time course of respiratory mechanics and pulmonary structural remodelling in acute lung injury

Respiratory Physiology & Neurobiology 143 (2004) 49–61

Time course of respiratory mechanics and pulmonarystructural remodelling in acute lung injury

Patricia R.M. Roccoa,∗, Livia D. Facchinettib, Halina C. Ferreirab, Elnara M. Negric,Vera L. Capelozzid, Debora S. Faffeb, Walter A. Zinb

a Laboratory of Pulmonary Investigation, Carlos Chagas Filho Institute, Federal University of Rio de Janeiro,Ilha do Fundao, Rio de Janeiro, 21949-900, Brazil

b Laboratory of Respiration Physiology, Carlos Chagas Filho Biophysics Institute, Federal University of Rio de Janeiro,Ilha do Fundao, Rio de Janeiro 21949-900, Brazil

c Department of Thoracic Medicine, Hospital A.C. Camargo, São Paulo, Brazild Department of Pathology, Faculty of Medicine, University of São Paulo, São Paulo, Brazil

Accepted 24 June 2004

Abstract

The aim of this study was to evaluate the time course of in vivo and in vitro respiratory mechanics and examine whetherthese parameters could reflect the temporal changes in lung parenchyma remodelling in paraquat (PQ)-induced lung injury.Measurements were done 1, 3 and 8 weeks after the intraperitoneal (i.p.) injection of saline (control) or paraquat (7 mg kg−1)i Q8, in ac-c number ofp n PQ3 andP e fractiono vementi astance andv©

K als; Rat;P

f teersy.

1

n rats. Airway and tissue resistances increased from control in PQ1 and PQ3 and returned to control values in Pordance with the magnitude of bronchoconstriction. Viscoelastic/inhomogeneous pressure, tissue elastance, theolymorphonuclear cells, and collagen fibre content in lung parenchyma increased in PQ1 and remained elevated iQ8. Static elastance increased in PQ1, returned to control values after 3 weeks, and was correlated with the volumf collapsed alveoli. In conclusion, there is a restoration of normal alveolar-capillary lung units with a gradual impro

n airway and tissue resistances and static elastance. However, the on-going fibrotic process kept elevated tissue eliscoelastic/inhomogeneous pressure.2004 Elsevier B.V. All rights reserved.

eywords: Mechanics of breathing; Elastance; Lung viscoelasticity; Hysteresivity; Extracellular matrix; Collagen; Elastin; Mammathology; Lung histopathology

∗ Corresponding author. Tel.: +55 21 2562 6557;ax: +55 21 2280 8193.

E-mail address:[email protected] (P.R.M. Rocco).

1. Introduction

The evolution of acute lung injury (ALI) is quivariable and has been the subject of great controv

569-9048/$ – see front matter © 2004 Elsevier B.V. All rights reserved.doi:10.1016/j.resp.2004.06.017

50 P.R.M. Rocco et al. / Respiratory Physiology & Neurobiology 143 (2004) 49–61

Acute lung injury elicits events responsible for restor-ing the tissue to its normal architecture and function.However, these events do not always result in repair,and might trigger tissue remodelling, leading to irre-versible damage (Meduri et al., 1998; Marshall et al.,2000).

Although ALI is typically described as presentingthree consecutive phases: exudative, proliferative, andfibrotic, recent evidence suggests that there is a muchgreater overlap of the inflammatory and fibroprolifer-ative phases than previously imagined (Marshall et al.,2000). Furthermore, the amount of pro-collagen typesI (Liebler et al., 1998) and III (Chesnutt et al., 1997;Meduri et al., 1998; Pugin et al., 1999), and the num-ber of collagen fibres (Rocco et al., 2001) increasesearly in the course of ALI, suggesting that the prolif-erative phase begins much sooner than had been previ-ously appreciated. Efforts to understand the pathogen-esis of pulmonary fibrosis have concentrated primarilyupon collagen turnover in the lung. Although elastinis a chief component of lung interstitium, and it is es-sential to lung morphology and function (Hoff, 1999);few studies have focused on elastin (Rocco et al., 2001,2003). We demonstrated that the various types of elas-tic fibres (elaunin, oxytalan or fully developed elasticfibres) have different impact on respiratory mechanicalbehaviour (Rocco et al., 2001). These findings suggestthat the different phases in the course of ALI couldpotentially have different impact over dynamic respi-ratory mechanical parameters. Thus, establishing thet od-e ingo theo

val-u an-i ec nd,b ndl at-i alm nicalfi al-t ing,a olarc me-c hys-i rk

within the connective tissue matrix (Fredberg andStamenovic, 1989; Yuan et al., 1997, 2000). Lungparenchyma remodelling was evaluated by light andelectron microscopy.

2. Methods

2.1. Animal preparation

Twenty Wistar rats (250–300 g) were randomly di-vided in four groups. In the control group (C) saline(5 ml kg−1 body weight (BW)) was intraperitoneally(i.p.) injected. In PQ group paraquat was injected(7 mg kg−1 BW i.p.). One, three and eight weeks af-ter the injection of either saline or paraquat the animalswere sedated with diazepam (5 mg i.p.), anaesthetisedwith pentobarbital sodium (20 mg kg−1 BW i.p.), anda snugly fitting cannula (1.7 mm i.d.) was introducedinto the trachea. Mechanical ventilation (model 683,Harvard Apparatus, Southnatick, MA, USA) was thenstarted with a frequency of 80 breaths min−1 and a tidalvolume of 6 ml kg−1.

A pneumotachograph (1.5 mm i.d., length = 4.2 cm,distance between side ports = 2.1 cm) was connectedto the tracheal cannula for the measurements of air-flow (V′) and changes in lung volume (VT) (Mortolaand Noworaj, 1983). The pressure gradient across thepneumotachograph was determined by means of aValidyne MP45-2 differential pressure transducer (En-g wr -ca ep ryr esenti witha En-g ino rc 0-c lesa dif-f ico).T thens osi-te sure

emporal consequences of lung parenchyma remlling in ALI is essential to both our understandf the key pathological processes involved and toptimal timing and targeting of therapies.

The aim of the present study was therefore, to eate the temporal evolution of respiratory mech

cs in ALI, in parallel with the analysis of the timourse of lung morphological changes. To that eoth in vivo and in vitro respiratory mechanics a

ung histology were studied in a model of paraqunduced mild lung injury. While in vivo mechanic

easurements can be more easily related to clinding, the in vitro method avoids the influence oferations in the surfactant system, alveolar floodnd of tissue inhomogeneities secondary to alveollapse/hyperdistention. The oscillatory tissuehanics provides a direct assessment of tissue pology, as well as the role of fibre–fibre netwo

ineering Corp., Northridge, CA, USA). The floesistance of the equipment (Req), tracheal cannula inluded, was constant up to flow rates of 26 ml s−1, andmounted to 0.12 cmH2O mL−1 s. Equipment resistivressure (=Req/V′) was subtracted from pulmonaesistive pressure so that the present results reprntrinsic values. Tracheal pressure was measuredValidyne MP-45 differential pressure transducer (ineering Corp, Northridge, CA, USA). Changesesophageal pressure (Pes), which reflects pleural ohest wall pressure (Pw), were measured with a 3m-long water-filled catheter (PE-200) with side hot the tip connected to a PR23-2D-300 Statham

erential pressure transducer (Hato Rey, Puerto Rhe catheter was passed into the stomach andlowly returned into the oesophagus; its proper pioning was assessed using the “occlusion test” (Baydurt al., 1982). The frequency responses of the pres

P.R.M. Rocco et al. / Respiratory Physiology & Neurobiology 143 (2004) 49–61 51

measurement systems (Ptr and Pes) were flat up to20 Hz, without appreciable phase shift between the sig-nals. All signals were conditioned and amplified in aBeckman type R Dynograph (Beckman Instruments,Schiller Park, IL, USA). Flow and pressure signalswere also passed through 8-pole Bessel filters (902LPF,Frequency Devices, Haverhill, MA, USA) with the cor-ner frequency set at 100 Hz, sampled at 200 Hz with a12-bit analogue-to-digital converter (DT2801A, DataTranslation, Marlboro, MA, USA), and stored on aPC-compatible computer. All data were collected us-ing LABDAT software (RHT-InfoData Inc., Montreal,Que., Canada).

2.2. Measurement of respiratory mechanics

Muscle relaxation was achieved with gallamine tri-ethyliodide (2 mg kg−1 BW i.v.) and artificial ven-tilation was provided by a constant flow ventilator(Samay VR15, Universidad de la Republica, Monte-video, Uruguay). During the test breaths the venti-lator was adjusted to generate a 5-s end-inspiratorypause, whereas during baseline ventilation no pausewas used. Special care was taken to keep tidal vol-ume (VT = 1 ml) and flow (V′ = 7 ml s−1) constant inall animals in order to avoid the effects of differentflows and volumes (Kochi et al., 1988) and inspira-tory duration (Similowski et al., 1989) on the measuredvariables.

The experiments did not last more than 40 min. Res-p tiono l.,1 reif int(t o thee� ys,p s inn sr andc n ofp 9;S epP ryp

culated by subtracting the chest wall data from the cor-responding values pertaining to the respiratory system.Total pressure drop (�Ptot) is equal to the sum of�P1and�P2, yielding the values of�Ptot,rs,�Ptot,L, and�Ptot,w. Respiratory system, lung, and chest wall staticelastances (Est,rs,Est,L, andEst,w, respectively) werecalculated by dividingPel,rs,Pel,L, andPel,w, respec-tively, by the inflation volume. Dynamic elastances ofthe respiratory system, lung, and chest wall (Edyn,rs,Edyn,L, andEdyn,w, respectively) were obtained by di-viding Pi ,rs,Pi ,L, andPi ,w, respectively, byVT. �Ewas calculated as the differenceEdyn − Est, yieldingthe values of�E,rs,�E,L, and�E,w. Respiratory me-chanics were measured 10 times in each animal.

The delay between the beginning and the end ofthe valve closure (10 ms) was allowed for by back-extrapolation of the pressure records to the actual timeof occlusion and the corrections in pressure, althoughvery minute, were performed as previously described(Bates et al., 1987).

All data were analysed using ANADAT data anal-ysis software (RHT-InfoData Inc., Montreal, Que.,Canada).

2.3. Measurement of tissue mechanics

Heparine (1000 IU) was intravenously injectedimmediately after the determination of respiratory me-chanics. The trachea was clamped 10 min later at end-expiration, and the abdominal aorta and vena cavaw thatq ede eith( MKC -cs pe-r y ina at3 %O

dr r.L om-e eb ndδ

iratory mechanics were measured by the end-inflacclusion method (Bates et al., 1988; Similowski et a989). Briefly, after end-inspiratory occlusion, the

s an initial fast drop in tracheal pressure (�P1,rs)rom the preocclusion value down to an inflection poPi ,rs) followed by a slow pressure decay (�P2,rs), un-il a plateau is reached. This plateau corresponds tlastic recoil pressure of the respiratory system (Pel,rs).P1,rs selectively reflects the combination of airwaulmonary, and chest wall Newtonian resistanceormal animals and humans, and�P2,rs reflects streselaxation, or viscoelastic properties, of the lunghest wall tissues, together with a small contributioendelluft (Bates et al., 1988; D’Angelo et al., 198imilowski et al., 1989; Saldiva et al., 1992). The samrocedures apply toPw yielding the values of�P1,w,i ,w, �P2,w, andPel,w, respectively. Transpulmonaressures (�P1,L, Pi ,L, �P2,L, andPel,L) were cal-

ere sectioned, yielding a massive haemorrhageuickly killed the animals. The lungs were removn bloc, and placed in a modified Krebs–HenselK–H) solution containing 118.4 mM NaCl, 4.7 mCl, 1.2 mM K3PO4, 25 mM NaHCO3, 2.5 mMaCl2·H2O, 0.6 mM MgSO4·H2O, and 11.1 mM gluose; at pH 7.40 and 6◦C. A 3 mm× 3 mm× 10 mmtrip of subpleural parenchyma was cut from theiphery of each left lung and suspended verticalln organ bath filled with K–H solution maintained7◦C, continuously bubbled with a mixture of 952–5% CO2.Lung strips were weighed (W), and their unloade

esting lengths (L0) were determined with a callipeung strip volume was measured by simple densittry, as:V=�F/δ, where�F is the total change in forcefore and after strip immersion in K–H solution ais the mass density of K–H solution (Lopez-Aguilar


and Romero, 1998; Faffe et al., 2001; Rocco et al.,2001, 2003).

Parenchymal strips were suspended vertically in aK–H organ bath maintained at 37◦C and continuouslybubbled with of 95% O2–5% CO2. Metal clips made of0.5 mm-thick music wire were glued to both ends of thetissue strip with cyanoacrylate. One clip was attachedto a force transducer (FT03, Grass Instruments Co.,Quincy, MA, USA), whereas the other one was fastenedto a vertical rod. This fibreglass stick was connected tothe cone of a woofer, which was driven by the ampli-fied sinusoidal signal of a waveform generator (3312AFunction Generator, Hewlett Packard, Beaverton, OR,USA). A sidearm of the rod was linked to a second forcetransducer (FT03, Grass Instruments Co., Quincy, MA,USA) by means of a silver spring of known Young’smodulus, thus allowing the measurement of displace-ment. Length and force output signals were conditioned(Gould 5900 Signal Conditioner Frame, Gould Inc.,Valley View, OH, USA), fed through 8-pole Besselfilters (902LPF Frequency Devices, Haverhill, MA,USA), analogue-to-digital converted (DT2801A, DataTranslation Inc., Marlboro, MA, USA), and stored on acomputer. All data were collected using LABDAT soft-ware (RHT-InfoData Inc., Montreal, Que., Canada).The frequency response of the system was dynamicallystudied by using calibrated silver springs with differ-ent elastic Young’s modulus. Neither amplitude depen-dence (<0.1% change in stiffness) nor phase changeswith frequency were detected in the range from 0.01t en-d ,2

w gth,a f0A thef 8;Fp forcea h be-t re-c tionat .

e inu-

soidal oscillation of the tissue during 30 min (frequency= 1 Hz; amplitude large enough to reach a final forceof 1 × 10−2 N). Thereafter the amplitude was adjustedto 5% L0 and the oscillation maintained for another30 min, or until a stable length-force loop was reached(Rocco et al., 2001). The isometric stress adaptationperiod resulted in a final force of 5× 10−3 N. After pre-conditioning, the strips were oscillated at a frequency(f) = 1 Hz (Rocco et al., 2001). The bath solution wasrenewed regularly (every 20 min) with 37◦C K–H so-lution.

To calculate tissue resistance (R), elastance (E), andhysteresivity (η) force-length curves were analyzed(Lopez-Aguilar and Romero, 1998; Faffe et al., 2001;Rocco et al., 2001, 2003). Instantaneous average cross-sectional area (Ai ) was determined asAi = V/Li (cm2),whereLi is instantaneous length. Instantaneous stress(σ i ) was calculated by dividing force (g) by Ai (cm2),σ i = F/Ai .

All mechanical parameters were measured cycle-by-cycle. Tissue resistance (R) was determined fromthe enclosed area of force-length loops:

R = 4 × H

π × ω × �.ε2

whereH is the stress–strain hysteresis area,ω is the an-gular frequency (ω = 2�f, rad s−1), and�.ε is the nor-malised strain or peak-to-peak change in length (�L)divided byLB. Tissue dynamic elastance was deter-mined as:

E

w rce,a ment[ db sv oursw

2

sedl ovalo yi y’ss byv ng

o 14 Hz. The hysteresivity of the system was indepent of frequency and had a value <0.003 (Faffe et al.001; Rocco et al., 2001).

Cross-sectional, unstressed area (A0) of the stripas determined from volume and unstressed lenccording toA0 =V/L0. Basal force (FB) for a stress o.1 N cm−2 was calculated asFB (N) = 0.1 (N cm−2).0 (cm2) and adjusted by vertical displacement of

orce transducer (Lopez-Aguilar and Romero, 199affe et al., 2001; Rocco et al., 2001, 2003). The dis-lacement signal was then set to zero. Once basalnd displacement signals were adjusted, the lengt

ween bindings (LB) was measured by means of a pision calliper. Instantaneous length during oscillaroundLB was determined by adding the value ofLB

o the measured value of displacement at any timeAfter the basal force was adjusted to 8× 10−3 N,

ach parenchymal strip was preconditioned by s

=(

�σi

�ε

)cosθ

here�σi represents the peak-to-peak change in fondθ is the phase lag between force and displaceθ = sin−1 (4·H/(�·�σι·�ε))]. Hysteresivity, defineyFredberg and Stamenovic (1989)as a dimensionlesariable coupling the dissipative and elastic behavias calculated as:η = tanθ.

.4. Lung morphometric analysis

Morphometric analysis was performed in exciungs at end-expiration. Immediately after the remf the lungs en bloc, the right lung was quick-frozen b

mmersion in liquid nitrogen and fixed with Carnoolution (ethanol:cloroform:acetic acid, 70:20:10olume) at−70◦C for 24 h. Progressively increasi


concentrations of ethanol at−20◦C were then sub-stituted for Carnoy’s solution until 100% ethanol wasreached. The tissue was maintained at−20◦C for 4 h,warmed to 4◦C for 12 h, and then allowed to reachand remain at room temperature for 2 h (Nagase et al.,1996). After fixation, the tissue was embedded inparaffin. Blocks were cut 4�m-thick by a microtome.The slices were stained with haematoxylin–eosin. Twoinvestigators, who were unaware of the origin ofthe material, performed the microscopic examination.Morphometric analysis was performed with an inte-grating eyepiece with a coherent system made of a100-point grid consisting of 50 lines of known length,coupled to a conventional light microscope (Axio-plan, Zeiss, Oberkochen, Germany). The volume frac-tion of collapsed and normal pulmonary areas and thefraction of the lung occupied by large-volume gas-exchanging air spaces (hyperinflated structures witha morphology distinct from that of alveoli and widerthan 120�m) were determined by the point-countingtechnique (Weibel, 1990), made at a magnification of40× across 10 random, non-coincident microscopicfields.

Lung tissue distortion was assessed by measuringthe mean linear intercept between alveolar walls (Lm)at a magnification of 100× (Nagase et al., 1996). Lmwas determined by counting the number of interceptsbetween the eyepiece lines and the alveolar septa ofeach microscopic field.Lm was expressed as the rela-tion between the line length (1250�m) and the totaln fi

asc ayl ande atedb tinge pro-c Thea erec eirv temw nd-it por-t ints( aya rac-

tion index (CI )] was computed by the relationship:CI= NI /

√NP (Sakae et al., 1994).

The number of polymorpho- and mononuclear cells,and pulmonary tissue were determined in each sampleby the point-counting technique (Weibel, 1990) across10 random noncoincident microscopic fields at 1000×magnification. Points falling on tissue area and notover air spaces were counted and divided by the to-tal number of points in each microscopic field. Thus,data are reported as the fractional area of pulmonarytissue.

2.5. Morphometric analysis of the parenchymalstrips

At the end of the experiments the organ bath wasremoved and the parenchymal strips were frozen byrapid immersion in liquid nitrogen at the force main-tained during the experiment. Frozen strips were fixedas aforementioned.

Morphometric analysis was performed with an in-tegrating eyepiece with a coherent system made ofa 100-point grid consisting of 50 lines of knownlength coupled to a light microscope (Axioplan, Zeiss,Oberkochen, Germany). Sections were examined at400× magnification, and the fractional areas of alveo-lar wall (AW), blood-vessel wall (BVW), and bronchialwall (BW) were determined by the point-countingtechnique (Weibel, 1990). All points falling on thesecomponents were counted and divided by the total num-b ran-d ndB liall sso-c r airs weree

s toc elas-tT Redd ob-s en-h thep ol-l :T rcinF

umber of intercepts (Lm =∑

line length/number ontercepts).

The internal diameter of the central airways womputed by counting the points falling on the airwumen and those falling on airway smooth musclepithelium. The perimeter of the airways was estimy counting the intercepts of the lines of the integrayepiece with the epithelial basal membrane. Thisedure was repeated four times for each airway.reas of smooth muscle and airway epithelium worrected in terms of airway perimeter by dividing thalues by the number of intercepts of the line sysith the epithelial basal membrane of the correspo

ng airway. Because the number of intercepts (NI ) ofhe lines with the epithelial basal membrane is proional to airway perimeter, and the number of poNP) falling on airway lumen is proportional to airwrea, the magnitude of bronchoconstriction [cont

er of points. This analysis was performed in 10om, non-overlapping fields in each strip. BVW aW were counted when a point fell on the endothe

ayer, the epithelial layer, the smooth muscle, or aiated connective tissue. Points falling on alveolapaces, blood-vessel lumen, and bronchial lumenxcluded.

The slices underwent specific staining methodharacterize the fibres of the collagenous andic systems present in the alveolar septa.Collagen:he tissue was stained in a solution of Siriusissolved in aqueous saturated picric acid, anderved under polarized light microscopy, as theancement of collagen birefringence promoted byicrosirius-polarization method is specific for c

agenous structures (Montes, 1996). Elastic fibreswo different methods were used: Weigert’s Resouchsin method (RF) (Weigert, 1898), which allows the


identification of elaunin and fully developed elastic fi-bres, and Weigert’s Resorcin Fuchsin method modifiedwith oxidation (ORF) (Fullmer et al., 1974), whichallows the identification of the three components ofthe elastic fibre (elaunin, oxytalan, and fully devel-oped elastic fibres). The oxytalan fibres were calcu-lated by subtracting the number of fibres given bythe RF method from the value provided by the ORFmethod. In each rat, 20 different microscopic fieldswere randomly selected in order to quantify collagenor elastic fibres. Quantification was carried out withthe aid of a digital analysis system and specific soft-ware (Bioscan-Optimas 5:1, Bioscan Incorporated, Ed-mond, WA, USA) under 200× magnification. The im-ages were generated by a microscope (Axioplan, Zeiss,Oberkochen, Germany) connected to a camera (SonyTrinitron CCD, Sony, Tokyo, Japan), fed into a com-puter through a frame grabber (Oculus TCX, CorecoInc., St. Laurent, PQ, Canada) for off-line processing.The thresholds for fibres of the collagenous and elasticsystems were established after enhancing the contrastup to a point at which the fibres were easily identified aseither black (elastic) or birefringent (collagen) bands.The area occupied by fibres was determined by digitaldensitometric recognition. Bronchi and blood vesselswere carefully avoided during the measurements. Toavoid any bias due to septal oedema or alveolar col-lapse the areas occupied by the elastic and collagenfibres measured in each alveolar septum were dividedby the length of each studied septum. The results weree bresp

2

s of2 s oft henfi ffer0 nr mict hedt icesw lacedi em-b ask o-t

2.7. Statistical analysis

The normality of the data (Kolmogorov–Smirnovtest with Lilliefors’ correction) and the homogeneityof variances (Levene median test) were tested. In allcases, both conditions were satisfied, and thus one-wayANOVA was used to determine the possibility of differ-ences among the groups. If multiple comparisons werethen required, Tukey test was applied. The relation-ships between mechanical parameters and the numberof collagen and elastic fibres were evaluated by Spear-man correlation. In all tests the significance level wasset at 5%. Statistical analyses were done with Sigmastat2.0 (Jandel Scientific, San Rafael, CA, USA).

3. Results

Because the mechanical and morphometrical valuesof control group were similar at one, three and eightweeks, the C group was considered as one. The meanconstant inspiratory flows (± S.E.M.) measured in C,PQ1, PQ3, and PQ8 groups amounted to 7.06± 0.03,7.05± 0.04, 7.03± 0.01, and 7.03± 0.07 ml s−1, re-spectively. The corresponding tidal volumes were: 1.2± 0.1, 1.1± 0.1, 1.1± 0.1, and 1.2± 0.04 ml. Nostatistically significant differences among the groupscould be detected.

Fig. 1 shows the mean values (+1 S.E.M.) of res-piratory system, lung, and chest wall�P’s, and statice oups.� nd6 ntrolvc PQ8.E re-t� s.

atafi eks.η

ol-l rceptb ni ictedi sei eeks,

xpressed as the number of elastic and collagen fier unit of septal length.

.6. Transmission electron microscopy

To obtain a stratified random sample, three slicemm×2 mm were cut from three different segment

he right lung (upper, middle, and lower lobes), and txed with glutaraldehyde 2.5% and phosphate bu.1M (pH 7.4) for 60 min at−4◦C. The slices were theinsed in phosphate buffer, post-fixed with 1% osetroxide in phosphate buffer for 30 min and rewashree times in the phosphate buffer. Finally, the slere dehydrated in an acetone series, and then p

n a mixture of 1:1 acetone:Epon overnight beforeedding in Epon for 6 h. After fixation the material wept for 48 h at 60◦C before undergoing to ultramicromy for transmission electron microscopy.

lastances obtained in C, PQ1, PQ3, and PQ8 grP1,rs and�P1,L increased from C to PQ1 (59 a7%) and PQ3 (45 and 58%) and returned to coalues in PQ8.�Ptot,rs, �Ptot,L �P2,rs,�P2,L, in-reased in PQ1 and remained elevated in PQ3 andst,rs andEst,L increased in PQ1 (51 and 61%) and

urned to control values after 3 weeks.�P1,w,�P2,w,Ptot,w, andEst,w were similar among the four groupE augmented from C to PQ1 (32%), and the d

rom PQ1, PQ3 and PQ8 were not different.Rincreasedn PQ1 (40%) and returned to control values at 8-wewas similar in all groups (Table 1).The mean (±S.E.M.) percentages of normal, c

apsed, and hyperinflated areas, mean linear inteetween alveolar walls (Lm), and bronchoconstrictio

ndex in C, PQ1, PQ3, and PQ8 groups are depn Table 2. The fraction of area of alveolar collapncreased from C to PQ1, and decreased after 3 w


Fig. 1. (A) Stacked bar chart plot data in which the gray bars represent the resistive pressures (�P1) and the white bars are the viscoelas-tic/inhomogeneous (�P2) pressure dissipations of the respiratory system, lung, and chest wall. The whole column represents the total pressurevariation in each group. (B) Static elastance (Est) of the respiratory system, lung and chest wall. In the control group (C) saline (5 ml kg−1) wasintraperitoneally injected. In PQ groups paraquat was injected intraperitoneally (7 mg kg−1) 1, 3, and 8 weeks before the measurements. Barsare means (+S.E.M.) of five animals (8–10 determinations/animal).Values significantly different from C (P < 0.05).

Table 1In vitro mechanical parameters

Groups E (104 N m−2) R (102 N s m−2) η

C 0.95± 0.09 0.88± 0.13 0.07± 0.010PQ1 1.25± 0.11a 1.23± 0.11ab 0.08± 0.017PQ3 1.21± 0.06a 1.20± 0.08ab 0.07± 0.006PQ8 1.17± 0.11a 0.97± 0.13 0.07± 0.004

Values are means (±S.E.M.) of five animals in each group. Tissueelastance (E), resistance (R) and hysteresivity (η) at 1 Hz. C, controlgroup; PQ1, PQ3 and PQ8 correspond to the groups with acute lunginjury induced by paraquat (7 mg kg−1 BW), 1, 3 and 8 weeks afterthe induction of acute lung injury.

a Values significantly different from C group (P < 0.05).b Values significantly different from PQ8 group (P < 0.05).

remaining higher than C. It can be seen that PQ groupspresented lower Lm than group C. The internal diam-eter of the central airways decreased in PQ1 and PQ3and returned to C values in PQ8.

Table 3shows the results of tissue strip morpho-metrical analysis. All groups showed similar anatomiccomposition. Total cell content and the number of poly-morphonuclear cells were higher in PQ1 in relationto C group and remained elevated in PQ3 and PQ8.Mononuclear cells were less frequent in PQ1, PQ3,and PQ8 than in C group.

Collagen fibre content was increased in PQ1, PQ3and PQ8 groups, with no difference among them(Fig. 2). Elastic fibre content (elaunin, oxytalan andfully developed elastic fibre) was not altered (Fig. 2).


Table 2Morphometrical parameters

Groups Normal areas (%) Alveolar collapse (%) Alveolar hyperinflation (%) Lm (�m) Broncho-constriction index

C 92.6± 0.8 6.0± 1.2 1.4± 0.8 44± 2 2.0± 0.1PQ1 61.8± 2.0ab 36± 2.5ab 2.2± 1.0 31± 1a 2.7± 0.1ab

PQ3 64.7± 2.7ab 32± 2.2ab 3.3± 1.4 32± 1a 2.8± 0.3ab

PQ8 77± 1.6a 18± 1.3a 5.0± 0.9 32± 1a 1.8± 0.2

Values are means (±S.E.M.) of five animals in each group (10 random, non-coincident microscopic fields were analyzed in each lung). Percentageof normal, collapsed and hyperinflated alveoli.Lm: mean linear intercept between alveolar walls. C, control group; PQ1, PQ3 and PQ8 correspondto the groups with acute lung injury induced by paraquat (7 mg kg−1 BW), 1, 3 and 8 weeks after the induction of acute lung injury.

a Values significantly different from C group (P < 0.05).b Values significantly different from PQ8 group (P < 0.05).

Table 3Volume proportions of alveolar, blood–vessel, and bronchial walls, plus cellularity in lung parenchymal strips of rats

Groups AW BVW BW Total cell PMN MN

C 90.2± 1.0 5.8± 0.5 4.0± 0.6 12.5± 0.6 3.3± 0.3 9.2± 0.4PQ1 90.4± 1.1 5.5± 0.7 4.1± 0.5 17.2± 1.0a 10.7± 1.3a 6.6± 0.9a

PQ3 90.9± 0.6 5.3± 0.3 3.8± 0.5 16.7± 1.0a 9.3± 0.8a 7.4± 1.0a

PQ8 90.7± 0.7 5.5± 0.3 3.8± 0.3 16.6± 0.2a 9.7± 0.5a 6.9± 0.5a

Values are means±S.E.M. (in %) of five strips in each group (10 random, non-coincident microscopic fields were analysed in each strip). C,control group; PQ1, PQ3 and PQ8 correspond to the groups with acute lung injury induced by paraquat (7 mg kg−1 BW), 1, 3 and 8 weeks afterthe induction of acute lung injury. AW, alveolar wall; BVW, blood vessel wall; BW, bronchial wall; total cell, total cellular fractional area; PMN,polymorphonuclear cell; MN, mononuclear cell.

a Values significantly different from C (P < 0.05).

Fig. 3 shows the ultramicroscopy of lungparenchyma. At 1 week there is lesion of epitheliumbasement membrane with alveolar collapse and neu-trophils. The alveolar interstitium was thickened due toincreased amounts of matrix elements, such as collagen

Fig. 2. Amounts of elastic and collagenous fibres in alveolar walls from the control (C) and PQ1, PQ3, and PQ8 groups (1, 3, and 8 weeksafter the injection of paraquat, respectively). Symbols represent means of five animals in each group (10 microscopic fields/rat); bars are±S.E.M. FDEF: fully developed elastic fibres. Considering each elastic or collagenous system fibres, different letters indicate values significantlydifferent (P < 0.05) among C, PQ1, PQ3, and PQ8 groups and same letters indicate no significant difference between data points.

fibres. The remarkable feature of this stage is the begin-ning of fibrosis with fibroblast migration into the alve-olar lumen and myofibroblast transformation togetherwith type III collagen fibre. Elastic fibre content wasnormal. At the third week electron microscopy showed


Fig. 3. Electron microscopy of lung parenchyma in control (A) and PQ1 (B), PQ3 (C), and PQ8 (D) groups (1, 3, and 8 weeks after the injectionof paraquat, respectively). Type III collagen fibre content (stars) was augmented and incipient type I collagen fibre synthesis is also evident after3 weeks. PII: type II pneumocyte, N: neutrophil, M: myofibroblast.

intra-alveolar proliferation of myofibroblast with oblit-erative fibrosis, and proliferation of type II epithelialcell to form the new alveolar septa. At this momentthere is an increased number of collagen fibres typesI and III. At 8 weeks there is a replication of type IIepithelial cell repopulating the denuded air-lung inter-face and myofibroblast proliferation determining a newalveolar septa formation. Interestingly, myofibroblastremain proliferating but elastic fibre content was nor-mal at this stage.

Considering PQ and C groups together,Est,L waswell correlated with the fraction of alveolar col-lapse,�P2,L was correlated withLm, and �P1,Lwas correlated with bronchoconstriction index. In ad-dition, E was correlated with collagen fibre con-

tent and the number of polymorphonuclear cells(Table 4).

4. Discussion

In the present study, paraquat-induced ALI led toa time-dependent modification in in vivo and in vitrorespiratory mechanics and to an increase in collagenfibre content at 1 week that remained unaltered until8 weeks. Furthermore, the number of elastic fibres didnot increase in this mild model of ALI.

We used a model of ALI-induced by a small doseof paraquat, thus analyzing the effects of a mild degreeof ALI. Mildly abnormal parenchyma means thickened


Table 4Correlation matrix between physiologic and morphometric parameters in lung and parenchymal strips (R-values)

BI Collapse (%) Lm (�m) PMN (%)

Est,L (cmH2O ml−1) 0.53 (0.02) 0.70 (<0.0001) −0.36 (NS) 0.48 (0.03)�P2,L (cmH2O) 0.24 (NS) 0.47 (0.03) −0.77 (<0.0001) 0.49 (0.03)�P1,L (cmH2O) 0.68 (<0.0001) 0.80 (<0.0001) −0.54 (0.02) 0.37 (NS)

Collagen fibres (�m2 �m−1)

E (104N m−2) 0.74 (0.0001)R (102N s m−2) 0.24 (NS)

Est,L: lung static elastance;�P2,L: lung viscoelastic/inhomogeneous pressure;�P1,L: lung resistive pressure; BI: bronchoconstriction index;collapse: fraction of area of alveolar collapse;Lm; mean linear intercept between alveolar walls; PMN: polymorphonuclear;E (tissue elastance)andR (tissue resistance); NS: nonsignificant;P-values are shown in parentheses. The correlation was performed on data from control andparaquat (7 mg kg−1 BW, 1, 3 and 8 weeks after the induction of acute lung injury) groups.

alveolar membranes, increased cellularity and hyalinemembrane formation with no demonstration of haem-orrhage. Paraquat is an herbicide that accumulates pre-dominantly in the lung and induces alveolar epithelialdamage due to its action on type II pneumocytes. Thisis an experimental model of diffuse alveolar damage,which by virtue of its low cost, rapid effect and sim-plicity of administration has been used to study acutelung injury (Smith et al., 1974; Delaval and Gillespie,1985). In the present model of paraquat-induced ALI,although 100% of the animals survived during the firstweek, 10% of the rats died at 8 weeks. At autopsy, theseanimals presented acute tubular necrosis.

Respiratory mechanics were measured by the end-inflation occlusion method to provide data from thewhole lung. Acute lung injury induced by 7 mg kg−1

presented a mechanical behaviour similar to that in-duced with 10 mg kg−1 (Rocco et al., 2003). �P1,rsand�P1,L increased at 1 and 3 weeks, returning to con-trol values 8 weeks after ALI-induction. As previouslyreported,�P1,L is directly related to airway resistance(Saldiva et al., 1992). The internal diameter of the cen-tral airways reduced in PQ1 and PQ3 and returned to Cin PQ8 (Table 2), supporting the changes in airway re-sistance. The reduction of central airway calibre couldbe caused by oedema, fluid accumulation, reflex bron-choconstriction, and/or reduced lung volume. In thepresent study�P2,L increased significantly in PQ1 andremained elevated until eight weeks (Fig. 1). �P2,Lreflects pressure losses due to viscoelastic propertiesa ungh sedae mo-

geneities. There are some possibilities that account forthe dissipation of energy at the tissue level: (a) the in-crease in collagen fibre content (Fig. 2) (b) the changesin the rheological properties of the air–liquid interface(surfactant) (c) atelectasis, and (d) inflammation withneutrophil infiltration (Table 3). The overall respira-tory system and lung pressures (�Ptot,rs and�Ptot,L)were elevated in all ALI groups. Because chest wallpressures were not altered (Fig. 1), respiratory systemmechanical profile reflects solely its pulmonary com-ponent.Est,rs andEst,L, increased in PQ1 and returnedto control values after 3 weeks (Fig. 1) Prior studieshave described changes in lung elastance in ALI, re-sulting from surfactant dysfunction and/or loss of func-tional capacity due to alveolar flooding (Grossman etal., 1980; Gregory et al., 1991). Actually, mechanicaldysfunction can result from air–liquid interface and/ortissue changes, but after 3 weeks surface film proper-ties could be restored because of the repair of pneu-mocyte type II. Lung static elastance changes weresignificantly correlated with increased cellularity(PMN) and atelectasis (Table 4). Thus, an improve-ment in lung elastance does not necessarily indicateless fibrosis (Fig. 2), but expressed more accurately thereduction in alveolar collapse.

The method used in the in vitro study specificallyallows the analysis of tissue resistance, elastance, andhysteresivity in the absence of surfactant and interde-pendence effects, providing a direct assessment of tis-sue physiology (Fredberg and Stamenovic, 1989, Yuane ncew fterA ede se

nd/or mechanical inhomogeneities of the lung. Listology showed interstitial oedema, and increalveolar collapse in PQ1 group (Table 2), leading tolevated tissue viscoelasticity and mechanical inho

t al., 1997, 2000). Tissue elastance and resistaere significantly increased at 1 and 3 weeks aLI induction (Table 1), but at 8 weeks E remainlevated whereasR returned to control values. The


data suggest that parenchymal mechanical dysfunctionplayed an important role in ALI pathophysiology. Con-trary to our data, in a model of fibrosis induced bybleomycin, the maximal increase of tissue resistanceand elastance occurred at 14 days post bleomycin in-stillation (Ebihara et al., 2000). This difference couldbe attributed to the diversity in the components of theextracellular matrix found in their study.

The changes in tissue mechanics were accompaniedby deposition of collagen fibres in the alveolar septa(Table 4). We had described that collagen content wasalready elevated 1 day after tissue damage whatever thedose of paraquat used (between 10 and 25 mg kg−1), in-dicating that the biochemical processes implicated incollagen synthesis are indeed able to react very quicklyto the aggression (Rocco et al., 2001, 2003). Collagenfibre types were identified at electron microscopy. Al-though the total number of collagen fibre did not changewith the time-course of the lesion, type III collagen,which is more flexible and susceptible to breakdown(Raghu et al., 1985), appeared earlier (PQ1), whiletype I collagen, thicker and more resistant, appearedlate in the course of the disease (Fig. 3). Based onimmunocytochemical studies of human ARDS lungs,type III collagen increases early in the evolution ofthis fibrotic process, whereas, increases in type I col-lagen predominate later on (Raghu et al., 1985). Thepotential significance of these observations stems fromthe fact that type III collagen is believed to be moreflexible and more easily remodelled or degraded thant sish pastd vari-e andb -h ucedl sticp por-t atrix( nc aysa

tentd hee ents:o de-fi andfi l

of lung fibrosis, elastic fibre content was greater thancontrol at day 14 (Raghow et al., 1985; Ebihara et al.,2000). The absence of elastosis in this model of ALIcould be attributed to the intensity of lung injury. Inter-estingly, with a higher dose of paraquat (25 mg kg−1)elastogenesis appear early in the course of lung injuryand with 10 mg kg−1 there is an increment of elasticfibre content only at day 30 (Rocco et al., 2001, 2003).Thus, there is a degree of lung injury responsible forthe beginning of elastogenesis independent of the timecourse of ALI. Additionally, the mechanism triggeringfibrogenesis seems to be different from that of elasto-genesis.

The network of stress-bearing collagen and elas-tic fibres, proteoglycan and glycosaminoglycans, aswell as contractile elements present in parenchymaltissues potentially determine tissue viscoelastic be-haviour (Fredberg and Stamenovic, 1989; Mijailovichet al., 1993). In our study, tissue elastance changes werecorrelated with collagen fibre content. In accordancewith our results, Yuan and coworkers reported that col-lagen fibres contribute to tissue elasticity during normalbreathing (Yuan et al., 1997, 2000).

Hysteresivity is the ratio of energy dissipation toenergy storage in a cyclic deformation (Fredberg andStamenovic, 1989). According to the structural damp-ing hypothesis, tissue elastance and resistance changedtogether, and thus hysteresivity remained unchangedin the present model of mild lung injury. Similarly, inmild ALI induced by paraquat (10 mg kg−1), hystere-s in-dt es intW lasticfi f in-c didn xtra-c teri

ra-c es inl erei ngu ce,l ever,t elas-t

ype I collagen. Pulmonary remodelling and fibroave been under intensive investigation during theecade in experimental animals exposed to aty of agents including hyperoxia, mineral dusts,leomycin (Ebihara et al., 2000). In this context, Ebiara and colleagues observed that bleomycin-ind

ung fibrosis resulted in alterations in the viscoelaroperties of the lung parenchymal tissues with im

ant changes in the structure of the extracellular mEbihara et al., 2000). Contrary to our data, collageontent on the alveolar wall increased only at 28 dfter bleomycin instillation.

The present study showed that elastic fibre conid not increase with the time course of mild ALI. Tlastic system is composed of three different elemxytalan, elaunin and fully developed elastic fibres,ned according to increasing amounts of elastinbril orientation (Montes, 1996). In bleomycin mode

ivity did not increase at 24 h, but severe lesionuced by a higher dose of paraquat (25 mg kg−1) led

o an increase in hysteresivity secondary to changhe collagen–elastin fibre network (Rocco et al., 2001).

e hypothesized that the absence of changes in ebre content together with the small percentage orease in collagen fibre in the present mild lesionot induce a derangement of the tridimensional eellular matrix organization, which would had a greampact on tissue mechanics.

In conclusion, the structural modification of extellular matrix induced by paraquat caused changung tissue mechanics both in vivo and in vitro. Ths a restoration of the normal alveolar-capillary lunits with a gradual improvement of airway resistan

ung static elastance, and tissue resistance. Howhe on-going fibrotic process kept elevated tissueance and viscoelastic/inhomogeneous pressure.


Acknowledgement

The authors would like to express their grati-tude to Antonio Carlos Quaresma and Alaine Pru-dente for their technical assistance. Supported by: TheCentres of Excellence Program (PRONEX-MCT andMCT/FAPERJ), The Brazilian Council for Scientificand Technological Development (MCT/CNPq), TheCarlos Chagas Filho Rio de Janeiro State ResearchSupporting Foundation (FAPERJ), The Sao Paulo StateResearch Support Foundation (FAPESP), and TheHoward Hughes Medical Institute (departmental shar-ing of grant # 55003669).

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