Journal of Surgical Research 166, e71–e81 (2011)doi:10.1016/j.jss.2010.10.022

Comparison of ‘‘Open Lung’’ Modes with Low Tidal Volumes

in a Porcine Lung Injury Model

Scott Albert, M.D.,* Brian D. Kubiak, M.D.,* Christopher J. Vieau, B.A.,* Shreyas K. Roy, M.D.,*,1

Joseph DiRocco, M.D.,* Louis A. Gatto, Ph.D.,† Jennifer L. Young, Ph.D.,‡ Sudipta Tripathi, Ph.D.,*Girish Trikha, M.D.,§ Carlos Lopez, M.D.,k and Gary F. Nieman, B.A.*

*Department of Surgery, Upstate Medical University, Syracuse, New York; †Department of Biological Sciences, SUNY Cortland,Cortland,NewYork; ‡Department of Pediatrics, University of Rochester, Rochester, NewYork; §Division of Pulmonary andCritical Care,Upstate Medical University, Syracuse, New York; and kDepartment of Anesthesiology, Upstate Medical University, Syracuse, New York

Submitted for publication July 26, 2010

Background. Ventilator strategies that maintain an‘‘open lung’’ have shown promise in treating hypox-emic patients. We compared three ‘‘open lung’’ strate-gies with standard of care low tidal volumeventilation and hypothesized that each would dimin-ish physiologic and histopathologic evidence of venti-lator induced lung injury (VILI).Materials andMethods. Acute lung injury (ALI) was

induced in 22 pigs via 5% Tween and 30-min of injuri-ous ventilation. Animals were separated into fourgroups: (1) low tidal volume ventilation (LowVt -6 mL/kg); (2) high-frequency oscillatory ventilation(HFOV); (3) airway pressure release ventilation(APRV); or (4) recruitment and decremental positive-end expiratory pressure (PEEP) titration (RMDOP)and followed for 6 h. Lung and hemodynamic functionwas assessed on the half-hour. Bronchoalveolar lavagefluid (BALF) was analyzed for cytokines. Lung tissuewas harvested for histologic analysis.Results. APRV andHFOV increased PaO2/FiO2 ratio

and improved ventilation. APRV reduced BALF TNF-aand IL-8. HFOV caused an increase in airway hemor-rhage. RMDOP decreased SvO2, increased PaCO2,with increased inflammation of lung tissue.Conclusion. None of the ‘‘open lung’’ techniques

were definitively superior to LowVt with respect toVILI; however, APRV oxygenated and ventilatedmore effectively and reduced cytokine concentrationcompared with LowVt with nearly indistinguishablehistopathology. These data suggest that APRV may beof potential benefit to critically ill patients but other

1 To whom correspondence and reprint requests should be ad-dressed at Department of Surgery, Upstate Medical University, 750East Adams Street, Syracuse, NY 13210. E-mail: roys@upstate.edu.

e71

‘‘open lung’’ strategies may exacerbate injury. � 2011

Elsevier Inc. All rights reserved.

Key Words: ventilator induced lung injury; porcine;acute lung injury; airway pressure release ventilation;recruitment maneuver; high frequency oscillatoryventilation.

INTRODUCTION

Mechanical ventilation is necessary to sustain lungfunction in critically ill patients of diverse etiologies,however improper mechanical ventilation may causeventilator induced lung injury (VILI), a mechanicaland inflammatory injury to the lung, and exacerbatethe underlying pulmonary pathophysiology. Continuedinsult and unchecked inflammation of the lung mayprogress to acute respiratory distress syndrome(ARDS), which carries an extremely high mortalityrate. An epidemiologic study by Rubenfeld and Her-ridge proposed that an estimated 17,000–43,000 livescould be saved per year if VILI were eliminated [1].

The current standard of care for mechanically venti-lated patients is low tidal volume ventilation (LowVt)with moderate positive-end expiratory pressure(PEEP) per the ARDSnet protocol. By recommendinga tidal volume of 6 mL/kg predicted body weight, thisstrategy is based on the assumption that low tidal vol-umes and plateau pressures below 30 cmH2O [2] pre-vent overdistension of heterogeneously injuredalveoli. In clinical trials and animal studies, the lowtidal volume approach has met with success in termsof a reduction inmarkers of inflammation and,most im-portantly, overall mortality compared with ventilation

0022-4804/$36.00� 2011 Elsevier Inc. All rights reserved.

JOURNAL OF SURGICAL RESEARCH: VOL. 166, NO. 1, MARCH 2011e72

with high tidal volumes [3, 4]. The ARDSnet clinicaltrial demonstrated that lung protective approachesimproved patient outcomes as shown by thesignificant reduction in mortality from 40% to 31%with the use of low tidal volume ventilation [2].

Despite the success of ARDSnet, alternative ventila-tion regimes have been proposed, claiming superior oxy-genation, ventilation, and lung protection [5–7]. Theterm ‘‘open-lung’’ has been coined as an umbrella forstrategies that promote and maintain full lung inflationby first increasing distending pressures to ‘‘recruit’’collapsed units, then maintaining adequate positive endexpiratory pressure (PEEP) to prevent alveolar collapse.In theory, open lung modes create a more homogenousdistribution of airflow, reducing the stress/strainassociated with cyclic alveolar opening and collapse.This study focuses on three open lung strategies: highfrequency oscillatory ventilation (HFOV), airwaypressure release ventilation (APRV), and a strategyutilizing aggressive recruitment maneuvers (RM) witha decremental titration of PEEP to determine theminimum PEEP necessary to prevent lung collapse thatwe have designated as the ‘‘optimal PEEP’’ (OP).

Small animal models have corroborated theoreticalreports by showing HFOV, APRV, and recruitment ma-neuvers to be beneficial to the injured lung [8–10],however translation to the clinical setting has beenlimited. Therefore, further investigation is required toelucidate the benefits and disadvantages betweendifferent ventilation strategies in clinically relevantmodels. In this large animal model of acute lung injury(ALI), we hypothesized that each open lungventilation mode (HFOV, APRV, and RMþOP) woulddiminish the physiologic and histologic evidence ofVILI compared with low tidal volume ventilation, thusdemonstrating that they offer superior lung protection.

METHODS

Vertebrate Animals

Experiments described in this study were performed in accordancewith the National Institutes of Health guidelines for the use of exper-imental animals in research. The Committee for the Humane Use ofAnimals at Upstate Medical University approved the experimentalprotocol (approval no. 040).

Surgical Preparation

Yorkshire pigs (n ¼ 22) were pretreated with glycopyrrolate (0.01mg/kg, intramuscular), telazol (tiletamine hydrochloride and zolaze-pam hydrochloride (5 mg/kg, intramuscular), and xylazine (2 mg/kg,intramuscular). A ketamine (3mg/1mL)/xylazine (0.003/1mL) contin-uous infusion via an infusion pump (3M model 3000) was used tomaintain anesthesia throughout the experiment. A Galileo ventilator(Hamilton Medical, Reno, NV) was used. Baseline ventilator settingswere as follows: conventional volume-controlled ventilation (CMV)with a tidal volume 12 cc/kg; PEEP 3 cmH20; inspiration-to-

expiration ratio 1:2, FiO2 of 21% and respiratory rate 15 breaths/min. A left carotid artery, left internal jugular, and left external jug-ular catheter were placed for hemodynamic monitoring, blood sam-pling, and administration of fluids. Blood gas analysis wasperformed (model ABL5’ Radiometer Inc., Copenhagen, Denmark)to ensure pulmonary health. A right internal jugular pediatricSwan-Ganz catheter was placed for cardiac output and filling pres-sures. An open tracheostomy was performed with placement of a 7.5French cuffed endotracheal tube. ECG monitoring, pulse oximetry,central venous pressure, pulmonary artery pressure, and arterialpressure were continuously monitored (CMS-2001; Agilent, SantaClara, CA, USA). A Foley catheter was placed through a cystotomyfor urine output monitoring.

Fluid Administration

Each pig received a 1 L lactated ringers fluid bolus during surgicalpreparation to maintain end-organ perfusion. A slow infusion2cc/kg/h wasmaintained throughout the experiment. Additional fluidboluses were given in response to hypotension (MAP < 65 mmHg).

Lung Injury

Pigs were placed in a right decubitus position for instillation of 5%Tween-20 in normal saline (1.5 cc/kg/lung) through a 2 mm i.d. cath-eter threaded down the endotracheal tube. During instillation, theventilator settings were changed to pressure controlled ventilation(PCV) with a Pcontrol 25 cm H2O, PEEP 10 cmH2O, and FiO2 1.0for 10 min. This was repeated with the pig in a left decubitus position.Immediately following Tween instillation, the pig was placed supinefor 30min of injurious ventilation (baseline settings, see above). Acutelung injury caused by Tween instillation has been characterized byprevious work in our laboratory [11].

Experimental Groups

Pigswere separated into four groups immediately following lung in-jury as follows. LowVt: (n ¼ 6, 24.4 6 2.7 kg) pigs were placed onvolume-controlled ventilation with a Vt of 6 mL/kg, PEEP14 cmH2O, and FiO2 of 0.8. Settings were adjusted according to theARDSnet sliding scale FiO2 and PEEP protocol [2]. Briefly, bloodgases were obtained every 30 min and the FiO2 and PEEP were ad-justed tomaintain a PaO2 between 55 and 80mmHg [2]. Plateau pres-sure (Pplat) was maintained <30 cmH2O. If Pplat >30cmH2O, thetidal volume was reduced by 1 cc/kg. If pH fell below 7.30, respiratoryrate was increased until pH > 7.30 or PaCO2 < 25 mmHg.

Recruitment Maneuver þ Optimal PEEP Group (RMþOP): (n ¼ 5,21.4 6 1.9 kg); this strategy was based on the method described byBorges et al. [12]. Briefly, an aggressive recruitment maneuver (RM)wasperformed followedbyadecrementalPEEPtrial tofind theamountof PEEP necessary to keep the newly recruited alveoli open. Fora detailed description of this procedure, please refer to Figure 1.

HFOV: (n¼ 5, 33.76 1.9 kg). Animalswere switched toHFOV (Sen-sormedics 3100B oscillator, San Diego, CA, USA) and a RM was per-formed by increasingmean airway pressure (Pmean) to 40 cmH2O for40 s with a FiO2 of 1.0. The initial oscillator settings were: Pmean 30cmH2O, amplitude (DP)¼ 50 cmH2O if pH> 7.25 orDP¼ 70 cmH2O ifpH < 7.25 cm H2O. Frequency ¼ 7 Hz (if animal < 25 kg) or 6 Hz (ifanimal> 25kg), percent inspiratory time 33%, and a power of 4.3. Ad-justments were made to DP (minimum DP of 3) to achieve a pH targetof 7.30 to 7.45.

APRV: (n ¼ 6, 23.17 6 0.5 kg). The ventilator was switched to theDuoPAP-Plus mode (Galileo Ventilator; Hamilton Medical, Reno,NV) with a FiO2 of 1.0 and baseline ventilator settings determinedby the adult recommended values: High pressure (Phigh)30 cmH2O, low pressure (Plow) 0 cmH2O, the time at high pressure(Thigh) set to 5 s, and the time at low pressure (Tlow) 0.2 s. A RMwas performed immediately after switching to APRV. Recruitment

FIG. 1. RMþOP protocol. Immediately following lung injury, thelung was recruited by increasing PEEP to 25 cmH2O for four minutesand an arterial blood gas was taken (arrows). If the PaO2 þ PaCO2

was less than 400 mmHg, the PEEP was increased by 5 cmH2O for2 min, then returned to 25 for 2 min, and an arterial blood gas taken.This was repeated until the PaO2 þ PaCO2 was greater then 400mmHg (asterisk arrow). The maximum PEEP used was termed the‘‘recruited’’ PEEP. After recruitment, a decremental PEEP trial wasperformed by lowering the PEEP in 2 cmH2O increments every2 min until the PaO2 þ PaCO2 was less than 380 mmHg (arrowhead).The PEEP 2 cmH2O greater than the PEEPwhere PaO2þPaCO2wasless than 380 mmHg was termed the ‘‘optimal’’ PEEP. Following thePEEP trial, the lung was re-recruited using the ‘‘recruited PEEP’’for 4 min and then the ventilator was set using low tidal volume(6mL/kg) with the ‘‘optimal’’ PEEP for the duration of the experiment.

ALBERT ET AL.: HFOV, APRV, AND RM+OP VERSUS LOWVT e73

was performed by increasing the Phigh to 40 cmH2O with a Thigh of40 s. After the recruitmentmaneuver was completed, the peak expira-tory flow rate (PEFR) was measured at Tlow. Expiratory flow was an-alyzed using the freeze function and the tracer on the Galileo. Tlowwas adjusted to resume CPAP phase before the lung fully exhaled(i.e., between 50% and 75% PEFR). Animals were allowed to sponta-neously breathe while on APRV.

Hemodynamic and Lung Function Measurements

Arterial and venous blood gases (ABG), heart rate (HR), mean arte-rial blood pressure (MAP), central venous pressure (CVP), cardiacoutput (CO), and urine output (UOP) were recorded at baseline (aftersurgical preparation), immediately post-injury (T0) and every 30 minfor 6 h (T360). CO was measured using a thermodilution technique(5F catheter, Edwards Lifesciences, Irvine, CA, USA) and recordedas the average of three measurements. Pulmonary parameters (respi-ratory rate (RR), peak airway pressure (Ppeak), mean airway pres-sure (Pmean), plateau pressure (Pplat), tidal volume (Vt), PEEP,static compliance (Cstat), were measured and calculated inline bythe Galileo ventilator at each 30 min time point.

Necropsy

Euthanasia was performed by barbiturate overdose (150 mg/kg).The heart and lungs were removed en bloc. The heart was then

removed and the bronchus to the right middle lobe was exposed, can-nulated with a small endotracheal tube, and the balloon was inflatedto secure the tube. The lobe was lavaged with 60 mL normal saline(three 20 mL injections) and collected. The bronchoalveolar lavagefluid (BALF) was centrifuged and frozen for measurement of inflam-matory mediators.

Wet/Dry Weight Ratio

Representative samples of lung tissue were minced, placed in pre-weighed tin dishes, weighed, and placed in an oven to dry. The disheswere weighed daily until there was no weight change for 24 h, atwhich time the tissue was determined to be completely dry and waterfree. The wet to dry weight ratio (W/D) was calculated by dividing thewet weight by the dry after subtracting the weight of the dishes.

Histology

The lung was immersed in formalin for a minimum of 48 h prior tohistologic sectioning. A constant airway pressure of 25 cmH2O wasused to fix the lung. Samples were obtained from the lower left lobeand were scored by a blinded histologist using a 0–4 scale, with 4 rep-resenting the most profound injury. Samples were evaluated for evi-dence of fibrinous deposits, red blood cells in the alveolar space(airspace hemorrhage), vessel congestion, alveolar wall thickness,and leukocyte infiltration. A detailed description of the histologicmethodology and lesion descriptions can be found in previous workby our lab [13].

BALF TNF-a and IL-8

Bronchoalveolar lavage fluid (BALF) levels of TNF-a and IL-8 weredetermined by porcine-specific enzyme linked immunosorbent assay(ELISA) kits (R andDSystems,Minneapolis,MN) using themanufac-turer’s specified protocol.

Apoptosis

A terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL) assay was used to measure the number of apoptotic cellsin the lung tissue on the surface of each histologic slice. Eight-micron sections were cut, de-parinized, and then stained using withChemiCon’s ApopTag Red in situ. The counts were made usinga CY3 filter at 203 magnification.

Statistics

Physiologic variables were analyzed following group assignmentusing a repeatedmeasures ANOVA (RMANOVA) to determine differ-ences between groups and within groups. To determine differencesat specific time points, Dunnet’s post-hoc test was performed. Gradedhistologic data was analyzed to determine a c2 value via a likelihoodratio calculation. Comparisons were limited to the aims of this study,which were to compare the respective open lung strategies to low tidalvolume ventilation, and were not extended to compare open lunggroups with each other. Analyses were performed on ver. 5.0.1.2 ofJMP (SAS Institute, Cary, NC). All data are presented as mean 6standard error mean. An a level of 0.05 or less was considered statis-tically significant.

RESULTS

There were no significant differences between groupsin any of the hemodynamic or respiratory parametersat baseline or immediately following lung injury. All

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animals developed a severe lung injury (PaO2/FiO2 ra-tio 151.8 6 25.5) following instillation of Tween-20 andnonprotective ventilation (Fig. 2A) regardless of groupassignment.

Respiratory Parameters

Average Vt was similar in the RMþOP and LowVtgroups (6.6 6 0.1 mL/kg versus 6.3 6 0.5 mL/kg) whilethe experimentally derived ‘‘optimal’’ PEEP in theRMþOP group was significantly greater (15.7 6 0.2cmH2O versus 9.0 6 0.4 cmH2O, P > 0.0001). AlthoughPEEP was significantly different in the RMþOP(Fig. 3C), there was no difference in static compliancecompared with the LowVt (Table 1). Tidal volume wassignificantly greater with APRV (15.4 6 0.3) than

FIG. 2. Blood gas analysis between LowVt (filled circles), APRV (fi(A) PaO2/FiO2: lung injury was uniform between all groups followinHFOV (P ¼ 0.0567 by group; 0.0060 by time*group), APRV (P ¼ 0.02oxygen saturation was significantly greater with HFOV (P ¼ 0.0118 byby time*group) thanLowVt. SvO2was significantly lower in the RMþOP(C) PaCO2: arterial CO2 content was effectively maintained at normagroup*time) and APRV (P ¼ 0.0012 by group; <0.0001 by groumarked hypercapnia. (D) Arterial pH: pH was significantly lower i(P < 0.0001 by time*group) group compared with LowVt. Data mean 6

with LowVt. Per protocol, the mean airway pressure(mPaw) was lowest in the LowVt, and maintained ata constant pressure of 30 cmH2O in the HFOV andAPRV groups; mPaw in the RMþOP group was signifi-cantly greater than LowVt (Fig. 3D). Following injury,the PaO2/FiO2 ratio improved significantly in all ofthe ‘‘open lung’’ groups (RMþOP, HFOV, APRV) com-pared with LowVt (Fig. 2A). Systemic oxygen utiliza-tion measured by SvO2 was significantly greater withAPRV and HFOV versus LowVt; with RMþOP it wassignificantly reduced (Fig. 2B). CO2 clearance was sig-nificantly improved in the HFOV and APRV groupscompared with LowVt, whereas the RMþOP displayedhypercapnia more severe than LowVt, albeit not statis-tically significant (Fig. 2C). These changes in PaCO2

were reflected in the arterial pH, which was in the

lled triangles), HFOV (open circles), and RMþOP (open triangles):g lung injury and subsequently improved with the application of46 by group) and RMþOP (P ¼ 0.0260 by group). (B) SvO2: venousgroup; 0.0229 by time*group) and APRV (P ¼ 0.0399 by group; 0.0019group over the duration of the experiment (P¼ 0.0278 by time*group).l levels with application of HFOV (P ¼ 0.029 by group; <0.0001 byp*time) whereas animals in the LowVt and RMþOP showedn the OP (P < 0.0001 by time*group) and greater in the APRVSEM.

FIG. 3. Physiologic data between LowVt (filled circles), APRV (filled triangles), HFOV (open circles), and RMþOP (open triangles).(A) Mean arterial pressure: all groups exhibited progressive decline in MAP over the course of these experiments. The differences in MAP be-tween APRV versus LowVt were statistically significant at the P ¼ 0.05 level; this is denoted by asterisks **. The differences in MAP betweenOP and LowVt were statistically significant at the 0.05 level. The differences in MAP between HFOV and LowVt were not statistically signi-ficant. (B) Fluids given, urine output, fluid balance – of the three experimental groups (APRV, HFOV, and OP) only the OP group was signi-ficantly different in fluid requirements and fluid balance compared with the LowVt Group. The OP group required significantly more fluidresuscitation in order tomaintainMAPand as a result hadmuchhigher fluid balance. (C) Peak end expiratory pressure (PEEPwasmeasurableonly in LowVt and OP groups over the course of the entire experiment; PEEP was significantly higher in the OP group than the LowVt group.For groups in which PEEP was not measured (HFOV, APRV), PEEP values were identical to LowVt at baseline and immediately post-injurytime points. (D) Mean airway pressure (mPaw): All experimental groups had significantly higher mPaw than the LowVt group.

ALBERT ET AL.: HFOV, APRV, AND RM+OP VERSUS LOWVT e75

normal range and significantly greater by time*treat-ment with APRV, while the RMþOP group showed sig-nificant acidosis compared with LowVt (Fig. 2D).Neither hemoglobin saturation nor expired minvolume showed significant differences between groups(Table 1).

Hemodynamics

Themean arterial pressure was greater in the LowVtcompared with all three open lung techniques; thesedifferences were significant in the RMþOP and theAPRV groups (Fig. 3A). Most notably, the RMþOPgroup developed hypotension < 65 mmHg at 6 h. Car-diac output was not significantly different betweengroups (Table 1). Since fluids were given in response

to hypotension, the RMþOP group received signifi-cantly more fluids than the LowVt group and retainedsignificantly more fluids, as urine output was similarin all groups (Fig. 3B). There were no significant differ-ences between LowVt and the experimental groups inlung wet:dry weight ratio (data not shown); thereforeit is unlikely that the increased fluid load in theRMþOP significantly impaired lung function by in-creasing edema.

BALF Cytokines

IL-8 and TNF-awere significantly lower in the APRVgroup compared with LowVt. There were no differencesin cytokine concentration between LowVt and the othertwo groups (Fig. 4).

TABLE 1

Hemodynamic and Respiratory Function

Group Pre-Injury Injury 90 180 270 360 P < 0.05

HR (b/min) LowVt 126 6 11 138 6 12 107 6 8 95 6 7 95 6 6 95 6 5APRV 135 6 16 120 6 5 98 6 9 92 6 16 96 6 13 95 6 12HFOV 120 6 15 107 6 5 82 6 7 89 6 13 76 6 3 81 6 7RMþOP 115 6 8 107 6 8 92 6 16 127 6 18 102 6 13 105 6 24

Hemoglobin saturation (%) LowVt 94.34 6 2.5 81.58 6 19.2 96.74 6 3.04 97.42 6 2.3 95.9 6 2.8 93.84 6 3.0APRV 96.83 6 2.0 94.33 6 4.6 100.0 6 0 99.0 6 2.4 99.0 6 2.40 97.67 6 5.7HFOV 98.23 6 1.16 94.52 6 5.71 99.93 6 0.1 99.93 6 0.1 99.93 6 0.1 99.93 6 0.1RMþOP 95.16 6 2.9 79.92 6 10.2 98.53 6 3.8 98.18 6 1.9 98.58 6 0.8 98.5 6 0.9

PAP (mmHg) LowVt 27.8 6 3.6 23.3 6 1.9 27.5 6 3.1 30.8 6 5.5 31.8 6 6.8 37.0 6 10.6APRV 35.0 6 4.5 29.8 6 2.4 30.2 6 1.3 35.3 6 2.6 38.2 6 3.4 39.7 6 3.8HFOV 26.8 6 5.3 22.6 6 1.2 27.8 6 1.6 29.2 6 404 30.4 6 3.7 34.0 6 6.7RMþOP 31.2 6 5.9 19.5 6 5.5 30.5 6 2.5 26.0 6 3.4 33.8 6 3.7 42.3 6 7.7

Cardiac output (L/min) LowVt 5.08 6 0.91 5.06 6 0.82 4.69 6 0.95 3.61 6 0.59 3.43 6 0.58 3.24 6 0.54APRV 4.66 6 0.66 4.60 6 0.64 3.32 6 0.32 2.40 6 0.35 2.67 6 0.53 2.77 6 0.49HFOV 5.38 6 0.94 5.04 6 1.08 3.06 6 0.69 2.20 6 0.46 2.75 6 0.76 1.79 6 0.46RMþOP 4.44 6 0.52 4.99 6 0.55 3.74 6 0.24 2.30 6 0.39 2.31 6 0.43 2.48 6 0.52

Vt (mL/kg) Low Vt 12.1 6 0.25 10.6 6 1.3 6.3 6 0.6 6.2 6 0.17 6.3 6 0.13 6.3 6 0.14APRV 13.4 6 1.04 12.1 6 0.32 15.8 6 0.99 15.6 6 0.53 14.9 6 0.71 15.2 6 0.70 x

RMþOP 11.8 6 0.27 12.1 6 0.31 6.8 6 0.6 6.3 6 0.35 6.9 6 0.43 6.7 6 0.24Cstat (mL/cmH2O)* LowVt 20.2 6 1.6 11.0 6 0.7 12.3 6 0.8 16.2 6 3.3 12.3 6 1.2 11.6 6 1.4

APRV 20.2 6 1.6 11.6 6 0.6 14.1 6 1.3 14.1 6 1.8 14.6 6 1.5 15.6 6 1.8RMþOP 13.6 6 0.5 10.3 6 1.1 11.6 6 0.4 10.2 6 0.7 11.9 6 1.8 13.3 6 3.3

EMV (L/min) LowVt 3.9 6 0.2 3.75 6 0.2 4.8 6 0.6 5.2 6 0.6 5.3 6 0.5 5.5 6 0.5APRV 4.3 6 0.28 4.9 6 0.38 4.4 6 0.2 4.1 6 0.2 4.2 6 0.1 4.2 6 0.1RMþOP 3.4 6 0.3 3.4 6 0.1 4.7 6 0.4 4.7 6 0.3 4.8 6 0.3 4.5 6 0.3

Data mean 6 SEM.HR ¼ heart rate; PAP ¼ pulmonary artery pressure; Vt ¼ tidal volume; Cstat ¼ static compliance; EMV ¼ expiratory minute volume.*Cstat values in groups in which it was not measured (HFOV, APRV) were identical to LowVt at baseline and immediately post-injury time

points.xP < 0.05 versus LowVt following RM ANOVA.

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Apoptosis

The number of apoptotic cells in the lung was not sig-nificantly different between the LowVt group and anyof the three experimental groups (Table 2).

Histologic Assessment

APRV significantly reduced vessel congestion and in-creased alveolar wall thickness compared with LowVt.HFOV significantly increased airspace hemorrhaging.The RMþOP group demonstrated significantly greaterleukocyte infiltration, vessel congestion, and fibrinousdeposits compared with LowVt (Table 2, Fig. 5).

DISCUSSION

The most important findings for this study were thatnone of the ‘‘open lung’’ techniques dramatically re-duced lung histopathology or inflammation comparedwith ARDSnet. Despite improving oxygenation,HFOV increased airspace hemorrhage and RMþOPcaused an increase in pulmonary neutrophil sequestra-tion, vessel congestion, and fibrin deposition, indicating

VILI. These results support numerous clinicalstudies that have not shown a reduction in morbidityor mortality using techniques designed to ‘‘open thelung’’ [14, 15].

Although our results have failed to demonstrate thatthe ‘‘open lung’’ approach is superior to LowVt in reduc-ing lung pathology, this is not unequivocal evidencethat the low tidal volume ventilation is superior at pre-venting VILI. Indeed, our results with APRV were fa-vorable to LowVt, improving oxygenation, ventilation,and cytokine concentration with a similar pattern ofhistopathology.

APRV Versus LowVt

The protective effects of APRV are derived from itsunique pattern of ventilation, which alternate extendedperiods of continuous positive airway pressure (CPAP)with brief, abrupt pressure releases. In theory, the ex-tended high-pressure phase serves as a continuous re-cruitment maneuver, while the intermittent releasesfacilitate carbon dioxide release. Our laboratory hasshown that lung homogeneity increases as a functionof time when continuous airway pressure is applied,

FIG. 4. APRV demonstrated a significant reduction in IL- 8 (openboxes) and TNF-a (solid boxes) in BALF compared with Low Vt. xP <0.05 compared with Low Vt. Data mean 6 SEM.

ALBERT ET AL.: HFOV, APRV, AND RM+OP VERSUS LOWVT e77

therefore, continuously recruiting alveoli over a settime of high pressure (Thigh) may increase the func-tional homogeneity of interconnected alveolar units,thereby reducing shearing forces associated with atel-ectasis [16, 17]. Ventilation to perfusion matchingwould improve due to the increase in alveolar surfacearea available to pulmonary capillaries, thuspromoting the observed improvement in gas exchange[18].

APRV is most effective when the patient’s ability tospontaneously breathe throughout the ventilator cycleis preserved [17, 19]. The benefits of spontaneousbreathing include enhancing the movement of gastowards dependent lung regions, reducing over-distension of nondependent regions through the appli-cation of pleural pressure, and an overall reduction inthe number of mechanical breaths [18]. The fact thatAPRV reduced expiratory minute volume, while im-proving CO2 exchange exemplifies the notion that thecost of ventilation is lower with ARPV. In conventionalmodes, either the airway pressure or the rate wouldhave to be increased in response to changes in oxygen-ation and CO2. Over time, the cost of ventilation wouldbe exacted by damage to the lung due to repetitivestress/strain, as it has been shown that increased respi-ratory rates alone can promote VILI [20]. Therefore,

TABL

Histological Assessment

Group Fibrin PMNs Alveolar wall thickness

LowVt 0.48 6 0.1 0.57 6 0.1 0.97 6 0.2APRV 0.75 6 0.1 0.85 6 0.1 1.38 6 0.2x

HFOV 0.62 6 0.1 0.68 6 0.1 0.88 6 0.1RMþOP 0.86 6 0.2x 1.0 6 0.1x 0.90 6 0.2

Data mean 6 SEM.xP < 0.05 compared with LowVt following determination of c2 value v

APRV accomplishes the dual goal of promoting more ef-fective and less injurious breathing.

In this vein, we speculate that the combined effect offewer ventilator imposed breaths and the increase infunctional homogeneity of the lung account for the dif-ference in TNF-a and IL-8 concentration between theAPRV and LowVt groups. Therefore, a gross mecha-nism for the reduction in cytokine concentration inthe APRV group may be a decrease in the incidenceand severity of shear stress on alveolar units [21].A mechanism for the elevated cytokine concentrationof the LowVt group may be associated with over-distension of nondependent regions. Independent ob-servations by Terragni et al. and Grasso et al. havedemonstrated that a tidal volume of 6 mL/kg may causehyperinflation in a subset of ARDS patients [22, 23].However, our data cannot confirm or reject theseobservations since we did not differentiate betweendependent and nondependent histology for ourhistologic analyses.

A significant histologic finding was that APRV in-creased alveolar wall thickness. Increased alveolarwall thickness may be caused by edema, inflammation,or the aggregation of alveolar epithelial cells due to col-lapse. Since themorphology is not typical of edema (i.e.,nuclear density is normal) and there is only moderateleukocyte infiltration, the increase in wall thickness ismost suggestive of alveolar collapse. Tschumperlinand colleagues have shown that alveolar epithelial sur-face area positively correlates with inflation volumeand pressure [24]. At low volumes and pressures, thesurface area to the airspace is low, and the alveolar ep-ithelium resembles a ‘‘folded paper bag.’’ Inflation pres-sures cause the epithelial surface to unfold, providinga greater surface area for gas exchange. We speculatethat due to the model of surfactant deactivation, thecombined effect of Phigh and Thigh was not enough toovercome critical opening thresholds. Despite the areasof collapse, the overall histologic, physiologic, and cyto-kine data suggest that APRV and ARDSnet were simi-lar in preventing VILI, with APRV promoting moreefficient gas exchange. Furthermore, these data de-mand amore nuanced understanding of the role of tidal

E 2

of Lung Parenchyma

Vessel congestion Airspace hemorrhage Apoptosis (cells/field)

1.33 6 0.1 0.68 6 0.1 9.6 6 1.81.25 6 0.2 x 0.72 6 0.1 12.7 6 1.31.22 6 0.1 1.78 6 0.1x 9.8 6 2.01.44 6 0.2x 0.92 6 0.1 10.6 6 1.7

ia likelihood ratio.

FIG. 5. Histologic features representative of each treatment condition. LowVt: lowest leukocyte infiltration and least hemorrhage. APRV:significantly greater alveolar wall thickness (arrowheads), and less, albeit moderate, vessel congestion. HFOV: demonstrated significantlygreater airspace hemorrhage (arrow) than LowVt. RMþOP: evidence of significant elevations in the incidence of leukocyte infiltration, vesselcongestion (arrow), and alveolar fibrin deposition (asterisks). All photomicrographs are routine H an E at high magnification (bar ¼ 25 mm).

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volume in VILI; APRV was set to deliver over 15 mL/kgtidal volume, a volume deemed injurious by conven-tional standards.

HFOV Versus LowVt

The clinical role of HFOV is largely restricted to a res-cue therapy in patient populations where conventionalventilation has failed [25]. Our study sought to addressthe use of HFOV as an early or primary treatment forALI, much like the recent TOOLS Trial Pilot Study andthe randomized controlled trials conducted by Fergusonet al., Derdak et al. and Bollen et al. [26–28]. In thesetrials, HFOV was associated with similar complicationrates as conventional ventilation, however none of thethree studies were able to prove a significant benefit inpatient care with HFOV [27, 28]. Our results weresimilar to these trials, as we were unable to showhistologic lung protection with HFOV despitesignificant improvements in oxygenation and CO2

exchange. A major difference between our approachand the previous clinical trials is that we utilized a lowtidal volume, ARDSnet approach as a control group.

Our results differed from small animal studiesshowing definitive lung protection with HFOV [8, 9].The reported protective benefit of HFOV is derivedfrom tidal volumes that are extremely small, oftenless than the anatomical deadspace. Sedeek et al.[29] have challenged the notion of small tidal volumesin HFOV by showing that tidal volume is directly pro-portional to the pressure amplitude (DP) and in-versely proportional to frequency (Hz). For example,a frequency of 4 Hz and DP of 60 cmH2O yielded tidalvolumes of over 120 mL, approximately 4 mL/kg intheir porcine subjects. Applying the results of Sedeeket al. to this study, our settings (DP w 50 cmH2O anda frequency w 5 Hz) probably produced tidal volumescloser to the recommended 6 mL/kg than the tidalvolumes used in most rodent models. Therefore,a larger tidal volume, and greater risk of VILI, maybe the reason why we did not demonstrate the re-duced pathologic and immunologic injury that otherstudies have shown with HFOV [8, 9]. The use ofan animal model with anatomy and physiologysimilar to adult patients thus yielded results moresimilar to previous clinical trials, showing no benefit

ALBERT ET AL.: HFOV, APRV, AND RM+OP VERSUS LOWVT e79

in morbidity and mortality with HFOV comparedwith conventional strategies.

HFOV increased airspace hemorrhage, which has notbeen described previously in clinical or animal studies.One possible explanation is related to the pulmonaryperfusion pressure (i.e., PAP), which was elevated togreater than 30 mmHg. The relatively high pulmonaryvascular pressure, combined with the increased meanairway pressures duringHFOV,may have been respon-sible for vascular leakage. Hotchkiss and colleaguesdemonstrated that either high-sustained airway pres-sures or vascular pressures can lead to hemorrhagicairway edema when the other is held constant [30].Other experiments have shown that increased respira-tory rate during conventional ventilation positively cor-relates with vascular leakage [20, 30]; howeverdecreasing tidal volume attenuated the injuriouseffect of a high rate. If the tidal volumes in our studywere more similar to conventional tidal volumes, thehigh respiratory rate likely played a role in ourresults, ostensibly promoting airspace edema byincreasing the frequency of mechanical stress onalveolar epithelium and limiting the time-window forcellular repair. Although the relationship between air-way edema, vascular pressure, and respiratory rate hasnot been tested with HFOV inmind, these factors likelyplayed a role in our results.

Aggressive Recruitment Maneuvers and ‘‘Optimal PEEP’’

(RMDOP) Versus LowVt

Like the clinical study by Borges et al., we showedthat the lungs of our animals could be recruited. How-ever, Borges et al. only investigated whether collapsedlung units could be opened in ARDS patients and thedegree to which oxygenation would improve [12], notwhether the ‘‘maximum recruitment maneuver’’ pro-tected against VILI. Therefore, our results duplicatethe results of the previous clinical study showing im-provement in oxygenation secondary to lung recruit-ment. However, our study adds to Borges et al. byshowing that RMþOP increased lung inflammation(i.e., increased leukocyte infiltration) and impaired he-modynamic function, ventilation, and systemic oxygen-ation. Similar to previous studies, arterial oxygenationwas shown to be a poor indicator of underlying lungpathophysiology.

The protection offered by open lung techniques isbased on the assumption that opening closed alveoliwill decrease the number of junctional interfaces be-tween heterogeneous lung tissue, thereby reducing me-chanical stress during each breath [31]. Theoretically,recruitment maneuvers and high PEEP accomplishlung homogeneity, however evidence from animal stud-ies of ALI have shown that recruiting injured lung may

be accompanied by serious limitations, most promi-nently the risk of over-distending compliant lung units(volutrauma) [6, 32, 33]. Even if over-distension had notbeen a concern, Grasso et al. [34] showed that unstablealveoli that became re-aerated following a recruitmentmaneuver had an increased elastance compared withalveoli that were healthier (the ‘‘baby lung’’) beforerecruitment. Thus, collapsed units that had been re-aerated did not resume normal lung mechanics follow-ing recruitment despite being fully inflated.

While we improved oxygenation, thus demonstratinga successful re-aeration of collapsed tissues, we likelycaused concomitant over-distension in normal tissuethrough a direct and excessive application of PEEP.Over-distension and excessive PEEP can cause a redis-tribution of pulmonary perfusion and increase in deadspace ventilation [31], which is the most likely causeof the observed increase in arterial PCO2.

Although we chose to define our PEEP in terms of ar-terial blood gases (PaO2þ PaCO2> 400 mmHg, a valueshown by CT to correspond to recruited alveoli [12]),there are alternative methods to determine this value.Specifically, optimal PEEP has been identified by thefollowing physiologic markers following a decrementalPEEP trial: (1) maximum dynamic compliance [35],(2) minimum elastance [36], (3) maximal PaO2 þPaCO2 [12], and (4) lowest PEEP needed to maintainPaO2 above a threshold level [37]. Despite these differ-ences in approach, it was demonstrated that the opti-mal PEEP determined from any of these approacheswere statistically indistinguishable in a sheep modelof ALI [37]. Accordingly, our optimal PEEP(15.7 cmH2O) was nearly identical to the PEEP em-ployed in a similar porcine model of surfactant deacti-vation by Suarez-Sipmann et al. (16 cmH2O, maximaldynamic compliance method) [35]. Our study adds tothese studies by suggesting that the application of an‘‘optimal’’ PEEP can have significant negative conse-quences on hemodynamics and ventilation, in additionto promoting VILI, and may not be the optimal ap-proach in managing a hypoxemic patient.

Critique of Study

The results of our experiments may have differedfrom those of other studies due to the injury model,Tween-20, and the use of large animals rather than ro-dents. Tween-20 induced injury, as characterized byprevious work in our lab [11], causes surfactant deacti-vation and alveolar instability, but does not cause an in-flammatory injury to the lung unless combined withVILI [38]. Regardless of this limitation, surfactant de-activation is a hallmark of ARDS pathophysiology andis believed to be a key component in the mechanism ofVILI. Thus, our model effectively recreates the alveolar

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instability integral to the pathogenesis of ARDS andVILI. With this model, we have shown that APRV canbe used effectively to improve gas exchange with mini-mal changes in histopathology, where HFOV andRMþOP showed signs of VILI.

An outstanding criticism of HFOV and APRV is thelack of concrete evidence that the settings chosen bythe clinician are the optimal settings for providing ade-quate gas exchange and reducing VILI [18]. Since de-termining these settings was not the aim of thisstudy, we chose our ventilator settings according to suc-cessful accounts in literature [17, 39]. Our resultsshowing improved gas exchange suggest that oursettings and management were proficient.

Our protocol included the use of recruitment maneu-vers, a controversial topic in mechanical ventilation.We included RMs as part of our protocol to ‘‘normalize’’the level of aerated alveoli in each animal before groupassignment. It remains to be seen if APRV and HFOVare more effective when used in conjunction with RMs(as the TOOLS trial suggests with HFOV) and, there-fore, if the inclusion of RMs positively affected our re-sults. Furthermore, it is well known that certainsubgroups of ALI and ARDS patients do not respond op-timally to RMs. For this reason, the direct clinical appli-cability of our methods must come under scrutiny;however, it by nomean invalidates our results and theirinterpretation.

CONCLUSION

We have demonstrated in a large animal model ofsurfactant deactivation that ‘Open Lung’ ventilationmodes are not superior to ARDSnet in preventingVILI when used as primary treatment modalities.APRV increased oxygenation and carbon dioxide ex-change, and significantly reduced BALF cytokine con-centration with a histopathological footprint nearlyindistinguishable from LowVt, warranting future labo-ratory and clinical investigation as an early treatmentfor ARDS. HFOV improved gas exchange but causedsignificant airspace hemorrhage. The aggressive lungrecruitment strategy improved oxygenation but pro-duced significant impairments in CO2 elimination,SvO2 and mean arterial pressure, as well as increasedhistological evidence of inflammation (i.e. leukocyte in-filtration, vessel congestion and fibrin deposition).

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