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Simulated Reflux and Laryngotracheal Reconstruction
A Rabbit Model
Jeffrey D. Carron, MD;
John H. Greinwald, MD;
James P. Oberman, MD;
Alice L. Werner, MD;
Craig S. Derkay, MD
Arch Otolaryngol Head Neck Surg. 2001;127:576-580.
ABSTRACT
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Objectives (1) To test the feasibility of a rabbit model using a pharyngostomy
tube to simulate gastroesophageal reflux and (2) to study the effects of gastroesophageal
reflux on laryngotracheal reconstruction using a new rabbit model.
Design Prospective randomized trial.
Subjects Thirty-three New Zealand white rabbits.
Interventions Anterior cartilage laryngotracheoplasty and pharyngostomy tube placement
into the pyriform sinus were performed in 33 rabbits, 22 of which are included
in this analysis. Beginning postoperative day 1, hydrochloric acid at a pH
of 1.5 with pepsin (n = 7) or at a pH of 4.0 with pepsin (n = 8) was irrigated
twice daily through the pharyngostomy tube to simulate gastroesophageal reflux,
and a control group received twice-daily isotonic sodium chloride solution
irrigations (n = 7).
Main Outcome Measures Specimens were scored by a pathologist masked to individual groups using
a newly modified inflammation scoring system. In addition, cross-sectional
areas of the cartilage grafts and subglottic airway lumina were compared.
Results Inflammation scores were significantly higher in rabbits receiving hydrochloric
acid and pepsin irrigations at a pH of 4.0 (P = .04)
but not in those in the pH 1.5 group. Cartilage necrosis was prominent in
all groups, and airway sizes and cross-sectional areas of the grafts were
not significantly different among the 3 groups.
Conclusions Cartilage necrosis is prominent during the early stages after laryngotracheoplasty.
Inflammation can be increased using hydrochloric acid and pepsin irrigations
but is difficult to predict based on this study. Although we confirmed the
feasibility of this model, further modifications of this study are proposed
to improve animal survival and data collection.
INTRODUCTION
GASTROESOPHAGEAL reflux (GER) in children is a significant health problem
that has been implicated in a variety of disease entities, including hoarseness,1 chronic cough,2 sinusitis,3 stridor or recurrent croup,2, 4
central or obstructive apnea,4 aspiration pneumonia,4 bronchospasm,4 and
idiopathic subglottic stenosis (SGS).5 Because
there has been difficulty in producing a reliable animal model, research on
the effects of GER on the airway has largely been limited to clinical observations.
In adults, treatment of GER has been shown to have a markedly beneficial
effect in the treatment of chronic cough, reflux laryngitis, globus pharyngeus,
and laryngeal and tracheal stenosis.6 Furthermore,
in adults and children, asthmatic attacks have been linked to the presence
of acid in the esophagus.3, 7 In
children with GER and respiratory symptoms, the severity of attacks has been
directly linked to the acidity of the gastric contents.7
Since the advent of mechanical ventilation in neonates, the number of
acquired cases of SGS after endotracheal intubation has grown significantly,
with reported rates ranging from 1% to 8% of neonates requiring prolonged
intubation.8 During the past 20 years, surgical
correction of SGS has focused on increasing the cross-sectional area via laryngotracheal
reconstruction (LTR) by placing anterior and/or posterior autogenous cartilage
grafts to expand the upper trachea and larynx.
The role of GER in the success of LTR remains controversial. Retrospective
studies by Yellon et al9 and Gray et al10 have suggested improved results after LTR with GER
treatment. However, a retrospective analysis by Zalzal et al11
showed no obvious benefit. Experimental studies6, 12, 13, 14
on dogs and pigs showed that gastric juices are deleterious to the healing
of injured mucosa in the subglottic airway and that SGS can be induced by
applying acid to injured mucosa. The main drawback of these experiments is
that they do not mimic the physiologic characteristics of GER.
The focus of this experiment was to study the fate of anterior cartilage
grafts placed in rabbits while simulating GER in a recently developed rabbit
model.15 After anterior laryngotracheoplasty
and pharyngostomy tube placement with postoperative acid exposure, a newly
modified histologic scoring system was used to grade the degree of inflammation
in addition to looking at cartilage resorption.
MATERIALS AND METHODS
All experiments were approved by the institutional animal care and use
committees at Eastern Virginia Medical School, Norfolk, and the Naval Medical
Center–Portsmouth, Portsmouth, Va.
Healthy male Pasteurella-free New Zealand white rabbits weighing approximately
2.5 kg (approximate age, 15 weeks) were preanesthetized intramuscularly with
acepromazine maleate, 0.75 to 1.0 mg/kg; xylazine hydrochloride, 3 to 5 mg/kg;
and ketamine hydrochloride, 35 to 44 mg/kg. Anesthesia was maintained with
inhaled isoflurane administered by cone mask. Endotracheal intubation was
avoided so that further subglottic injury would not confound results. Perioperative
combination trimethoprim and sulfamethoxazole was administered the morning
of surgery and for 3 days after surgery.
Surgery was performed as follows: Cartilage from the left costal margin
was harvested from an upper midline abdominal incision and placed in isotonic
sodium chloride solution (normal saline). Gastrostomy was then performed through
the same incision with a 20F Foley catheter brought out through a separate
incision. The abdomen was closed in layers with absorbable sutures (Figure 1).
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Figure 1. Intraoperative photographs. A,
Forceps under the pharyngostomy tube going into the left pyriform sinus, with
the thyroid cartilage retracted by a skin hook. B, Rabbit near the end of
surgery, with the abdominal incision closed and gastrostomy in place. The
graft has been sewn in place anteriorly, with the pharyngostomy tube tunneled
under the skin flap.
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Access to the larynx was obtained through a vertical midline neck incision.
The strap muscles were separated in the midline, and dissection was carried
down to the pretracheal fascia. The pharyngostomy catheter was composed of
polyethylene tubing (Intramedic; Clay Adams, Parsippany, NJ) (outside diameter,
0.95 mm), chosen for its tissue nonreactivity. The left pyriform sinus was
entered lateral to the thyroid ala, and the pharyngostomy catheter was secured
via a 4-0 silk purse-string suture. The catheter was then tunneled subcutaneously
and brought out posterolaterally, well away from the midline incision, and
anchored to the skin with a 2-0 nylon suture (Figure 1A-B).
An incision was made into the upper 3 tracheal rings and cricoid cartilages;
laryngotracheoplasty was performed using a spindle-shaped rib graft, attempting
to maintain a uniform size of 2.5 x 2.5 x 11.0 mm. Perichondrium
was preserved along the luminal surface of the grafts, which were sewn into
place using 6-0 polypropylene sutures. The strap muscles and skin were closed
with absorbable sutures. Small Penrose drains were occasionally placed if
an air leak was apparent.
After graft placement, endoscopy was performed by inserting a 4.0 ventilating
bronchoscope with an appropriately sized telescope into the supraglottic area.
The glottis was anesthetized with topical 1% lidocaine, and the position of
the pharyngostomy cuff and the patency of the tube were confirmed by flushing
saline through the tube. Subglottic luminal diameter in the anteroposterior
and transverse dimensions was measured using optical grasping forceps. Animals
were then placed in a nonrestrictive jacket to prevent chewing at the catheters.
Postoperative pain relief was provided by subcutaneous administration
of buprenorphine hydrochloride, 0.05 mg/kg, every 12 hours for 3 days. Animals
were offered rabbit chow and water ad libitum after surgery, with supplemental
feedings of "blenderized" rabbit chow and water given 4 times daily if animals
were unable to maintain adequate energy intake (5 g of chow per 100 g of body
weight per day) by postoperative day 4. Water was also given if inadequate
oral intake was noted (<5-10 mL per 100 g of body weight per day).
Pharyngostomy catheter irrigations were initiated on postoperative day
1 and continued for approximately 2 weeks. (For the sake of uniformity, the
protocol was shortened to 2 weeks because the catheter extrusion rate was
high at 2-3 weeks.) Rabbits were randomly assigned to 3 groups: those receiving
acid, pH 4.0, with pepsin, 0.3 mg/mL (group 1, n = 8); those receiving acid,
pH 1.5, with pepsin, 0.3 mg/mL (group 2, n = 7); and those receiving saline
(control group, n = 7). These hydrochloric acid concentrations are the same
as those used by Koufman6 and were prepared
in the same manner and mixed with purified porcine pepsin (Sigma-Aldrich Corp,
St Louis, Mo), 3900 U/mL. All animals were irrigated twice daily with solution,
1 mL/kg, and observed for swallowing response, coughing, tachypnea, stridor,
or other signs of respiratory distress. Cough response was graded using an
objective scoring scale developed by the authors (Table 1).
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Table 1. Cough Response Scoring System
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Animals that survived for 2 weeks were humanely killed via a lethal
dose of pentobarbital sodium (65 mg/kg intravenously), and their larynges
were harvested. Measurements at the narrowest point of the graft were taken
to calculate airway cross-sectional area. Larynges were then forwarded to
a pathologist (A.L.W.) masked to experimental groups. The degree of inflammation
for the graft was scored using a modification of the scale proposed by Koufman6 (Table 2).
Mean group inflammation scores were compared using the unpaired t test (1-tailed distribution).
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Table 2. Cartilage Graft Histologic Scoring System*
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The degree of cartilage graft resorption was examined in a manner similar
to that used by Cotton.8 Whole-mount photomicrographs
taken at the subglottic level were digitally scanned and analyzed on a desktop
computer using NIH Image 1.61 (National Institutes of Health, Bethesda, Md);
this program can calculate the area of any object outlined. Cross-sectional
areas of cartilage grafts were then computed, and mean cross-sectional areas
of cartilage grafts were then compared using 1-way analysis of variance, with
the Tukey-Kramer multiple comparison procedure to compare all possible pairs
of means.
Cross-sectional areas of the subglottic lumina were calculated from
measurements taken endoscopically immediately after surgery and directly at
the time the rabbits were killed. Cross-sectional area is calculated using
the formula for the area of an ellipse: area =  (A + B). Mean reductions
in cross-sectional areas were compared using 1-way analysis of variance as
well. Uniform error from the 2 different methods of measurement was assumed.
RESULTS
Pharyngostomy, gastrostomy, and LTR with rib graft were performed in
33 animals. One animal died of a pharyngeal leak on postoperative day 2. Seven
animals were killed prematurely: 2 on postoperative days 3 and 5 for severe
abdominal wall infection, 2 on postoperative days 7 and 10 for premature catheter
extrusion, 1 on postoperative day 3 for excessive pain and distress, 1 for
severe enterocolitis, and 1 for a gastrostomy leak and abscess on postoperative
day 10. In all, 3 animals were eliminated from data analysis for gastrostomy-related
problems, and 1 was eliminated for unilateral vocal cord paralysis and persistent,
severe coughing believed to be due to severe aspiration; its larynx was submitted
for histologic examination after death. This left 22 animals for analysis:
7 controls, 8 in group 1, and 7 in group 2.
Complications were mainly those listed in the previous paragraph; 2
animals that were excluded had their pharyngostomy tubes replaced on the first
and second postoperative days. Diarrhea occurred in 4 animals, and subcutaneous
emphysema occurred in 1. Another animal required operative replacement of
the gastrostomy tube on postoperative day 2 after chewing through it.
All animals except 5 (2 in group 1, 2 in group 2, and 1 control) resumed
full oral feeds within 1 week of surgery. The control animal that had difficulty
was later graduated to full oral feeds. When the graded responses of the animals
were compared on postoperative days 7 and 14, there was no significant difference
in the reaction among the groups.
Histologic scoring was done by a single pathologist (A.L.W.) masked
to the experimental groups. Mean ± SD inflammation scores are shown
in Figure 2. The mean inflammation
score for group 1 was significantly higher than that for the control group
(8.4 vs 6.3; P = .04), whereas the mean score for
group 2 was lower, but not to statistical significance (P = .15).
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Figure 2. Mean pathologist inflammation
scores by group. Group 1 received hydrochloric acid, pH 4.0, with pepsin;
group 2, hydrochloric acid, pH 1.5, with pepsin; and the control group, isotonic
sodium chloride solution. Asterisk indicates P<.05.
Error bars represent SD.
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Analysis of the cartilage graft showed extensive necrosis in all specimens.
The cross-sectional areas of the spindle-shaped grafts were not significantly
different among groups (P = .44). Units for area
measurement of the graft are not given because all images were uniformly magnified
with microscopy and photographing. Photomicrographs of the grafts are shown
in Figure 3A-B. In rabbits with
unilateral vocal fold paralysis, no cartilage elements were seen, and the
grafts were replaced with fibrous tissue (Figure 3C). Both of these animals were irrigated with 4.0-pH hydrochloric
acid solution with pepsin.
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Figure 3. A, Hematoxylin-eosin–stained
section of control group cartilage graft. Note the epithelial overgrowth (long
arrow) and the peripheral cartilage necrosis (short arrow) (original magnification
x40). B, Cartilage graft from a group 1 animal (hydrochloric acid, pH
4.0, with pepsin) showing necrosis and no epithelial overgrowth. In addition,
granulation tissue is seen at the edge (arrow) (original magnification x40).
(Some prolapse of the graft has occurred with tissue handling.) C, Airway
section from a rabbit that had a paretic vocal fold and was irrigated with
acid. There are no chondroidal elements seen in the cartilage graft, and its
framework has been replaced by fibrous tissue (original magnification x40).
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There was no significant difference among groups with respect to initial
airway size, final airway size, and reduction in airway size.
COMMENT
The question of the effects of GER on the success of LTR remains in
some dispute. Cotton,8 Yellon et al,9 and Gray et al10 maintain
that aggressive treatment of GER improves outcomes, whereas Zalzal et al11 suggest that there is no objective proof that GER
affects the ultimate success of LTR. These groups have published studies based
on retrospective reviews and clinical observations of patients treated in
a milieu of perioperative treatments, so the effects of antireflux treatment
are difficult to isolate. Further evidence for the importance of controlling
GER before LTR is presented in recent articles by Halstead16, 17
and Walner et al,18 which offer convincing
supporting evidence that the development of SGS is in many cases linked to
GER.
The lack of a reliable animal model remains an obstacle to GER research.
To date, the animal model that seems to most closely simulate GER was developed
by dividing the oblique fibers at the gastroesophageal junction in pigs; this
species was chosen because the muscular arrangement at this junction is identical
to that in humans.14 Although GER was documented
by extensive pH probe studies and esophageal manometry, this model has yet
to be duplicated and is not practical for widespread use.
The model proposed in this study is based on previous experiments by
Ludemann and others at McGill University.15
The major strength of the pharyngostomy model is the delivery of acid to the
hypopharynx, thus mimicking the effect of acid on the upper airway. Other
variables, such as reflex-induced bronchospasm, are not shown. Also, the infiltration
of acid into the subglottis and trachea cannot automatically be assumed with
intact laryngeal reflexes; thus, microaspiration is anticipated and relied
on to show a difference among groups.
Another obvious shortcoming of the presented model is that it does not
replicate the pathophysiologic characteristics of SGS and thus does not entirely
mimic an airway that receives a cartilage graft for SGS. Certainly, concentric
scarring and altered structural support in actuality affect the ultimate outcome.
The presence of the small pharyngostomy tube did not seem to impair
the rabbits' ability to eat and drink; if tube feeding was withheld, the rabbits
would generally resume their diet within 4 days. Three animals were eliminated
from data analysis for gastrostomy-related infections, and several chewed
through the tubes as well. Considering the fact that only 4 animals could
not resume full oral feeds and the extra time and complications associated
with operating on the stomach, it seems that gastrostomy is an unnecessary
component in this model.
We believe that the proposed scoring system, which was modified from
that designed by Koufman,6 is useful in evaluating
airway cartilage grafts in the early postoperative period. While measuring
the degree of polymorphonuclear cell infiltration, it also includes epithelial
regrowth as a factor for success. Also, glandular hyperplasia might not be
relevant during early healing phases.
The relative lack of differences among groups can be attributed to several
reasons. As mentioned earlier, we relied on microaspiration of the acid, which
is impossible to control, to incite subglottic inflammation. In addition,
because of the high catheter extrusion rate after 2 to 3 weeks, the postoperative
irrigation period was shorter than we had hoped. A longer period might have
better reflected healing, with more mucosal covering, scar tissue formation,
stenosis, and additional cartilage resorption.
Animals irrigated with the 4.0-pH solution (group 1) had higher inflammation
scores than those irrigated with the 1.5-pH solution (group 2). Because rabbits
receiving the stronger acid solution were not noted to cough more than the
others, a possible explanation for the lower inflammation scores in the 1.5-pH
group is that the more acidic solution encouraged an immediate swallow response
to clear the pharynx and allowed less time for microaspiration to occur. If
this model is to be used again, one possible way to counteract this response
might be to infuse the acid slowly with a pump over several hours. An interesting,
surreptitious finding is the extensive resorption of the grafts in the rabbits
with paretic vocal folds. Practically, this finding might be relevant to LTR
in children because the presence of an endotracheal tube for several days
after sugery can cause glottic incompetence and aspiration of secretions onto
the surgical site.
The degree of cartilage necrosis might seem surprising, but this has
been shown to be an expected phase of the healing process. In a study on LTR
in a rabbit model published by Jacobs and others,19
extensive cartilage necrosis followed by neochondrification occurred during
the 10 weeks after surgery. The cartilage remodeling had no apparent effect
on the outcome of the graft or the size of the airway lumen. This finding
supports those of other studies that have shown early necrosis and neochondrification8 and preservation of the graft size after long-term
healing.20, 21
CONCLUSIONS
Cartilage necrosis is prominent during the early healing stages after
laryngotracheoplasty. Inflammation can be increased by administration of hydrochloric
acid and pepsin but is difficult to predict; glottic incompetence most likely
exacerbates the inflammatory effects. Further studies are needed to determine
whether placing acid into the hypopharyngeal region reliably simulates the
effects of GER on airway surgery.
AUTHOR INFORMATION
Accepted for publication September 22, 2000.
Presented at the 15th Annual Meeting of the American Society of Pediatric
Otolaryngology, Orlando, Fla, May 17, 2000.
The views expressed in this article are those of the authors and do
not reflect the official policy of the US Department of the Navy, US Department
of Defense, or US government.
From the Department of Otolaryngology–Head and Neck Surgery,
Eastern Virginia Medical School, Norfolk (Drs Carron and Derkay); the Department
of Otolaryngology–Head and Neck Surgery, Naval Medical Center–Portsmouth,
Portsmouth, Va (Drs Greinwald and Oberman); and Anatomic and Laboratory Pathology,
Children's Hospital of the King's Daughters, Norfolk (Dr Werner). Dr Greinwald
is now with the Department of Pediatric Otolaryngology, Children's Hospital
of Cincinnati, Cincinnati, Ohio.
Corresponding author: Jeffrey D. Carron, MD, Department of Otolaryngology–Head
and Neck Surgery, Eastern Virginia Medical School, 825 Fairfax Ave, Norfolk,
VA 23507 (e-mail: jdcarron{at}yahoo.com).
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