 

 

! LIBRARY
Michigan State
University

 

 

 

This is to certify that the
thesis entitled

IMPROVEMENTS TO THE NERVE MUSCLE PEDICLE
GRAFT TECHNIQUE FOR THE TREATMENT OF LEFT
RECURRENT LARYNGEAL NEUROPATHY IN THE HORSE

presented by

Philip Andrew Cramp

has been accepted towards fulﬁllment
of the requirements for the

 

 

 

 

MS. degree in Large Animal Clinical Sciences
T M
M Professor’s Signature
07/01/2009
Date

MSU is an Afﬁrmative Action/Equal Opportunity Employer

 

PLACE IN RETURN BOX to remove this checkout from your record.
TO AVOID FINES return on or before date due.
MAY BE RECALLED with earlier due date if requested.

 

DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

5’08 K:IProj/Aoc&PresIClRC/DateDue.indd

 

IMPROVEMENTS To THE NERVE MUSCLE PEDICLE GRAFT TECHNIQUE FOR
THE TREATMENT OF LEFT RECURRENT LARYNGEAL NEUROPATHY IN THE
HORSE
By

Philip Andrew Cramp

A THESIS

Submitted to
Michigan State University
in partial fulﬁllment of the requirements
for the degree of

MASTER OF SCIENCE
Large Animal Clinical Sciences

2009

L.

ABSTRACT
IMPROVEMENTS TO THE NERVE MUSCLE PEDICLE GRAFT TECHNIQUE FOR
THE TREATMENT OF LEFT RECURRENT LARYNGEAL NEUROPATHY
IN THE HORSE
By

Philip Andrew Cramp

Five larynges were harvested from horses free from upper airway disease. Using
increments of 0.98 N, a dead-weight force generator applied a force of 0 N through 14.70
N for 1 minute each to the leﬁ muscular process at O, 10, 20, 30, 40, 50, 60, and 70
degree angles. The rima glottis was digitally photographed one minute after each force
had been applied. Increasing force from O N to 14.70 N progressively and signiﬁcantly
increased the length of all lines and right to left angle quotient, indicating abduction.
Applying forces in the direction of O to 30 degrees, which correspond with the direction
of pull exerted by the lateral compartment of the cricoid arytenoideus dorsalis muscle,
resulted in a signiﬁcantly greater degree of laryngeal abduction.

Four horses with induced left laryngeal hemiplegia were treated with nerve
muscle pedicle graft. Two horses were controls and 2 were subjected to
electrostimulation of the ﬁrst cervical nerve. Inspiratory pressure measurements, upper
airway videoendoscopy on the treadmill and histology at post mortem were used to assess
degree of improvement and effect with or without electrostimulation. No conclusions

could be made due to technical complications.

DEDICATION

To my parents Dudley and Andrea Cramp, who have been a source of tremendous
support and have made it possible for me to realize my ambition of becoming a

veterinary surgeon.

iii

ACKNOWLEDGEMENTS

I would like to acknowledge Dr Fred Derksen for his advice and support. Throughout
this project he has kept me on the ‘straight and narrow’ introducing me to, and educating
me with the principles of scientiﬁc research. In particular I would like to thank him for
his enthusiasm even when things were not going well, his positive nature is a huge
resource. I would like to acknowledge Dr John Stick and Dr Ian Fulton for their surgical

expertise, advice and assistance.

This project would not have been possible without the Pulmonary Laboratory at
Michigan State University and I would like to highlight the inﬂuence and expertise of Dr
Robinson, who has provided a great deal of guidance throughout this project.
Furthermore, I would like to thank Cathy Bemey, Sue Eberhart-Wisner and Heather de-
Feijter Rupp for all of their technical assistance and support, without them this would not
have been possible. I would also like to thank Ashley and Jenny for their good sense of
humor at ﬁve o’clock in the morning helping me to electrostimulate the horses in the
depths of a Michigan winter. Drs Stephanie Valberg and Kurt Williams also deserve
thanks for their expertise in the histologic assessment of the nerves and muscles. I would
like to acknowledge the ﬁnancial support of the Freeman Fund and the Matilda R Wilson

Endowment Fund for funding this research.

iV

TABLE OF CONTENTS

LIST OF TABLES ............................................................................................................ Vii
LIST OF FIGURES ......................................................................................................... viii
KEY TO ABBREVIATIONS .............................................................................................. x
CHAPTER 1
INTRODUCTION ............................................................................................................... 1
CHAPTER 2
ANATOMY OF THE EQUINE LARYNX ......................................................................... 6
CHAPTER 3
EQUINE RECURRENT LARYNGEAL NEUROPATHY .............................................. 11
a. A Historical Disease. ..................................................................................... 11
b. The Present Day ........................................................................... 14
i. Incidence ....................................................................... 14
ii. Etiopathogenesis .............................................................. 15
iii. Diagnosis ....................................................................... 22
iv. Progression of Disease ....................................................... 29
CHAPTER 4
TREATMENTS FOR EQUINE RECURRENT LARYNGEAL
NEUROPATHY ..................................................................................... 31
a. Ventriculectomy ........................................................................ 34
b. Ventriculocordectomy ................................................................. 36
c. Laser vocal cordectomy, ventriculectomy and ventriculocordectomy ......... 39
d. Laryngoplasty .......................................................................... 42
i. Post operative complications of laryngoplasty ................................ 49
e. Partial arytenoidectomy ............................................................... 53
f. Nerve muscle pedicle graﬁ ........................................................... 57
CHAPTER 5

EFFECT OF MAGNITUDE AND DIRECTION OF FORCE ON LARYNGEAL
ABDUCTION: IMPLICATIONS FOR THE NERVE MUSCLE PEDICLE GRAFT

TECHNIQE .......................................................................................... 62
a. Introductions and Aims ............................................................... 62
b. Materials and Methods ................................................................ 63
c. Results ................................................................................... 68

(1. Discussion and Conclusions ......................................................... 68

CHAPTER 6

EFFECT OF ELECTROSTIMULATION ON UPPER AIRWAY FUNCTION IN
LARYGENAL HEMIPLEGIA AFFECTED HORSES TREATED WITH A NERE

NUSCLE PEDICLE GRAFT ..................................................................... 79
a. Introductions and aims ................................................................ 79
b. Materials and Methods ................................................................ 82
c. Results ................................................................................... 89
(1. Discussion and Conclusions ......................................................... 94

CHAPTER 7:

LESSON LEARNT: A LOOK TO THE FUTURE ........................................... 102

REFERENCES .................................................................................... 105

Vi

 

LIST OF TABLES

Table 1. ' Weight and volume of the left and right
cricoarytenoideus dorsalis muscle of the 4
horses95

Tabe 2. Mean neuromuscular histology score for
each muscle region analyzed ...................................................... 96

Vii

LIST OF FIGURES

Figure 1. Rostral View of larynx showing biomarkers

used to assess degree of left arytenoid abduction ........................ 65
Figure 2. Dorsal View of larynx showing direction of force

(0-70 degrees) placed on the muscular process of

the left arytenoid cartilage ................................................... 66
Figure 3a. Effect of force (N ewtons) and angle (degrees) on

the length of Line 1. O 0 degrees; I 10 degrees;
A 20 degrees; X 30 degrees; * 40 degrees; 0 50
degrees; + 60 degrees; and — 70 degrees ................................. 69

Figure 3b. Effect of force (N ewtons) and angle (degrees) on
the length of Line 2. O 0 degrees; I 10 degrees;
A 20 degrees; X 30 degrees; * 40 degrees; 0 50
degrees; + 60 degrees; and - 70 degrees ................................. 70

Figure 30. Effect of force (N ewtons) and angle (degrees) on
the length of Line 3. O 0 degrees; I 10 degrees;
A 20 degrees; X 30 degrees; * 40 degrees; 0 50
degrees; + 60 degrees; and — 70 degrees ................................. 71

Figure 3d. Effect of force (N ewtons) and angle (degrees) on
the length of Line 4. O 0 degrees; I 10 degrees;
A 20 degrees; X 30 degrees; >l< 40 degrees; 0 50
degrees; + 60 degrees; and — 70 degrees ................................. 72

Figure 4. Effect of force (N ewtons) and angle (degrees) on
Right to Left Quotient (RLQ). O 0 degrees; I 10
degrees; A 20 degrees; X 30 degrees; * 40 degrees;
0 50 degrees; + 60 degrees; and — 70 degrees ......................... 73

Figure 5. Effect of angle (degrees) on to Left Quotient (RLQ)
at a force of 14.7 Newtons. * Signiﬁcant difference

viii

Figure 6.

Firgure 7.

Figure 8.

from 10, 20, and 30 degrees ................................................ 74

Timeline of procedures performed throughout the
experiment ..................................................................... 84

Mean inspiratory pressure measurements (cmHZO)

for the 4 horses recorded at maximum heart rate on

the treadmill. 0 days is baseline prior to left

laryngeal neurectomy, then 30 days after leﬁ laryngeal

neurectomy and then every 14 days thereafter ........................... 90

A) Longitudinal histologic section of normal peripheral
nerve, Horse No. 3 Luxol Fast Blue (LFB) histochemistry.
The nerve is comprised of numerous individual myelinated
nerve ﬁbers. The blue represents the myelin sheaths
surrounding the nerve ﬁbers (arrow). Bar = IOOum.

B) Histologic cross-section of normal skeletal muscle

(left CAD), horse No. 3 Hematoxylin and eosin histochemisty.
The density, size, and shape of the individual myoﬁbers is
normal within the muscle. Note the normal peripheral
localization of the myoﬁber nuclei. Bar = 100nm.

C) Longitudinal histologic section of abnormal peripheral
nerve histology, Horse No. 1 Luxol Fast Blue (LFB)
histochemistry. There are no normal nerve ﬁbers present,
and no histochemically detectable myelin in the nerve.

The tissue is comprised of numerous linear bundles of
proliferating Schwann cells (‘Bungner’s bands, arrow).

Bar = 100nm. D) Histologic cross-section of abnormal
skeletal muscle (left CAD). Hematoxylin and eosin
histochemisty. There are numerous markedly angular,

and atrophic muscle cells (arrow) interspersed with scattered
hypertrophic myocytes with internal nuclei (arrowhead).

Bar = lOOum .................................................................. 97

ix

KEY TO ABBREVIATIONS

RLN Recurrent Laryngeal Neuropathy

LH Laryngeal Hemiplegia
CAD Cricoid Arytenoideus Dorsalis Muscle
NMP Nerve Muscle Pedicle

ILH Idiopathic Larygneal Hemiplegia
EMG Electrical Myography
CAL Cricoid Arytenoideus Lateralis Muscle
MRI Magnetic Resonance Imaging

CT Computed Tomography

NszAG Neodymium Yttrium Aluminium Garnet Laser

PDS Polydiaxonone Suture Material
N Newtons
RLQ Right-to-left Quotient
NMC Neuromuscular Compartment
i.V. Intravenous
i.m. Intramuscular
b.i.d. Twice daily

s.i.d. Once daily
NADH Nicotinic Acid

Adenine Dinucleotide Dehydrogenase

xi

1. Introduction

Recurrent laryngeal neuropathy (RLN) has a number of synonyms. It is also referred
to as idiopathic laryngeal hemiplegia, laryngeal paralysis and more colloquially as

“roaring”. For the purposes of this thesis, I shall be referring to this disease as RLN.

Recurrent laryngeal neuropathy is an important problem in the equine industry and is
one of the most common causes of poor performance (Dixon et al. 2001; Russell and
Slone 1994). The incidence of LH, as high as 35% in draft breeds (Brakenhoff et al.
2006), has been reported to range from 2.6 to 11% in lighter breeds (Lane 1987;

Morris and Seeherman 1990; Raphel 1982).

Whilst the exact etiopathogenesis of the disease remains elusive, the term RLN refers
to a unilateral (typically the left side) paralysis of the cricoid arytenoideus dorsalis
muscle (CAD) resulting from a neuropathy of the left recurrent laryngeal nerve
(Duncan et al. 1978; Duncan et al. 1974). During exercise, under the inﬂuence of
increased negative inspiratory pressure, the paralyzed left arytenoid cartilage cannot be
abducted and collapses into the airway, causing obstruction and noise production

(Derksen et al. 2001; Morris and Seeherrnan 1990; Shappell et al. 1988).

Regardless of the cause, treatment is required to improve athletic performance. A
multitude of treatments have been used including tracheostomy (Cook 1970)
ventriculectomy (Hobday 1935), ventriculocordectomy (Shappell et al. 1988),
arytenoidectomy (Haynes et al. 1984), laryngoplasty (Marks et al. 1970) and
neuromuscular pedicle graft (Ducharme et al. 1989; Fulton et al. 2003) alone or in
combination. Currently, laryngoplasty with or without a ventriculocordectomy is the
treatment of choice despite a high complication and failure rate. The reported
complications associated with laryngoplasty include nasal discharge of food, chronic
coughing, and prosthesis failure (Hawkins et al. 1997; Russell and Slone 1994).
Although efforts have been made to modify the technique, it still suffers from a
number of these potentially devastating complications (Rossignol et al. 2006;

Schumacher et al. 2000).

Laryngeal reinnervation is used to treat people with laryngeal denervation (Kingham et
al. 2006; Su et al. 2007; Tucker 1989; Tucker and Rusnov 1981). Fulton et al. (1991)
described the use of the ﬁrst cervical nerve that supplies the omohyoideus muscle in a
neuromuscular pedicle (NMP) graft technique in horses with laryngeal hemiplegia
(Fulton et al. 1991; Fulton et al. 2003). The technique demonstrates the same level of
success as the laryngoplasty but suffers from fewer complications and is a more

physiologic treatment.

Three to 4 pedicles are usually available for transplantation in an individual horse, but

the optimal location for placement of the pedicles within the CAD muscle is currently

unknown. Recently, an elegant study described the neuroanatomy of the equine CAD
muscle (Cheetham et al. 2008). There is a medial and lateral neuromuscular
compartment and the lateral compartment plays a greater role in abduction of the
arytenoid cartilage. Therefore, optimal placement of neuromuscular pedicle grafts
may improve surgical efﬁcacy, including improved laryngeal abduction and

shortening of the recovery time.

Regardless, the procedure has failed to gain widespread acceptance due to the long
recovery time associated with the process of reinnervation; currently it is believed that
about 9 months are required before a horse will be returned to normal respiratory
soundness for athletic endeavor (Fulton et al. 2003). It is important to try and develop

techniques to shorten this time period.

Electrostimulation of skeletal muscle after denervation has been studied extensively in
humans and laboratory animals (Gondin et al. 2006; Mafﬁuletti et al. 2006; Marqueste
et al. 2004; Poortmans and Wyndaele 2002; Vitenzon et al. 2005). Within 5 weeks,
denervated muscle has a dramatic loss of size and ability to contract and carry out its
function.‘ For example, within 5 weeks, denervated extensor digitorum longus muscles
of rats lose 66% of mass, 91% of force, and 76% of ﬁber cross-sectional area (Dow et
al. 2004). Electrostimulation of these muscles can completely prevent these
impairments (Dow et al. 2004). Atrophy of denervated muscle impairs its ability to
become reinnervated (Cole and Gardiner 1984; Fu and Gordon 1995); therefore the

process of electrostimulation may aid the reinnervation process. This possibility has

important implications in equine LH, where the denervated CAD muscle is responsible

for the loss of upper airway function.

The electrostimulation protocol, muscle ﬁber type, and species differences have
important effects on muscle responses to stimulation (Dow et al. 2004). Generally,
frequent submaximal stimulation encourages slow ﬁber type growth, while less
frequent maximal contractions encourage fast twitch ﬁbers, muscle ﬁber hypertrophy,
and faster force recovery. For example, in the denervated extensor digitorum longus
muscle of rats, a typical fast twitch muscle, near normal force was maintained with
100 contractions per day for 5 weeks, while 800 contractions per day caused muscle
damage (Dow et al. 2004). In another study involving denervated (for 6 to 24 weeks)
human quadriceps muscle, a mixed ﬁber type muscle, 80 contractions per week for 8
weeks were sufﬁcient to generate a 20% increase in muscle mass (Dudley et al. 1999).
The equine CAD muscle is a mixed fast (55%) and slow twitch (45%) skeletal muscle

(Hoh 2005), similar to the human quadriceps muscle (Green et al. 1999).

To assist the surgeon in choosing the optimal placement of nerve-muscle pedicle
grafts, one of the aims of my thesis research was to systematically determine force-
laryngeal abduction relationships at various locations within the CAD muscle. This
study provided information for a pilot study, which was designed to investigate if, in
horses with LH, postoperative electrostimulation of the reinnervated muscle allows
earlier return to athletic performance without risk of coughing, aspiration, and

respiratory infection.

In the remainder of this Thesis I shall review equine laryngeal anatomy and function,

RLN, its treatment and report on the ﬁndings of my research.

2. Anatomy of the Equine Larynx

The larynx is a complex anatomical structure that connects the pharynx to the
tracheobronchial tree. It is a short tube that is responsible for not only the transit of air
from the atmosphere and nasopharynx to the trachea and lungs, but it regulates the
volume of air inspired and expired, prevents aspiration during swallowing and also is
the organ responsible for phonation. It is composed of cartilage structures, ligament,
muscles and tendons as well as afferent and efferent nerves, which determine how this

complicated little organ will carry out its diverse responsibilities.

The larynx is situated ventrally and relatively superﬁcially (Getty, 1975). It is
positioned dorsal to the stemohyoideus and omohyoideus muscles, and ventral to the
pharynx and proximal extent of the esophagus. In a normal head carriage, the rostral
half of the larynx lies between the mandibular rami and it is ﬂanked on either side by
the stylohyoideus muscle and the digastricus muscle, the parotid salivary glands and
the linguofacial vein (Getty, 1975). The larynx is connected to the tongue and hyoid

apparatus via the basihyoid and thyrohyoid bones.

There are three unpaired cartilages that make up the framework of the larynx, namely;
the cricoid, the thyroid and the epiglottic along with three paired sets of cartilages,

namely; the arytenoid, the comiculate and the cuneiform cartilages, which are fused

with the epiglottic cartilage (Getty, 1975). The epiglottic cartilage is the most rostral
and is described as an oblanoceolate leaf (Getty, 1975. The base is referred to as a
small stalk that is embedded between the root of the tongue, the basihyoid, and the
body of the thyroid cartilage. Attached to either side of the base of the epiglottic
cartilage are the paired cuneiform cartilages. These are thin elastic bars that originate
at the base of the epiglottic cartilage and support the mucosal folds passing ﬁ'om the
epiglottis to the aytenoids (Getty, 1975). Moving caudally, the thyroid cartilage is the
largest of the laryngeal cartilages (Getty, 1975). It consists of two lateral plates that
fuse medially to a varying degree producing a slight prominence. It is related dorsally
to the base of the epiglottic cartilage via an elastic ligarnentous attachment. The
rostral cornu of the thyroid cartilage articulates with the thyrohyoid bone. The caudal
comu of the thyroid cartilage articulates with the cricoid cartilage, the ventral border
joins the body rostrally, and diverges caudally to form a triangle, which is covered by
the cricothyroid membrane (Getty, 1975). The Signet ring shaped cricoid cartilage is
the next structure encountered as we move caudally. The dorsal aspect of the cricoid
cartilage possesses a-median ridge and it is within the shallow concaves of the dorsal
surface that the cricoid arytenoideus dorsalis muscle is situated. The caudal aspect of
the dorsal cricoid cartilage is thin and overhangs the ﬁrst tracheal ring; the rostral
aspect is much thicker and possesses two facets for the arytenoid cartilage
articulations. Ventrally, there is attachment of the cricothyroid membrane and the
cricoarytenoideus lateralis (Getty, 1975). Caudally from the ventral aspect, the cricoid
cartilage is attached to the ﬁrst ring of the trachea by the cricotracheal membrane. The

paired arytenoid cartilages on either side of the rostral aspect of the cricoid cartilage,

just medial to the thyroid cartilage and are best described as pyramidal in form (Getty,
1975). Each arytenoid cartilage possesses three processes; a vocal process, which
attaches to the vocal cord; a muscular process, which extends laterally and is the site
of insertion for the cricoid arytenoideus dorsalis; and a comiculate process, which is
orientated caudomedially and forms the dorsomedial and dorsolateral boundaries of

the rima glottidis. The comiculate processes are also named the comiculate cartilages.

These cartilages are joined together by synovial diarthrodial joints (cricothyroid

articulations, cricoarytenoid articulations and throhyoid articulations) all of which
possess a thin synovial capsule. The cricotracheal ligament is elastic and attaches the
ﬁrst ring of the trachea to the cricoid cartilage, the cricothyroid ligament is elastic and
is found between the rostral border of the ventral aspect of the cricoid cartilage and the
caudal border of the lamina of the thyroid cartilage. The ventral part of this ligament
is the cricothyroid membrane. The cricoarytenoid ligament supports the ventral aspect
of the capsule and the transverse arytenoid ligament connects the dorsomedial angles
of the opposing arytenoid cartilages. The thyroepiglottic and hyoepiglottic ligaments
run from the base of the epiglottic cartilage to the inner surfaces of the thyroid
cartilages and to the basihyoid and lingual process of the hyoid bone respectively. The

vestibular and vocal ligaments run from the arytenoid cartilages ventrad.

The muscles of the larynx can be divided into two groups, namely intrinsic and
extrinsic (Getty, 1975). The extrinsic muscles of the larynx are made up of the

thyrohyoideus and hypoepiglotticus muscles, which receive their efferent nervous

supply from the hypoglossal nerveandthe stemothyrohyoideus and omohyoideus that
receive their innervation via branches of the ﬁrst and second cervical nerves. These
muscles are responsible for location and movement of the larynx in its entirety. The
intrinsic muscles consist of the cricothyroideus, cricoarytenoideus dorsalis,
cricoarytenoideus lateralis, arytenoideus transversus and the thyroarytenoideus. These
muscles are responsible to the movement of the cartilages of the larynx, below is a
description of their movement (taken from Getty (1975) in Sisson’s and Grossman’s

The Anatomy of Domestic Animals, Volume 1, page 509):

“The cricothyroideus tenses the vocal fold and ligament by approximating the ventral
parts of the cricoid and the thyroid cartilages, thereby increasing the diameter of the
rima glottidis. This action also has the effect of adducting the vocal folds. The
thyroarytenoideus, arytenoideus transverses, and the cricoarytenoideus lateralis
adduct the vocal processes of the arytenoid cartilages and narrow the rima glottidis.
The cricoarytenoideus dorsalis and the arytenoideus transverses abduct the vocal

processes. ”

The cricothyroideus muscle is supplied by motor ﬁbers in the external branch of the
cranial laryngeal nerve whereas all of the other intrinsic musculature is supplied by

motor ﬁbers in the recurrent laryngeal nerve (Getty, 1975).

The mucus membrane of the larynx is contiguous with the mucus membrane of the

laryngopharynx and the trachea (Getty, 1975). The aryepiglottic folds are formed by

the membranous reﬂection from the borders of the epiglottic cartilage. Within the
larynx, on either side of the laryngeal wall, the mucus membrane covers the vocal
ligament and the vocalis muscle forming the basis of the vocal cord. Furthermore, this
mucus membrane forms another fold as it covers the cuneiform cartilage, the
vestibular ligament and the underlying part of the vestibularis muscle, this is
commonly known as the vestibular fold. Lying between this fold and the vocal cord is

a 2.5-5 cm deep reﬂection known as the lateral ventricle, or laryngeal saccule.

The blood supply to the larynx arrives in the form of the caudal laryngeal arteries and
branches from the ascending pharyngeal arteries, which can arise from either the
cranial thyroid artery or the common carotid artery (Getty, 1975). The venous
drainage is into the external jugular vein. The lymphatic vessels draining the larynx

run into the retropharyngeal and cranial deep cervical lymph nodes (Getty, 1975).

10

3. Equine Recurrent Laryngeal Neuropathy

a. A Historical Disease

One of the ﬁrst complete reports on RLN was published in 1882 by Fleming, it
was entitled Laryngismus paralytica (“roaring”) and since then there has been
much in the literature about this common equine disease. However, in a review
article by MacQueen in 1896 reference is made to earlier reports of “roaring”
dating back to 1664 and 1807 (Dupuy). Indeed, in the later reference, an
association between degeneration of the muscles on the left side of the larynx and
horses that “roared” had been established. 80, even at that time it was recognized
by veterinarians that this disease arose from a paralyzed larynx that affected
ﬁrnction and produced a “roaring” noise. Furthermore, it was appreciated that this
“roaring” noise was a symptom of the disease and not in itself a disease. In
Flemming’s article in 1882 he makes mention of the observation made in 1866 by
Gunther that 96% of horses with clinical signs of chronic “roaring” had wasting of

the muscles of the left side of the larynx.

In 1905, Professor Wortley provided a review of “Roaring and Whistling”. He
noted that this was a symptom and that chronic roaring related to wasting of the

dilator muscles of the larynx. It was suggested that this would be the result of

11

strangles, inﬂuenza or some affection of the chest that results in damage and
paralysis of the nerve supply the affected muscles. He goes on to commentate on
the fact that it is invariably the left side (left recurrent branch of the pneumogastric
nerve) that is affected because it winds around the base of the aorta. Whilst
Professor Wortley could not discover any microscopic differences between nerves
he does make the observation that the right side is far less frequently affected.
Furthermore, he also notes that the disease is seldom seen in equidae less than 14
hands and that height and length of the neck are proportional to the risk of
acquiring the disease. This is a statement that echoed the sentiments Fleming
and McCall, who in 1889 noted that the large breeds such as saddle horses and
draught horses are most commonly affected and that it usually occurs in younger

horses (three to ﬁve years of age).

Despite the fact that F lemming (McCall, 1889) stated that during inspiration, the
left lateral ventricle ﬁlls with air and billows into the lumen of the larynx and that
during exercise the arytenoid cartilage may also contribute in affected horses
diagnosis was based on clinical observation. If noise was heard at exercise by an
experienced car it was considered to be diagnostic. Symptoms such as grunting
were a common ﬁnding among clinically affected roarers and something that horse
dealers became very familiar with. Often times, threatening an animal with a stick
to see if they grunt in fear, and pinching the throat to evoke a cough were used to
help diagnose the condition. Professor Wortley (1905) comments on the

characteristic nature of the cough. He states that the clinical “roarer” will have a

12

deep cough, which is a mixture of a groan and a cough, the subtlety of which can

be easily appreciated by an experienced listener.

Due to the fact that a number of celebrated racehorses were susceptible to this
disease, treatment was sought. Between the years of 1860 and 1906, a number of
surgical treatments were attempted. These included vocal cordectomy,
ventriculectomy and partial and total arytenoidectomy. Initially, these attempts
were unsuccessful due to infection and aspiration pneumonia and it was not until
1905 when Professor Wortley suggested that whilst arytenoidectomy cannot be
considered as a success it should also not be regarded as an unqualiﬁed failure.
However, at this time tracheostomy was still considered to be the treatment of
choice despite it’s recognized limitations of exuberant granulation tissue formation
and irritation. In addition to surgery, management in 1905 consisted of keeping
horses in high condition, the application of “counter-irritants” to the throat, such as
mustard or turpentine liniment, not to mention, of course, the early application of

nux vomica!

In 1906, Williams began treating horses with ventriculectomy and after ﬁve years
of modiﬁcations reported that 71% of roarers produced no noise after the
procedure. Sir Frederick Hobday reported on 405 cases in 1911 and in 1935 he
commented that 85-95% of roarers were returned to useful function after a
ventriculectomy, however only 20% produced no noise. So successful was the

procedure perceived to be that it’s popularity in the United Kingdom led the

13

ventriculectomy being known, as it is still today, as the “Hobday” procedure.
However, a treatment was born and from this point modiﬁcations continued and

improvements made culminating in the Laryngoplasty by Marks (1970).

b. The Present Day

i. Incidence

This has been covered in part in the Introduction section of this Thesis,
however, to reiterate; RLN is an important problem in the equine industry
and is one of the most common causes of poor performance (Dixon et al.
2001; Russell and Slone 1994). The incidence of LH, as high as 35% in
draft breeds (Brakenhoff et al. 2006), has been reported to range from 2.6
to 11% in lighter breeds (Lane 1987; Morris and Seeherrnan 1990; Raphel
1982). However, in the study by Brakenhoff et al. (2006) only 17% of
Clydesdales were affected compared with 42% and 31% for the Belgians
and Percherons respectively. This ﬁgure compares more favorably with
Goulden et al. (1985) who determined a prevalence of only 9% in a
Clydesdale population. Furthermore, Clydesdale horses did not have a
signiﬁcant association of disease occurrence with height, where as Belgian
and Percheron horses did (Brakenhoff et al., 2006), which suggests some
degree of breed variation. There is no known reason why this discrepancy

exists with Clydesdales, Brakenhoff et al. (2006) offered potential reasons

14

ii

to be associated with sample size and genetic differences among and within

each breed.

It is important to check ourselves at this point because RLN is disease of at
least four grades and the numbers listed above refer to grades III and IV,
which are severe forms of the disease. However, if one looks at the lesser
grades of laryngeal paralysis then it is a more generalized distribution of
the disease with 80-90% of horses are affected (Cook, 1988). Whilst this
piece of work does not stand up to scrutiny, mainly because diagnosis was
based on palpation alone, it does generate some provoking thoughts and we
should bear that in mind. However, for simplicity’s sake, RLN is a disease
that affects predominantly young (1-5 years) horses that are tall with long

necks but the disease is by no means limited to this conformation of horse.

Etiopathogenesis

This has remained a challenging and elusive concept since the disease was
ﬁrst reported over 300 years ago. Once the veterinary world agreed that
this disease was in fact the result of disease or damage affecting the
nervous supply to the cricoarytenoideus dorsalis muscle and that the
“roaring” sound was not a product of mucus secretion the etiology could be
sought. Essentially, there are two broad categories, the ﬁrst is iatrogenic

damage to the recurrent laryngeal branch of the vagus nerve. The second is

15

the more common and is an idiopathic neuropathy hence the alias of

idiopathic laryngeal hemiplegia (ILH).

There are a number of speciﬁc reports detailing cases where the actual

etiology of the RLN could be found and diagnosed, such as:

o Laceration involving the jugular vein resulting in right-sided
laryngeal paralysis (Gilbert, 1972).

o Thiamin deﬁciency resulting in left-sided laryngeal
hemiplegia (Loew, 1973).

o Arteriopuncture following catheter placement resulting in
right-sided laryngeal hemiplegia (Cramp, unpublished).

o A large abscess (Staphylococcus aureus) overlying the
caudal larynx and cranial trachea resulted in left-sided
laryngeal paralysis (Barber, 1981).

o Abscessation of the cranial mesenteric lymph nodes
resulting in left-sided laryngeal hemiplegia (Rigg et al.,
1985)

- Retropharyngeal abscessation leading to left-sided laryngeal
hemiplegia (Todhunter et al., 1985).

0 Guttural pouch mycosis with ipsilateral laryngeal

hemiplegia (Church et al., 1986).

16

0 Lead poisoning resulting in laryngeal hemiplegia in foals
(Willoughby, 1972)

0 Acute laryngeal paralysis was reported in Arabian foals
from organophosphate poisoning (Rose, 1978).

0 Oral haloxon administration resulting in bilateral laryngeal
hemiplegia in Arabian foals (Rose, 1981).

o Perivascular injection of an irritant drug (for example;
Phenylbutazone) resulting in left-sided laryngeal hemiplegia
(Goulden and Anderson, 1981).

o Bite wound to the neck leading to left-sided laryngeal
hemiplegia (Goulden and Anderson, 1981).

o Blunt trauma to the neck from running through a rope fence
(Goulden and Anderson, 1981).

o Hepatic dysfunction leading to bilateral laryngeal
dysfunction and paresis (Hughes et al., 2009).

0 Organophosphate poisoning whilst dosing with
contaminated mineral oil led to bilateral laryngeal
hemiplegia (Duncan and Brook, 1985).

o Laryngeal dysfunction was noted in 10 out of 11 horses that

were diagnosed with Australian stringhalt (Huntingdon et

al,1989)

However, in the majority of cases there is no obvious cause and in these

17

cases of idiopathic laryngeal hemiplegia there is no established
etiopathogenesis. There would almost certainly appear to be a hereditary
link and this has been recognized since the early 19008 but more work
needs to be done. The very fact that RLN occurs in taller horses and is
more prevalent in certain breeds would indicate a familial factor is involved
(Cook 1988; Poncet et al. 1989; Duncan 1992; Harrison et al. 1992).
However, familial or not, this still does not explain the etiology of the

disease.

One theory is based on a set of mechanical possibilities for why and how
this disease occurs and may also go some distance to explaining why this
nerve (left) is affected more than the right. The left recurrent laryngeal
nerve is approximately 20 cm longer than it’s counterpart because it runs
around the base of the aorta. In 1970, Rooney postulated that the damage
to the left recurrent laryngeal nerve was due to the mechanical effects of
stretching when the head and neck moved. He suggested that this could
cause ischemia and therefore result in nerve damage. However, there are
some concerns with this hypothesis, ﬁrstly; if tension and stretching does
occur, it would have to be greater than 8% of the nerve’s existing length
before any ischemia can occur (Lundborg, 1988). Furthermore, nerves are
relatively robust in the face of ischemic damage and Lundborg (1988)
demonstrated that nerves can retain their function for up to 6 hours after

total loss of their blood supply. It seems unlikely that even an extreme

18

postural movement would be maintained for that length of time. This is
supported by the fact that Duncan (1987) did not ﬁnd microscopic lesions

that would indicate ischemic nerve injury.

RLN has been characterized histologically and ultrastructurally (Cole 1946;
Duncan et al. 1978; Cahill and Goulden 1986 (parts I—V), 1987, 1989;
Duncan 1987; Duncan and Baker 1987; Duncan and Hammang, 1987;
Hahn et al., 2009). The characteristic lesions associated with RLN are
characterized by a loss of myelinated ﬁbers that is most profound distally
and decreases in amount proxirnally (Hahn et al., 2009). Histological
lesions demonstrate digestion chambers containing axon fragments, axonal
atrophy, collapsed myelin sheaths, no axis cylinder and paranodal and
intemodal accumulations of axonal debris and organelles, which indicates a
primary axonal lesion. However, some confusion arises because there is

also evidence of extensive myelin damage.

There is some debate as to whether or not RLN is a polyneuropathy or not.
The changes are greatest in the distal portions of the left recurrent laryngeal
nerves (Duncan et al. 1978; Cahill and Goulden 1986a; Hahn et al., 2009),
however milder lesions are also present in the right recurrent laryngeal
nerve (Duncan et al. 1978; Cahill and Goulden 1986a; Hahn et al., 2009).
If RLN is a polyneuropathy then it is possible that the myelin damage could

be the primary lesion (Hahn etal., 2009), however, if not it is unlikely.

19

Until the recent work by Hahn et al. (2009) this topic had largely be left
unchallenged for 15 years. Hahn et al. set out to determine whether or not
RLN was indeed a mononeuropathy or a polyneuropathy. Horses do not
demonstrate clinical signs of polyneuropathy, whereas other species
affected by myelinopathies and inherited and metabolic primary
axonopathies more typically demonstrate clinical signs affecting multiple
nerves. However, there is no evidence of RLN affected horses suffering
from megaesophagus, tetrapareisis, muscle atrophy and hyporeﬂexia as we
see in dogs with RLN. Interestingly, stringhalt is seen in Australia with
RLN (Huntingdon et al., 1987), so perhaps there is a polyneuropathic
element in some species or geographical regions. Furthermore, there have
been isolated reports of pathologic changes of other long peripheral nerves
in RLN cases (Cahill and Goulden 1986c; Hahn and Mayhew, 2007) but
these are uncontrolled studies, which do not account for concurrent effects
such as toxin-associated disorders. 80, there is evidence that it is a
polyneuropathy, however it is something that we do not see clinically. The
questions that Hahn et al. (2009) asked were 1) whether other similarly
long nerves have the same pathology and2) is RLN indeed a

polyneuropathy or is it a mononeuropathy?

It is fair to comment that Hahn et al. (2009) only used 4 horses and only

one of these was a grade IV (complete paralysis) affected patient. They

20

investigated the distal portions of the median, peroneal and phrenic nerves,
which were chosen because they are the longest somatic motor nerves in
the horse apart from the recurrent laryngeal nerves. There is no evidence to
suggest that these nerves would be the more or less likely affected ones in
the case of puolyneuropathy. However, their ﬁndings were interesting,
they stated that RLN is not a polyneuropathy. This is based lack of
evidence of axonal lesions in any other long nerves and absence no
neurogenic atrophy of the intrinsic musculature of the muscles that these
nerves were supplying. Interestingly, they also noted that the right
recurrent laryngeal nerve was also signiﬁcantly affected in RLN cases so
the term ‘mononeuropathy’ should be more correctly applied as a ‘bilateral
mononeuropathy’. This piece of work by Hahn et al. (2009) is the tip of

the iceberg and needs to followed up with more in depth studies.

Reference was made to ‘neurogenic atrophy’, which is the term used to
describe the histologic appearance of the muscles affected by RLN. This
term represents typical changes, which include atrophied and hypertrophied
ﬁbers, centrally placed nuclei, scattered angular ﬁbers, ﬁber type grouping
and increased perifasicular fat (Duncan and Grifﬁths 1974; Cahill and
Goulden1986d; Duncan et al. 1991; Harrison et al. 1992). Most
commonly, these ﬁndings are seen to be more severe in the laryngeal
adductor muscle groups, a point that will be discussed further in the next

section.

21

iii.

Diagnosis

No diagnosis should ever be attempted without a full and complete history
and physical examination. Details surrounding the type of work that the
horse is performing, how ﬁt the horse is and at what stage of exercise is the
horse suffering. One of the most frequent complaints from owners and
riders is that a horse affected with RLN is suffering from poor performance
and making a respiratory noise. It is important to get as much detail
surrounding the type of noise that is heard. Whilst none of these factors are
in themselves diagnostic for RLN they are useful in establishing a sensible

list of differential diagnoses.

During a thorough clinical examination, attention should be paid to the
cardiovascular and respiratory system, as frequently these horses present
for poor performance. Close attention should be paid to the nasal passages,
starting at the nares and assessing facial symmetry and looking for signs of
nasal discharge and unequal airﬂows from either nostril. The
submandibular lymph nodes should be assessed for enlargement and the
jugular grooves and trachea should be palpated for signs of trauma or
damage. The trachea and larynx should be auscultated carefully looking

for evidence of respiratory disease. The heart and lungs should also be

22

carefully auscultated to rule out any arrhythmias or primary lower

respiratory tract disease as a cause of the poor performance.

Assuming a normal physical examination particular attention should be
paid to the larynx. This can be effectively palpated transcutaneously, a
diagnosis of RLN can be made by experienced palpators when the
muscular process of the arytenoid can be palpated more clearly on one side
when compared with the other (Cook, 1988). It is also possible to perform
an arytenoid depression test as reported by Marks et al. (1970), which
consists of producing an axial displacement of the left arytenoid cartilage to
produce an inspiratory noise. Another test that can be performed to try and
diagnose RLN is the “slap test”. The “slap test” involves slapping the
whithers on one side and observing or palpating adduction of the
contralateral arytenoid cartilage and it tests the integrity of the thoraco-
laryngeal reﬂexes. However, this diagnostic test is unreliable because it is
unable to detect reduced adductor function, which is often the ﬁrst sign of
neurologic deﬁcit (Newton-Clarke et al., 1994). This sentiment has been
echoed by Hawe et al. (2001) who demonstrated that this diagnostic test
was not an accurate test for the diagnosis of RLN. So, whilst palpation
may not be the answer for deﬁnitive diagnosis it is very important as it may
allow the examiner to detect other abnormalities such as arytenoid
chondritis or the presence of scars indicating previous upper airway surgery

(Sﬁck,2006)

23

Deﬁnitive diagnosis is made via Videoendoscopy, this can be done in the

standing horse and it can be used to assess the normal structures of the

larynx, observe any abnormalities such as chondritis or subepiglottic cysts

for example. Because it is a dynamic instrument it can also be used to

assess function and movement of the structures of the larynx. Various

grading systems for evaluating resting laryngeal function have been

established. The system used most consistently grades laryngeal function

from I to IV (Rakestraw et al., 1991). This grading system is as follows:

Grade 1:

Grade 11:

Grade 111:

Grade IV:

Symmetric and synchronous movement of
the arytenoid cartilages.

Symmetric but asynchronous movement of
the arytenoid cartilages, abduction
maintained.

Asymmetric and asynchronous movement of
the arytenoid cartilages, abduction cannot be
maintained.

No perceptible movement of the arytenoid

cartilages.

It is important to stress that these grades are used to describe horses that are

evaluated at rest. The Videoendoscope can be used in horses whilst they

24

are exercising on the treadmill in an effort to mimic real exercise scenarios,

and despite the fact that it is not the same, it has allowed for huge advances

in diagnosis of upper airway conditions (Stick & Derksen, 1989; Dart et al.,

2001; Durando et al., 2002; Parente et al., 2002; Dart et al., 2005; Tan et

al., 2005; Franklin et al., 2006; Lane et al., 2006 (part 1 and 2)). Due to

subtle differences that could be seen on treadmill examination of the upper

airway tract, subdivision of the grade III affected horses were put in place

(Hammer et al., 1998). These subdivisions are as follows:

Grade IIIA:

Grade HIB:

Grade IIIC:

Asymmetric and asynchronous movement of
the arytenoid cartilage but .it is able to
maintain abduction at exercise.

Asymmetric and asynchronous movement of
the arytenoid cartilage but it is unable to
maintain abduction at exercise and the
affected arytenoid remains in a ﬁxed but
incompletely abducted position.

Asymmetric and asynchronous movement of
the arytenoid cartilage that leads to severe

collapse during exercise.

Subsequently, this scale meant that horses that fell into Grades IIIB, IIIC

and IV were surgical candidates.

25

In addition to palpation, Videoendsocopy at rest and at exercise; sound
analysis has been used to help diagnose RLN as well as some other
diseases of the upper airway (Derksen et al., 2001; Burn & Franklin, 2006).
Spectrum sound analysis is used in human medicine in the ﬁelds of speech
therapy and voice recognition. In 2001 , Derksen et al. used this software to
analyze the respiratory sounds of horses with experimentally induced
dorsal displacement of the soft palate and LH. The group identiﬁed that
inspiratory sounds of horses with LH were characterized by 3 frequency
bands (formants). Of particular interest, the second formant, which lay
between 0.9 i 0.06 kHz and 2.4 i 0.06 kHz (Derksen et al., 2001). The
reason that this is of importance is that it lies within the range of human
hearing (0.02 to 20 kHz) (Strong & Plintnik, 1992) furthermore; spectrum
analysis indicated that the timing, frequency and amplitude of theses
sounds had an easily recognizable pattern. Not only has this developed into
a very useful research tool but it can also be used clinically for horses that

do not demonstrate airway obstruction during exercise on the treadmill.

Electromyographical (EMG) analysis has been used to study RLN. It is a
technique for evaluating and recording the activation signal of muscles.
EMG is performed using an instrument called an electromyograph, to
produce a record called an electromyogram. An electromyograph detects

the electrical potential generated by muscle cells when they contract, and

26

also when the cells are at rest. It was ﬁrst used to study the larynx by
Goulden et al. (1976), since then it has been used to study a number of
muscles in the upper airway (Tessier et al., 2005; Holcombe et al., 2002
and 2007). It has failed to gain widespread acceptance as a diagnostic tool
due to its invasive nature and the need for specialist equipment and

training.

Ultrasound of the laryngeal structures was ﬁrst discussed in 1997 but was
not really revisited for another nine years other than sporadic case reports.
In 2006, Chalmers et al. described a technique for ultrasonographic
examination of the equine larynx. In this paper the author comments on the
limitations of the Videoendoscope because it can only see structures from
within the lumen of the larynx and thus non-luminal structures including
the intrinsic and extrinsic laryngeal muscles and hyoid bones are not
evaluated. Therefore although the function of the larynx can be
determined, many anatomic components of the larynx and related
structures are largely not assessed except by gross palpation. Whilst
videoendoscopy is the gold standard for diagnosis of RLN, ultrasonography
may provide an adjunctive diagnostic tool. Furthermore, it may provide the
general practitioner with a more practical diagnostic aid. Indeed, Chalmers
et al. (2006) report the diagnosis in one of the cases with grade IV RLN
based on the observation that the arytenoid on that side was not moving

during ultrasound. In particular reference to RLN, ultrasonography is

27

useful for the examination of the cricoiarytenoideus lateralis muscle
(CAL), which is one of the adductor muscles of the larynx. The lateral
window provides the best avenue for ultrasound examination when
assessing the CAL. The importance of this ﬁnding is that the CAL is often
ﬁrst to be affected so if this disease is a bilateral mononeuropathy it should
be possible to detect some atrophy on the contralateral side. Whilst still in

its infancy, laryngeal ultrasound does show some promise.

The advent of modern imaging modalities, such as magnetic resonance
imaging (MRI) and computed tomography (CT), has revolutionized many
areas of veterinary diagnostic imaging. In the case of the upper airway
their use is usually cost prohibitive if the deterrent of general anesthesia is
not sufﬁcient. There is a CT machine for use in the standing horse in the
United Kingdom but its use is by no means widespread although the
obvious beneﬁts are clearly apparent. So, to date the use of MRI and CT to
diagnose upper respiratory tract disease is usually limited to research and
isolated case reports. CT has been used to develop a computational ﬂow
model of the upper airway at exercise and this has been used to evaluate the
degree of abduction of the arytenoid cartilages that is required at various
pressures (Rakesh et al., 2008). However, at this stage the use of these

imaging modalities is not clinically relevant.

So, what lies ahead? The most tangible of products is the telemetric

28

iv.

endoscope, which allows for endoscopy of the upper airway whilst the
horse is exercising in its normal environment (Franklin et al., 2008, Pollock
et al., 2009). This is a tremendous advance as it removes some of the valid
concerns that are associated with treadmill examination failing to replicate
actual exercise conditions. Indeed, we have used our own prototype (Dr.
Derksen, unpublished) to diagnose vocal fold collapse in a Belgian mare
that could not be Viewed with normal videoendoscopic techniques. This is
likely to revolutionize diagnostic techniques in the upper airway and it will
help elucidate the pathogenesis of the complex disease that is RLN. We

eagerly await further data.

Progression of disease

This is a difﬁcult and, as yet, unanswered aspect of the disease. An
endoscopic survey of 109 Thoroughbreds all under the age of two years old
was performed and then repeated 16 months later. At the second
examination, 12% of horses had endoscopic examinations consistent with
laryngeal hemiplegia suggesting a progression of the disease (Anderson et
al., 1997). Another interesting ﬁnding from this piece of work was that in
29% of the horses examined, a better grade of laryngeal function was
awarded indicating that some horses could recover. More recently, a study
looking at older national hunt and sport horses demonstrated that 15%

showed progression of disease over a median of 12 months with the onset

29

of progression occurring at a median age of seven years. In this study there
was no sign in improvement in horses with clinical signs (Dixon et al.,
2003a). More recently, 197 foals were studied endoscopically and then
again one year later, of the 187 foals that were available at this time point
only one demonstrated progression to paralysis (0.63%) indicating that
RLN was not progressive (Lane JG, 2004). However, this population of
very young seems not to represent the more typical clinical population. A
series of 3 adult horses with RLN were demonstrated to have progression

of the disease using successive dynamic endoscopy (Davidson etal., 2007).

It would appear that 'whilst RLN can progress, this progression is

inconsistent and unpredictable.

30

4. Treatments for Equine Recurrent Laryngeal Neuropathy

When considering treatment of RLN it is important to remember that this is not a life
threatening disease unless it is bilateral in nature. It is a performance limiting disease
both in terms of athletic function and also for producing an undesirable clinical sign
in the form of noise, which is offensive to the show circuit judges. In the show ring

being ‘unsound of wind’ can result in deduction of points and penalization.

This is an important concept to consider because the decision to perform upper
airway surgery should be based on clear evidence that it is needed. As such, each of
the procedures that will be talked about below should be targeted to treat a particular
problem. Likewise, it is vital that the owner is fully informed of the various
advantages and disadvantages of the various procedures prior to performing laryngeal
surgery. This relationship between clinician and owner is absolutely Vital for a
successful outcome. It is important that the correct criteria are used to assess the
selection of the procedure, for example, elimination of upper airway noise does not
indicate success if restoration of normal upper airway mechanics i was the
requirement. There is a poor correlation between upper airway noise level and degree
of arytenoid abduction as measured by inspiratory pressure, in fact it was found that
the greater the abduction the greater the noise produced (Brown et al., 2004). There

has also been recognition of the fact that combining some of these procedures is

31

successful, such as combining ventriculectomy with vocal cordectomy, or
ventriculocordectomy with laryngoplasty. The latter of which is commonplace
nowadays as both the problems of unwanted noise production and poor performance

are addressed.

There is much discussion within the profession as how best to treat RLN. Not only
does this discussion revolve around which procedure is best for what horse but also at
what stage of the disease one should intervene. It is generally accepted that grade I
and II RLN affected horses are clinically normal but that grade III and IV RLN
affected horses will require treatment (Stick et al., 2001). Indeed, performing
laryngoplasty on grade HI RLN affected horses has been suggested as a source of
potential failure of the laryngoplasty suture (Hammer et al., 1998). This is suspected
to be due to repeated residual contraction of the cricoarytenoideus dorsalis muscle in
concert with the adductor muscles of the larynx in horses treated for grade III RLN
causing the prosthesis to loosen or cut through the cartilage, resulting in gradual loss
of arytenoid abduction or complete laryngoplasty failure due to cyclical loading
(Hawkins et al., 1997). Furthermore, the presence of larger CAD muscle mass means
that there is more soft tissue for the suture to “cut down” through and thereby loosen
to a greater degree (Stick, personal communication). A study demonstrated that
eliminating contraction of the cricoarytenoideus dorsalis muscle by performing a
recurrent laryngeal neurectomy in conjunction with laryngoplasty and unilateral
ventriculocordectomy did not improve the postoperative outcome of Thoroughbred

horses treated for left RLN (Davenport-Goodall & Parente, 2003 and Hammer et al.,

32

1998). Interestingly, a recent paper has disposed of the popular concem that grade III
RLN-affected horses are a much higher risk of failure suggesting that there is no
difference in terms of success if a laryngoplasty is performed on a grade IV RLN
affected horse or a grade HI RLN affected horse (Witte et al., 2009). Classifying the
grade of RLN is not easy. It may be possible to diagnose grades I, II and IV via
endoscopy of the standing horse however determining whether or not a horse as grade
II or III RLN is more difﬁcult. This demonstrates the need to be thorough when
evaluating these patients as well as the need to observe the laryngeal movements at
exercise either via a telemetric endoscope or using a treadmill. Furthermore, there
may be concurrent dysfunction of other structures in the upper airway confounding
the diagnosis of RLN. This point was identiﬁed in a study by Lane et al. (2006b)
where 7 % of horses that had been graded as having normal laryngeal ﬁrnction
showed dynamic collapse of the left arytenoid or vocal fold when exercised on the

treadmill.

Once the correct diagnosis has been reached, and following sufﬁcient discussion with
the owner with regards to their expectations of the horse’s requirements, one, or a

combination, of the following can be performed.

33

a. Ventriculectom y

Ventriculectomy has been performed to treat “roarers” for well over 100 years and
was made popular by Sir Frederick Hobday in the early to mid 19005, colloquially
becoming known as the “Hobday” procedure (Hall et al., 1990). It refers to the
surgical procedure whereby the lateral ventricle (saccule) is removed, it can be
performed unilaterally or bilaterally and in combination with any of the other

procedures.

Ventriculectomy can be performed via a laryngotomy in the standing sedated horse or
under general anesthesia with the horse placed in dorsal recumbency (Stick, 2006;
Cramp et al., 2009). It involves the removal of the mucosal lining of the lateral
ventricle, which creates a ﬁbrous adhesion between the vocal fold, thyroid and
arytenoid cartilages when it heals. This mucosal lining is everted by inserting a
“roaring burr” in to the ﬁrll depth of the ventricle, rotating it to engage the mucosal
lining and everting it out of the ventricle. Next, Carmalt forceps can be used to grip
the everted mucosa and continue to stretch it out of the saccule where it can be
excised using a pair of Metzenbaum scissors. There are reports where the incised
edges of the ventricle can be sutured (Tetens et al., 1996) but this is not considered
necessary. This adhesion, or scar, may have two effects; one is to make the paralyzed
arytenoid cartilage ‘stiffer’ and prevent it from collapsing across the airway under the
negative pressure inﬂuence of inspiration (Shappell et al., 1988). The second is that

it reduces upper airway noise by reducing the turbulence created by the ventricle

34

when it is left in place (Brown et al., 2003). Typically, the laryngotomy incision will
heal within 3-4 weeks and requires basic wound management consisting of cleaning
the site 2-3 times per day. The cricothyroid membrane can be closed with absorbable
suture for a more aesthetically pleasing ﬁnish but this is largely down to surgeon
preference (Beroza, 1994 and Boulton et al., 1995). It is recommended based on the
work by Brown et al. (2004) that horses are rested for 90 days post-operatively,
however if the procedure is combined with a laryngoplasty then only 45 days rest is

required.

Typically this procedure is performed with a laryngoplasty on the ipsilateral side to
reduce the noise associated with laryngeal hemiplegia (Russell & Slone, 1994;
Hawkins et al., 1997; Kidd & Slone, 2002; Kraus et al., 2003; Brown et al., 2004;
Taylor et al., 2006 and Henderson et al., 2007). However, it can also be performed
alone and the effects of ventriculectomy on the equine upper airway were studied by
Shappell et al. (1988). In this study, the horses had left laryngeal hemiplegia induced
by transecting the left recurrent laryngeal nerve and then exercised on a treadmill 30
days after left ventriculectomy. Post-operative inspiratory impedance, airﬂow, and
upper airway pressures were compared to pre-operative values. There was no
difference between the two groups, subsequently these horses underwent
laryngoplasty and baseline values were restored. Ventriculectomy alone has been
successful in treating draft horses with laryngeal hemiplegia (Bohanon et al., 1990).

Either way it is generally accepted that ventriculectomy alone should be reserved for

35

those horses in which the major complaint is that of noise production (Adreani &

Parente, 2007).

b. Ventriculocordectom y

This is the same procedure as a ventriculectomy except for the additional removal of
the vocal cords. The vocal cords are grasped and a crescent piece of the rostral edge
of the vocal cord is resected using a pair of Metzenbaum scissors. Both studies by
Tetens et al. (1996) and Shappell et a1. (1988) failed to demonstrate any beneﬁt on
upper airway ﬂow mechanics, especially when compared with laryngoplasty.
However, limitations to these studies are that they did not evaluate respiratory sound
production and the horses were only exercised on the treadmill, which cannot

generate enough speed to mimic accurately racehorse performance.

The most convincing work supporting the use of ventriculocordectomy procedures is
that carried out by Brown et al. (2003). In this experimental study, bilateral
ventriculocordectomy reduced indices of upper airway sound to those pre-neurectomy
(before laryngeal hemiplegia was induced) after 90 days post-operatively. They also
analyzed inspiratory pressure data and discovered that, whilst still higher than pre-
neurectomy levels, the inspiratory pressure had signiﬁcantly decreased by 90 days
post ventriculocordectomy. It is interesting to note what the effect of removing the
vocal cord really is. A good explanation can be found in the work done by Brown et

al. (2005). This study compared sound data and inspiratory impedance in

36

standardbred horses exercised on the treadmill before and after induction of laryngeal
hemiplegia following left recurrent laryngeal nerve transection that was treated with
laser vocal cordectomy. Their ﬁndings indicated that removing the vocal cords
improves upper airway ﬂow mechanics to the same degree as ventriculocordectomy
but does not reduce noise to the same extent. Improvements in noise reduction were
noted but not to the same degree as recorded for ventriculocordectomy. Taken
together, these studies would suggest that in order to improve sound data, the
ventricles should be removed, however, in order to improve upper airway ﬂow
mechanics, albeit not back to baseline values, the vocal cords need to be removed.
Consequently, bilateral ventriculocordectomy, which can be performed in the
standing sedated patient, or under general anesthesia, with a laser or via a
laryngotomy, is the treatment of choice when upper airway noise is the chief

complaint.

This statement is further supported by the retrospective data analyzed and reported by
Taylor et al. (2006), where a unilateral ventriculocordectomy in mild cases of RLN
(median grade IIIA) saw 66% of horses make no noise after surgery and 86% of
owners were satisﬁed with the outcome. These data indicate that unilateral
ventriculocordectomy is a suitable procedure for horses with low grades of RLN
(these included 64 out of 92 National Hunt racehorses) or higher grades of RLN in
non-performance horses (Taylor et al., 2006). The obvious advantage of this is that
the need for general anesthesia can be avoided with it’s associated risks as well as

avoiding the not insigniﬁcant risk of complication associated with the laryngoplasty

37

procedure (Dixon et al., 2003a; Kraus et al., 2004; Taylor et al., 2006). Nothing is
ever that simple, however, with Kraus et al. (2003) ﬁnding, a few years earlier, that
seven out of 104 draft horses that had been treated with ventriculectomy or
ventriculocordectomy alone required a laryngoplasty to alleviate the clinical signs.
Their conclusion is that ventriculectomy and ventriculocordectomy are insufﬁcient to
alleviate the clinical signs .RLN in draft horses and that a laryngoplasty should be
performed in conjunction for best results. They agree that the risk of general
anesthesia in draft breeds is higher than in lighter breeds and that the complications
associated with laryngoplasty are much greater compared with ventriculectomy or
ventriculocordectomy but they argue that the beneﬁts of laryngoplasty outweigh the
risks. This led to a study by Cramp et al. (2009) to compare the effects of bilateral
ventriculocordectomy with bilateral ventriculectomy on upper airway noise in draft
horses. Thirty competitive hitch or pulling draft horses were diagnosed with grade IV
RLN Via videoendoscopy in the standing horse. Sound data was collected prior to
receiving either a bilateral ventriculectomy (n=11) ~ or a bilateral
ventriculocordectomy (n=19). All procedures were performed in the standing sedated
animal via a laryngotomy incision. Eighty four percent of owners were satisﬁed with
the outcome both in terms of noise production and performance after
ventriculocordectomy compared with only 64% of owners statisﬁed after
ventriculectomy. In terms of sound data, both procedures improved indices of upper
airway noise (100% of the horses showed improvement) but bilateral
ventriculocordectomy improved the inspiratory sound level associated with the

second formant, that which is most acutely audible to the human ear (Strong &

38

Plintnik, 1992). Subsequently, it was concluded that bilateral ventriculectomy and
bilateral ventriculocordectomy signiﬁcantly reduce upper airway noise and indices of
airway obstruction in draft horses with RLN, but ventriculocordectomy is more

effective than ventriculectomy (Cramp et al., 2009 (in press)).

On the basis of these data, this author concluded that bilateral ventriculocordectomy
is the treatment of choice for horses suffering from either milder forms of RLN or
severe forms of RLN that do not require a maximum airway for high levels of
performance. By reducing the need of larygnoplasty, the morbidity associated with
upper airway surgery is reduced as complications associated with ventriculectomy
and/or ventriculocordectomy are much less severe and frequent (Dixon et al., 2003a;
Kraus et al., 2003; Cramp et al., 2009. It is imperative that a 0.5 to 1 cm length of
vocal fold tissue be left at the most ventral aspect, leaving the fomix intact to prevent
against laryngeal cicatrix formation, which is a rare but serious complication (Cramp

et al. 2009).

c. Laser vocal cordectom y, ventriculectom y and ventriculocordectom y

Due to the unsightly and crude appearance of the laryngotomy incision, surgeons
have looked to perform these procedures transendoscopically with the use of lasers.
The initial work was performed by Shires et al. (1990) attempting laser
ventriculectomy and whilst the procedure was quick and easy, the results were less

than promising with a mucosal remnant being left in the ventricle and thermal

39

damage of the adjacent structures present. In 2001, Hawkins & Andrews-Jones
performed a study evaluating a technique of ventriculocordectomy using a transnasal
Nd:YAG laser in a non-contact fashion in 6 horses. The surgical sites had healed by
47 days but there were a small number of complications; latent thermal necrosis of

the arytenoid cartilage and mucocele formation due to small remnants of the ventricle

being left behind.

In 2006, Robinson et al. reported on the effects of unilateral laser-assisted
ventriculocordectomy in horses with laryngeal hemiplegia. Six standardbred horses
were used that had laryngeal hemiplegia induced by transection of the left recurrent
laryngeal nerve. They were assessed for inspiratory obstruction and respiratory noise
post neurectomy and then at 60, 90 and 120 days post-operatively. The surgery was
well tolerated and successful, it reduced inspiratory pressures to pro-neurectomy
levels but the sound results were less convincing. There was an initial reduction but
then the sound level returned to the post neurectomy level indicating no effect. They
speculated that this was due to the fact that it was a unilateral procedure, lending
further support to the notion of a bilateral ventriculocordectomy should be the
treatment of choice. The same group followed up this studied by evaluating the
histological effects of the procedure (Robinson et al., 2006) and found that it
effectively removed the laryngeal ventricle (although care must be taken to ensure
that all is removed as some was left in one of the specimens) and that there was no
long-tenn damage to surrounding laryngeal cartilage. An important consideration is

that this study was performed with a diode laser, as compared to the Nd:YAG laser,

40

which has been used in other studies. A diode laser requires less energy than an
Nd:YAG laser and therefore there is less risk of latent thermal necrosis of nearby

StTllCtlll'CS .

Henderson et al. (2007) followed up this work with a long-term retrospective analysis
of the laser assisted ventriculocordectomy technique as described by Robinson et al..
Out of 22 horses that had unilateral laser assisted ventriculocordectomy, twenty
(91%) horses returned to their intended use. Excessive airway noise was eliminated
after surgery in 18 (82%) horses; exercise improved postoperatively in 8 of 10 horses.
Three racing Thoroughbreds returned to racing; 1 additional racehorse returned to
racing but required a laryngoplasty 1 year later to continue racing. Complications
occurred in 3 horses; one had laryngeal swelling, another had exuberant granulation
tissue and the third developed right sided arytenoid chondritis, all responded well to

ﬁirther treatment.

So where does this leave us with regard to laser use for ventriculocordectomy? It is a
safe and effective procedure so long as the surgeon is careful, uses the correct laser
system and everts and ablates the entire ventricle. The technique described by
Robinson et al. (2006) using a transnasal bur is the most effective but it is more
cumbersome and takes more time. The technique described by Hawkins & Andrews-
Jones (2001) using a pair of grasping forceps to grab the ventricle is simpler and
faster, however, it is likely that some of the ventricle is left in place. At the largest

equine hospital in the world, they routinely use this procedure and do not report many

41

complications, however, it should be noted that the majority of their cases have a
laryngoplasty performed as well (Woodie personal communication). How effective
this technique would be as a stand alone procedure is still unknown although

anecdotal evidence is promising and there is a high level of success.

d. Laryngoplasty

This technique was ﬁrst described by Marks et al. (1970) as an alternative to
ventriculectomy and arytenoidectomy, which the authors had determined was

ineffective for treatment for athletic horses.

Laryngoplasty aims to replace the function of the atrophied cricoid arytenoideus
dorsalis muscle to maintain the “laryngeal lumen in its anatomic position of maximal
inspiratory dilatation” Marks et al. (1970). Therefore the prosthesis runs from the
dorsocaudal edge of the cricoid cartilage to the muscular process of the arytenoid and,
when tightened, maintains the muscular process of the arytenoid cartilage in a caudad
retraction. Hence it’s colloquial name of the “tieback”. The choice of suture is very
much surgeon dependent but large diameter, non-absorbable, monoﬁlament or coated
suture material is preferable (Adreani & Parente, 2007). Initially, Marks et al. (1970)
recommended using a material that had “beneﬁcial elastic properties” and used
braided Lycra® but this has since been replaced with more stout materials (Adreani

& Parente, 2007).

42

Horses are placed under general anesthesia in right lateral recumbency with the left
side uppermost. The surgical site is routinely prepared and draped, and a 10-12 cm
skin incision is made ventral and parallel to the linguofacial vein centered over the
palpable edge of the cricoid cartilage. Using careful sharp and blunt dissection, the
lateral and dorsal aspects of the larynx are exposed. Retractors are used to maintain
exposure whilst the aponeurosis between the cricopharyngeus and thyropharyngeus
muscles is transected to expose the muscular process of the arytenoid cartilage. The
type of suture and the type of needle is down to surgeon preference, but a large
reverse cutting swaged on needle is recommended (this author recommends No. 5
Ticron, Sherwood-Davis & Geek, St. Louis, Mo). The suture is passed from the
caudal aspect of the cricoid cartilage, avoiding the lumen of the larynx, and rotated so
that it exits the cricoid cartilage 2-3 cm cranial to its caudal border and 1 cm lateral to
the dorsal ridge (spine). Next, the needle is removed and the suture is drawn under
the muscle belly of the cricopharyngeus muscle. A smaller reverse cutting needle
(this author recommends a no.6 Martin uterine reverse cutting needle) is threaded and
used to pass through the muscular process of the arytenoid cartilage in a craniolateral
direction (Stick 2006). Typically a second suture is placed in the same manner as the
ﬁrst and then they are both tied. An endoscope can be passed so that of the arytenoid
cartilage can be observed to assess the degree of abduction, although this is not
essential. The thyropharyngeus and cricopharyngeus muscles are sutured together
using small diameter monoﬁlarnent (3-0 PDS) in a simple continuous pattern. The

incision is closed in two layers; the ﬁrst joins the fascia adjacent to the linguofacial

43

vein to the omohyoideus muscle with small diameter monoﬁlarnent suture (2-0 PDS)

and then the skin is closed in any of the preferred methods.

Since its advent in 1970, the laryngoplasty technique has endured much scrutiny.
Arterial blood gas analysis performed before laryngoplasty and 48 hours after the
procedure and demonstrated that the hypoxemia and hypercapnia observed pre-
operatively after galloping were reversed postoperatively (Bayly et al., 1984).
Treadmill experiments demonstrated that experimentally induced laryngeal
hemiplegia decreased inspiratory ﬂow and increased inspiratory resistance that could
be reversed with laryngoplasty (Derksen et al., 1986). Similarly, in 1988, Shappell et
al. demonstrated that experimentally induced laryngeal hemiplegia increased
inspiratory impedance and trans-upper airway pressure that was reversed following
laryngoplasty. The submaximal performance on a treadmill compared with real
exercise has. always cast some doubt on the usefulness of these studies and in 1990
Williams et al. performed two studies to address this. The ﬁrst looked at racehorses
galloping on a track with experimentally induced laryngeal hemiplegia, they
measured inspiratory pressures before and after laryngoplasty. They found that whilst
laryngoplasty did reduce the pressures it did not reduce them to pre-neurectomy
levels. In the second study, horses with clinical (naturally occurring) RLN were
evaluated and they were found to have similar increases in upper airway pressures to
experimentally induced horses. Furthermore, laryngoplasty did return the upper
airway pressures to within the normal range. Tate et a1. (1993) looked again at blood

gas data and acid-base balance of exercising horses with RLN before and after

44

laryngoplasty. Their data supported that of Bayly et al. (1984) nine years previously
but also demonstrated that, whilst improved, values did not return to those of normal
horses. Again, looking at submaximally exercising horses Tetens et al. (1996) found
that horses exercising at 75% and 100% of their maximum heart rate demonstrated
that theincreased inspiratory impedance and decreased ﬂow associated with laryngeal
hemiplegia were reversed following larygnoplasty. In conclusion, laryngoplasty can

be considered to improve upper airway ﬂow mechanics towards the normal range.

The addition of a ventriculectomy procedure to a laryngoplasty in racehorses does not
appear to improve post-operative racing performance compared with laryngoplasty
alone (Hawkins et al., 1997). However, if the primary objective is to reduce noise
production, or if this is a complaint by the owner then either ventriculectomy or
ventriculocordectomy should be performed either unilaterally or bilaterally in

addition to the laryngoplasty (Brown et al., 2004).

Most importantly, clinical success needs to be deﬁned. This is difﬁcult to do as
reported success rates vary depending the criteria used, which is different for different
horses used for different events (Adreani & Parente, 2007). Indeed, Russell & Slone
(1994) reported on the success of 70 horses following laryngoplasty and found that
93% of non-racing breeds were improved satisfactorily compared with 48% of

racehorses.

45

If we consider racehorses, it is always difﬁcult to assess racing data as there are so
many confounding variables associated with success or apparent lack thereof.
However, a number of well-constructed studies have attempted to investigate the
success rates of laryngoplasty in racing populations. When 100 thoroughbred
racehorses that had laryngoplasty were compared with 400 controls (similar in terms
of age, breed, sex and occupation) there was no signiﬁcant difference between groups
(Spiers et al., 1983). Russell & Slone (1994) noted that whilst only 48% of
racehorses raced post laryngoplasty, 70% of those less than three years old raced
compared with only 25% of those horses older than three years. A large retrospective
study of 174 thoroughbred racehorses and 56 standardbred racehorses sought to
evaluate success with relation to a number of variables such as breed (Thoroughbred
V Standardbred), endoscopic grade of laryngeal function, ventriculectomy versus no
ventriculectomy, type of prosthetic suture used, and number of prostheses placed
(Hawkins et al., 1997). The ﬁndings of this study were that laryngoplasty with or
without ventriculectomy allowed 77% of the horses to race at least one time after
surgery, improved racing performance in 56% of the horses that completed three
races before and after surgery, and improved subjectively evaluated racing
performance in 69% of the horses. In 2000, Strand et al. used a statistical method to
evaluate racing performance of inexperienced and experienced groups of racehorses
after laryngoplasty with bilateral ventriculectomy. Their data demonstrated that
approximately 60% of all horses won one race after surgery and that 25% won at least
three races after surgery, indicating that laryngoplasty is a successful procedure.

However, experienced horses never returned to their baseline levels, in other words,

46

they were improved by the surgery but not returned as good as new. Therefore, their
conclusion was that “it may be prudent to provide a guarded prognosis for full
restoration of racing performance in older horses, unless they are especially talented

and are free of other racing-related problems” (Strand et al. , 2000).

There has always been some controversy over whether or not horses with grade III
RLN respond as well has horses with grade IV RLN (Hawkins et al., 1997). In 2001,
Davenport et al. set out to investigate the effects of recurrent laryngeal neurectomy in
combination with laryngoplasty and ventriculocordectomy on the postoperative
performance of Thoroughbred race-horses treated for grade III left laryngeal
hemiparesis. Fifty-ﬁve horses with grade III RLN as diagnosed via videoendoscopy
in the standing horse were treated with laryngoplasty and unilateral ispsilateral laser
assisted ventriculocordectomy, 39 had a recurrent laryngeal neurectomy performed at
the same time and 16 did not. The idea behind this is that laryngoplasty in grade III
RLN affected horses is less successﬁrl than in grade IV RLN affected horses due to
residual muscle movement that cycles the suture more quickly, the fact that the
cricoarytenoideus dorsalis muscle atrophies and loosens the laryngoplasty or that the
suture cuts through the larger mass of muscle becoming more loose. Davenport et al.
(2001) performed the neurectomy so that there would be no cycling of the muscle,
however, there was no beneﬁt to neurectomy suggesting that cycling has no effect.
Witte et al. (2008) recently demonstrated that horses treated by laryngoplasty and
unilateral laser ventriculocordectomy for grade III RLN performed better than those

with grade IV RLN. It would appear that there is no evidence that laryngoplasty is

47

doomed in horses with grade III RLN and is still recommended as an appropriate

treatment by many surgeons.

In non-racing horses, the results of laryngoplasty are unsurprisingly reported to be
better than for racehorses (Adreani & Parente, 2007). In 2003, Dixon et al.
performed a large study on 200 mixed breed horses that had larygnoplasty performed
and 86% of owners considered the procedure to have been worthwhile. In the same
study, 91% of were able to resume training six weeks postoperatively. This group
also noted that when noise reduction was the major barometer for success, and when
combined with ventriculocordectomy, 73% were considered to make no abnormal
respiratory noise postoperatively. This compares well with the 73% of draft horses
where noise was eliminated following laryngoplasty with ventriculocordectomy
(Kraus et al. 2003). These percentages are markedly higher than the 25-47% range
quoted for racehorses (Russell & Slone (1994) and Hawkins et al. (1997)). It would
appear from these data that laryngoplasty is a very successful procedure in non-
racehorses probably because these horses do not require the full arytenoid abduction
necessitated by the athletic endeavours of a racehorse. In fact, the more abduction
achieved, the higher the risk of postoperative complications, which begs the question:

Is it worthwhile performing a laryngoplasty on non—racehorses at all?

Post-operative care consists of stall rest for 30 days with handwalking only, then two

weeks of small paddock turnout or light exercise. After 45 days, training can be

48

resumed. Recommendations for diet are limited to feeding hay from the ground to

minimize post-operative coughing (Hawkins et al., 1997; Dixon. et al., 20033).

i. Postoperative Complications of Laryngoplasty

The complications associated with this procedure have always been the
disadvantage of the procedure. A balance needs to be found between the
performance beneﬁts of laryngoplasty and the risk of serious complications
developing. This is the main reason why there is a substantial amount of continued

research into ﬁnding alternative and ‘better’ treatment options than laryngoplasty.

Coughing is the most likely postoperative complication. This results from a degree
of dysphagia and the greater the degree of arytenoid abduction is signiﬁcantly
associated with postoperative coughing (Russell & Slone (1994) and Dixon et al.
(2003a)). During deglutition the arytenoid cartilages adduct in concert with the
vocal cords. This movement combined with the dorsal position of the epiglottis
protect the upper airway from being contaminated. However, if a prosthesis is
preventing the left arytenoid from adducting, or worse still, this prosthesis is
maintaining the arytenoid in a position of exaggerated abduction, this protective
function will not be possible. The larynx and carina of the trachea are highly
sensitive to light touch or small amounts of foreign material (Guyton & Hall,
2006). If stimulated, afferent nerve impulses pass via the vagus nerve to the brain

and initiate a series of autonomic sequences resulting in a cough where by the

49

unwanted material is rapidly exhaled. Furthermore, there is some speculation that
damage to the cranial laryngeal nerve during surgery (during dissection between
the cricopharyngeus and thyropharyngeus muscles) may result in disruption to the
pharyngeal constrictor muscles thereby interrupting the swallowing reﬂex
permanently, a separate issue to that of the abducted arytenoid physically
obstructing the esophagus (Greet, 1979). The incidence of post-operative
coughing has been reported to be as high as 43% (Dixon et al., 2003a). It is not
uncommon for coughing to be present in the initial postoperative period but it
should resolve in a matter of days to weeks. In the above paper, the 43% referred
to coughing for less than 6 months, 24% of which coughed when eating and 19%
had no association with eating. Fourteen percent of these horses had a cough,
which persisted for longer than 6 months (Dixon et al., 2003a). The chronic cough
can be more of an irritation to the owner and horse in some cases (approximately
half of the these reported by Dixon et al.). However, in some, it can develop into
pulmonary disease and occasionally into life threatening aspiration pneumonia.
Thankfully, the number of cases that develop fulminant bacterial pulmonary
infection is few (Dixon et al., 2003a) especially when on considers that 3-10% of
horses have endoscopic evidence of food in the trachea postoperatively (Russell &

Slone (1994) and Strand et al., 2000).

As with any permanent implant, the prosthesis and the wound can become
infected. Since its advent in 1970, the prosthesis was performed using braided

Lycra®, which was associated with a high rate of infection, up to 15% being

50

reported in the literature (Adreani & Parente, 2007). Since then monoﬁlament or
coated braided multiﬁlarnent suture materials have been used and this percentage
has decreased. Currently, the reported incidence of wound infection is between 0.5
and 6% (Hawkins et al., 1997; Strand et al., 2000; Davenport et al., 2001, Kidd &
Slone, 2002; Dixon et al., 2003a; Kraus et al., 2003). However, whilst this range
is not dissimilar for any elective, clean surgical wound the frequent presence of a
laryngotomy incision increases the risk (Dixon et al., 2003a). The incidence of
postoperative swelling associated with the laryngoplasty wound is 17% (Dixon et
al., 2003a), however more than half of these resolved within a couple of weeks.
Despite the fact that most of the of the incisional infections resolve by themselves,
meticulous wound care of the laryngotomy incision, if present, is vital and careful
hemostasis and dissection around lymph vessels is important during surgery.
Suture penetration into the airway, through the airway mucosa, can result in
inﬂammation and infection. This is best avoided by intraoperative endoscopy so
that the suture can be replaced at the time of surgery. A far less common
complication is that of granuloma formation and chondritis, which occur in 1% of

(Dixon et al., 2003a).

The last major complication associated with this procedure is loss of arytenoid
abduction. This phenomenon occurs in every case to some degree (Dixon et al.,
2003a), however, it results in failure of the procedure in 2-15% of cases (Marks et
al., 1970; Hawkins et al., 1997; Dixon et al., 2003a; Kraus et al., 2003). In these

cases where failure of the prosthesis has resulted in total loss of arytenoid

51

abduction, it is usually the result of failure of the cartilage at the muscular process
of the arytenoid cartilage (Dean et al., 1990; Dixon et al., 2003a). A number of
different prosthetic materials. and techniques have been tested and whilst some
have demonstrated increased strength none have solved the issue of cartilage
failure (Dean et al., 1990, Scherzer et al., 2005 and Rossignol et al., 2006). One of
the most promising studies was that of Schumacher et al. in 2000. This group
performed an in vitro study investigating properties of a prosthesis that is
composed of a steel cable and stress-reducing washers. Whilst the in vitro leg of
the study provided encouraging results, it has failed to gain widespread acceptance

clinically due to its impractical technical application.

In a computational ﬂow model study investigating decreasing degrees of abduction
of the left arytenoid cartilage it was noted that as the left arytenoid lost abduction
different ﬂow of air through the larynx was encountered (Rakesh et al., 2008).
They found that when the left arytenoid had relaxed to the grade 3 position (Dixon
et al., 2003b) there was increased airﬂow indicating collapse associated with the
right vocal cord and aryepiglbttic fold. Subsequently, a recommendation to
remove the right aryepiglottic fold in concert with a laryngoplasty was made in the
discussion section of this paper. This raises the question of which additional
procedures should be performed along with a laryngoplasty. Laryngoplasty will
return upper airway mechanics to normal levels and therefore should improve the
performance of the horse, however, without an additional procedure upper airway

noise will still be produced. Removal of the ipsilateral vocal cord and lateral

52

ventricle should reduce this noise, however, if the right vocal cord is also involved
as indicated and we know that the lateral ventricles cause noise then both of these
should also be removed. So, the recommendation would be bilateral
ventriculocordectomy with a laryngoplasty. According to the work by Rakesh et
al., 2008 we should also consider the removal of the right aryepiglottic fold,
however, one wonders whether their results would have been the same if the
laryngoplasty had been performed with any other procedure at the same time?

These issues still warrant further investigation.

e. Partial Arytenoidectom y

Typically this technique is more commonly performed to treat unilateral or bilateral
arytenoid chondritis. However, it has also been used to treat RLN and whilst there
are many forms of arytenoidectomy, partial arytenoidectomy has been shown to
reduce postoperative obstruction the most (Belknap et al., 1990; Williams et al.,

1990; Lumsden et al., 1994, Radcliffe et al., 2006).

The technique is performed Via a ventral laryngotomy incision with the horse in
dorsal recumbency. It is necessary to place the endotracheal tube through a
tracheotomy incision so that the surgeon can work freely on the larynx. The
comiculate process of the arytenoid is removed en bloc with its mucosa. Next, the
arytenoid cartilage is exposed by dissecting away the overlying mucosa using a

scalpel blade. The incision is made along the caudal border of the laminar portion of

53

the arytenoid from the dorsal midline ventrad and then continued craniad along its
ventral border. The mucosa is carefully elevated using a periosteal elevator and the
now exposed laminar portion of the arytenoid cartilage is freed from its deep
muscular attachments. The loose arytenoid cartilage is dissected free from the
muscular process via the articulation. Next a ventriculocordectomy is performed in
the standard manner and the mucosal ﬂap is closed using a simple continuous pattern
of absorbable suture material (Stick, 2006). Primary closure of the mucosa is debated
but it is recommended in order to reduce the risk of developing excessive
postoperative granulation tissue (Parente et al., 2008). However Barnes et al. (2004)
argue that the procedure is as effective without closure of the mucosa and that this is
advantageous due to the reduced surgical time and avoidance of the associated
complications such as suture line dehiscence, hematoma formation, thickening and
ﬁbrosis. To close the mucosa or not to close the mucosa likely comes down to
surgeon preference and it is this author’s recommendation to close the mucosa in an

effort to speed healing and make the airway as smooth as possible.

More recently, partial arytenoidectomy has been investigated for the treatment of
RLN as well as arytenoid chondritis (Barnes et al., 2004; Radcliffe et al., 2006;
Parente et al., 2008; Witte et al., 2009). Barnes et al., 2004 reported that partial
arytenoidectomy without mucosal closure would return 61-78% of racehorses to
successful racing. They stated that these horses had a fair prognosis and their results
were much better than the previously reported results of 45% (Tulleners et al., 1988).

They also reported no complications associated with the surgery, however, only 55%

54

of those horses raced three or more times implying some degree of exercise
intolerance (Parente et al., 2008). In 2006, Radcliffe et al. compared laryngoplasty
and unilateral ventriculocordectomy to partial arytenoid in experimental horses
exercising submaximally and at maximum heart rate. They found that at submaximal
speeds there was no difference between the two procedures but that at maximum
heart rate laryngoplasty demonstrated a slight improvement. It should be noted that
the treadmill does not perfectly mimic natural exercise and that the partial
arytenoidectomy was performed on the same animal after the laryngoplasty and
ventriculcordectomy and there was likely some effect of the ﬁrst procedure
superimposed on the results. It is also possible that the low power of the study makes
the difference between the two procedures less obvious. However, partial
arytenoidectomy can yield very positive results, 82% of horses that had partial.
arytenoidectomy raced after surgery and 64% raced more than ﬁve times (Parente et
al., 2008). Not all of these horses were being treated for RLN, 22 out of the 76 were
treated for failed laryngoplasty, the rest for arytenoid chondropathies, however, the
procedure still shows some promise. It should be noted that 17% of these horses have
a second procedure to laser debride via an endoscope excessive granulation tissue at
the surgery site despite primary mucosa closure in all of the cases. The median time
from surgery to racing was six months, which may be considered a lengthy lay off but
these cases are repeat procedures due to a failed ﬁrst attempt. The notion that partial
arytenoidectomy is a successful procedure with a favorable prognosis for racing is
supported by a recent study by Witte et al., 2009 comparing partial arytenoidectomy

with laryngoplasty and ipsilateral ventriculocordectomy. They suggest that a partial

55

arytenoidectomy may be less technically demanding than a larygnoplasty and because
there is no prosthesis it may be an attractive alternative to the laryngoplasty if the
outcome is no different. Furthermore, it may be a preferable procedure in grade III
RLN cases where surgeons are concerned about the anecdotal notion of cyclical
fatigue and failure due to residual movement of the arytenoid, or loosening of the
suture due increased muscle mass and subsequent atrophy and “cut down” of the
suture material (Ducharme & Hackett, 1991; White 1992). This study demonstrated
that horses with grade IH RLN treated with laryngoplasty and ipsilateral
ventriculocordectomy showed postoperative earnings comparable to controls and was
actually better than those horses with grade IV RLN. This dispatches the theory that
grade III RLN should be treated with a partial arytenoidectomy because a
laryngoplasty procedure is more likely to fail (Witte et al., 2009). This group also
noted that whilst both procedures lead to similar rates of return to racing,
laryngoplasty with ipsilateral ventriculocordectomy beneﬁts from higher
postoperative earnings and therefore should still be recommended as the treatment of

choice (Whitte et al., 2009).

Partial arytenoidectomy should be used as a secondary procedure to treat those horses
where laryngoplasty has failed and in these instances it can be recommended with a
good degree of conﬁdence. It should not be used as a ﬁrst line of treatment due to the
complications associated with the procedure, furthermore, it should not be
recommended for those horses where noise reduction is the primary goal of the

procedure. Postoperative noise production frequently occurs following partial

56

arytenoidectomy due to residual mucosa including the aryepiglottic fold that is no
longer held in place by the comiculate process of the arytenoid cartilage (Adreani &
Parente, 2007). Parente et al., 2008 identiﬁed the need to endoscopically examine
patients one month post partial arytenoidectomy due to the 17% likelihood of
developing granulation tissue, which can be easily debrided Via the endoscope using a
laser on an outpatient basis. However, the most serious complications associated with
partial arytenoidectomy are those of dysphagia and. aspiration resulting in coughing
and potential pneumonia. The biggest disadvantage of this procedure and why
another procedure should be performed ﬁrst in cases of RLN is that it is irreversible.
If a laryngoplasty results in dysphagia, the prosthesis can be loosened or removed but
if an arytenoidectomy results in dysphagia and coughing there is nothing that can be

done other than management practices to resolve this.

Postoperative management consists of broad-spectrum antibiotics for 3-5 days and
anti-inﬂammatories for 7-10 days as well as feeding from the ground with soaked hay
until horses can demonstrate that they can eat normally without coughing. Horses
should be stall rested for 4-6 weeks prior to complete healing, which usually occurs

within 8 to 10 weeks (Stick, 2006).

f Nerve Muscle Pedicle Graft (NMPG)

This procedure consists of implanting nerve muscle pedicles from the omohyoideus

muscle (the ﬁrst cervical nerve) and implanting them into the affected CAD muscle.

57

The main advantage to laryngeal reinnervation using a nerve muscle pedicle graft is
the preservation of the normal architecture of the larynx and thereby minimizing
postoperative complications compared with other techniques such as laryngoplasty
and partial arytenoidectomy (Fulton, 2007). Typically, this author performs a
bilateral ventriculocordectomy in the standing sedated horse a number of days after
the NMPG. The reason for this additional procedure is two-fold; ﬁrstly, it is
performed to reduce the noise production satisfactorily for the owner. Secondly, it is
performed to improve performance especially in the non-racing breeds. Whilst
evidence of reinnervation is noted after 3-4 months postoperatively, complete return
to function may take up to 12 months. However, useful endeavor by the horse may
be possible within this time frame with a bilateral ventriculocordectomy resulting in

greater owner satisfaction.

The postoperative management is very important and very speciﬁc to this procedure.
The following is taken from Fulton, 2003 and this regime was based on experiments
performed on dogs, humans and horses (Hengerer & Tucker, 1973; Tucker, 1976;
Fulton, 2001). Postoperatively, the horses are conﬁned to a stall for a period of 2
weeks. A neck bandage should be placed at surgery and horses should be in stalls
where they cannot put their head over the door and rub the neck incisiOn. The
bandage can be removed 2-3 days postoperatively and sutures are removed after 14
days. The prophylactic antibiotics penicillin and gentarnicin are routinely
administered for 3—5 days. Anti-inﬂammatory medication is administered for 7-10

days after surgery (Fulton et al., 2003).

58

After stall confinement, a further period of 2 weeks in a small yard or paddock
followed by normal paddock turnout for 12 weeks (Fulton et al., 2003). At this stage
the horse should go into training (16 weeks after surgery, 12 weeks is the earliest time
that reinnervation has been seen). When the horses are returned to exercise, it is
advised that episodes of fast exercise are introduced as early and as frequently as
possible. The omohyoideus muscle is an accessory muscle of respiration,
considerable respiratory effort must be undertaken to activate the ﬁrst cervical nerve.
Therefore, it is recommended that Thoroughbreds gallop over 400 m every second

day of training (Fulton et al., 2003).

After 6 weeks of training, trainers/owners should present the horse for endoscopic
assessment of the larynx. At rest, the left arytenoid cartilage most commonly looks
exactly as it did before surgery because the omohyoideus is not functioning at rest
and therefore the ﬁrst cervical nerve is not stimulated. There are two diagnostic
reﬂexes that can be performed, one involves stretching the horse’s head and neck
upward and if reinnervation has occurred, there is brief abduction of the left arytenoid
cartilage. The second consists pulling back sharply on the commissure of the horse’s
lips resulting in a sudden and brief abduction of the left arytenoid cartilage. If
reinnervation has occurred then the trainer is to continue training toward a return to
racing. If there is no evidence of reinnervation the horse should be turned out again
for another 8 weeks and repeat the same exercise and series of evaluation procedures.

At the second visit a period of approximately 9 months has elapsed since surgery and

59

if there is no improvement then clinical experience suggests that it is unlikely (Fulton
et al., 2003). However, Fulton comments that horses can take up to 12 months to

show evidence of successful reinnervation (Fulton et al., 1991).

The only real report of results is from Fulton et al., 2003 and again from Fulton, 2007
as a follow up with more horses having been studied. Fulton, 2007 reports that 76%
of unraced horses and 84% of raced horses demonstrated evidence of reinnervation.
Of these reinnervated racehorses 54% won one or more races after surgery, 58% had
overall improved performance rankings after surgery, 51% had higher overall prize
money after surgery while 57% had more money per start after surgery. Of the
reinnervated unraced horses 66% started in one race, of these 63% won at least one
race. Out of 11 standardbred horses that have undergone nerve muscle pedicle graft
surgery six have raced, three were retired with unrelated problems, one died and one
was a failure (Fulton, 2007). Three out of the six horses earned more money after
surgery (but raced in more races) and ﬁve out of six owners were satisﬁed with the
procedure. Seven warrnbloods have been reported on and ﬁve have performed at a
higher level postoperatively, which compares .more favorably than the 57% success
rate that Tyler, 2000 reported. The procedure compares favorably with laryngoplasty

but suffers due to the long recovery time.

The postoperative complications are limited to seroma, hematoma and infection of the

surgical site but in general complications occur infrequently. Three out of 182 horses

60

that have demonstrated successful reinnervation that later became denervated for

unknown reasons (Fulton, 2007).

This is a promising technique, however, the limiting factor is the time it takes for
reinnervation to result in the cricoid arytenoideus dorsalis muscle being able to
perform it’s ﬁrnction. This in part due to the process of reinnervation and in part due
to the mechanical effort that the cricoid arytenoideus dorsalis muscle has to overcome
to abduct the arytenoid. So far we have identiﬁed the need for a superior technique to
larygnoplasty due to the high complication rate associated with the procedure. We
have also identiﬁed a procedure (NMPG) that has a similar success rate and avoids
the serious complications associated with larygnoplasty. However, as
aforementioned, the time period taken for return to function is unattractive to owners
and trainers alike and this is a sever limitation to the NMPG. The rest of this Thesis
looks at two attempts to shorten this convalescence period. The ﬁrst study
investigates the effect of angle and force with regards to laryngeal abduction in an
effort to determine whether the medial or lateral muscle belly of the CAD muscle are
more important in laryngeal abduction. NMPGs placed in a location that confers
signiﬁcant mechanical advantage may be able to return the CAD to it’s appropriate
function sooner. The second study investigates the effect of electrostimulation of the
implanted ﬁrst cervical nerve in an effort to improve reinnervation speciﬁcally and to
produce muscular hypertrophy of the CAD. The aim is to be able to shorten the
recovery time of the NMPG procedure so that it is competitive with the current gold

standard (larygnoplasty).

61

5. Effect of magnitude and direction of force on laryngeal abduction: Implications

for the nerve-muscle pedicle graft technique

a. Introduction and Aims

By way of reminder, the nerve-muscle pedicle graft procedure needs to be modiﬁed
and improved before there will be widespread acceptance. When performing a NMP
graft, three to 4 pedicles are usually available for transplantation, but the optimal
location for placement of the pedicles within the CAD muscle is currently unknown.
Recently, an elegant study described the neuroanatomy of the equine CAD muscle
(Cheetham et al. 2008). There is a medial and lateral neuromuscular compartment and
the lateral compartment plays a greater role in abduction of the arytenoid cartilage.
Therefore, optimal placement of neuromuscular pedicle grafts may improve surgical

efﬁcacy, including improved laryngeal abduction and shortening of the recovery time.

The aim of this study was to determine force-abduction relationships at various lines

of action on the muscular process of the arytenoid cartilage. The medium-term goal

was to assist surgeons in choosing the optimal placement of NMPGS.

62

The hypothesis of this study was that the magnitude and direction of force placed on
the muscular process of the left arytenoid cartilage affects the magnitude of laryngeal

abduction.

b. Materials and Methods

Five larynges were collected from horses that had been euthanased for reasons
unrelated to upper airway disease. This study was approved by the All-University

Committee on Animal Use and Care at Michigan State University.

Specimen preparation

Immediately after euthanasia, the larynges were removed from the cadavers by sharp
dissection. Visual inspection of the larynges failed to reveal any abnormalities. The
pharyngeal musculature was dissected from each of the specimens prior to storage at
0°C in a 2% solution of 2-phenoxyethanol, a solution known to preserve tissue
pliability (Lussier et al. 1996). Specimens were tested within 24 hours of harvesting
and were allowed to return to room temperature before each experiment. Larynges
were placed into a custom-made jig and were secured using a polymer resin].
Furthermore, 1 screw was placed vertically through the epiglottis, and a second screw
was placed through the centre of the cricoid cartilage into the jig. To tie back the right

arytenoid cartilage in full abduction, a # 5 Ticron2 suture was placed through the

 

' Hydroplastic, TAK SystemsZO Kendrick Rd. Wareham, MA 02571
2 Syneture, United States Surgical, Customer Service, 150 Glover Avenue, Norwalk, CT
06856

63

caudal-most aspect of the cricoid cartilage and through the muscular process of the
right arytenoid cartilage, and secured under maximal tension. With the right arytenoid
cartilage ‘tied back’ maximally, another length of # 5 Ticron suture was secured to the
muscular process of the left arytenoid cartilage and attached via a mechanical force
transducer3 to a dead-weight force generator. Biomechanical markers were placed on
the rostral face of the rima glottis at the ventral aspects of the left and right comiculate
processes (points A and B, respectively); halfway between the most dorsal and ventral
aspects of the left comiculate process (point C); and at the 2 points where the left and
right aryepiglottic folds join the epiglottis (points D and B, respectively) (Figure 1). A
digital camera was mounted on a tripod and was positioned so that its view was

orthogonal to the plane of the rima glottis.

Experimental protocol

Using increments of 0.98 N, the force generator applied a force of 0 N through 14.70
N for 1 minute each to the left muscular process at 0, 10, 20, 30, 40, 50, 60, and 70
degree angles (Figure 2). The 14.70 N endpoint was chosen because in preliminary
experiments visual inspection revealed that additional force did not further abduct the
left arytenoid cartilage, and because this force is signiﬁcantly less then the 55.8 - 219.6
N force required to cause failure of the laryngoplasty at the muscular process
(Schumacher et al., 2000). Zero degrees was deﬁned as a line through the muscular
process of the left arytenoid cartilage and parallel to the wing of the thyroid cartilage.

The sequence of angles studied was randomized by use of a random numbers table.

 

3 Transducer Techniques Inc., CA, USA
64

"" ........-.l.liinmrrv""' """ltturptix...,».
.. ' I" 'I, ”1.”:

i l
l .

i)‘: .I
I All”??? it, H i

'i
l

 

. . ., ,
_ .. r
1. 'II. 'lu..tlu..» I). '.' .

N. Ilu.“

Figure 1: Rostral view of larynx showing biomarkers used to assess degree of left
arytenoid abduction.

65

 

0 10° 20°. 30° 40°

Figure 2: Dorsal view of larynx showing direction of force (0-70 degrees) placed
on the muscular process of the left arytenoid cartilage.

66

A digital image was captured of the rima glottis one minute after each force had been
applied. In each digital image the distance between points A and B (linel), D and C
(line 2), D and B (line 3), and B and E (line 4) was measured by use of Olympus
Biological Suite software4. For each larynx, the software was calibrated by use of a

ruler that was incorporated into the ﬁrst digital photograph.

From the photographs, the right to left angle quotient (RLQ) was calculated using a
modiﬁcation of a previously described method (Jansson et al. 2000). Brieﬂy, a line
was drawn from the ventral-most aspect of the vocal fold fomix to the dorsal-most
junction of the left and right comiculate processes of the arytenoid cartilages. This
line was then extended dorsad by one third of its original length. From the top of this
line, tangential lines were drawn to the dorsal aspects of the left and right comiculate
processes. The angles between the dorsoventral line and the tangents were measured.

The RLQ was calculated by dividing the right angle by the left angle.

Statistical analysis
Data were analyzed by three-factor ANOVA with the ﬁxed effects of tension and
angle and the random effect of horse. Differences between the means were determined

by post-hoc Tukey comparisons. The level of signiﬁcance was set at P _< 0.05.

 

4 SIS MicroSuiteS Biological Software, Olympus America, Inc., Center Valley,
Pennsylvania

67

c. Results

Increasing force from 0 N to 14.70 N progressively and signiﬁcantly increased the
length of lines 1-4 (Figures 3a-d) and RLQ (Figures 4 and 5) (P <0.001). Furthermore,
there was a signiﬁcant interaction between force and angles (line 1 [Figure 3a] P
<0.016; lines 2-4 [Figures 3b-d] and RLQ [Figures 4 and 5] P <0.001). Differences in
the amount of abduction of the left arytenoid cartilage caused by angle became
signiﬁcant at a force of 7.84 N for lines 2 (Figure 3b), 3 (Figure 3c), and 4 (Figure 3d),
and RLQ (Figure 4), and at a force of 11.76 N for line 1 (Figure 3a). At 14.70 N mean
abduction achieved at angles 10° or 30° was always greatest while abduction at angle
70° was always least. At this force the differences between angles 10° or 30° and
angle 70° were signiﬁcant for all indices of abduction, whilst differences between

angles 0 through 30, and angles 40 through 70 were not signiﬁcant.

d. Discussion and Conclusions

While the NMP graft technique used in the treatment of RLN has been described in
detail, little attention has been paid to the optimal placement of the NMPs within the
CAD muscle. Reinnervation of the CAD muscle emanates from the grafted NMPs,
and occurs gradually. Results of the current study demonstrate that direction of force

applied to the muscular process of the arytenoid cartilage is an important factor in

68

54.00 -

53.00 -

52.00, A / v _

 

Length of Line 1 (mm)

 

 

47.00 _I_ U T f ﬂ r j
o oo 00 v N o oo \0 St N O
o as 3 Si Si 8 oo 3 oo 00 00 l‘ R " Z S
o o _ or to <1- tn o l\ 00 Ox 2 : Q ,_ .—

Force

2

Figure 3a: Effect of force (Newtons) and angle (degrees) on the length of Lines 1
(a), 2 (b), 3 (c), and 4 (d). O 0 degrees; I 10 degrees; A 20 degrees; X
30 degrees; * 40 degrees; 0 50 degrees; + 60 degrees; and - 70
degrees.

69

62.00 -

 

 

A 61.00 ' l/ I
E 60.00‘ /
E /
N 59.00- /
0 .
E 58.00 ./
A 57.00- /
r...
9 56.00- /
e -
01) 55.001
= /
3 54.00- ///
53.00- [IL/ 4’
52.00 325:“
51.00 . A . s
agesasassaaﬁ’ﬁﬁﬁﬁ
o o —« N m sr In \0 [\ oo 0\ S :1 Q {2 E,
Force(N)
Figure 3b: Effect of force (N ewtons) and angle (degrees) on the length of Line 2.

O 0 degrees; I 10 degrees; A 20 degrees; X 30 degrees; * 40
degrees; 0 50 degrees; + 60 degrees; and — 70 degrees.

70

 

 

 

' oo \5 <1- N :3
o 00 o <- N o 00 so <1- N o
o C» as ox O\ as oo oo oo oo oo 2 f: i: 2 2
o o ~ N m v in \o l\ 00 ax ._ ._ _. ._. ._
Force (N)

Figure 3c: Effect of force (Newtons) and angle (degrees) on the length of Line 3.
O 0 degrees; I 10 degrees; A 20 degrees; X 30 degrees; * 40 degrees;
I 50 degrees; + 60 degrees; and - 70 degrees.

71

27.00 -

 

 

 

A 26.00‘
E
E
v
V- 25.00-
C)
.E
A
a 24.004
C
.5
I510
= 23.00;
0
l-l

22.00-

21.00. . j I 1
sassaaassaaaeaeﬁ
ooﬂmvivwxorx'oomg:fj23

Force‘(N)

Figure 3d: Effect of force (Newtons) and angle (degrees) on the length of Line 4.
O 0 degrees; I 10 degrees; A 20 degrees; X 30 degrees; * 40 degrees;
0 50 degrees; + 60 degrees; and — 70 degrees.

72

1.60

1.40 -

1.30 .

RLQ

1.20 -

 

1.10 -

 

 

1 00 I r t I T l ‘T I I I i I I I I
o oo v: v N o 00 v N O
o m a. or o S 3 3 6‘5 Q 00 r: ‘19 l\ 1\ xx
O C v-' N M q- rn \O I\' oo 05 0 -~ N M V
v-t H — c-II ’—
Force (N)

Figure 4: Effect of force (N ewtons) and angle (degrees) on Right to Left Quotient
(RLQ). O 0 degrees; I 10 degrees; A 20 degrees; X 30 degrees; * 40
degrees; 0 50 degrees; + 60 degrees; and — 70 degrees.

73

1.40 - *

 

 

 

1.35 . *
1.30 -
O
a 1.25 .
1.20 -
1.15 -
1.10-
0 10 20 30 4o 50 6O
Angle (Degrees)

Figure 5: Effect of angle (degrees) on to Left Quotient (RLQ) at a force of 14.7
Newtons. * Significant difference from 10, 20, and 30 degrees.

74

abduction, and suggest that proper placement of NMPs in the CAD muscle may result

in earlier and greater arytenoid abduction.

The CAD muscle has a medial and lateral neuromuscular compartment (NMC) (Figure
2) (Cheetham et al. 2008). In our study, angles of 0-30 degrees correspond to the
lateral NMC whilst angles 40-70 degrees correspond to the medial compartment. In
dorsal (line 4), lateral (linel), and dorsolateral (lines 2 and 3) directions, force applied
at angles 0-30 degrees produced a greater degree of abduction than angles 40-70
degrees. This was conﬁrmed by the RLQ. At 0 N of force applied there was no
signiﬁcant difference in abduction as measured by any of the lines and RLQ,
demonstrating that in the experiment, previous abduction of the left arytenoid did not
affect subsequent abductions. At 7.84 N for lines 2, 3, and 4, and RLQ, and at a force
of 11.76 N for line 1, the differences in abduction of the left arytenoids became
signiﬁcant. Greatest abduction of the arytenoid required 14.70 N of force applied at
an angle of 30 degrees. This same force applied at 40-70 degrees resulted in
submaximal abduction. These data imply that placement of a nerve-muscle pedicle
graft in the lateral NMC would be more effective in terms of arytenoid abduction than
placing the grafts in the medial NMC. This conﬁrms previous ﬁndings in a
neuroanatomical study of the CAD muscle (Cheetham et al. 2008). Because nerve
sprouting does not cross fascial planes (Tucker 1979), and the equine CAD has two
distinct NMCs separated by connective tissue (Cheetham et al. 2008), nerve sprouts
from a NMP placed in the medial compartment will likely not cross over and

reinnervate the lateral compartment.

75

We are not suggesting that placement of a NMP in the medial compartment is of no
value. Indeed, it is likely that reinnervation of both NMCS is required for optimal
CAD function. During abduction most of the movement occurs in a lateral and dorsal
direction; however, the arytenoid cartilage can also move in a caudal direction
(Cheetham et al. 2008). We do not have information about arytenoid movement in a
caudal direction, therefore it is possible that the medial and lateral NMCs have
differing actions on the movement of the arytenoid in a caudal direction. Further
studies are required to evaluate the effect of force-angle interactions on three-

dimensional movement of the left arytenoid.

In the experience of our surgeons, in the majority of horses, four nerve-muscle
pedicles are available for grafting and there is sufﬁcient space for two nerve-muscle
pedicle grafts in the lateral NMC. To place more than 2 of these grafts, or use pedicles
that are too large, in the lateral NMC could result in ﬁbrosis and excessive scar tissue,
negatively affecting successful reinnervation and the muscle’s ability to abduct the left
arytenoid cartilage (Tucker 2006). Therefore it would be more beneﬁcial to place the
remaining nerve-muscle pedicles in the lateral portion of the medial NMC. It is
fortunate that the approach to the lateral NMC and the lateral portion of the medial
NMC of the CAD muscle is easier than the approach to the more medial aspects of the
CAD muscle. We have found anatomic variability in the size of the medial and lateral
NMCs between horses. This means that surgeons must adapt to these variations in

each case, bearing in mind the importance of the lateral NMC.

76

It has been suggested that in the case of neurotization, reinnervation of muscle by
transected nerves, the reinnervation occurs most easily at the site of the native motor
endplates (Goding Jr. 2005). In the equine CAD muscle, the 2 NMCs each have a
single motor end plate band arranged along the caudal aspect of the lateral
compartment and the medial aspect of the medial compartment (Cheetham et al.
2008). Thus, placement of the NMP in the middle of the lateral compartment and on
the lateral aspect of the medial compartment as recommended above, creates a
distance between some of the nerve pedicles and the native motor and plates. When a
NMP is placed in a denervated muscle, most reinnervation occurs by axon sprouting
from motor end plates located in the donor pedicle (Gray et al. 1997). A muscle
pedicle of 2-3mm2 contains approximately 60% of the motor end plates for that nerve
(Tucker 2006). Reinnervation of motor endplates in the recipient muscle by nerve
endings transected at the edge of the muscle pedicle is also possible (Gray et al. 1997).
In both cases, the nerve ”sprouts” must either reinnervate native motor end plates or
generate new (ectopic) motor end plates. When a transected nerve is implanted near
native motor end plates, most new axons innervate these native motor end plates,
while nerve implantation distant to native motor end plates results in signiﬁcantly
more ectopic motor end plates (Payne and Brushart 1997). The total number of motor
end plates is independent of implant location (Payne and Brushart 1997). Taken
together, these data suggest that the location of NMPs relative to native motor

endplates is unimportant.

77

Results of the current study also have relevance to the placement of the prosthetic
laryngoplasty suture in the treatment of RLN. Cranially, the suture penetrates the
muscular process of the left arytenoid cartilage, and caudally the suture is placed
around the posterior aspect of the cricoid cartilage and into a position 2-3 cm cranial to
its caudal border and 1 cm lateral to the spine of the cricoid cartilage (Stick 2006).
Placed in this manner, the force generated by the suture is at angles between 20 and 40
degrees. All of our measurements indicate that this direction of pull resulted in
maximum arytenoid abduction, probably contributing to the success of this procedure.
While a more lateral placement of the suture through the posterior edge of the cricoid
cartilage would also result in maximum abduction, this is not advised because of
anatomical considerations. The curvature of the caudal border of the cricoid cartilage
allows slipping of the suture along the posterior aspect of the cartilage and subsequent
loosening of the entire prosthesis. In the clinical setting, lateral placement has been

associated with prosthetic failure.

To summarize, this study demonstrates that forces applied to the muscular process of
the arytenoid cartilage in a direction corresponding to the lateral NMC of the CAD
results in greater arytenoid abduction than when these same forces are applied in a
more medial direction. This suggests that NMP grafts should be placed in a manner as
to confer most success in reinnervating the lateral NMC. These data should be helpful
to surgeons when performing NMP grafts and should result in a more successful

procedure.

78

6. Effect of electrostimulation on upper airway function in laryngeal hemiplegia

affected horses treated with a nerve muscle pedicle graft.

a. Introduction and Aims

Laryngeal reinnervation is used to treat people with laryngeal denervation
(Kingharn et al. 2006; Su et al. 2007; Tucker 1989; Tucker and Rusnov 1981). In
horses, laryngeal reinnervation using a ﬁrst cervical nerve-omohyoideus muscle
pedicle graft technique can be as effective in the restoration of racing performance
as prosthetic laryngoplasty and offers a more physiologic treatment with fewer
complications (Fulton et al. 1991; Fulton et al. 2003). However, the technique has
not gained wide acceptance due to the extended recovery time (Fulton et al. 2003).
The overall goal of this research was to determine if, in horses with LH,
postoperative electrostimulation of the reinnervated muscle allows earlier return to
athletic performance without risk of coughing, aspiration, and respiratory

infection.

Electrostimulation of skeletal muscle after denervation has been studied
extensively in humans and laboratory animals (Gondin et al. 2006; Mafﬁuletti et
al. 2006; Marqueste et al. 2004; Poortmans and Wyndaele 2002; Vitenzon et al.

2005). Within 5 weeks, denervated muscle has a dramatic loss of size and ability to

79

contract and carry out its ﬁrnction' For example, within 5 weeks, denervated
extensor digitorum longus muscles of rats lose 66% of mass, 91% of force, and
76% of ﬁber cross-sectional area (Dow et al. 2004). Electrostimulation of these
muscles can completely prevent these impairments (Dow et al. 2004). Atrophy of
denervated muscle impairs its ability to become reinnervated (Cole and Gardiner
1984; Fu and Gordon 1995); therefore the process of electrostimulation may aid
the reinnervation process. This possibility has important implications in equine
LH, where the denervated CAD muscle is responsible for the loss of upper airway

function.

The electrostimulation protocol, muscle ﬁber type, and species differences have
important effects on muscle responses to stimulation (Dow et al. 2004). Generally,
frequent submaximal stimulation encourages slow ﬁber type growth, while less
frequent maximal contractions encourage fast twitch ﬁbers, muscle ﬁber
hypertrophy, and faster force recovery. For example, in the denervated extensor
digitorum longus muscle of rats, a typical fast twitch muscle, near normal force
was maintained with 100 contractions per day for 5 weeks, while 800 contractions
per day caused muscle damage (Dow et al. 2004). In another study involving
denervated (for 6 to 24 weeks) human quadriceps muscle, a mixed ﬁber type
muscle, 80 contractions per week for 8 weeks were sufﬁcient to generate a 20%
increase in muscle mass (Dudley et al. 1999). The equine CAD muscle is a mixed
fast (55%) and slow twitch (45%) skeletal muscle (Hoh 2005), similar to the

human quadriceps muscle (Green et al. 1999). In this study we have chosen an

80

electrostimulation protocol that would be expected to increase muscle mass, force,
and ﬁber cross-sectional area in denervated fast and mixed ﬁber-type muscles in

rats and humans respectively (Dow et al. 2004; Dudley et al. 1999).

Hypothesis

The central hypothesis of this study is that post-operative electrostimulation of the
nerve muscle pedicle graft and recipient cricoarytenoideus dorsalis muscle results
in greater and earlier decrease in upper airway obstruction in exercising horses

with laryngeal hemiplegia in comparison to unstimulated controls.

Speciﬁc Aims

1. To measure trans-upper airway pressure in exercising horses before and after

induction of LH

2. To measure trans-upper airway pressure in exercising horses with LH before

and 30 days after placement of neuromuscular pedicle grafts

3. To determine if, relative to controls, postoperative electrostimulation of the
nerve muscle pedicle grafts and recipient cricoarytenoideus dorsalis muscle
decreases trans-upper airway pressure and improves laryngeal function at 44,

58, 72, 86, 100, 114, 128, 142, 156 and 170 days after nerve muscle pedicle

graft surgery.

81,

b. Materials and Methods

Horses

Four adult horses were used in this study. All horses were administered an
antihelmintic and vaccinated against eastern and western equine encephalitis,
tetanus, equine inﬂuenza, and rhinopneumonitis before the start of the study.
Endoscopic evaluation of the upper airway, including the trachea, was performed
when horses were at rest and during high-speed exercise. To be included in the
study, all horses had endoscopically normal upper airways. This study was
approved by the Institutional Animal Care and Use Committee at Michigan State

University.

Experimental Protocol

The four horses were randomly divided into two groups of two horses: an
electrostimulation group (principals) and a control group. Upper airway
endoscopy was performed at rest, and before and after surgical procedures to
document the induction of LH or postoperative complications. Horses in the
electrostimulation group received stimulation of the nerve muscle pedicle graft as
described below. The control group did not receive stimulation. Between
measurement periods, all horses were maintained on pasture. I planned to, make
measurements at 13 time periods: before (baseline) and 30 days after left recurrent
laryngeal neurectomy; and at 2 week intervals (30, 44, 58, 72, 86, 100, 114, 128,

142, 156 and 170 days) after nerve muscle pedicle graft surgery. Principal horses
82

were supposed to receive electrostimulation for 6 months, starting at 30 days after
nerve muscle pedicle graft surgery, however this could not be achieved in any of
the horses for technical reasons, which will be discussed later. The tirneline of
measurements and electrical stimulation actually used is shown in ﬁgure 6. During
each measurement period, datum were collected from horses exercising at their
individual maximum heart rate determined as previously described (Tetens et al.
1996). Following a 4-minute warm-up period, horses were exercised at a treadmill
speed corresponding to their individual maximum heart rate for 2 minutes or until
the horse could no longer maintain its position on the treadmill. Trans-upper
airway pressure measurement was performed and endoscopy was recorded on 2
separate days. After the last measurement was taken, horses were euthanized.
Each of the larynges and implanted ﬁrst cervical nerves were subjected to a
postmortem evaluation to assess the degree of denervation, degeneration and

reinnervation.

83

 

END

Figure 6:

30

60

74

88

102

116

130

144

158

Baseline

30d post NMP graft

Euthanized horses 1 & 2

Euthanized horses 3 & 4

 

Tests and/or Procedures Performed

Left recurrent laryngeal neurectomy
Endoscopy (standing and on treadmill)
Inspiratory Pressure

Nerve muscle pedicle graft (NMP
graft)

Endoscopy (standing and on treadmill)
Inspiratory Pressure

Start of electrostimulation protocol
Endoscopy (standing and on treadmill)
Inspiratory Pressure

Endoscopy (standing and on treadmill)
Inspiratory Pressure

Endoscopy (standing and on treadmill)
Inspiratory Pressure

Endoscopy (standing and on treadmill)
Inspiratory Pressure
Endoscopy (standing and on treadmill)

Inspiratory Pressure

Endoscopy (standing and on treadmill)
Inspiratory Pressure

Endoscopy (standing and on treadmill)
Inspiratory Pressure

Endoscopy (standing and on treadmill)
Inspiratory Pressure

Timeline in days from day 0 through to the end of the
electrostimulation research project.

84

Measurement of T rans- Upper Airway Pressure

A fenestrated polyethelene catheter, placed via the right nostril into the mid-
cervical region, was used to measure tracheal pressure. Inspiratory and expiratory
trans upper airway pressure was measured as the pressure difference between

tracheal and atmospheric pressure (Robinson et al. 2006).

Surgical Procedures

For all surgical procedures, all horses were administered peri-operative antibiotics
(potassium penicillin 22,000 IU/kg i.V. q.i.d. and gentamicin sulphate 6.6 mg/kg
i.V. s.i.d.) and an anti-inﬂammatory agent (ﬂunixin meglumine 1.1 mg/kg i.V.

b.i.d.).

Left Recurrent Laryngeal Neurectomy

Each horse was induced into general anesthesia. The left, mid-cervical region was
clipped and prepared for aseptic surgery. An incision was made immediately
ventral to the jugular vein and the left recurrent laryngeal nerve was identiﬁed and
isolated. Identiﬁcation of the correct nerve was conﬁrmed by endoscopic
visualization of abduction of the left comiculate process during nerve stimulation
by use of a hemostatic forceps. After isolation, a 2.5 cm segment of the nerve was

transected and removed (Derksen et al. 2001).

85

Nerve Muscle Pedicle Graft Procedure

The nerve muscle pedicle graft was inserted by use of a technique described by
Fulton et a1. (1991). A 12-cm linear skin incision was made along the ventral
border of the linguofacial vein. Blunt dissection between the omohyoideus muscle
and linguofacial vein was used to identify the ﬁrst cervical nerve as it travels over
the left cricopharyngeus muscle to its insertion in the omohyoideus muscle.
Cranial retraction of the cricopharyngeus muscle allows visualization of the
cricoarytenoideus dorsalis muscle in which three l-cm openings were created
between and parallel to the muscle ﬁbers in the lateral compartment of the CAD
muscle. Three nerve muscle pedicle grafts were created at the point of entry of a
branch of the ﬁrst cervical nerve into the omohyoideus muscle. Pedicles of muscle
approximately 5 x 5 mm were removed with the nerve intact and transposed to the
previously created openings in the cricoarytenoideus dorsalis muscle. Two simple
interrupted sutures of 4-0 polydioxanone were used to secure the cranial and
caudal aspect of the pedicle. A length of insulated wire (Teﬂon-coated stainless
steel , 36 AWG) was wrapped around the nerve and the two ends clamped close to
the nerve to ensure the wires stayed in place. The wire was tunneled underneath
the skin and exited in the midcervical region. This region was bandaged for 96
hours post-operatively and changed when indicated. The omohyoideus muscle and
lingual facial vein was apposed with 2-0 polydioxanone suture, and the skin was

closed in a routine manner.

86

 

Electrostimulation Protocol

Thirty days after placement of the nerve muscle pedicle grafts, the left cricoid-
arytenoideus dorsalis (CAD) muscle was stimulated every other day for a target
period of 60 days as follows: The ends of the electrodes were attached to a N EMS
+2 neuromuscular stimulator (Rehabilicare, New Brighton, MI). An endoscope
was placed to View the larynx. A 50 Hz, 0.4 milli-seconds duration, 0.5 V biphasic
stimulus was applied for 5 seconds. Eight sets of 12 stimuli were administered
(total of 96 stimuli) using a 5 second/Ssecond contraction /rest ratio with 2 minutes
of rest between sets. This stimulation protocol took 30 minutes to complete, which

is brief enough to be practical in a future ﬁeld setting.

Post-mortem Evaluation

The left and right CAD muscles were dissected out, measured, weighed and their
volume was calculated. The CAD muscle pairs were then packed in ice and sent
away for neuromuscular evaluation. When evaluating the tissue samples for this
study several approaches were taken according to the criteria of interest. The
presence of angular atrophy, anguloid atrophy, central nuclei, acute necrosis,
macrophages, myotubes, pyknotic nuclei, vasculitis, ﬁbrosis, and ﬁber size
variation were determined by evaluating H&E stained tissues. Each slide was
analyzed in 12 consecutive sections at 20 X power of 3 rows and 4 columns to
ensure completeness of data collection. The individual criteria was ranked from 0
to 3, 0 indicating no change, 1 indicating the presence of change, 2 indicating mild

to moderate change and 3 indicating severe change. Each of the 12 sections was

87

then averaged to give a ﬁnal value for each of the criteria. A number of other
criteria were evaluated on the same number scale but as a whole slide rather than
in 12 sections. These included the presence of weird glycogen and nerve de-
myelination using the periodic acid-Schiff (PAS) stain, the degree of ﬁber-type
grouping using the ATPase stain, the presence of moth-eaten ﬁbers and targets
using the NADH stain, the presence of intracellular and extracellular lipids using
the Oil Red 0 (ORO) stain, the presence of red stippling using the
Acidphosphatase stain, and the presence of end plates using the esterase stain.
Minimum ﬁber diameter was evaluated by viewing three sections on each slide.
Sections were determined by the general appearance of one large, the next largest
and one of the smallest group of ﬁbers and measuring the minimum diameter all
ﬁbers present on each of these individual Views at 20X. Then an average was taken
of these three views to give a ﬁnal score. Finally a score was determined for
denervation by assessing angular atrophy, anguloid atrophy, pyknotic nuclei nerve
bundle de-myelination. Degeneration was evaluated by assessing acute necrosis,
macrophages, ﬁbrosis red. stippling (acidphosphatase). Reinnervation was
evaluated by assessing central nuclei, myotubes, ﬁber-type grouping, moth-eaten
ﬁbers, target ﬁbers and end plates. By adding the averages for the categories and
dividing them by the number of categories a score could be attributed to each

category for the purposes of comparison.

88

c. Results

Endosc0pic evaluation at rest and on the treadmill conﬁrmed that all horses

became grade IV left laryngeal hemiplegia affected horses following left recurrent

laryngeal neurectomy.

i.

ii.

Inspiratory Pressure

In all horses there was an increase in inspiratory pressure following induced

left sided laryngeal hemiplegia. The results of this can be seen in ﬁgure 7.

Endoscopic Observations

None of the horses evaluated experienced a return to normal ﬁrnction and
only one horse (horse 3) had any evidence of reinnervation. This was
observed 128 days after the nerve muscle pedicle graft procedure had been
performed. When lifting the head during endoscopic examination of the
larynx there was a small ﬂicker from the left arytenoid cartilage.
Furthermore, there was slight abduction during inhalation when the
exercised at maximum heart rate on the treadmill. During exercise the
arytenoid abducted initially but then returned to the position as it was 30

days post NMP graft.

89

-15

Inspiratory Pressure (cmHZO)
115
o

Figure 7:

 

Time(Days)
0 30 60 74 88 102116130144158

  

I GINGER n PITT ' MAJOR I MAGGIE

Mean inspiratory pressure measurements (cmHZO) for the 4 horses
recorded at maximum heart rate on the treadmill. 0 days is baseline
prior to left laryngeal neurectomy (at 0 days), then 30 days after left
laryngeal neurectomy (at 30 days), then 30 days following NMPG (at 60
days) and then every 14 days thereafter.

90

iii.

Complications:

There were a number of unforeseen technical problems that prevented me
from completing the experiment as planned. In both of the stimulated
horses, the wires were pulled out or transected. The Teﬂon coated wires that
exited the skin in horse 1 were inadvertently cut during a bandage change in
the hospital. This was an unscheduled bandage change and was not
witnessed by any of the team conducting the experiment. However, given
the extensive damage to the ﬁrst cervical nerve (see later section) I think
that a signiﬁcant amount of traction may have been applied to the wires
resulting in tightening and damage to the nerve that they encircled. Initially,
this was not recognized and horse I became a non-stimulated control. Horse
2 became the stimulated principal. Horse 2 lost the wires 102 days (72 days
after nerve muscle pedicle graft) into the experiment. There was no
explanation for this, but it was later discovered that horse 3 had developed a
penchant for rubbing and nibbling on the exposed wires of her pasture

mates.

Horses 3 and 4 lasted for longer in the study, it was not until day 158 that
horse 4 lost the wires (128 days after nerve muscle pedicle graft), an
eyewitness account clearly identiﬁed horse 3 as the saboteur! At this point
in time both horses were euthanized and sent for post-mortem examination

and sample collection.

91

 

iv.

Post Mortem Findings

At post-mortem the left and right CAD muscles were dissected out and
visually inspected, all of the NMP grafts were in place and covered with a
ﬁbrous tissue layer. The ﬁrst cervical nerve was dissected free and
histologically evaluated. Results of the histological evaluation of the ﬁrst

cervical nerve were as follows, as reported by the pathologist:

Horse 1 :

“There was marked collagen deposition within the perineurirun and
epineurium, and subdivision of nerve fascicles. Most of the fascicles were
disrupted by collagen, with extensive loss of nerve ﬁbers; many of the
individual nerve ﬁbers are no longer within an apparent fascicle. There is
essentially no myelin surrounding the nerve ﬁbers, and there are abundant
‘Bungrrer’s bands’ present in the remnant fascicles. Within the most intact

fascicles there are scattered markedly enlarged nerve ﬁbers (spheroids).”

Horse 2:
“There are multiple nerve fascicles present, with a normal number and

density of myelinated ﬁbers present.”

92

Horse 3:

“The epineurium is moderately expanded by mature collagen. Multiple
nerve fascicles are evident, and the majority of these fascicles contain a
normal number and density of myelinated nerve ﬁbers within each fascicle.
Other fascicles have a moderate loss of myelinated ﬁbers, with scattered
foci of proliferating Schwann cells forming ‘Bungner’s bands’, and
inﬁltration with small numbers of macrophages. There are two foci of
inﬂammation, comprised of moderate numbers of eosinophils and

lymphocytes, with fewer epithelioid macrophages.”

Horse 4:

“The epineurium is moderately expanded by mature collagen. Multiple
nerve fascicles are evident, and the majority of these fascicles contain a
normal number and density of myelinated nerve ﬁbers within each fascicle.
Adjacent to this the nerve bundle, there are several fascicles that are
markedly atrophied, and enmeshed within abundant collagen. The
remaining nerve ﬁbers within the remnant fascicles are devoid of myelin.
Along the edge of the sample, there is a thin rim of epithelioid macrophages,

admixed with small numbers of lymphocytes and occasional eosinophils.”

The CAD muscles were weighed and their volume calculated by measuring
the volume of water displaced in a 250 ml ﬂask. The results are shown in

Table 1, horse 1 demonstrated a loss of half of the weight of the muscle and

93

 

 

a loss of half of the volume of the muscle compared with the right side
(control), both horses 3 and 4 demonstrated a mild decrease in both weight
and volume of the CAD muscle when compared with the right CAD muscle
(control). Horse 2 had no differences between the left and right CAD

muscle.

The CAD muscle pairs were sent for neuromuscular tissue evaluation and
the results are summarized in Table 2. All horses showed signs of
denervation, degeneration and reinnervation when compared with the right
CAD muscle (control). Horse I experienced the largest amount of
denervation and horse 3 showed the least. Horse 1 and 4 suffered from the
largest amounts of muscle degeneration and horse 3 experienced the least
amount. When the degree of reinnervation was evaluated, horse 3 showed
the largest amount, followed by horse 1 with horse 4 experiencing the least
amount of reinnervation. Please see ﬁgure 8 for examples of these

histological ﬁndings in the muscle and the nerve.

d. Discussion and Conclusions

Technical complications involving the wires and the ﬁrst cervical nerve including

premature severing of the wires and inappropriate placement around the nerve

94

 

Horse 1 Horse 2 Horse 3 Horse 4

CAD Wt (g) Vol (cc) Wt (g) Vol (cc) Wt (g) Vol (cc) Wt (g) Vol
(CC)

 

Left 6.13 3.50 7.92 5.50 8.45 8.00 8.55 9.00
Right 8.60 6.50 7.79 5.50 9.45 9.50 11.15 12.00

 

Wt: Weight; (g): grams; Vol: Volume; (cc): centiliters; CAD: Cricoarjytenoideus dorsalis
muscle

Table 1: Weight and volume of the left and right cricoarytenoideus dorsalis
muscle of the 4 horses

95

 

Muscle Horse 1 Horse 2 Horse 3 Horse 4
Region
DN DG RN DN DG RN DN DG RN DN DG RN

 

LCraLat 1.42 0.85 1.00 0.96 1.25 0.49 0.83 0.50 0.67 1.40 1.37 0.35
LCau Med 1.36 1.42 0.76 1.29 0.73 0.85 0.65 0.50 1.37 1.02 0.94 0.26

Combined
for]. CAD 2.78 2.27 1.76 2.25 1.98 1.34 1.48 1.00 1.14 2.42 2.31 0.6]

 

RCraLat 0.02 0.25 0.50 0.00 0.27 0.17 0.27 0.05 0.35 0.00 0.27 0.51
RCau Lat 0.02 0.29 0.83 0.00 0.50 0.17 0.04 0.33 0.17 1.14 0.27 0.40

Combined
fork CAD 0.04 0.54 1.33 0.00 0.77 0.34 0.31 0.38 0.52 1.14 0.54 0.9!

 

Arytenoid None None Yes at the end of the None but no collapse
Stimulation experiment seen
Endoscopy No change No change None No change

UA 11’ No change No change Some abduction at No change

exercise

 

 

 

 

Where L is left; R is right; Cra is cranial; Cau is caudal. CAD is cricarytenoideus dorsalis
muscle; UA IP is upper airway inspiratory pressure; DN is denervation score; DC is
degeneration score; RN is reinnervation score

Table 2: Mean neuromuscular histology score for each muscle region analyzed.

96

 

0

"h. :34.

1‘.‘ " H.101“ “11“”

t

 

Figure 8: A) Longitudinal histologic section of normal peripheral nerve, Horse No. 3
Luxol Fast Blue (LFB) histochemistry. The nerve is comprised of numerous
individual myelinated nerve fibers. The blue represents the myelin sheaths
surrounding the nerve ﬁbers (arrow). Bar = 100nm. B) Histologic cross-section of
normal skeletal muscle (left CAD), horse No. 3 Hematoxylin and eosin histochemisty.
The density, size, and shape of the individual myofibers is normal within the muscle.
Note the normal peripheral localization of the myoﬁber nuclei. Bar = 100um. C)
Longitudinal histologic section of abnormal peripheral nerve histology, Horse No. l
Luxol Fast Blue (LFB) histochemistry. There are no normal nerve ﬁbers present,
and no histochemically detectable myelin in the nerve. The tissue is comprised of
numerous linear bundles of proliferating Schwann cells (‘Bungner’s bands, arrow).
Bar = 100nm. D) Histologic cross-section of abnormal skeletal muscle (left CAD).
Hematoxylin and eosin histochemisty. There are numerous markedly angular, and
atrophic muscle cells (arrow) interspersed with scattered hypertrophic myocytes
with internal nuclei (arrowhead). Bar = 100um.

97

doomed the experiment. Loss of the wires in all horses led to early euthanasia of
the horses. In horses 1 and 2 this resulted in the there being insufﬁcient time for
the NMP graft to reinnervate the CAD and it was not long enough for any
reasonable evaluation of the efﬁcacy of the electrostimulation protocol. In horses
3 and 4, loss of wires occurred later. This left enough time for the NMP graft to

reinnervate the CAD muscle in horse 3 (128 days).

Placement of the wires around the ﬁrst cervical nerve also resulted in signiﬁcant
complications. To secure the wire we used a small stainless steel clamp
(Securosss) to secure the loop. In horse 1 histologic evaluation of the nerve at the
level of the clamp showed that the nerve was severely damaged. In horse 2
however, there were few histologic abnormalities indicating that the wire
placement can be effective. These 2 horses are interesting because horse 1 had a
completely damaged nerve and consequently a CAD muscle with no neural
stimulation. In contrast, horse 2 had a CAD muscle with a ﬁrnctioning NMP graft,
which had been in place for 74 days. When these 2 pairs of CAD muscle were
examined, there was a signiﬁcant difference. Horse 1 had a much smaller (volume
and weight) atrophied left CAD muscle compared to the right (control) CAD,
whereas horse 2 had similar volume and weight of the right and left muscles
(Table 2 and Figure X). The results of the histological neuromuscular evaluation
generally support the gross observations. Horse 2 had mild evidence of

degeneration and denervation but very little signs of reinnervation (Table 1). This

 

5 Securos 443 Main Street, PO Box 950, Fiskdale, MA 01518
98

 

is what would be expected given the time frame; 12 weeks is the earliest time that
reinnervation has been seen (Fulton et al., 2003) and the grafts had only been in
place for 74 days (10 weeks) so it is unlikely that any reinnervation would have
been seen. Apparently, effective placement of the NMP graft was sufﬁcient to
prevent signiﬁcant degeneration and denervation in the left CAD muscle of horse
2. However, horse 1 that had no neurologic supply to the CAD muscle had lost
approximately half of the weight and volume of the left CAD muscle when
compared to the right side, which is what would be expected for a muscle that has
not had any innervation for 102 days. For example, within 5 weeks, denervated
extensor digitorum longus muscles of rats lose 66% of mass, 91% of force, and
76% of ﬁber cross-sectional area (Dow et al. 2004). Furthermore, this horse had

more evidence of denervation and degeneration than any of the other horses.

In horses 3 and 4, the left CAD weighed less than the right and possessed a smaller
volume than its control (Table 2), horse 3 demonstrated a smaller discrepancy than
horse 4. This difference between the two was reﬂected by the fact that there was
more damage to the nerve in horse 4 than horse 3. On histologic neuromuscular
evaluation, horse 3 showed small amounts of degeneration and denervation of the
CAD muscle with the highest amount of reinnervation occurring. In contrast,
horse 4 showed the least amount of reinnervation, the most degeneration and a
substantial amount of denervation. These data would suggest that horse 3 was
beginning to beneﬁt from a healthy NMP graft and the associated reinnervation

whereas the more signiﬁcant damage to the ﬁrst cervical nerve in horse 4 was

99

sufﬁcient to prevent any reinnervation. It is apparent that horse 3 was beginning to

see a return to function and acceptance of the NMP graft.

At no point did electrostimulation of horses 2 and 4 result in abduction of the left
arytenoid as reported in Fulton’s pilot study (Fulton et al., 2003). Instead, we
observed other muscles such as the cricopharyngeus, cricothyroideus and
unidentiﬁed muscles of neck contracting when the electrostimulation was applied.
This would indicate that the current was not applied speciﬁcally to the ﬁrst
cervical nerve and was instead also transmitted to the surrounding musculature.
There is a remote possibility that the lack of abduction could have resulted from

synkinesis (Tucker, 2005) but this seems unlikely due to the short time frame.

As expected, inspiratory pressure increased in all horse demonstrating that left
recurrent laryngeal neurectomy caused upper airway obstruction. Inspiratory
pressure did not change following NMP graft in any of the horses. In horses 1 and
4 this was probably due to ﬁrst cervical nerve damage due to electrode placement.
However, in horses 2 and 3 this is probably because there was insufﬁcient time
between NMP graft placement and measurements. Furthermore, videoendoscopy
indicated that there was no active left arytenoid movement in all horses except
horse 3. In this horse there was a small ‘ﬂicker’ with head elevation and an active
arytenoid abduction during exercise. This correlates well with the histologic
evidence of an undamaged ﬁrst cervical nerve and histologic evidence of

reinnervation. It should be noted that horse 4 demonstrated some persistent

100

 

 

 

resistance to dynamic collapse of the left arytenoid, which could have resulted
from excessive ﬁbrous tissue at the surgery site. A ﬁnding that could be used to
explain the lack of success associated with the procedure in this horse (Tucker,
1989). Horse 3 began to show some promise at the end of the experiment,
response to head lift and also resistance to inspiratory collapse of the left arytenoid
at the initial period of exercise, which soon became overcome by the negative
inspiratory pressure. This supports the theory that horse 3 had a successful NMP

graft that would have gone on to do well given enough time.

In conclusion, unfortunately, it was not possible for us to test our hypothesis. We
cannot draw conclusions on whether electrostimulation speeds up the time taken
for a NMP graft to reduce upper airway obstruction associated with laryngeal

hemiplegia.

101

7. Lessons Learnt: A Look to the Future

In two out of the four horses placement of the electrodes in the manner that we used
resulted in signiﬁcant damage to the ﬁrst cervical nerve rendering it dysfunctional.
Furthermore, the electrodes that we used were not sufﬁciently well insulated, making
it impossible to direct electrostimulation accurately. We did not tie the Teﬂon wires as
Fulton reports instead we used a small metal clamp to hold the two ends together as
they wrapped around the nerve. I believe that the metal clamp provided two problems;
ﬁrstly, it was not insulated so it allowed a current to ﬂow to whatever structure it lay
in contact with. Secondly, it was relatively heavy and would put traction on the nerve,
which may have caused damage, alternatively, any swelling of the nerve or slipping of
the wire as the head and neck moved may have lead to signiﬁcant tightening around
the nerve and subsequent damage. The size of the clamp may have been responsible
the signiﬁcant amount of ﬁbrous tissue that was found at post-mortem, which is
disrupting to the process of reinnervation (Tucker, 1989). Perhaps tying the nerves
would not have resulted in the damage to the nerve and we may have seen more
meaningful results. It is my recommendation that a well-insulated, lightweight and
purpose-built electrode should be used, however, these can be ineffective and
expensive (Fulton et al., 2003). The search for suitable device would be valuable, it is
important that we can demonstrate that electrostimulation of the ﬁrst cervical nerve in

a NMP graft results in abduction of the left arytenoid. Once this circuit is established

102

then the effects of the electrostimulation can be evaluated. It is a technique of promise
that warrants further investigation but it is vital that the surgical technique of electrode
implantation is deliberate and proper. A potentially suitable device may have been
identiﬁed by a Belgian group (Vanschandevijl et al., 2009). A Cybertronics vagal
nerve stimulation electrode (3.00 mm, model 302) was wound around the left recurrent
laryngeal nerve and connected to a pulse generator (Cybertronics, model 102), which
allowed stimulation parameters to be adjusted using a telemetric wand
(Vanschandevijl et al., 2009). Abduction of the left arytenoid cartilage was noted with
stimulation frequencies ranging from 2-30 Hz and stimulation intensity of 1 mA (pulse
width 250 usec during 30 sec). The horse that this stimulation device was implanted
in could be stimulated whilst standing and unsedated. A similar device should be

tested on the ﬁrst cervical nerve following a NMP graft procedure.

In this experiment, we tunneled the wires out of the skin at the level of the distal
aspect of the proximal third of the cervical region. This was a mistake as it allowed
the wires too much exposure and, in hindsight, their damage and loss was unavoidable.
More attention should be paid to their location, tunneling and placement under the
mandible would be appropriate (Fulton et al., 2003) or maintenance in a tissue pocket

may be a solution.

I believe that there is a great deal of potential in the use of electrostimulation of NMP
grafts in horses. It has the possibility of providing an affordable and manageable

method to improve the technique. Due to our inability to test our hypothesis, this

103

piece of research cannot provide any real information on the technique for
electrostimulation other than to note the technical nuances and difﬁculties with placing

electrodes around the ﬁrst cervical nerve. In conclusion, I leave you with a quotation:

“Don 't be discouraged by a failure. It can be a positive experience. Failure is, in a
sense, the highway to success, inasmuch as every discovery of what is false leads us to
seek earnestly after what is true, and every fresh experience points out some form of

error which we shall afterwards carefully avoid. ”

John Keats

English lyric poet (1795 - 1821)

104

References

1.

10.

ll.

Adrearri, CM. and Parente, EJ. (2007) Surgical Treatment of Laryngeal
Hemiplegia and Hemiparesis. In: Equine Respiratory Medicine and Surgery
Eds: McGorum, B.C., Dixon, P.M., Robinson, N.E., Schumacher, J. Elsevier, St.
Louis pp497-508.

Anderson, B.H., Kannegieter, N.J., Goulden, BE. (1997) Endoscopic
observations on laryngeal symmetry and movements in young racing horses. N Z
Vet J. 45: 188-92.

Barber, SM. (1981) Paralaryngeal abscess with laryngeal hemiplegia and
ﬁstulation in a horse. Can Vet J. 22; 389-392.

Barnes, A.J., Slone, D.E., Lynch, TM. (2004) Performance after partial
arytenoidectomy without mucosal closure in 27 Thoroughbred racehorses. Vet
Surg. 33: 398-403.

Bayly, W.M., Grant, B.D., Modransky, P.D. ( 1984) Arterial blood gas tensions
during exercise in a horse with laryngeal hemiplegia, before and after corrective
surgery. Res Vet Sci. 36: 256-8.

Belknap, J .K., Derksen, F.J., Nickels, F.A., Stick, J .A., Robinson, NE. (1990)
Failure of subtotal arytenoidectomy to improve upper airway ﬂow mechanics in

exercising standardbreds with induced laryngeal hemiplegia. Am J Vet Res. 51:
1481-7.

Beroza, GA. (1994) Partial closure of laryngotomies in horses. J Am Vet Med
Assoc. 1994 204: 1227-9.

Bohanon, T.C., Beard, W.L. and Robertson, IT (1990) Laryngeal hemiplegia in
draft horses. A review of 27 cases. Vet Surg. 19: 456-459.

Boulton, E.P., Seeherrnan, H.J., Kirker-Head, C.A., Steckel, RR. (1995) Primary
closure of equine laryngotomy incisions: a review of 42 cases. Vet Surg. 24: 226-
30.

Brakenhoff, J .E., Holcombe, S.J., Hauptrnan, J.G., Smith, H.K., Nickels, F .A.
and Caron, J .P. (2006) The prevalence of laryngeal disease in a large population
of competition draft horses. Vet Surg 35: 579-583.

Brown, J.A., Derksen, F.J., Stick, J .A., Hartrnann, W.M., Robinson, NE. (2003)

Ventriculocordectomy reduces respiratory noise in horses with laryngeal
hemiplegia. Equine VetJ. 35: 570-4.

105

12.

l3.

14.

15.

l6.

l7.

l8.

19.

20.

21.

22.

23.

24.

25.

26.

Brown, J.A., Derksen, F.J., Stick, J .A., Hartmann, W.M. and Robinson, NE.
(2004) Effect of laryngoplasty on respiratory noise reduction in horses with
laryngeal hemiplegia. Equine Vet J. 36: 420-425.

Brown, J .A., Derksen, F.J., Stick, J .A., Hartmann, W.M., Robinson, NE. (2005)
Laser vocal cordectomy fails to effectively reduce respiratory noise in horses
with laryngeal hemiplegia. Vet Surg. 34: 247-52.

Burn, J .F ., Franklin, SH. (2006) Measurement of abnormal respiratory sounds
during over-ground exercise. Equine Vet J. 38: 319-23.

Cahill, J .I. and Goulden, BE. (1986) Equine laryngeal hemiplegia. I. A light
microscopic study of peripheral nerves. New Zealand Vet J 34: 161-169.

Cahill, 1.1. and Goulden, BE. (1986) Equine laryngeal hemiplegia. II. An
electron microscopic study of peripheral nerve. New Zealand Vet J 34: 170-175.

Cahill, J .I. and Goulden, BE. (1986) Equine laryngeal hemiplegia. III. A teased
ﬁbre study of peripheral nerves. New Zealand Vet J 34: 181-185.

Cahill, II. and Goulden, BE. (1986) Equine laryngeal hemiplegia. IV. Muscle
pathology. New Zealand Vet J34: 186-190.

Cahill, J .I. and Goulden, BE. (1986) Equine laryngeal hemiplegia. V. Central
nervous system pathology. New Zealand Vet J34: 191-193.

Cahill, J .I. and Goulden, BE. (1987) The pathogenesis of equine laryngeal
hemiplegia-a
review. New Zealand Vet J35: 82-90.

Cahill, J.I. and Goulden, BE. (1989) Further evidence for a central nervous
system component in equine laryngeal hemiplegia. New Zealand Vet J 37: 89-90.

Chalmers, H.J., Cheetham, J ., Yeager, A.E., Ducharme, NC. (2006)
Ultrasonography of the equine larynx. Vet Radio] Ultrasound. 47: 476-81.

Cheetham, J ., Radcliffe, C.R., Ducharme, N.G., Sanders, I., Mu, L. and
Hennanson, J.W. (2008) Neuroanatomy of the equine dorsal cricoarytenoid
muscle: Surgical implications. Equine Vet J 40: 70-75.

Church, S., Wyn-Jones, G., Parks A.H., Ritchie, HE. (1986) Treatment of
guttural pouch mycosis. Equine Vet J. 18: 362-5.

Cole, CR. (1946) Changes in the equine larynx associated with laryngeal
hemiplegia. Am. J. Vet. Res 7: 69-77.

106

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

Cole, BC. and Gardiner, PF. (1984) Does electrical stimulation of denervated
muscle, continued after reinnervation, inﬂuence recovery of contractile function?
Exp Neurol 85: 52-62.

Cook, W.R. (1970) A comparison of idiopathic laryngeal paralysis in man and
horse. Journal of laryngology and otolaryngology 84: 819-835.

Cook, W.R. (1988) Diagnosis and grading of hereditary recurrent laryngeal
neuropathy in the horse. J Eq Vet Sci. 8: 432-455.

Cramp, P., Derksen, F.J.D., Stick, J.A., Nickels, F .A., Brown K.E., Robinson, P.,
Robinson, NE. (2009) Effect of ventriculectomy versus ventriculocordectomy

on upper airway noise in draft horses with recurrent laryngeal neuropathy.
Equine VetJ. 41 (fastrack)

Dart, A.J., Dowling, RA, Smith, CL. (2005) Upper airway dysﬁrnction
associated with collapse of the apex of the comiculate process of the left
arytenoid cartilage during exercise in 15 horses. Vet Surg. 34: 543-7.

Dart, A.J., Dowling, B.A., Hodgson, D.R., Rose, R]. (2001) Evaluation of high-
speed treadmill videoendoscopy for diagnosis of upper respiratory tract
dysfunction in horses. Aust VetJ. 79: 109-12.

Davenport, C.L., Tulleners, E.P., Parente, El. (2001) The effect of recurrent
laryngeal neurectomy in conjunction with laryngoplasty and unilateral
ventriculocordectomy in thoroughbred racehorses. Vet Surg. 302417-21.

Davenport-Goodall, C.L., Parente, EJ. (2003) Disorders of the larynx. Vet Clin
North Am Equine Pract. 19: 169-87.

Davidson, E.J., Martin, B.B. Jr, Parente, El. (2007) Use of successive dynamic
videoendoscopic evaluations to identify progression of recurrent laryngeal
neuropathy in three horses. J Am Vet Med Assoc. 230:555-8.

Dean, P.W., Nelson, J .K., Schumacher, J. (1990) Effects of age and prosthesis
material on in vitro cartilage retention of laryngoplasty prostheses in horses. Am
J Vet Res. 51: 114-7.

Derksen, F]. (2006) Overview of Upper Airway Function. In: Equine surgery,
Eds: J.A. Auer and J .A. Stick, Elsevier, St. Louis. pp516-522.

Derksen, F]. (2007) Evaluation of upper respiratory tract sounds. In: Equine
Respiratory Medicine and Surgery Eds: McGorum, B.C., Dixon, P.M.,
Robinson, N.E., Schumacher, J. Elsevier, St. Louis pp249-255.

107

39.

40.

41.

42.

43.

44.

45.

46.

47.

48.

49.

Derksen, F.J., Stick, J .A., Scott, E.A., Robinson, N.E., Slocombe, RF. (1986)
Effect of laryngeal hemiplegia and laryngoplasty on airway ﬂow mechanics in
exercising horses. Am J Vet Res. 47: 16-20.

Derksen, F .J ., Holcombe, S.J., Hartmann, W., Robinson, NE. and Stick, J .A.
(2001) Spectrum analysis of respiratory sounds in exercising horses with

experimentally induced laryngeal hemiplegia or dorsal displacement of the soft
palate. Am J Vet Res 62: 659-664.

Dixon, P.M., McGorum, B.C., Railton, D.I., Hawe, C., Tremaine, W.H., Pickles,
K. and McCann, J. (2001) Laryngeal paralysis: a study of 375 cases in a mixed-
breed population of horses. Equine Vet J33: 452-458.

Dixon, P.M., McGorum, B.C,. Railton, D.I., Hawe, C., Tremaine, W,H,, Dacre
K, McCann J. (2003a) Long-tenn survey of laryngoplasty and
ventriculocordectomy in an older, mixed-breed population of 200 horses. Part 1:

Maintenance of surgical arytenoid abduction and complications of surgery.
Equine VetJ. 35:389-96

Dixon, P.M., McGorum, B.C., Railton, D.I., Hawe, C., Tremaine, W.H., Dacre,
K. and McCann, J. (2003b) Long-term survey of laryngoplasty and
ventriculocordectomy in an older, mixed-breed population of 200 horses. Part 2:
Owners' assessment of the value of surgery. Equine Vet J. 35: 397-401.

Dow, D.E., Cedema, P.S., Hassett, C.A., Kostrominova, T.Y., Faulkner, J .A. and
Dennis, RC. (2004) Number of contractions to maintain mass and force of a
denervated rat muscle. Muscle Nerve 30: 77-86.

Ducharme, N.G., Hackett, RP. (1991) The value or surgical treatment of
laryngeal hemiplegia in horses. Comp. Cont. Edu. Pract. Vet. 13: 472-474.

Ducharme, N.G., Homey, F.D., Partlow, CD. and Hulland, T.J. (1989) Attempts
to restore abduction of the paralyzed equine arytenoid cartilage. I. Nerve-muscle
pedicle transplants. Can J Vet Res. 53: 202-209.

Dudley, G.A., Castro, M.J., Rogers, S. and Apple, D.F., Jr. (1999) A simple
means of increasing muscle size after spinal cord injury: a pilot study. Eur J Appl
Physiol Occup Physiol 80: 394-396.

Duncan, ID. (1992) Determination of the early age of onset of equine recurrent
laryngeal neuropathy. 2. Nerve pathology. Acta Neuropathol. 84: 316-21.

Duncan, ID. and Baker, G.J. (1987) Experimental crush of the equine recurrent

laryngeal nerve: a study of normal and aberrant reinnervation. Am J Vet Res 48:
431-438.

108

50.

51.

52.

53.

54.

55.

56.

57.

58.

59.

60.

61.

62.

Duncan, ID. and Brook, D. (1985) Bilateral laryngeal paralysis in the horse.
Equine VetJ. 17: 228-33.

Duncan, I.D., Grifﬁths, LR. and Madrid, RE. (1978) A light and electron
microscopic study of the neuropathy of equine idiopathic laryngeal hemiplegia.
Neuropathol Appl Neurobiol 4: 483-501.

Duncan, I.D., Griffths, I.R., McQueen, A. and Baker, GO. (1974) The pathology
of equine laryngeal hemiplegia. Acta Neuropathol (Ber!) 27: 337-348.

Duncan, 1D. and Hammang, J .P. (1987) Ultrastructural observations of organelle
accumulation in the equine recurrent laryngeal nerve. J Neurocytol. 16: 269-80.

Durando, M.M., Martin, B.B., Hammer, E.J., Langsarn, S.P., Birks, E.K. (2002)
Dynamic upper airway changes and arterial blood gas parameters during
treadmill exercise. Equine Vet J Suppl. 34: 408-12

Fleming, G. Laryngismus paralytica (“Roaring”). (1882) Vet J of Comp Path. 14:
1-12.

Franklin, S.H., Usmar, S.G., Lane, J .G., Shuttleworth, J ., Burn, J .F. (2003)
Spectral analysis of respiratory noise in horses with upper airway disorders.
Equine Vet J. 35: 264-8.

Franklin, S.H., Naylor, J .R., Lane, J .G. (2006) Videoendoscopic evaluation of
the upper respiratory tract in 93 sport horses during exercise testing on a high-
speed treadmill. Equine Vet J Suppl. 36: 540-5.

Franklin, H., Burnt, J .F., Allen, K]. (2008) Clinical trials using a telemetric
endoscope for use during over-ground exercise: a preliminary study. Equine Vet
J. 40: 712-5.

F u, S.Y. and Gordon, T. (1995) Contributing factors to poor functional recovery
after delayed nerve repair: prolonged denervation. J Neurosci 15: 3886-3 895.

Fulton, LC. (2007) Equine Laryngeal Reinnervation. In: Equine Respiratory
Medicine and Surgery Eds: McGorum, B.C., Dixon, P.M., Robinson, N.E.,
Schumacher, J. Elsevier, St. Louis pp509-515

Fulton, I.C., Stick, J .A. and Derksen, F .J . (2003) Laryngeal reinnervation in the
horse. Vet Clin North Am Equine Pract 19: 189-208.

Fulton, I.C., Derksen, F.J., Stick, J .A., Robinson, NE. and Walshaw, R. (1991)

Treatment of left laryngeal hemiplegia in standardbreds, using a nerve muscle
pedicle graft. Am J Vet Res 52: 1461-1467.

109

63.

64.

65.

66.

67.

68.

69.

70.

71.

72.

73.

74.

75.

Getty, R. ed. (1975) Sisson and Grossman’s The anatomy of the domestic
animals. 5th ed. WB Saunders Co. Philadelphia. 376-454, 498-524, 650-670.

Gilbert, G.H. (1972) laryngeal hemiplegia following jugular injury. J Am Vet
Med Assoc. 171: 1686-1687.

Goding Jr., GS. (2005) Laryngeal Reinnervation. In: Cummings Otolaryngology
Head & Neck Surgery, 4 edn., Ed: P.W.F. C.W. Cummings, B.H. Haughey, K.T.
Robbins, J. R. Thomas, L.A. Harker, M.A. Richardson, and DE. Schuller,
Elsevier, Philadelphia. pp 2207-2221.

Gondin, J ., Guette, M., Ballay, Y. and Martin, A. (2006) Neural and muscular
changes to detraining after electrostimulation training. Eur J Appl Physiol 97:
165-173.

Goulden, B.E., Barnes, G.R., Quinlan, T.J. (1976) The electromyographic
activity of intrinsic laryngeal muscles during quiet breathing in the anaesthetized
horse. N Z Vet J. 24: 157-62.

Goulden, B.E., Anderson, L.J. (1985) Equine laryngeal hemiplegia, Part 1:
Physical characteristics of affected animals. N Z Vet J. 29: 151-4.

Goulden, B.E., Anderson, L.J. (1981) Equine laryngeal hemiplegia part 11: Some
clinical observations. N Z Vet J. 29: 194-8.

Goulden, B.E., Anderson, L.J. (1982) Equine laryngeal hemiplegia. Part 111.
Treatment by laryngoplasty. N Z Vet J. 30: 1-5.

Gray, W.P., Keohane, C. and Kirwan, WC. (1997) Motor nerve transplantation.
J Neurosurg 87: 615-624.

Green, H., Goreham, C., Ouyang, J ., Ball-Bumett, M. and Ranney, D. (1999)
Regulation of ﬁber size, oxidative potential, and capillarization in human muscle
by resistance exercise. Am J Physiol. 276: 591-596.

Greet, T.R., Baker, G.J., Lee, R. (1979) The effect of laryngoplasty on
pharyngeal function in the horse. Equine Vet J. 11: 153-8.

Hahn, C.N., Matiasek, K., Dixon, P.M., Molony, V., Rodenacker, K., Mayhew,
LG. (2008) Histological and ultrastructural evidence that recurrent laryngeal

neuropathy is a bilateral mononeuropathy limited to recurrent laryngeal nerves.
Equine VetJ. 40: 666-72.

Hahn, C.N., Mayhew, LG. (2007) Idiopathic recurrent laryngeal neuropathy:
etiopathogenesis. In: Equine Respiratory Medicine and Surgery Eds: McGorum,

110

76.

77.

78.

79.

80.

81.

82.

83.

84.

85.

86.

87.

BC, Dixon, P.M., Robinson, N.E., Schumacher, J. Elsevier, St. Louis pp473-
477.

Hall, L.W., Young, 8.8., Franklin, R.J., Jefferies, A.K., Corke, M.J. (1990) Use
of the forced oscillating airﬂow technique to measure the resistance of the equine

upper airway: effects of laryngoventriculectomy and laryngoplasty. Res Vet Sci.
49: 229-35.

Hammer, E.J., Tulleners, E.P., Parente, E.J., Martin, B.B. Jr. (1998)
Videoendoscopic assessment of dynamic laryngeal function during exercise in

horses with grade-III left laryngeal hemiparesis at rest: 26 cases (1992-1995). J
Am Vet Med Assoc. 212: 399-403.

Harrison, G.D., Duncan, I.D., Clayton, MK. (1992) Determination of the early

age of onset of equine recurrent laryngeal neuropathy. 1. Muscle pathology. Acta
Neuropathol. 84: 307-15.

Hawkins, J .F., Tulleners, E.P., Ross, M.W., Evans, LH. and Raker, CW. (1997)
Laryngoplasty with or without ventriculectomy for treatment of left laryngeal
hemiplegia in 230 racehorses. Vet Surg 26: 484-491.

Hawkins, J .F., Andrews-J ones, L. (2001) Neodymiumzyttrium aluminum garnet
laser ventriculocordectomy in standing horses. Am J Vet Res. 62: 531-7.

Haynes, P.F., McClure, J .R. and Waters, J .W. (1984) Subtotal arytenoidectomy
in horses: an update. Proceedings, American Association of Equine Practitioners
30: 21-23.

Henderson, C.E., Sullins, KB. and Brown, J .A. (2007) Transendoscopic, laser-
assisted ventriculocordectomy for treatment of left laryngeal hemiplegia in

horses: 22 cases (1999-2005). J Am Vet Med Assoc. 231: 1868-1872.

Hengerer, A.S., Tucker, HM. (1973) Restoration of abduction in the paralyzed
canine vocal cord. Arch 0tolaryngol. 97 : 247-50.

Hobday, F. (1911) The merits of the Williams’ operation for roaring. Proc 48’”
Ann Conv ofA VMA. 291-198.

Hobday, F. (1935) The surgical treatment of roaring in horses. Veterinary
Research 15: 1535-1539.

Hoh, J .F. (2005) Laryngeal muscle ﬁbre types. Acta Physiol Scand 183: 133-149.
Holcombe, S.J., Comelisse, C.J., Bemey, C., Robinson, NE. (2002)

Electromyographic activity of the hyoepiglotticus muscle and control of
epiglottis position in horses. Am J Vet Res. 63: 1617-21.

111

88.

89.

90.

91.

92.

93.

94.

95.

96.

97.

98.

Holcombe, S.J., Derksen, F .J ., Robinson, NE. (2007) Electromyographic
activity of the palatinus and palatopharyngeus muscles in exercising horses.
Equine Vet J. 39: 451-5.

J ansson, N., Ducharme, N.G., Hackett, RP. and Mohammed, H0. (2000) An in
vitro comparison of cordopexy, cordOpexy and laryngoplasty, and laryngoplasty
for treatment of equine laryngeal hemiplegia. Vet Surg 29: 326-334.

Hughes, K.J., McGorum, B.C., Love, S., Dixon, RM. (2009) Bilateral laryngeal
paralysis associated with hepatic dysfunction and hepatic encephalopathy in six
ponies and four horses. Vet Rec. 164: 142-7.

Huntington, P.J., Jeffcott, L.B., Friend, S.C., Luff, A.R., Finkelstein, D.I., Flynn,
R.J. (1989) Australian Stringhalt--epidemiological, clinical and neurological
investigations. Equine Vet J. 21: 266-73.

Kidd, J .A., Slone, DE. (2002) Treatment of laryngeal hemiplegia in horses by
prosthetic laryngoplasty, ventriculectomy and vocal cordectomy. Vet Rec. 150:
481-4.

Kingham, P.J., Terenghi, G. and Birchall, M.A. (2006) Tissue engineering
strategies for reinnervation of the larynx. Clin Otolaryngol. 31: 245.

Kraus, B.M., Parente, E]. and Tulleners, ER (2003) Laryngoplasty with
ventriculectomy or ventriculocordectomy in 104 draft horses (1992-2000). Vet
Surg 32: 530-538.

Lane, J .G. (1987) F ibreoptic endoscopy of the equine upper respiratory tract: a
commentary on progress. Equine Vet J. 19: 495-499.

Lane, J .G., Bladon, B., Little, D.R., Naylor, J .R., Franklin, S.H. (2006a)
Dynamic obstructions of the equine upper respiratory tract. Part 1: observations

during hi gh-speed treadmill endoscopy of 600 Thoroughbred racehorses. Equine
Vet J. 38: 393-9.

Lane, J .G., Bladon, B., Little, D.R., Naylor, J .R., Franklin, S.H. (2006b)
Dynamic obstructions of the equine upper respiratory tract. Part 2: comparison of
endoscopic ﬁndings at rest and during high-speed treadmill exercise of 600
Thoroughbred racehorses. Equine Vet J. 38: 401-7.

Loew, F.M. ( 1973) Thiamin and equine laryngeal hemiplegia. Vet Rec. 97 : 372-
373.

112

99. Lumsden, J .M., Derksen, F.J., Stick, J .A., Robinson, NE. and Nickels, FA.
(1994) Evaluation of partial arytenoidectomy as a treatment for equine laryngeal
hemiplegia. Equine VetJ 26: 125-129.

100. Lundborg, G. (1988) Intraneural microcirculation. Orthop Clin North Am. 19: 1-
12.

101. Lussier, B., Flanders, J .A. and Erb, H.N. (1996) The effect of unilateral
arytenoid lateralization on rima glottidis area in canine cadaver larynges. Vet
Surg 25: 121-126.

102. MacQueen, J. (1896) Roaring, whistling and grunting. J Comp Path and
Therapy. 9: 112-119.

103. Mafﬁuletti, N.A., Zory, R., Miotti, D., Pellegrino, M.A., Jubeau, M. and
Bottinelli, R. (2006) Neuromuscular adaptations to electrostimulation resistance
training. Am J Phys Med Rehabil. 85: 167-175.

104. Marks, D., Mackay-Smith, M.P., Cushing, LS. and Leslie, J .A. (1970) Use of a
prosthetic device for surgical correction of laryngeal hemiplegia in horses. J Am
Vet Med Assoc. 157: 157-163.

105. Marlin, D. and Nankrvis, K. (2003) The Respiratory System. In: Equine Exercise
Physiology. Blackwell Publishing, Oxford UK pp42-55.

106. Marqueste, T., Alliez, J .R., Alluin, 0., J ammes, Y. and Decherchi, P. (2004)
Neuromuscular rehabilitation by treadmill running or electrical stimulation after
peripheral nerve injury and repair. J Appl Physiol 96: 1988-1995.

107. McCall, J. (1889) A review of: Roaring in horses (laryngimus paralyticus). Its
history, nature, causes, prevention and treatment. London: Balliere, Tindall and
Lex. The Vet J. 28: 265-271.

108. Morris, EA. and Seeherman, H.J. (1990) Evaluation of upper respiratory tract
function during strenuous exercise in racehorses. J Am Vet Med Assoc 196: 431-
438.

109. Newton-Clarke, M.J., Divers, T.J., Delahunta, A., Mohammed, H0. (1994)
Evaluation of the thoraco-laryngeal reﬂex ('slap test') as an aid to the diagnosis
of cervical spinal cord and brainstem disease in horses. Equine Vet J. 26: 358-61.

110. Parente, E]. (2007) Laser surgery of the upper respiratory tract. In: Equine

Respiratory Medicine and Surgery Eds: McGorum, B.C., Dixon, P.M.,
Robinson, N.E., Schumacher, J. Elsevier, St. Louis pp533-543.

113

111.Parente, E.J., Martin, B.B., Tulleners, E.P., Ross, M.W. (2002) Dorsal
displacement of the soft palate in 92 horses during hi gh-speed treadmill
examination (1993-1998). Vet Surg. 31: 507-12.

112. Parente, E.J., Tulleners, E.P., Southwood, LL. (2008) Long-term study of partial
arytenoidectomy with primary mucosal closure in 76 Thoroughbred racehorses
(1992-2006). Equine VetJ. 40: 214-8.

113. Payne, S.H., Jr. and Brushart, T.M. (1997) Neurotization of the rat soleus
muscle: a quantitative analysis of reinnervation. J Hand Surg [Am] 22: 640—643.

114. Pollock, P.J., Reardon, R.J.M., Parkin, T.D.H., Johnston, M.S., Tate, J ., Love, S.
(2009) Dynamic respiratory endoscopy in 67 Thoroughbred racehorses training
under normal ridden exercise conditions. Equine Vet J. 41 (fastrack)

115. Poncet, P.A., Montavon, S., Gaillard, C., Barrelet, F., Straub, R., Gerber, H.
(1989) A preliminary report on the possible genetic basis of laryngeal
hemiplegia. Equine VetJ. 21: 137-8.

116. Poortmans, A. and Wyndaele, J .J . (2002) Preventing fatigue of fast striated
muscles of the pelvic ﬂoor and slow striated muscles of the limb by manipulating
the on-off time of electric stimulation. Arch Phys Med Rehabil. 83: 550-554.

117. Radcliffe, C.H., Woodie, J .B., Hackett, R.P., Ainsworth, D.M., Erb, H.N.,
Mitchell, L.M., Soderholm, L.V., Ducharme, NC. (2006) A comparison of
laryngoplasty and modiﬁed partial arytenoidectomy as treatments for laryngeal
hemiplegia in exercising horses. Vet Surg. 35: 643-52.

118. Rakesh, V., Ducharme, N.G., Cheetham, J ., Datta, A.K., Pease, AP. (2008)
Implications of different degrees of arytenoid cartilage abduction on equine
upper airway characteristics. Equine Vet J. 40: 629-35.

119. Rakestraw, P.C., Hackett, R.P., Ducharme, N.G., Nielan, G.J., Erb, H.N. (1991)
Arytenoid cartilage movement in resting and exercising horses. Vet Surg. 20:
122-7.

120. Raphel, CF. (1982) Endoscopic ﬁndings in the upper respiratory tract of 479
horses. J Am Vet Med Assoc. 181: 470-473.

121 . Rigg, D.L., Ramey, D.L., Reinertson, EL. (1985) Tracheal compression
secondary to abscessation of cranial mediastinal lymph nodes in a horse. J Am
Vet Med Assoc. 186: 283-4.

122. Robinson, NE. (2007) How horses breathe: the respiratory muscles and the
airways. In: Equine Respiratory Medicine and Surgery Eds: McGorum, B.C.,
Dixon, P.M., Robinson, N.E., Schumacher, J. Elsevier, St. Louis ppl9-33.

114

123. Robinson, P., Derksen, F.J., Stick, J .A., Sullins, K.E., DeTolve, PG. and
Robinson, NE. (2006) Effects of unilateral laser-assisted ventriculocordectomy
in horses with laryngeal hemiplegia. Equine Vet J. 38: 491-496.

124. Rooney, JR. and Delaney, EM. (1970) A hypothesis on the causation of
laryngeal hemiplegia in the horse. Equine VetJ. 2: 35-37.

125. Rose, R.J. (1978) Acute laryngeal paralysis in Arabian foals. Aust Vet J. 54: 154.

126. Rose, R.J., Hartley, W.J., Baker, W. (1981) Laryngeal paralysis in Arabian foals
associated with oral haloxon administration. Equine Vet J. 13: 171-6.

127. Rossignol, F., Perrin, R., Desbrosse, F. and Elie, C. (2006) In vitro comparison
of two techniques for suture prosthesis placement in the muscular process of the
equine arytenoid cartilage. Vet Surg. 35: 49-54.

128. Russell, AP. and Slone, DE. (1994) Performance analysis after prosthetic
laryngoplasty and bilateral ventriculectomy for laryngeal hemiplegia in horses:
70 cases (1986-1991). JAm Vet Med Assoc. 204: 1235-1241.

129. Schumacher, J ., Wilson, A.M., Pardoe, C. and Easter, J .L. (2000) In vitro
evaluation of a novel prosthesis for laryngoplasty of horses with recurrent
laryngeal neuropathy. Equine VetJ. 32: 43-46.

130. Shappell, K.K., Derksen, F.J., Stick, J .A. and Robinson, NE. (1988) Effects of
ventriculectomy, prosthetic laryngoplasty, and exercise on upper airway function
in horses with induced left laryngeal hemiplegia. Am J Vet Res. 49: 1760-1765.

131. Scherzer, S., Hainisch, E.K. (2005) Evaluation of a canine cranial cruciate
ligament repair system for use in equine laryngoplasty. Vet Surg. 34: 548-53.

132. Shires, G.M., Adair, H.S., Patton, CS. (1990) Preliminary study of laryngeal
sacculectomy in horses, using a neodymiumzyttrium aluminum garnet laser
technique. Am J Vet Res. 51: 1247-9.

133. Stick, J .A., Peloso, J .G., Morehead, J .P., Lloyd, J ., Eberhart, S., Padungtod, P.,
Derksen, F.J. (2001) Endoscopic assessment of airway function as a predictor of
racing performance in Thoroughbred yearlings: 427 cases (1997-2000). J Am Vet
Med Assoc. 219: 962-7.

134. Stick, J .A. (2006) The larynx. In: Equine surgery, Eds: J .A. Auer and J .A. Stick,
Elsevier, St. Louis. pp.566-590.

115

 

135. Stick, J .A. and Derksen, F.J. (1989) Use of videoendoscopy during exercise for
determination of appropriate surgical treatment of laryngeal hemiplegia in a colt.
J Am Vet Med Assoc. 195: 619-22.

136. Strand, E., Martin, G.S., Haynes, P.F., McClure, J .R., Vice, JD. (2000) J Am Vet
Med Assoc. 217: 1689-96.

137. Strong, W.J. and Plintnik, GR. (1992) Music, Speech and Audio, Soundprint,
Provo UT.

138. Su, W.F., Hsu, Y.D., Chen, RC. and Sheng, H. (2007) Laryngeal reinnervation
by ansa cervicalis nerve implantation for unilateral vocal cord paralysis in
humans. J Am Coll Surg. 204: 64—72.

139. Tan, R.H., Dowling, B.A., Dart, A]. (2005) High-speed treadmill
videoendoscopic examination of the upper respiratory tract in the horse: the
results of 291 clinical cases. VetJ. 170: 243-8.

140. Tate, L.P., Corbett, W.T., Bishop, B.J., Foreman, J .H. (1993) Blood gas tensions,
acid-base status, heart rates, and venous proﬁles in exercising horses with
laryngeal hemiplegia before and after corrective surgery. Vet Surg. 22: 177-83.

141 . Taylor, S.E., Barakzai, S.Z., Dixon, P. (2006) Ventriculocordectomy as the sole
treatment for recurrent laryngeal neuropathy: long-term results from ninety-two
horses. Vet Surg. 35: 653-7.

142. Terenghi, G. (1998) Peripheral nerve regeneration and neurotrophic factors. J.
Anat. 194: 1—14.

143. Tessier, C., Holcombe, S.J., Stick, J .A., Derksen, F.J., Boruta, D. ( 2005)
Electromyographic activity of the stylopharyngeus muscle in exercising horses.
Equine VetJ. 37: 232-5.

1441Tetens, J ., Derksen, F.J., Stick, J .A., Lloyd, J .W. and Robinson, NE. (1996)
Efﬁcacy of prosthetic laryngoplasty with and without bilateral
ventriculocordectomy as treatments for laryngeal hemiplegia in horses. Am J Vet
Res. 57: 1668-1673.

145. Todhunter, R.J., Brown, C.M., Stickle, R. (1985) Retropharyngeal infections in
ﬁve horses. J Am Vet Med Assoc. 187: 600-4.

146. Tucker, HM. (1976) Human laryngeal reinnervation. Laryngoscope. 86: 769-79.

147. Tucker, HM. (1979) Reinnervation of the paralyzed larynx: A review. Head and
Neck Surgery 1: 235-242.

116

148. Tucker, HM. (1989) Long-term results of nerve-muscle pedicle reinnervation for
laryngeal paralysis. Ann Otol Rhinol Laryngol 98: 674-676.

149. Tucker, HM. (2006) Laryngeal reinnervation. In: Diagnosis and treatment of
voice disorders, Eds: J .S. Rubin, R.T. Sataloff and GS. Korovin, Plural
Publishing, San Diego. pp. 727-733.

150. Tucker, HM. and Rusnov, M. (1981) Laryngeal reinnervation for unilateral
vocal cord paralysis: long—term results. Ann Otol Rhinol Laryngol. 90: 457-459.

151.Tulleners, E.P., Harrison, I.W., Mann, P., Raker, C.W. ( 1988) Partial
arytenoidectomy in the horse with and without mucosal closure. Vet Surg. 17:
252-7.

152. Tyler, R. (2000) Experiences with the nerve muscle pedicle graft. Proceedings
of the British Equine Veterinary Association Congress. Equine Veterinary
Journal Ltd. Newmarket pp. 86-87.

153. Vanschandevijl, K., Nollet, H., Vonck, K., Boon, P., Raedt, R., Van Roost,
Martens, A., Deprez, P. (2009) Functional electrical stimulation of the left
recurrent laryngeal nerve with an implanted vagal nerve electrical stimulation

device in a normal horse. J Vet Int Med 23: 436 E CEIM 2009 Abstracts

154. Vitenzon, A.S., Mimonov, EM. and Petrushanskaya, K.A. (2005) Functional
electrostimulation of muscles as a method for restoring motor functions.
Neurosci Behav Physiol. 35: 709-714.

155. White, NA. (1992) Surgical treatment of laryngeal abnormalities: Current
recommendations. In: Proceedings of the American College of Veterinary
Surgeons Symposium. pp. 86-89.

156. Williams, J .W., Pascoe, J .R., Meagher, D.M., Homof, W.J. (1990) Effects of left
recurrent laryngeal neurectomy, prosthetic laryngoplasty, and subtotal
arytenoidectomy on upper airway pressure during maximal exertion. Vet Surg.
19: 136-41.

157. Willoughby, RA, MacDonald, E., McSherry, B.J., Brown, G. (1972) Lead and
zinc poisoning and the interaction between Pb and Zn poisoning in the foal. Can
J Comp Med. 36: 348-59.

158. Witte, T.H., Mohammed, H.O., Radcliffe, C.H., Hackett, R.P., Ducharme, NC.
(2009) Racing performance after combined prosthetic laryngoplasty and
ipsilateral ventriculocordectomy or partial arytenoidectomy: 135 Thoroughbred
racehorses competing at less than 2400 In (1997-2007). Equine Vet J. 41: 70-5.

117

159. Wortley, J. (1905) Diseases of the larynx. In: The horse its treatment in health
and disease with a complete guide to breeding training and management.
Divisional Volume H. The Gresham Publishing Company, London. pp. 507-512.

118

   

"111111111111lllljlllllllllllllll’s

3 62 8865