(Chest. 2000;117:299S-302S.)
© 2000
American College of Chest Physicians
The Combination of Elastase and Sulfur Dioxide Exposure Causes COPD-Like Lesions in the Rat*
Urmila P. Kodavanti, PhD;
Mette C. Jackson, BS;
Allen D. Ledbetter, BS;
Barry C. Starcher, PhD;
Paul A. Evansky, BS;
Adrian Harewood, BS;
Darrell W. Winsett, BS and
Daniel L. Costa, ScD
*
From the Pulmonary Toxicology Branch, Experimental Toxicology Division (Dr. Kodavanti, Ms. Jackson, Mr. Ledbetter, Mr. Evansky, Dr. Costa, and Mr. Winsett), National Health and Environment Effects Research Laboratory, United States Environmental Protection Agency, Research Triangle Park, NC; University of Texas Health Center (Dr. Starcher), Tyler, TX; and North Carolina Central University (Mr. Harewood), Durham, NC.
Correspondence to: Urmila Kodavanti, Research Biologist, MD-82, PTB/ETD/NHEERL, USEPA, Research Triangle Park, NC 27711
 |
Introduction
|
|---|
Abbreviations: BALF = BAL fluid; LDH = lactate
dehydrogenase; PPE = porcine pancreatic elastase;
SH = spontaneously hypertensive; SO2 = sulfur dioxide;
TLC = total lung capacity
Human
COPD is associated with chronic cigarette smoking and is characterized
by coexistent diseases of emphysema and bronchitis.1
2
3
The disease syndrome also encompasses many other pathologic
manifestations, such as inflammation, airway fibrosis, and pulmonary
infections. These manifestations occur at relatively different levels
in individuals, such that it is nearly impossible to denote any one
proportional combination in defining COPD. Clinically, all of these
pathologies are associated with a gradual and persistent decline in
FEV1. The disease develops in only ~ 10% of
smokers. and yet 80 to 90% of COPD patients are current or past
smokers, suggesting that genetic predisposition may play a role in the
pathogenesis of the disease. Involvement of a number of host
susceptibility factors/genes has been proposed to explain the
predisposition to COPD.4
Because of the complexities of
COPD, and the chronicity associated with its pathogenesis, no animal
model has been developed to represent the multiple components of the
disease. In addition, laboratory rodents seem to differ from humans
with respect to their host responses, such that their potential to
repair or regenerate lung tissue is higher and induced inflammation or
infections are not sustained over a long period of
time.5
6
A number of rodent models have been reported
which exhibit at least one of the attributes of COPD (eg,
emphysema or bronchitis).7
8
However, these chemically
induced rodent models of emphysema or bronchitis lack critical
pathobiological features of human COPD, such as persistent lung
inflammation and infection. The purpose of this study was to combine
emphysema and bronchitis together in the rat such that prior emphysema
developed by elastase instillation would potentiate persistence of
bronchitis induced by sulfur dioxide (SO2). We
hypothesized that the combination of elastase treatment followed by
SO2 exposure would result in COPD with sustained
inflammation in the rat.
|
Materials and Methods
|
|---|
Male Sprague-Dawley or Brown Norway rats (100-days old)
were intratracheally instilled with saline solution or porcine
pancreatic elastase (PPE), 800 U/kg (Elastin Products; Owensville, MO)
and 2 weeks later exposed to clean air or SO2,
250 ppm, 6 h/d, 5 d/wk for 7 weeks. The dose of PPE was based on a
pilot study in Sprague-Dawley rats that demonstrated moderate to
severe emphysema (emphysema score, mean ± SD: control,
0.67 ± 0.23; PPE, 5.8 ± 0.6). One week after PPE
instillation, rats were housed in Hazleton 2000 L stainless steel
chambers (Lab Products Inc; Maywood, NJ) in wire mesh cages and
acclimatized for 1 additional week. They were then exposed to either
filtered air or SO2. Anhydrous
SO2 was delivered into the inlet of the chamber.
The SO2 concentration in each chamber was
monitored by an infrared analyzer (250 ± 10 ppm). Chamber relative
humidity was 50 to 60%; temperature, 20 to 21°C; and flow was
~ 550 L/min. One day after the last SO2
exposure, all animals (saline solution + air; saline
solution + SO2; PPE + air;
PPE + SO2) were evaluated for the presence of
emphysema and/or bronchitis. Control and
PPE + SO2 rats were also evaluated at 4 days
and 7 days for postexposure recovery. All animals were anesthetized
with urethane, 2 mL/kg intraperitoneal, and underwent assessment
for static lung volume using a whole body pressure
plethysmograph.9
Vital capacity was measured between
airway pressures of - 15 and + 30 cm H2O.
Total lung capacity (TLC) was obtained by gas dilution methods, and
residual volume was computed by subtracting vital capacity from TLC.
Multibreath diffusion capacity of the lung for carbon monoxide was also
obtained by a modified gas dilution technique.10
11
Following lung volume measurements, the left lungs were inflated at a
pressure of 20 cm with filtered 4% paraformaldehyde in
phosphate-buffered saline solution (pH 7.2) for 15 to 20 min. After
fixation, the lung tissues were embedded in paraffin, and 4-µm thick
midsagittal sections were mounted and stained with hematoxylin and
eosin. Emphysema score was determined based on a severity grade of 1 to
10, with 1 being minimal and 10 being severe.
The right lung was lavaged using a phosphate-buffered saline solution
(pH 7.4) at a volume of 28 mL/kg body weight (approximately 75% TLC).
Three in-and-out washes were performed using the same fluid. One
aliquot of whole BAL fluid (BALF) was used for determining total cells
using a Coulter Counter (Coulter; Miami FL), and a second aliquot was
centrifuged (Cytospin-III; Shandon; Pittsburgh, PA) for preparing cell
differential slides. The slides were dried at room temperature and
stained with LeukoStat (Fisher Scientific; Pittsburgh, PA).
Macrophages, neutrophils, eosinophils, and lymphocytes were quantitated
using light microscopy (200 cells/slide). The remaining BALF was
centrifuged at 1,500g to remove cells, and the supernatant
fluid was analyzed for protein, albumin, and lactate dehydrogenase
(LDH) activity. Assays for protein, albumin, and LDH activity were
modified and adapted for use on a Hoffmann-La Roche Cobas Fara II
clinical analyzer (Roche Diagnostics; Branchburg, NJ). Total protein
content was determined using a Coomassie Plus Protein Assay Kit
(Pierce; Rockford, IL) and bovine serum albumin as a standard. BALF
albumin was measured using a commercially available kit and controls
(ICN Star Corporation; Stillwater, MN). LDH activity was determined
using a Kit 228 and standards (Sigma Chemical; St. Louis, MO). Data
were statistically analyzed using general linear models procedures (SAS
516; SAS Institute; Cary, NC) and Dunnett’s t test. The
type I error rate was set at 0.05 for significance.
|
Results and Discussions
|
|---|
Since an animal model of COPD representing the complete spectrum
of human pathologies is not available, this study endeavored to
simulate COPD in the rat by experimentally inducing emphysema and
bronchitis together. This was done by prior instillation of elastase
followed by several weeks of SO2 exposure.
SO2-induced bronchitis in the rat has been well
characterized and is considered to be a chronic disease
model.8
We hypothesized that prior induction of emphysema
by elastase instillation would establish permanent enlargement of
airspaces, and subsequent exposure to SO2 would
induce bronchitis in the rat, producing lesions similar to COPD. It was
also hypothesized that prior emphysema would result in prolongation of
the inflammatory response caused by SO2 on
termination of exposure. This hypothesis was tested in two rat strains:
(1) the Sprague-Dawley rat, for which both elastase-induced emphysema
and SO2-induced bronchitis are well
characterized7
8
; and (2) the Brown Norway rat, for which
airway responsiveness, allergic immune responses, and alveolar cell
types resemble that of humans.12
In general, lung volume,
morphometric, histologic, and BALF changes caused by PPE and
SO2 appeared to be more marked in Sprague-Dawley
rats when compared to Brown Norway rats; therefore, much of the
discussion is focused on Sprague-Dawley rats. The experimental protocol
consisted of elastase instillation first, since changes caused by
elastase are permanent. SO2 exposure was
initiated 2 weeks after elastase instillation, to allow the initial
hemorrhage and injury caused by elastase to resolve during nonexposure
periods. Consistent with lung volume changes in COPD, TLC and residual
volume were increased in rats exposed to PPE + air, saline
solution + SO2, and
PPE + SO2. A true measure of the decrease in
FEV1 cannot be accurately made in rodents;
however, changes in lung volumes along with pathology and
morphometric analysis of lung sections are consistent with the presence
of emphysema and COPD. Exposure to SO2 alone
caused bronchial epithelial hyperplasia and inflammation; however,
emphysema was not present in these rats. Histologic evaluation of lung
sections revealed areas of enlarged airspaces and airway mucus cell
hyperplasia in rats instilled with PPE and then exposed to
SO2 (Fig 1
). Peribronchiolar inflammation and alveolar macrophage accumulation
were also apparent in rats treated with PPE and
SO2 alone or in combination. Emphysema score (Fig 2
, top, A) and mean linear intercept were increased
in all PPE-instilled rats regardless of SO2. BALF
analysis for biochemical markers of pulmonary injury, and inflammation
(Fig 2
, bottom, B) revealed increased protein,
albumin, and LDH activity in rats pretreated with PPE + air, saline
solution + SO2, and
PPE + SO2. Alveolar macrophages were higher in
saline solution + SO2, PPE + air, and
PPE + SO2-exposed rats. Neutrophils in BALF
were also increased in all SO2-exposed rats when
analyzed 1 day after exposure. Increased macrophages and neutrophils
are prominent features of human COPD and thus, COPD and chronic asthma
broadly separate in terms of the prominency of inflammatory cell
types.3
Although neutrophilic inflammation in the rat was
apparent and is consistent with the human COPD, the degree of
inflammatory changes was milder in the induced rat model than that
reported to occur in patients.3
While individual treatment
with PPE or SO2 produced either emphysema or
bronchitis, the combined model exhibited both, with marked mucus
hypersecretion, alveolar destruction, and mild injury/inflammation.
View larger version (74K):
[in this window]
[in a new window]
[Download PPT slide]
|
Figure 1. Lung histologic changes consistent with emphysema
and bronchial cell hypertrophy/hyperplasia in Sprague-Dawley rats
exposed to PPE and SO2. Lung tissue sections were stained
and observed under light microscope. A representative area within the
section depicts broken alveolar septa and bronchial/bronchiolar changes
consistent with mucus hypersecretion and inflammation in rats exposed
to PPE and SO2. This micrograph is derived from a rat
sacrificed 1 day following the last SO2 exposure
(hematoxylin-eosin, original 80x).
|
|
View larger version (38K):
[in this window]
[in a new window]
[Download PPT slide]
|
Figure 2. Representative data showing increased emphysema
score (top, A) and BALF cell numbers
(bottom, B) in Sprague-Dawley rats
exposed to PPE and SO2, individually or in combination.
Each group represents mean ± SE of six rats and * show
significant difference from controls (p  0.05). Sal. = saline
solution.
|
|
Lack of persistency of bronchitis and neutrophilic inflammation on
termination of SO2 exposure is a weakness of this
model and suggests that there may be a susceptible subgroup within a
species that is predisposed to the disease, similar to humans where
only ~ 10% of smokers develop COPD. Humans with COPD are presumed
to have multigenetic abnormalities that underlie their susceptibility
to the development of the disease.4
These abnormalities
include increased oxidative burden, presence of activated T cells in
the circulation, a generalized immunosuppression, and vulnerability to
infections.4
Incidentally, our use of a genetic rat model
of human cardiovascular disease to examine enhanced susceptibility to
air pollution-induced cardiopulmonary injury has shown that these
underlying complications also occur in these predisposed
rats.13
Thus, based on the experimental evidence from our
lab and from the published literature,14
15
we
hypothesized that there may be common susceptibility traits
encompassing the cardiopulmonary system, and these genetically
predisposed spontaneously hypertensive (SH) rats may represent a
species that would develop COPD similar to humans, exhibiting
persistent inflammation and only partially reversible disease.
Preliminary study of PPE instillation in SH rats showed that this rat
model was almost twice as susceptible to PPE-induced alveolar
destruction compared to the Sprague-Dawley rat. Follow-up studies will
further refine this model to include the use of SH rats, with exposure
to cigarette smoke as well as exposures to elastase,
SO2, lipopolysaccharide, or infectious agents in
different combinations to enhance chronicity of the disease. In
summary, emphysema and bronchitis can be experimentally combined in a
rodent model to mimic some features of human COPD; however, the
potential for rodents to recover from airways disease limits the
progression and the chronicity of the disease. The use of genetically
predisposed rat strains may resolve this shortcoming in COPD model
development.
|
Acknowledgements
|
|---|
The authors acknowledge Judy H. Richards and James
R. Lehmann for technical help, and Donald L. Doerfler for statistical
analysis of the data.
|
Footnotes
|
|---|
The research described in this article has been reviewed by the
National Health and Environmental Effects Research Laboratory, United
States Environmental Protection Agency and approved for publication.
Approval does not signify that the contents necessarily reflect the
views and the policies of the Agency, nor does mention of trade names
or commercial products constitute endorsement or recommendation for
use.
|
References
|
|---|
-
Bartolome, RC, Snider, GL, Heffner, J, et al (1995) Standards for the diagnosis and care of patients with chronic obstructive pulmonary disease. Am Rev Respir Crit Care Med 152,S77-S120
-
Niederman, MS (1998) Mechanisms and management of COPD. Chest 113(Suppl),233S-287S[ISI][Medline]
-
O’Byrne, PM, Postama, DS (1999) The many faces of airway inflammation: asthma and chronic obstructive disease. Am J Respir Crit Care Med 159,S41-S66
-
Barnes, PJ (1999) Molecular genetics of chronic obstructive pulmonary disease. Thorax 54,245-252[Free Full Text]
-
Berger, LC, Burri, PH (1985) Timing of the quantitative recovery in the regenerating rat lung. Am Rev Respir Dis 132,777-783[ISI][Medline]
-
Gilmour, MI, Selgrade, MJK (1993) A comparison of the pulmonary defenses against streptococcal infection in rats and mice following O3 exposure: differences in disease susceptibility and neutrophil recruitment. Toxocol Appl Pharmacol 123,211-218[CrossRef][ISI][Medline]
-
Finlay, GA, O’Donnell, MD, O’Connor, CM, et al (1996) Elastin and collagen remodeling in emphysema: a scanning electron microscopy study. Am J Pathol 149,1405-1415[Abstract]
-
Shore, S, Kobzik, L, Long, NC, et al (1995) Increased airway responsiveness to inhaled methacholine in a rat model of chronic bronchitis. Am J Respir Crit Care Med 151,1931-1938[Abstract]
-
Raub, JA, Mercer, RR, Miller, FJ, et al (1982) Dose response of elastase-induced emphysema in hamsters. Am Rev Respir Dis 125,432-435[ISI][Medline]
-
Takezawa, J, Miller, FJ, O’Neil, JJ (1980) Single-breath diffusing capacity and lung volumes in small laboratory mammals. J Appl Physiol Respir Environ Exerc Physiol 48,1052-1059[Abstract/Free Full Text]
-
Costa, DL (1985) Interpretation of new techniques used in the determination of pulmonary functions in rodents. Fundam Appl Toxicol 5,423-434[CrossRef][ISI][Medline]
-
Schneider, T, van Velzen, D, Moqbel, R, et al (1997) Kinetics and quantitation of eosinophil and neutrophil recruitment to allergic lung inflammation in a brown Norway rat model. Am J Respir Cell Mol Biol 17,702-712[Abstract/Free Full Text]
-
Kodavanti UP, Schladweiler MCJ, Ledbetter AD, et al. The spontaneously hypertensive rat as a model of human cardiovascular disease: evidence of excerbated cardiopulmonary injury and oxidative stress from inhaled emisson particulate matter. Toxicol Appl Pharmacol 2000, in press
-
Schmidt-Schonbein, GW, Seiffge, D, DeLano, FA, et al (1991) Leukocyte counts and activation in spontaneously hypertensive and normotensive rats. Hypertension 17,323-330[Abstract/Free Full Text]
-
Suzuki, H, Swei, A, Zweifach, BW, et al (1995) In vivo evidence for micro vascular oxidative stress in spontaneously hypertensive rats: hydroethidine microfluorography. Hypertension 25,1083-1089[Abstract/Free Full Text]
This article has been cited by other articles:
|
|
|
|
 
U. P. Kodavanti, M. C. Schladweiler, A. D. Ledbetter, R. V. Ortuno, M. Suffia, P. Evansky, J. H. Richards, R. H. Jaskot, R. Thomas, E. Karoly, et al.
The Spontaneously Hypertensive Rat: An Experimental Model of Sulfur Dioxide-Induced Airways Disease
Toxicol. Sci.,
November 1, 2006;
94(1):
193 - 205.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. B. Soriano, K. J. Davis, B. Coleman, G. Visick, D. Mannino, and N. B. Pride
The Proportional Venn Diagram of Obstructive Lung Disease: Two Approximations From the United States and the United Kingdom
Chest,
August 1, 2003;
124(2):
474 - 481.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. P. Fishman
One Hundred Years of Chronic Obstructive Pulmonary Disease
Am. J. Respir. Crit. Care Med.,
May 1, 2005;
171(9):
941 - 948.
[Full Text]
[PDF]
|
|
|
TOP
Introduction
Materials and Methods
