High respiratory rate is associated with early reduction

High respiratory rate is associated with early reduction of lungedema clearance in an experimental model of ARDSJ. Retamal1,2, J. B. Borges1,3, A. Bruhn2, X. Cao4, R. Feinstein5, G. Hedenstierna6, S. Johansson4,F. Suarez-Sipmann1 and A. Larsson1

1Hedenstierna Laboratory, Department of Surgical Sciences, Section of Anaesthesiology & Critical Care, Uppsala University, Uppsala, Sweden2Departamento de Medicina Intensiva, Pontificia Universidad Cat�olica de Chile, Santiago, Chile3Cardio-Pulmonary Department, Pulmonary Divison, Heart Institute (Incor), University of S~ao Paulo, S~ao Paulo, Brazil4Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden5Department of Pathology and Wildlife Diseases, National Veterinary Institute, Uppsala, Sweden6Department of Medical Science, Clinical Physiology, Uppsala University Hospital, Uppsala, Sweden

Correspondence

Jaime Retamal, Hedenstierna Laboratory,

Department of Surgical Sciences, Section of

Anaesthesiology & Critical Care, Uppsala

University, 751 85 Uppsala, Sweden

E-mail: [email protected]

Conflicts of interest

The authors have no conflicts of interest.

Funding

Supported by Beca de Cotutela Doctoral

CONICYT, and by the Swedish Heart and Lung

Foundation and the Swedish Research Council

(K2015-99X-22731-01-4).

Submitted 8 July 2015; accepted 13 July 2015;

submission 15 June 2015.

Citation

Retamal J, Borges JB, Bruhn A, Cao X,

Feinstein R, Hedenstierna G, Johansson S,

Suarez-Sipmann F, Larsson A. High respiratory

rate is associated with early reduction of lung

edema clearance in an experimental model of

ARDS. Acta Anaesthesiologica Scandinavica

2015

doi: 10.1111/aas.12596

Background: The independent impact of respiratory rate on ven-

tilator-induced lung injury has not been fully elucidated. The aim

of this study was to investigate the effects of two clinically rele-

vant respiratory rates on early ventilator-induced lung injury evo-

lution and lung edema during the protective ARDSNet strategy.

We hypothesized that the use of a higher respiratory rate during a

protective ARDSNet ventilation strategy increases lung inflamma-

tion and, in addition, lung edema associated to strain-induced

activation of transforming growth factor beta (TGF-b) in the lung

epithelium.

Methods: Twelve healthy piglets were submitted to a two-hit

lung injury model and randomized into two groups: LRR

(20 breaths/min) and HRR (40 breaths/min). They were mechani-

cally ventilated during 6 h according to the ARDSNet strategy.

We assessed respiratory mechanics, hemodynamics, and extravas-

cular lung water (EVLW). At the end of the experiment, the lungs

were excised and wet/dry ratio, TGF-b pathway markers, regional

histology, and cytokines were evaluated.

Results: No differences in oxygenation, PaCO2 levels, systemic

and pulmonary arterial pressures were observed during the study.

Respiratory system compliance and mean airway pressure were

lower in LRR group. A decrease in EVLW over time occurred only

in the LRR group (P < 0.05). Wet/dry ratio was higher in the HRR

group (P < 0.05), as well as TGF-b pathway activation. Histologi-

cal findings suggestive of inflammation and inflammatory tissue

cytokines were higher in LRR.

Conclusion: HRR was associated with more pulmonary edema

and higher activation of the TGF-b pathway. In contrast with our

hypothesis, HRR was associated with less lung inflammation.

Editorial comment: what this article tells us

This study, performed in an animal model of ventilator-induced lung injury, tells us that higher

respiratory rate during mechanical ventilation may have potential positive effects by reducing lung

inflammation, but also negative effects by increasing lung edema.

Acta Anaesthesiologica Scandinavica 60 (2016) 79–92

ª 2015 The Acta Anaesthesiologica Scandinavica Foundation. Published by John Wiley & Sons Ltd 79

ORIGINAL ARTICLE

Mechanical ventilation is a life-saving support-

ive therapy for patients with acute respiratory

distress syndrome (ARDS) but it also may

induce lung injury, the so-called ventilator-in-

duced lung injury (VILI).1 The mechanisms of

VILI encompass distinct regional mechanical

stresses, including overstretching2,3 and repeti-

tive opening and closing of airspaces.4–7

Clinical studies have demonstrated the

importance of protective-ventilation strategies

to minimize VILI and improve outcomes.8–12

The National Institutes of Heart, Lung and

Blood ARDS Network low tidal volume

(ARDSNet) trial showed that use of low tidal

volume (VT) ventilation of 6 vs. 12 ml/kg pre-

dicted body weight significantly reduced mor-

tality.8 This strategy is the current standard for

mechanical ventilation of patients with

ARDS.13–17 Mitigation of the proinflammatory

process is proposed as the rationale for its ben-

efit.8,18–20

During the use of the ARDSNet strategy a

high respiratory rate (RR) may be necessary to

maintain adequate carbon dioxide removal and

aiming in maintaining a pH of > 7.30.21 How-

ever, there is no solid experimental data sup-

porting the safety of a high RR concerning

VILI.22 The product of the transpulmonary pres-

sure and the VT changes relative to the end-

expiratory lung volume (EELV) can be viewed

as the energy delivered by the ventilation

within the lungs and an estimate of how much

the ventilator can damage the lungs. In this

framework, the development of VILI may be

also relevantly modulated by the cyclic fre-

quency of stress application. However, the inde-

pendent and specific impact of the RR on VILI

has not been fully elucidated.22–26 Inappropriate

ventilation has been found to be associated with

increased inflammatory markers, but also with

edema formation. The edema may not exclu-

sively be caused by inflammation, but impor-

tantly by an independent mechanism, i.e.,

integrin activation of latent transforming growth

factor beta (TGF-b) by stretching the epithelial

cells. This will via downstream phosphorylation

of the signal transducer, Smad2, increase endo-

cytosis of the epithelial sodium channel com-

plex (ENaC), inhibiting edema resorption.27 In

addition, TGF-b also, is an important regulator

of inflammation and cell proliferation.28–30

The aim of the present study was to investi-

gate the effects of two clinically relevant respira-

tory rates on early VILI evolution and lung

edema during the protective ARDSNet strategy.

We hypothesized that a higher respiratory rate

increases lung inflammation and, in addition,

lung edema associated with strain-induced acti-

vation of the TGF-b pathway.

Materials and methods

The animal Ethics Committee in Uppsala (C 5/

14) approved the study and the animals were

treated in accordance with National Institute of

Health’s guidelines (NIH). We studied 12 pig-

lets (2–3 months old, weighing 27–32 kg) of

mixed Hampshire, Yorkshire, and Swedish

country breeds.

Anesthesia and instrumentation

Animals were premedicated by an IM injection

of xylazine (2.2 mg kg�1, Rompun�; Bayer, Lev-

erkusen, Germany) and tiletamine/zolazepam

(6 mg kg�1, Zoletil�; Virbac, Carros, France).

After 5–10 min, the animals were placed supine

on a table and an orotracheal intubation was

performed using a non-cuffed 8 mm internal

diameter endotracheal tube (Mallinckrodt Medi-

cal, Dublin, Ireland) and secured in place by a

tight strap.

At baseline, the lungs were ventilated with

volume-controlled mode with VT of 8 ml/kg,

RR 30 breaths per minute (bpm), positive end-

expiratory pressure (PEEP) 5 cm H2O, inspira-

tory to expiratory ratio (I:E) 1:2, and fraction of

inspired oxygen (FIO2) of 1. Just before the

endotracheal intubation, a bolus of fentanyl

0.02 mg/kg was given IV. Anesthesia was

maintained with fentanyl 0.04 mg/kg/h, keta-

mine 30 mg/kg/h, and midazolam 0.1 mg/kg/h,

and after checking that anesthesia was suffi-

cient to prevent responses to painful stimula-

tion between the front toes, muscle relaxation

was achieved by rocuronium 0.3 mg/kg/h. The

depth of anesthesia was monitored continually

by observing arterial blood pressure and heart

rate.

Thirty ml/kg/h of Ringer’s acetate was infused

IV during the first hour. Since the second hour

till the end of the establishment of the two-hit


80 ª 2015 The Acta Anaesthesiologica Scandinavica Foundation. Published by John Wiley & Sons Ltd

J. RETAMAL ET AL.

injury VILI model, Ringer’s acetate infusion rate

was 20 ml/kg/h. Thereafter, the infusion rate

was kept at 10 ml/kg/h till the end of the

protocol.

After open dissection of the neck vessels, a

central venous catheter was inserted via the

right external jugular vein. A pulmonary arterial

catheter (Edwards Life-Sciences, Irvine, CA,

USA) for measurement of cardiac output (CO)

and monitoring of pulmonary artery pressure

was introduced via the right external jugular

vein. Invasive systemic arterial pressure was

monitored through a femoral arterial catheter

(PV2015L20, Pulsion, Munich, Germany). ECG

electrodes were applied on the chest as well as

a pulse oximeter on the base of tail for continu-

ous monitoring. A lower midline mini-laparo-

tomy was performed and a bladder catheter was

inserted to measure hourly urine production.

After the instrumentation, a period of stabiliza-

tion of 15 min was implemented.

Volumetric capnography was performed with

a NICO-monitor (Philips, Wallingford, CT,

USA) using airway flow and CO2 signals from

mainstream sensors placed between the endotra-

cheal tube and the “Y” piece of the ventilator

circuit. Data were recorded by the Datacoll soft-

ware (Philips) and later handled by the Flow-

tool software (Philips). Respiratory mechanics

calculations were performed from the NICO-

monitor airway pressure and flow waveforms.

Gas exchange measurements were made using

conventional blood gas analysis (Radiometer

ABL 505; Radiometer OSM3) and derived

parameters were calculated according to the

standard formula.

VILI model

We established a two-hit injury VILI model:

repeated lung lavages with 30 ml/kg of warmed

isotonic saline were applied until a pressure of

arterial oxygen to fractional inspired oxygen

concentration (PaO2/FIO2) less than 27 kPa was

achieved. This was followed by 120 min of inju-

rious mechanical ventilation using low PEEP

(mean PEEP = 2 cmH2O), high inspiratory pres-

sures (mean plateau pressure = 36 cmH2O), RR

20 bpm, and I:E 1:2. This model has previously

been found to produce inflammatory changes

compatible with ARDS.31,32

Investigational protocol

Gas exchange, lung mechanics, and hemody-

namic parameters were assessed after the initial

stabilization period (Baseline), after the estab-

lishment of our lung injury model (H0), and

hourly during 6 h of application of the ARDS-

Net strategy (see below) (H1–H6). Plasma sam-

ples for cytokines measurements were taken at

Baseline, H0, H3, and H6. Post-mortem, regional

lungs samples were extracted to evaluate tissue

cytokines, TGF-b pathway biomarkers, wet/dry

weight ratio, and for histological analysis. Bron-

choalveolar lavage fluid (BAL) samples were

collected at the end of the protocol (Fig. 1).

ARDSNet strategy

During the first 15 min of application of the

ARDSNet strategy, the following settings were

used in all animals: VT 6 ml/kg, PEEP 14

cmH2O, Inspiratory time 0.5 s, FIO2 = 0.7, and

RR 30 bpm. At the end of this period, the H0

measurements were performed. Then, the ani-

mals were randomized to RR = 40 bpm (high

respiratory rate group; HRR) or RR = 20 bpm

(low respiratory rate group; LRR).

To keep similar PaCO2 levels between groups

and based on pilot data, we added adjustable

instrumental dead space (IDS) in the HRR and

cut the endotracheal tube in the LRR. The IDS

was inserted between the “Y” piece and the dis-

tal flow sensor, which was placed at the endo-

tracheal tube opening. This device allowed us to

adjust the amount of rebreathed gas (that con-

tained CO2) to keep PaCO2 at the desired level.

Then, the following mechanical ventilation

settings were applied and maintained till the

end of the study: VT of 6 ml/kg, PEEP

10 cmH2O, and FIO2 0.5.

Lung tissue analysis

After euthanasia, during the opening of the

chest wall, ventilation was maintained identical

to the protocol. Then, the inferior cava vein was

sectioned, the trachea was clamped at end-expi-

ratory lung volume, and heart and lungs were

excised in bloc. Right lungs were clamped at

the hilum, and lung tissue samples were

collected from the following regions: superior



RESPIRATORY RATE EFFECTS ON VILI

lobe-apical (SA), middle lobe-intermediate

(IM); and ventral (CV), intermediate (CI), and

dorsal (CD) regions of the inferior lobe. Samples

were immersed in 10% buffered formalin, pro-

cessed, and stained with hematoxylin–eosin for

histological analysis. The CV, CI, and CD

samples were evaluated histologically by a

pathologist blindly, which performed a semi-

quantitative analysis based on: alveolar collapse,

emphysema, alveolar edema, alveolar and alveo-

lar septa leukocytes infiltration, perivascular,

bronchiolar, peribronchiolar leukocytes infiltra-

tion, and interlobular and alveolar septa edema

and leukocytes infiltration. Each item was

scored based on the following scale: 0 = not

observed; 1 = mild; 2 = moderate; 3 = severe;

and 4 = very severe. Other samples of the same

regions were snap-frozen in liquid nitrogen,

kept at �80°C and later on processed for bio-

chemical and molecular biology analysis.

Wet-to-dry ratio

Wet-to-dry (W/D) ratio was measured in sam-

ples of the same lung regions from the right

lung. Briefly, samples of 2–4 g were weighted,

dried in a oven at 50°C by 72 h, and then

weighted again.

Bronchoalveolar lavage

BAL was performed in the left lung by intra-

bronchial injection of 50 ml of normal saline

solution and subsequent aspiration. The recov-

ered fluid was centrifuged and snap-frozen.

Cytokines

By using the ELISA method, the following

cytokines concentrations were measured in lung

homogenates, plasma, and bronchoalveolar

lavage (BAL): interleukin (IL)-1b, IL-6, IL-8, IL-10, and tumor necrosis factor alpha (TNFa).

TGF-b pathway assessment

Cryosections (5 lm) of formalin-fixed biopsies

from pig lungs were stained with 2 lg/ml of

rabbit polyclonal anti-phospho-Smad2 IgG [27].

Secondary goat anti-rabbit antibodies conju-

gated with Alexa Fluor 568 (Life Technologies)

were used at 1:1000 dilutions. Nuclei were visu-

alized with 1 lg/ml Hoechst 33342 (VWR Inter-

national, Radnor, PA, USA). Fluoromount-G

mounting medium (Southern Biotech, Birming-

ham, AL, USA) was used to mount stained sec-

tions. Pictures were with a Nikon Eclipse 90i

microscope, using the 209 objective at the same

exposure time for all samples.

Statistical analysis

We assessed that for a P < 0.05, with a power of

0.8, six animals in each group were needed if the

difference between the outcome variable W/D

would be 1 � 0.5.

We expressed values as means � standard

deviation (SD) or median � range where appro-

priate. The Shapiro–Wilk test was used to test

data for normality. Groups were compared using

Student’s t-test or Mann–Whitney U-test, one-

way (repeated-measures) analysis of variance

(ANOVA) or Kruskal–Wallis test. Interactions

between groups and time were assessed with

two-way repeated-measures ANOVA. The Bon-

ferroni adjustment for multiple tests was

applied for post hoc comparisons. The statistical

analyses were conducted by SPSS v.20.0.0 soft-

ware (SPSS, Inc, Chicago, IL, USA), and Graph-

Pad Prism version 5.0 (GraphPad Software, San

Diego, CA, USA). Statistical tests were carried

out with the significance level set at P < 0.05.

Preparation Lunglavages

Ventilator-induced lung injury(2 h)

RESP. RATE 20 bpm, ARDSNet ventilation (VT 6 ml/kg, PEEP 10 cmH2O, FIO2 0.5)

RESP. RATE 40 bpm, ARDSNet ventilation (VT 6 ml/kg, PEEP 10 cmH2O, FIO2 0.5)

H0 H6 H3

Fig. 1. Experimental timeline. Ventilator-induced lung injury (mean PEEP = 2 cmH2O, mean plateau pressure = 36 cmH2O). Black arrows

correspond to plasma sampling; white arrow corresponds to bronchoalveolar lavage and tissue sampling.



J. RETAMAL ET AL.

Results

At baseline measurements, no significant differ-

ences between groups were found in body weight,

hemodynamics, and gas exchange (Table 1).

Respiratory system mechanics

After establishment of the two-hit injury VILI

model, mean airway pressure increased (7 � 0.4

to 15 � 1.9 cmH2O), as well as plateau pressure

(12 � 1.1 to 25 � 2.1 cmH2O) and peak airway

pressure (14 � 1.1 to 27 � 2.4 cmH2O), whereas

static compliance decreased (28 � 4.3 to 15 �1.8 ml/cmH2O). These variables did not differ

between groups. The evolution of respiratory

system mechanics and mechanical ventilation

parameters during the ARDSNet strategy is pre-

sented in Table 2 and Fig. 2.

Gas exchange

Figure 2 exhibits the gas exchange data. After

establishment of the two-hit injury VILI model,

the PaO2/FIO2 decreased from 63.2 � 5.1 to

36.7 � 13.2 kPa. At the beginning of the ARDSNet

strategy, no difference between the HRR and LRR

groups in PaO2/FIO2 was evident, but the LRR

group presented an increase of PaO2/FIO2 over time

(P < 0.05). There was no difference in venous

admixture (%) between HRR and LRR groups

(P > 0.05): at baseline 13.4 (1.3) and 13 (1.4), H0:

19.5 (4.6) and 24 (7.8), and H6: 26 (8.3) and 23.1

(5). The pH values did neither show differences

between groups nor over time (HRR = H1: 7.16,

H6: 7.17; and LRR = H1: 7.19, H6: 7.17).

Hemodynamics

During the ARDSNet strategy, heart rate

increased significantly in both groups, but car-

diac output increased only in the HRR group

(P < 0.05; Table 3). Figure 3 exhibits the evolu-

tion of the extravascular lung water measure-

ments in both groups. During and at the end of

the experiment, fluid intake and diuresis were

not different between groups.

Wet-to-dry ratio

Pooling together all regional samples of each

animal and then comparing HRR vs. LRR, the

W/D ratio in the HRR group was greater than

LRR (P < 0.001; Fig. 4).

In the CV region, LRR presented higher W/D

than the rest of samples of the same group

(P < 0.05), and similar levels than HRR group.

Histological analysis

Histological findings are shown in Table 4.

There was a higher peribronchiolar and perivas-

cular leukocyte infiltration in the ventral region

samples from the LRR group than in samples

from the same region from the HRR group

(P < 0.05; (Fig. 5).

Cytokines analyses

IL-10, IL-8, and TNFa concentrations in lung

homogenates increased in response to LRR

(P < 0.05, two-way repeated-measures ANOVA

– effect of RR). IL-6 showed interaction between

RR and lung region (P < 0.05). RR did not influ-

ence IL-1b concentration in lung homogenates

(Fig. 6). BAL analysis showed higher concentra-

tions of IL-1b in LRR vs. HRR (P < 0.01). Quan-

tification of plasma cytokines demonstrated very

low values and in most samples under the

detection limit, not showing any difference

between groups.

TGF-b pathway assessment

Immunohistochemistry of lung tissue exhibited

higher pSmad2-stained cells in the HRR group

compared with the LRR group (P < 0.05;

Fig. 7).

Table 1 Baseline characteristics from LRR and HRR groups.

Variable LRR HRR

Body weight (kg) 30 (1.1) 29 (1.4)

Arterial pH 7.43 (0.02) 7.47 (0.05)

PaCO2 (kPa) 5.9 (0.3) 5.5 (0.5)

PaO2/FIO2 (kPa) 63.1 (4.1) 63.1 (6.2)

Mean arterial pressure (mmHg) 75 (7.2) 89 (11.7)

Cardiac output (l/min) 3.8 (1.0) 4.1 (0.8)

EVLW (ml) 296 (49.7) 287 (42.2)

LRR, low respiratory rate (20 bpm); HRR, high respiratory rate

(40 bpm); EVLW, extravascular lung water. No significant differ-

ences between groups were observed. Values expressed as

mean (SD).




Discussion

The present study evaluated the isolated and

independent effect of two clinically relevant RRs

on the evolution of early experimental VILI dur-

ing the protective ARDSNet strategy. HRR was

associated with increased pulmonary edema and

higher activation of the TGF-b pathway, whereas

LRR showed evidence of higher inflammation.

Our experimental model, saline lavage fol-

lowed by injurious mechanical ventilation with

high volumes and low PEEP, was developed to

Fig. 2. Evolution of some physiological measurements in both groups. Low respiratory rate group (20 breaths/min) and HRR: high respiratory rate

group (40 breaths/min). Symbols (*) refer to post hoc analysis (one-way ANOVA) from each respiratory rate group, hour 1 vs. hour 6. P < 0.05.

Two-way ANOVA analysis showed group effect for Cdyn, Driving pressure, and VD/VT, P < 0.05. No interaction (time vs. group) was observed on

these variables P < 0.01. Values expressed as mean (SD). Cdyn, Dynamic respiratory system compliance; Driving pressure, Plateau pressure –

positive end-expiratory pressure; VD/VT, dead space; Mean PAP, mean pulmonary arterial pressure.

Table 2 Respiratory variables.

Variable Group Hour 1 Hour 2 Hour 3 Hour 4 Hour 5 Hour 6

Group

effect

Group 9 time

effect

VT/kg (ml/kg) LRR 5.3 (0.2) 5.3 (0.2) 5.3 (0.2) 5.4 (0.2) 5.4 (0.1) 5.5 (0.2) NS NS

HRR 5.5 (0.1) 5.5 (0.1) 5.5 (0.1) 5.5 (0.1) 5.5 (0.1) 5.5 (0.1)

Minute volume

(ml/min)

LRR 3105 (228) 3101 (210) 3104 (200) 3142 (190) 3157 (168) 3179 (170) P < 0.05 NS

HRR 6414 (379) 6415 (353) 6425 (368) 6457 (371) 6452 (380) 6476 (390)

Pmean (cmH2O) LRR 11.8 (0.5) 11.9 (0.5) 11.9 (0.5) 11.6 (0.3) 11.5 (0.3) 11.6 (0.3) P < 0.05 NS

HRR 13.4 (0.8) 13.0 (0.4) 13.0 (0.3) 12.9 (0.3) 12.9 (0.3) 12.8 (0.3)

Ppeak (cmH2O) LRR 26.4 (1.6) 26.6 (1.9) 26.8 (2.1) 26.7 (2.3) 26.3 (2.4) 26.0 (2.4) NS NS

HRR 25.6 (1.2) 25.4 (1.3) 25.5 (1.0) 25.2 (1.3) 24.7 (1.0) 25.3 (1.5)

PEEPi (cmH2O) LRR 0.1 (0.1) 0.1 (0.2) 0.1 (0.1) 0 (0.1) 0 (0) 0.1 (0.2) NS NS

HRR 0.1 (0.1) 0.1 (0.1) 0.1 (0.1) 0.1 (0.1) 0.1 (0.1) 0.1 (0.1)

Tidal alveolar

ventilation (ml)

LRR 48 (4.2) 49.7 (5.4) 48.1 (5.9) 49.3 (6) 51.2 (6.1) 50.6 (6.7) NS NS

HRR 55.8 (14) 46 (11.1) 42.7 (15.1) 44.6 (15) 38 (22.4) 37.5 (17.2)

LRR, low respiratory rate; HRR, high respiratory rate; VT, tidal volume; Pmean, mean airway pressure; PEEPi, intrinsic positive end-expiratory

pressure.



J. RETAMAL ET AL.

reproduce as faithfully as possible the full char-

acteristics of early human ARDS. Surfactant

depletion by saline lavage results in a decrease

in lung compliance and hypoxemia33 but not in

alveolar epithelial injury or neutrophilic alveoli-

tis.34 When saline lavage is followed by injuri-

ous mechanical ventilation, it results in a type

of lung injury, which is very similar to ARDS in

humans.35 Thus, the used model results in

heterogeneously aerated lungs with significant

amounts of non-aerated lung tissue, tidal R/D,

hyperinflated lung tissue, tidal hyperinflation,

and, regionally distributed lung inflamma-

tion.31,32 In accordance with previous studies,

we found that lung edema and inflammation

develop within six hours after establishing a

lung injury model.31,32,36–38

To date, the effect of RR has not been studied

in clinical trials of protective ventilation strate-

gies. In these trials, RR was assigned only on

basis of pH levels, and/or the presence or not of

intrinsic PEEP, and/or an arbitrary upper limit

for RR. Importantly, none of these criteria can

discriminate any independent and specific effect

of RR on VILI.

Lung inflammation assessment

Our data show a higher concentration of some

cytokines in lung parenchyma from the LRR

group. However, the concentrations of cytokines

from most plasma samples were under the

detection threshold suggesting that the inflam-

mation was minor and/or very compartmental-

ized. Moreover, the cytokine levels reflect only

the early phase of ARDS, and not the whole

evolution of ARDS.

The higher degree of inflammation in the LRR

compared with HRR group could be explained

Table 3 Hemodynamic variables.

Variable Group Hour 1 Hour 2 Hour 3 Hour 4 Hour 5 Hour 6

Group

effect

Group 9 time

effect

Heart rate (beats/

min)

LRR 102 (15.9)* 113 (17.8) 122 (19.3) 130 (18.7) 131 (17.2) 126 (17.4)* NS NS

HRR 105 (9.9)* 114 (13.5) 117 (14.2) 118 (13.8) 121 (16.4) 125 (19.5)*

MAP (mmHg) LRR 84 (5.8) 83 (8) 82 (9.8) 82 (9.6) 80 (10.9) 78 (11.7) NS NS

HRR 82 (4.8) 83 (10.3) 82 (10.3) 82 (12) 81 (10.3) 81 (11.6)

PCWP (mmHg) LRR 10 (1.6) 9 (1.5) 9 (2) 8 (1.5) 8 (1.1) 8 (1) NS NS

HRR 10 (1.2) 10 (1.2) 9 (0.6) 9 (1.7) 9 (0.6) 9 (0.7)

Hemodynamic variables are shown for each time and each respiratory rate. LRR, low respiratory rate; HRR, high respiratory rate; MAP, mean

arterial pressure; PCWP, pulmonary capillary wedge pressure. Symbols (*) refer to post hoc analysis, hour 1 vs. hour 6 (one-way ANOVA)

from each respiratory rate group. Values expressed as mean (SD). *P < 0.05.

Fig. 3. Evolution of the extravascular lung water (EVLW)

measurements in both groups. Black circles correspond to low

respiratory rate group (LRR, 20 breaths/min), and white squares

correspond to high respiratory rate group (HRR, 40 breaths/min).

Symbol * refers to P < 0.05 (Bonferroni’s post hoc analysis). Also,

there is interaction between LRR and HRR group P < 0.01 (two-way

ANOVA).

Fig. 4. Comparison of wet-to-dry (W/D) weight ratio between both

respiratory rates setting at the end of the experiment. LRR

corresponds to low respiratory rate group (20 breaths/min), and HRR

corresponds to high respiratory rate group (40 breaths/min). Symbol *

refers to P < 0.05.




by two possible mechanisms; (1) by possible

cyclic recruitment/decruitment (R/D) and the

somewhat higher driving pressures inducing

inflammation in the LRR group,6,39 or (2) TGF-b“context-dependent” actions and/or anti-inflam-

matory effects induced by the higher TGF-b con-

centration downregulating inflammation in the

HRR group. Thus, “context-dependent” mecha-

nism consists in that TGF-b can either inhibit or

enhance effector function over inflammatory

cells depending on the environmental cytokines

profile. High concentrations of TGF-b, as we

observed in the HRR group, can suppress and

antagonize production of several cytokines from

macrophages and NK cells. The importance of

TGF-b in limiting inflammation is clearly

demonstrated in TGF-b knockout mice, which

die at 3–4 weeks of age from a wasting syn-

drome associated with multifocal inflammatory

disease.30

In contrast to our study, several experimental

studies have suggested that low respiratory rate

reduces inflammation, edema, and alveolar dam-

age.22–26 However, most of these studies were

performed in rodents and furthermore, did not

fully control tidal volumes, inspiratory times,

and importantly, inspiratory flows, which were

lower in the LRR group. Indeed, limiting inspi-

ratory flow has been shown to be highly lung

protective when using a volume-controlled pres-

sure-limited mode.25 In addition, most of these

studies examined extreme values of RR.22,25,26,40

Table 4 Histological analysis.

Region

Alveolar

Collapse Emphysema

Alveolar

Edema

Alveolar

Leukocytes

Perivascular and peribronchial

leukocytes

Interlobular edema and leukocytes

infiltration

Non-dependent

LRR 0 (0–1) 1 (1–3) 4 (0–4) 1 (1–3) 4 (3–4) 3 (3–4)*

HRR 0 (0–1) 2 (1–3) 4 (0–4) 2 (1–3) 4 (2–4) 2 (2–3)*

Intermediate

LRR 2 (0–3) 2 (1–2) 0 (0–0) 2 (1–2) 3 (1–3) 2 (1–3)

HRR 3 (2–3) 1 (1–2) 0 (0–4) 1 (1–2) 3 (2–3) 2 (2)

Dependent

LRR 4 (0–4) 1 (0–1) 0 (0–0) 1 (0–1) 1 (1–3) 2 (1–3)

HRR 3 (3–4) 1 (1–2) 0 (0–4) 1 (1–2) 2 (2–3) 2 (2)

Quantitative and morphometric analysis of lung samples from LRR = low respiratory rate groups; HRR = high respiratory rate groups. Non-

dependent (CV region), intermediate (CI region), and dependent (CD region). Values expressed as median (min-max). *P < 0.05.

* *

*

A B

Fig. 5. Lung histological preparations. Panel A shows a section from caudal ventral (CV) region (10X) of a representative animal in the high

respiratory rate group. Pulmonary edema appears as an amorphous, feathery eosinophilic material, barely visible, in the alveoli and the

interlobular septum (black arrow). Not also a dilated lymphatic capillary in the septum (§) and low numbers of inflammatory cells in the alveoli and

the septum. Two contracted bronchioles are visible (*). Panel B shows a section from CV region (10X) of a representative animal in the low

respiratory rate group. Severe acute inflammation: alveoli display numerous leukocytes, predominantly polymorphs and macrophages, and also

amorphous proteinaceous contents, consistent with a serous inflammatory exudate. A bronchiovascular unit displaying a collapsed bronchiolus is

visible (*).



J. RETAMAL ET AL.

In our study, the only difference between the

groups was a clinically relevant RR. However,

as both groups had identical inspiratory flow,

inspiratory time, PEEP, and tidal volumes, min-

ute ventilation was doubled in the HRR group

compared with the LRR group. Hypercapnic aci-

dosis resulted as expected by the low minute

ventilation associated with LRR strategy. Hyper-

capnia might reduce inflammation and lung

injury as found in several experimental mod-

els.41,42 Therefore, to keep PaCO2 and alveolar

ventilation similar in both groups, we added an

extra instrumental dead space in the HRR

group, eliminating the confounding effect of

hypercapnic acidosis. Another option had been

to add exogenous CO2 in the inspired gas,

instead of increasing the rebreathed CO2 to the

HRR group. This would have eliminated the

need of instrumental dead space, but in other

regards not influenced the study design or

results

Hyperventilation has been shown to have

deleterious effects, not only during mechanical

ventilation but also in spontaneously strenu-

ously breathing healthy animals43 Moreover,

ultraprotective mechanical ventilation strategies

suggest that “less ventilation, less injury”.44–46

However, in these experiments, it is not possi-

Fig. 6. Levels of cytokines on lung

homogenates. Levels IL-6, IL-8, IL-10, and

tumor necrosis factor alpha (TNFa) on tissue

homogenates separated by lung regions:

Non-dependent region (Non-D), intermediate

region (Interm), and dependent region (Dep).

Dark boxes correspond to low respiratory

rate, and white boxes correspond to high

respiratory rate. IL-10, IL-8, and TNFa

showed main effect of respiratory rate,

P < 0.05 (two-way ANOVA). IL-6 showed

interaction between RR and lung region

(P < 0.05).

A B

Fig. 7. Representative lung sections from superior lobe (SA) from (A) LRR, low respiratory rate groups, and (B) HRR, high respiratory rate groups.

Determination of pSmad2 as determinants of TGF-b pathway activation was done through immunostaining. In blue: cellular nuclei (Hoechst 33342),

and in red pSmad2 (+) nuclei (Alexa Fluor 568). pSmad2: phosphorylated mothers against decapentaplegic-2/3.




ble to separate the effects of VT, RR, minute

ventilation, and hypocapnia as the igniting

cause of VILI development.

Baumgardner et al. have shown that the mag-

nitude of PaO2 oscillations decreases with

increasing RR,47 suggesting that the dynamics

of the lungs, i.e., RR and expiratory time and

not only the statics, i.e., the PEEP levels, are

important for cyclic recruitment/derecruitment.

The dynamics is a function of regional alveolar

tissue mechanics, regional compliance, regional

airway resistance, and gas/liquid interfacial

mechanics determining the time constants for

regional lung collapse. Thus, a high RR reduces

the available time for expiratory collapse and

thus, prevents cyclic R/D at low extrinsic

PEEP.47 In addition, a possible explanation is

that in the HRR group intrinsic PEEP occurred

in lung regions with longer time constants

reducing regional derecruitment. In fact, Neu-

mann et al. showed by dynamic computed

tomography scanning in oleic acid injury in pigs

that by reducing the expiratory time, time-de-

pendent collapse was prevented.48 Recently, the

same group found in a surfactant-depleted

model that the reverse was true, i.e., that pro-

longed expiratory time promoted alveolar col-

lapse, cyclic R/D, and also regional ventilation

redistribution to non-dependent region.49 In our

study, the expiratory time was almost doubled

in the LRR group, and it is conceivable that this

caused a higher degree of collapse that is also

indicated by the lower respiratory compliance.

This implies that the end-expiratory lung vol-

ume was lower in the LRR group, and therefore

the VT was distributed in a smaller lung volume

generating higher dynamic strain,50 and thus

injury in the ventilated lung regions. In fact, in

the LRR group, inflammation was concentrated

to the ventral, non-dependent, i.e., ventilated

lung regions and not to the collapsed, i.e., not

ventilated, lung regions.49 Notwithstanding,

another possible mechanism for more inflamma-

tion in the low LRR group is a difference in

driving pressures (DP). DP, the difference

between cyclic end-inspiratory and end-expira-

tory pressure, may be an especially interesting

clinical estimate of lung stress as it is inversely

proportional to respiratory system compliance.

When analyzed during constant flow ventilation,

DP contains implicit information regarding on

how changes in PEEP modulate cyclic stretch.

Tidal volume/dynamic strain, when normalized

for lung compliance (i.e., DP), contains addi-

tional useful information regarding the interplay

between VT, plateau pressure, and PEEP and

their role in VILI.50,51 In our study, the LRR

was associated with significant higher DP.Amato et al. examined DP as an independent

variable associated with survival in ARDS

patients. Their analyses indicated that reduc-

tions in VT or increases in PEEP were beneficial

only if associated with drops in DP and no other

ventilation variable exhibited such a mediating

effect.39

Lung water content assessment

The HRR lungs presented higher water content

at the end of experiment (Fig. 3). But, observing

the temporal changes of EVLW (Fig. 2), we

interpret these findings not just as a gaining of

water but also as a decrease in the ability to

clear lung edema. The primary mechanism

responsible for the resolution of alveolar edema

is active vectorial sodium transport across the

alveolar epithelium.52 In ARDS, factors that

may impair the resolution of alveolar edema are

hypoxia, high tidal volume, and several cytoki-

nes such as IL-1b, IL-8, and TGF-b. TGF-b is a

cytokine implicated in inflammation, fibrosis,

and wound repair processes, and it is activated

by, e.g., mechanical strain. TGF-b signaling pro-

teins pSmad2/3 promptly reduce transepithelial

sodium transport by inducing endocytosis of

abc epithelial sodium channel complex (ENaC),

then promoting alveolar flooding and persis-

tence of pulmonary edema. Recently, Peters

et al. demonstrated that BAL fluids from ARDS

patients rapidly led to ENaC internalization on

lung epithelial cell cultures.27 This pathway

drives a rapid (within 30 min) and dramatic

(> 80%) reduction in the cell-surface abundance

of ENaC. Administration of TGF-b to rabbits

and rats blocked the sodium transport and

caused fluid retention.27,53 Corroborating the

role of TGF-b in ARDS, two studies demon-

strated increased TGF-b levels in lung fluids

from patients with ARDS and, furthermore,

lower TGF-b levels correlated with more venti-

lator-free and intensive care unit-free days.54,55

We assessed the activation of this pathway in



J. RETAMAL ET AL.

both RR groups. By immunohistochemistry

analysis, a higher activation in the HRR group

was evident (Fig. 7). The continuous higher

degree of mechanical strain in the HRR group

might be reason for this finding and suggest a

possible mechanism for the impairment of lung

water clearance associated with HRR.

The HRR group presented higher respiratory

system compliance and consequently larger lung

volume. Egan et al. studied alveolar permeabil-

ity at different levels of lung inflation in adult

sheep.56 Their results demonstrated that the

epithelium became permeable to larger mole-

cules as inflation volume was increased. More-

over, the inflation changes in epithelial

permeability appeared restricted to situations in

which only a fraction of the lung expands in

response to high pressures,56–58 such as in the

clinical situation of respiratory failure as well as

our experimental study, where much of the air

space is collapsed. In the same direction, Bhat-

tacharya et al. suggested that high lung inflation

contracts the interstitial liquid channels in ede-

matous lungs and, consequently, decreases the

ability to clear edema.59 Therefore, the higher

water content of our HRR lungs may also be

related to a larger, more inflated, lung volume.

Our study has limitations. First, it is an ani-

mal ARDS model with all its inherent limita-

tions and although it mirrors many of the

characteristics of ARDS, it is not possible to

translate our findings directly to patients. Sec-

ond, 6 h of mechanical ventilation according to

the ARDSNet strategy may be insufficient to

comprehensively evaluate the complete inflam-

matory and lung edema formation/reabsorption

processes. Thus, our results mainly reflect the

very early changes associated with the use of

two respiratory rates, and we cannot assure that

these results remain the same during a longer

protocol. Finally, different tissue samples were

used for immunohistology of the TGF-b path-

way and for analyses of lung tissue cytokines.

Conclusions

In conclusion, high respiratory rate was associ-

ated with more pulmonary edema and higher

activation of the TGF-b pathway, whereas low

respiratory rate was associated with higher lung

inflammation.

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Documents

High respiratory rate is associated with early reduction