Endomicroscopic analysis of time- and pressure-dependent area of subpleural alveoli in mechanically ventilated rats

Endomicroscopic analysis of time- and pressure-dependent area of subpleural alveoli in mechanically ventilated rats

Respiratory Physiology & Neurobiology 203 (2014) 1–8 Contents lists available at ScienceDirect Respiratory Physiology & Neurobiology journal homepag...

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Respiratory Physiology & Neurobiology 203 (2014) 1–8

Contents lists available at ScienceDirect

Respiratory Physiology & Neurobiology journal homepage: www.elsevier.com/locate/resphysiol

Endomicroscopic analysis of time- and pressure-dependent area of subpleural alveoli in mechanically ventilated rats Hanna Runck a,∗ , David Schwenninger a , Jörg Haberstroh b , Josef Guttmann a a b

Department of Anesthesiology and Intensive Care Medicine, Division of Experimental Anesthesiology, University Medical Center Freiburg, Germany Experimental Surgery, CEMT, University Medical Center Freiburg, Germany

a r t i c l e

i n f o

Article history: Accepted 4 August 2014 Available online 20 August 2014 Keywords: Recruitment Compliance Endoscopy Alveolar mechanics Respiratory mechanics

a b s t r a c t We investigated the effects of recruitment maneuvers on subpleural alveolar area in healthy rats. 36 mechanically ventilated rats were allocated to either ZEEP-group or PEEP – 5 cmH2 O – group. The subpleural alveoli were observed using a transthoracal endoscopic imaging technique. Two consecutive low-flow maneuvers up to 30 cmH2 O peak pressure each were performed, interrupted by 5 s plateau phases at four different pressure levels. Alveolar area change at maneuver peak pressures and during the plateau phases was calculated and respiratory system compliance before and after the maneuvers was analyzed. In both groups alveolar area at the second peak of the maneuver did not differ significantly compared to the first peak. During the plateau phases there was a slight increase in alveolar area. After the maneuvers, compliance increased by 30% in ZEEP group and 20% in PEEP group. We conclude that the volume insufflated by the low-flow recruitment maneuver is distributed to deeper but not to subpleural lung regions. © 2014 Elsevier B.V. All rights reserved.

1. Introduction A lot of effort has been made to investigate respiratory mechanics under mechanical ventilation and to link global pulmonary mechanics with the status of alveolar recruitment (DiRocco et al., 2007; Gatto and Fluck, 2004; Schiller et al., 2003). Hence the relationship between global and local respiratory mechanics came into the focus of interest. In the severely injured lung, alveoli may open and collapse with almost every breath, leading to an exaggeration of lung injury during necessary mechanical ventilation. The reason is transfer of too high mechanical energy from the ventilator to the lung’s alveolar tissue. The mechanical behavior of alveoli in the mechanically ventilated healthy lung is largely unexplained. Open alveoli are essential for an efficient gas exchange. From the lack of knowledge about dynamic alveolar mechanics follows that the mechanism

∗ Corresponding author at: Department of Anesthesiology and Intensive Care Medicine, Division of Experimental Anesthesiology, University Medical Center Freiburg, Universitätsklinikum Freiburg, Hugstetter Strasse 55, 79106 Freiburg, Germany. Tel.: +49 761 27023320. E-mail addresses: [email protected] (H. Runck), [email protected] (J. Haberstroh). http://dx.doi.org/10.1016/j.resp.2014.08.010 1569-9048/© 2014 Elsevier B.V. All rights reserved.

of intrapulmonary volume changes during tidal ventilation is not clear. Different mechanisms are being discussed, such as distension and unfolding of the alveolar walls, opening and closing of single alveoli or volume changes of the airways (Hajari et al., 2012; Nieman, 2012; Smaldone and Mitzner, 2012). Supposedly more than one of these mechanisms is involved at the same time. Unlike in the injured lung, opening and closing of single alveoli does probably not contribute significantly to volume changes in the healthy lung (Mertens et al., 2009). However, there are still mechanisms that can contribute to injury in healthy lungs during mechanical ventilation. While unfolding of alveolar walls happens without a rise in mechanical wall tension at normal tidal breathing, further increase in pulmonary gas volume may lead to an increase in strain of the alveolar walls, which is likely a main cause for ventilator associated lung injury (Gattinoni et al., 2003). Because of the inconsistent results of past studies, further knowledge of alveolar mechanical behavior is important to estimate its dependence on ventilator strategies and is a prerequisite to developing numerical models of the lung which can help to improve lung-protective ventilation strategies. Recently, several methods to visualize alveoli have been established. Some of them use open chest preparations (Carney et al., 1999; Pavone et al., 2007), others lung window preparations, for

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example in combination with dark-field intravital microscopy and optical coherence tomography (Mertens et al., 2009). For analysis of intravital properties of subpleural alveoli we introduced a method based on endoscopic microscopy, allowing direct optical analysis of subpleural alveoli via a minimally opened thorax (Schwenninger et al., 2010). It is well known that respiratory system compliance increases with pressure or volume respectively up to the range of pulmonary gas volume where overdistension occurs (Gattinoni and Pesenti, 2005; Grasso et al., 2004; Schumann et al., 2011). In the present study we wanted to investigate if in the healthy mechanically ventilated lung an intrapulmonary volume increase (and lung recruitment respectively) comes along with an increase in subpleural alveolar area. To differentiate which factor, pressure or time, is more significant for imposing stress and strain on alveolar walls in the healthy lung, we investigated the dynamic compliance within small volume portions of the tidal volume and the alveolar area within small time-windows during 5-s-periods of relatively low static pulmonary pressure. 2. Methods The experiments were approved by the review board for the care of animal subjects of the government executive (Regierungspräsidium, Freiburg, Germany, G-12-078) and were carried out in accordance with the German law for animal protection and in compliance with the animal care guidelines of the European Community (86/609/EC). 2.1. Protocol 36 male Wistar rats (Charles River, Sulzfeld, Germany) with an average body weight of 411 ± 42 g were anesthetized with an intraperitoneal injection of 100 mg/kg ketamine (Ketavet® , Pfizer, Karlsruhe, Germany) plus 0.2 mg/kg medetomidine (Domitor® , Pfizer, Karlsruhe, Germany). A venous catheter was placed in a dorsal pedal vein for volume resuscitation (3 ml/h 0.9% NaCl). The animals then were tracheotomised and tracheally intubated. For measurement of arterial blood pressure and for blood gas sampling (i-STAT portable clinical analyzer, Heska Corporation, Loveland, CO), a catheter (Portex Non Sterile Polythene Tubing, 0.58 mm ID, 0.96 mm OD, SIMS Portex Ltd., Kent, UK) was placed in the arteria carotis communis. Subsequently, volume-controlled, pressure-limited ventilation was applied via a small animal ventilator (FlexiVent, Scireq, Montreal, Canada). Ventilation was started with 70 breaths per minute and a tidal volume of 10 ml/kg bodyweight. FiO2 was 1.0 and positive end-expiratory pressure was set to one of the randomized levels 0 or 5 cmH2 O. Inspiratory and expiratory gas flow rates were measured via two separate flow sensors (Fleisch pneumotachograph 000, Dr. Fenyves and Gut GmbH, Hechingen, Germany). Pressure and flow values were recorded with a sampling rate of 500 Hz using custom software. For introduction of the endoscope, the intercostal space between the fifth and sixth rib was opened dorsally at the left side of the thorax and a trocar for guiding the endoscopic system was inserted. This trocar was anchored inside the thorax between the ribs and fixed with a screw nut from outside the thorax. The animal was then placed into supine position, and the endoscopic system was inserted through the trocar until its tip touched the surface of the lung (Fig. 1). In both groups, PEEP 5 cmH2 O and ZEEP group, following a 10 min period for hemodynamic and respiratory stabilization, measurement maneuvers to analyze mechanics of subpleural alveoli and respiratory mechanics of the whole lung were performed every 30 min. O2 partial pressure (paO2 ) was analyzed before and after

Fig. 1. Schematic view of the experimental setup of the endoscopic in vivo recording of subpleural alveoli in the rat model. The lungs are ventilated via tracheotomy tube by a small animal ventilator. Inspiratory (V in) and expiratory flow (V ex) are measured separately. Airway pressure (P) is measured at the proximal end of the endotracheal tube (ET). The endoscope is introduced through a tube (T) that is mechanically fixed with a locknut (N) between two ribs (R). The endoscope has channels for pressure measurement in the field of view (not displayed) and for fluid flushing (FI) and fluid removal (FO), and a connector for a fiber-optic light guide (LG).

each respiratory maneuver and the mean arterial pressure was noted. At the end of the protocol, after a total experimental time of 120 min, the rat was killed by exsanguination. 2.2. Measurement maneuvers A “double low-flow-manoeuvre” was programmed in the FlexiVent software, consisting of two consecutive pressure controlled low-flow-maneuvers with a peak pressure of 30 cmH2 O, interrupted by a 5 s plateau phase at different pressures (2, 4, 8 and 12 cmH2 O) (Fig. 2). The same maneuvers were conducted in both groups. In the FlexiVent system ventilation is driven by a piston pump connected to a pressure transducer, measuring the absolute pressure applied and controlling the piston position. The expiratory valve was closed during the maneuver, making plateau pressures lower than PEEP possible. The maneuvers were performed every 30 min in a randomized order. 2.3. Endoscopic system The endoscopic system consists of a rigid endoscope (Schölly Fiberoptic GmbH, Denzlingen, Germany) inserted in two concentric trocars (6.5 mm o.d.). The system was designed to guide a controlled fluidic flow from the outer toward the inner trocar to create a defined negative pressure at its tip (Fig. 1). Images were recorded using a charge-coupled device camera (UI-5550HEC-HQ, iDS, Obersulm, Germany) connected to the eyepiece of the endoscope. The pressure in the endoscope’s field of view was adjusted by controlled flushing and suction through the endoscope so that subpleural

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Fig. 2. Schematic airway pressure-time diagram of the double low-flow-maneuver with 30 cmH2 O peak pressure and a 5-s-plateau in between. Gray boxes with black X-es mark points at which alveolar area was analyzed.

alveoli stayed in focus during ventilation (average pressure −5 cmH2 O). Suction pressure was maintained at a constant level throughout the measurement maneuvers. 2.4. Data analysis 2.4.1. Alveolar area Alveolar area at the three frames around peak pressures of the maneuver and of all 50 frames during the plateau levels was analyzed (Fig. 2) from the recorded videos. 10 alveoli with clear outlines that were trackable throughout these frames were chosen for further analysis. The alveolar outline of these 10 alveoli was marked manually frame by frame (Fig. 3). Then, the area of the marked polygon was used as alveolar area. Mean of the area of the 10 marked alveoli was calculated for each frame. Values for the 3 frames at each peak pressure and for 5 consecutive frames respectively during the plateau phases were averaged to compensate for variations due to cardiogenic oscillations visible in the videos and inaccuracies caused by manual marking of the alveolar outlines. This resulted in

one value for each low-flow-peak and 10 values in the course of the plateau phases (one value for every 0.5 s of duration). To compensate for variations in zoom factor that resulted from focusing on the alveoli, changes in alveolar area were calculated as percentage, not as absolute value. For plateau phases, the mean value of the analyzed frames was taken as the 100% reference. For peak pressures, mean area at the first peak was taken as 100% reference. 2.4.2. Respiratory system compliance Respiratory system compliance (Crs ) was determined as the ratio between the expiratory tidal volume and the difference of end-inspiratory and end-expiratory alveolar pressure (two-point compliance). Dynamic volume-dependent respiratory system compliance (Crsdyn ) was determined using the gliding slice method (Schumann et al., 2009) which is a modification of the slice method (Guttmann et al., 1994). Each tidal pressure-volume loop is divided into adjoining volume portions (slices) of equal size. In the gliding slice method the number of slices is arbitrary and the slices can overlap. For each slice, compliance (C) is calculated via multiple linear regression analysis using the equation of motion: P = P0 +

1 · V + R · V˙ C

where P is the tracheal pressure (difference between airway pressure and the pressure drop across the endotracheal tube), P0 the dynamic pressure base, C the compliance, V the volume, V˙ the flow rate and R the resistance. From this analysis, the intratidal course of the compliance can be obtained by drawing the slice-compliance values over the corresponding volume portions. Crs and Crsdyn were determined during normal tidal ventilation breaths before and after the double low-flow-maneuver. For quantification of recruitment in the global pulmonary scale, Crs -values before the respiratory maneuver were compared with Crs -values after the maneuver. 2.5. Statistical methods

Fig. 3. Endoscopic frame showing subpleural alveoli with three individually marked alveoli. The alveoli reveal different areas which were related to a relative area scale (100% being the mean of the measured areas); the relative areas of the marked alveoli are 80% in alveolus 1, 152% in alveolus 2 and 84% in alveolus 3.

Statistical analysis was performed using paired t-tests for comparison of compliance values and peak pressure alveolar area. For alveolar area during plateau pressures linear regression analysis was performed, including ANOVA.

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Table 1 Change in alveolar area at the second peak pressure of the double low-flowmaneuvers (with different plateau pressures) in ZEEP and PEEP 5 group. Alveolar area at the first peak was normalized to 100%. Plateau pressure

% Second peak ZEEP

SE

% Second peak PEEP 5

SE

2 cmH2 O 4 cmH2 O 8 cmH2 O 12 cmH2 O

101.9 106.1 97.7 98.1

2.5 3.3 0.9 3.7

103.3* 106.0 98.6 101.6

0.9 4.0 2.9 1.8

*

3. Results Some of the recorded videos had to be excluded from analysis because of blurriness or too much movement of the lungs during the endoscopic recordings, so that the outlined alveoli could not be traced through the frames. One animal died at the start of the experiment. 3.1. Maneuver peaks Analysis of the alveolar area revealed that alveolar size at the second peak of the double low-flow-maneuver does not differ significantly compared to the first peak in all but one plateau pressure (2 cmH2 O) in the PEEP 5 group (Table 1). 3.2. Plateau pressures Linear regression showed that during the plateau phases there is a slight increase in alveolar area. The effect is more distinct in the ZEEP group (Fig. 4). 3.3. Compliance Paired t-test revealed that after the maneuver two-point compliance increased significantly to a mean of 137.6% in ZEEP group and 119.9% in PEEP 5 group (all p < 0.05) (Table 2). Results for volume-dependent Crsdyn are shown in Fig. 5 (a) and (b). In both groups intratidal compliance increased after recruitment. While in ZEEP group compliance-volume course changed from a concave shape before recruitment to an inclining shape after recruitment, compliance changed from a declining shape before recruitment to an almost linear shape after recruitment in PEEP group (Fig. 5a and b). Table 2 Respiratory system compliance increase after the different double low-flowmaneuvers in ZEEP and PEEP 5 group. Compliance before the maneuvers was normalized to 100%. Plateau pressure

Compliance increase after maneuver (%) ZEEP

SE

Compliance increase after maneuver (%) PEEP 5

SE

2 cmH2 O 4 cmH2 O 8 cmH2 O 12 cmH2 O

134.7** 142.8** 132.3* 141.1**

8.3 4.5 9.7 4.2

122.8** 119.4** 118.2** 119.2**

3.0 1.7 2.4 2.6

*

p < 0.05. p < 0.001.

Plateau pressure

PaO2 increase after maneuver (%) ZEEP

SE

PaO2 increase after maneuver (%) PEEP 5

SE

2 cmH2 O 4 cmH2 O 8 cmH2 O 12 cmH2 O

111.3* 104.6 107.9 106.4

2.9 4.0 4.5 3.0

104.0 100.8 100.0 106.7

2.5 2.6 3.5 4.0

*

p < 0.05.

To analyze the relation between the increase in alveolar area and increase in Crs after recruitment maneuvers, linear correlation of these two parameters was calculated. All data are presented as mean ± SD, unless indicated otherwise. Statistics software (IBM SPSS Statistics 19, IBM Corporation, Armonk, NY) was used for all statistical analyses.

**

Table 3 paO2 increase after the different double low-flow-maneuvers in ZEEP and PEEP 5 group. paO2 before the maneuvers was normalized to 100%.

p < 0.05.

Correlation between increase in alveolar area and increase in Crs was r = 0.5. 3.4. Oxygenation paO2 slightly increased in ZEEP group after the maneuvers, but the increase was only significant at a plateau level of 2 cmH2 O (p < 0.05). In PEEP 5 group, increase in paO2 was not significant at all plateau levels (Table 3). 4. Discussion We investigated subpleural alveolar area in mechanically ventilated healthy rats with closed chest. By manual tracking of alveolar contours we measured the average change in alveolar area during a specially created double-low-flow recruitment maneuver with four different levels of plateau pressure between the low-flow phases. The main finding of this study is that repeated recruitment maneuvers do not lead to further significant distension and hence recruitment of subpleural alveoli in the healthy rat lung, but to an increase in respiratory system compliance. Nevertheless, there is a slight increase in alveolar area during plateau pressure phases suggesting alveolar expansion that is timedependent. Although there is no obvious recruitment manifesting at the lung surface, respiratory system compliance increased significantly during the recruitment maneuvers. In ZEEP group, intratidal compliance-volume course changes from concave shape before recruitment to ascending shape after recruitment, while compliance after recruitment is overall higher. Ascending compliance courses indicate intratidal opening of atelectasis. Due to the higher compliance and pressure limited ventilation, the applied tidal volume is higher after recruitment maneuvers (pressure limit was adapted to match a tidal volume of 10 ml/kg shortly after, but not immediately after the maneuvers). The high tidal volume is very likely to open up previously atelectatic regions that are not kept open by ZEEP. In contrast, in PEEP 5 cmH2 O group, before recruitment maneuvers, compliance-volume courses are descending, indicating overdistension. However, after recruitment, they change to a less descending, almost linear course, indicating that recruited areas are kept open by the applied PEEP. There is some overall increase in compliance, but it is less pronounced than in ZEEP group. Regarding the poor correlation of increase in subpleural alveolar area and Crs after recruitment maneuvers (r = 0.5) we can probably state that the pressure-dependent increase in pulmonary gas volume which is responsible for the compliance increase does not significantly affect the subpleural alveoli but very likely the applied volume is distributed to large degrees in more central regions of the lung. Alveolar microscopy has been used in multiple forms to investigate alveolar mechanics in healthy as well as in injured lungs (Carney et al., 1999; Daly et al., 1975; Mertens et al., 2009; Namati et al., 2013). The observations made concerning the mechanisms of alveolar recruitment and derecruitment during lung volume change are diverging. In a study with healthy mongrel dogs, Carney

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Fig. 4. Alveolar area during 5-s-plateau phases at different plateau pressures (PP) 2, 4, 8 and 12 cmH2 O. Values are given every 500 ms (black dots). Upper part: ZEEP group, lower part: PEEP 5 group. Red: regression line, B = slope of linear regression line, R = R-value, SD = Root-MSE, P = p-value. Mean alveolar area was standardized to 100%.

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Fig. 5. (a) and (b): Red curves: intratidal compliance-volume profiles during tidal ventilation before and after double low-flow-maneuvers with different plateau phases (PP 2, 4, 8 and 12 cmH2 O) in ZEEP (a) and PEEP 5 cmH2 O (b) group. The curves are averaged for all animals included in each group. To standardize for volume (due to variations in total tidal volume caused by differences in body weight), the x-axis is presented in slice numbers and not absolute volume. Horizontal blue line marks level of two-point compliance.

and coworkers found that with an inflation from opening volume (described as the volume when alveoli first recruit), change in alveolar volume was minimal, but the number of alveoli increased significantly (Carney et al., 1999). In contrast Pavone and coworkers observed in a study in rats that normal alveoli are very stable at tidal ventilation (Pavone et al., 2007), which supports the results of our study. Only few newer investigations reported heterogeneity of alveolar expansion. For example Namati et al. found that some alveolar air spaces expand and contract during ventilation, while others maintain minimal volume change (Namati et al., 2013). While Allen et al. focus on lasting effects of recruitment maneuvers and how they have to be applied in lung injury (Allen et al., 2002) and Albert and coworkers studied timely effects of alveolar expansion in the isolated injured lungs (Albert et al., 2009), we investigated in the present study if normal alveoli further expand during repeated recruitment maneuvers and how they behave when constant pressure is applied, latter giving information about the time-dependence of alveolar recruitment even under completely static, i.e. no-flow conditions. What the above mentioned studies have in common with respect to the methods used is that the thorax has to be opened large scale to apply the microscope and are thus extensively invasive. Consequently the impact of these invasive methods on

respiratory system mechanics may not be negligible and therefore the results may be biased. In previous experiments the invasiveness of our method has been evaluated (Schwenninger et al., 2011), revealing that while providing an almost air-sealed system with a minimally open thorax, respiratory system mechanics were only minimally affected. To keep the lung in place, i.e. within the optical focus during mechanical ventilation, a small suction pressure of −3 to −5 cmH2 O was applied at the tip of the endoscope with only slight influence on the subpleural alveolar mechanics in the field of view (Schwenninger et al., 2011). So it can be assumed that alveoli in this area are behaving in a physiological way. Because cardiogenic oscillations transmitted from the beating heart to the lung tissue also led to optical variations in alveolar area mainly at the plateau phases, alveolar area of single frames over a time span of 5 frames was averaged to compensate for these optical faults. To compensate for variations in zoom factor that resulted from focusing on the alveoli, we calculated changes in alveolar area as percentage, not as absolute area. In our previous experiments, insertion of the endoscope led to an initial drop in compliance in healthy lungs. It is possible that the drop resulted from air streaming into the thoracic cavity when the chest was opened for insertion of the endoscope. To

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meet these concerns and to equalize volume history in all animals, we excluded the first recruitment maneuver from further analysis. Expansion of the lungs during recruitment and suction at the tip of the endoscope supports removal from remaining air from the thoracic cavity, restoring physiological conditions. Albert et al. showed in their experiments, that alveolar recruitment in the injured rat lung is time-dependent, assuming recruitment to be a result of increase in the number of alveoli (Albert et al., 2009). This was not the case in our healthy lung experiments, where opening of previously closed subpleural alveoli was not observed. But it has to be considered that we used much lower plateau pressures and a considerably shorter plateau phase compared to the setting of Albert et al. During the experiments performed by Albert et al. the biggest enlargement in alveolar area occurred during the first 5 s of the plateau phase. After that the amount of alveolar recruitment was similar to the overall amount observed in our experiments. The relatively small amount of alveolar recruitment during plateau phases in our experiments is probably due to two facts. First, the pressure applied during the plateau phases was considerably lower in our investigation compared to the study of Albert et al.: we applied a maximum plateau pressure of 12 cmH2 O, compared to 30 cmH2 O in their experiments. Second, the plateau phase in our experiments was preceded by a low-flow-maneuver with a peak pressure of 30 cmH2 O, so the lung was already recruited at the time the plateau phase started. Although alveolar area did not differ between the peak pressure points of the two consecutive low-flow-maneuvers there was still a marked increase of compliance and a slight improvement in oxygenation, indicating lung recruitment that was not visible in our fields of view. So lung recruitment could have simply taken place in another, not observed lung surface area or somewhere in central regions of the lungs. Previous experiments using alveolar microscopy support the view that volume increase during lung inflation occurs to large parts in terminal airways like alveolar ducts (Daly et al., 1975; Storey and Staub, 1962). A recent study comparing dead space in mechanically ventilated and spontaneously breathing rats showed that dead space is increased during mechanical ventilation (Dassow et al., 2013), indicating distension of conducting airways, which also supports our hypothesis. During pressure decline of the descending part of the low-flow maneuvers some derecruitment of the previously recruited lung might have happened, maybe even through absorption of oxygen from the alveoli, so that the slight increase in alveolar area that could be observed during the following plateau phase may have resulted from rerecruitment. It is noticeable, that in both groups, ZEEP and PEEP 5, at a plateau pressure of 4 cmH2 O the alveoli remain the most stable in size. This plateau pressure level seems to be closest to a critical opening/closing or “resting” pressure (Salmon et al., 1981) of our observed subpleural alveoli, keeping the alveoli stable without letting them collapse or expand further. Also remarkable is the observation that alveolar area increased at a plateau pressure as low as 2 cmH2 O, at which the amount of area increase was much bigger in the ZEEP group. 2 cmH2 O was the lowest applied plateau pressure in the ZEEP group as well as in the PEEP 5 group. One reasonable explanation is that there is some redistribution of volume inside the lung. The pressure is not high enough to provide lung recruitment, but time would also be needed to allow reallocation of volume from “high pressure zones” which are probably located in deeper lung regions toward “lower pressure zones” in the lung periphery, very similar to the pendelluft-phenomenon (Bates et al., 1985). This would explain the observed enlargement of subpleural alveoli at a comparatively low plateau pressure. One could speculate about the distribution of respiratory gas within the lungs: Our observation in subpleural alveoli of

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healthy rat lungs could be generally valid for all alveoli of the lung parenchyma. This would indicate that a recruitment maneuver does not influence the alveolar volume and the gas volume insufflated by the maneuver remains within the airways. But it is known from early investigations about the geometry of peripheral airways that terminal airways begin branching deep inside the lungs and that acini are located in a broad range of generations of airway branchings, especially in the mammalian lung (Sznitman, 2009; Weibel et al., 2005). So the subpleural alveoli probably represent that part of the acini that is farthest away from the conducting airways. Further investigation of central lung regions would be necessary to illuminate gas volume distribution and redistribution in the lungs. An alternative mechanism of alveolar recruitment would be the increase of the number of alveoli participating in gas exchange. Several authors have observed that phenomenon in injured, but also in healthy lungs (Carney et al., 1999). In our study we investigated subpleural alveoli of healthy lungs after a recruitment maneuver and come to the conclusion that any further recruitment if occurred at all must have happened in more central lung regions, since the subpleural alveoli remained comparatively stable in size despite an increase in respiratory system compliance after recruitment. Although we did not observe an increase in the number of alveoli in the healthy lungs, we nevertheless have observed an increase of number of alveoli in surfactant depleted lungs before (Schwenninger, 2010). It is well known that in injured lungs such as ALI or ARDS recruited and overinflated lung regions coexist at the same time (Yang et al., 2014; Zick et al., 2013). Even in the healthy lung during mechanical ventilation, cyclic lung recruitment and derecruitment occur with low PEEP and high tidal volume, mainly in the dorsocaudal lung regions. The effect is ameliorated when PEEP is applied (Sinclair et al., 2010; Zick et al., 2013). The plateau phases in between the two low-flow maneuvers in our experiments of course also resemble different applied PEEP levels without the impact of tidal ventilation dynamics on top. The lack of flow during the static plateau phases emphasizes the importance of the factors time and pressure for alveolar expansion. In our experiments the increase in alveolar area during the plateau phases was more distinct at higher plateau pressure levels, but was overall lower in PEEP 5 group, especially at a plateau pressure of 12 cmH2 O. This indicates a more even distribution of volume in the lungs of animals ventilated with PEEP compared to a probably uneven distribution in lungs ventilated with ZEEP. It would have been interesting to see if there is any change in alveolar area between the peak and plateau pressures. But because of some movement of the lung tissue during inflation and deflation, it was not possible to trace individual alveoli throughout the whole maneuver. It was only possible to compare the area at the maneuver peaks and to observe the change in area during the plateau phases. Another limitation of our study is that our method does not allow for three-dimensional measuring of alveolar volume, but only for two-dimensional measuring of subpleural alveolar area. Mertens et al. nevertheless showed that there is a correlation between volume and area in subpleural alveoli of mice (Mertens et al., 2009), allowing us to some extent to interpret increase in subpleural alveolar area as volume gain. From our study we conclude that a double low-flow recruitment maneuver with different levels of plateau pressure in between does not significantly increase the area of subpleural alveoli in healthy rat lungs. But at the same time the respiratory system compliance increased by more than 30% in the ZEEP group and by 20% in the group mechanically ventilated with a PEEP of 5 cmH2 O. We assume that the volume insufflated by the low-flow recruitment maneuver is distributed to deeper lung regions not visible by the endoscopic

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