Airway obstruction due to bronchial vascular injury after sulfur mustard analog inhalation.
ABSTRACT Sulfur mustard (SM) is a frequently used chemical warfare agent, even in modern history. SM inhalation causes significant respiratory tract injury, with early complications due to airway obstructive bronchial casts, akin to those seen after smoke inhalation and in single-ventricle physiology. This process with SM is poorly understood because animal models are unavailable.
To develop a rat inhalation model for airway obstruction with the SM analog 2-chloroethyl ethyl sulfide (CEES), and to investigate the pathogenesis of bronchial cast formation.
Adult rats were exposed to 0, 5, or 7.5% CEES in ethanol via nose-only aerosol inhalation (15 min). Airway microdissection and confocal microscopy were used to assess cast formation (4 and 18 h after exposure). Bronchoalveolar lavage fluid (BALF) retrieval and intravascular dye injection were done to evaluate vascular permeability.
Bronchial casts, composed of abundant fibrin and lacking mucus, occluded dependent lobar bronchi within 18 hours of CEES exposure. BALF contained elevated concentrations of IgM, protein, and fibrin. Accumulation of fibrin-rich fluid in peribronchovascular regions (4 h) preceded cast formation. Monastral blue dye leakage identified bronchial vessels as the site of leakage.
After CEES inhalation, increased permeability from damaged bronchial vessels underlying damaged airway epithelium leads to the appearance of plasma proteins in both peribronchovascular regions and BALF. The subsequent formation of fibrin-rich casts within the airways then leads to airways obstruction, causing significant morbidity and mortality acutely after exposure.
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ABSTRACT: Background: Inhalation of sulfur mustard (SM) and SM analog, 2-chloroethyl ethyl sulfide (CEES), cause fibrinous cast formation that occludes the conducting airways, similar to children with Fontan physiology-induced plastic bronchitis. These airway casts cause significant mortality and morbidity, including hypoxemia and respiratory distress. Our hypothesis was that intratracheal heparin, a highly cost effective and easily preserved rescue therapy, could reverse morbidity and mortality induced by bronchial cast formation. Methods: Sprague-Dawley rats were exposed to 7.5% CEES via nose-only aerosol inhalation to produce extensive cast formation and mortality. The rats were distributed into three groups: non-treated, phosphate-buffered saline (PBS)-treated, and heparin-treated groups. Morbidity was assessed with oxygen saturations and clinical distress. Blood and bronchoalveolar lavage fluid (BALF) were obtained for analysis, and lungs were fixed for airway microdissection to quantify the extent of airway cast formation. Results: Heparin, given intratracheally, improved survival (100%) when compared to non-treated (75%) and PBS-treated (90%) controls. Heparin-treated rats also had improved oxygen saturations, clinical distress and airway cast scores. Heparin-treated rats had increased thrombin clotting times, factor Xa inhibition and activated partial thromboplastin times, indicating systemic absorption of heparin. There were also increased red blood cells (RBCs) in the BALF in 2/6 heparin-treated rats compared to PBS-treated control rats. Conclusions: Intratracheal heparin 1 hr after CEES inhalation improved survival, oxygenation, airway obstruction, and clinical distress. There was systemic absorption of heparin in rats treated intratracheally. Some rats had increased RBCs in BALF, suggesting a potential for intrapulmonary bleeding if used chronically after SM inhalation. Pediatr Pulmonol. © 2014 Wiley Periodicals, Inc.Pediatric Pulmonology 04/2014; 50(2). · 2.38 Impact Factor
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ABSTRACT: Mustard gas (sulfur mustard [SM], bis-[2-chloroethyl] sulfide) is a vesicating chemical warfare agent and a potential chemical terrorism agent. Exposure of SM causes debilitating skin blisters (vesication) and injury to the eyes and the respiratory tract; of these, the respiratory injury, if severe, may even be fatal. Therefore, developing an effective therapeutic strategy to protect against SM-induced respiratory injury is an urgent priority of not only the US military but also the civilian antiterrorism agencies, for example, the Homeland Security. Toward developing a respiratory medical countermeasure for SM, four different classes of therapeutic compounds have been evaluated in the past: anti-inflammatory compounds, antioxidants, protease inhibitors and antiapoptotic compounds. This review examines all of these different options; however, it suggests that preventing cell death by inhibiting apoptosis seems to be a compelling strategy but possibly dependent on adjunct therapies using the other drugs, that is, anti-inflammatory, antioxidant, and protease inhibitor compounds.International Journal of Toxicology 05/2014; · 1.23 Impact Factor
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ABSTRACT: Statins have anti-inflammatory effects in patients with chronic obstructive pulmonary disease (COPD). This study designed to evaluate the effects of atorvastatin on serum highly sensitive C-reactive protein (hs-CRP) and pulmonary function in sulfur mustard exposed patients with COPD. Fifty patients with chronic obstructive pulmonary disease due to sulfur mustard and high serum hs-CRP entered in this study. Participants were randomized to receive 40 mg atorvastatin or placebo in a double-blind clinical trial. Forty-five patients completed the study (n = 23 atorvastatin and n = 22 placebo). Pulse oximetry (SpO2), pulmonary function test (PFT), and 6 min walk distance test (6MWD) was measured. COPD assessment test (CAT) and St. George's respiratory questionnaire (SGRQ) were also completed by patients at the beginning of trial and after 9 weeks of prescription of 40 mg/day atorvastatin or placebo. At fourth week, SpO2, PFT, and 6MWD were again measured. After 9 weeks serum hs-CRP was re-measured. There was no significant difference between atorvastatin and the placebo group in SpO2, FEV1, and 6MWD after fourth week (P = 0.79, P = 0.12, P = 0.12, respectively). The difference between baseline and ninth week was calculated for two groups of trial and control in term of serum hs-CRP, SpO2, FEV1, and 6MWD. Significant improvement was not observed between two groups in above mentioned variables (P = 0.35, P = 0.28, P = 0.94, P = 0.43, respectively). However, the quality of life was improved by administration of atorvastatin using the CAT score (P < 0.001) and SGRQ total score (P = 0.004). Atorvastatin does not alter serum hs-CRP and lung functions but may improve quality of life in SM-injured patients with COPD.Journal of research in medical sciences 02/2014; 19(2):99-105. · 0.61 Impact Factor
Airway obstruction due to bronchial vascular injury after sulfur mustard analog inhalation
Livia A Veress1,3, Heidi C. O’Neill2,3, Tara B. Hendry-Hofer3, Joan E. Loader3, Raymond C.
Rancourt3, Carl W. White1,2,3
1Department of Pediatrics, 2Department of Pharmaceutical Sciences, University of Colorado
Health Sciences Center, Denver, Colorado; 3Department of Pediatrics, National Jewish Health,
Corresponding author: Carl W. White, M.D., Phone: 303-322-4002, Fax: 303-270-2189, Email:
Descriptor number: 6.11 Inhalational Disasters Science and Health
This work was supported by the National Institutes of Health grant 3U54ES015678.
Correspondence and requests for reprints should be addressed to Carl W. White, M.D.,
Department of Pediatrics, Pulmonary Division, National Jewish Medical and Research Center,
1400 Jackson Street, Denver, CO 80206. Email: email@example.com
Short running head: Airway obstruction by sulfur mustard analog
Word count for body of manuscript: 6531
At a Glance Commentary
Scientific Knowledge on the Subject: Among the effects of sulfur mustard (SM) on the
respiratory tract, airway obstructive cast development after acute exposure remains poorly
What This Study Adds to the Field: We demonstrate a rat model for a SM surrogate agent-
induced bronchial cast formation, and show that bronchial vascular injury occurring early after
exposure results in extravasation of plasma proteins, and leads to the development of airway
This article has an online data supplement, which is accessible from this issue’s table of content
online at www.atsjournals.org.
Page 1 of 72
Media embargo until 2 weeks after above posting date; see thoracic.org/go/embargo
AJRCCM Articles in Press. Published on July 16, 2010 as doi:10.1164/rccm.200910-1618OC
Copyright (C) 2010 by the American Thoracic Society.
Rationale: Sulfur mustard (SM) is a frequently used chemical warfare agent, even in modern
history. SM inhalation causes significant respiratory tract injury, with early complications due to
airway obstructive bronchial casts, akin to those seen after smoke inhalation and in single
ventricle physiology. This process with SM is poorly understood because animal models are
Objectives: To develop a rat inhalation model for airway obstruction due to SM analog, 2-
chloroethyl ethyl sulfide (CEES), and investigate the pathogenesis of bronchial cast formation.
Methods: Adult rats were exposed to 0, 5 or 7.5% CEES in ethanol via nose-only aerosol
inhalation (15 min). Airway microdissection and confocal microscopy were used to assess cast
formation (4 and 18 h postexposure). Bronchoalveolar lavage fluid (BALF) retrieval and
intravascular dye injection were used to evaluate vascular permeability.
Measurements and Main Results: Bronchial casts, composed of abundant fibrin and lacking
mucus, occluded dependent lobar bronchi within 18 h after CEES exposure. BALF contained
elevated concentrations of IgM, protein, and fibrin. Accumulation of fibrin-rich fluid in
peribronchovascular regions (4 h) preceded cast formation. Monastral blue dye leakage
identified bronchial vessels as the site of leakage.
Conclusion: Following CEES inhalation, increased permeability from damaged bronchial vessels
underlying damaged airway epithelium leads to the appearance of plasma proteins in both
peribronchovascular regions and BALF. The subsequent formation of fibrin-rich casts within the
Page 2 of 72
airways then leads to airways obstruction, causing significant morbidity and mortality acutely
Keywords: fibrin; pseudomembrane; plastic bronchitis; vascular permeability; microdissection
Abstract word count: 236
Page 3 of 72
Sulfur mustard, bis (2-chloroethyl) sulfide (SM), is a chemical agent used in modern
warfare, most recently by Iraq in the 1983-1988 Iran-Iraq war (1-6). It is a vesicant, affecting
mainly the skin, eyes, and respiratory systems shortly after direct contact (3, 7, 8). In high doses,
SM exposure can lead to multiorgan involvement (3, 4, 8, 9) and can result in death (3, 6, 10-12).
There are 40-50,000 surviving victims of SM inhalation in Iran and Iraq alone, many with
permanent pulmonary disabilities such as bronchiolitis obliterans (13, 14).
Respiratory effects of SM exposure have long been investigated, focusing mainly on
chronic respiratory effects in survivors (1, 2, 13-16). Regarding the acute effects of SM on the
human respiratory tract, scarce data is available other than a few case reports (2-4, 6, 8, 10, 11,
17). Injury appears concentration-dependent (5, 7), with low level SM exposure affecting mainly
the upper respiratory tract. This can result in nasal mucosal injury, rhinorrhea, loss of smell and
taste, pharyngeal mucosal injury, and laryngitis (2, 3, 7, 10). Moderate SM exposure results in
varying degrees of tracheobronchial mucosal injury, leading to a painful and forceful cough (2, 3,
7, 15). By contrast, high level SM exposure can often lead to more severely disabling respiratory
lesions that may cause death (6, 8, 10, 12). These effects include severe airways edema and
ulceration, tracheobronchial mucosal sloughing, and airway occlusive pseudomembranes (2, 4-7,
10-12, 15, 17-19).
Although high dose SM can cause fatality from multiorgan failure alone (2, 6), numerous
case reports have also documented sudden deaths occurring from acute airways obstruction
due to pseudomembrane formation (6, 7, 10, 12, 20). The etiology of this airway occlusion
remains poorly understood, as human exposure in recent years has been fortunately infrequent,
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and no animal model has yet been developed to specifically mimic this phenomenon. Therefore,
no therapies have been evaluated in this process, and none exist to prevent or alleviate this
potentially fatal event. As SM continues to be a potential agent in bioterrorism (5, 21), an
improved understanding of this disorder could allow development of effective therapeutic
2-Chloroethyl ethyl sulfide (CEES) is a surrogate agent used to mimic SM injury in
laboratories (22, 23). It is less toxic than SM due to the absence of one of two terminal chloride
groups (that crosslink DNA and proteins), and due to its considerably shorter half-life in aqueous
solution. While safer to handle than SM, CEES possesses many of the same damaging properties
as an alkylating agent (22, 23) as does SM, making it suitable for use in experiments to mimic
SM-induced respiratory tract injury without the need for a specialized containment facility. In
this paper, we provide an animal model for potentially fatal airway obstruction seen acutely
after SM exposure using the SM surrogate agent, CEES. In addition, we examine the
composition of these pseudomembranous casts, and demonstrate the significant role that early
injury to the bronchial circulation plays in their formation.
Words: 442 (+ 51 numbers in references)
Materials and Methods
An expanded methods description can be found in the online supplement.
Page 5 of 72
2-chloroethyl ethyl sulfide (CEES, 8.41 M) was obtained from TCI America (Portland, OR). All
other chemicals were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO) unless
The Institutional Animal Care and Use Committee (IACUC) of National Jewish Medical and
Research Center approved this study. Adult male (275-350 g) Sprague-Dawley rats (Harlan Co.,
Indianapolis, IN) were used.
Inhalation Exposure to CEES
Rats were anesthetized with a cocktail of ketamine (75 mg/kg), xylazine (7.5 mg/kg), and
acepromazine (1.5 mg/kg), and placed in polycarbonate tubes with sealing plungers. Tubes
containing animals were mounted in a nose-only inhalation system (CH Technologies, NJ), and
were delivered compressed air with the aerosolized compound (ethanol, 5 or 7.5% CEES in
ethanol) for 15 minutes. Aerosolization was conducted via a Razel syringe pump (Razel
Scientific, St.Albans, VT) connected to a BioAerosol Nebulizing Generator (BANG; CH
Technologies, NJ). After 15 minutes of exposure, rats were removed from polycarbonate tubes,
and were observed in their cages until fully recovered from anesthesia.
Animals were euthanized at 4, 18, or 72 h after exposure as per experimental design. If rats
became moribund, as demonstrated by weight loss >25% body weight, inability to eat or drink,
Page 6 of 72
etc., they were euthanized prior to the planned study termination as per IACUC protocol. Rats
within experimental groups were terminally anesthesized with pentobarbital (Sleepaway, Fort
Dodge Animal Health, Fort Dodge, IA), the tracheas were cannulated, and lungs were fixed at 20
cm H2O with either Karnovsky’s fixative or 4% paraformadehyde in phosphate-buffered saline
(PBS) for 10 and 30 min, respectively. Whole lungs were then removed by gross dissection.
The protocol for microdissection as described by Postlethwait et al (24, 25) was followed.
Microdissection was carried out on a petri dish. Beginning at the main lobar bronchus
(generation 3), the axial pathway was exposed by cutting the airway lumen at 3 and 9 o’clock
positions. Main daughter branches, or side branches, were also exposed during the
microdissection. A map of airway generations was drawn during microdissection (Figure 1).
Confocal Microscopy Using EthD-1 and YOPRO-1
Distribution of airway injury was assessed by using a three-dimensional imaging technique
modified after Postlethwait et al (24, 25). Main modifications to the previously described
protocol included the use of 20 ml/kg 6 µM ethidium homodimer-1 (EthD-1) (Molecular Probes
Inc., Eugene, OR) in phenol-red free RPMI medium used for vital staining of airways and their
contents. In addition, we also used 2 µM YOPRO-1 (Molecular Probes Inc., Eugene, OR) solution
in PBS for staining of right middle lobe airways after microdissection but prior to imaging. A
Zeiss Two Photon LSM 510 confocal microscope was used to obtain images and z-stacks. For
bronchial cast imaging, casts were removed via microdissection from their airway locations after
Page 7 of 72
EthD-1 labeling in situ, and then incubated with YOPRO-1 solution and imaged with the confocal
Differential Cell Counts in Bronchoalveolar Lavage Fluid
BALF was pooled and centrifuged, the pellet was washed in 2 ml PBS, then resuspended in 2 ml
PBS, and then centrifuged in a Cytospin (Shandon Scientific) followed by staining using a
modified Wright–Giemsa stain (Protocol Hema 3; Fisher Scientific, Fair Lawn, NJ, USA). Cell
counts were then obtained via hemacytometer, counting 200 cells minimum in three random
IgM and Total Protein Measurement in Bronchoalveolar Lavage Fluid
Two lavages with 5 ml of PBS each were instilled into the lungs via the tracheal cannula and
subsequently withdrawn and then pooled. IgM concentrations were quantitatively measured in
the BALF using standard ELISA protocol (Bethyl Laboratories, Inc., Montgomery, TX). Total
protein was measured in BALF using the bicinchoninic Acid (BCA) protein assay (Pierce, Rockville,
β-Fibrin Detection by Western Blot
β-Fibrin was detected in BALF by Western blot using polyclonal rabbit anti-human fibrinogen
(DAKO Cytomation, Denmark) or mouse monoclonal β-actin (Sigma, St. Louis, MO), followed by
an incubation with HRP-conjugated goat anti-rabbit IgG (Bio-Rad, Hercules, CA) or HRP-
conjugated rabbit anti-mouse IgG (Sigma), respectively. Rat fibrinogen (Sigma-Aldrich Chemical
Page 8 of 72
Co., St. Louis, MO) was used to generate fibrin standards. Results were normalized to protein
level within each sample.
Fibrinogen/β-Fibrin and Acetylated Tubulin Immunohistochemistry (IHC)
Avidin-biotin complex peroxidase methods were used on tissue sections for staining based on
VectaStain Elite ABC kit (Vector Laboratories, Burlingame, CA). The primary antibody used for
fibrin(ogen) IHC was polyclonal rabbit anti-human fibrinogen (DAKO Cytomation, Denmark), and
for acetylated tubulin IHC was monoclonal mouse acetylated alpha tubulin (Abcam, Cambridge,
MA), for 60 min incubations each. Secondary antibody used was biotinylated horse anti-mouse
IgG. Counterstaining was performed using hematoxylin.
Myeloperoxidase (MPO) activity assay
Snap-frozen lung tissue was homogenized, centrifuged, supernatant withdrawn, and steps
repeated until clear. The 1-ml reaction cuvette (with PBS, H2O2, and TMB) was followed for
3 min at 652 nm using a Beckman DU-64 spectrophotometer (Beckman Coulter, Fullerton, CA,
USA). Calculated milliunits of activity was normalized to milligrams of protein using the BCA
protein assay (Thermo Scientific).
Tissue Preparation for Histology
Paraffin embedded tissues were sectioned at 5 µm thickness, and then stained with hematoxylin
and eosin. Additional slides were also stained with a combined Alcian blue (AB) and periodic
acid Schiff (PAS) stain for the localization of acidic and neutral mucins, respectively, and
Page 9 of 72
counterstained with hematoxylin. In addition, Movat’s pentachrome staining was performed on
72 h lung sections for assessment of collagen deposition.
Evans Blue Dye Extravasation
Vascular permeability changes were assessed by monitoring the extravasation of Evans blue dye
(Sigma-Aldrich Chemical Co., St. Louis, Mo) using a method modified after Evans et al (26, 27).
Briefly, 45 min before they were killed and their lungs collected, the animals were injected via
tail vein with 30 mg/kg of Evans blue dye. After fixation, lungs were microdissected and airway
images obtained with a Nikon SMX 1500 camera (Nikon Instruments, Inc.).
Monastral Blue B-Labeling of Permeable Vessels
Thirty minutes before they were killed, the animals were injected via a tail vein with 30 mg/kg
Monastral blue B suspension (Sigma-Aldrich Chemical Co., St. Louis, Mo), a compound used to
label sites of vascular leak, at a concentration of 1 mg/ml (28). Animals were euthanized 4 h
after exposure, lungs were harvested, fixed, microdissected and imaged as described.
For statistical analysis, Prism 5.01 software (GraphPad, La Jolla, CA) was used. Results are
presented as mean ± SEM in the text and figures. Groups were subjected to one-way analysis of
variance (ANOVA), and when significance was found, Tukey’s post-hoc analysis was applied. A p
value < 0.05 was considered significant.
Page 10 of 72
Assessment of airway cast formation
At 18 h after CEES inhalation, we found bronchial casts within all lobes, especially within
dependent lung regions such as the right lower lobe, the lower portion of the left lobe, and the
accessory lobe (Figure 2B). Cast formation with 5% CEES was inconsistent in both location and
degree of obstruction. By contrast to 5% CEES, 7.5% CEES inhalation produced reliable cast
formation and a more severe injury, with larger bronchial casts that often caused complete
airways obstruction of some lobes. Again, this was particularly evident within the dependent
lobes. Complete occlusion of all lobes was incompatible with survival and was noted during
necropsy of several non-surviving animals. Mortality rate with 7.5% CEES was 25% at 18 h, and
67% at 72 h, while 5% CEES caused no mortality at all time points examined (Table 1). With
ethanol exposure alone, no cast formation was observed in any airways (Figure 2A). Detailed
mapping of bronchial casts within the airways revealed that such casts extended from the
tracheal bifurcation to, at most, airway generation 15 of the axial pathway (Figure 1). Major
daughter generations also contained extensions of same casts for up to an additional 4 distal
Composition of airway casts
Since bronchial cast composition is likely related to underlying mechanism(s) resulting in
their formation, we next sought to classify the casts formed after CEES exposure. After rats
were exposed to 5% CEES for 18 h, the lungs were fixed and microdissection was performed on
the right middle lobe. Bronchial casts were then carefully removed in their entirety, and
Page 11 of 72
processed for immunohistochemistry, histology, or confocal microscopy. Immunohistochemical
examination revealed fibrin(ogen) in great abundance as a component of these casts (Figure 3, A
and B). Periodic acid Schiff/Alcian blue staining of cast sections did not demonstrate mucus
staining at 18 h (Figure 3C), indicating that casts formed after CEES exposure were not mucin-
based. Hematoxylin and eosin staining of airway cast sections demonstrated scattered
inflammatory cells dispersed throughout entire casts. The inflammatory cells appeared to be
concentrated heavily along the edges of the casts, particularly by 72 h after exposure (Figure 3E).
Collagen deposition was noted by 72 h, with the appearance of spindle cells suggestive of
myofibroblasts or fibroblasts within the casts (Figure 3F). We also noted occasional clumps of
ciliated epithelial cells by 18 h (Figure 3, C and D), deeply embedded within the periphery of the
casts. The presence of ciliated epithelial cells was confirmed via immunohistochemical staining
for acetylated tubin present in cilia (see Figure E1 in the online data supplement).
In order to assess if the cells within these casts and their adjacent airways were still
viable, we employed a confocal microscopy double staining technique using YOPRO-1 and EthD-
1 nuclear dyes to indicate live (green) or dead (red) cells, respectively (24, 25) . YOPRO-1 nuclear
dye was used to stain all cell nuclei as a “background” stain by study design, and EthD-1 nuclear
dye was used to indicate dead or dying cells with compromised cell membrane integrity. We
found that the majority of cells within the casts were YOPRO-1-positive, or live cells, especially
those cells located at the periphery of the casts (see Figure E2 in the online data supplement).
Dead cells, which stained positively with EthD-1, were only sporadically noted, and were seen
mostly within the core of the cast, where cells identified by histology appeared to be mainly
inflammatory in origin.
Page 12 of 72
Plasma-derived protein quantitation in BALF
The presence of fibrin-rich casts within airways after CEES implied leakage of the fibrin
precursor protein, fibrinogen (340 kDa as dimer), from surrounding vasculature into the airway
lumen, since fibrinogen is normally found only within blood plasma. Therefore, we next sought
to quantify the amount of fibrin(ogen) within the BALF after different inhaled CEES
concentrations, using Western blotting and densitometry for quantitation. We also assessed the
concentration of total protein present in the BALF, as well as that of IgM, a high molecular
weight immunoglobulin normally confined to the circulation (700 kDa pentamer). At both 4 and
18 h, we found a significant dose-related increase in BALF protein, IgM and most notably β-fibrin
at both CEES concentrations tested. As compared to levels in ethanol-exposed rats, with 5%
CEES exposure we observed a 3-fold increase at 4 h and a 6-fold increase at 18 h in BALF total
protein concentration (Figure 4A). After 7.5% CEES inhalation, protein in BALF was increased 4-
fold at 4 h and 10-fold at 18 h over ethanol. BALF obtained from naïve rats contained total
protein at concentrations similar to those found after ethanol exposure, which were minimal.
When IgM concentrations were measured in BALF, there was a 3-fold increase with 5% CEES at 4
h, and a 19-fold increase at 18 h as compared to ethanol (Figure 4B). The BALF IgM levels
further increased with 7.5% CEES to 4-fold at 4 h and 31-fold at 18 h. IgM was not detected in
BALF from naïve rat lungs. The concentration of β-fibrin (normalized to protein content) also
was significantly increased in the BALF after CEES exposure (Figure 4C). Inhalation of 5% CEES
resulted in a 4-fold increase at 4 h and a 10-fold increase at 18 h of BALF β-fibrin concentration
compared to ethanol levels. After 7.5% CEES inhalation, this increase was 5-fold at 4 h and 12-
Page 13 of 72
fold at 18 h over ethanol levels. Again, β-fibrin concentrations in BALF from naïve rats were
comparable to those observed after diluent (ethanol) exposure.
Cell differential counts and myeloperoxidase activity
To assess the role of inflammation in cast formation, we analyzed differential cell counts
of inflammatory cells in BALF of both 5 and 7.5% CEES-exposed rat lungs at 4 and 18 h (see
Figure E3, in the online data supplement). Macrophage levels gradually declined over time and
with higher CEES concentrations. The BALF absolute macrophage counts with 7.5% CEES showed
a 2-fold (4 h) and a 3-fold (18 h) reduction over ethanol-exposed levels, while with 5% CEES no
change in macrophages was observed at 4 h, and only a modest decrease at 18 h (1.3-fold). In
contrast, the BALF percent and absolute polymorphonuclear leukocyte (PMN) count increased in
a time dependent fashion but without significant CEES dose-response (Figure 5A; see Figure E3
in the online supplement). Only a minimal increase in PMNs was detected in the BALF at the 4 h
time point with either 5 or 7.5 % CEES. However, at 18 h there was a very significant 15-fold
increase in percent BALF PMNs with both 5 and 7.5% CEES. Ethanol exposure caused no
measurable increase in BALF PMNs, and showed comparable macrophage levels to naïve (data
In order to assess whole lung inflammation, we next evaluated the levels of
myeloperoxidase (MPO) in lung homogenates after CEES exposure. MPO is a peroxidase enzyme
predominantly present in neutrophils, thereby serving as a useful marker for the presence of
these granulocytes. Relative to ethanol exposure, 5% CEES inhalation resulted in a 3-fold (4 h)
and a 19-fold (18 h) increase in MPO, while 7.5% CEES inhalation resulted in a 2-fold (4 h) and a
Page 14 of 72
14-fold (18 h) increase. Levels from naïve animals were comparable to ethanol exposure (data
Assessment of vascular permeability by Evans blue dye
As localization of fibrin within the airway implies vascular injury, we next sought to
examine vascular permeability after CEES inhalation by tracing the extravasation of Evans blue
dye from permeable vessels. Evans blue dye binds to serum albumin (66 kDa), and its leakage
implies that blood vessels are permeable to proteins of this size or greater. Since casts were
‘well formed’ by 18 h, and plasma proteins were a major component of the casts, we assumed
that vascular leakage must precede cast formation. Therefore, we assessed for increased
vascular permeability at 4 h via the Evans blue dye extravasation method, prior to appearance of
any casts. Animals were injected by tail vein with Evans blue dye (30 mg/kg) 45 minutes prior to
necropsy after exposure to CEES or ethanol. Microdissection of all lobes was then performed in
order to localize dye leakage. Following CEES exposure, we noted extravasation of Evans blue
dye around the distal trachea and central bronchi (see Figure E4, C and D, in the online data
supplement), but no dye was detected after ethanol-only exposure (see Figure E4, A and B, in
the online data supplement). This effect was CEES concentration-dependent, in that dye
extravasation was greater in the 7.5% CEES group (data not shown). In addition to peribronchial
and peritracheal staining, regions around the central pulmonary vessels (both arteries and veins)
also demonstrated increased Evans blue staining after CEES inhalation compared to both
ethanol and naïve controls (see Figure E5, A and B, in the online data supplement). No
parenchymal staining was noted at any concentration tested, indicating that increased
permeability did not occur in the pulmonary microcirculation.
Page 15 of 72