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doi:10.1152/ajplung.00528.2007294:L974-L983, 2008. First published 14 March 2008;Am J Physiol Lung Cell Mol Physiol

Possmayer, James F. Lewis and Ruud A. W. VeldhuizenAdam A. Maruscak, Daniel W. Vockeroth, Brandon Girardi, Tanya Sheikh, Freddeterioration during high tidal volume ventilationAlterations to surfactant precede physiological

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Alterations to surfactant precede physiological deterioration during high tidal

volume ventilation

Adam A. Maruscak,1,3 Daniel W. Vockeroth,1,3 Brandon Girardi,1,3 Tanya Sheikh,1,3 Fred Possmayer,1,4,5

James F. Lewis,1,2,3 and Ruud A. W. Veldhuizen1,2,3

1 Lawson Health Research Institute, and Departments of2Medicine, 3Physiology and Pharmacology, 4Biochemistry,and 5Obstetrics and Gynecology, The University of Western Ontario, London, Ontario, Canada

Submitted 19 December 2007; accepted in final form 9 March 2008

Maruscak AA, Vockeroth DW, Girardi B, Sheikh T, Pos-smayer F, Lewis JF, Veldhuizen RA. Alterations to surfactantprecede physiological deterioration during high tidal volume ventila-tion. Am J Physiol Lung Cell Mol Physiol 294: L974L983, 2008.First published March 14, 2008; doi:10.1152/ajplung.00528.2007.Lung injury due to mechanical ventilation is associated with animpairment of endogenous surfactant. It is unknown whether thisimpairment is a consequence of or an active contributor to thedevelopment and progression of lung injury. To investigate this issue,the present study addressed three questions: Do alterations to surfac-

tant precede physiological lung dysfunction during mechanical ven-tilation? Which components are responsible for surfactants biophys-ical dysfunction? Does exogenous surfactant supplementation offer aphysiological benefit in ventilation-induced lung injury? Adult ratswere exposed to either a low-stretch [tidal volume (VT) 8 ml/kg,positive end-expiratory pressure (PEEP) 5 cmH2O, respiratory rate(RR) 5456 breaths/min (bpm), fractional inspired oxygen (FIO2)1.0] or high-stretch (VT 30 ml/kg, PEEP 0 cmH2O, RR 1416bpm, FIO2 1.0) ventilation strategy and monitored for either 1 or 2 h.Subsequently, animals were lavaged and the composition and functionof surfactant was analyzed. Separate groups of animals receivedexogenous surfactant after 1 h of high-stretch ventilation and weremonitored for an additional 2 h. High stretch induced a significantdecrease in blood oxygenation after 2 h of ventilation. Alterations insurfactant pool sizes and activity were observed at 1 h of high-stretch

ventilation and progressed over time. The functional impairment ofsurfactant appeared to be caused by alterations to the hydrophobiccomponents of surfactant. Exogenous surfactant treatment after aperiod of high-stretch ventilation mitigated subsequent physiologicallung dysfunction. Together, these results suggest that alterations ofsurfactant are a consequence of the ventilation strategy that impair thebiophysical activity of this material and thereby contribute directly tolung dysfunction over time.

mechanical ventilation; biophysical activity; ventilation-induced lunginjury

MECHANICAL VENTILATION IS an essential therapeutic interventionemployed in all patients with acute lung injury (3). However,

it has been well established that mechanical ventilation canalso propagate the existing lung injury (5, 30, 37). This dam-aging effect is caused, in part, by collapse and overdistention oflung regions that occur during ventilation of a heterogeneouslyinjured lung. This phenomenon can be studied in healthyanimals by utilizing extremely high tidal volumes and zeroend-expiratory pressure during ventilation of normal lungs(37). The injury in these animal models has been termedventilation-induced lung injury (VILI). Alterations to the

endogenous pulmonary surfactant system are an importantcomponent of the pathophysiology of VILI (31, 34, 37).

Surfactant is a mixture of phospholipids (80%), neutrallipids (510%), and surfactant-associated proteins (SP-A,SP-B, SP-C, and SP-D) that is secreted into the alveolar airspace from type II cells (11). Once secreted, surfactant formshighly organized lipid-protein structures that are capable offorming a surface tension-reducing surface film (38). Thesebiophysically active structures are called the large aggregates

(LA). During respiration an inactive form of surfactant isproduced, designated the small aggregates (SA). The mainfunction of surfactant is the reduction of surface tension at theair-liquid interface of the alveoli, which allows the lung toinflate with minimal effort, thereby contributing directly tolung function.

As can be deduced from these important biophysical prop-erties of surfactant, impairment or deficiency of this materialwould result in impaired lung function. The prototypic clinicalexample of this latter situation is the neonatal respiratorydistress syndrome, in which a developmental deficiency ofsurfactant causes severe lung dysfunction unless exogenoussurfactant is administered (27). Impairments of surfactant func-tion in mature lungs contributes to the lung dysfunction asso-ciated with acute lung injury; however, to date, exogenoussurfactant therapy has not proven to be consistently beneficialin this setting (20, 21).

Alterations of surfactant due to mechanical ventilationhave been studied in a variety of animal models of VILI (31,34, 36). The specific alterations observed include a reduc-tion in the amount, composition, and structure of the LAsubfraction and an impairment in the surface activity ofthese LA, possibly due to the inhibition by serum proteinsthat had leaked into the lung (7). Interestingly, these obser-vations have been made at a time point at which the lunginjury due to ventilation was severe. As such, it is unknownwhether alterations of the surfactant system are the cause

or a consequence of the lung dysfunction present in theseanimals.On the basis of the above information, the present study

utilized an animal model of VILI to address three specificquestions: 1) Do alterations to pulmonary surfactant precedephysiological dysfunction during the development of VILI?2) Which components of the surfactant system are responsiblefor its biophysical dysfunction? and 3) Does surfactant supple-mentation offer a physiological benefit in VILI?

Address for reprint requests and other correspondence: R. A. W. Veldhui-zen, Lawson Health Research Institute F4-117, 268 Grosvenor St., London,ON, Canada, N6A 4V2 (e-mail: [email protected]).

The costs of publication of this article were defrayed in part by the paymentof page charges. The article must therefore be hereby marked advertisementin accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Am J Physiol Lung Cell Mol Physiol 294: L974L983, 2008.First published March 14, 2008; doi:10.1152/ajplung.00528.2007.

1040-0605/08 $8.00 Copyright 2008 the American Physiological Society http://www.ajplung.orgL974


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METHODS

Animal Experimentation

A total of 82 male Sprague-Dawley rats (350 450 g, CharlesRiver, St. Constant, PQ, Canada) were utilized. These animals wereused for three separate animal experiments. All procedures wereapproved by the animal use subcommittee at the University of West-

ern Ontario under the guidelines of the Canadian Council of AnimalCare.For all three experiments rats were acclimated for a minimum of 3

days in an animal housing facility, during which time they were grouphoused with free access to water and standard chow. The mass of eachrat was recorded and animals were anesthetized with an intraperito-neal injection of 75 mg/kg ketamine and 5 mg/kg xylazine in sterile0.15 M NaCl. Following anesthesia, the animal received a subcuta-neous injection of analgesic (0.050.1 mg/kg buprenorphine). The leftcarotid artery and both jugular veins were exposed and cannulatedwith PE-50 tubing. The carotid line was used to obtain arterial bloodsamples for blood gas measurements (model ABL500, Radiometer,Copenhagen, Denmark), monitor heart rate and blood pressure, anddeliver fluid (sterile NaCl and 100 IU heparin/l) by use of an infusionpump set at 0.5 ml 100 g1 h1. The right jugular line was used to

deliver additional anesthetic (0.52.0 mg

100 g

1

h

1

propofol), andthe left jugular line was used to deliver additional fluid (0.5 ml 100g1 h1). The trachea was exposed and a 14-gauge plastic endotra-cheal tube was secured with 2-0 surgical silk. The animal wasadministered a neuromuscular paralytic agent intravenously (2 mg/kgpancurium bromide) to stop spontaneous breathing and was immedi-ately connected to a volume-cycled rodent ventilator (Harvard Instru-ments, St. Laurent, PQ, Canada) set at a baseline tidal volume (VT) of8 ml/kg, a positive-end expiratory pressure (PEEP) of 5 cmH 2O, arespiratory rate (RR) of 5456 breaths/min (bpm), and a fractionalinspired oxygen (FIO2) of 1.0. All rats were ventilated by this strategyfor 10 min prior to the first blood gas measurement. Airway pressureswere measured throughout ventilation via an airway pressure monitor(Sechrist Industries, Anaheim, CA) connected to the inspiratory limbof the ventilator. Inclusion criteria for all experiments required ani-mals to have an arterial partial pressure of oxygen (PaO

2

) to fractionalpercentage of inspired oxygen ratio (PaO2/FIO2) 400 mmHg, arterialpartial pressure of carbon dioxide (PaCO2) between 3540 mmHg, peakinspiratory pressure (PIP) 15 cmH2O, and mean systolic bloodpressure between 80 and 120 mmHg.

Experiment 1

The first experiment utilized 20 animals to examine the effects oftwo different ventilation strategies employed for two different timeperiods on physiology, surfactant pool sizes, cholesterol and surfac-tant protein levels in the LA, total protein concentrations in the lunglavage, and concentrations of IL-6 and TNF- in the lavage.

Upon reaching inclusion criteria, animals were randomized toreceive mechanical ventilation for 1 or 2 h, with either a noninjurious,low-stretch (LS) ventilation strategy with PEEP (VT 8 ml/kg,

PEEP 5 cmH2O, RR 54 56 bpm) designated as LS1h and LS2hgroups, or an injurious high-stretch (HS) strategy with no PEEP (VT 30ml/kg, PEEP 0 cmH2O, RR 1416 bpm) designated as HS1h andHS2h. During randomization, all animals were briefly disconnectedfrom the ventilator prior to initiation of the respective ventilationstrategy; whereas animals in the LS groups were reconnected to theventilator with the baseline settings, HS animals were reconnected toa second ventilator calibrated to a VT of 30 ml/kg. Measures of bloodpressure and PIP were recorded every 15 min, with blood gasmeasurements taken at 15 min and then every half hour followingrandomization.

After the 1- or 2-h ventilation period, animals were euthanized withan intravenous overdose of pentobarbital sodium (110 mg/kg). Amidline sternectomy was performed to expose the lungs and animals

were exsanguinated by transection of the descending aorta. Lungcompliance was assessed via pressure-volume curve measurements aspreviously described (7). Briefly, lungs were allowed to deflatepassively and connected to a U-tube manometer attached to a pressuregauge. The lung was inflated in stepwise increments of 2 cmH2Opressure to a maximal pressure of 26 cmH2O while the administeredvolume was recorded at each pressure value. The same stepwiseprocedure was performed on deflation (7).

Surfactant, total protein, and cytokine analysis. Following pres-sure-volume curve analyses, lungs were lavaged with 10 ml of sterile0.15 M NaCl, which was instilled and withdrawn three times (7). Onemilliliter of the first lavage was aliquoted for cytokine analysis. Theremaining lavage was combined with the fluid from four additional10-ml lavages. Total lavage volume was recorded and centrifuged at150 g for 10 min to remove cellular debris. The supernatant of thiscentrifugation was termed total surfactant (TS). A 3-ml aliquot of TSwas retained for analysis. Centrifugation at 40,000 g for 15 minseparated the remaining TS into the supernatant containing the smallaggregate subfraction (SA) and pellet containing the large aggregatesubfraction (LA). The LA pellet from each animal was then resus-pended in 2 ml of sterile 0.15 M NaCl. All samples were stored at20C following separation into the TS, LA, and SA subfractions.

A chloroform-methanol lipid extraction was performed on aliquots

of TS, LA, and SA samples, followed by a Duck-Chong phosphorusassay to determine the amount of phospholipids within these subfrac-tions (2, 8). A Micro BCA protein assay was used to determine theamount of total protein in the TS and LA subfractions, by followingthe manufacturers instructions (Pierce Biotechnology, Rockford, IL).Total cholesterol present in the LA subfraction was quantified by anenzymatic colorimetric method (Wako Chemicals, Richmond, VA) aspreviously described (24).

SP-A measurements. A volume equivalent to 3 g of phospholipidfrom LA samples were loaded into a stacking gel and the proteinswere separated via 2 h of electrophoresis at 100 V (Bio-Rad Labora-tories, Mississauga, ON, Canada) through a 10% polyacrylamideseparating gel containing 0.01% SDS. The samples were run along-side 10 l of human SP-A standard (0.19 g/l) and a 10-lmolecular weight marker (Kaleidoscope prestained standards). The

proteins were then transferred to a nitrocellulose membrane (Bio-RadLaboratories). The membranes were blocked overnight at 4C in 5%blocking milk in PBS (pH 7.4). The membranes were then incubatedfor 2 h in rabbit anti-rat SP-A IgG primary antibody with a 1:1,000dilution in 1 PBS, prior to being washed for 15 min, then 2 5 minin 1 PBS with 0.05% Tween-20, followed by 5-min washing in 5%blocking milk in PBS. Following the washing, the nitrocellulosemembranes were incubated for 2 h in donkey anti-rabbit IgG second-ary antibody in 1 PBS (1:1,000), prior to being washed again for 15min, then 3 5 min in 1 PBS with 0.05% Tween-20. Finally,supersignal (Biolynx OptiBlaze WEST picoLUCENT, G-Biosciences,St. Louis, MO) was applied to all nitrocellulose membranes andchemiluminescence was observed. Densitometry was performed andimmunoreactivity was expressed as a ratio to the LS1h samples oneach respective membrane.

SP-B measurements. A volume equivalent to 10 g of phospholipidfrom LA samples were added to 5 l of 33% SDS and loaded into thewells of a 1020% Tricine Gel (Invitrogen, Carlsbad, CA), and theproteins were separated via 2 h of electrophoresis at 100 V (Bio-RadLaboratories, Mississauga, ON Canada). The samples were run along-side 10 l of human SP-B standard (0.23 g/l) and 10-l molecularweight marker. The proteins were then transferred to an Immobilon-PSQ transfer membrane (Millipore, Bedford, MA), and the membraneswere blocked overnight at 4C in NAP-blocker blocking agent (G-Biosciences, St. Louis, MO) at a 1:1 dilution in 1 Tris-bufferedsaline (TBS). The membranes were then incubated for 2 h in SP-Bmonoclonal antibody (generously supplied by Dr. Suzuki, KyotoUniversity, Kyoto, Japan) at a 1:1,000 dilution in 1 TBS, prior tobeing washed for 15 min, then 2 5 min in 1 TBS with 0.05%

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AJP-Lung Cell Mol Physiol VOL 294 MAY 2008 www.ajplung.org


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Tween-20, followed by 5-min washing in NAP-blocker. Followingthe washing, the nitrocellulose membranes were incubated for 2 hwith goat anti-mouse horseradish peroxidase-IgG secondary antibodyin 1 TBS (1:1,000), prior to being washed again for 15 min, then3 5 min in 1 TBS with 0.05% Tween-20. Finally, supersignal(Biolynx OptiBlaze WEST picoLUCENT, G-Biosciences) wasapplied to all nitrocellulose membranes and chemiluminescencewas observed. Densitometry was performed and immunoreactivity

was expressed as a ratio to the LS1h samples on each respectivemembrane.

Cytokine analysis. The 1-ml aliquot recovered from the first lavagewas centrifuged at 4C, 200 g, for 10 min. The supernatant wasseparated into 240-l aliquots and snap frozen in liquid nitrogenbefore being stored at80C until cytokine analysis. IL-6 and TNF-were measured by using opti-EIA ELISA kits following the manu-facturers specifications (Pharmigen, San Diego, CA).

Experiment 2

A separate cohort of 42 animals was utilized in experiment 2 tocollect sufficient amounts of LA samples from the four experimentalgroups described in experiment 1, to examine the effects of thedifferent ventilation strategies on the biophysical properties of surfac-

tant. Animal experimentation and collection of lavage material wasidentical as described for experiment 1. The entire lavage volumefrom each animal was centrifuged at 150 g and subsequently at 40,000g to obtain a LA pellet, which was resuspended in 2 ml of sterile 0.15M NaCl. Subsequently, LA samples from each group of animals werepooled and further processed into four different preparations: crudeLA, purified LA, extracted LA, and extracted 5% SP-A LA.

Crude LA samples required no additional preparation after centrif-ugation and resuspension as described above. purified LA sampleswere prepared via sucrose gradient separation (7). Briefly, samples ofcrude LA were divided into 2-ml aliquots and then placed onto 15 mlof 0.8 M sucrose, followed by centrifugation at 40,000 g for 15 min.The pellicle was removed from the surface of the sucrose, diluted with0.15% NaCl, and then recentrifuged at 40,000 g for 15 min to removeremaining sucrose. This washing process was performed twice and the

final pellet was resuspended in 0.15 M NaCl.Preparation of extracted LA samples was performed via lipid

extraction of separate samples of crude LA samples by the method ofBligh and Dyer (2) using chloroform-methanol (2:1 vol/vol). Thechloroform layer, containing the hydrophobic surfactant components,was dried under nitrogen followed by reconstitution of the driedsample in 0.15 M NaCl. Finally, extracted 5% SP-A LA sampleswere prepared by supplementation of lipid extracted LA with 5%human SP-A (wt/wt). Human SP-A was obtained from a patient withalveolar proteinosis as described elsewhere (1, 15).

Each LA preparation from the four groups was analyzed forphospholipid phosphorous as described above, to obtain a phospho-lipid concentration. A total protein assay was also performed on crudeand purified LA samples to verify the removal of nonsurfactantproteins.

Biophysical functional analysis. A custom-designed captive bubblesurfactometer was used to study the biophysical function of pooledLA samples obtained in experiment 2 (28). All samples were resus-pended in a buffer (0.15 mM NaCl, 2 mM Tris HCl, 1.5 mM CaCl2,pH 7.4) at a phospholipid concentration of 300 g/ml and incu-bated at 37C for 1 h.

Briefly, a gas-tight chamber was filled with a surfactant suspensionand allowed to equilibrate to a temperature of 37 1C. An air bubbleof8 mm in diameter was introduced into the suspension and theshape of the bubble was recorded by digital photography as a functionof time. The digital images of the bubble were used to measure thearea and volume and to calculate the surface tension at the air-liquidinterface inside the bubble. Immediately following introduction of thebubble, the change in shape was recorded as a function of time to

monitor adsorption of the surfactant material to the air-liquid inter-face. After an equilibrium surface tension of23 mN/m was attained,the chamber was sealed, and one quasi-static compression-expansioncycle was performed to establish the surface area reduction required toobtain minimum surface tension prior to dynamic cycling. Dynamiccycling of the air-liquid interface within the bubble was conducted byfirst allowing the surfactant to readsorb to equilibrium surface tension,followed by compression and expansion of the bubble at 30 cycles/

min between 100 and 110% of the original surface area and the arearequired to achieve minimum surface tension established during thesingle quasi-static cycle.

Experiment 3

The third experiment was performed on 20 rats to address the roleof the surfactant system in the physiological development and pro-gression of VILI. Once inclusion criteria (similar to experiment1) were met, rats were randomized to receive 1 h of either a LS or aHS mechanical ventilation strategy as described for experiment 1.Following the 1-h period of ventilation rats were further randomizedto receive treatment with 20 mg phospholipids/kg body wt of acommercially available exogenous surfactant preparation, BLES (Bo-vine Lipid Extract Surfactant, BLES Biochemicals, London, ON,

Canada), or no treatment as a control group. This dose of surfactantwas chosen on the basis of previous studies (1) and preliminaryexperiments, to increase the surfactant pool size by three- to four-fold. This resulted in four separate experimental groups: 1) 1 h of LSmechanical ventilation with no treatment (LS-NT), 2) 1 h o f L Smechanical ventilation with BLES treatment (LS-BLES), 3) 1 h ofHS mechanical ventilation with no treatment (HS-NT), and 4) 1 h of HSmechanical ventilation with BLES treatment (HS-BLES).

Surfactant administration involved disconnecting the rat from theventilator and injecting the BLES solution through the endotrachealtube directly into the lung via a 3-ml syringe. Each instillation wasfollowed by administration of an air bolus and, upon reconnection tothe ventilator, a sigh was performed by blocking the expiratory linefor two breaths to optimize peripheral distribution. Control ratsunderwent the same procedure but were administered an air bolus. All

rats were subsequently ventilated for an additional 2 h by using onlythe LS strategy. During ventilation, PIP and mean arterial pressurewere monitored and blood gas samples were taken at 15 min intervals.Following this ventilation period animals were euthanized and la-vaged as described for experiment 1.

Statistical Analysis

All values are reported as means SE. Statistical analysis wasperformed by a two-way ANOVA to explore any interactive effectsfollowed by a one-way ANOVA with Tukeys post hoc test forpairwise comparison of groups. Changes over time were comparedbetween groups by repeated-measures ANOVA with a Tukeys posthoc test. All statistical analysis was performed utilizing SPSS 11.0.Differences between and within groups were considered statisticallysignificant at probability values of0.05.

RESULTS

Experiment 1

Physiology. Figure 1 shows the PaO2/FIO

2ratio during me-

chanical ventilation for the four experimental groups. Withinthe LS groups there was no significant effect of time on PaO

2

values, with all values remaining above 420 mmHg. Animalsventilated with the HS strategy had PaO

2/FIO

2values that were

not significantly different from their respective baseline mea-surements up to the 90-min time point. At 120 min, the PaO

2

values had significantly decreased compared with all otherearlier time points. Statistical comparisons between ventilation

L976 SURFACTANT IN VENTILATION INJURY



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strategy groups revealed no significant differences in PaO2/FIO

2

values between the LS1h and HS1h groups. However, at 120min, there was a significantly lower PaO

2/FIO

2value measured

in the HS2h group compared with the LS2h group.Values of other physiological outcomes recorded during

ventilation are shown in Table 1. PaCO2

values did not changeover time within any group but were significantly lower in bothHS groups compared with values recorded from the LS groups.PIP values recorded in both LS ventilation groups also did notchange significantly over time. Ventilation with HS resulted inhigher PIP values than the LS groups and increased within theHS2h group during the last 30 min of ventilation, such that PIPvalues after 2 h of HS ventilation were significantly higher

compared with all earlier time points. Arterial blood pressureremained constant for all animals at all time points with theexception of the HS2h group, which had significantly lowerblood pressure values at 120 min compared with the LS2hgroup and compared with earlier time points within the HS2hgroup.

Figure 2 shows the pressure-volume curves measured after

euthanasia of animals in the four experimental groups. Therewere no significant differences in the pressure-volume curvesof lungs in animals from the LS1h and LS2h groups. Lungcompliance was lower in the HS2h group compared with theHS1h group as determined by the significantly lower volumesat 26 cmH2O (Vmax). Further comparison between the twoventilation strategies revealed that there were no significantdifferences between the LS1h and HS1h groups; however, theVmax was significantly lower in the HS2h group compared withthe LS2h group.

Surfactant analysis. Values for total surfactant, LA, SA, and%LA are shown in Fig. 3. There was no significant differencein any of these four outcomes when comparing the LS1h andLS2h groups. Comparison of the two HS groups revealed nosignificant differences in total surfactant and SA pool sizes;however, LA and percent LA were significantly lower in theHS2h group compared with the HS1h group. Comparisonsbetween the two ventilation strategies revealed that, in general,HS ventilation resulted in higher TS pools compared with LSventilation, which was statistically significant for HS1h com-pared with LS1h (Fig. 3A). SA pools were also greater and%LA lower in the HS groups vs. LS groups. There were nodifferences in LA pool sizes between the LS1h and HS1h;however, there was a significantly smaller LA pool obtainedfrom the HS2h group compared with the LS2h group.

Further analysis of the LA fraction was performed to assessthe levels of SP-A, SP-B, and cholesterol relative to the amount

of phospholipid in this subfraction (Table 2). Comparison ofthe two time points revealed no significant differences betweenthe two LS groups. Within the HS ventilation groups, the 2-htime point had significantly lower SP-B levels, and signifi-cantly higher cholesterol values, compared with the 1-h timepoint. These SP-B and cholesterol values in the HS2h werealso significantly different than the values in the LS2h group.

Fig. 1. Arterial partial pressure of oxygen (PaO2)-to-fractional inspired oxygen(FIO2) ratio values of animals ventilated with either a low-stretch [LS, tidalvolume (VT) 8 ml/kg, positive end-expiratory pressure (PEEP) 5 cmH2O,respiratory rate (RR) 5456 breaths/min (bpm)] or high-stretch (HS, VT 30 ml/kg, PEEP 0 cmH2O, RR 1416 bpm) strategy, for either 1 or 2 h(LS1h, LS2h, HS1h, and HS2h, respectively). dP 0.05 for HS2h vs. LS2h.

Fig. 2. Pressure-volume curves obtained after ventilation in the experimentalgroups described in Fig. 1. BW, body weight. cP 0.05 for HS2h vs. HS1h;dP 0.05 for HS2h vs. LS2h.

Table 1. PaCO2 , PIP, and blood pressure values at different

times during ventilation in the experimental groupsdescribed in Fig. 1

Low Stretch High Stretch

1 h 2 h 1 h 2 h

PaCO2, mmHgBaseline 42.20.7 39.40.9 41.60.8 42.20.830 min 39.91.0 39.81.0 31.71.1* 33.31.460 min 41.31.0 39.20.8 32.11.0* 31.61.290 min 39.61.1 31.01.2120 min 39.41.0 33.41.5

PIP, cmH2O

Baseline 13.10,2 13.30.2 13.10.2 12.90.330 min 13.20.3 13.50.3 26.11.0* 25.80.660 min 13.50.2 13.30.2 26.41.0* 25.90.690 min 13.30.2 28.81.2120 min 13.50.2 36.21.2

Blood pressure, mmHgBaseline 88.92.3 87.92.7 92.31.9 88.72.330 min 84.23.0 84.43.0 96.92.5 93.22.660 min 91.22.6 90.63.8 95.82.3 92.93.490 min 97.92.7 88.83.5120 min 99.02.8 65.34.9

Values are means SE. PaCO2, arterial partial pressure of CO2; PIP, peakinspiratory pressure. *P 0.05 for 1-h high-stretch group (HS1h) vs. 1-hlow-stretch group (LS1h); P 0.05 for 2-h high-stretch group (HS2h) vs. 2-hlow-stretch group (LS2h).




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There were no significant differences in SP-A levels among thegroups.

Total protein and cytokine levels. Lung lavage material wasanalyzed for total protein as well as inflammatory cytokinesTNF- and IL-6 (Table 3). There were no differences betweenthe two LS groups in either the total amount of proteinrecovered from the lavage or the concentrations of inflamma-tory cytokines. In comparing the two HS groups, there wassignificantly more protein, TNF-, and IL-6 in the lavage fromanimals ventilated for 2 h compared with the 1-h group.

Comparison between the two ventilation strategies revealed nosignificant difference at the 1-h time point, but significantlymore protein, TNF-, and IL-6 in the HS group after 2 h ofventilation.

Experiment 2

Experiment 2 investigated the biophysical consequences ofthe alterations to surfactant observed in experiment 1. Func-tional assessment of the crude (the resuspended 40,000 gpellet) LA samples from the four experimental groups is shownin Fig. 4. The surface tension during adsorption was notsignificantly different among the four experimental groups

Fig. 3. Pulmonary surfactant values for the 4 experimental groups described in Fig. 1. A: amount of total surfactant in the lung lavage. B: amount of largeaggregates (LA) in the lung lavage. C: amount of small aggregates (SA) in the lung lavage. D: percentage of LA. PL, phospholipid. bP 0.05 for HS1h vs. LS1h;cP 0.05 for HS2h vs. HS1h; dP 0.05 for HS2h vs. LS2h.

Table 2. Values of SP-A, SP-B and cholesterol in the LA

subfraction for the 4 experimental groups describedin Fig. 1


1 h 2 h 1 h 2 h

SP-A 1.000.21 1.110.20 0.790.16 0.450.16SP-B 1.000.04 1.160.08 1.150.19 0.360.07*Cholesterol,

% wt of PL 8.310.47 8.220.38 8.621.2 13.01.6*

Values are means SE. LA, large aggregates. Surfactant protein-A (SP)-Aand SP-B are relative densitometry values from Western blots that had equalamounts of phospholipids (PL) loaded, relative to the LS1h group. *P 0.05for HS2h vs. HS1h, P 0.05 for HS2h vs. LS2h.

Table 3. Lavage concentrations of 3 markers of lung injury,total protein, IL-6, and TNF- of the 4 experimentalventilation groups described in Fig. 1


1 h 2 h 1 h 2 h

Total protein, mg/kg body wt 192 276 221 21640*IL-6, pg/ml 704 15223 10114 1480380*TNF-, pg/ml 5125 6434 5922 29198*

Values are means SE. *P 0.05 for HS2h vs. HS1h, P 0.05 for HS2hvs. LS2h.




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(Fig. 4A). The results of the surface tension reduction duringdynamic compression-expansion cycles are shown in Fig. 4B.Comparison of the two LS groups revealed that the surfacetension after one compression was significantly higher in theLS2h group compared with the LS1h group, but no othersignificant differences were noted between these two groups.Comparisons between the two HS groups revealed surfacetension values that were not significantly different for cycles 1,5, and 10 but were significantly higher in the HS2h groupduring cycle 21 compared with the HS1h group. Comparisonbetween the two ventilation strategies revealed higher surface

tensions values in general for the two HS groups comparedwith the LS groups with statistically significant differencesnoted at cycles 1 and 5 for the 1-h groups and cycle 5, 10, and21 for the 2-h groups.

Figure 5 shows the biophysical function of LA obtained aftera sucrose gradient purification procedure (purified LA). Thesucrose gradient removed contaminating serum proteins fromthe crude LA as determined by protein measurements on thosesamples (data not shown). The adsorption of the purified LAsamples was not significantly different among the four exper-imental groups (Fig. 5A). Surface tension reduction duringcompression-expansion cycles was also not significantly dif-ferent between the two LS groups (Fig. 5B). All samples from

the HS2h group had significantly higher surface tensions ateach cycle compared with both the LS2h and HS1h groups.

Surface tension reduction of chloroform extracted samples isshown in Fig. 6 (extracted LA). These samples represent theactivity of only the hydrophobic components of the LA. Com-parison of the adsorption curves (Fig. 6A) revealed significantdifferences among samples. Adsorption was significantly

slower in the LS2h group compared with the LS1h group asreflected by longer time period to reach a surface tension below25 mN/m. The two HS groups were not significantly differentfrom each other but were both significantly slower than theirrespective LS groups. Surface tension reduction during dy-namic compression was not significantly different within boththe two LS groups and within the two HS groups. Comparisonbetween the HS and the LS groups revealed significantlyhigher surface tensions at the first, fifth, and tenth compressioncycles, in the two HS groups compared with their respectiveLS groups.

Figure 7 shows the functional properties of extracted LAsamples supplemented with 5% SP-A for each of the experi-mental groups (extracted SP-A). Adsorption was not signif-icantly different between the two LS groups but was signifi-cantly slower in the HS2h group compared with the HS1hgroup (Fig. 7A). Adsorption of samples from the HS2h groupwas also significantly slower than the adsorption in the LS2h

Fig. 4. Surface activity of crude LA of the 4 experimental groups described inFig. 1. A: surface tension during adsorption. B: surface tension during differentdynamic compression-expansion cycles. aP 0.05 for LS2h vs. LS1h; bP 0.05 for HS1h vs. LS1h; cP 0.05 for HS2h vs. HS1h; dP 0.05 for HS2hvs. LS2h.

Fig. 5. Surface activity of purified LA of the 4 experimental groups describedin Fig. 1. A: surface tension during adsorption. B: surface tension duringdifferent dynamic compression-expansion cycles. cP 0.05 for HS2h vs.HS1h; dP 0.05 for HS2h vs. LS2h.




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group. In general, surface tension reduction of the extracted LAplus SP-A (Fig. 7B) showed a similar pattern as the purifiedsamples shown in Fig. 5B. There were no significant differ-ences between the two LS groups. Samples from the HS2hgroup had significantly higher surface tensions at each cyclecompared with both, the LS2h and HS1h groups.

Experiment 3

Having established alterations and biophysical impairmentof surfactant after 1 h of HS ventilation, experiment 3 evalu-ated the physiological consequences of these alterations bysupplementing animals with exogenous surfactant at this time

point. Physiological responses over a subsequent 2-h period ofLS ventilation were recorded.

Figure 8 shows the PaO2/FIO

2values of the four experimental

groups in this experiment. During the first hour of mechanicalventilation all animals had PaO

2/FIO

2values that were not

significantly different among groups. In the subsequent 2 h ofventilation, animals in the LS-NT and LS-BLES groups main-tained PaO

2/FIO

2values above 400 mmHg. Animals that re-

ceived 1 h of HS ventilation and were subsequently switched to2 h of LS ventilation (HS-NT) had significantly lower PaO

2

values compared with the LS-NT. Treatment with BLES after1 h of HS mitigated this effect since HS-BLES had signifi-cantly higher PaO

2/FIO

2values compared with the HS-NT group

and these values were not significantly different than theLS-BLES group.

Analysis of the lavage material obtained from these animalsconfirmed the increase in surfactant LA pools in the BLES-treated animals, with no significant difference between theamounts of LA recovered between the LS-BLES and theHS-BLES groups (data not shown).

DISCUSSION

The majority of studies to date that have investigated alter-ations to the surfactant system in the setting of VILI haveperformed measurements when lung injury was severe (7, 31,36). In this context it is unknown whether the alterations are aconsequence of or an active contributor to the development andprogression of lung injury. This distinction has importantimplications for therapeutic interventions such as exogenoussurfactant administration. To address this issue we performedthree separate experiments. First, we observed alterations ofthe surfactant system of rats ventilated with a HS strategy at atime before physiological lung dysfunction was apparent and

noted that these changes increased in magnitude as lung dys-function occurred. Second, we determined that there werebiophysical consequences of the alterations to surfactant atthese two time points and that alterations to the hydrophobiccomponents of surfactant were responsible for this dysfunc-

Fig. 6. Surface activity of extracted LA of the 4 experimental groups describedin Fig. 1. A: surface tension during adsorption. B: surface tension duringdifferent dynamic compression-expansion cycles. aP 0.05 for LS2h vs.LS1h; bP 0.05 for HS1h vs. LS1h; dP 0.05 for HS2h vs. LS2h.

Fig. 7. Surface activity extracted LA 5% SP-A of the 4 experimental groupsdescribed in Fig. 1. A: surface tension during adsorption. B: surface tensionduring different dynamic compression-expansion cycles. cP 0.05 for HS2hvs. HS1h; dP 0.05 for HS2h vs. LS2h.




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tion. Finally, we administered exogenous surfactant after aperiod of HS mechanical ventilation and observed a mitigationof subsequent physiological lung dysfunction. It is concludedthat alterations of surfactant occur during ventilation prior tolung injury and that, over time, the alterations and impairmentin the biophysical activity of this material may contribute tolung dysfunction.

The specific alterations to surfactant observed after 1 h ofHS ventilation included an increase in SA in the lavage and adecreased percentage of LA. Furthermore, the biophysicalanalysis showed that these LA had an impaired surface tension-

reducing ability. These changes continued over time, such thatafter 2 h of HS ventilation, when lung dysfunction was evident,all relative changes had increased in magnitude including asevere functional impairment of the LA fraction. At this 2-htime point, the amounts of SP-B and cholesterol present in theLA were also significantly altered. Furthermore, markers oflung injury, such as total lavage protein, IL-6, and TNF-,were significantly increased after 2 h of HS ventilation, but notafter 1 h. Thus it appears that high VT initially changes thesurfactant system by changing the pool sizes of the surfactantsubfractions. Several mechanisms may account for suchchanges. For example, it has been shown that HS ventilationcan rapidly affect the activity or numbers of alveolar macro-phages in the alveolar space (10, 26). These cells are very

important in surfactant metabolism and may therefore be indi-rectly responsible for the altered pool sizes observed in thepresent study. Another potential mechanism would be throughthe effects of surfactant secretion and conversion. Previousreports have demonstrated that high-VT ventilation increasedsecretion of surfactant in the short term (9, 22). This increasedsecretion would increase the LA pool. However, studies on thealveolar metabolism of surfactant would suggest that these LArapidly converted to SA because of the large changes inalveolar surface area associated with the high-VT ventilation,leading to the observed increase in SA (12, 32, 33). Over time,this decrease of LA, combined with the impaired function ofthese LA, would contribute to an impaired surface tension

reduction within the lung leading to decreased lung function.This would then progress over time with further alterations ofthe surfactant system and the upregulation of other indexes oflung injury, such as increases in inflammatory mediators.

To further assess the effects of HS on surfactant function,the second experiment examined the biophysical properties ofthe LA. Most previous studies have utilized the crude LA

fraction to determine surfactant activity (14, 31). We examinedthis fraction as well as a sucrose gradient-purified LA, achloroform-extracted LA, and the extracted LA with SP-A.One of the main mechanisms by which surfactant impairmentoccurs is thought to be via protein inhibition (7, 16, 29). Manyin vitro and some in vivo studies have shown that addition ofserum protein to surfactant results in poor biophysical functionand can impair lung function (7, 16, 18, 23, 29, 35). In thepresent study we examined this phenomenon by purifying theLA over a sucrose gradient, thereby removing serum proteinfrom the samples. Purification of the crude HS1h samples wassufficient to rescue the surface tension-reducing ability tofunctional levels similar to LA from both LS samples that werepurified. These findings, in conjunction with the data shown inFigs. 6 and 7 showing abnormal surface tension properties inthe extracted HS1h samples and rescue of these alterationsin the extracted SP-A HS1h samples, suggest that theimpaired activity of the LA from the HS1h group is likely dueto protein inhibition in conjunction with a functional alterationto the hydrophobic components of surfactant. Interestingly, removalof serum protein from the HS2h samples did not rescue function.

The complete analyses of the various LA samples isolatedfrom these lungs at both time points revealed that alterations tothe hydrophobic components of surfactant significantly con-tributed to surfactant impairments noted at both time points ofHS mechanical ventilation. As noted, lipid extraction wasperformed to isolate the hydrophobic components of surfac-

tant. This process removed all hydrophilic components, includ-ing serum protein as well as the surfactant-associated proteinSP-A, from the crude LA samples of each group (39). Ingeneral the lipid-extracted samples had slower adsorptiontimes and were less surface active over consecutive compres-sion expansion cycles compared with the crude and purifiedLA samples. The hydrophobic components of surfactant in-clude the surfactant-associated proteins SP-B and SP-C and alllipid components of surfactant, including the neutral lipidcholesterol. The observed impairments following lipid extrac-tion in the HS samples implicates these proteins and lipids ascontributing to the observed biophysical dysfunction.

Two of the hydrophobic components may be specificallyinvolved in the observed surfactant impairment in the HS2h

samples: cholesterol and SP-B. Several recent studies haveindicated that increased cholesterol levels, as observed in theLA from the HS2h group, can impair surfactant function (13,17, 19). The source of this cholesterol is unknown in thepresent study but could come from membrane breakdown ofinflammatory cells and/or plasma contamination in the moreseverely injured HS2h group. Although the increase in choles-terol was relatively modest at the 2 h mark, these changes, inconjunction with the decreased SP-B levels and the increasedprotein levels at this time point, could have significant conse-quences.

The reduced amounts of SP-B, as noted in the HS2h group,combined with the results from the measurements of lipid

Fig. 8. PaO2/FIO2 values of animals ventilated with either a LS (VT 8 ml/kg,PEEP 5 cmH2O, RR 5456 bpm) or a HS (VT 30 ml/kg, PEEP 0cmH2O, RR 1416 bpm) strategy for 1 h. Subsequently, animals wererandomized to either no treatment (NT) control or a surfactant administration(BLES) procedure, followed by an additional 2 h of LS ventilation. eP 0.05

for HS-NT vs. HS-BLES;f

P 0.05 for HS-NT vs. LSNT.




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extracts supplemented with SP-A, suggests that SP-B is also animportant factor at 2 h. Past studies have shown that thehydrophobic protein SP-B is necessary for SP-A to enhance thebiophysical function of surfactant (6, 25). In the present study,addition of SP-A to extracted LA samples resulted in animprovement in function of the LS and HS1h extracted sam-ples compared with extracted samples lacking SP-A, indicating

that these extracted samples contained functional SP-B thatfacilitated the enhancement of biophysical properties whenSP-A was added. Conversely, the poor function observed in theHS2h samples following addition of SP-A provides evidencesupporting alterations to SP-B as contributing to surfactantdysfunction in the latter HS time point. The mechanismsresponsible for the decreased SP-B in the present study areunknown but are currently being investigated.

To further address the cause or effect issue of the surfactantalteration in VILI, the final series of experiments were per-formed in which animals were switched from HS ventilation toLS ventilation, with or without surfactant treatment after thefirst hour of mechanical ventilation. The subsequent 2 h of LSventilation, as performed in experiment 3, led to an immediatedecrease in PaO

2values in animals not receiving exogenous

surfactant. This result demonstrated that the 1 h of ventilationwith the HS strategy resulted in a subsequent deterioration inoxygenation despite utilization of a more protective ventilationstrategy over the latter time period. Furthermore, since theadministration of exogenous surfactant mitigated these subse-quent effects of 1 h of HS, it is concluded that the alterationsof the surfactant system at the 1-h time point represent animportant mechanism by which HS ventilation affected thelung over time.

The experimental model we utilized to examine the role ofsurfactant during ventilation was a rat model of VILI. Thespecific ventilation strategy implemented was based on prelim-

inary studies designed to develop a consistent decrease inoxygenation within a 2-h time period and involved a VT of 30ml/kg without PEEP. This HS ventilation strategy was com-pared with a LS strategy. One apparent limitation of the presentstudy is that the high VT values utilized are much greater thanthose used in clinical settings (4). Nevertheless, the application ofeven relatively low VT values to injured lungs with a heteroge-neously distributed pattern of injury may result in regional over-stretching of the more compliant areas of the lung, similar tothe overstretching observed in the entire lung in our model. Inaddition, the high concentrations of oxygen in conjunction withthe ventilation strategies used could also have contributed tothe changes noted in this study, although animals in all groupsreceived the same concentrations of oxygen, and the durations

of exposure were relatively short.In conclusion, previous studies have shown that surfactant

obtained from severely injured lungs of both patients withacute respiratory distress syndrome and animals with VILIexhibited impaired biophysical function compared with surfac-tant obtained from healthy lungs. The present study has ex-panded on this knowledge by demonstrating that alterations tothe pulmonary surfactant system occur prior to evidence oflung dysfunction, suggesting that these changes are a conse-quence of the ventilation itself. We have also demonstrated thatthese relatively early alterations of surfactant directly contrib-ute to lung dysfunction. Finally, we have demonstrated thatalthough the initial impairment of surfactant function is pri-

marily caused by inhibition by serum protein, as suggested inthe literature, alterations to the hydrophobic components ofsurfactant contribute to the dysfunction and are in fact the mainmechanisms for surfactant impairment at the stage of severelung dysfunction in our model of VILI.

ACKNOWLEDGMENTS

The authors thank Dr. Nils Petersen for helpful discussions. We alsoacknowledge Lynda McCaig and Dr. Li-Juan Yao for technical assistance andNevena Markovic for proofreading the manuscript.

GRANTS

These studies were supported by a grant from the Canadian Institute ofHealth Research.

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