Download Histological Examination of the Relationship between Respiratory

Exp. Anim. 60(2), 141–150, 2011
—Original—
Histological Examination of the Relationship between
Respiratory Disorders and Repetitive Microaspiration
Using a Rat Gastro-Duodenal Contents Reflux Model
Keisuke OUE1, 2), Ken-ichi MUKAISHO1), Tomoki HIGO1, 2),
Yoshio ARAKI1), Masanori NISHIKAWA2), Takanori HATTORI1),
Gaku YAMAMOTO2), and Hiroyuki SUGIHARA1)
Departments of 1)Pathology and 2)Oral and Maxillofacial Surgery, Shiga University of
Medical Science, Seta-tsukinowa-cho, Otsu, Shiga 520-2192, Japan
Abstract: Microaspiration due to gastroesophageal reflux (GER) has been suggested as a
factor contributing to the development and exacerbation of several respiratory disorders. To
explore the relationship between GER and respiratory disorders, we histologically examined
the bilateral lungs of a rat gastroduodenal contents reflux model, which was previously used
to investigate the histogenesis of Barrett’s esophagus and esophageal carcinoma. GER was
surgically induced in male Wistar rats. The bilateral lungs of the reflux rats were examined
with hematoxylin and eosin (HE), PAS-Alcian blue, and Azan staining at 10 and 20 weeks
after surgery. Immunohistochemical staining of CD68 and α-SMA was also performed.
Aspiration pneumonia with severe peribronchiolar neutrophilic and lymphocytic infiltrates,
goblet cell hyperplasia, prominence of blood vessels, and increased thickness of the smooth
muscle layer were detected. Bronchiolitis obliterans (BO)-like lesions comprising granulation
tissue with macrophages, spindle cells, and multinucleated giant cells in the lumen of
respiratory bronchioles were observed in the bilateral lungs of the reflux animals. These
findings suggest that the severe inflammation and the BO-like lesions may play a role in
exacerbation of the forced expiratory volume in 1 second (FEV 1) in human cases. In
conclusion, we speculate that repetitive microaspiration due to GER may contribute to the
exacerbation of various respiratory diseases, particularly asthma and chronic obstructive
pulmonary disease (COPD), and the development of BO syndrome following lung
transplantation. The reflux model is a good tool for examining the causal relationships
between GER and respiratory disorders.
Key words: bronchiolitis obliterans, gastro-duodenal reflux, microaspiration, rat reflux model,
respiratory disorder
(Received 12 August 2010 / Accepted 25 November 2010)
Address corresponding: K. Mukaisho, Department of Pathology, Shiga University of Medical Science, Seta-tsukinowa-cho, Otsu, Shiga 520-2192,
Japan
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K. Oue, ET AL.
Introduction
Gastroesophageal reflux disease (GERD) has become
a common disorder in the USA and Western Europe in
recent decades [16]. The manifestations can be divided
into esophageal and extraesophageal syndromes, which
affect various tissues and organ systems in addition to
the esophagus [28, 41]. Among the various extraesophageal manifestations, causal relationships between
GERD and cough, laryngitis, asthma, and dental erosion
have been strongly suggested in the Montreal definition
[42]. In addition, gastroesophageal reflux (GER) accompanied by regurgitation and microaspiration has been
suggested as a contributory factor in several respiratory
disorders. Idiopathic pulmonary fibrosis, cystic fibrosis,
connective tissue disease, and obstructive lung disease
have all been reported to be associated with GER [19,
39, 42]. Moreover, GER has been shown to worsen
asthma through esophagobronchial reflex, and to heighten bronchial reactivity and microaspiration [2, 14, 20].
GER has also been reported to be accompanied by neutrophilic airway inflammation [10] and it has been suggested that patients with chronic obstructive pulmonary
disease (COPD) along with GERD symptoms are more
likely to experience exacerbations than those lacking
these symptoms. GERD may also increase the tracheobronchial aspiration of gastric juice directly and/or disturb the clearance of swallowed contents from the pharynx to the esophagus indirectly, leading to frequent
exacerbations. Therefore, GER may act as a confounding factor in COPD exacerbations through mechanisms
similar to those seen in asthma and/or by increasing
airway inflammation [38].
Microaspiration due to GER has been most commonly observed in lung transplant patients in recent
years. Lung transplantation has become a therapeutic
option for appropriately selected patients with end-stage
lung diseases in the last 20 years. However, successful
approaches for managing chronic rejection of pulmonary
allografts have not yet been developed. Long-term survival of patients with pulmonary allografts is currently
hindered by bronchiolitis obliterans syndrome (BOS), a
form of chronic rejection that is unique to lung transplantation. Clinical studies suggest that both immuneand non-immune-mediated factors contribute to BOS
development. High GER levels with resultant aspiration
have been implicated as a non-immune risk factor for
BOS development following lung transplantation [15,
22, 31, 34, 35, 43]. GER is promoted after lung transplantation probably because of the potential for iatrogenic vagal nerve injury during lung transplantation and
due to the use of immunosuppressive drugs such as calcineurin inhibitors, cyclosporine and tacrolimus that
prolong gastric emptying [5, 15, 35]. In addition, bile
acids in bronchoalveolar lavage fluid at three months
after transplant were found to be associated with BOS
development in a time- and dose-dependent manner [17].
These findings suggest that bile acids, not gastric juice
in reflux contents, play an important role in BOS development following lung transplantation.
In order to establish experimental evidence for causal
relationships between GER and respiratory disorders,
we examined both lungs of the reflux model, which had
previously been used to investigate the histogenesis of
Barrett’s esophagus and esophageal carcinoma [11, 12]
and was recently used to examine the mechanism of
development of extraesophageal syndromes including
laryngitis and dental erosion [26, 30].
Materials and Methods
All procedures complied with the ethical guidelines
for animal experimentation and the care and use of
laboratory animals at Shiga University of Medical Science, Japan.
Animal models
Gastro-duodenal reflux was induced in eight-week-old
male Wistar rats (CLEA, Tokyo, Japan) according to a
previously reported procedure [11, 12]. Shortly afterwards, the esophagogastric junction was transected, the
distal cut end was closed, and the proximal cut end was
anastomosed end-to-side to the upper jejunum, approximately 2 cm distal to the origin. After the esophagojejunostomy, a serosal suture (interrupted 7-0 nylon) was
placed between the esophagus and jejunum to support the
afferent loop side of the anastomosis. As a result, the
serosal suture allowed ingested food to easily enter the
efferent loop and prevented food from entering the afferent loop. The gastro-duodenal contents flowed through
RESPIRATORY DISORDERS AND MICROASPIRATION IN RATS
143
the esophagojejunal stoma and back into the esophagus
[11, 12] (Fig. 1). Sham-operated rats, which underwent
a laparotomy with blunt manipulation of the abdominal
contents under diethyl ether anesthesia, were used as control animals. The reflux animals that survived were euthanized with an overdose of diethyl ether at postoperative
week 10 (n=7) or 20 (n=7). Sham-operated control rats
were euthanized at similar time points (n=7 per group).
Histological examination
Resected bilateral lung tissue samples were weighed
and fixed in 10% formalin in phosphate-buffered saline
for 4 h and embedded in paraffin. Four-micrometer sections were made and stained with hematoxylin and eosin
(HE). PAS-Alcian blue staining for detection of goblet
cell metaplasia and Azan staining for fibrosis were also
performed on the lung samples. The degree of positive
cells for PAS-Alcian blue staining was recorded as weak
(0), mild (1+), moderate (2+), or strong (3+).
Immunohistochemical stainings of CD68 and α-SMA
Immunohistochemical staining was performed with
mouse monoclonal antibodies: CD68 (a marker for macrophages, Clone:ED1, 1:500, AbD Serotec, Oxford, UK)
and α-SMA (a marker for α-smooth-muscle isoform of
actin, Clone:ASM-1, 1:200, Progen Biotechnik, Heidelberg, Germany). The staining was performed on a Discovery XT Automated IHC Stainer using the Ventana
DABMap detection kit (catalog No. 760-124, Ventana
Medical Systems, Tucson, AZ, USA). Each step of the
Ventana DABMap detection kit procedure was optimized
on the Discovery XT instrument and preset. Antigen
retrieval of tissue sections was performed with enzyme
for CD68 and with heat for α-SMA. The sections were
counterstained with haematoxylin. Slides of the negative
control without the primary antibody and those of the
positive control were processed in parallel.
Thickness of smooth muscle layer of bronchioles
The thickness of the bronchiolar smooth muscle layer
was quantified by measuring the thickness of α-SMA
positive layer at 4 points in randomly selected bronchiolar cross sections of the smallest diameters, 250–500
µm. The mean of the measurements in each groups was
recorded.
Fig. 1. Gastro-duodenal contents reflux model. A
single serosal suture between the esophagus
and jejunum was added after the esophagojejunostomy. B, Bile duct; E, esophagus;
F, fore stomach; G, glandular stomach; D,
duodenum; J, jejunum.
pH analysis of the esophageal and gastric contents
Following euthanasia, pH of the esophageal and gastric contents was estimated using a Compact pH meter
(Horiba, Kyoto, Japan).
Statistical analysis
Absolute lung weights, the pH of esophageal and gastric contents, and the thickness of the smooth muscle
layer of bronchioles are expressed as mean ± SE. Student’s t-test or Welch’s t-test were used for comparisons.
P<0.05 was considered statistically significant.
Results
pH of the esophageal and gastric contents
The pH of the gastric contents in the reflux rats was
3.75 ± 0.19 after 10 weeks and 4.11 ± 0.34 after 20 weeks
compared to control rat values of 4.06 ± 0.30 after 10
weeks and 4.32 ± 0.49 after 20 weeks. The pH of the
esophageal contents in the reflux rats was 7.07 ± 0.11
after 10 weeks and 6.48 ± 0.08 after 20 weeks. However, no comparable pH measurements were made in
control rats at 10 and 20 weeks due to the small volume
of esophageal contents in these animals. There were no
significant differences in the pH of the stomach contents
between the groups (Table 1).
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K. Oue, ET AL.
Table 1. Esophageal and gastric content pH
Gastric pH
Esophageal pH
Control (10 w)
Reflux model (10 w)
Control (20 w)
Reflux model (20 w)
4.06 ± 0.30
7.29 ± 0.06
3.75 ± 0.19
n.d.
4.32 ± 0.49
6.40 ± 0.08
4.11 ± 0.34
n.d.
The data are shown as the mean ± SE. n.d., not determined; due to very little fluid in the esophagus of
control animals.
Table 2. Lung weights
R. lung (g)
L. lung (g)
Control (10 w)
Reflux model (10 w)
Control (20 w)
Reflux model (20 w)
0.812 ± 0.03
0.440 ± 0.02
0.822 ± 0.05
0.466 ± 0.04
0.864 ± 0.03
0.467 ± 0.01
0.854 ± 0.11
0.536 ± 0.09
The data are shown as the mean ± SE.
Bilateral absolute lung weights
Bilateral absolute lung weights are summarized in
Table 2. At both 10 and 20 weeks, no significant increase
of bilateral absolute lung weights from both control rats
and reflux rats were noted. However, the left lung
weights from reflux rats at 20 weeks were slightly higher than those of the controls. The lung weights appeared
to correlate with the degree of inflammatory cell infiltrates in the lungs of the reflux animals.
Macroscopic and under-loupe findings
Both lungs of the reflux animals were partially consolidated compared to those of the control animals at 10
and 20 weeks following surgery (Fig. 2). HE-stained
sections showed several areas of densely stained inflammatory cell infiltrates, and atelectasis was detected in
the bilateral lung fields of the reflux animals (Fig. 3).
These areas grew in size in a time-dependent manner.
Microscopic findings
Low-power views of both lungs showed partial consolidation in areas surrounding the bronchi and bronchioles of the reflux animals (Fig. 4a). High-power views
of the lung alveolar spaces of reflux animals showed
partly severe lymphocytic and neutrophilic infiltrates,
macrophages, and multinucleated giant cells within the
alveolar space (Fig. 4b and 4c). In addition, bile acids
and food contents were occasionally found within multinucleated giant cells. In severe cases, the bronchioles
were partially plugged with neutrophilic exudates (Fig.
4c and 4d). Neither congestion nor pulmonary edema
was detected in any animal.
A markedly increased number of goblet cells positive
for PAS-Alcian blue and an increased number of peribronchiolar blood vessels were detected in the reflux
animals compared to the control animals (Fig. 5a–d).
PAS-Alcian blue staining showed a time-dependent increase in the number of goblet cells. Goblet cells accounted for a significantly greater number of bronchial
and bronchiolar epithelial cells at 20 weeks after surgery
than at 10 weeks. We detected strong (3+) proliferation
of goblet cells, i.e., goblet cell hyperplasia, in the bronchial and bronchiolar epithelium in three of seven specimens at 10 weeks and all seven specimens at 20 weeks
in the left lung, and five of seven specimens at 10 weeks
and all seven specimens at 20 weeks in the right lung
(Table 3). The thickness of the smooth muscle layer of
the bronchiole in the bilateral lungs of the reflux animals
was significantly greater than that of the control animals
at 10 and 20 weeks after surgery (Table 4). Bronchioles
of 250–500 µm representatives of the smallest diameters
in the reflux and control animals are shown in Fig. 6.
We also observed that many reflux animals had BO-like
lesions, characterized by peribronchiolar lymphocytic
infiltrates and luminal narrowing of the respiratory bronchioles due to polypoid granulation tissue composed of
numerous macrophages positive for CD68, multinucleated giant cells, and spindle cells (Fig. 7a and 7b). Fibrous connective tissue, partially positive for Azan stain,
was observed in the granulation tissue (Fig. 7c). BO-like
RESPIRATORY DISORDERS AND MICROASPIRATION IN RATS
145
Fig. 2. Macroscopic views of lungs at 20 weeks postoperatively. a) sham-operated animal, b) reflux animal. Gross figure shows that the color of bilateral
lungs in the reflux animals is markedly red and bilateral lungs in the reflux
animals are partly consolidated compared to those of the control animals.
Fig. 3. Loupe views of HE sections. a–c, left
lung; d–f, right lung; a and d, control
animal at 20 weeks; b and e, reflux animal
at 10 weeks postoperatively; c and f, reflux animal at 20 weeks postoperatively.
Several parts with dense inflammatory
cell infiltrates were detected in bilateral
lung fields. These infiltrates were more
severe at 20 weeks than at 10 weeks after
operation.
Fig. 4. Microscopic findings in HE sections from
the reflux animals at 20 weeks. a, Lowpower view; b–d, high-power view. a)
Severe lymphocytic and neutrophilic infiltrates, macrophages, and multinucleated giant cells were detected. b) Many
multinucleated giant cells were found in
the alveolar spaces. c and d) Many neutrophilic infiltrates were found within the
alveolar spaces and in the small airway.
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Fig. 5. Goblet hyperplasia and an increased number of blood vessels developed in the reflux animals at
20 weeks. a and d, HE staining; b and e, PAS-Alcian blue staining; c, and f, immunohistochemical
staining of α-SMA; a–c, control animal; d–f, reflux animal. Marked goblet cell hyperplasia positive for PAS-Alcian blue was observed in the reflux animals compared to the control animals (a,
b, d, e). Not only bronchiolar smooth muscle layer but also peribronchiolar blood vessels were
positive for α-SMA (c and f). An increased number of blood vessels, and inflammatory cell infiltrates were also found in the reflux animals (a, c, d, f).
Fig. 6. Comparison of the thickness of the bronchiolar smooth muscle layer of reflux
animals and controls at 20 weeks after surgery. a) Control animal, b) Reflux
model. We measured the thickness at 4 points in each cross-section of the smallest diameter bronchioles, 250–500 µm. The thickness of the smooth muscle
layer in the reflux animals was significantly greater than in the controls.
Fig. 7. Bronchiolitis obliterans-like lesions developed in the reflux animals at 20 weeks. a) HE
staining, b) CD68, c) Azan staining. Bronchiolitis obliterans-like lesions comprising
granulation tissue with macrophages positive for CD68, spindle cells, and a multinucleated giant cell in the lumen of respiratory bronchioles were observed in the reflux animals
(a and b). Partially fibrous connective tissue positive for Azan staining was noted in the
granulation tissue (c).
147
RESPIRATORY DISORDERS AND MICROASPIRATION IN RATS
Table 3. Incidence of bronchiolitis obliterans-like lesion and goblet cell metaplasia in
control and reflux animals
Bronchiolitis obliterans-like lesion
Control (10 w)
Control (20 w)
Reflux model (10 w)
Reflux model (20 w)
L. lung
R. lung
0/7
0/7
4/7
7/7
0/7
0/7
2/7
7/7
Goblet cell metaplasia (3+)
L. lung
R. lung
0/7
0/7
3/7
7/7
0/7
0/7
5/7
6/7
Table 4. Thickness of smooth muscle layer
Diameter of bronchiole
Control (10 w)
Control (20 w)
Reflux model (10 w)
Reflux model (20 w)
L. lung
R. lung 290.0 ± 24.2
313.7 ± 16.4
335.2 ± 16.7
295.0 ± 11.3
300.4 ± 10.0
297.5 ± 12.7
329.1 ± 21.6
321.6 ± 20.2
Thickness of smooth muscle bundles
L. lung
12.7 ± 0.47*
10.9 ± 0.31**
17.5 ± 0.98*
17.3 ± 1.14**
R. lung
11.7 ± 0.52*
9.52 ± 0.43**
21.3 ± 0.88*
15.3 ± 0.58**
Data are shown as the mean ± SE. There were no significant differences among the groups in
the thickness of the smooth muscle layer in the smallest diameter bronchioles examined. The
mean thickness of bronchiolar smooth muscle layer was significantly greater in the reflux animals
than in the control animals (* and **, P<0.0001; Student’s t-test).
lesions were detected in four of seven specimens at 10
weeks and in all seven specimens at 20 weeks in the left
lung, and in two of seven specimens at 10 weeks and all
seven specimens at 20 weeks in the right lung (Table
3).
Discussion
We detected bronchopneumonia with foreign body
reaction, namely aspiration pneumonia and peribronchial atelectasis and fibrosis following purulent inflammation with severe peribronchiolar neutrophilic and
lymphocytic infiltrates in our reflux model. Goblet cell
hyperplasia, prominence of blood vessels, and increased
thickness of the smooth muscle layer were also detected
in association with the continuous chronic inflammatory
stimulation of GER. The most important findings of the
present study were BO-like lesions characterized by
luminal narrowing of the respiratory bronchioles due to
polypoid granulation tissue composed of numerous macrophages, multinucleated giant cells, and spindle cells.
Based on the abovementioned results, we hypothesize
that GER may play an important role in the exacerbation
of various respiratory diseases, especially asthma,
COPD, and BOS following lung transplantation.
In human asthma cases, GER appears to be associated
with airway inflammation and has been recognized as
an important causative factor in asthma attacks [21, 23].
However, the mechanism by which GER may provoke
airway inflammation is doubtful. A key role seems to
be played by the vagally-mediated neurogenic reflex or
by the microaspirated refluxate that probably mediates
airway inflammation [9, 24]. Irrespective of the active
mechanism, airway inflammation in GER can be the
linkage by which GER eventually exacerbates asthma.
A recent study showed that GER associated with asthma
is characterized by an increase in the numbers of eosinophils and neutrophils, while GER alone presents a neutrophilic pattern of inflammation. In addition, GER
appears to exacerbate already existing oxidative stress
[10]. These findings are consistent with the pattern of
aspiration pneumonia with severe peribronchiolar neutrophilic and lymphocytic infiltrates found in the present
animal model. Therefore, the inflammation caused by
GER can be a causative factor of exacerbation of asthma.
Moreover, it has also been reported that a marked increase in the goblet cells in the airways is a characteristic feature of patients with bronchial asthma who have
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K. Oue, ET AL.
died of severe acute attacks. The discovery of goblet
cell hyperplasia could be used as a marker to identify
patients with bronchial asthma who are at risk of severe
attacks [1]. Considering these results, the goblet cell
hyperplasia observed in the present study may also play
a role in worsening asthma in human cases.
COPD exacerbations are characterized by acute and
chronic deterioration in respiration [3, 7]. One of the
most common causes of COPD exacerbations is tracheobronchial infection. The most frequent route for
bacterial infection of the tracheobronchial tree is the
silent aspiration of oropharyngeal secretions [36]. Studies on the relationship between the swallowing function
and GERD have revealed that subjects presenting with
cough and GERD have significantly reduced laryngopharyngeal sensitivity, potentially resulting in an increased risk of aspiration via the impairment of the
swallowing reflex [32]. A recent study suggested that
swallowing abnormalities, probably via GERD comorbidity, were associated with frequent COPD exacerbations [37]. Our previous study using the same rat reflux
model as the present study showed that the reflux of
gastro-duodenal contents causes severe laryngitis [30].
Inflammation of the larynx must play a role in impairment of the swallowing reflex.
BOS is a major long-term complication and a leading
cause of death after lung transplantation; it affects 50–
70% of transplant recipients [4, 6, 25]. The histological
hallmark of BOS is BO [8]. BOS was originally defined
as a decrease in forced expiratory volume in 1 second
(FEV 1) [13]. In the present study, we detected BO-like
lesions associated with repetitive microaspiration caused
by GER. These lesions may play an important role in
exacerbation of FEV 1 subsequent to COPD exacerbations, unlike true constrictive BO that is fused to the
bronchiolar wall and obliterates the lumen. In a recent
experimental study, Li et al. [29] reported histological
evidence consistent with BO development in lung allografts after inducing chronic gastric fluid aspiration in
a rat model that utilized immunosuppression to avoid
acute rejection. However, the BO-like lesions observed
in the present reflux animal lungs did not result from
lung transplants or cyclosporine treatments. Thus, as
previously suggested by Li et al., our results imply that
GER plays an important role in BO-like lesion develop-
ment and that alloimmune injury following bone marrow
transplantation may promote BO development.
Another issue to be investigated with regard to refluxate in GER involves the determiation of the degree of
harm and importance of gastric juice and bile acids in
the pathogenesis of BO. The refluxates of the reflux
animals in the present study contained not only gastric
juices but also duodenal contents including bile acids.
D’Ovidio et al. examined bile acids in the bronchoalveolar lavage fluid of 120 post-transplant patients in a
cross-sectional study and found a 17% prevalence of
elevated bile acid concentrations. Furthermore, the highest concentrations were found in patients with early
onset BOS [18]. Bile aspiration secondary to duodenoGER has been associated with severe pulmonary injury.
Cytotoxicity may result in disruption of cellular membranes or alteration of cellular cationic permeability
depending on the bile acid concentration [27, 33, 40].
These findings suggest that refluxates, including bile
acids, play an important role in BO pathogenesis in lung
transplant patients.
In summary, we detected bronchopneumonia with
foreign body reaction and peribronchial atelectasis and
fibrosis in conjunction with purulent inflammation with
severe peribronchiolar neutrophilic and lymphocytic
infiltrates in our reflux-induced rats. We also noted goblet cells and smooth muscle hyperplasia. From these
findings, we speculate that repetitive microaspiration due
to GER contributes to the exacerbation of various respiratory diseases, especially asthma and COPD. BO-like
lesions were also observed in reflux animal lungs without
lung transplantation or cyclosporine treatment. These
findings confirm that GER including bile acids plays an
important role in the development of BO as a nonimmune-mediated factor in lung transplant patients. The
gastro-duodenal contents reflux model was originally
used in studies of Barrett’s esophagus; however, the
model is a good tool for studying the development of
extraesophageal syndromes and respiratory disorders
based on GER.
Acknowledgment
We thank Mr. Michiharu Nasu for excellent technical
assistance.
RESPIRATORY DISORDERS AND MICROASPIRATION IN RATS
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