Effect of Corticosteroid on Lung Parenchyma Remodeling at an Early Phase of Acute Lung Injury

EFFECT OF CORTICOSTEROID ON LUNG PARENCHYMA

REMODELING AT AN EARLY PHASE OF ACUTE LUNG INJURY

PATRICIA R.M. ROCCO1, ALBA B. SOUZA1, DEBORA S. FAFFE1, CAROLINE P.

PÁSSARO1, FLÁVIA B. SANTOS1, ELNARA M. NEGRI2, JANUÁRIO G.M. LIMA1,

RENATA S. CONTADOR1, VERA L. CAPELOZZI3, WALTER A. ZIN1

1Laboratory of Respiration Physiology, Carlos Chagas Filho Biophysics Institute, Federal

University of Rio de Janeiro, Centro de Ciências da Saúde, Ilha do Fundão, 21949-900, Rio

de Janeiro, Brazil; 2LIM59 and 3Department of Pathology and Department of Clinical

Emergencies, University of São Paulo, São Paulo, Brazil.

Correspondence address: Walter Araujo Zin

Universidade Federal do Rio de Janeiro Instituto de Biofísica Carlos Chagas Filho - C.C.S. Laboratório de Fisiologia da Respiração Ilha do Fundão 21949-900 - Rio de Janeiro - RJ Brazil e-mail: [email protected] tel: (+5521) 2562-6557 fax: (+5521) 2280-8193

Supported by: Centers of Excellence Program (PRONEX-MCT), Brazilian Council for

Scientific and Technological Development (CNPq), Financing for Studies and Projects

(FINEP), Rio de Janeiro State Research Supporting Foundation (FAPERJ), São Paulo State

Research Support Foundation (FAPESP).

Running head: Steroid effects on lung parenchyma remodeling

Subject of the manuscript: 1

Word count: 4253

This article has an online data supplement, which is accessible from this issue’s table

content online at www.atsjournals.org.

Copyright (C) 2003 by the American Thoracic Society.

AJRCCM Articles in Press. Published on July 3, 2003 as doi:10.1164/rccm.200302-256OC

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ABSTRACT

In vivo [lung resistive and viscoelastic pressures, and static elastance] and in vitro [tissue

resistance, elastance, and hysteresivity] respiratory mechanics were analyzed 1 and 30 days

after saline (control) or paraquat [P (10 and 25 mg/kg, i.p.)] injection in rats. Additionally,

P10 and P25 were treated with methylprednisolone (2 mg/kg, i.v.) at 1 or 6 hours after

acute lung injury (ALI) induction. Collagen and elastic fibers were quantified. Lung

resistive and viscoelastic pressures, and static elastance were higher in P10 and P25 than in

control. Tissue elastance and resistance augmented from control to P10 (1 and 30 days) and

P25. Hysteresivity increased only in P25. Methylprednisolone at 1 or 6 hours attenuated in

vivo and in vitro mechanical changes in P25, while P10 parameters were similar to control.

Collagen increment was dose- and time-dependent. Elastic fibers increased in P25, and at

30 days in P10. Corticosteroid prevented collagen increment, and avoided elastogenesis. In

conclusion, methylprednisolone led to a complete maintenance of in vivo and in vitro

respiratory mechanics in mild lesion, whereas minimized the changes in tissue impedance

and extracellular matrix in severe ALI. The beneficial effects of the early use of steroids in

ALI remained unaltered at day 30.

Word count: 200

Key words: Tissue mechanics, acute lung injury, corticosteroid, hysteresivity, elastin

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The use of corticosteroid in the treatment of acute lung injury (ALI)/acute

respiratory distress syndrome (ARDS) has been subject of great controversy and debate

over the years. Although the exact mechanism of action remains unknown, corticosteroids

inhibit a host of potent inflammatory mediators and have been shown to improve morbidity

and mortality in animal models of ALI (1-4). Because of the great difficulties in designing

and interpreting studies in humans, much of our knowledge about ALI/ARDS

pathophysiology comes from animal experimentation.

Corticosteroid therapy in ARDS has been studied in three main different situations:

a) prevention in high risk patients (5), b) early treatment with high dose, short course

therapy (6), and c) prolonged therapy in unresolved cases (7-10). Unfortunately, trials of

short term, high dose steroid therapy failed to show an improvement in mortality of patients

at risk of or with early ARDS (5-6). Despite the unfavorable experience with steroids at the

early phase of ALI, there has been a recent resurgence in enthusiasm for their use in late

ALI (fibroproliferative phase) (8). The late phase of ALI is characterized by progressive

pulmonary fibrosis and lung restriction (11). In addition, pulmonary fibrosis contributes to

the unremitting respiratory failure and death in a significant proportion of patients with ALI

(12). Several investigators have suggested that the use of corticosteroids in the late phase

improved lung function and survival (7-10). Steroids prevent excessive collagen deposition

and increase collagen breakdown (9, 13), however, its effect on the elastic system in ALI is

much less understood.

Some authors have recently observed an increased number of myofibroblasts and

cells producing procollagen types I and III in the early course of ALI (14-18), suggesting

that the proliferative phase begins much sooner than had been previously appreciated.

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Complementary, we observed pronounced mechanical changes at tissue level and

fibroelastogenesis at an early phase of acute lung injury, even in mild abnormal lung

parenchyma (19). Thus, in the present study we tested the hypothesis that low-dose

corticosteroid when used at an early phase of acute lung injury could modify: a) in vivo and

in vitro respiratory mechanics, b) lung histology, and c) the structural remodeling of lung

parenchyma in two different degrees of paraquat-induced ALI. These parameters were

studied at 1 and 30 days after the induction of lung injury. We also examined whether

oscillatory tissue mechanical data were correlated with collagen or elastic system fiber

contents.

Some of the results of this study have been previously reported in the form of

abstract (20, 21).

METHODS (Word count: 497)

Animal Preparation

Seventy-eight Wistar rats (250-300 g) were divided into 13 groups of 6 animals each. In the

control group (C) saline (5 ml/kg BW) was administered intraperitoneally (i.p.). In P

groups two different doses of paraquat were injected i.p. (10 and 25 mg/kg BW), 1 or 30

days prior to the measurements. P10 and P25 groups were treated with methylprednisolone

(M, 2 mg/kg, i.v.) at 1 or 6 hours after paraquat administration. Animals were sedated

(diazepam 5 mg i.p.), anesthetized (pentobarbital sodium 20 mg/kg i.p.), and a snugly

fitting cannula (1.7 mm ID) was introduced into the trachea. Airflow, volume, tracheal,

transpulmonary, and esophageal pressures were registered. Respiratory system (rs), lung

(L), and chest wall (w) resistive (∆P1), and viscoelastic/inhomogeneous pressures (∆P2),

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∆Ptot (∆P1+∆P2), and static elastance (Est) were computed by end-inflation occlusion

method (22, 23). Lungs were removed en bloc, and placed in a modified Krebs-Henseleith

(K-H) solution [in mM: 118.4 NaCl, 4.7 KCl, 1.2 K3PO4, 25 NaHCO3, 2.5 CaCl2.H2O, 0.6

MgSO4.H2O, and 11.1 glucose] at pH = 7.40 and 6°C (11-13), bubbled with 95% O2-5%

CO2. Strips (3x3x10 mm) were cut from the periphery of the left lung, and suspended

vertically in a K-H organ bath maintained at 37°C and continuously bubbled with 95% O2-

5% CO2. One clip was attached to a force transducer, while the other one was fastened to a

vertical rod. This fiberglass stick was connected to a woofer cone, which was driven by a

waveform generator. A side arm of the rod was linked to a second force transducer by

means of a silver spring of known Young’s modulus, allowing the measurement of

displacement (19, 24).

Strips were preconditioned by sinusoidally oscillating the tissue during 30 min

(frequency = 1 Hz; amplitude large enough to reach a final force of 1x10-2 N). Thereafter

the amplitude was adjusted to 5% of the strip’s resting length (L0), the strips were unloaded

to a force of 8x10-3 N, and the oscillation maintained for another 30 min, or until a stable

length-force loop was reached. The final basal force was approximately 5x10-3 N. After

stress adaptation, strips were oscillated at a frequency = 1 Hz.

Tissue resistance (R), elastance (E), and hysteresivity (η) were calculated from the

oscillatory recordings according to Fredberg and Stamenovic (25).

Morphometric analysis

The right lung was fixed at the end of expiration with glutaraldeyde and submitted to

ultramicrotomy for transmission electron microscopy. Left lung and parenchymal strips

were quick-frozen by immersion in liquid nitrogen and fixed with Carnoy's solution (26,

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27). Slices 4-µm-thick were cut and underwent hematoxylin-eosin and specific staining

methods to quantify the collagenous [Picrosirius-polarization method (28)] and elastic

system [Weigert's resorcin fuchsin method modified with oxidation (29)] fibers in the

alveolar septa.

Statistical analysis

SigmaStat 2.0 statistical software package (Jandel Corporation, CA, USA) was used.

Differences among groups were assessed by one-way analysis of variance and the Tukey

test. Correlation between mechanical and histological data was determined by Spearman

correlation test. A p value < 0.05 was considered significant.

RESULTS

The survival rate in P10 and P25 groups was 85% and 50%, respectively, within the first

day after injection. At day 30, survival was 33% in P10. Steroid led to 100% survival in

P10 group, but presented no beneficial effect in rats from P25 group, which died within 48

hours after ALI induction (Table 1).

In vivo mechanics

Respiratory system and lung static elastances (Figure 1), resistive,

viscoelastic/inhomogeneous, and total pressures (Figure 2) increased significantly with the

severity of lung injury (from P10 to P25). Respiratory system and lung static elastances and

resistive pressures returned to control values at 30 days in P10, while respiratory system

and lung viscoelastic and total pressure variations remained higher than control. P10 group

treated with steroid presented values of mechanical parameters similar to control group.

The beneficial effects of methylprednisolone were similar when given 1 or 6 hours after

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ALI induction, and remained unaltered at day 30. In P25 group, corticosteroid prevented

the modifications in Est,rs, Est,L, ∆P1,rs, and ∆P1,L, and attenuated ∆P2,rs, ∆P2,L,

∆Ptot,rs, and ∆Ptot,L changes. Chest wall total and resistive pressures increased in P25

group, and steroid did not modify these changes (Figures 1 and 2).

In vitro mechanics

E and R augmented from C to P10 and P25. In P10, E was higher at 30 days than at the first

day after ALI induction, while R was similar independently of the time course (Figure 3).

The use of methylprednisolone, either 1 or 6 hours after ALI induction, yielded E and R

values similar to control in P10 group, and attenuated the changes in P25. η increased only

in P25 group (Figure 3).

Lung histology

Typical photomicrographs of lung parenchyma from control, P10 at 1 and 30 days, and P25

groups with and without steroid 6 hours after ALI induction are shown in Figure 4. Because

lung histology was similar in groups M1 and M6, we depicted only one treatment (6 hours).

Lung histological changes included atelectasis, interstitial edema, and inflammation with

polymorphonuclear (PMN) cells in P10 group (Figure 4B). Lung parenchyma in P25

presented an increment in all histological changes described above, together with alveolar

edema and intra-alveolar hemorrhage (Figure 4D). P10 lung parenchyma at day 30 showed

only thickened alveolar membranes and increased cellularity in comparison to control

group (Figure 4F). Both P10 groups treated with steroid presented less interstitial edema

and tissue cellularity than P10 independent of the time of analysis (Figures 4C and G). In

P25 group, alveolar modifications were prevented by corticosteroid, while interstitial

edema and inflammatory cells, although lessened, remained increased (Figure 4E).

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Morphometrical examination of the lung demonstrated significant increase in alveolar

collapse in all P groups in relation to control (Table 2). Steroid reduced alveolar collapse in

P10 and P25 groups, although they remained higher than control at day 1 and similar to

control group at day 30. Alveolar hyperinflation was not present in any group. Total and

polymorphonuclear cell contents augmented from control to P10 and P25, but there was no

difference in cell count between P10 at days 1 and 30 (Table 2). Steroid presented anti-

inflammatory effects on acute lung injury independently its severity. Mononuclear cell

content was smaller in P25 and P10 than in control group. Mononuclear cells increased

with the time course of the lesion (Table 1).

Figure 5 shows the amounts of collagen and elastic system fibers in the alveolar

septa from lung tissue strips of control (C) and acute lung injury groups (P10 and P25), at

day 1 and 30, and after methylprednisolone (2 mg/kg) injection 1 or 6 hours after ALI

induction. Collagen fiber content increased with the severity of lung injury and with the

temporal evolution of ALI (Figure 5). Corticosteroid attenuated the increment in collagen

content independently of ALI degree. Additionally, its beneficial effects were present until

30 days after steroid injection. Elastic system fiber content increased only at 30 days after

ALI induction in P10, augmenting earlier in the more severe lesion (P25). Steroid

administration maintained the elastic fiber content equal to control value independently of

the dose (severity of the lesion) at day 1 and time (P10, 30 days) (Figure 5).

Figure 6 shows the ultramicroscopy of lung parenchyma. In P10 group

inflammatory cells including lymphocytes, plasma cells, macrophages, fibroblasts, and

primitive mesenchymal cell (PMC) were seen in the interstitium. The alveolar interstitium

was thickened due to both edema and increased amounts of matrix elements, such as

collagen fibers. The alveolar spaces had a mixed inflammatory infiltrate consisting of PMN

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and alveolar macrophages. Fibroblasts were frequently found together with types I and III

collagen fibers and elastin (Figure 6B). Steroid decreased the number of fibroblasts and

type III collagen fiber content. At day 1, elastic fiber content was normal (Figure 6C).

However, in P10 group 30 days after ALI induction and in P25 there was an increased

number of types I and III collagen fibers and elastin, thus characterizing fibroelastosis

(Figures 6D and F). Steroid treatment inhibited the aggregation process of type III collagen

fibers into type I (Figures 6C, E, and G). In addition, the amount of elastic fibers was also

normal (Figures 6E and G).

Considering all groups together, tissue elastance and resistance were correlated

significantly with total cell count [p=0.007 (r=0.37) and p=0.001 (r=0.44), respectively],

and with polymorphonuclear cell count [p=0.002 (r=0.42) and p=0.002 (r=0.42),

respectively]. Additionally, tissue elastance and resistance values were correlated

significantly with collagen [p<0.001 (r=0.65) and p<0.001 (r=0.57), respectively] and

elastic fiber content [p=0.01 (r=0.36) and p=0.01 (r=0.34), respectively). Hysteresivity was

correlated only with elastic fiber content [p=0.02 (r=0.33)].

DISCUSSION

In the present study, low-dose corticosteroid administered at an early phase of acute lung

injury kept unaltered in vivo and in vitro mechanical parameters in mildly abnormal lung

parenchyma, whereas it minimized the changes in tissue viscoelastic properties in a severe

lesion. Paraquat-induced ALI led to a time- and dose-dependent increase in the amount of

collagen and elastic fibers. Furthermore, the amount of elastic system fibers augmented

later in the course of mild ALI. Corticosteroid treatment acted on the remodeling process,

significantly reducing collagen fibers deposition in mild and severe ALI, and also

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preventing elastogenesis. In addition, the use of steroids at the early phase of ALI showed

beneficial effects 24 hours after lung injury induction, which lasted until the 30th day post-

injection in the mild form.

It has been previously described that corticosteroid, when given in large doses

before or shortly after endotoxin administration in sheep, prevent the subsequent increase in

lung vascular permeability, but do not reverse the abnormality once established (30). In

addition, these abnormalities resolve within 24 hours even if no treatment is given. These

results differ from ours, where 50% of the animals with 25 mg/kg paraquat-induced ALI

died at the first 24 hours and 100% at 48 hours. At autopsy, these animals presented acute

tubular necrosis, esteatosis, carditis, and diffuse alveolar damage. The use of corticosteroid

did not change survival rate in the severe lesion. On the other hand, steroid increased

survival in 10 mg/kg paraquat-induced ALI (Table 1).

Methylprednisolone was chosen as steroid therapy because it is commonly used as

an anti-inflammatory agent in the treatment of human pulmonary fibrosis and acute

respiratory distress syndrome (7-10). Corticosteroids modulate the host defence response at

virtually all levels, protecting the host from immune system overreaction. Corticosteroids

inhibit nuclear factor (NF)-κB and activator protein (AP)-1, blocking NF−κB dependent

pro-inflammatory gene expression (31, 32), and the transcription of several cytokines

relevant to ARDS pathology. In addition to its anti-inflammatory properties,

methylprednisolone is also known to inhibit proliferation of fibroblasts and synthesis of

collagen by fibroblasts in tissue culture (13, 33).

The initial enthusiasm for the use of corticosteroids to prevent and treat ARDS was

based on animal and human studies (13, 34). Several investigators suggested that the use of

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corticosteroids in the late phase of ARDS improved lung function and survival (7-10),

while trials of short term, high dose steroid therapy failed to show an improvement in

mortality in patients at risk of or with early ARDS (5-6). However, the studies on early

ARDS showed some potential limitations: the population studied, which varied

heterogeneously in terms of case mix and patient management, and the use of high doses of

steroids, leading to negative effects due to profound immunosuppression or other side

effects counterbalancing positive effects. Corticosteroid therapy could also be ineffective if

many of the patients, who were considered to have ARDS based on clinical definitions, did

not develop activation of inflammatory cascades in their lungs. Furthermore, the use of

steroid at the late phase of ARDS was based on the assumption that the fibroproliferative

phase began 7-10 days after the onset of the insult. However, different experiments

reported that the proliferative phase begins much sooner than had been previously

appreciated (14-19). Thus, inflammatory and repair mechanisms occur simultaneously

rather than subsequently.

In the present study, a controlled model of ALI induced by two different doses of

paraquat was used, allowing the analysis of different reproducible degrees of severity. In

addition, the ALI model selected was confirmed by light and electronic microscopy, with

great potential to induce fibrogenesis (19, 35). The single low dose of methylprednisolone

(2 mg/kg) administered early in the course of ALI (1 or 6 hours after lung injury induction)

minimized the negative side effects related to immunodepression. This study design could

potentially bypass the drawbacks observed in previous reports (5, 6). It is interesting to note

that histological lung changes observed at 24 hours in mild and severe ALI were already

present as early as 6 hours after ALI induction, although less intense. Thus,

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methylprednisolone was used before the beginning of functional changes (1 hour) and after

the establishment of the lung injury process (6 hours).

Our results showed that corticosteroid treatment at the early phase of ALI improved

in vivo and in vitro respiratory mechanics (Figures 1-3). The method used for determination

of in vivo respiratory mechanics allows the identification of its elastic, resistive, and

viscoelastic and/or inhomogeneous components (22, 23). Respiratory system and lung

static elastances, resistive, viscoelastic/inhomogeneous pressures increased significantly

with the severity of lung injury at day 1 (Figures 1 and 2), but only respiratory system and

lung viscoelastic pressures remained higher than control values at day 30 in P10. Prior

studies described changes in lung resistance and elastance in ALI, resulting from surfactant

dysfunction and/or loss of functional capacity due to alveolar flooding (36, 37). Actually,

mechanical dysfunction can result from air-liquid interface and/or tissue changes (38). In

the present work, the increase in lung resistive pressure probably reflects a reduction in

bronchial caliber caused by fluid in the airways, reflex bronchoconstriction, and/or reduced

lung volume. The augment in lung viscoelastic and/or inhomogeneous pressure, suggests

the presence of heterogeneities that can be due to many different factors, e.g., alveoli

collapse and overdistension, distortion of patent alveoli, edema, inflammation with

neutrophils and mononuclear cells infiltration (Table 2, Figure 4), and changes in collagen

and elastic fiber contents (Figure 5). The maintenance of the changes in lung viscoelastic

pressure at day 30 suggests that parenchymal dysfunction dominates the later ALI, since

alveolar inhomogeneities and edema were less remarkable in this period (Figure 4F). Lung

static elastance in P10 and P25 groups was significantly increased compared to control, in

accordance with previous results (39, 40).

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Corticosteroid prevented the in vivo mechanical changes in P10 group

independently of the time of injection and of the moment of analysis (1 or 30 days after

ALI induction), while in P25 group corticosteroid prevented the modifications in lung static

elastance and resistive pressure, and attenuated viscoelastic changes (Figures 1 and 2).

These modifications could be attributed to the effect of corticosteroid on inflammatory (41-

43) and fibroproliferative processes (9, 13, 44, 45), leading to less atelectasis, cellular

influx, and fluid transudation in the groups treated with steroid (Figure 4, Table 2). It has

been demonstrated that in experimental acute lung injury corticosteroid treatment is

effective in decreasing lung collagen content and edema formation as long as treatment is

prolonged, whereas steroid withdrawal rapidly reverses this positive effect (13, 44, 45).

However, recent evidence suggests that the inflammatory and fibrotic processes appear to

be separately regulated (46), thus offering the possibility for early-directed treatments

against fibrosis independently of the effects on inflammation. Recently, the beneficial

effects of early low-dose steroid treatment was also observed in patients with septic shock,

where low-dose hydrocortisone treatment inhibited systemic inflammation and prevented

overwhelming compensatory anti-inflammatory response (47).

Parallel to the in vivo analysis, oscillatory tissue mechanics was also addressed. The

importance of alterations in the biophysical properties of the surfactant system in

ARDS/ALI pathophysiology (36, 37), as well as the relevant role of tissue inhomogeneities

secondary to alveoli collapse/hyperdistension are well established. The method used to

determine lung tissue mechanics avoids the influence of kinetics of surface-active molecule

absorption-desorption to the surface film and of recruitment-derecruitment (25, 48, 49).

This method specifically allows the analysis of tissue resistance, elastance, and

hysteresivity after the induction of ALI in the absence of surfactant and interdependence

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effects, providing a direct assessment of tissue physiology (38). Additionally, a direct

analysis of the role of fiber-fiber network within the connective tissue matrix on tissue

mechanical properties is ensured (25, 48, 49). The present study is the first analysis of

oscillatory tissue mechanical properties of lung parenchyma in animals with acute lung

injury treated with corticosteroid. Elastance and resistance of lung parenchyma of paraquat-

treated rats were significantly increased in relation to control (Figure 3), supporting

previous findings that parenchymal mechanical dysfunction plays an important role in ALI

pathophysiology (19, 38). Hysteresivity increased only in P25 group.

The changes in tissue mechanics were accompanied by deposition of collagen and

elastic fibers in the alveolar septa (Figure 5). The kinetics of paraquat-induced fibrogenesis

showed a continuous transition among C, P10 at day 1, and P10 at day 30, as previously

reported (50). In addition, the present results disclosed that collagen content was already

elevated 1 day after tissue damage whatever the dose of paraquat used, indicating that the

biochemical processes implicated in collagen synthesis are indeed able to react very quickly

to the aggression. Conversely, several studies (9, 14-16, 18) have reported that elevated

procollagen aminoterminal propeptide levels in the BALF reflect collagen synthesis at the

site of disease, and may be used as markers of the reparative process. Newly synthesized

procollagen is cleaved by specific endopeptidases at the amino and carboxy termini,

forming collagen molecules (51). The amino and carboxy propeptides are soluble proteins,

sampled relatively ease by BAL, and used as good markers of collagen synthesis without

resorting to invasive tissue sampling (52). Thus, these studies (9, 14-16, 18) demonstrate

the presence of increased collagen synthesis, with no indication of matrix deposition, which

would be possible only through direct histological analysis. In this line, our findings

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demonstrated that the increased collagen synthesis previously observed (9, 14-16, 18) was

actually followed by matrix deposition.

Collagen types were identified by electron microscopy (Figure 6). Type I collagen

fibrils closely aggregate to form thick fibers of 74 nm, while type III collagen fibrils present

a mean fibrillary diameter of 45 nm (53). Type III collagen appeared early in mild lesion

(P10), while type I collagen appeared late in P10 group (at 30 day) and in the severe lesion

(P25). In this context, Armstrong et al. (52) hypothesized that an imbalance between

synthesis and degradation may contribute to the net accumulation of type I collagen in

ARDS, and that the profibrotic response occurred early in the course of disease, being

associated with the severity of the lung injury and mortality. In our study steroid treatment

was administered before fibroproliferation advanced to end-stage acellular fibrosis, when

the more resistant type I collagen predominates. Methylprednisolone prevented the

increment in tissue elastance and resistance of paraquat-treated rats (Figure 3). The

improvement in tissue mechanics could be explained by the effect of steroid preventing

excessive collagen deposition, increasing collagen breakdown, or inhibiting the aggregation

process of type III collagen fiber into type I (9). Corticosteroid also minimized epithelial

and endothelial cell damage, fibroproliferation, and extracellular matrix deposition (Figure

6). The beneficial effects of steroid could be attributed to its inhibitory effects on host

defense response, including modulation of macrophage and fibroblast activity (7-10, 43,

54).

The present study also disclosed the effects of corticosteroid on elastic system.

Although there are some studies describing collagen changes after corticosteroid treatment

in ALI, the elastic system is scantly dealt with. Mild ALI was followed by a late increase in

elastic fiber content, while the severe lesion presented early elastogenesis. In bleomycin

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models of lung fibrosis, elastic fiber content increased at 14 and 30 days (55, 56). However,

in severe paraquat-induced lung injury, the amount of elastic fibers was augmented as early

as 24 hours after ALI induction.

Methylprednisolone modified elastogenesis both in mild and severe ALI (Figure 5).

Recently, we quantified the amount of different types of elastic fibers in the alveolar septa

(elaunin, oxytalan, and fully developed fibers), and observed that oxytalan fibers were

increased in paraquat-induced ALI (19). Oxytalan fibers appear early in the development of

the elastic system, and consist of microfibrils without elastin, rigid under mechanical stress

(28). In our study, the amount of oxytalan fibers in the presence of steroid did not increase;

supporting the hypothesis that steroid could probably diminish elastase activity,

consequently interfering in the resynthesis of the elastic system fiber.

The anatomic elements that potentially determine tissue viscoelastic behavior

include the network of stress-bearing collagen and elastic fibers, proteoglycan and

glycosaminoglycans, as well as the contractile elements present in parenchymal tissues (25,

57). Other authors (58, 59) have suggested that the pulmonary parenchyma can be modeled

as an interconnected network of elastic elements, presumably composed of collagen and

elastic fibers, which determine the mechanical behavior of the system. In our study, tissue

E and R changes were correlated with collagen and elastic fiber contents, while

hysteresivity was correlated only with elastic fibers. Similarly, Yuan et al reported (48, 49)

that collagen and elastic fibers contribute to tissue elasticity during normal breathing.

According to the structural damping hypothesis, induced changes in hysteresivity

with ALI must reflect changes in the kinetics of the stress-bearing process, e.g., the

extracellular fiber matrix, the surface lining layer, and the contractile apparatus (25). In P25

group, a significant modification of collagen-elastin fiber network was present and was

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probably the main determinant of the increase in hysteresivity. We hypothesized that the

increment in the amount of elastic fibers, primarily oxytalan fibers, could be responsible for

a derangement of the tridimensional extracellular matrix organization, which would had a

greater impact on tissue mechanics than the effective amount of each fiber component.

These findings are supported by the observation that tissue hysteresivity is not determined

by collagen and elastic fibers alone, being most likely a network effect (49). It is interesting

to observe that in mild ALI the increment in collagen and tissue cellularity was not large

enough to affect hysteresivity.

In conclusion, single low-dose methylprednisolone led to a complete maintenance

of in vivo and in vitro respiratory mechanics in mild ALI, and minimized the changes in

tissue impedance and extra-cellular matrix components in a severe lesion. In addition, the

beneficial effects of the early use of steroids in ALI were also seen at 30 days after lung

injury. We should be extremely cautious in extrapolating these data to the more complex

clinical situation, since this is an animal model of ALI induced by paraquat, and, thus, the

direct extrapolations to the clinical syndrome is unwarranted. Additionally, our animals

were not ventilated or treated with fluid management, thwarting the comparison of the

animal outcome with the human’s. However, the present study has an important clinical

relevance, i.e., the treatment with a single low dose of methylprednisolone at the early

phase of ALI may help to prevent fibroelastogenesis, avoiding the side effects related to

prolonged and high doses of steroid. Finally, this study may help the design of a clinical

trial focused on diminishing clinical lung injury with steroid.

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Acknowledgement: We would like to express our gratitude to Mr. Antonio Carlos de

Souza Quaresma and Mrs. Alaine Prudente for their skillful technical assistance, and to Dr.

Ana Paula B Barbosa for her help with the initial analysis of tissue mechanics.

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Legends:

Figure 1. Static elastance (Est) of respiratory system (rs), lung (L), and chest wall (w). In

control group (C) saline (5 ml/kg) was intraperitoneally (i.p.) injected. In P groups,

different doses of paraquat (10 and 25 mg/kg) were injected i.p. 1 and 30 days prior to the

measurements. M1 and M6 correspond to the groups that received methylprednisolone (2

mg/kg) 1 and 6 hours, respectively, after the induction of acute lung injury with different

doses of paraquat. Values are means (+SEM) of 6 animals. *Values significantly different

from C (p<0.05); **values significantly different from P at the same dose and time

(p<0.05); #values significantly different from P10 at day 1 (p<0.05).

Figure 2. Stacked bar chart plot data where the gray bars represent the resistive pressures

(∆P1) and the white bars are the viscoelastic/inhomogeneous (∆P2) pressure dissipations of

the respiratory system (rs), lung (L), and chest wall (w). The whole column represents the

total pressure variation in each group. In control group (C) saline (5 ml/kg) was

intraperitoneally (i.p.) injected. In P groups, different doses of paraquat (10 and 25 mg/kg)

were injected i.p. 1 and 30 days prior to the measurements. M1 and M6 correspond to the

groups that received methylprednisolone (2 mg/kg) 1 and 6 hours, respectively, after the

induction of acute lung injury with different doses of paraquat. Values are means (+SEM)

of 6 animals. *Values significantly different from C (p<0.05); **values significantly

different from P at the same dose and time (p<0.05); #values significantly different from

P10 at 1 day (p<0.05).

Blue-200302-2560C-R1-Clean version 27

Figure 3. Tissue elastance, resistance, and hysteresivity at 1 Hz. In control group (C) saline

(5 ml/kg) was intraperitoneally (i.p.) injected. In P groups, different doses of paraquat (10

and 25 mg/kg) were injected i.p. 1 and 30 days prior to the measurements. M1 and M6

correspond to the groups that received methylprednisolone (2 mg/kg) 1 and 6 hours,

respectively, after the induction of acute lung injury with different doses of paraquat.

Values are means (SEM) of 6 animals.*Values significantly different from C (p<0.05);

**values significantly different from P at the same dose and time (p<0.05); #values

significantly different from P10 at 1 day (p<0.05).

Figure 4. Photomicrographs of lung parenchyma stained with hematoxylin-eosin in control

(A) and paraquat treated lungs [P10 (B) and P25 (D)] at day 1, and P10 at day 30 (F).

Panels C, E, and G correspond to paraquat groups (as in B, D, and F, respectively) treated

with methylprednisolone 6 hours after ALI induction. Scale bars=200 µm.

Figure 5. Amounts of collagen and elastic system fibers in alveolar septa. In control group

(C) saline (5 ml/kg) was intraperitoneally (i.p.) injected. In P groups, different doses of

paraquat (10 and 25 mg/kg) were injected i.p. 1 and 30 days prior to the measurements. M1

and M6 represent the groups that received methylprednisolone (2 mg/kg) 1 and 6 hours,

respectively, after the induction of acute lung injury with different doses of paraquat.

Values are means (+SEM) of 6 animals.*Values significantly different from C (p<0.05);

**values significantly different from P at the same dose and time (p<0.05); #values

significantly different from P10 at 1 day (p<0.05).

Blue-200302-2560C-R1-Clean version 28

Figure 6. Electron microscopy of lung parenchyma in control (A) and paraquat treated

lung at day 1 [P10 (B) and P25 (D)] and 30 (F). In P10 and P25 groups, paraquat (10 and

25 mg/kg, respectively) was injected intraperitoneally. Panels C, E, and G correspond to

paraquat groups treated with methylprednisolone (2 mg/kg, i.v.) 6 hours after the induction

of ALI. Normal ultrastructure in untreated lung (A). Note the type II pneumocyte (PII) with

lamellar bodies (lb) (Panel A). In P10 (Panel B) there were apoptotic changes in PII

characterized by condensation of chromatin (arrows). Type III collagen content (stars) was

augmented and incipient type I collagen fiber (col I) synthesis is also evident. A randomly

orientated, interconnected network of elastic fibers is depicted (arrowheads). PMC:

primitive mesenchymal cell. In panel D, types I and III collagen syntheses are evident in

P25. Arrowheads indicate elastin distributed throughout the interstitium, stars represent

type III collagen fibers. Mφ: macrophage. In P10 group 30 days after ALI induction

fibroelastosis was present (Panel F). In panels C and E, hyaline membranes (HM) are still

evident after methylprednisolone treatment in both P10 and P25 groups, respectively, as

well as edema of the basement membrane (BM) (Panel C). Panels C, E, and G show that

steroid treatment modulates the increment in types I and III collagen fibers (stars) and

elastogenesis. Photomicrographs are representative of data obtained from lung sections

derived from six animals.

Blue-200302-2560C-R1-Clean version 29

TABLE 1. EFFECT OF STEROID ON THE SURVIVAL OF PARAQUAT-

CHALLENGED RATS

Groups Number of surviving rats Survival (%)

DAY 1

CONTROL 6(6) 100

P10 6(7) 85

P10M1 6(6) 100

P10M6 6(6) 100

P25 6(12) 50

P25M1 6(10) 60

P25M6 6(12) 50

DAY 30

P10 6(9) 67

P10M1 6(6) 100

P10M6 6(6) 100

Data represent the number of surviving rats, with the total number of rats in parentheses. In

control group, saline was intraperitoneally (i.p.) injected. In P groups, different doses of

paraquat (10 and 25 mg/kg) were injected i.p. 1 and 30 days prior to the measurements. M1

and M6 correspond to the groups that received methylprednisolone (2 mg/kg) 1 and 6

hours, respectively, after the induction of acute lung injury with different doses of paraquat.

Rats from P25 group independent of the use of steroid died within 48 hours after ALI

induction

Blue-200302-2560C-R1-Clean version 30

TABLE 2. MORPHOMETRICAL PARAMETERS

Groups Normal (%) Alveolar Collapse (%)

Total cell (%) PMN (%) MN (%)

DAY 1

CONTROL 97.9±0.8 2.1±0.8 18.2±1.1 3.7±0.7 14.5±0.9

P10 85.9±0.7* 13.9±0.7* 25.6±1.5* 23.3±2.5* 2.3±0.4*

P10M1 91.8±0.3* ** 8.2±0.3* ** 17.7±1.2** 16.2±1.4* ** 1.6±0.2*

P10M6 91.1±0.8* ** 8.9±0.8* ** 16.4±2.1** 14.8±2.1* ** 1.6±0.3*

P25 74.6±1.9*# 25.4±3.4*# 32.8±1.8*# 32.2±1.8*# 0.7±0.1*#

P10M1 84.0±2.0* ** 16.7±1.9* ** 22.8±1.4** 21.0±1.5* ** 1.8±0.3* **

P10M6 77.2±0.8* ** 22.8±0.8* ** 24.8±1.8** 22.5±1.7* ** 2.3±0.3* **

DAY 30

P10 78.6±1.9*# 21.4±1.9*# 26.3±0.6* 20.4±1.1* 5.9±0.6*#

P10M1 94.3±0.8** 5.8±0.8** 20.1±0.3** 15.5±0.7* ** 4.6±0.5*

P10M6 93.3±2.0** 6.9±2.0** 21.7±1.0** 15.6±0.9* ** 6.1±0.3*

Values are means ± SEM of 6 lungs (rats) in each group (10 random, non-coincident

microscopic fields were analyzed in each lung). In control group, saline was

intraperitoneally (i.p.) injected. In P groups, different doses of paraquat (10 and 25 mg/kg)

were injected i.p., 1 and 30 days prior to the measurements. M1 and M6 correspond to the

groups that received methylprednisolone (2 mg/kg) 1 and 6 hours, respectively, after the

induction of acute lung injury with different doses of paraquat. Total cell: total cellular

fractional area, PMN: polymorphonuclear cell fractional area, MN: mononuclear cell

fractional area. *Values significantly different from C (p<0.05); **values significantly

different from P at the same dose and time (p<0.05); #values significantly different from

P10 at day 1 (p<0.05).

Blue-200302-2560C- R1 31

Est

(cm

H2O

/ml)

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4

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8

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C P10

1 day

****

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Blue-200302-2560C- R1 32

Figure 2.

∆∆ ∆∆P (

cmH

2O)

0

1

2

3

4

5

6

7

8 ∆∆∆∆P2∆∆∆∆P1

rs∆∆ ∆∆P

(cm

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)

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2

3

4

5

6

∆∆ ∆∆P (

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** **

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*#

*#

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P M1M6 P M1M6 P M1 M6

Blue-200302-2560C- R1 33

Figure 3.

C P10 P25 P10

1 day 30 days

Ela

stan

ce (

104 N

/m2 )

0.0

0.5

1.0

1.5

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2.5

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(10

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0.02

0.04

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Blue-200302-2560C- R1 34

Figure 4.

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Blue-200302-2560C- R1 35

Figure 5.

C P10 P25 P10

1 day 30 days

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llag

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iber

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m( µµ µµ

m2 / µµ µµ

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Blue-200302-2560C- R1 36

Figure 6.

*

B C

*

Alb

*

**

EFFECT OF CORTICOSTEROID ON LUNG PARENCHYMA REMODELING

AT AN EARLY PHASE OF ACUTE LUNG INJURY

PATRICIA R.M. ROCCO1, ALBA B. SOUZA1, DEBORA S. FAFFE1, CAROLINE P.

PÁSSARO1, FLÁVIA B. SANTOS1, ELNARA M. NEGRI2, JANUÁRIO G.M. LIMA1,

RENATA S. CONTADOR1, VERA L. CAPELOZZI3, WALTER A. ZIN1

ONLINE DATA SUPPLEMENT

Blue-200302-2560C- R1 - Online-only repository

1

METHODS

Animal preparation

Seventy-eight Wistar rats (250-300 g) were randomly divided into 13 groups of 6 animals each.

In the control group (C), saline (0.9% NaCl, 5 ml/kg BW) was injected intraperitoneally (i.p.). In

P groups different doses of paraquat were injected i.p. (10 and 25 mg/kg BW), 1 or 30 days prior

to the measurements. Each group (P10 or P25) was divided into 3 subgroups: animals that

received solely paraquat or were additionally treated with methylprednisolone (M, 2 mg/kg, i.v.)

at 1 or 6 hours after paraquat administration. The animals were sedated with diazepam (5 mg

i.p.), anesthetized with pentobarbital sodium (20 mg/kg i.p.), and a snugly fitting cannula (1.7

mm ID) was introduced into the trachea. Mechanical ventilation (model 683, Harvard Apparatus,

Southnatick, MA, USA) was then started with a frequency of 80 breaths.min-1 and a tidal volume

of 6 ml/kg.

A pneumotachograph (1.5 mm ID, length = 4.2 cm, distance between side ports = 2.1 cm)

was connected to the tracheal cannula for the measurements of airflow (V’) and changes in lung

volume (VT) (1). The pressure gradient across the pneumotachograph was determined by means of a

Validyne MP45-2 differential pressure transducer (Engineering Corp, Northridge, CA, USA). The

flow resistance of the equipment (Req), tracheal cannula included, was constant up to flow rates of

26 mL.s-1, and amounted to 0.12 cmH2O.mL-1.s. Equipment resistive pressure (= Req/V’) was

subtracted from pulmonary resistive pressure so that the present results represent intrinsic values.

Tracheal pressure was measured with a Validyne MP-45 differential pressure transducer

(Engineering Corp, Northridge, CA, USA). Changes in esophageal pressure (Pes), which reflects

chest wall pressure (Pw), were measured with a 30-cm-long water-filled catheter (PE-240) with side

holes at the tip connected to a PR23-2D-300 Statham differential pressure transducer (Hato Rey,

Puerto Rico). The catheter was passed into the stomach and then slowly returned into the

Blue-200302-2560C- R1 - Online-only repository

2

esophagus; its proper positioning was assessed using the "occlusion test" (2). The frequency

responses of the pressure measurement systems (Ptr and Pes) were flat up to 20 Hz, without

appreciable phase shift between the signals. All signals were conditioned and amplified in a

Beckman type R Dynograph (Beckman Instruments, Schiller Park, IL, USA). Flow and pressure

signals were also passed through 8-pole Bessel filters (902LPF, Frequency Devices, Haverhill, MA,

USA) with the corner frequency set at 100 Hz, sampled at 200 Hz with a 12-bit analogue-to-digital

converter (DT2801A, Data Translation, Marlboro, MA, USA), and stored on a PC-compatible

computer. All data were collected using LABDAT software (RHT-InfoData Inc., Montreal, Quebec,

Canada).

Measurement of respiratory mechanics

Muscle relaxation was achieved with gallamine triethyliodide (2 mg/kg i.v.) and artificial

ventilation was provided by a constant flow ventilator (Samay VR15, Universidad de la

Republica, Montevideo, Uruguay). During the test breaths the ventilator was adjusted to generate

a 5-s end-inspiratory pause, whereas during baseline ventilation no pause was used. Special care

was taken to keep tidal volume (VT = 1 ml) and flow (V’= 7 ml/s) constant in all animals in order

to avoid the effects of different flows and volumes (3) and inspiratory duration (4) on the

measured variables.

The experiments did not last more than 40 minutes. Respiratory mechanics were measured by

the end-inflation occlusion method (5, 6). Briefly, after end-inspiratory occlusion, there is an initial

fast drop in tracheal pressure (∆P1,rs) from the preocclusion value down to an inflection point

(Pi,rs) followed by a slow pressure decay (∆P2,rs), until a plateau is reached. This plateau

corresponds to the elastic recoil pressure of the respiratory system (Pel,rs). ∆P1,rs selectively

reflects the combination of airways, pulmonary, and chest wall Newtonian resistances in normal

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animals and humans (5, 6), and ∆P2,rs reflects stress relaxation, or viscoelastic properties, of the

lung and chest wall tissues, together with a small contribution of pendelluft (6, 7). The same

procedures apply to the chest wall pressure (Pw) yielding the values of ∆P1,w, Pi,w, ∆P2,w, and

Pel,w, respectively. Transpulmonary pressures (∆P1,L, Pi,L, ∆P2,L, and Pel,L) were calculated by

subtracting the chest wall from the corresponding values pertaining to the respiratory system. Total

pressure drop (∆Ptot) is equal to the sum of ∆P1 and ∆P2 yielding the values of ∆Ptot,rs, ∆Ptot,L,

and ∆Ptot,w. Respiratory system, lung, and chest wall static elastances (Est,rs, Est,L, and Est,w,

respectively) were calculated by dividing Pel,rs, Pel,L, and Pel,w, respectively, by VT. Dynamic

elastances of the respiratory system, lung, and chest wall (Edyn,rs, Edyn,L, and Edyn,w,

respectively) were obtained by dividing Pi,rs, Pi,L, and Pi,w, respectively, by VT. ∆E was calculated

as the difference Edyn - Est, yielding the values of ∆E,rs, ∆E,L, and ∆E,w. Respiratory mechanics

were measured 10 times in each animal.

The delay between the beginning and the end of the valve closure (10 ms) was allowed for by

back-extrapolation of the pressure records to the actual time of occlusion and the corrections in

pressure, although very minute, were performed as previously described (8).

All data were analyzed using ANADAT data analysis software (RHT-InfoData Inc.,

Montreal, Quebec, Canada).

Measurement of tissue mechanics

Tissue preparation

Heparine (1000 IU) was intravenously injected immediately after the determination of

respiratory mechanics. The trachea was clamped ten minutes later at end-expiration, and the

abdominal aorta and vena cava were sectioned, yielding a massive hemorrhage that quickly

killed the animals. The lungs were removed en bloc, and placed in a modified Krebs-Henseleith

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(K-H) solution containing (mM) 118.4 NaCl, 4.7 KCl, 1.2 K3PO4, 25 NaHCO3, 2.5 CaCl2.H2O,

0.6 MgSO4.H2O, and 11.1 glucose; at pH = 7.40 and 6°C. A 3x3x10 mm strip of subpleural

parenchyma was cut from the periphery of each left lung and suspended vertically in an organ

bath filled with K-H solution maintained at 37°C, continuously bubbled with a mixture of 95%

O2-5% CO2.

Lung strips were weighed (W), and their unloaded resting lengths (L0) were determined

with a caliper. Lung strip volume was measured by simple densitometry, as: Vol = ∆F/δ, where

∆F is the total change in force before and after strip immersion in K-H solution and δ is the mass

density of K-H solution (9-12).

Apparatus

Parenchymal strips were suspended vertically in a K-H organ bath maintained at 37ºC and

continuously bubbled with of 95% O2-5% CO2. Metal clips made of 0.5 mm-thick music wire

were glued to both ends of the tissue strip with cyanoacrylate. One clip was attached to a force

transducer (FT03, Grass Instruments Co., Quincy, MA, USA), whereas the other one was

fastened to a vertical rod. This fiberglass stick was connected to the cone of a woofer, which was

driven by the amplified sinusoidal signal of a waveform generator (3312A Function Generator,

Hewlett Packard, Beaverton, OR, USA). A sidearm of the rod was linked to a second force

transducer (FT03, Grass Instruments Co., Quincy, MA, USA) by means of a silver spring of

known Young’s modulus, thus allowing the measurement of displacement. Length and force

output signals were conditioned (Gould 5900 Signal Conditioner Frame, Gould Inc., Valley

View, OH, USA), fed through 8-pole Bessel filters (902LPF Frequency Devices, Haverhill, MA,

USA), analogue-to-digital converted (DT2801A, Data Translation Inc., Marlboro, MA, USA),

and stored on a computer. All data were collected using LABDAT software (RHT-InfoData Inc.,

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Montreal, Quebec, Canada). The frequency response of the system was dynamically studied by

using calibrated silver springs with different elastic Young’s modulus. Neither amplitude

dependence (<0.1% change in stiffness) nor phase changes with frequency were detected in the

range from 0.01 to 14 Hz. The hysteresivity of the system was independent of frequency and had

a value <0.003 (11, 12).

Preconditioning

Cross-sectional, unstressed area (A0) of the strip was determined from volume and unstressed

length, according to A0 = vol/L0. Basal force (FB) for a stress of 0.1 N/cm2 was calculated as FB

(N) = 0.1 (N/cm2).A0 (cm2) and adjusted by vertical displacement of the force transducer (11-

14). The displacement signal was then set to zero. Once basal force and displacement signals

were adjusted, the length between bindings (LB) was measured by means of a precision caliper.

Instantaneous length during oscillation around LB was determined by adding the value of LB to

the measured value of displacement at any time.

After the basal force was adjusted to 8x10-3 N, each parenchymal strip was

preconditioned by sinusoidal oscillation of the tissue during 30 min (frequency = 1 Hz;

amplitude large enough to reach a final force of 1x10-2 N). Thereafter the amplitude was adjusted

to 5% L0 and the oscillation maintained for another 30 min, or until a stable length-force loop

was reached (12). The isometric stress adaptation period resulted in a final force of 5x10-3 N.

After preconditioning, the strips were oscillated at a frequency (f) = 1 Hz (11, 12). The bath

solution was renewed regularly (every 20 min) with 37° C K-H solution.

Measurements of parenchymal mechanics

To calculate tissue resistance (R), elastance (E), and hysteresivity (η) force-length curves were

analyzed (10-12). Instantaneous average cross sectional area (Ai) was determined as Ai = Vol/Li

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(cm2), where Li is instantaneous length. Instantaneous stress (σi) was calculated by dividing

force (g) by Ai (cm2), σi= F/Ai.

All mechanical parameters were measured cycle-by-cycle. Tissue resistance (R) was

determined from the enclosed area of force-length loops:

R = (4⋅H)/ [ π⋅ω⋅(∆ε)2]

where H is the stress-strain hysteresis area, ω is the angular frequency (ω = 2πf, rad/s), and ∆ε is

the normalized strain or peak-to-peak change in length (∆L) divided by LB. Tissue dynamic

elastance was determined as:

E = (∆σi/∆ε) cosθ

where ∆σi represents the peak-to-peak change in force, and θ is the phase lag between force and

displacement [θ = sin-1 (4⋅H/(π⋅∆σi⋅∆ε))]. Hysteresivity, defined by Fredberg and Stamenovic

(15) as a dimensionless variable coupling the dissipative and elastic behaviors was calculated by

using the following equation: η = tan θ.

Histology and morphometry

Lung

Morphometric analysis was performed in excised lungs at end-expiration. Immediately after the

removal of the lungs en bloc, the right lung was quick-frozen by immersion in liquid nitrogen

and fixed with Carnoy's solution (ethanol:cloroform:acetic acid, 70:20:10 by volume) at −70°C

for 24 hours. Progressively increasing concentrations of ethanol at -20°C were then substituted

for Carnoy's solution until 100% ethanol was reached. The tissue was maintained at -20°C for 4

hours, warmed to 4°C for 12 hours, and then allowed to reach and remain at room temperature

for 2 hours (16). After fixation, the tissue was embedded in paraffin. Blocks were cut 4−µm-

thick by a microtome. The slices were stained with hematoxylin-eosin. Two investigators, who

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were unaware of the origin of the material, performed the microscopic examination. Morphometric

analysis was performed with an integrating eyepiece with a coherent system made of a 100-point

grid consisting of 50 lines of known length, coupled to a conventional light microscope

(Axioplan, Zeiss, Oberkochen, Germany). The volume fraction of collapsed and normal

pulmonary areas and the fraction of the lung occupied by large-volume gas-exchanging air

spaces (hyperinflated structures with a morphology distinct from that of alveoli and wider than

120 µm) were determined by the point-counting technique (17), made at a magnification of x40

across 10 random, noncoincident microscopic fields.

Polymorpho- and mononuclear cells, and pulmonary tissue were determined in each

sample by the point-counting technique (17) across ten random noncoincident microscopic fields

at x1000 magnification. Points falling on tissue area and not over alveolar spaces were counted

and divided by the total number of points in each microscopic field. Thus, data were reported as

the fractional area of pulmonary tissue (17). The same method was applied to determine

polymorpho- and mononuclear cells.

Parenchymal strips

At the end of the experiments the organ bath was removed and the parenchymal strips were

frozen by rapid immersion in liquid nitrogen at the force maintained during the experiment.

Frozen strips were fixed as aforementioned.

The slices underwent specific staining methods to characterize the fibers of the collagenous

and elastic systems present in the alveolar septa. Collagen: The tissue was stained in a solution

of Sirius Red dissolved in aqueous saturated picric acid, and observed under polarized light

microscopy, as the enhancement of collagen birefringence promoted by the picrosirius-

polarization method is specific for collagenous structures (18). Elastic fibers: Two different

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methods were used: Weigert's Resorcin Fuchsin method (RF) (19), which allows the

identification of elaunin and fully developed elastic fibers, and Weigert's Resorcin Fuchsin

method modified with oxidation (ORF) (20), which allows the identification of the three

components of the elastic fiber system (elaunin, oxytalan, and fully developed elastic fibers). The

oxytalan fibers were calculated by subtracting the amount of fibers given by the RF method from

the value provided by the ORF method. In each rat twenty different microscopic fields were

randomly selected in order to quantify collagen or elastic fibers. Quantification was carried out

with the aid of a digital analysis system and specific software (Bioscan-Optimas 5:1, Bioscan

Incorporated, Edmond, WA, USA) under x200 magnification. The images were generated by a

microscope (Axioplan, Zeiss, Oberkochen, Germany) connected to a camera (Sony Trinitron

CCD, Sony, Tokyo, Japan), fed into a computer through a frame grabber (Oculus TCX, Coreco

Inc., St Laurent, PQ, Canada) for off-line processing. The thresholds for fibers of the collagenous

and elastic systems were established after enhancing the contrast up to a point at which the fibers

were easily identified as either black (elastic) or birefringent (collagen) bands. The area occupied

by fibers was determined by digital densitometric recognition. Bronchi and blood vessels were

carefully avoided during the measurements. To avoid any bias due to septal edema or alveolar

collapse the areas occupied by the elastic and collagen fibers, measured in each alveolar septum,

were divided by the length of each studied septum. The results were expressed as the amount of

elastic and collagen fibers per unit of septal length.

Transmission electron microscopy

To obtain a stratified random sample, three slices of 2x2 mm were cut from three different

segments of the right lung (upper, middle, and lower lobes), and then fixed with glutaraldehyde

2.5% and phosphate buffer 0.1M (pH = 7.4) for 60 min at –4°C. The slices were then rinsed in

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phosphate buffer, post-fixed with 1% osmic tetroxide in phosphate buffer for 30 min and

rewashed three times in phosphate buffer. Finally, the slices were dehydrated in an acetone

series, and then placed in a mixture of 1:1 acetone:Epon overnight before embedding in Epon for

6 hours. After fixation the material was kept for 48 hours at 60°C before being submitted to

ultramicrotomy for transmission electron microscopy.

Statistical analysis

The normality of the data (Kolmogorov-Smirnov test with Lilliefor’s correction) and the

homogeneity of variances (Levene median test) were tested. In all cases, both conditions were

satisfied, and thus one-way ANOVA was used to determine the possibility of differences among

the groups. If multiple comparisons were then required, Tukey test was applied. The

relationships between mechanical parameters and the amount of collagen and elastic fibers were

evaluated by Spearman correlation. In all tests the significance level was set at 5%. Statistical

analyses were done with Sigmastat 2.0 (Jandel Scientific, San Rafael, CA, USA).

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