Structure and function of the esophagus of the American alligator (Alligator mississippiensis)
1 Department of Biology, 257 South 1400 East, University of Utah, Salt Lake
City, UT 84112, USA
2 Department of Medicine and Pathology, Salt Lake City Veteran's Hospital,
500 Foothill Blvd, Salt Lake City, UT 84148, USA
* Author for correspondence (e-mail: uriona{at}biology.utah.edu)
Accepted 13 June 2005
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Summary |
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Key words: lower esophageal sphincter, peristalsis, animal model, alligator, esophagus
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Introduction |
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The aim of our study was to examine the structure and function of the
esophagus of juvenile American alligators (Alligator
mississippiensis) and to compare this anatomy and physiology with that of
typical mammalian models. We found that the underlying function and control of
the esophagus is similar in alligators and mammals (including humans) but that
the esophageal musculature is thicker and the strength of esophageal
peristaltic waves considerably stronger in the alligator when compared with
the human. Furthermore, regulatory responses of the lower esophageal sphincter
(LES) were larger by several orders of magnitude in alligators than in
mammals. These large responses and anatomical differences (e.g. the lack of a
mammalian-type diaphragm) may render the alligator a useful model species to
study the regulation of esophageal performance, particularly the LES.
Understanding the mechanisms required to coordinate esophageal function with
other organ systems is of clinical importance because of the high incidence of
the co-existence of gastroesophageal reflux disease (GERD) and respiratory
diseases (e.g. asthma, emphysema) (Harding,
1999; Theodoropoulos and
Ledford, 2000
).
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Materials and methods |
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Histology
One alligator (2.5 kg) was euthanized and immediately exsanguinated and
embalmed with 10% neutral-buffered formalin. Tissues were sampled from the
proximal (pharyngeal or cranial), middle and distal (gastric or caudal)
esophagus. Samples were embedded in paraffin and cut in longitudinal sections
4 µm thick. The sections were stained with hematoxylin and eosin. The
distal sample included the lower esophageal sphincter (LES). Samples were
analyzed using a light microscope. Photographs were taken with a digital
camera.
Esophageal peristalsis
A Polygraph HR (Medtronics, Minneapolis, MN, USA) water perfusion system,
inserted orally, with a four-port probe (each port 1 cm apart) was used to
measure peristaltic waves in the esophagus. Water flowed from the ports at 1
ml s1. The speed of propagation of each peristaltic wave was
determined as the time that the leading edge of each wave passed from the
proximal (pharyngeal or cranial end) to distal (gastric or caudal end) port (4
cm). Data were recorded with a Polygram Windows software package
(Medtronics).
Esophageal reflux
Two solid-state pH electrodes (Medtronics), inserted orally, were used to
measure the pH of the esophagus 3 cm proximal (craniad) to the LES and of the
stomach 3 cm distal (caudad) to the LES. Each probe was calibrated with pH 4
and 7 buffers prior to insertion into the animals. At the end of the
experiment, the probe was removed and the calibration checked using pH 4 and 7
buffers. After fasting for one week, the pH was monitored for a period of 24
h. The sensors were then removed for approximately 10 min while each of the
animals consumed a meal of mice weighing 3% of the alligator's mass. The
probes were reinserted and esophageal and gastric pH were monitored for 48 h
postprandially. A bout of reflux was defined as a drop in esophageal pH below
4.
Ventilation
Ventilation through a mask placed over the nares was measured with a
pneumotach (model 8311; Hans Rudolph, Inc., Kansas City, MO, USA). The mouth
was sealed except for a small port through which the pH electrodes and
pressure transducers were passed.
LES manometric technique
Two pressure transducers (model SPR-524; Millar MikroTip, Houston, TX, USA)
1 cm apart were attached to a pH electrode (Medtronics). The caudad pressure
transducer was adjacent to the pH sensor. PE 90 tubing was also attached 4 cm
craniad to the pH sensor in order to administer a bolus of water into the
esophagus. The sensors were slipped through the mouth and past the velum
palatinum, a transverse fold descending from the palate that completely shuts
off the oral cavity from the esophagus
(Reese, 1915), and into the
stomach. Pressure and pH measurements were made at 1-cm intervals while the
transducer was pulled cephalad until reaching the LES, at which time pressure
and pH measurements were made at 0.5-cm intervals. After passing through the
LES, measurements at 1-cm intervals were resumed. Once the position of the LES
was determined from the pressure profile, the transducers were returned to the
following four positions to collect data during both apneic and ventilatory
periods: (1) the distal pressure transducer was located in the stomach while
the proximal transducer was located in the LES, (2) both transducers were
positioned within the LES, (3) the distal transducer was positioned within the
LES whereas the proximal transducer was positioned in the esophagus and (4)
both transducers were positioned within the esophagus. A 2 ml intraesophageal
bolus of water was used to stimulate deglutition and relaxation of the
LES.
Data acquisition
For all experiments except the studies of peristalsis, analog signals were
converted to digital using a BioPac System (Goleta, CA, USA) and analyzed with
AcqKnowledge software (BioPac). Data were collected at 50 Hz.
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Results |
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Esophageal and gastric pH of fasting alligators
pH of the esophagus and stomach of fasting alligators (N=5) was
6.33±0.06 and 3.31±0.23, respectively.
Esophageal reflux
The percentage of time that esophageal pH was less than or equal to 5.5,
5.0, 4.5 and 4.0 is reported in Table
2 for both the 24 h preprandial period and the 48 h postprandial
period. One of five animals showed esophageal reflux where the pH dropped to
4.0 or less, which occurred for 11.3% of the postprandial period.
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LES, gastric and esophageal pressures, and ventilation
Nonventilatory, resting pressures of the LES were measured to be either
lower or within the range of normal human LES pressures (1.34.7 kPa).
During a period of ventilation, LES pressure increased significantly. A sample
of the data collected when both the proximal and distal pressure sensors were
within the LES is provided in Fig.
5, and these data illustrate the large increase in LES pressure
seen during ventilation. Fig. 6
illustrates data collected when the distal pressure transducer was within the
stomach and the proximal transducer was within the LES. In contrast to the
increase in pressure seen within the LES during ventilation, gastric pressure
declined during ventilation compared with apnea. Similarly, esophageal
pressures decreased rather than increased during ventilation compared with
apnea. Additionally, Fig. 6
illustrates that when a bolus of water was given during a ventilatory bout,
the reflex to swallow and relax the LES predominated over a rise in LES
pressure.
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Although a large rise in LES pressure (ranging from 200% to 3000%) was nearly always seen during ventilation, we did on rare occasions observe a bout of ventilation that was not accompanied by any increase in LES pressure.
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Discussion |
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The esophagus of alligators exhibited characteristic peristaltic waves, as found for humans. However, the mean velocity of esophageal peristalsis (0.56 cm s1) is considerably slower than that of humans (35 cm s1). Also, peak esophageal pressures were considerably higher in the alligator (mean of 19.3 kPa in the distal and 25.3 kPa in the proximal esophagus; Table 1) than in humans (typically 1.21.4 kPa in the distal esophagus and 0.91.1 kPa in the proximal esophagus).
The LES functions similarly in alligators and humans. For both, boli of water in the esophagus stimulate peristalsis and relaxation of the sphincter simultaneously. After a short period, the sphincter regains its tone, and pressure rises to levels equal to or greater than those observed before boli delivery and swallowing, as is observed with water boli in the human esophagus. We found episodes of reflux to be relatively rare in fasting alligators. As observed for mammals, the incidence of reflux increased in the postprandial period.
Coordination of respiration with LES function
Alligators experience large but variable increases in LES pressure during
bouts of ventilation compared with apnea. The increase in pressure generally
ranged from 200% to 3000%. However, on rare occasions, no pressure increase
was seen coincident with ventilation. Fluctuations in LES pressure during
apnea and ventilation have also been observed in anesthetized piglets
(Kiatchoosakun et al., 2002).
Kiatchoosakun et al. (2002
)
used hypoxia to stimulate respiratory rate and found a peak increase in LES
pressure of 49% (from a baseline of 0.65±0.09kPa to
1.0±0.12kPa). This increase in LES pressure was blocked by atropine,
indicating cholinergic control of LES tone. A decline in LES pressure
accompanied the development of apnea during a subsequent hyperoxic exposure.
These authors suggest that a loss of respiratory neural output might
contribute to the loss of LES tone. A cholinergic-dependent neuromuscular
control of the LES is also observed in humans. Thus, the changes in pressure
observed within the LES of the alligator may well be due to
cholinergic-dependent neural control.
Although the patterns for neural control of ventilation and LES tone appear to be similar in mammals and alligators, alligators may have certain advantages as a model organism to study this phenomenon for the following reasons.
(1) Because alligators lack a crural diaphragm, it is unlikely that LES
tone is greatly influenced by diaphragmatic contractions. The mammalian LES is
closely associated with the crural part of the diaphragm, and diaphragmatic
contraction exerts a sphincteric action on the LES (Mittal et al.,
1988,
1990
), although the
diaphragmatic contribution to LES pressure has been controversial.
Crocodilians have a muscle called the `diaphragmaticus' but it is not
homologous to the mammalian diaphragm. The crocodilian diaphragmaticus
attaches the liver to the pelvic girdle and the posterior-most gastralia and
facilitates inspiration; it does not insert on the esophagus
(Gans and Clark, 1976
;
Reese, 1915
). Yet some of the
muscle fibers of this diaphragmaticus connect with an aponeurosis that passes
over the upper border of the liver and binds the liver to the esophagus
(Reese, 1915
). Whereas a
contribution of the alligator diaphragmaticus to LES pressure seems unlikely,
it cannot be ruled out. The amphibian diaphragm, which is not homologous to
either the mammalian diaphragm or the crocodilian diaphragmaticus, originates
on the pelvic girdle (the ilium) and inserts on the esophagus at the level of
the LES. Contraction of the amphibian diaphragm increases LES pressure
(Pickering et al., 2004
;
Pickering and Jones, 2002
).
Thus, further research is warranted to fully address the importance of the
crocodilian diaphragmaticus to LES tone. Be that as it may, it is very clear
from the data that the rise in LES pressure during ventilation is not due to
thoraco-abdominal pressure fluctuations associated with ventilation. On the
contrary, gastric (see Fig. 6)
and proximal esophageal pressures decreased during bouts of ventilation
compared with apnea.
(2) Long apneas are natural for alligators. Respiration in reptiles is
characterized by intermittent bouts of ventilation and long periods of apnea
(Hicks, 1998). In the study by
Kiatchoosakun et al. (2002
),
consecutive hypoxia and hyperoxia induced apnea in only eight out of 12
piglets. The period of apnea was short (in the order of a few minutes)
compared with the natural apneas occurring in alligators, which at room
temperature can easily last for 2030 min (C.G.F., personal
observation).
(3) Alligators tolerate instrumentation extremely well, without the requirement of anesthesia, when instrumented through the oral cavity. It is desirable to avoid the use of anesthetics because they can interfere with smooth muscle tone and control. Furthermore, anesthetics can influence central nervous control of both ventilation and LES function.
(4) Alligators exhibit much greater magnitudes of response than mammals. While the maximum increase in LES tone during hyperventilation in piglets was 49%, we measured increases in LES pressure during bouts of ventilation of up to 3000% in the alligator.
Complex phenomena can often best be studied by a judicious choice of model organisms. For example, to study the regulation of capillaries, August Krogh, 1920 Nobel laureate in medicine, used the tongues of frogs, which are translucent and enabled him to make visual observations of small arteries, veins and capillaries. An understanding of evolution, excitable membranes and hox genes has benefited from the study of finches, squid giant axons and fruit flies, respectively. Gastroesophageal reflux disease is a complex phenomenon, with largely unknown etiology, but dysregulation of the lower esophageal sphincter may lie at the core of this common disease. Both humans and alligators share peristaltic-motor waves as a means of propelling foodstuffs distally and an LES that relaxes upon swallowing and prevents gastric acid from refluxing proximally. In addition, the clear relationship between ventilation and rise in LES pressure in both mammals and alligators indicates that reflexes exist to coordinate the gastrointestinal and pulmonary systems; however, this reflex is not yet fully understood in humans. The vastly greater magnitude of this reflex in alligators compared with mammals recommends them as a useful model system to study the regulation of LES function.
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Acknowledgments |
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