth noting that SB activity, whether in combination with higher or lower PEEP, did not increase total lung inflammation. This is likely explained by our observation that regional transpulmonary pressures from ventral to dorsal-caudal regions were comparable between mechanical ventilation with and without SB activity at the same level of PEEP. Previous studies have shown conflicting data regarding the potential of SB to worsen lung injury. In rabbits, SB activity exacerbated lung damage compared to controlled mechanical ventilation during artificially high respiratory drive (30), and also in severe but not mild to moderate ARDS (4). Based on this observation, authors suggested that the potential of SB activity to cause lung injury depends on the underlying severity of lung damage (4). We hypothesized that not only the degree of lung injury, but also the ventilatory settings, especially the levels of transpulmonary pressure at end-expiration, determines whether SB activity may be injurious or not (5). In accordance to this hypothesis, it was demonstrated that in severe experimental ARDS, a PEEP level titrated to keep lungs open according to electrical impedance tomography was associated with no pendelluft and less tidal recruitment, two potential mechanisms of VILI (6). In the present study, we observed that during SB activity with lower, but not higher PEEP, the level of spontaneous efforts correlated with inflammation in dorsal areas, even when peak transpulmonary pressures in dependent regions are significantly lower than previously reported (7). These findings seem to confirm that stabilization of lung units at end-expiration might be important also in mild to moderate ARDS in the presence of SB activity (5), even in the absence of pendelluft. We cannot completely rule out the possibility that a reduced respiratory drive and improved electromechanical coupling contributed to less regional inflammation at higher PEEP.

Potential implications of the findings

Mechanical ventilation modes that permit or support SB activity are used in patients with mild to moderate ARDS (8), and at risk for ARDS (31). The present findings suggest that even in patients with non-severe lung injury, avoidance of instability of lung units at end-expiration might be important within a protective ventilation strategy that allows SB activity. Such stability could be achieved by the use of PEEP levels able to maintain a positive transpulmonary pressure in dorsal-caudal, i.e. the most dependent, lung zones. Further studies are necessary to identify which tools available at the bedside could be helpful to accomplish this task, for example esophageal manometry, electrical impedance tomography and lung ultrasound.

Limitations

The present study knows different limitations. First, although we used a double-hit model of lung injury, the full picture of clinical ARDS in patients was likely not reproduced. Second, animals were placed in supine position, increasing the pleural gradients of transpulmonary pressures. Third, we used a measure of regional metabolic activity as a surrogate of VILI. Different studies have shown that, in acutely injured lungs, the [¹⁸F]FDG uptake is a reliable marker of neutrophilic inflammation (24, 25, 32–35). Fourth, we used the APRV mode for mechanical ventilation, which allows but not actively assist SB activity. Also, this ventilation mode is associated with relatively high airway pressures and need for fluids. Since the mode of mechanical ventilation seems to affect the potential of injury due to SB activity (36), our findings cannot be directly extrapolated to other ventilatory strategies.

Conclusions

In this experimental model of mild to moderate ARDS, PEEP levels able to stabilize dependent lung regions at end-expiration reduce lung inflammation during mechanical ventilation with and without SB activity. Even in non-severe ARDS, SB activity has the potential to increase regional inflammation in dorsal zones when inappropriate low PEEP is used.