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The Laryngoscope
C 2016 The American Laryngological,
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Rhinological and Otological Society, Inc.
Are the Interarytenoid Muscles Supplied by Branches of Both the
Recurrent and Superior Laryngeal Nerves?
Ar
an Pascual-Font*, PhD; Luis Cubillos*, MS; Teresa V
azquez, PhD; Steve McHanwell, MD;
Jose R. Sa~
nudo, MD; Eva Maranillo, MD
Objectives/Hypothesis: It has been generally accepted that the branches of the internal branch of the superior laryngeal nerve to the interarytenoid muscle are exclusively sensory. However, some experimental studies have suggested that
these branches may contain motor axons, and therefore that the interarytenoid muscle is supplied by both the superior and
recurrent laryngeal nerves. The aim of this work was to determine whether motor axons to the interarytenoid muscles are
present in both laryngeal nerves.
Study Design: Basic research.
Methods: Twelve human internal branches of the superior laryngeal nerve were dissected, and its branches to the interarytenoid muscle were removed and processed for choline-acetyltransferase immunohistochemistry, a method not used previously in studying the nerve fiber composition of the laryngeal nerves.
Results: The internal branch of the superior laryngeal nerve divided into two to five branches to the interarytenoid
muscle. All branches contained motor axons, with the proportion of motor axons varying from 6% to 31%.
Conclusion: The present study confirms that the internal branch of the superior laryngeal nerve provides a motor
innervation to the interarytenoid muscles.
Key Words: Internal laryngeal nerve, internal branch of the superior laryngeal nerve, recurrent laryngeal nerve, superior laryngeal nerve, internal branch, ChAT, larynx, motor fibers.
Level of Evidence: N/A.
Laryngoscope, 126:1117–1122, 2016
INTRODUCTION
In humans, the internal branch of the superior laryngeal nerve (ibSLN) divides into three terminal branches.
The posterior branch, called the ramus communicans,
runs over the interarytenoid muscle and divides into a
variable number of branches that pierce it.1–3
Traditionally, it has been thought that the sole
source of motor innervation to the interarytenoid
muscles (oblique and transverse) is from the recurrent
laryngeal nerve (RLN). However, these muscles also
receive a variable number of branches from the ibSLN,
which can be observed entering the muscular bellies.
These branches have always been considered as being
From the Department of Human Anatomy and Embryology I,
School of Medicine, Complutense University of Madrid (A.P–F., L.C., T.V.,
J.R.S., E.M.), Madrid, Spain; and the School of Medical Education, Newcastle University (S.M.), Newcastle, United Kingdom
Editor’s Note: This Manuscript was accepted for publication April
20, 2015.
This work was supported by a grant FIS10–02721 (Fondo de
Investigaciœn Sanitaria) of the institute of Health Carlos III (ISCIII)
Institute of Health Carlos III (ISCIII) of the Spanish government and by
funds obtained through postgraduate training courses by the
UCM920547 Group. The authors have no other funding, financial relationships, or conflicts of interest to disclose.
*Ar
an Pascual-Font, and Luis Cubillos, contributed equally to this
work.
Send correspondence to Eva Maranillo, Departamento de Anatomia y Embriologia Humanas, Facultad de Medicina, Universidad Complutense de Madrid, Ciudad Universitaria s/n, 28040 Madrid, Spain.
E-mail: [email protected]
DOI: 10.1002/lary.25375
Laryngoscope 126: May 2016
exclusively sensory, either to the mucosa of the larynx or
proprioceptive to the muscles themselves or to both
structures.4–9
However, there have been several anatomical studies
in humans that suggest the ibSLN may provide a secondary
motor innervation to the interarytenoid muscles, and therefore that these muscles receive a dual motor innervation
from both the RLN and the ibSLN1,10–13 (Table I).
A variety of methods have been employed in attempts
to determine the function of the nerve fibers in the ibSLN
that enter the interarytenoid muscles. A significant number of studies have employed dissection either with or
without prestaining of the nerve fibers using Sihler’s
stain35–7,9–16 (Table I). Other studies have employed a
variety of histological methods to determine the functional
nature of these nerve fibers, which have included silver
impregnation, gold chloride stain, Wrete impregnation,
Romanes’ method, acetylcholinesterase stain, and horseradish peroxidase labeling7,8,10–12,17,18 (Table II). Other
techniques that have been used are electromyography or
studying the morphometry of the nerve.7,19–22 However,
none of the studies employing the methods described
above have produced results conclusive to establishing
whether any of the ibSLN fibers that enter the interarytenoid muscles are motor in function.
This article reports the results of a study employing
an immunohistochemical method to investigate whether
choline acetyltransferase (ChAT) is present in these
ibSLN fibers. This technique selectively labels motor
axons by using a primary antibody against ChAT, the
Pascual-Font et al.: Neural Sources for the Interarytenoid Muscle
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TABLE I.
Anatomical Studies on the Innervation of Interarytenoid Muscle by Internal Branch of the Superior Laryngeal Nerve (ibSLN) in Humans.
Number of ibSLN
Author (date)
Number of Branches to
Interarytenoid
Method of Study
Right
Left
Right
Left
Dilworth (1921)1
Dissection
33
33
1–2
1–2
Lemere (1932)5
Todd (1938)10
Dissection
Dissection
12
96
12
96
Winckler (1948)6
Dissection
Williams (1951)7
Vogel (1952)11
Dissection
Dissection
30
16
30
16
Gupta et al. (1959)12
thi (1962)13
Re
Dissection
25
25
Dissection
Dissection
Not specified
11
11
Rueger (1972)8
Not specified
Not specified
3–8
2
Sensory 1 Motor
Sensory
Sensory 1 Motor
2
Not specified
Not specified
1–2
Function
Sensory
Sensory Proprioception
Sensory 1 Motor
1–2
Sensory 1 Motor
Not specified
0–3
0–3
Sensory 1 Motor
Sensory
Sanders et al. (1993)14
Sihler’s stain
10
10
Mu et al. (1994)15
Sanders and Mu (1998)16
Sihler’s stain
Sihler’s stain
10
5
10
5
2–4
2–3
Maranillo et al. (2005)3
Dissection
75
75
1–4
Olthoff et al. (2007)9
Dissection
30
30
Not specified
2–4
2–3
1–4
Multiple
Not discernible
Not discernible
Not discernible
Not discernible
Sensory
ibSLN 5 superior laryngeal nerve.
enzyme responsible for synthesis of the neurotransmitter
acetylcholine, which is the neurotransmitter released at
the motor neuron terminals.23–25
MATERIALS AND METHODS
Six human larynges (12 ibSLN) without known laryngeal
pathology were obtained from necropsies (4 males and 2
females, age range from 69–93 years), from cadavers that were
donated to the Human Anatomy and Embryology Department
of the Faculty of Medicine, Complutense University of Madrid,
Spain, in accordance with the legal procedures established for
body donation in Spain. The larynges were fixed in 10% formalin and were microdissected by means of a surgical Zeiss-OPM1
microscope (Carl Zeiss AG, Oberkochen, Germany) using 43 to
63 magnification. The number and distribution of branches
innervating each interarytenoid muscle were studied.
The ibSLN was sampled at three sites. A segment of the
posterior terminal branch of the ibSLN, the ramus communicans,
and the collateral branches entering into the interarytenoid
muscles were removed, embedded in paraffin, and serially sec-
tioned at 10 lm. The segment of the posterior terminal branch of
the ibSLN was next to its origin from the main nerve trunk. The
piece of the ramus communicans was at the level of the superior
third of the posterior cricoarytenoid muscle, and pieces of the collateral branches were immediately before the point at which they
pierced the interarytenoid muscle.
After treatment with sodium citrate buffer (10.2 mM, pH
6.1) for 20 minutes at 958C for antigen retrieval,26 the nerve
sections were blocked with horse serum (Vector Laboratories,
Peterborough, UK) (10%) and Bovine Serum Albumin (BSA)
(Sigma-Aldrich, Steinheim, Germany) (3%) in Tris-phosphate
buffer (TBS) with 0.3% in (Merck, Darmstadt, Germany) for
1 hour at 48C. They were incubated for 3 days with goat antiChAT primary polyclonal antibody (ChAT; 1/75; AB144P; Millipore), to identify motor axons. The primary antibody was
diluted in TBS containing 0.3% Triton X-100, 5% serum. After
washes with TBS containing 0.3% Triton X-100 (7 3 10
minutes), sections were incubated for 24 hours at 48C with
biotin-conjugated horse anti-goat Immunoglobulin G antibody
(Vector Laboratories), diluted 1:200 in TBS with 0.3% Triton X100. To visualize the presence of ChAT, the ABC-peroxidase kit
TABLE II.
A Summary of Results of Histological Studies on Innervation of Interarytenoid Muscle by ibSLN in Humans.
Author (date)
Todd (1938)10
Williams (1951)7
Vogel (1952)11
Method
Number Interarytenoid Muscles
End-Plates From ibSLN
Function
Gold chloride stain
Wrete impregnation and
Romanes’ method
7
5
Yes
No
Sensory 1 Motor
Sensory Proprioception
Gold chloride stain
19
Yes
Sensory 1 Motor
Gupta et al. (1959)12
Carey’s gold chloride
method
2
Yes
Sensory 1 Motor
Rueger (1972)8
Freije et al. (1987)17
Gold chloride stain
Acetylcholinesterase stain
6
4
No
No
Sensory
Sensory
Wustrow et al. (1988)18
Horseradish peroxidase
Silver impregnation
Acetylcholinesterase stain
4
No
Sensory
ibSLN 5 superior laryngeal nerve.
Laryngoscope 126: May 2016
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Pascual-Font et al.: Neural Sources for the Interarytenoid Muscle
TABLE III.
Innervation of Interarytenoid Muscle From ibSLN (posterior branch
and ramus communicans). Distribution by Sides and Sex.
Larynx
Sex
Branches
Right ibSLN
Branches
Left ibSLN
Total
Branches
1
Male
1
1
2
2
Male
2
3
5
3
4
Male
Female
2
2
2
2
4
4
5
Male
2
1
3
6
Female
3
2
5
ibSLN 5 superior laryngeal nerve.
Fig. 1. Posterior view of the larynx. The pharyngeal mucosa has
been removed. White arrowhead: branches from ibSLN to interarytenoid muscle; black arrowhead: branches from RLN to internarytenoid muscle. ia 5 interarytenoid muscle; ibSLN 5 internal branch of
the superior laryngeal nerve; pca 5 posterior cricoarytenoid muscle;
pb 5 posterior branch of ibSLN; rc 5 ramus communicans (Galen’s
loop); rln 5 recurrent laryngeal nerve. [Color figure can be viewed in
the online issue, which is available at www.laryngoscope.com.]
with Diaminobenzidine (Vector Laboratories) was used. Slides
were dehydrated through a series of alcohols and mounted in
DPX (Sigma Aldrich, St. Louis, MO). All of the samples were
subjected to the immunohistochemical procedure in pairs, with
one sample of each pair not incubated with the primary antibody to act as a negative control.
The samples were photographed at 2003 to 4003, and
analysis of the fibers was carried out manually with ImageJ
1.44p software (National Institutes of Health, Bethesda, MD).
Each sample was quantified, taking as a reference the corresponding negative control of each pair to eliminate any nonspecific staining of nerve fibers. Nonmyelinated nerve fibers of less
than 1.5 mm were excluded from the counts to ensure that type
C fibers were not included in the final totals of nerve fibers
counted.27 The mean and standard deviation (SD) of the number of ChAT-positive axons, as well as the total number of axons
in the main trunk and in each branch, were calculated.
RESULTS
In humans, the ibSLN divides into three terminal
branches. The posterior branch courses over the interarytenoid muscle and connects with the RLN over the surface of the posterior cricoarytenoid muscle via a branch
called the ramus communicans (Fig. 1).
The posterior branch and the ramus communicans
on each side divded into a variable number of branches
that entered the muscular belly (Fig. 1) (Table III).
Laryngoscope 126: May 2016
The motor branch of the RLN to the interarytenoid
muscle ascended deep to the belly of the posterior cricoarytenoid to reach the interarytenoid muscle at the level of
the superior border of the cricoid cartilage (Fig. 1).
ChAT-positive axons were observed in all the segments of the ibSLN that were examined (Table IV).
These axons were distributed in a random way throughout all of the nerve branches that were studied (Fig. 2).
The number of ChAT-positive axons that were presumptively identified as motor axons in each of the different branches of the ibSLN innervating the
interarytenoid muscles can be summarized as follows:
The posterior terminal branch of the ibSLN was found
to contain an average of 290 6 225 (SD) ChAT-positive
axons (Fig. 2A). The mean total number of axons in the
nerve was 1967 6 995; therefore, these ChAT-positive
axons represented 15% of the axonal population present
in the nerve. The number of branches of the ibSLN varied from one to three, although it was only in a minority
of specimens that three branches of the ibSLN were
found to be entering the interarytenoid muscle. Consequently, the total number of axons in all three branches
was calculated. The average total number of motor
axons in those branches of the nerve was 342 6 159, representing 17% of the axonal population (Fig. 2C, D). The
ramus communicans was found to contain an average of
127 6 101 ChAT-positive axons out of a total axonal population of 995 6 649 axons, representing 12% of the total
axonal population (Fig. 2B).
DISCUSSION
The precise details of nerve supply of the interarytenoid muscles and how the motor supply to the muscle
is distributed in humans is an aspect of the neuroanatomy of the larynx that remains unclear despite numerous attempts at resolving this issue.
There are a number of experimental studies using
animal models that have investigated the contributions
of the recurrent and superior laryngeal nerves to the
innervation of the intrinsic laryngeal muscles.28–30 However, these studies only examined the innervation of cricothyroid, thyroarytenoid, or posterior cricoarytenoid
muscles. Furthermore, there are anatomical differences
between the intrinsic laryngeal muscles in rat and
mouse, for example, compared to humans. In the mouse,
interarytenoid muscles are not present (although an
Pascual-Font et al.: Neural Sources for the Interarytenoid Muscle
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TABLE IV.
Distribution of Motor Axons in ibSLN.
ibSLN
PB
B1
B2
B3
Total (B1 1 B2 1 B3)
RC
1 right
5.90% (75/1271)
9.98% (53/531)
–
–
9.98% (53/531)
6.06% (20/330)
1 left
2 right
8.40% (78/929)
8.21% (168/2047)
14.43% (58/402)
8.80% (33/375)
–
4.01% (16/399)
–
–
14.43% (58/402)
6.33% (49/774)
12.37% (24/194)
5.58% (60/1075)
9.81% (173/1763)
10.08% (286/2837)
11.42% (81/709)
10.17% (540/5309)
not assessable
7.39% (180/2435)
5.37% (164/3056)
8.27% (123/1488)
12.82 (140/1092)
–
–
22.86% (469/2052)
31.01% (431/1390)
13.20% (334/2530)
8.33% (100/1200)
2 left
26.44% (87/329)
3 right
3 left
7.57% (207/2735)
8.37% (257/3069)
4 right
23.06% (712/3088)
25.33% (57/225)
16.20% (144/889)
–
28.83% (352/1221)
4 left
5 right
23.84% (711/2982)
16.20% (353/2179)
13.53% (131/968)
17.14% (413/2409)
21.67% (91/420)
21.77% (133/611)
–
–
18.08% (546/3020)
7.33% (304/4148)
12.14% (75/618)
10.52% (100/951)
9.55% (127/1330)
5 left
21.61% (424/1962)
28.83% (352/731)
–
–
7.72% (303/3923)
20.39% (210/1030)
6 right
6 left
11.82 (121/1024)
14.64% (292/1994)
25.49% (157/616)
31.05% (227/504)
20.62% (214/1038)
30.96% (204/659)
24.62% (98/398)
–
15.99% (222/1388)
18.04% (201/1114)
23.61% (265/1118)
13.56% (77/568)
Mean
14.7% (290/1967)
16.4% (167/1228)
16.3% (150/1048)
18.0% (90/554))
16.6% (342/2434)
12.3% (127/995)
6 SD
7.4% (225/995)
9.0% (118/1016)
8.4% (77/779)
9.3% (12/244)
8.9% (159/1566)
5.5% (101/649)
B1–3 5 branches to interarytenoid muscle; ibSLN 5 superior laryngeal nerve; PB 5 posterior branch; RC 5 ramus communicans; SD 5 standard deviation.
anatomical counterpart is present), whereas in the rat,
an alar cricoarytenoid muscle and superior cricoarytenoid muscle can be found that are absent in
humans.31,32 Also, there are always uncertainties in
relation to precise muscle homologies between species,
meaning that any direct comparisons between apparently similar muscles always have to be made with caution. There are also significant differences in the
Fig. 2. Immunohistochemical labelling for the enzyme ChAT in axial sections of the nerves. The stain revealed the presence of motor axons
in all the samples. (A) Posterior branch of the internal branch of the superior laryngeal nerve. (B) Ramus communicans (Galen’s loop). (C, D)
Branches to the interarytenoid muscle. Arrowhead: an example of ChAT-positive axon (motor). Scale bars: 100 mm. ChAT 5choline acetyltransferase. [Color figure can be viewed in the online issue, which is available at www.laryngoscope.com.]
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Pascual-Font et al.: Neural Sources for the Interarytenoid Muscle
composition of the laryngeal nerves between rat and
human because afferent axons are absent from the RLN
in the rat.33
The total number of branches that enter the interarytenoid muscle has been reported as varying from four
to eight, with two branches contributed by the RLN and
two to six branches contributed by the ibSLN or by the
ramus communicans that joins the two nerves.3
Branches from RLN have been considered as exclusively
motor, whereas branches from the ibSLN have been
thought by some to be purely sensory4 or proprioceptive7,9 or by others to be mixed.1,10–13
A variety of methodologies have been employed in
attempting to resolve this issue (Table I). Dissection,
sometimes in combination with Sihler’s stain, has been
extensively used by many authors, but we consider these
methods to simply lack any degree of selectivity that
would allow one to determine the functional character of
axons that reach the interarytenoid muscle.14–16 The
interpretation of the results of any dissection studies is
further complicated by the fact of the existence of a complex neural plexus within the interarytenoid muscle,
which receives contributions from both the RLN and the
ibSLN. This means that it is simply not possible to be
certain that any axons observed within the muscle itself
have originated from the ibSLN or from the RLN.2
Studies using standard histological techniques,7,8,10–12
anterograde tracers,18 and histochemical methods17,18 also
fail to provide conclusive evidence in favor of one or other
hypothesis. Again, this is because of the above-mentioned
existence of this complex neural plexus, which prevents
the precise source of the axons within the muscle and ending as motor end plates to be identified as originating from
one laryngeal nerve or the other (Table II).
In the present study, axons within the branches of
the ibSLN were stained using an immunohistochemical
method to determine whether they contained ChATpositive axons. Significant numbers of ChAT-positive
axons were seen in all segments of the ibSLN that
entered the interarytenoid muscles.
Somatic motor axons express ChAT, but ChAT
expression is not confined to this functional category of
axons. ChAT expression can also be seen in the preganglionic axons of the autonomic nervous system, both
sympathetic and parasympathetic divisions; in all the
postganglionic parasympathetic fibers; and in the postganglionic axons of the sympathetic nervous system
innervating the sweat glands and blood vessels that provide a vascular supply to skeletal muscles.34–36
However, only large myelinated ChAT-positive axons
within the branches of the ibSLN, which were characterized as Aa motor axons, were included in the counts. The
smaller type C nonmyelinated fibers of the autonomic
nervous system were excluded. Furthermore, it was considered highly unlikely that any postganglionic sympathetic fibers to sweat glands would be included in the
counts because these are confined to skin, are not found
in the mucosa of the larynx, and are also nonmyelinated.
Thus, it seems possible to conclude that, using the criteria
outlined above, only somatic motor axons were included
in the counts of ChAT-positive axons.
Laryngoscope 126: May 2016
The finding in this study of motor axons in
branches of the ibSLN could also provide an explanation for a number of previously difficult-to-explain findings in cases of RLN palsies. In these cases, the
reports of the presence of motor-unit potentials in
denervated laryngeal muscles,37,38 the findings on the
position of the vocal folds by neurotization of the adjacent muscles,39 and the phenomenon of synkinesis40,41
could all be attributed to the presence of motor axons
in the ibSLN.
CONCLUSION
Based on our results and the considerations
described above, it can be concluded that the ibSLN does
contain a small but significant number of motor fibers
distributed to the interarytenoid muscles. This dual
innervation, previously described by other authors and
confirmed in the present study, needs be taken into
account for reinnervation and denervation procedures
and to understand the consequences of recurrent laryngeal nerve damage.
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Pascual-Font et al.: Neural Sources for the Interarytenoid Muscle