Oral N-Acetylcysteine Attenuates
Elastase-Induced Pulmonary
Emphysema in Rats*
Maria L. Rubio, PhD; M. Carmen Martin-Mosquero, MS; Mercedes Ortega, MS;
German Peces-Barba, PhD; and Nicolás González-Mangado, PhD
Study objective: To study the effect of the antioxidant N-acetylcysteine (NAC) in the development
of elastase-induced emphysema in rats.
Materials and methods: Wistar rats (n ⴝ 72) were orotracheally instilled with 75 IU elastase or
saline solution. Eighteen rats from each group received the antioxidant NAC from 2 days before
induction of the lesion until they were killed 2, 8, and 28 days after instillation. The effects of
treatment were assessed by measuring collagen content for the left lung, a histopathology
evaluation (ie, mean alveolar internal surface area (AIA) and mean linear intercept measurement), and lung function.
Results: Twenty-eight days after elastase instillation, rats treated with NAC showed significant
attenuation of the lesion in comparison with rats treated only with elastase, including the
following: normalization of mean (ⴞ SEM) collagen content (1.23 ⴞ 0.09 vs 1.51 ⴞ 0.10 mg per
left lung, respectively; p < 0.05); partial inhibition of mean AIA (14,860 ⴞ 1,135 vs
19,622 ⴞ 1,294 ␮m2, respectively; p < 0.05) and mean linear intercept (108.8 ⴞ 3.7 vs
123.0 ⴞ 4.2 ␮m, respectively; p < 0.05); and increases and improvement in expiratory flows
(27.8 ⴞ 1.2 vs 23.4 ⴞ 1.3 mL/s, respectively; p < 0.05). NAC was not able to avoid the compliance
increase in the elastase-plus-NAC group.
Conclusion: Consistent with the results of anatomic, pathologic, and functional studies, NAC is
able to attenuate the lesions induced by elastase in rats, which is in accordance with previous data
supporting the idea that oxidant injury could contribute to the development of elastase-induced
emphysema.
(CHEST 2004; 125:1500 –1506)
Key words: elastase; emphysema; lung function; N-acetylcysteine; rats
Abbreviations: AIA ⫽ alveolar internal surface area; CL ⫽ lung compliance; F75 ⫽ expiratory flow at 75% of FVC;
HYP ⫽ hydroxyproline; IC ⫽ inspiratory capacity; Lm ⫽ mean linear intersection; Lmc ⫽ computerized mean linear
intersection; NAC ⫽ N-acetylcysteine; PMN ⫽ polymorphonuclear cell
ntratracheal elastase administration induces a leI sion
that resembles human panacinar emphysema.1 In hamsters, elastase initially provokes a severe
decrease in the elastin content of the lung that is
gradually recovered while the emphysema worsens.2
There also appears to be an increase in collagen
synthesis during the development of emphysema.2
These changes in connective tissue after elastase
*From the Laboratorio Neumologı́a Experimental, Servicio de
Neumologı́a, Fundación Jiménez Dı́az, Universidad Autónoma,
Madrid, Spain.
Supported in part by Zambón SA and Red Respira (grant RTIC
C03/11,FIS,ISCIII).
Manuscript received March 25, 2003; revision accepted September 1, 2003.
Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (e-mail:
[email protected]).
Correspondence to: Nicolás González Mangado, MD, PhD, Servicio de Neumologı́a, Fundación Jiménez Dı́az Avda/ Reyes
Católicos, 2 28040-Madrid, Spain; e-mail: [email protected]
administration, observed not only biochemically but
also by electron microscopy,3 reflect the repair reaction of the lung after elastase-induced injury.
Functionally, the lesion produces an increase of
inspiratory capacity (IC) and lung distensibility, and
a decrease of expiratory flows. Morphologically, elastase provokes a disruption of the alveolar walls that
leads to the enlargement of the airspace regularly
distributed throughout all the parenchyma, which is
reflected in an increase of the mean linear intercept.4
Furthermore, intratracheal elastase induces an
early inflammatory response with neutrophils and
macrophages,2 which is still present 1 month later.3
These cells could be the source of proteases and
oxidants that can contribute to the destruction of
lung connective tissue as well as of inflammatory mediators that exacerbate elastase-induced
emphysema.5
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Laboratory and Animal Investigations
N-acetylcysteine (NAC) is a precursor of glutathione molecules and has oxygen radical-scavenging
properties.6,7 The effectiveness of NAC administration in animal models of lung fibrosis as well as in
patients with idiopathic pulmonary fibrosis has been
reported. In this respect, it has been demonstrated8 –10 that NAC is able to attenuate cellular infiltration and collagen deposition in a model of bleomycin-induced lung fibrosis. When administered
together with steroids, it improves the lung function
index in patients with idiopathic pulmonary fibrosis.11 It also has been reported that NAC has an
anti-inflammatory role because of its capacity to
regulate the production of some inflammatory mediators in fibrosis induced in vitro12 and in vivo.10 To
our knowledge, there are no studies about the effect
of NAC administration on the evolution of induced
pulmonary emphysema. The aim of this study was to
investigate whether oral administration of antioxidant/anti-inflammatory NAC has any effect on the
course of development over time of elastase-induced
emphysema in rats in terms of repair, lung function,
and morphometry.
Materials and Methods
Materials and Subjects
The Animal Research Committee of the center approved all
animal experimentation. Male Wistar rats (weight range, 180 to
200 g) were classified into the following four groups: a control
group (n ⫽ 18) orotracheally instilled with saline solution; an
elastase group (n ⫽ 18) orotracheally instilled with 75 IU porcine
pancreatic elastase (Roche Diagnostics; Basel, Switzerland); a
control group treated with NAC (n ⫽ 18); and an elastase group
treated with NAC (n ⫽ 18). After instillation, the rats were
returned to their cages. The rats that were to receive NAC
(Laboratorios Zambón SA; Barcelona, Spain) were given 200 mg
NAC per rat per day mixed with powdered food divided into two
doses (at 9:00 am and at 6:00 pm) beginning 2 days before the
instillation until the study day. Food was provided ad libitum,
checking that there was as little food as possible remaining
between dosages in the groups receiving NAC. The dosage,
which was equivalent to the human dosage in terms of body
surface, was the same that proved to be effective ameliorating
cigarette smoke-induced lung lesions in rats when used previously.13 Rats were killed 2, 8, and 28 days after instillation.
Lung Function Tests
The functional study was performed in a breathing assembly
for small animals, as described elsewhere.4 The rats were anesthetized with sodium pentobarbital (60 mg/kg intraperitoneally),
tracheotomized, put into a plethysmograph, and fitted to a
cannula that allowed communication with the breathing assembly. As soon as the rat was connected to the breathing equipment,
it was paralyzed with 0.2 mg pancuronium bromide and then
artificially ventilated. Changes in IC, lung compliance (CL), and
expiratory flow at 75% of FVC (F75) were assessed.
Morphometry
After lung function study, the chest was opened. The left lung
was weighed and immediately frozen for the biochemical determination of collagen (see below). The right lung was fixed by
filling it with 10% formalin to an airway pressure of 25 cm H2O
for 24 h. Five-micrometer sections were stained with hematoxylin-eosin. The mean linear intersection (Lm) was measured as
previously described.4 Using computer-assisted imaging analysis,
the mean of the horizontal and vertical alveoli lengths (ie, the
mean computerized linear intersection [Lmc]), equivalent to the
Lm, as well as the mean alveolar internal surface area (AIA) were
calculated in each field. Images were visualized by a video camera
(Leica DC 100; Leica Microsystems; Cambridge, UK) with a
resolution of 782 ⫻ 582 pixels adapted to a microscope (model
BX40; Olympus; Tokyo, Japan). Routine imaging analysis software (Qwin; Leica Microsystems) adapted for the purpose of this
study was applied for AIA and Lmc determinations. Fields (12 to
18 per rat) were quantified under a ⫻4 objective and a ⫻0.5
reducing video camera adapter. For each animal, the AIA and
Lmc were averaged from all the fields measured.
Collagen Analysis
Collagen quantification was performed by measuring hydroxyproline (HYP). Briefly, the left lung was homogenized, and,
after acid hydrolysis, chloramine T (Merck; Darmstadt, Germany) was added to the hydrolyzate to allow for oxidation. After
the addition of the Ehrlich reagent (Sigma; St. Louis, MO) and
reaction development, absorption of each sample was read at 560
nm.14 The results are expressed in milligrams of HYP per left
lung.
Statistical Analysis
All data are expressed as the mean ⫾ SEM. Comparisons were
made with analysis of variance. Multiple range tests (least
significance differences method) were used for the analysis of
differences among means. Pearson correlations also were
calculated.
Results
Collagen Content
The sequential results of collagen changes over
time are represented in Figure 1. Collagen measured
by HYP was significantly higher in elastase-treated
rats at 8 days, with the difference increasing with
respect to the control group after 28 days. Treatment
with NAC significantly hindered the increase of HYP
from the first days after instillation, which was
significantly lower than that in the elastase group on
the 28th day (1.23 ⫾ 0.09 vs 1.51 ⫾ 0.1 mg, respectively) and was not significantly different from that of
the control group at any point in time.
Morphometry
In terms of airspace enlargement, the lesion was
still not evident on or before the 8th day. For this
reason, we have focused on Lm and area values after
28 days (Table 1). Rats treated with elastase pre-
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CHEST / 125 / 4 / APRIL, 2004
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Figure 1. The amount of HYP (in milligrams) per left lung for
the control, control-plus-NAC, elastase, and elastase-plus-NAC
groups at 2, 8, and 28 days after elastase instillation. * ⫽ HYP was
significantly increased in the elastase group with respect to the
control group at 8 and 28 days (p ⬍ 0.05); † ⫽ HYP was significantly decreased in the elastase-plus-NAC group at 28 days with
respect to the elastase group (p ⬍ 0.05).
sented significantly increased AIA compared with
that of the control group. As shown in Figure 2, the
elastase-plus-NAC group showed significantly lower
percentages of Lmc and AIA than the elastase group.
Figure 3 shows histopathologic images of lung
sections from the control, elastase, and elastase-plusNAC groups obtained 28 days after induction of the
lesion. The Lm followed the same tendency and
correlated highly with Lmc (r ⫽ 0.96; p ⬍ 0.05).
The correlation between AIA and HYP also was
calculated. As can be seen in Figure 4, 28 days after
elastase administration, a significant positive correlation was found between AIA increase and HYP
level in the elastase and control groups (r ⫽ 0.46;
p ⬍ 0.05), but not in the elastase-plus-NAC group.
Functional Study
The course of function deterioration over time in
the control, control-plus-NAC, elastase, and elastaseplus-NAC groups for IC, CL, and F75 percentages
are represented in Figures 4 and 5. IC was significantly lower in the first days after elastase adminis-
Figure 2. AIA and Lmc measured by computer-assisted image
analysis 28 days after instillation, expressed as a percentage of the
control value in the elastase and elastase-plus-NAC groups.
* ⫽ AIA and Lmc were significantly lower in the elastase-plusNAC group compared to the elastase group (p ⬍ 0.05).
tration, but was not different from the saline control
group after 28 days (Fig 5, left, A). CL (Fig 5, right,
B) was not different from the control group in the
first days after elastase administration, but was significantly higher in both elastase groups on the 28th
day. As can be seen in Figure 6, F75, expressed as
the percentage of decline with respect to the control
group, was significantly decreased in the elastase and
elastase-plus-NAC groups after 2, 8, and 28 days.
However, on the 28th day the decline was significantly lower in the elastase-plus-NAC group compared with the elastase group. Correlations between
functional and morphometric parameters after 28
days were calculated for the elastase and elastaseplus-NAC groups. As shown in Table 2, AIA correlated positively with CL and negatively with F75 in
both elastase groups. In the elastase-plus-NAC
group, the correlation with CL was higher and that
with F75 was lower than those obtained in the
elastase group, in absolute values. Lmc followed the
same tendency.
Discussion
Treatment with oral NAC partially attenuates lung
emphysema induced by elastase in rats. Improve-
Table 1—Histopathologic Findings at 28 Days*
Variables
Control Group
Control ⫹ NAC Group
Elastase Group
Elastase ⫹ NAC Group
AIA, ␮m2
Lmc, ␮m
Lm, ␮m
6,982.9 ⫾ 1,181.6
79.38 ⫾ 3.82
95.38 ⫾ 5.55
6,009.1 ⫾ 1,181.6
74.79 ⫾ 3.82
89.75 ⫾ 6.02
19,621.6 ⫾ 1,294.4†
122.97 ⫾ 4.18†
151.77 ⫾ 6.41†
14,859.7 ⫾ 1,135.3†‡
108.78 ⫾ 3.67†‡
130.53 ⫾ 5.34†‡
*Values given as mean ⫾ SEM.
†Significantly different from the control group (p ⬍ 0.05 by analysis of variance).
‡Significantly different from the elastase group (p ⬍ 0.05 by analysis of variance).
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Laboratory and Animal Investigations
Figure 4. Positive correlation between AIA and HYP content for
the control and elastase groups (r ⫽ 0.46; p ⬍ 0.05).
Figure 3. Light microscopic photomicrograph of lung parenchyma 28 days after the induction of the lesion. Top left, A:
control group (Lm, 89 ␮m). Top right, B: elastase group (Lm, 175
␮m). Bottom, C: elastase-plus-NAC (Lm, 118.5 ␮m) [hematoxylin-eosin, original ⫻100]. The enlargement of airspaces in the
elastase group (top right, B) is partially inhibited by the administration of NAC (bottom, C).
ment is observed in the amelioration of airspace
enlargements, the partial recovery of expiratory
flows, and the normalization of lung collagen content.
We found that after elastase administration there
was an evident increase of collagen from the 8th day.
The higher collagen content in elastase-treated ani-
mals was initially found by Kuhn et al2 in hamsters,
and later this event was described with detail using
scanning electron microscopy in rats.3 The alveolar
structures of the lung seem to initiate repair responses after structural injury. In this way, elastin
and collagen gene expression is up-regulated after
elastase administration.15 There is some evidence
suggesting that inadequate repair contributes to the
worsening of emphysema. For example, starvation,
which generally inhibits anabolic responses, can exacerbate the development of elastase-induced emphysema in animals.16 In addition, the inhibition of
elastin synthesis by ␤-aminopropionitrile, a lathyrogen that inhibits elastin maturation, worsens emphysema in elastase-exposed animals.17 Our data demonstrate that the repair reaction started after elastase
administration does not, however, efficiently restore
tissue integrity. Morphometric determinations after
28 days showed that elastase provoked an enlargement of alveolar spaces reflected in an increase of
AIA of 280% with respect to the control group,
which is compatible with the increase in Lmc.
Moreover, we found that the higher amount of HYP
in the elastase group correlated positively with airspace enlargement, quantified in terms of AIA
(r ⫽ 0.46) [Fig 3]. In accordance with these results,
Vlahovic et al18 found a positive correlation between
Lm and alveolar interstitial thickness in patients with
emphysema. Thus, there seems to be a relationship
between the degree of parenchyma destruction and
the synthesis of collagen for reparation. Regarding
lung function, after 2 and 8 days there was a decrease
of IC, CL, and F75, probably reflecting lung edema
produced after elastase instillation. Thereafter, the
lesions developed, and, on the 28th day, there was an
increase of CL and a fall in expiratory flows without
changes in IC, which was similar to the results
obtained in previous studies.4,19
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Figure 5. IC (left, A) and compliance (right, B) of the control, control-plus-NAC, elastase, and
elastase-plus-NAC groups 2, 8, and 28 days after elastase instillation. * ⫽ significantly different from
the control group (p ⬍ 0.05).
Interestingly, the daily administration of NAC for
28 days after elastase instillation significantly reduced lung collagen content compared to the elastase group. Morphometrically, the emphysema was
less severe, with both AIA and Lmc being significantly lower than that in the elastase group (reduction of 25% for AIA and 12% for Lmc compared to
elastase group). Functionally, expiratory flow was
significantly improved after NAC treatment, although values still remained lower than those in the
control group. However, CL was not influenced by
NAC and was equally higher in both elastase groups,
suggesting that NAC does not interfere in the elastolytic process. Nevertheless, the correlation between CL and AIA (Table 2) not only persisted in the
elastase-plus-NAC group but was even higher than
that for the elastase group. This could mean that in
the elastase-plus-NAC group the increased distensibility is mainly explained by alveolar enlargement. In
contrast, in the elastase group several other factors
could be involved that may interfere in the relationship between the increase in AIA and distensibility.
Taking into account the lack of change in CL, the
improvement of expiratory flow in the elastase-plusNAC group only could be explained by an increase in
airway conductance in rats treated with NAC. In a
similar study, retinoic acid was administered daily for
2 weeks, 25 days after elastase instillation. However,
despite notable lung morphology results,20 retinoic
acid was not able to reverse either increased CL or
expiratory flows21 and anatomic-functional correlations were not stated.
Is difficult to believe that NAC prevents the direct
enzymatic effect of elastase. It must be acting at a
different level. Previous studies have demonstrated
Table 2—Pearson Correlations Between Morphometry
and Lung Function Parameters at 28 Days*
Figure 6. F75 reduction at 2, 8, and 28 days after elastase
instillation for the control-plus-NAC, elastase, and elastase-plusNAC groups, expressed as a percentage of the control value. F75
was significantly decreased in the elastase and elastase-plus-NAC
groups compared to the control group at 2, 8, and 28 days
(p ⬍ 0.05). * ⫽ significantly different from the control group
(p ⬍ 0.05); † ⫽ F75 decline was significantly smaller in the
elastase-plus-NAC group than in the elastase group at 28 days
(p ⬍ 0.05).
Elastase Group
Variables
IC
CL
F75
Elastase ⫹ NAC
Group
AIA
Lmc
AIA
Lmc
NS
⫹0.55
⫺0.70
NS
⫹0.59
⫺0.68
⫹0.45
⫹0.72
⫺0.48
⫹0.48
⫹0.74
⫺0.43
*Correlations are significant at p ⬍ 0.05. NS ⫽ not significant.
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Laboratory and Animal Investigations
that 2 h after elastase administration its activity is
markedly reduced22 and is cleared from the lung
within 24 h.23 However, the development of emphysema is gradual over a span of at least 2 months,22
suggesting that endogenous mechanisms must be
involved.
It has been reported that during the first 48 to 72 h
after elastase administration there is a moderate
acute inflammatory response with neutrophils and
macrophages,2 and that 4 weeks later there is still a
markedly elevated total cell count.3 Activated inflammatory cells can release oxygen radicals and proteases that can directly damage components of the
lung matrix. Moreover, it has been demonstrated24
that several proteases induce a direct rise in reactive
oxygen species in bronchial epithelial cells and fibroblasts. On the other hand, Leff et al25 described a
decrease in neutrophil influx into lung lavage fluid
after NAC administration in an animal model treated
with interleukin-1␣.
In addition, genes for many inflammatory mediators, some of them involved in the recruitment of
inflammatory cells and, therefore, in the development of emphysema,5 are regulated by transcription
factors such as nuclear factor ␬B. Nuclear factor ␬B
activation can be induced by oxidants,26 and NAC
previously has been demonstrated to be efficient in
decreasing this activation in vivo.27 In the elastaseinduced emphysema model, NAC could be acting at
this level by hindering the overexpression of genes
for inflammatory cytokines.
Thus, lesions induced in rats by elastase instillation
seem to be not only the consequence of the initial
enzymatic damage but, more probably, the result of
several secondary stimuli that lead to the worsening
of the disease over time. Lucey et al5 recently stated
that tumor necrosis factor-␣ and interleukin-1␤ account for about 80% of the emphysema that develops after elastase treatment, pointing out that these
proinflammatory mediators (polymorphonuclear cell
[PMN] chemoattractants) are taking part in the
development of the lesion. But actually, there is no
evidence showing that the elastase-induced emphysema is accompanied by an increase in the number
of PMN cells. According to these data, Cantor et al28
found that 1 week after the induction of the lesion
with elastase the number of PMNs was normal. In
our experiment, it is not possible to know whether
NAC treatment completely abolishes the oxidative
stress occurring after elastase, but we think that
oxidants can explain at least part of the emphysematous lesion, taking into account the possible antiinflammatory effect of NAC throughout the diminution of PMN influx into the lung.
The effectiveness of NAC in attenuating bleomycin-induced lung fibrosis is unquestionable,8 –10 al-
though the mechanism by which it limits fibrosis is
also unclear. Bleomycin damage consists of the
following two phases: an early inflammatory phase,
characterized by cellular infiltration; and a late fibrotic phase. Reactive oxygen species and proteases
generated from infiltrated cells are considered to
injure the lung tissue, and excessive fibrosis occurs as
a reparative process. These two phases also seem to
be present in emphysema development, the second
chronic severe reparative phase establishing the different pathophysiologies of both diseases.
In patients with COPD, the oxidant burden is also
increased. Cigarette smoke, the major etiologic factor in COPD, is a rich source of oxidant molecules.
Oxidative stress is also a critical feature in the
pathogenesis of COPD since it results in the inactivation of antiproteinases, airspace epithelial injury,
mucus hypersecretion, increased influx of neutrophils into the lungs, transcription factor activation,
and gene expression of proinflammatory mediators.
Antioxidants, therefore, should not only protect
against the direct injurious effects of oxidants, but
may also fundamentally alter the inflammatory
events that have a central role in the pathogenesis of
COPD.29
Fibrosis, emphysema, and COPD seem to have
the oxidant/antioxidant imbalance in common. The
site and specific characteristics of the inflammatory
responses may differ in each of these diseases.
In conclusion, oxidant/antioxidant imbalance
seems to contribute to the development of elastaseinduced emphysema, and treatment with antioxidant
NAC could attenuate or slow down the process.
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