Brazilian Journal of Medical and Biological Research (2015) 48(5): 458-464, http://dx.doi.org/10.

1590/1414-431X20144305
ISSN 1414-431X

Contractile profile of esophageal and

gastric fundus strips in experimental
doxorubicin-induced esophageal atresia
F.A. Capeto1, F.J.B. Lima2, W. Okoba2, F.L. Ramos1, T.F.A. Messias1, G.A. Rigonatto1,
L. Sbragia3, P.J.C. Magalhães2 and A.A. Melo-Filho1
1
Departamento de Cirurgia, Faculdade de Medicina, Universidade Federal do Ceará, Fortaleza, CE, Brasil
2
Departamento de Fisiologia e Farmacologia, Faculdade de Medicina, Universidade Federal do Ceará, Fortaleza, CE, Brasil
3
Departamento de Cirurgia e Anatomia, Faculdade de Medicina de Ribeirão Preto,
Universidade de São Paulo, Ribeirão Preto, SP, Brasil

Abstract

Esophageal atresia (EA) is characterized by esophageal and gastric motility changes secondary to developmental and postsurgical
damage. This study evaluated the in vitro contractile profile of the distal esophagus and gastric fundus in an experimental model of EA
induced by doxorubicin (DOXO). Wistar pregnant rats received DOXO 2.2 mg/kg on the 8th and 9th gestational days. On day 21.5,
fetuses were collected, sacrificed, and divided into groups: control, DOXO without EA (DOXO– –EA), and DOXO with EA (DOXO+ +EA).
Strips from the distal esophagus and gastric fundus were mounted on a wire myograph and isolated organ-bath system, respectively,
and subjected to increasing concentrations of carbamylcholine chloride (carbachol, CCh). The isolated esophagus was also stimulated
with increasing concentrations of KCl. In esophagus, the concentration-effect curves were reduced in response to CCh in the
DOXO+ +EA and DOXO– –EA groups compared to the control group (P,0.05). The maximum effect values (Emax) for DOXO+ +EA and
DOXO– –EA were significantly lower than control (P,0.05), but the half-maximal effective concentration (EC50) values were not
significantly different when the three groups were compared (P.0.05). In response to KCl, the distal esophagus samples in the three
groups were not statistically different with regard to Emax or EC50 values (P.0.05). No significant difference was noted for EC50 or Emax
values in fundic strips stimulated with CCh (P.0.05). In conclusion, exposure of dams to DOXO during gestation inhibited the
contractile behavior of esophageal strips from offspring in response to CCh but not KCl, regardless of EA induction. The gastric fundus
of DOXO-exposed offspring did not have altered contractile responsiveness to cholinergic stimulation.

Key words: Esophageal atresia; Doxorubicin; Experimental; Esophageal contractility

Introduction
Esophageal atresia (EA) is a primary structural due to technical limitations (6-10). Therefore, it is difficult to
anomaly that results from incomplete esophageal- separate postsurgical and embryological alterations.
tracheal septation during intrauterine development, with Experimentally induced EA has been useful for identifying
associated varied lesions in the distal esophagus and and understanding esophageal and gastric motility changes
stomach during its development or after surgical correc- attributable to development. The teratogenesis of doxo-
tion. Dysmotility-associated symptoms such as gastro- rubicin (DOXO) is useful for this purpose (11,12).
esophageal reflux (GER) and dysphagia are frequent in The main objective of this work was to study in vitro
the affected population, persisting through adulthood and the gastric and esophageal contractility in an experimental
thus negatively impacting quality of life (1-3). model of EA induced by DOXO in rats to determine if it is
Experimental and clinical studies have previously a useful model for dysmotility-associated symptoms.
shown the existence of altered intrinsic innervation in EA
(4,5). However, data on esophageal and gastric motility Material and Methods
profiles are scarce. Furthermore, the few available pub-
lished clinical studies on patients with EA with tracheoeso- Animals
phageal fistula (TEF) have failed to show the contractile The experimental protocol was approved by the Ethics
nature of the esophagus after surgical correction, probably and Research on Animals Committee (CEPA #27/2011)

Correspondence: A.A. Melo-Filho: ,aamelofilho@gmail.com..

Received September 1, 2014. Accepted December 11, 2014. First published online March 6, 2015.

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Esophageal and gastric contractility in esophageal atresia 459

at the Universidade Federal do Ceará. Female albino bubbled with 5% CO2 and 95% O2, and the pH was adjusted
Wistar rats (, 250 g) were obtained from the Central to 7.4. A basal period of 1.5 h was established before
Vivarium, Universidade Federal do Ceará. They were initiating chemical stimulation. During this period, the perfus-
maintained under an acclimatized day/night ambience, ing liquid was changed after every 15 min and the basal
with free access to food and water. Under this condition, tension was monitored. Consequently, a concentration-effect
females were mated with males and natural reproduction curve (CEC) was obtained after cumulatively pipetting several
was allowed, with verification carried out on the subse- concentrations of carbamylcholine chloride (carbachol, CCh,
quent morning. Rats that showed a positive sperm-test on 0.01-100 mM) or KCl (10-100 mM). The maximum effect
vaginal lavage were considered pregnant, with the date (Emax) of a CEC was determined when a given concentration
determined as day 0 (term=22 days). Pregnant rats were of the contractile agent did not elicit a contraction higher than
separated into single cages with the same conditions as that provoked by the preceding concentration. The half-
prior to pregnancy. maximal effective concentration values (EC50) of contractile
agents were derived for each CEC.
Experimental procedure
The technique used for the induction of EA was In vitro gastric contractility protocol
described by França et al. (13). Doxorubicin chloride Fundic tissues were obtained as ring-like strips from
(2.2 mg/kg) was administered (intra-peritoneal injections the fetal stomach cut in the coronal plane. Two non-
using a solution of 2 mg/mL DOXO in saline) once daily extensible cotton purse strings were used: one to maintain
on days 8 and 9 of pregnancy (n=26). Another group of the tissue fixed on a metal haste, and the other to
pregnant rats (n=13) received only the saline vehicle. maintain the ring on a highly sensitive force isometric
transducer (ML870B60/CV, AD Instruments, New South
Data collection Wales, Australia) that was subsequently connected to a
On day 21.5, pregnant rats were anesthetized with an digital data acquisition system (PowerLabTM 8/30, AD
intramuscular injection of 1.25 mL/kg ketamine (50 mg/ Instruments). Each tissue bath contained 5 mL of Tyrode
mL) and 0.25 mL/kg xylazine (10 mg/mL) and subse- solution (pH 7.4), heated to 376C and constantly oxygen-
quently subjected to caesarian surgery to remove the ated with 5% CO2 in 95% O2. Each successful set up was
fetuses. The bicornuate uterus was exposed, and the re- calibrated to a basal tension of 1 g over 1 h. During this
spective fetuses were identified numerically from the most period, the perfusing fluid was changed every 15 min, and
proximal to the cervix to the most distal. Afterward, the the basal tension was adjusted. To test the viability of
fetuses were weighed before being sacrificed by occipital each tissue set-up after the basal period, two successful
puncture and subjected to a cervico-thoraco-laparotomy contractions were induced by adding 80 mM KCl into the
using a surgical magnifying lens (Magnifier Lamp, Model bath. Thereafter, each strip was stimulated with increas-
TL1092, TÜV Rheinland1, Germany) to detect EA and ing concentrations of CCh (0.01-300 mM) to obtain a
associated TEF. Thereafter, the distal esophageal tissue respective CEC. At the end of each protocol, each isolate
and gastric fundic strips were obtained for in vitro was weighed, and the contractile force was expressed as
contractile studies. Tissues were obtained from animals its relationship to the respective tissue mass (g tension/
divided into three groups: control (rats that received only mg of tissue). The respective Emax and EC50 values were
vehicle during pregnancy), DOXO– –EA (dams injected with derived at the end of the experiment.
DOXO during pregnancy, but the fetus did not develop EA)
and DOXO+ +EA (dams injected with DOXO during Statistical analysis
pregnancy and the fetus developed EA). Data for esophageal and gastric contractility are
reported as means±SE, with n indicating the number of
In vitro esophageal contractility protocols experiments. Statistical analysis was determined with
The isolated esophagus was cut transversally to produce two-way variance analysis (ANOVA), followed by Student-
ring-like segments (approximately 2-mm length) that were Newman-Keuls tests when results were significant. The
mounted horizontally onto a wire myograph (610M-DMT; respective EC50 values were obtained by semi-logarithmic
DMT, Aarhus, Denmark). Mechanical tension was applied to interpolation and are reported as the geometric mean
each segment by two tungsten wires (40 mm) that were [95% confidential interval] and the number of experi-
extended parallel through the esophageal lumen. One wire ments. We compared the respective EC50 values using
was connected to a highly sensitive force transducer to record Mann-Whitney U-tests. The Emax values were determined
isometric contractions, while the other was connected to a after obtaining the maximum contractile response.
micrometer that allowed basal tension adjustments (set at P,0.05 was considered statistically significant.
8 mN). The tissue was constantly bathed with a physiological
Tyrode solution (136.0 mM NaCl, 5.0 mM KCl, 0.98 mM Results
MgCl2, 2.0 mM CaCl2, 0.36 mM NaH2PO4, 11.9 mM
NaHCO3, and 5.5 mM glucose) at 376C that was continually Twenty-six DOXO-exposed pregnant rats produced

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460 F.A. Capeto et al.

164 fetuses, with 17 (10.36%) showing macroscopic EA Evaluation of the contractile behavior of fundic
with distal TEF. gastric strips in response to CCh
The mean weights of fundic strips were 3.80±0.33,
Evaluation of the contractile behavior of distal 4.10±0.35, and 3.77±0.44 mg, for the control, DOXO– –
esophageal strips in response to CCh EA, and DOXO+ +EA groups, respectively. These values
In isolated strips from the control group, cumulative were not significantly different (P.0.05, one-way
concentrations of CCh (0.01-100 mM) produced a CEC ANOVA). Exposure of fundic strips from the control group
with an Emax of 5.97±0.58 mN (n=11) that was obtained to increasing concentrations of CCh (0.01-300 mM)
at 100 mM (Figure 1). It was significantly higher than that resulted in contractile responses with an E max of
obtained from isolated tissues from animals exposed to 0.084±0.016 g/mg of tissue (n=7) at a concentration of
DOXO (P,0.05, two way ANOVA, Student-Newman- 100 mM (Figure 3). However, the respective values in the
Keuls; Table 1). The decreased E max value under DOXO– –EA and DOXO+ +EA groups were not significantly
cholinergic stimuli was independent of the presence of different (P.0.05, two-way ANOVA; Table 2). Similarly,
EA; Emax values observed for both the DOXO– –EA and the respective EC50 values were not statistically different
DOXO+ +EA groups were significantly lower than that between the three groups (P.0.05, Mann-Whitney U-test;
measured in the control group, but they were not Table 2).
significantly different from each other (P.0.05, ANOVA;
Table 1). Regarding the EC50 values for CCh-induced Discussion
contractions, there was no significant difference among the
three groups (P.0.05, Mann-Whitney U-test; Table 1). It is well known that even after surgical correction,
babies born with EA continue to experience functional
Evaluation of the contractile behavior of distal repercussions. The most common complication is gastro-
esophageal strips in response to KCl esophageal reflux disease (GERD), with varied symptom
After exposure to increasing concentrations of KCl intensity. GERD-related symptoms seem to stem from the
(10-100 mM), esophageal strips in the control group initial EA-induced lesions on two primordial protective
produced a CEC with an Emax of 1.31±0.14 mN (n=5), barriers to reflexive episodes: the mechanical and physio-
which was not significantly different from the values in logical entity of the esophageal-gastric junction and the
groups pre-treated with DOXO (P.0.05, two-way esophagus clearance mechanism (3,6-8). Particularly, the
ANOVA; Table 1 and Figure 2). lack of distal esophageal contractions is significantly
correlated with the development of GERD (14). This may
be demonstrated by a conventional or a high-resolution
postsurgical esophageal manometry that may reveal
alterations including absence of or uncoordinated peristal-
tic waves, simultaneous contractions, low amplitude con-
tractions, and high base-line pressure (9,10).
It is thought that surgical intervention of the esopha-
gus, even for therapeutic purposes, is capable of
negatively impacting esophageal motility, probably due
to vagal denervation whether by operational traction-
mechanisms or cranial dislocation of the esophagogastric
junction due to surgery (3). However, these mechanisms
do not negate the fact that congenital alterations affect
motility mechanisms prior to diagnosis and therapy.
Clinical presurgical evaluation of neonates with EA
routinely reveals esophageal dysmotility (15). Moreover,
esophageal motility is also severely affected in children
with isolated TEF prior to surgery (16).
There are three types of animal models used to study
Figure 1. Comparison of the effect of carbachol (CCh) on the EA: teratogen-induced, surgically created, and knockout
distal esophagus of fetus with esophageal atresia (EA). The models. Among surgical models, the chick embryo does
graph shows the mean tension of the distal esophageal strips in not reliably mimic EA and TEF. Another surgical model is
response to increasing concentrations of CCh (0.01-100 mM). producing esophageal lesions in puppies to study post-
The tension was verified considering difference in peak and the natal surgical correction techniques. On the other hand,
amplitude expressed in mN. Data are reported as means±SE.
knockout mice are useful for studying genetic influences.
DOXO: doxorubicin. *P,0.05, treated groups compared to
controls (two-way ANOVA, followed by a Student-Newman- The DOXO rodent model is the most useful for investigat-
Keuls test). ing the morphology and faulty organogenesis associated

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Esophageal and gastric contractility in esophageal atresia 461

Table 1. Values of Emax for CCh and KCl and values of EC50 for CCh in segments of the distal esophagus.

Control –EA
DOXO– DOXO+
+EA

CCh (n=11) (n=11) (n=8)

Emax (mN) 5.97 ± 0.58* 4.48 ± 0.34 4.42 ± 0.68
EC50 (nM) 190 [96-379] 228 [125-418] 439 [206-936]
KCl (n=5) (n=7) (n=7)
Emax (mN) 1.31 ± 0.14 1.27 ± 0.42 1.21 ± 0.20

Data are reported as means±SE or geometric mean [95%CI]. DOXO– –EA: doxorubicin without
esophageal atresia; DOXO+ +EA: doxorubicin with esophageal atresia; CCh: carbachol; Emax: maximum
effect values in mN; EC50: half-maximal effective concentration. * P,0.05 (two-way ANOVA, Student-
Newman-Keuls test).

with gene-expression regulation in EA. Even though the intrinsic nerve complex size and limited distribution and
anomaly is secondarily induced by an external agent (a immaturity of nerve tissue at the distal esophagus tissue,
teratogenic drug) and its incidence in treated fetuses is especially where there is an association with TEF (4,5).
variable (which necessitates a higher number of animals), Midrio et al. (21) demonstrated that in neonates with EA/
the model is clinically relevant with regard to phenotypic TEF, there is a lower density of the interstitial cells of Cajal
similarity. In addition, the rat model provides fetal samples at the tip of the trachea.
that can be macroscopically evaluated (13,17,18). With the goal of elucidating the pathophysiology of
With the intent of clarifying the primary etiology of this motor dysfunctions, in vitro experimental studies may
anomaly, the DOXO experimental model has shown that provide a way to explain this phenomenon at the tissue
the length of the intra-abdominal esophagus is shorter, level. However, few studies that employed the methodol-
but the size of the esophageal hiatus is larger, than in ogy of the present study are available, in particular
controls (19). Using the same model, Qi et al. (20) found exploiting DOXO teratogenicity and analyzing the contrac-
fewer extrinsic innervations with respect to both of the tility of the esophagus and stomach of rat fetuses (11,12).
recurrent laryngeal nerves, deviation of the left vagus In the studies published by Tugay et al. (11,12), the effects
from its normal course to form a single nerve trunk on the of specific agonists such as CCh, serotonin, and isoproter-
right side of the esophagus, and relatively few branches enol were evaluated, and the effects of unspecific
from the single vagal nerve trunk. compounds such as KCl and papaverine on smooth
Diverse studies have correlated primary motility muscle cells were also addressed. No significant differ-
dysfunction with intrinsic innervation alteration, including ences were noted in relation to CCh-elicited contractile

Figure 2. Comparison of the effect of KCl on the distal Figure 3. Comparison on the effect of carbachol (CCh) on the
esophagus of fetus with esophageal atresia (EA). The graph gastric fundic strips of fetus with EA. The graph shows the mean
shows the mean tension of the distal esophageal strips in tension of the gastric fundic strips in response to increasing
response to increasing concentrations of KCl (10-100 mM). The concentrations of CCh (0.01-300 mM). The tension was verified
tension was verified considering difference in peak and the considering difference in peak and the amplitude expressed as g
amplitude expressed in mN. Data are reported as means±SE. (force)/mg (tissue mass). Data are reported as means±SE.
DOXO: doxorubicin. There were no statistical differences in DOXO: doxorubicin. There were no statistical differences in
contractile activity in response to KCl comparing treated groups contractile activity in response to CCh comparing treated groups
to controls (P.0.05, two-way ANOVA, followed by a Student- to controls (P.0.05, two-way ANOVA, followed by a Student-
Newman-Keuls test). Newman-Keuls test).

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462 F.A. Capeto et al.

Table 2. Values of Emax and the EC50 for carbachol (mM) in gastric fundic strips.

Control DOXO–
–EA DOXO+
+EA

CCh (n=7) (n=12) (n=6)

Emax 0.084 ± 0.016 0.068 ± 0.006 0.070 ± 0.022
EC50 2.17 [1.03-4.58] 1.47 [0.83-2.61] 3.26 [1.90-5.60]

Data are reported as means±SE or geometric mean [95%CI]. DOXO– –EA: doxorubicin without
esophageal atresia; DOXO+ +EA: doxorubicin with esophageal atresia; CCh: carbachol; Emax: maximum
effect values in g/mg tissue mass; EC50: half-maximal effective concentration.

response in lower esophageal strips from fetus pretreated is reasonable to hypothesize that pretreatment with DOXO
with DOXO, despite the fact that a significantly lower did not interfere with the capacity of the esophageal cells to
capacity of muscle relaxation in response to serotonin was recruit VOC channels in inducing motility. These findings
noted in this tissue (11). corroborate those of Tugay et al. (11).
In the present study, the muscarinic agonist CCh Few published studies have analyzed gastric motility in
elicited concentration-dependent contraction in esopha- neonates with EA. Some have identified antral hypomotility
geal strips, even in tissues from the DOXO group. and reduced gastric emptying by utilizing electrogastrog-
However, esophageal strips of animals treated with raphy, gastric cintilography, and manometry, but such
DOXO demonstrated a smaller maximum force of con- events were not directly related to GERD episodes (24,25).
traction than the control group. The decrease in the This renders the etiologies of these alterations and their
pharmacological capacity of CCh to produce a contractile roles in gastric motility obscure. However, it is debatable
response does not appear to be due to EA development. whether the intrinsic nervous system of the stomach is also
Notably, both groups subjected to DOXO (with or without affected in cases of embryologic EA or if such alterations
EA) showed reduced contractility in response to choliner- are consequential to surgical interventions that involve
gic stimulus. This difference may be due to treatment with gastric tractions. For instance, Zhou et al. (19) found that
DOXO, and it was not extended to gastric tissues the EA fetuses of DOXO-treated rats possessed smaller
because fundic rings reacted similarly to CCh-induced stomachs than controls.
stimulation, as discussed below. With regard to CCh stimulation, we did not find any
It is worth noting that Qi et al. (22) employed a similar significant difference in contractility profiles when compar-
model and detected reductions in the density and ing the fundic strip responses of the three groups.
distribution of the myoenteric plexus, specifically regard- However, Tugay et al. (12) demonstrated that stomach
ing vasoactive intestinal peptide- (VIP-) and substance strips of rat fetuses with EA due to DOXO exhibited
P-producing neurons in DOXO pretreated animals. Such significant differences in contractility. While the contractile
findings revealed the deleterious effects of DOXO in the response to CCh was increased, the contraction induced
intrinsic innervations of the esophagus, which are able to by serotonin was blunted. This difference may be due to
change the mechanisms of signal transduction in affected methodological variability between the two studies. Our
tissues independently of morphological alterations due to isolated fundic preparations were mounted after obtaining
its teratogenicity. Notwithstanding, it is important to continuous circular muscle strips in the form of tissue
mention that while Tugay et al. (11) utilized whole-mount rings. In contrast, Tugay et al. (12) adopted the method
preparations that allowed contractile recordings of the suggested by Vane (26) and registered longitudinal
longitudinal smooth muscle layer of the lower esophagus, contractions of the gastric fundus. This difference raises
we employed esophageal strips arranged to record the issue that in the gastric fundus, contrary to what is
circular smooth muscle contractions. Thus, it is possible seen in the distal esophagus, the longitudinal layer is
that the decreased responsiveness of the distal esopha- more responsive to cholinergic stimulation (12).
gus to cholinergic stimulus may be restricted to functional The DOXO treatment regimen was based on a previous
changes in the circular smooth muscle layer. protocol that induced EA in 91% of fetuses (13). The two
In our studies, the KCl concentration-effect curves of the studies published by Tugay et al. (11,12) used a very
three groups were not significantly different in esophageal similar dose (2 mg/kg for the same two gestational days)
strips. Unlike CCh, which causes contractions mainly after and only reached rates of 42.85% and 57.69% of fetuses
intracellular release of Ca2+ +
stores and subsequent influx with EA. Notably, our rate was just 10.36%. It is possible
from the extracellular medium, KCl-induced contraction that the use of different rat strains may have contributed to
results from its depolarizing properties on the cell mem- variations among the studies. While the present approach
brane that stimulate the opening of voltage-operated Ca2+ +
was developed based on Wistar rats, other studies (11-13)
(VOC) channels and Ca2+ +
influx into the cytosol (23). So, it utilized Sprague Dawley rats. Although the teratogenic

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Esophageal and gastric contractility in esophageal atresia 463

effect of DOXO appeared less toxic, this dose was Acknowledgments

sufficient to induce EA and inhibit the in vitro contractile
behavior of esophageal strips in response to CCh. Despite The authors would like to thank Armênio A. Santos
this, we acknowledge that more studies are needed to and Ronaldo A. Ribeiro for their valuable and constructive
identify which factors contribute to functional alterations. suggestions regarding this research. This study was
Correlations of in vitro results with detailed histological supported by CNPq (#2011/163018-1 and #2009/
evaluations of the esophageal and gastric tissues may 479853-6).
improve our understanding about motility in EA.

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