. _r, \ b1 ' 111.1 ““11“. “ figmwl11‘1“‘§1:’i‘g ‘-‘ ”1:1. ‘1' "“ ::"“- 1-,“ .3”. . ”‘I1-3<‘_ - :,T||‘,‘_,'S_" '. , . 1L“ '333‘1‘331 ”“1, H01!" 111'; :1' 11:1“:"1 . :':‘o‘1'3:!‘}"‘1‘ 21““. 1211‘ . 'i- “—1. ‘1 4‘ " ~ I‘ I in , _.,, 1,1; , 1.1, . ,33 q, 13311.11 ~ - ~19. 1111,13 In. MW 2 1 . 1.. . "I' 31.3,, ‘J, L ”1:“- 111; L :n 31135111131141”: 1.1 “11211-3; 3 '333‘ 313,-.,,111111:;:L-11 331114113331?L113M11V11c11'31421g “It? mvh112bflhlgi; 1'1‘1‘ I" "' 1:11:11, 11111311. 111135“? :2]. qua 1?“, 3,336,111. ,itggtfi’é'g’k“ 1" 1;; “15' 3171:1113 '1 1111‘ Il1q£$1"‘,‘"% 1 21M :3” ¥;11W“;;1‘j§sh;11fi:k'1y" : 1| ‘3}. ‘V n 139'1 ,11113'333 §1331w111r [511, 3:24:21“ 1%? .. . 1 ' 157‘ . 1'1 311111131311 . ., 3., >11 I 11.5712," , 1 . .. -.| 21.1?‘1 , 3 11254;} ,1 1. m~ .1. , 11.31.,.1..s*-1~11111-- " 1:.1: ’ 51‘11‘113' 3,3,1, I ‘0 I l ,1, 1.1-1.1 -, 3 ., .1 3, 11 '. "‘1‘ A. 1911' ,9 1.131;,1 11111,, 1‘1" 3,3. X" "1:5: 3.4,: .‘al'i:!?!11 - “1.113.312.5115 123,119-111in "’ .1111 “I“ II " ‘:I§‘i1}‘._'1_11m”_3 .. 4‘ “9 111- 11.1? :1 .‘ 113I' 3* 2111111 I 1 1 . . I'M- '- 1 4 -. fry—3‘ n: “ma 3". .113 133i, xI '1 ’ 3 "11,1711 ‘1 x 111:. . l. 21:? 533113;“ ‘ 1,111”; - 12:,“ w “W" -..':_.:.: . .5. a.-i .7» 'T‘:- - :_I ‘1"11"1.‘,f1‘ l'.,.‘ ” -‘ a ‘111111‘fi‘t'iil‘1' 1 ‘2 _ . V .3. 1‘41 .1 . 3 3 _w ‘. ' . 9’5'151‘3. ‘M‘ I t "“1011 11’" 1 " :’-’} 'f’R-1I‘1,'.§ru'r .11: 1*1 ’1.'Jo'- “‘A. "31‘1"‘{‘ H . 1 1.11,, “I“ 1 ”$1.61,.“ . 120.3,, LI.“ 1.. * ‘---.= 11 “ , 3., A .1 ‘1‘}: 1 "111111 3'33, 13111164132151. " - . *1}; .:,1~~.;=1111,.1~11- {,3 £1 11! 321.13, .1‘ W1 1 H; 13% 11:? WW (,1 «114’! ,g ‘1’}! 11"“!11214 If; 1 11311,; .- .23 .. . .a'. . . ... 1...- wfi."1ip; $311,131; . 3‘: .3112” .‘;o']. 2'») v '4 . 1111121; I r‘X " 4112,1311’ -‘ . I ..‘.1 'iflx‘. 111 3,6: ~11, ‘ ffu‘Qfi " ,,.-i:» 112‘111, 331111,:ij n Jun: 1‘1in .111 ~: 1 - 1 1 1 :" ' '. ‘2 .1“: 1 ":1: . '1 ‘35:; ,1) 3 3.23% 1' :23: “’1, .,,€,.'-..' 133113“, 1‘ , ‘ , 3 33 3:226:31 13,3533, ,1}, 1 -:;;,, 9% 11.1,, “:11 114%,: ,1. ”111 ». 13131,"... 15131.1” ‘ ”IE1 1% 1“"1‘ ' . 11111111: 3’! ,1 . 1'11 11-11,. ~ ' , , 111111,! ‘ ' ‘ 1 ‘. _...__,‘ ...-—-_1 . “ ‘. . . a 15.1. ,.-v . 9... _. .. , ..._. E?“ I 0‘ .. . . J. \I. 4.. &.. __2.. _ .4. '53:. 1“ . . . . .. ‘ .7: f .fi m. ., ‘5 I :~’ I 1f, ' I Ev}; _ (3!!!“ $ 1 Max-33 3.131339. . "VJ.“ 113,311,111 ' 1.1111 ‘11; #135, "‘32.!" .E‘fi1q33 , “(‘11 13,531. 1 J. r ”-3?“ 1 1-11. I ,1145.‘ . 1:111 1‘.‘ ,- THESlS lulllllllllfilllllllllillgll l 293 844 1 LIBRARY Michigan State University This is to certify that the dissertation entitled The Neuromuscular Regulation of the Nasopharynx of the Horse presented by Susan J. Holcombe has been accepted towards fulfillment of the requirements for PhD degree in Large Animal Clinical Sciences Jag— Major professor ‘ Date [gig/a7 MSU is an Affirmative Action/Equal Opportunity Institution 0-12771 PLACE IN RETURN BOX to remove this checkout from your record. To AVOID FINE return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE MAY 11,2006 J ; c. fl? 1m acumen-mu THE NEUROMUSCULAR REGULATION OF THE NASOPHARYNX OF THE HORSE By Susan J. Holcombe A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Large Animal Clinical Sciences 1977 ABSTRACT THE N EUROMUSCULAR REGULATION OF THE NASOPHARYNX OF THE HORSE By Susan J. Holcombe Soft palate dysfunction that causes dorsal displacement of the soft palate is an obstructive upper airway condition that occurs in exercising horses. The soft palate assumes a position dorsal to the epiglottis and obstructs airflow through the rima glottis. This syndrome commonly affects race horses and is performance limiting, and at times, life threatening. The obstructive lesion that occurs during episodes of dorsal displacement of the soft palate is very similar to the velopharyngeal obstruction that occurs during episodes of sleep apnea in people, although the mechanisms that cause either obstruction are yet unknown. However, because obstructive sleep apnea is a severe, debilitating syndrome in people, extensive research has been conducted evaluating upper respiratory muscle function, coordination, and activation. A synopsis of this information is presented in the introduction of this dissertation, and has been used to develop a series of experiments directed toward understanding the pathophysiology and etiology of dorsal displacement of the soft palate in horses, which we hypothesize to be an abnormality in the neuromuscular regulation of the nasopharynx. Nasopharyngeal stability and dilation results from intricate neuromuscular coordination that relies on central and peripheral reflex stimulation. Negative inspiratory pressure that occurs during inhalation stimulates upper airway muscle contraction that expands the nasopharynx. Pressures achieved during nasal occlusion trials and maximal exercise trials are identified and compared in Chapter 1. Nasopharyngeal patency is produced by muscles that control the position of the tongue, the hyoid apparatus, and the soft palate. Therefore, based on the hypothesis that dorsal displacement of the soft palate results from abnormal neuromuscular integration, upper respiratory mechanics were measured and soft palate position was assessed following interventions that selectively removed the function of nerves and muscles of the stabilizing and dilatory apparatus. This dissertation is dedicated to all animals whose lives have been affected by science, especially the wonderful horses who worked in the project: Tavish, Eli, Joplin, Neli, Miss Avenger, Carmen, Mr. Emory Yoder, Story, Stevie, Cameo, Maurie Povich, Kurt, Mollie, Rex, Bob, Johnny, Harley, Crystal, Otto, and Bitsie ACKNOWLEDGMENTS I would like to thank the members of my guidance committee: Drs. Frederik Derksen, Dorothy Ainsworth, James Galligan, N. Edward Robinson, and John Stick. I would especially like to thank Drs. Derksen, Robinson, and Stick for their enthusiasm, support, kindness, wisdom, and sense of humor. None of this work could have been accomplished without the invaluable assistance of Cathy Bemey, Deb Boehler, Sue Eberhart, Ann, Mark, Maggie, Tracey, Mike, Rodney, Dennis, Dave, and Vonnie. I would also like to thank Jerry Bailey, George Bohart, John Caron, Tom Evans, William Jackson, Tim Lynch, John Peroni, Sheila Robertson, Phyllis Shance, Joanne Tetens, and Debbie Wilson for their help. For assistance with formatting this dissertation and answering numerous questions, I would like to thank Victoria Hoelzer-Maddox. TABLE OF CONTENTS LIST OF TABLES ................................... x LIST OF FIGURES .................................. xi LIST OF ABBREVIATIONS ............................ xiv INTRODUCTION ................................... 1 Dorsal displacement of the soft palate ................... l Afferent nerve receptors in the upper airway .............. 3 Central and peripheral control of upper airway dilating muscles . . 7 Nasopharyngeal muscles and the hyoid apparatus .......... 9 Genioglossus muscle ........................... 10 Geniohyoideus muscle .......................... l3 Hyoepiglotticus muscle ......................... 14 H ypoglossal nerve .............................. 15 Stemohyoideus and stemothyroideus muscles ............. 16 Soft palate ..................................... 18 Tensor veli palatini muscle ....................... .. . 19 Levator veli palatini muscle ........................ 20 Palatinus and palatopharyngeus muscles ................ 21 Chapter 1 EFFECT OF NASAL OCCLUSION ON TRACHEAL AND PHARYNGEAL PRESSURES IN HORSES ....... 23 Abstract ...................................... 23 Objective .................................... 23 Design ..................................... 23 Animals .................................... 23 Results ..................................... 24 Conclusion ................................... 24 Clinical relevance .............................. 24 vi Introduction .................................... Materials and methods ............................. Horses Instrumentation ................................ Experimental design ............................. Nasal occlusion ................................ Data analysis ................................. Results . . Chapter 2 EFFECT OF BILATERAL TENSOR VELI PALATINI MUSCLE TENECTOMY ON SOFT PALATE FUNCTION IN HORSES ............................... Abstract ...................................... Objective .................................... Animals OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO Procedure ................................... Results Conclusions .................................. Clinical relevance .............................. Introduction .................................... Materials and methods ............................. Instrumentation ................................ Experimental design ............................. Surgical procedure .............................. Postoperative evaluation .......................... Data analysis ................................. Results ....................................... Discussion 0000000000000000000000000000000000000 vii 24 26 26 26 27 28 28 28 30 33 33 33 33 33 34 34 34 34 36 37 37 38 $888 Chapter 3 BILATERAL HYPOGLOSSAL AND GLOSSO- PHARYNGEAL NERVE BLOCKS CAUSED EPIGLOTTIC RETROFLEXION WITH NO EFFECT ON SOFI‘ PALATE POSITION IN EXERCISING HORSES ............. Abstract ...................................... Objective .................................... Design ..................................... Animals .................................... Procedure ................................... Results ..................................... Conclusions and clinical relevance .................... Introduction .................................... Materials and methods ............................. Instrumentation ................................. Experimental design ............................... H ypoglossal and glossopharyngeal nerve block ............ Data analysis ................................. Results ....................................... Chapter 4 BILATERAL NERVE BLOCKADE OF THE PHARYNGEAL BRANCH OF THE VAGUS NERVE PRODUCES PERSISTENT SOFI‘ PALATE DYSFUNCTION IN HORSES ............................... Abstract ...................................... Objective .................................... Animals .................................... Procedure ................................... Results ..................................... Clinical relevance .............................. Introduction .................................... Materials and methods ............................. Experiment One ............................... Instrumentation .............................. Experimental protocol .......................... viii 47 47 47 47 47 47 48 48 49 5 1 52 53 53 55 55 58 61 61 61 61 61 62 62 62 63 65 66 67 68 Experiment Two ............................... Instrumentation .............................. Experimental design ........................... Experiment Three .............................. Instrumentation .............................. Experimental design ........................... Data analysis ................................... Results ....................................... Experiment One ............................... Experiment No ............................... Experiment Three .............................. Discussion ..................................... Chapter 5 ELECTROMYOGRAPHIC ACTIVITY OF THE PALATINUS AND PALATOPHARYNGEUS MUSCLES IN EXERCISING HORSES ....................... Abstract ...................................... Introduction .................................... Materials and methods ............................. Experimental protocol ............................ EMG measurements ............................. Data analysis ................................... Results ....................................... SUMMARY AND CONCLUSIONS ........................ REFERENCES ..................................... APPENDICES ...................................... 68 68 69 69 69 7O 70 71 71 71 72 73 78 78 79 8O 82 82 83 83 84 92 98 Table 1 Table 2 Table 3 Table 4 Table 5 Table 6 LIST OF TABLES Tracheal and pharyngeal pressures during exercise and nasal occlusion in five horses ...................... Tracheal and pharyngeal pressures (:t STD) in cm of H20 during exercise in five horses before and after bilateral tensor veli palatini muscle tenectomy .................. Mean tracheal and pharyngeal pressures and respiratory frequency during exercise in five horses before and after bilateral hypoglossal nerve block ................. Mean tracheal expiratory and inspiratory peak pressures and impedance, expiratory and inspiratory airflows, tidal volume, minute ventilation, and Rf:Sf ratios (j: STD) in five horses before and after bilateral pharyngeal branch of the vagus nerve block .................................. Respiratory and stride frequency, Rf :Sf ratios, and inspiratory and expiratory times in five horses with and without bilateral pharyngeal branch of the vagus nerve block .......... Mean electrical activity (i STD) reported as the percent of activity at 6 M/s for the palatinus and palatopharyngeus muscles in three horses exercising on a treadmill ....... 29 41 57 72 73 84 Figure 1. Figure 2. Figure 3. Figure 4. Figure 5. LIST OF FIGURES Videoendoscopic image of a normal larynx and nasopharynx of a horse with the soft palate (S) positioned ventral to the epiglottis (E) ............................. Videoendoscopic image of the nasopharynx of a horse with the soft palate (S) displaced dorsal to the epiglottis ........ Videoendoscopic image of the nasopharynx of a horse with DDSP during high-intensity treadmill exercise. Notice how the soft palate (S) billows aeross the airway, obstructing the rima glottis .............................. Computer graphic illustration of the anatomical relationship between the hyoid apparatus, the tongue, and the muscles that attach to the basihyoid bone. (SH) stylohyoid bone; (CH) ceratohyoid bone; (BH) basihyoid bone ............. Computer graphic illustration of the hyoid apparatus illustrat- ing the angle between the stylohyoid and ceratohyoid bones. The diameter of the nasopharynx is determined by the angle (A) between the stylohyoid and ceratohyoid bones. The vertical distance between the base of the cranium and the basihyoid bone determines the vertical diameter of the pharynx ................................ xi 99 100 101 102 103 Figure 6. Figure 7. Figure 8. Figure 9. Figure 10. Computer graphic illustration of the change in position of the hyoid apparatus during contraction of the genioglossus muscles. Contraction of the genioglossus muscle or cranial traction on the tongue is causing ventral displacement of the basihyoid, which increases the angle formed by the stylohyoid and ceratohyoid bones, expanding the longitudinal dimension of the nasopharynx ......................... Computer graphic illustration of a transverse section through the larynx and nasal and oral pharynx of a horse. The hamulus of the pterygoid bone, muscles, and tissue layers of the soft palate are labeled. The m. tensor veli palatini and m. levator veli palatini are emphasized with hatch marks to indicate that these muscles course beneath the nasopharyngeal mucosa ................................ Computer graphic illustration of the ventral aspect of the horse’s skull. Notice the muscles of the soft palate, the palatine aponeurosis, and the hamulus of the pterygoid bone (arrow) ................................ Videoendoscopic view of the nasopharynx of a horse following bilateral tensor veli palatini muscle tenectomy during an inspiratory effort with both nostrils occluded. The soft palate billows dorsally into the nasopharynx. (1) nasal septum, (2) soft palate, and (3) junction of the hard and soft palate Videoendoscopic view of the nasopharynx of a horse following bilateral tensor veli palatini muscle tenectomy during an expiratory effort with both nostrils occluded. The soft palate collapses ventrally, developing a concave conformation. (1) nasal septum, (2) soft palate, and (3) junction of the hard and soft palate ............................... xii 104 105 106 107 108 Figure 11. Figure 12. Figure 13. Computer graphic illustration of a transverse section of a horse’s head. The basihyoid bone and the muscles attaching to the basihyoid bone are labeled ................. Videoendoscopic image of the ventro-medial aspect of the guttural pouch. A bleb of mepivicaine (arrow) is injected between the hypoglossal nerve (X11) and the glossopharyngeal nerve (IX) ............................... Videoendoscopic image of the nasopharynx of a horse exercising on a treadmill at the speed resulting in HRM. The ventral surface of the epiglottis (E) is seen because the epiglottis is retroflexed into the rima glottis. The caudal free margin of the soft palate (arrow) can also be seen. The insertion of the hyoepiglotticus muscle (H) is seen beneath the mucosa covering the ventral surface of the epiglottis ..... xiii 109 110 111 DDSP EMG HRmax HanS0 HRmax75 Rf:Sf LIST OF ABBREVIATIONS dorsal displacement of the soft palate electromyographic maximal heart rate 50% of maximal heart rate 75% of maximal heart rate respiratory frequencyzstride frequency coupling xiv INTRODUCTION Dorsal displacement of the soft palate Dorsal displacement of the soft palate (DDSP) is a performance-limiting upper airway condition in horses that has been identified in 1.3% of 479 resting horses examined endoscopically.29'3‘"63 However, the prevalence of this condition is probably much higher because palate displacement is a dynamic condition that occurs most frequently during intense exercise, making diagnosis in the resting horse difficult. The horse is an obligate nasal breather, perhaps to allow olfaction during deglutition, and therefore cannot breath through the mouth. The epiglottis is normally positioned dorsal to the soft palate and contacts its caudal free margin. The soft palate forms a tight seal around the base of the epiglottis and extends along the lateral aspect of the larynx, forming the pillars of the soft palate (Figure 1).25 The pillars converge dorsally, forming the palatopharyngeal arch.25 Using fiberoptic endoscopy, the epiglottis cannot be seen within the nasopharynx when the soft palate displaces dorsally, because it is positioned within the oropharynx (Figure 2). When displaced dorsally, the caudal free margin of the soft palate billows across the rima glottis during exhalation, creating airway obstruction (Figure 3). The etiology of DDSP is unknown, and investigating the cause of this condition was the purpose of the experiments described in this dissertation. Many anatomical and 2 functional abnormalities have been attributed to DDSP, including excessively negative nasopharyngeal pressure, excessive caudal retraction of the larynx, epiglottic hypoplasia or malformation, caudal retraction of the tongue and opening the mouth, an overly long soft palate, and neuromuscular dysfunction.‘3'“‘~'5-2"'3“-“5'84 These conditions may prevent the soft palate from maintaining its position ventral to the epiglottis as exercise intensity increases. In order to understand the potential etiologies of DDSP, a review of nasopharyngeal and soft palate anatomy is necessary and will be included in this introduction. It is important to understand that the soft palate forms the floor of the nasopharynx and, therefore, the position of the nasopharynx and the role of the nasopharynx in breathing is intimately associated with the function of the soft palate during breathing. The pharynx is a musculomembranous tubular structure that functions during breathing, deglutition, vocalization, and eructation, and connects the nasal cavity to the larynx. It is attached to the pterygoid, palatine, and hyoid bone, and to the laryngeal cricoid and thyroid cartilages by nasopharyngeal muscles that cause pharyngeal dilation and constriction.25 The nasopharynx is not directly supported by cartilage or bone, yet contraction of these pharyngeal muscles allows the nasopharynx to withstand large changes in intraluminal pressures that occur during tidal breathing at rest and during exercise. Dilation and stabilization of the nasopharynx during alterations in intraluminal pressure is achieved by neuromuscular activation that is synchronous with breathing.“’“ This synchronization is coordinated by sensory receptors that are located within the mucosa of the nasopharynx and larynx and within the lung that communicate changes in pressure, flow, temperature, and tension to the brain. In response to afferent 3 nerve inputs, efferent nerves signal alterations in muscle activity that control the size and shape of the naSOpharynx. Several groups of muscles alter the size and configuration of the nasopharynx, including the muscles that alter the shape and position of the tongue, the muscles that control the position of the hyoid apparatus, a constrictor group of muscles located in the dorsal pharynx, and a group of muscles that regulate the position of the soft palate.”94 In horses, little is known about the neuromuscular circuitry that controls the dimensions of the nasopharynx, but we can infer from information obtained in laboratory species about the contributions of these receptors, nerves, and muscles to nasopharyngeal stability and patency. First, I will discuss the afferent receptors in the upper airways and the lower airways that signal changes in muscle function. Then I will discuss other factors that are important in nasopharyngeal patency. Finally, I will discuss the groups of muscles that control nasopharyngeal patency and some of the corresponding efferent motor control. As the anatomical discussion continues, I will attempt to clarify why in my research I asked specific questions concerning the functional anatomy of the nasopharynx and soft palate and how I evaluated their potential relationship to DDSP in horses. Afferent nerve receptors in the upper airway Sensory receptors in the larynx and the pharynx include pressure, temperature, I drive, and irritant receptors.7 Especially relevant to dilation and stability of the upper airway during exercise are the pressure receptors. When subatmospheric pressures are applied to the larynx and pharynx there is a marked augmentation of activity of the upper 37.65.71.72.77.X3,3lt-‘)2 airway dilating muscles. Pressure receptors account for 63.3% of the 4 laryngeal receptors and are also present in the pharynx.71 These receptors increase firing with upper airway occlusion, as pressures become more positive and more negative in the larynx and pharynx during nasal occlusion, and less so with tracheal occlusion, due to the absence of pressure changes within the nasopharynx. These receptors respond most to collapsing pressures that occur during inspiration, but some respond also to positive pressure. Pressure receptors in the larynx are innervated by branches of the vagus nerve, specifically the superior laryngeal nerve. Topical anesthesia of the luminal surface of the larynx or bilateral superior laryngeal nerve section markedly reduces the response to changes in upper airway pressure, indicating that superior laryngeal nerve afferents are the primary mediators of these reflex responses.“49 Receptors in the nasopharynx are innervated by branches of the glossopharyngeal and trigeminal nerves.71 Pressure changes in the upper airway affect contraction of upper airway dilating muscles.“"“"'90 For example, studies in many species, including dogs, cats, rabbits, monkeys, and humans, unequivocally show that reflex augmentation of muscle contraction by negative pressure occurs in the genioglossus, geniohyoideus, sternothyroideus, stemohyoideus, and tensor veli palatini muscles.“‘0"‘6-38-9"93 The time of application of negative pressure during the breathing cycle is an important variable in determining the magnitude of the response, and will be discussed later in more detail. Specifically, upper airway motor neurons are more responsive during early inspiration to negative pressure changes that would promote the maintenance of upper airway patency and facilitate recovery from airway obstruction. “’“ 5 Because negative pressure induces contraction of upper airway muscles in order to maintain airway patency as airflows increase, a nasal occlusion test was developed in horses to challenge the upper airway with more negative pressures during resting Videoendoscopic examination, in order to mimic pressure changes that might occur during intense exercise. By occluding the nares at rest, perhaps dynamic obstructive pathologies, such as DDSP, could be induced. However, the pressures achieved in the airway during nasal occlusion were unknown. Therefore, the purpose of the first experiment was to measure the tracheal and pharyngeal pressures achieved during 60 seconds of nasal occlusion and compare these pressures to those achieved during intense exercise in horses. Flow receptors make up about 12.7% of the laryngeal receptors. These receptors respond to temperature as well and may detect changes in flow based on changes in laryngeal temperature. Flow receptors are active during upper airway breathing and silent during tracheostomy breathing in cats.71 Drive receptors represent 21.8% of the laryngeal receptor population and this receptor subtype is a mechanical type of receptor. Drive receptors are stimulated in the absence of pressure or flow, and respond to alterations in work of breathing." Receptors located in the lower airways and lungs, whose afferents are carried primarily via the vagus nerve, have a profound influence on upper airway muscle activity.7l Lower airway afferents alter both respiratory timing and pattern of activation of upper airway muscles. Lung inflation causes slowly adapting receptor stimulation, which terminates inspiration and prolongs expiration. Prevention of lung expansion during end expiratory airway occlusion reduces slowly adapting receptor activity due to 6 decreased volume-related feedback, and prolongs inspiratory duration. Prolonged inspiratory duration is reflected in prolonged and increased activity of upper airway dilating muscles.”94 Withholding lung inflation causes increased cranial nerve activity, specifically increased firing of the hypoglossal and recurrent laryngeal nerves, and mild increases in phrenic nerve activity. Lung deflation increases the activity of both inspiratory and expiratory upper airway muscles. The inhibitory influence of lung volume-related afferent information accounts for much but not all of the early peaking pattern of upper airway muscle electrical activity during inspiration.9"°‘ Many of the upper airway muscles are electrically active in phase with the respiratory cycle.‘00 The extent of activity varies considerably depending on the muscle. Some muscles are consistently active, whereas others are either quiescent or intermittently active during resting breathing and are recruited as work of breathing increases. Muscles also differ with respect to the portion of the respiratory cycle during which they are active. Those that dilate the upper airways tend to be active predominantly during inspiration, whereas those that narrow the upper airways tend to be active predominantly during expiration. These muscles are sometimes active exclusively during one phase of the respiratory cycle, although more commonly they are predominantly active during one phase and have lower levels of activity during other phases. Examples of muscles that are more active during inspiration than during expiration are the genioglossus and the geniohyoideus muscles. ""8" Examples of muscles that are predominantly active during expiration are the pharyngeal constrictors.24 Some muscles may change their predominant phase of activation in response to differing chemical or mechanical stimuli. 7 The onset of inspiratory upper airway muscle activity often precedes that of the diaphragm, and is modulated by chemical drive, and mechanical afferent input from the upper airways, which is primarily vagally mediated."l Many of the upper airway muscles are maximally active during early to mid-inspiration, with a subsequent decrement in activity during the remainder of inspiration. Inspiratory activation of upper airway muscles prior to the diaphragm will dilate or stiffen the upper airway, promoting upper airway patency, prior to the onset of inspiratory airflow, and hence produce an early inspiratory stabilization of upper airways.91 The degree of negative pressure established in the upper airway will increase the amount of muscular preactivation.90 The pattern of contraction of upper airway muscles during inspiration resembles the pattern of inspiratory airflow, thereby stabilizing the upper airway during inspiration.“6'83“?”'94 This pattern occurs because of both vagal and nonvagal mechanisms.9'27-9495'97 Central and peripheral control of upper airway dilating muscles Upper airway dilating muscles are affected by central and peripheral factors. Central factors include anesthesia, posture, state of arousal, and chemical stimuli. Anesthesia suppresses upper airway muscle function, due to its suppressive effects on the reticular formation in the brain stem. Halothane, isoflorane, and enflurane, as well as barbiturates and az-agonists, suppress motor unit firing via reticular formation suppression. Diazepam does not cause brain stem suppression.80 The specific neural network that leads to decreased muscular response is unknown. State of arousal alters upper airway muscle responsiveness. Specifically, stages of sleep, especially REM sleep, decrease phasic and tonic activity of the genioglossus 8 muscle, geniohyoid muscle and, in some sleep apnea patients, the tensor veli palatini muscle."2'72'7"'98 In humans, phasic and tonic activity of the genioglossus muscle may be abolished for over 90 seconds, predisposing these patients to the pharyngeal collapse that occurs during sleep apnea."2 Posture or head position alters upper airway muscle activity. Head flexion or cervical spine flexion increases genioglossal activity in humans, rats, and cats-“"77”“93 Therefore, altered upper airway muscle activity in response to postural changes may be due to stimulation of joint and muscle receptors in the head and neck and/or stimulation of pressure receptors in the pharynx and larynx, perhaps due to the alterations in pharyngeal pressures that occur with head and neck flexion. Chemical stimuli such as hypercapnia and hypoxia increase the activity of both thoracic and upper airway respiratory muscles. "9'50”" The carbon dioxide threshold of the upper airway muscles tends to be higher than the threshold for the diaphragm."9'5"'7" Hypoxia also influences the activation of upper airway muscles, with mild to moderate degrees of hypoxia-stimulating activity of upper airway muscles. Hypoxia and hypercarbia stimulate inspiratory and expiratory motor neuron activity.”'5"'7°'87 Twenty- three percent of the hypoglossal motor neurons are active at normocarbia.” During hypercarbic breathing, inspiratory motor neuron fiber recruitment increases and the motor neurons fire earlier, whereas hyperoxia inhibits inspiratory motor neuron activity and has no effect on expiratory motor activity.”38 The neural mechanism by which central and peripheral chemoreceptors affect cranial motor neuron activity and the role of vagal afferents in these responses is unknown. 9 In summary, I explained that negative intraluminal upper airway pressure increases upper airway dilating muscle activity. Lung inflation terminates inspiratory activity and prolongs expiratory activity of upper airway dilating muscles. Contraction of these muscles is altered by head position and respiratory drive (CO2 and 02 levels), and they are depressed to varying degrees by anesthetic agents. The neural circuitry responsible for these responses is within the reticular formation of the brain stem, outside the dorsal and ventral respiratory groups. Next, I will describe the muscles that control nasopharyngeal patency. Nasopharyngeal muscles and the hyoid apparatus The relationships between upper airway muscle electrical activity and the size and configuration of the upper airway lumen are complex. The upper airway can be dynamically altered at multiple sites, and each segment is unique with regard to the ana- tomic arrangement and mechanical actions of the muscles. Muscles can act directly or indirectly on the upper airway. The genioglossus acts directly to move one of the struc- tures that makes up the pharyngeal wall, the tongue, whereas the hyoid muscles act on the hyoid arch, and it is only via soft-tissue connections between the hyoid arch and the pharyngeal wall that changes in pharyngeal size occur.“"25'°3 Contraction of muscles that alter the position of the hyoid apparatus increase upper airway size and stability by in- creasing the diameter and stiffness of the nasopharynx.9'77"“""2'93 Muscles that apply traction cranially on the basihyoid bone work in a coordinated fashion with muscles that apply caudal traction (Figure 4). The sum of these vector forces is a net ventral displacement of the basihyoid bone.77 This motion increases the angle at the 10 ceratohyoid-stylohyoid joint, increasing the dorsal ventral dimension of the nasopharynx (Figure 5). In doing so, the lateral walls of the nasopharynx expand slightly and become taut. The specific muscles involved in positioning the hyoid apparatus include genioglossus muscle, geniohyoideus muscle, stemohyoideus muscle, and sternothyroideus muscle, and these will now be discussed. Genioglossus muscle Alterations in the position of the tongue affect the position of the hyoid apparatus.10 The muscles of the tongue are classified into two types: intrinsic and extrinsic. The intrinsic muscles are located entirely within the tongue; they alter the shape and increase the rigidity of the tongue.” Genioglossus and hyoglossus are two extrinsic tongue muscles that are important during respiration.‘°'” The genioglossus is a fan-shaped muscle that lies within and parallel to the median plane of the tongue. It is separated from the muscle of the opposite side by a layer of fat and areolar tissue.” The genioglossus muscle originates from the medial surface of the mandible just caudal to the symphysis. A large tendon runs through the muscle and from this tendon muscle fibers radiate, some curving rostrally toward the tip of the tongue, dorsally, and toward the root of the tongue.” Some muscle fibers pass from the caudal end of the tendon to the basihyoid and ceratohyoid bones, forming a direct attachment to the hyoid apparatus.” The hyoglossus muscle is a flat, wide muscle that lies in the lateral portion of the root of the tongue.” The hyoglossus originates from the lateral aspect of the basihyoid bone, from the lingual process to the oral extremity of the stylohyoid and thyrohyoid bones.” Contraction of the hyoglossus results in tongue retraction. 11 However, hyoglossus and genioglossus activity are synchronous during respiration, when the hyoglossus acts like a “tether,” strengthening the attachment of the genioglossus muscle to the hyoid apparatus. Contraction of the genioglossus muscle protracts the tongue and pulls the basihyoid bone rostrally while the genioglossus also acts with the hyoglossus muscle to depress the tongue (Figure 6). This activity correlates well with increases in pharyngeal airway size during breathing.'”'47°5" Contraction of the genioglossus muscle improves upper airway patency both by dilating the supraglottic airway and by stiffening its walls,57 thereby opposing collapsing negative nasopharyngeal pressures.10 The function of the genioglossus muscle has been extensively studied because of its potential role in obstructive sleep apnea syndrome. During room air and C02 rebreathing, nasopharyngeal resistance was maintained constant in awake males while nasal resistance and genioglossus EMG activity increased, suggesting that the stability of pharyngeal resistance resulted from compensatory activation of the genioglossus as flow rates change.“ The genioglossus muscle was electrically stimulated in anesthetized dogs and found to increase the critical collapsing pressure of the nasopharynx as well as to reduce upper airway resistance during augmented breaths.“O Transmucosal electrical stimulation of the genioglossus muscle in awake normal males decreases pharyngeal resistance. In sleeping males, increases in mean genioglossal EMG activity was correlated with increases in critical collapsing pressure, which may be important in the treatment of sleep-related obstructive airway disorders.73 Electromyographic activity of the genioglossus muscle was measured in anesthetized rabbits and dogs, and increased genioglossus activity was correlated with augmented inhalation, produced by hypoxia, hypercapnia, and non-specific respiratory 12 stimuli.'“'“" In awake normal men, sustained isocapnic hypoxia (20 minutes) augments genioglossus activity, whereas with repetitive isocapnic hypoxia (10 x 2 min episodes) early augmentation is followed by marked suppression.” By the tenth hypoxic episode there was no augmentation.50 Suppression of genioglossus activity during episodes of repetitive hypoxia may contribute to airway obstruction that occurs during obstructive sleep apnea.50 The genioglossus muscle, unlike the diaphragm. maintained increased activity during prolonged hypoxic breathing in neonatal pigs, suggesting that central inhibition during neonatal hypoxia is primarily distributed to the motor neuron pools regulating the diaphragm activation and that peripheral chemoreceptor stimulation and/or central disinhibition induced by hypoxia preferentially influence those motor neuron pools that regulate upper airway muscle activation, causing the different hypoxic responses of these muscle groups in neonatal piglets.62 This information may be critically important in determining the pathophysiology and preventative therapies for sudden infant death syndrome. Caudal tongue retraction has been implicated as a cause of DDSP. The caudal aspect of the tongue may push the soft palate away from the epiglottis. However, anatomically, this would not seem to be a plausible explanation for the syndrome. The caudal aspect of the tongue contacts the mid and rostral portions of the soft palate, while it is the most caudal portion of the soft palate that displaces dorsal to the epiglottis. Instability of the rostral portion of the soft palate can result in upper respiratory obstruction, but not cause DDSP.34 Protraction of the tongue and securing it in the interdental space of the mandible is the accepted treatment for the proposed caudal tongue retraction. Also, a treatment for horses that exhibit DDSP is to tie the tongue to the 13 mandible. Protracting the tongue will advance the hyoid apparatus and larynx, and indeed may improve nasopharyngeal patency and alter epiglottic position. Opening the mouth or swallowing during exercise may induce DDSP. A special head harness, called a figure-eight nose band, is used to keep the horse’s mouth shut. Interestingly, closing the mouth does protrude the jaw, altering hyoid position and stabilizing the nasopharynx. Protrusion of the jaw moves many of the oral and pharyngeal structures forward, especially the tongue, thereby dilating the pharynx.94 In addition, closure of the mouth produces an anteriorly directed traction on the hyoid arch via its muscular and soft tissue connections with the mandible.94 Muscles that protract the jaw are the medial and lateral pterygoid muscles.”94 Some of these muscles have been shown to be rhythmically active in phase with the respiratory cycle, and their degree of activity appears to correlate with stabilization of the pharyngeal upper airway. Geniohyoideus muscle Several members of the hyoid muscle group dilate and stabilize the pharynx and there may be considerable synergy when both the suprahyoid and infrahyoid muscles contract together. The geniohyoideus muscle is a fusiform paired muscle that lies on the ventral surface of the tongue.” Geniohyoideus, a suprahyoid muscle, originates on the oral surface of the mandible near the symphysis, in conjunction with the genioglossus, and inserts on the basihyoid bone.” Its action is to draw the hyoid bone rostrally.” The geniohyoid has a primary role as a pharyngeal dilator in the spontaneously breathing cat, with the sternohyoid muscle acting in an accessory capacity. The volume of the upper airway increases during inspiration and decreases during expiration. which appears to be 14 in part related to activity of the geniohyoid and sternohyoid muscles.93 During inspiration, the action of the diaphragm and trachea impose a caudal force on the hyoid apparatus. This caudal force is counteracted by a cranial force produced by contraction of the geniohyoideus muscles. Both traction on the hyoid arch and electrical stimulation of the geniohyoid and sternohyoid muscles produced a reduction in flow resistance in anesthetized dogs during inspiration and expiration” and increased critical closing pressure in the nasopharynx of anesthetized dogs." Based upon information in laboratory animals the geniohyoid muscle likely plays a role in modulating upper airway patency in humans during sleep.98 Hyoepiglotticus muscle Contraction of the geniohyoideus and genioglossus muscles moves the hyoid apparatus cranially, and in doing so, also moves the larynx cranially. This action may improve the epiglottic/soft palate articulation. The hyoepiglotticus muscle is a bilobed muscle that attaches the ventral epiglottic base to the basihyoid bone. Little information exists about the function of this muscle, because humans do not have a hyoepiglotticus muscle. In the horse, contraction of the hyoepiglotticus muscle may also improve the epiglottic/soft palate contact, by pulling the epiglottis against the soft palate. Epiglottic hypoplasia or dysfunction has been implicated as the cause of DDSP in horses.”'36"5'8"” The epiglottis is thought to function as a “claw” to hold the soft palate down. The hyoepiglotticus, geniohyoideus, and genioglossus muscles all receive efferent motor innervation from the hypoglossal nerve. If these muscles were made dysfunctional by 15 blocking the hypoglossal nerves bilaterally, what would happen to the epiglottic/soft palate contact? This experiment is described in Chapter 2 of this dissertation. Although epiglottic dysfunction has not been proven to cause DDSP in horses, a surgical therapy known as epiglottic augmentation was developed to treat epiglottic hypoplasia and/or flaccidity in an attempt to treat DDSP. Epiglottic augmentation is performed by injecting polytetrafluoroethylene paste along the ventral surface of the epiglottis within the aryepiglottic tissue, in order to enhance the rigidity of the epiglottis if this structure is malformed or flaccid.”85 A granulomatous reaction ensues and structurally thickens the epiglottis. The efficacy of this surgical procedure in horses with DDSP has not been documented, although anecdotal reports suggest that this procedure may be helpful. Hypoglossal nerve The genioglossus, geniohyoideus, and hyoepiglotticus muscles receive efferent motor innervation from the hypoglossal nerve.” Electrostimulation of the hypoglossal nerve decreases the critical closing pressure of the pharynx by protruding the tongue and advancing the hyoid apparatus, thereby increasing airway stiffness and dilating the upper airway.74 Activity of single hypoglossal nerve fibers was monitored in anesthetized cats. The majority of hypoglossal neuronal activity was inspiratory, discharging during a period approximating that of the phrenic nerve. Smaller numbers of hypoglossal fibers have inspiratory and expiratory, or solely expiratory, activity.38 Many fibers were not active at normocapnea but were recruited in hypercapnea or hypoxia.38 After recruitment, discharge frequencies rose quickly to maximal levels in early to mid- l6 inspiration, significantly increasing with further augmentation of drive.38 Thus, the respiratory-related efferent activity of the hypoglossal nerve has four major features: maximal to near-maximal activity during early inspiration; an augmentation of the maximal level in hypercapnia and hypoxia; earlier activity as ventilatory drive increases; and small bursts of expiratory activity at high levels of respiratory drive-‘8'39 The afferent pathway responsible for increased genioglossus muscle activity during negative pressure challenge originates from the pressure receptors in the mucosa of the larynx, innervated by the superior laryngeal nerve, and receptors in the pharynx, innervated by the trigeminal and glossopharyngeal nerves.""‘8 Increases in hypoglossal activity, stimulated by augmented negative pressure in the upper airway, were greatly reduced after bilateral sections of the superior laryngeal nerve, and further diminished after the pharyngeal branches of the glossopharyngeal nerves were sectioned. Additional bilateral destruction of the trigeminal nerves almost entirely eliminated responses to pressure changes.”39 Furthermore, the hypoglossal nerve has increased activity during inspiration that is augmented by hypoxia and hypercapnia, which is dependent on carotid sinus chemoreceptor input. ”-97 Stemohyoideus and sternothyroideus muscles The sternothyroideus and stemohyoideus are infrahyoid muscles and accessory respiratory muscles that originate on the manubrium and extend cranially.” The sternothyroideus inserts on the caudal abaxial aspect of the thyroid cartilage, and the stemohyoideus inserts on the basihyoid bone and the lingual process of the hyoid apparatus.” Contraction of these muscles results in caudal traction of the hyoid 17 apparatus and larynx, resulting in dilation of the nasopharynx."5"“"77 The sternothyroideus and stemohyoideus exhibit phasic inspiratory activity and increase their activity with increasing negative upper airway pressure to resist pharyngeal collapse in dogs, rabbits, and cats."°“8"’5"’° The specific muscle coordination, chemical or physical stimulation, and neuronal drive for coordinated function of these muscles are unknown. There is no information to suggest the hierarchy of importance of these muscles in controlling airway patency and minimizing resistance during respiration at rest and during exercise. It is postulated that caudal retraction facilitates the laryngopalatal displacement that occurs during DDSP.”36 In effect, caudal traction of these muscles on the larynx may pull the epiglottis off of the soft palate. This caudal pull of the sternothyroideus and stemohyoideus muscles is opposed by rostral traction of the geniohyoid and genioglossus muscles. The-stemothyroideus and stemohyoideus muscles are sometimes transected as a surgical treatment for DDSP in the horse. The proposed effect of stemothyrohyoid myectomy is that resection of the laryngeal retractor muscles will abolish their activity, thereby preventing caudal retraction of the larynx and the tendency for the epiglottis to displace ventral to the soft palate. Transection of these muscles in normal horses causes increased tracheal and translaryngeal inspiratory resistance in exercising horses.32 Increased translaryngeal and tracheal inspiratory pressures and resistances after sternothyrohyoid myectomy suggest that the sternothyroideus and stemohyoideus muscles act to increase or maintain upper airway patency and stability in normal horses.32 Other pharyngeal-dilating and -stabilizing muscles may make up for the loss of function of these muscles at rest. Loss of function of these dilating muscles during exercise may result in abnormal upper airway function as inspiratory pressures decrease from -1 to —50 cm 18 H20 in horses exercising at 14 m/s.“"77 However, performing this surgery on horses has produced no clinically documented detrimental effects to upper airway patency and breathing. Sternothyrohyoid myectomy has had a reported successes rate ranging from 58 to 60%.3'26 Soft palate The soft palate extends caudally from the hard palate to the base of the larynx. The soft palate consists of the oral mucous membrane, which contains ductile openings of the palatine glands, the palatine glands, the palatine aponeurosis, palatinus and palatopharyngeus muscles, and the nasopharyngeal mucous membrane.” The caudal free margin of the soft palate continues dorsally, on either side of the larynx, forming the lateral pillars of the soft palate. These pillars unite dorsally, forming the posterior pillar of the soft palate or the palatopharyngeal arch.” In humans, unlike in horses, the soft palate and uvula are movable tissues suspended from the posterior border of the hard palate and extending down and back toward the oropharynx. Raising the palate closes the pharyngeal isthmus, promoting oral breathing, whereas moving the palatoglossal arches toward midline narrows the oral pharyngeal isthmus and favors nasal 43.51 breathing. Movements of the palate toward and away from the posterior nasopharyngeal wall appear to be important determinants of airway obstruction in sleep apnea in humans and bulldogs, in obstructive syndromes in neonatal infants, and during episodes of DDSP in horses.2"'~"’"’""’7"”'82 No studies have been done to assess soft palate conformation or length in horses with DDSP, and therefore no critical information is available to support why DDSP 19 might be caused by redundant soft palate tissue. Despite this fact, staphylectomy or trimming the caudal free margin of the soft palate is a surgical treatment for horses with DDSP that may be performed alone or in concert with other surgical procedures. Staphylectomy supposedly is performed to stiffen the free edge of the soft palate, thereby preventing DDSP, or to increase the size of the pharyngeal ostium, thus decreasing the degree of obstruction, should DDSP occur.3 Staphylectomy has a reported success rate of 59%.3 The position of the soft palate is determined by the coordinated function of four muscles (Figure 5).” The tensor veli palatini muscle is innervated by the mandibular branch of the trigeminal nerve (cranial nerve V). The levator veli palatini muscle, the palatinus muscle, and the palatopharyngeus muscle are innervated by the pharyngeal branch of the vagus nerve. Tensor veli palatini muscle The tensor veli palatini muscle is a fusiform muscle that originates at the muscular process of the petrous part of the temporal bone, the pterygoid bone, and the lateral lamina of the auditory tube, and travels rostroventrally, lateral to the levator, along the lateral wall of the nasopharynx.” The tendon of the tensor veli palatini muscle courses around the hamulus of the pterygoid bone and ramifies in the palatine aponeurosis.” The tensor veli palatini muscle tenses the soft palate and retracts the soft palate away from the dorsal pharyngeal wall.”"‘3 This muscle expands the nasopharynx during inspiration by tensing the rostral half of the soft palate and depressing it, slightly, ventrally.”51 Because the tensor veli palatini muscle tenses the soft palate during breathing, 20 dysfunction of this muscle might be implicated as the cause of DDSP in horses. Chapter 3 describes an experiment that was performed in which tensor veli palatini dysfunction was created by transecting the tendons of the tensor veli palatini muscle bilaterally. Soft palate position and function were assessed and upper airway pressure measurements were made before and after this surgical procedure. Levator veli palatini muscle The levator veli palatini muscle arises from the muscular process of the petrous part of the temporal bone and from the lateral lamina of the auditory tube and passes along the lateral wall of the nasopharynx. The muscle turns medially and meets within the soft palate, dorsal to the glandular layer, caudal to the hamulus of the pterygoid bone.” The levator veli palatini muscle elevates the soft palate during swallowing, and facilitates oral ventilation in non-obligate nasal breathers.”43 The levator veli palatini has been shown to exhibit phasic inspiratory and expiratory activity during augmented breathing and during chemical stimulation.7"’°'""‘3'5‘53'37'88 In addition, in some studies, the levator has been shown to have no respiratory related activity.” Palatinus and palatopharyngeus muscles The palatinus muscle consists of a paired fusiform muscle that originates at the caudal aspect of the palatine aponeurosis and courses through the middle of the soft palate, just beneath the nasal mucosa, to ramify in the caudal free margin of the soft palate.” Fibers of the palatinus muscle continue dorsally in the lateral pillars of the soft palate, but terminate prior to the formation of the palatopharyngeal arch.” The 21 palatOpharyngeus muscle arises from the palatine aponeurosis, lateral to the attachment of the palatinus muscle, and also from the palatine and pterygoid bones.” The fibers continue caudally, on the lateral wall of the pharynx, and are inserted in part into the upper edge of the thyroid cartilage. The rest of the muscle continues dorsally and inserts at the median fibrous raphe.” Contraction of the palatinus and palatopharyngeus muscles shortens the soft palate and depresses it toward the tongue”:51 The palatinus has synchronous respiratory activity during augmented breathing, increased by negative pressure stimulation and hypercapnic hypoxic breathing.7°”°88 Uvula mucosal edema has been documented in patients with obstructive sleep apnea.” Soft—palate inflammation has been implicated in the pathophysiology of airway occlusion observed during sleep in obstructive sleep apnea patients.” There is no information about the respiratory function of the palatopharyngeus muscle. However, palatopharyngeus muscle biopsy specimens from obstructive sleep apnea patients showed abnormalities.” Atrophy with a fascicular distribution, increased number of angulated atrophic fibers, multiple peak distribution the fiber size spectra, and an abnormal distribution of fiber types in many muscle fascicles corresponding to type grouping all point to neurogenic lesions that may be a primary phenomenon or secondary to trauma of repetitive and prolonged stretching of the pharyngeal structures during obstructive episodes.” A disturbance of the function of the soft-palate stabilizing muscles may be important in causing the abnormal airway collapse seen in obstructive sleep apnea.” Because the palatinus and palatopharyngeus muscles control the position of the caudal soft palate and because dysfunction of these muscles has been implicated as the cause of other upper respiratory obstructive syndromes, we hypothesized that dysfunction 22 of the palatinus and palatopharyngeus muscles in the horse would result in DDSP. This dysfunction was created by blocking the pharyngeal branch of the vagus nerve and this experiment is described in Chapter 4. Historically, neuromuscular pathology has been suggested as a possible etiology of DDSP. Dorsal displacement of the soft palate was historically referred to as soft palate paresis, “which in racehorses, is characterized by the sudden onset of respiratory obstruction during a race. The horse makes a gurgling noise, like a death rattle, and in lay parlance, the horse is said to have swallowed its tongue. ”'4 The paresis was thought to result from guttural pouch diphtheria, which is currently referred to as guttural pouch mycosis.“ Palatal myositis was confirmed in horses with DDSP, based on tissue specimens obtained from horses with DDSP treated with staphylectomy.5 From the above literature review, it is clear that there is little concrete evidence supporting any of the current theories that describe the etiology or the pathophysiology of DDSP. Surgeons have no treatment options that are more than 60% successful, yet we operate on affected horses daily. The information presented in this introduction was used to plan a series of experiments that focused on identifying the pathogenesis of DDSP in the horse. Chapter 1 EFFECT OF NASAL OCCLUSION ON TRACHEAL AND PHARYNGEAL PRESSURES IN HORSES Abstract Objective The objective was to compare tracheal and pharyngeal inspiratory and expiratory pressures achieved during 60 seconds of nasal occlusion in standing horses with pressures achieved in horses during intense exercise. Design Tracheal and pharyngeal inspiratory and expiratory pressures were obtained from 5 horses during 60 seconds of nasal occlusion and compared with tracheal and pharyngeal pressures achieved during incremental treadmill exercise tests in which horses ran at 50, 75, and 100% of the speed that resulted in maximal heart rate (HR,,,,,). Animals Five Standardbreds were used. 23 24 Results Significant difference was not detected between peak tracheal inspiratory pressure during nasal occlusion and peak tracheal inspiratory pressure at HR,” Peak pharyngeal inspiratory pressure was significantly more negative, and peak tracheal and peak pharyngeal expiratory pressures were significantly more positive during 60 seconds of nasal occlusion than those observed in horses running at HRW. Conclusion During upper airway endoscopy in standing horses, 60-second nasal occlusion induced tracheal and pharyngeal inspiratory pressures that equaled or exceeded pressures achieved during high-intensity exercise. Clinical relevance Nasal occlusion is useful to simulate upper airway pressures achieved during high- intensity exercise. Introduction During inspiration, the upper airway is subjected to negative pressures generated by the thorax. Inspiratory tracheal pressures in horses range from -1.9 :1: 0.2 cm of H20 during normal tidal breathing to -38.6 i 3.9 cm of H20 while exercising at HRW.” Extrathoracic airway patency is maintained in the face of negative inspiratory pressure by upper airway muscle contraction, and neuromuscular response increases with Ill increasing negative airway pressure.” Specifically, in all species studied thus far, 25 negative pressure applied to the larynx and pharynx causes reflex activation of upper airway dilator muscles, including the genioglossus, geniohyoideus, thyrohyoideus, stemohyoideus, and sternothyroideus, to prevent airway collapse.37"‘7~57"’5'77-”:90-loo Dynamic upper airway obstructive diseases, such as idiopathic laryngeal hemiplegia, DDSP, and pharyngeal collapse, are performance-limiting conditions in horses.52 Techniques currently available to assess the upper airway include physical examination, endoscopy of the larynx and pharynx at rest and after exercise, videoendoscopy of the upper airway during high-speed treadmill exercise, and radiography. Because dynamic obstructive upper airway disease may be difficult to diagnose in standing horses, videoendoscopy of the larynx and pharynx during high-speed treadmill exercise often is performed. However, high-speed treadmill access is limited and, instead, nasal occlusion is frequently used in standing horses during endoscopy of the upper airway to simulate the increased respiratory effort that occurs during high- intensity exercise. However, tracheal and pharyngeal pressures achieved during nasal occlusion are unknown. When nasal occlusion in resting horses is used to evaluate function of the upper airway dilator muscles it is assumed that pressures generated during this maneuver are similar to those generated in exercising horses. Thus, the objectives of the study reported here were to evaluate this assumption and test the hypothesis that nasal occlusion in standing horses results in peak upper airway pressures similar to peak upper airway pressures achieved in the same horse during intense exercise. 26 Materials and methods The experiments were approved by the All-University Committee for Animal Use and Care at Michigan State University. Horses Five Standardbreds, two castrated males and three females (three to five years old), weighing between 410 and 489 kg were studied. Horses were maintained at pasture for 30 days prior to the study. All horses were vaccinated against equine influenza, rhinopneumonitis, and Eastern and Western equine encephalitis virus and Streptococcus equi infections. Physical examination of the horses and endoscopy of the larynx and pharynx at rest and during high-speed treadmill exercise failed to reveal any abnormalities. All horses were trained to run on a treadmill 4 weeks prior to the experiment. Heart rate was measured during an incremental exercise test to determine the speed that induced HR,” for each horse. The speeds corresponding to 50% (HRmaxso) and 75% (HRM75) of maximal heart rates were interpolated from this data.46 Heart rate was recorded using a telemetry system (Digital VHF Telemetry System, M1403 Andover, MA). Instrumentation For studies in standing horses, tracheal and pharyngeal sidehole catheters were passed through the left naris and secured to the muzzle with tape. The tracheal catheter was placed between the proximal and middle thirds of the cervical portion of the trachea and the tip of the pharyngeal catheter was positioned at the level of the opening of the 27 left diverticulum of the auditory tube. Two ISO-cm polyethylene side-hole catheters (polyethylene tubing, 2.15 mm 1D,3.25 mm OD) (Baxter Scientific Products, McGaw Park, IL) were used. Each catheter was made with 6 side holes beginning a distance of 8 catheter diameters from the sealed tip.54 In experiments with exercising horses, the transnasal tracheal catheter was replaced with a percutaneously placed lateral tracheal catheter to avoid the soft palate dysfunction that transnasal tracheal catheters may induce. Catheters were phase-matched at 5, 10, and 15 Hz.‘7 Tracheal and pharyngeal pressures were measured using differential pressure traducers (DP-45-22, Validyne engineering Sale, Northridge, CA), and were recorded on a respiratory function computer (Buxco LS-l4, Buxco Electronics, Inc. , Sharon, CT). The differential pressure transducers were calibrated using a water manometer before each experiment. Peak inspiratory and expiratory tracheal and pharyngeal pressures were determined from the pressure tracings. Eight to ten consecutive breaths were averaged to determine each data point. Experimean design Horses had tracheal and pharyngeal catheters placed, then were exercised on the treadmill for three minutes at a speed of 4 m/s and for one minute at HRMW. Subsequently, a videoendoscope was inserted through the right naris and was secured to the horse’s halter with adhesive strips. Horses were then exercised for one minute each, at HRmaxSW HRmaOS! and HRmax' 28 Nasal occlusion Horses were restrained using a lip twitch, and the nares were manually occluded for 60 seconds. Pressure measurements were taken during this procedure. The effect of stage of the respiratory cycle on nasal occlusion-induced tracheal and pharyngeal pressures was tested by performing nasal occlusion on each horse: by occluding the nares at the end of inhalation and at the end of exhalation. The end of inhalation and exhalation was determined by watching the motion of the thorax. The treatment order was randomized. Data analysis Data were analyzed by use of ANOVA with a repeated measures design, and post hoc comparisons were made, using the Student-Newman—Keuls’ test. The effect of respiratory timing on airway pressures obtained during nasal occlusion was tested by use of a paired, two-tailed Student’s T-test. Tracheal and pharyngeal inspiratory pressures during nasal occlusion were compared using paired, two-tailed Student’s T-test. Tracheal and pharyngeal expiratory pressures during nasal occlusion were compared, using a paired, two-tailed Student’s T-test. Significance value of P < 0.05 was chosen. Results Peak tracheal and pharyngeal pressures were compared when nasal occlusion occurred at the end of inhalation and the end of exhalation. The stage of the respiratory cycle when occlusion occurred had no significant effect on any of the pressure measurements. Therefore, we only report the pressures obtained when occlusion 29 occurred at the end of exhalation. Peak tracheal inspiratory pressure during nasal occlusion was significantly (P = 0.001) more negative than peak tracheal inspiratory pressure at rest, HR,,,,,5(,, and HR,,,,,,75. but was not significantly different from peak tracheal inspiratory pressure at HR“, (Table 1). Table 1. Tracheal and pharyngeal pressures during exercise and nasal occlusion in five horses. I Speed TIP TEP PIP PEP I (%HR....) Rest —1.6 i 0.5* -0.8 j; 03* -1.6 i0.5* 1.2 :l: 0.4* 50 -l3.2 i 28* 8.3 :t 1.3* -lO.6 i0.9* 5.1 i 1.9* 75 19.0 i 2.8* 10.9 :t 1.8* -12.0 3210* 8.4 -_l~ 3.0* 100 -25.6 :t 2.8* —13.8 :t 2.1* 17.5 i2.1* 11.3 j: 18* Nasal occlusion -24.9 i 3.0 29.0 i 0.6 -28.9 3; 4.9 35.8 i 5.3 * Significant difference from nasal occlusion pressure; TIP = tracheal inspiratory pressure; TEP = tracheal expiratory pressure; PIP = pharyngeal inspiratory pressure; PEP = pharyngeal expiratory pressure; HRmax = maximal heart rate; Data are expressed as mean :1; SD cm of H20 Peak pharyngeal inspiratory pressure was significantly (P < 0.000) more negative during nasal occlusion than at rest and at all speeds. Peak tracheal and pharyngeal inspiratory pressures were not significantly different during 60-second nasal occlusion. Peak tracheal and pharyngeal expiratory pressures were significantly (P < 0.000) higher during nasal occlusion than those at all speeds. Peak pharyngeal expiratory pressure during nasal occlusion was significantly (P = 0.001) higher than peak tracheal expiratory pressure during nasal occlusion. 30 Discussion Peak tracheal inspiratory pressure during nasal occlusion (-24.9 i 3 cm of H20) was not significantly different from peak inspiratory pressure while horses were exercising at HRW (-25.6 i 2.7 cm of H20), and peak pharyngeal inspiratory pressure was significantly more negative (-28.9 i 4.9 cm of H20) during nasal occlusion than while horses were exercising at HRmax (~17.5 i 2.1 cm of H20). These data clearly indicate that 60-second nasal occlusion in standing horses results in pharyngeal and tracheal inspiratory pressures that equal or exceed those that are generated during exercise at HRW. This suggests that nasal occlusion in standing horses may be a clinically useful test for evaluating the ability of laryngeal and pharyngeal muscles to maintain upper airway patency in the face of negative airway pressures that occur during intense exercise. The tendency for dynamic collapse of the unsupported structures of the upper airway is increased during intense exercise because of greater negative inspiratory pressure generated by thoracic expansion as peak inspiratory air flow increases from 5.09 1: 0.34 US at rest to 75 :1; 9.35 US while exercising at HRW.46 In all species studied thus far, the larynx and nasopharynx are supplied with specialized mechanoreceptors that detect changes in transmural pressure and initiate respiratory reflexes that maintain upper airway patency.7| Upper airway dilating muscles, such as the alae nasi, genioglossus, postier cricoarytenoid, geniohyoid, thyrohyoid, and sternohyoid, are activated by upper airway negative pressures, and the degree of muscle contraction is proportional to the negative pressure generated.“5‘77'”'” Dysfunction of the upper airway dilating muscles may lead to dynamic obstructive conditions, such as laryngeal hemiplegia and DDSP. 31 Therefore, generating negative inspiratory upper airway pressure by nasal occlusion may simulate neuromuscular events that occur during dynamic upper airway obstruction in exercising horses. Although negative inspiratory pressures generated during nasal occlusion and during intense exercise are similar, pressure flow relations differ. Airflow is not generated during nasal occlusion , but inspiratory airflow exceeding 75 US is generated while exercising at HR,,,,,,."" Mechanoreceptors sensitive to airflow have been identified in the larynx of other species studied thus far and may contribute to upper airway dilating muscle activation during high-intensity exercise.71 Furthermore, tension in thoracic and diaphragmatic muscles and chemical stimuli, including hypercarbia and hypoxemia, stimulates contraction of upper airway dilator muscles.47 Although blood gas tensions were not measured in this study, horses exercising at HRW become hypercarbic (PaCO2 of 50.2 mmHg) and hypoxemic (PaO2 of 56.1 mmHg).“0 These values are unknown for 60-second nasal occlusion. Thus, 60-second nasal occlusion is an easily applied test that gives an indication of the ability of upper airway dilator muscles to resist physiologically relevant collapsing pressures; however, the test does not fully mimic receptor stimulation obtained during high-intensity exercise. Nasal occlusion resulted in pharyngeal inspiratory pressures that were not significantly different from tracheal inspiratory pressures. This was expected because during nasal occlusion there is no airflow. In contrast, tracheal inspiratory pressures were more negative than pharyngeal inspiratory pressures in exercising horses. This has been reported and is caused by inspiratory laryngeal flow resistance.” 32 Interestingly, tracheal and pharyngeal expiratory pressures were significantly higher during nasal occlusion than during exercise at HR,,,,,,_ During exhalation in exercising horses, positive pressures dilate the upper airway and reduce resistance to flow. Thus, horses generate high expiratory flows, using low driving pressures during exercise. Why horses generate higher expiratory pressures during nasal occlusion is unclear. In four horses, pharyngeal expiratory pressure was 8 to 10 cm of H20 higher than tracheal expiratory pressure during the last three to four breaths of the nasal occlusion trial. As seen endoscopically, some horses narrow and close the glottis during forced expiration when the nares are occluded. Breathing with an expiratory restive load decreases glottal width during expiration, stimulated by the prolonged expiratory duration or the volume-time course of the lung.8 Horses closed their glottis during forced expiration during the nasal occlusion maneuver. Also, during expiration, pharyngeal size decreases, owing to contraction of pharyngeal constrictor muscles, as positive pressure increases in the upper airway.8 Therefore, in this study, pharyngeal expiratory pressure increase above tracheal expiratory pressure may be explained by glottis closure and pharyngeal constrictor muscle contraction during exhalation with nasal occlusion. In conclusion, tracheal and pharyngeal pressures generated during 60-second nasal occlusion are equal to or exceed the pressures generated while horses run at Han. Negative airway pressure causes reflex pharyngeal and laryngeal dilator muscle activation to stabilize the airway and prevent dynamic collapse. Therefore, nasal occlusion is a useful tool for assessing neuromuscular upper airway function in horses. Chapter 2 EFFECT OF BILATERAL TENSOR VELI PALATINI MUSCLE TENECTOMY ON SOFT PALATE FUNCTION IN HORSES Abstract Objective The objective of this research was to determine the effect of bilateral tensor veli palatini tenectomy on soft palate and nasopharyngeal function in exercising horses. Animals Five Standardbreds were used. Procedure Treadmill videoendoscopy was performed on 5 Standardbreds exercising at the speed that produced HRmxSO, HRmam, and HRmax while tracheal and pharyngeal pressures were measured before and after surgery. Tensor veli palatini muscle tenectomy was performed, bilaterally, on each horse under general anesthesia, using a transoral approach. 33 34 Results Peak inspiratory tracheal pressures were significantly (P = 0.016) more negative and there was a trend (P = 0.06) for peak pharyngeal inspiratory pressure to be less negative after bilateral tensor veli palatini muscle tenectomy compared with preoperative values. The rostral half of the soft palate was unstable and collapsed dorsally into the nasopharynx during inspiration, causing partial obstruction of the nasopharynx. The caudal free margin of the soft palate remained ventral to the epiglottis and DDSP did not occur in any horse. Conclusions Bilateral tensor veli palatini tenectomy muscle did not cause DDSP in horses while exercising at HRm, but resulted in collapse of the nasopharynx during inspiration. Clinical relevance Results of our study suggest that the tensor veli palatini muscle functions to support and dilate the nasopharynx during intense inspiratory efforts in the horse by tensing the palatine aponeurosis. Introduction Dorsal displacement of the soft palate is a dynamic upper airway obstructive disease that limits exercise performance in horses.” In clinically normal horses, the epiglottis is positioned dorsal to the soft palate and contacts the caudal free margin of the soft palate, forming an airtight seal and rendering horses obligate nasal breathers.” In 35 horses with DDSP, the soft palate intermittently displaces dorsal to the epiglottis during intense exercise, causing partial obstruction of airflow to the lungs.” The cause of DDSP is unknown but proposed mechanisms include damage to the innervation of the soft palate, epiglottic hypoplasia, excessive mucosa at the caudal free margin of the soft palate, excessive caudal retraction of the larynx, and primary muscle dysfunctions-‘3'29 The position of the soft palate is determined by the coordinated function of four muscles in humans and presumably also in horses. The muscles include the tensor veli palatini, levator veli palatini, palatinus, and the palatopharyngeus.43 The levator veli palatini muscle elevates the soft palate during swallowing and facilitates oral ventilation in non-obligate nasal breathers.43 The palatinus and palatopharyngeus muscles shorten the soft palate and depress the soft palate toward the tongue.“3 The tensor veli palatini muscle tenses the soft palate and retracts the soft palate away from the dorsal pharyngeal wall.“3""7 Despite the recognition of the soft palate as an important respiratory structure in maintaining nasopharyngeal patency, there is no information on the respiratory activity of these soft palate muscles and how their activity is regulated in the horse and little information in other species. The tensor veli palatini muscle is thought to be a secondary muscle of respiration, because it is quiescent during resting breathing but has increased activity during pressure stimulation in the pharynx and in response to chemical respiratory challenges in dogs and human subjects."7'”'88 Specifically, the phasic and tonic electromyographic activity of the tensor veli palatini muscle increases in response to augmented negative pressure in the pharynx and in response to hypoxic hypercapnia.”'” Therefore, the tensor veli palatini muscle likely contracts during high-intensity exercise in horses, expanding the 36 nasopharynx and stabilizing the soft palate during respiration. We hypothesized that loss of function of the tensor veli palatini muscle would lead to soft-palate instability and dynamic collapse of the soft palate during inspiration, resulting in DDSP in horses during maximal exercise. In the study reported here, we examined the effect of bilateral tensor veli palatini muscle tenectomy on tracheal and nasopharyngeal pressures and on soft palate function in five Standardbreds exercising at the speed that produced HRW. Materials and methods Five Standardbreds, (one castrated male and four mares) three to five years old, weighing 410 to 489 kg were used in the study which was approved by the All-University Committee for Animal Use and Care at Michigan State University. Horses were maintained at pasture for 30 days prior to the study. All horses were vaccinated against equine influenza, rhinopneumonitis, Eastern and Western equine encephalitis, and Streptococcus equi infections. Physical examinations of the horses and endoscopic examinations of the larynx and pharynx at rest and during high-speed treadmill exercise failed to reveal any abnormalities. All horses were trained to run on a treadmill with 0° incline four weeks prior to the experiment. Heart rate was measured during an incremental exercise test to determine the speed that produced HR,,,,,, for each horse. The speeds corresponding to Hanso. and HRW7S were interpolated from these data."6 Heart rate was recorded by use of a telemetry system (Digital VHF Telemetry System, M1403A, Hewlett Packard, Palo Alto, CA). 37 Instrumentation A lateral tracheal catheter was percutaneously placed at the junction of the proximal and middle thirds of the cervical portion of the trachea.‘7 A pharyngeal sidehole catheter was passed through the left naris. The tip of the catheter was positioned at the level of the left guttural pouch opening in the nasopharynx. The pharyngeal catheter was made with 150 cm of polyethylene (polyethylene tubing, 2.15 mm ID, 3.25 mm OD) (Baxter Scientific Products, McGraw Park, IL) with six side holes beginning a distance of eight catheter diameters from the sealed tip.54 Catheters were phase-matched up to 10 Hz.18 Tracheal and pharyngeal pressures were measured by use of differential pressure transducers (DP-45-22, Validyne Engineering Sales, Northridge, CA) and were recorded on a respiratory function computer (Buxco LS-14, Buxco Electronics, Inc., Sharon, CT). The differential pressure transducers were calibrated with a water manometer at 10 cm of H20 before each experiment. Peak inspiratory and expiratory tracheal and pharyngeal pressure and respiratory frequency were determined from the pressure tracings. Values obtained from 10 consecutive breaths were averaged to determine each datum point. Experimental design Horses were exercised on the treadmill with 0° incline for three minutes at four m/s and at one minute at HRSOW. Subsequently, a videoendoscope was placed through the right naris and was secured to the horse’s halter with adhesive strips. Horses were then exercised at HR“,,,5(,, HR,,,,,,75, and HRW for one minute at each speed. then at four m/s for two minutes, and again at HR for one minute. This exercise protocol was 1113!! 38 chosen because investigators have observed that DDSP occurs during changes in treadmill velocity, especially as the treadmill speed is reduced, as well as during high speeds.”-61 The exercise protocol was repeated a total of four times, with two-four days between protocols. During the fourth exercise protocol, horses were instrumented with tracheal and pharyngeal catheters, in addition to the videoendoscope, so that tracheal and pharyngeal pressures could be measured. Subsequently, bilateral tensor veli palatini muscle tenectomy was performed on each horse. Endoscopic examination of the larynx and pharynx was performed during resting breathing and with nasal occlusion on each horse before and two weeks after bilateral transection of the tendon of the tensor veli palatini muscle. The exercise protocol described above was repeated 4 times at 2 day intervals after tensor veli palatini muscle tenectomy. Surgical procedure Ten cadaver specimens (horse heads) were used to investigate the anatomy of the soft palate and to establish a transoral approach to the tendon of the tensor veli palatini muscle. To accomplish this objectives of the study, we developed a novel surgical procedure to transect the tendon of the tensor veli palatini muscle as it coursed around the hamulus of the pterygoid bone prior to inserting in the palatine aponeurosis (Figures 7 and 8). The objectives of the surgical procedure were to transect the tendon of the tensor veli palatini muscle with minimal tissue trauma, minimal postoperative complications, and timely return to normal activity, including eating, drinking, and running. Bilateral tensor veli palatini muscle tenectomy was performed on each horse under general 39 anesthesia using a transoral approach. Horses received antibiotics and nonsteroidal anti- inflammatory medication before and for 48 hours after the surgical procedure. Potassium penicillin (22,000 iu/kg of body weight, IV, q 6 hrs), gentamicin (6.6 mg/kg, IV, q24 hrs), and flunixin meglumine (1 mg/kg, IV, q 12 hrs) were administered to each horse. Horses were sedated with xylazine (0.5 mg/kg, IV), anesthetized with ketamine (1.0 mg/kg, IV) and diazepam (0.1 mg/kg, IV), and positioned in sternal recumbency. Anesthesia was maintained by use of an intravenous infusion of 5% guaifenesin containing 0.5 mg xylazine/ml and 2.0 mg ketamine/ml delivered at 2 ml/kg/hr. The horse’s head was elevated and the mouth opened, using a mouth speculum. The hamulus of the pterygoid bone was palpated approximately 3 cm caudal and slightly axial to the third molar. A 2-cm vertical incision was made directly over the hamulus, through the oral mucosa. A surgical hook was used to grasp the tendon of the tensor veli palatini as it coursed rostral to the hamulus, before it ramified in the palatine aponeurosis. The tendon was observed and then palpated to ensure that the entire tendon was within the hook. The tendon was transected by use of laparoscopic scissors. The rostral edge of the hamulus of the pterygoid bone was palpated again to ensure that the entire tendon had been cut. The incision in the oral mucosa was closed with 2-0 polydioxinan in a cruciate pattern. The procedure was performed bilaterally. Postoperative evaluation Horses were evaluated immediately after recovery from surgery for any discomfort associated with breathing or swallowing. Horses were fed hay soaked in water and bran mash two hours after recovery from anesthesia. Physical examination 40 and endoscopic examination of the nasopharynx and larynx were performed on each horse the day after surgery. Videoendoscopic examination of the larynx and pharynx was performed two weeks postoperatively on each horse during resting breathing and nasal occlusion. To evaluate whether the horse was able to tense the soft palate, the tip of the endoscope was positioned in the right naris just rostral to the junction of the hard and soft palate while the nostrils were manually occluded and the position of the soft palate was evaluated during inspiratory and expiratory efforts. Data analysis Data were analyzed by two-way analysis of variance with a 2 X 6 repeated measures design and post hoc comparisons were made, using the Student-Newman-Keuls’ test. A significance level of P < 0.05 was chosen. Results The surgical procedure (bilateral tensor veli palatini muscle tenectomy) was easily performed with minimal tissue trauma and required 15 to 25 minutes to perform bilaterally. None of the horses exhibited apparent postoperative complications. All horses were able to breath and swallow normally immediately after the procedure. The main effects in the two-way analysis of variance were treadmill speed and surgery. As expected, there was a significant effect of speed on peak tracheal and pharyngeal inspiratory and expiratory pressures. Increasing speed resulted in significantly (P < 0.0001) more negative peak tracheal and pharyngeal inspiratory pressures and more positive peak tracheal and pharyngeal expiratory pressures at all speeds (Table 2). 41 Table 2. Mean tracheal and pharyngeal pressure (:1; STD) in cm of H20 during exercise in five horses before and after bilateral tensor veli palatini muscle tenectomy. SPEED TIP TEP P1P PEP ll Rest 4.6 4.9 1.16 1.6 4.6 4.76 0.8 1.3 1 0.5 1; 0.8 .t 0.4 i 0.9 i 0.5 i 0.7 i 0.4 :1; 0.6 50 43.2 45.9 8.3 10.3 40.6 40.1 5.1 7.9 i 1.7 11.8 i151 1 2.5 t 1.0 $1.7 11.9 :t 4.6 75 49.0 -22.6 10.8 14.0 42.0 41.8 8.4 9.6 i 2.8 :l: 2.6' i 1.8 i 3.5 i 2.9 i 2.5 i 3.0 i 3.8 100 -25.6 -28.6 13.8 16.5 47.5 44.4 11.3 12.8 $2.7 :1; 1.2‘ 1 2.1 i 3.5 i 2.1 i 2.7 i 1.8 i 3.9 4 m/s 44.5 45.4 7.4 9.0 -9.1 -8.4 6.5 7.9 i 4.3 :1: 1.4 i 1.6 :t 1.4 1 3.2 i 1.5 1 3.0 i 1.3 100 -25.8 -28.4 13.9 16.3 45.3 43.4 11.6 13.0 i 2.3 :1; 1.0‘ i 1.9 i 4.2 i 4.0 i 2.1 :t 4.2 i 3.6 * Significantly different from preoperative value; Pre = preoperative pressures; Post = postoperative pressures; Bolded values are those obtained after tenectomy; TIP = peak tracheal inspiratory pressure; TEP = peak tracheal expiratory pressure; PIP = peak pharyngeal inspiratory pressure; PEP = peak pharyngeal expiratory pressure Peak tracheal inspiratory pressure was significantly (P = 0.016) more negative after bilateral tensor veli palatini muscle tenectomy at all speeds compared with preoperative values (Table 2). There was a trend (P = 0.06) for peak pharyngeal pressures to be less negative compared with pre-operative values. A significant effect of bilateral tensor veli palatini muscle tenectomy was not detected for peak tracheal and pharyngeal expiratory pressures. Dorsal displacement of the soft palate did not occur during any of the four preoperative or four post-operative exercise protocols. However. after bilateral tensor veli palatini muscle tenectomy, the rostral half of the soft palate was no longer able to tense, and collapsed dorsally into the nasopharynx during inspiration, resulting in nasopharyngeal collapse and partial nasopharyngeal obstruction while horses were 42 exercising on the treadmill. Inspiratory or expiratory noise was not heard. the larynx and pharynx appeared to function normally during resting breathing during Videoendoscopic examination performed two weeks post-operatively at rest the larynx and pharynx appeared to function normally. However, when the nares were manually occluded to simulate the increased respiratory effort that develops during maximal exercise,” the rostral half of the soft palate collapsed dorsally in the nasopharynx during inspiration and developed a slight concave conformation during expiration in all five horses (Figures 8 and 9). These,results are in contrast to the conformation of the soft palate during nasal occlusion in horses with intact tensor veli palatini muscles: during inspiratory efforts the rostral half of the soft palate moved ventral, deveIOping a concave conformation and expanding the nasopharynx, and during expiratory efforts the soft palate returned to a neutral position. Discussion Bilateral tensor veli palatini muscle tenectomy did not produce DDSP but did cause instability of the rostral half of the soft palate and partial airway obstruction during inhalation in five horses exercising at HR,,,,,,. The rostral half of the soft palate collapsed dorsally into the nasopharynx, causing partial obstruction of the nasopharynx, and significantly more negative peak tracheal inspiratory pressures in five horses exercising at HRm. This suggests that the tensor veli palatini muscle functions to support and dilate the nasopharynx during intense inspiratory efforts in the horse by tensing the rostral soft palate. The caudal aspect of the soft palate is supported by the palatinus and 43 palatopharyngeus muscles. Perhaps dysfunction of the palatinus or palatopharyngeus muscles produces instability of the caudal aspect of the soft palate, leading to DDSP. The tensor veli palatini muscle is a fusiform muscle that arises from the muscular process of the petrous temporal bone, the pterygoid bone, and the lateral lamina of the auditory tube (Figures 7 and 8).” The muscle courses rostroventrally, through the dorsal region of the guttural pouch, along the lateral wall of the nasopharynx, lateral to the levator veli palatini muscle and the pterygoid pharyngeus muscle. The tendon of the tensor veli palatini courses at a 90° angle around the hamulus of the pterygoid bone and ramifies in the palatine aponeurosis. The tendon is supported as it reflects around the hamulus by a fibrous sheath and is lubricated by a bursa.” Contraction of the tensor veli palatini muscles pulls the aponeurotic fascia taut, tensing the portion of the soft palate containing the palatine aponeurosis and depressing the soft palate toward the tongue.43 The function of the tensor veli palatini muscle to support and position the soft palate was observed in clinically normal horses by positioning the videoendoscope just rostral to the junction of the hard and soft palate and manually occluding the nares. The rostral half of the soft palate became tense and depressed ventrally toward the tongue during inspiratory efforts and resumed a normal resting position during expiratory efforts. After bilateral tensor veli palatini muscle tenectomy, the rostral half of the soft palate was flaccid and collapsed dorsally into the nasopharynx during inspiratory efforts and moved ventrally, developing concavity during expiratory efforts. As a result of dynamic collapse of the rostral portion of the nasopharynx during inspiration, peak inspiratory tracheal pressures were significantly more negative after bilateral tensor veli palatini muscle tenectomy compared with preoperative values. The 44 tendency for dynamic collapse of unsupported structures of the nasopharynx increases during intense exercise because of greater negative inspiratory pressure generated by thoracic expansion as peak airflow increases from 5.09 i 0.34l/s at rest to 75 i 9.35 US while exercising at HR,,,,,,.46 Presumably in horses in our study. inspiratory tracheal impedance increased as a result of decreased airway caliber within the nasopharynx. More negative peak tracheal inspiratory pressure was required to maintain airflow at each treadmill speed. Peak pharyngeal inspiratory pressure likely tended to decrease because the pharyngeal catheter was positioned at the level of the nasopharyngeal opening of the guttural pouch, which was within the region of the nasopharyngeal collapse. Therefore, the pharyngeal inspiratory pressure tended to decrease compared with preoperative values, at the position in the nasopharynx where the diameter was narrowed. In dogs, the tensor veli palatini muscle has inspiratory and tonic electro- myographic (EMG) activity that is augmented during nasal breathing and when negative pressure is applied to the pharynx.88 In human beings, the tensor veli palatini EMG activity is augmented during deep nasal breathing.” Augmentation of the EMG activity of the tensor veli palatini muscle during increased inspiratory effort suggests that the tensor veli palatini muscle functions to support and dilate the naSOpharynx during increased respiratory activity. The augmented inspiratory activity of the tensor veli palatini muscle may, as in other pharyngeal dilating muscles, result from reflexes arising from negative pressure receptors in the pharynx.88 Also, phasic inspiratory and tonic EMG activity of the tensor veli palatini muscle increases during hypoxic hypercapnia in dogs, suggesting that there is also centrally mediated respiratory-related regulation of the tensor veli palatini muscle, independent of local reflex control. 45 In our study, despite causing dynamic instability of the soft palate, bilateral tensor veli palatini muscle tenectomy did not result in DDSP in five horses exercising at HR,m during four exercise trials. The tensor veli palatini muscle tenses the rostral half of the soft palate, but it is the caudal portion of the soft palate that may become dysfunctional and displace dorsal to the epiglottis. Three other muscles are involved in positioning the caudal half of the soft palate: the levator veli palatini, the palatinus, and the palatopharyngeus muscles. However, in 1965, Cook suggested that the most common evidence of DDSP obtained via endoscopic examination of the nasopharynx was excess mobility of the palate, and that the rostral half billowed dorsally on inspiration.l3 This suggests that Cook may have observed tensor veli palatini muscle dysfunction as a component of DDSP, and perhaps, although sole loss of function of the tensor veli palatini muscle did not lead to DDSP in our study, the tensor veli palatini muscle may still be involved in the pathogenesis of the disease. Interestingly, a four-year-old Standardbred racehorse was admitted to the teaching hospital for evaluation and treatment of alleged DDSP. The horse had a history of exercise intolerance and made a loud respiratory noise, especially at slow speeds or during deceleration. Endoscopic examination of the larynx and nasopharynx at rest and during treadmill exercise initially revealed instability of the rostral half of the soft palate, mimicking the appearance of the soft palate during the respiratory cycle in the horses of this study after bilateral tensor veli palatini muscle tenectomy. In order to detect instability of the rostral part of the soft palate in the horse admitted for suspected DDSP. the tip of the endoscope was positioned in the right naris just rostral to the junction of 46 the hard and soft palate. During examination on the treadmill, DDSP was observed in the horse as tension was placed on the reins and the treadmill was decelerated. The pathophysiology of DDSP remains unknown, but loss of function of the tensor veli palatini muscle in the study reported here did not cause intermittent DDSP in exercising. However, partial inspiratory nasopharyngeal obstruction did develop with loss of function of the tensor veli palatini muscles. Therefore, the tensor veli palatini muscle may be an important respiratory muscle in the horse, producing stability and dilation of the nasopharynx by tensing the soft palate during maximal exertion, which is ultimately important in an obligate nasal breather such as the horse. Chapter 3 BILATERAL HYPOGLOSSAL AND GLOSSOPHARYNGEAL NERVE BLOCKS CAUSED EPIGLOTTIC RETROFLEXION WITH NO EFFECT ON SOFI‘ PALATE POSITION IN EXERCISING HORSES Abstract Objective The objective of this research was to determine the effect of bilateral hypoglossal and glossopharyngeal nerve block on epiglottic and soft palate position and tracheal and pharyngeal pressures in exercising horses. Design A 2 X 5 repeated measures design was used. Animals Five Standardbreds were used. Procedure Tracheal and pharyngeal pressures were measured in five Standardbreds exercising at the speed at which the horses achieved HR,,,,,,5(,, HR,,,,,,,_,, and HRmax after 47 48 bilateral hypoglossal and glossopharyngeal nerve block and without nerve block. Videoendoscopy was performed during each exercise trial. Nerve block was performed by injecting 1 to 2 cc of 2% mepivicaine hydrochloride between the glossopharyngeal and hypoglossal nerves, as they coursed through the medial compartment of the guttural pouch, using Videoendoscopic guidance and an injection apparatus. Results Peak inspiratory tracheal pressure was significantly (P = 0.02) more negative and peak pharyngeal inspiratory pressure was significantly (P = 0.004) less negative after bilateral hypoglossal and glossopharyngeal nerve block compared to control values. Respiratory frequency was significantly (P = 0.024) lower after nerve block compared to control values. The epiglottis was unstable and retroflexed through the rima glottis during inspiration after bilateral hypoglossal and glossopharyngeal nerve block. Despite loss of contact between the epiglottis and the caudal free margin of the soft palate, DDSP did not occur. Conclusions and clinical relevance Loss of contact of the epiglottis with the soft palate did not affect soft palate position, suggesting that when the soft palate is normal, the epiglottis does not function as a support to hold the soft palate in a ventral position. Therefore, epiglottic dysfunction is not solely responsible for intermittent DDSP in horses, and a neuromuscular dysfunction involving the hyoepiglotticus muscle, geniohyoideus muscle, or the hypoglossal nerve may cause epiglottic retroflexion in horses. 49 Introduction The epiglottis is composed principally of elastic cartilage and has the form of an oblanceolate leaf.” The epiglottis rests on the dorsal surface of the body of the thyroid cartilage and is held there by the thyroepiglottic ligaments, bands of elastic fibers extending from the base of the epiglottic cartilage to the inner surfaces of the laminae of the thyroid cartilage.” The position of the epiglottis is controlled by the position of the hyoid apparatus and larynx and by contraction of the hyoepiglotticus muscle.”86 Rostral movement of the hyoid apparatus, a function of the geniohyoid muscle, moves the epiglottis rostrally as well.”92 Contraction of the hyoepiglotticus muscle pulls the epiglottis toward the basihyoid bone, depressing the epiglottis against the caudal free margin of the soft palate. In the horse, the epiglottis is positioned dorsal to the soft palate and the caudal free margin of the soft palate closely approximates the larynx at the base of the epiglottis.13 The epiglottis may function as a rigid support to hold the soft palate ventral to the epiglottis. If the epiglottis holds the soft palate down, and prevents the soft palate from displacing dorsally, this support function would be especially important during intense exercise when nasopharyngeal and tracheal pressures become more negative, and tend to cause dynamic collapse of unsupported structures in the airway. Because a short, flaccid, or malformed epiglottis may be unable to maintain the soft palate in a subepiglottic position,”'8" epiglottic hypoplasia, malformation, and dysfunction have been implicated in the pathogenesis of DDSP in exercising horses.”'”'”"‘5 The function of the epiglottis during breathing has not been investigated in exercising horses despite the implication (by some investigators) that epiglottic 50 dysfunction is principally responsible for intermittent DDSP.”'”'”"” If the epiglottis is responsible for maintaining the position of the soft palate, then destabilizing the epiglottis by impairing the function of the hyoepiglotticus and geniohyoid muscles should cause intermittent DDSP in exercising horses. The hyoepiglotticus and geniohyoid muscles, as well as the styloglossus, hyoglossus, and genioglossus muscles receive motor innervation from the hypoglossal nerve, the XIIth cranial nerve (Figure 11).” Blockade of the hypoglossal nerve results in dysfunction of the muscles that cause cranial movement of the basihyoid bone and of the muscle that approximates the epiglottis and the basihyoid bone. The majority of the motor neurons in this nerve exhibit phasic discharge coincident with breathing, generally discharging early in the inspiratory phase.”54 The early peak discharge pattern activates muscles that widen and stiffen the nasopharynx before it is subjected to the transmural forces produced by thoracic expansion. ‘7"8'”'4°'5‘ Specifically, contraction of the genioglossus and geniohyoid muscles stabilizes and enlarges the pharynx by altering the position of the hyoid apparatus.”92 The geniohyoideus muscle mechanically displaces the hyoid bone cranially, tensing the lateral walls of the pharynx, whereas the genioglossus enlarges the upper airway by protruding the tongue, which places rostral traction on the hyoid bone?”16 The hyoepiglotticus muscle, the only muscle to insert on the epiglottis, is a bilobed muscle that originates on the basihyoid bone, is enclosed in an elastic sheath called the hyoepiglottic ligament, and inserts on the ventral body of the epiglottis.” Contraction of the hyoepiglotticus muscle in the horse approximates the basihyoid bone and the epiglottis, thereby pulling the epiglottis against the caudal free margin of the soft palate, expanding the nasopharynx and the rima glottis. The gloss0pharyngeal nerve 51 provides sensory innervation to the nasopharynx, specifically to the dorsal and lateral walls of the pharynx as well as to the naSOpharyngeal surface of the proximal half of the soft palate, and only supplies motor innervation to the stylopharyngeus muscle, a muscle that contracts during swallowing.” Therefore, in this study we examined the effect of bilateral hypoglossal and glossopharyngeal nerve block on epiglottic and nasopharyngeal function and position and on trachea] and nasopharyngeal pressures in exercising horses. Materials and methods Five Standardbreds (three geldings and two mares), two to seven years old, weighing between 400 and 427 kg were used in the study, which was approved by the All-University committee for Animal Use and Care at Michigan State University. Horses were maintained at pasture for 90 days prior to the study. All horses were vaccinated against tetanus, equine influenza rhinopneumonitis, Eastern and Western equine encephalitis, and Streptococcus equi. Physical examinations of the horses and endoscopic examinations of the larynx and pharynx at rest and during high-speed treadmill exercise failed to reveal any abnormalities. All horses were trained to run on the treadmill prior to the experiment. Horses were unfit before the experiment and only ran on the treadmill during the protocol. Three horses paced and two horses trotted. None of the horses ever galloped. Although the horses exercised at different gaits, each horse exercised at the same gait before and after nerve block, meaning specifically, that if a horse paced with the bilateral hypoglossal and glossopharyngeal nerve block, that horse also paced without the nerve block. Heart rate was measured using a telemetry system (Digital VHF Telemetry System, MI403A, Hewlett Packard, Palo Alto, CA) during an 52 incremental exercise test to determine the speed that produced HR,,,,, for each horse.” The speeds corresponding to HR,,,,,,5(, and HRHWS were interpolated from these data. Instrumentation A tracheal catheter that measured lateral pressure was percutaneously placed at the junction of the proximal and middle thirds of the cervical trachea.95 The tracheal catheter was made with 150 cm of polyethylene tubing (2.15 mm ID, 3.25 mm OD) (Baxter Scientific Products, McGraw Park, IL). A pharyngeal sidehole catheter was passed through the left naris and the tip of the catheter was positioned at the level of the left guttural pouch opening. The pharyngeal catheter was made with 150 cm of polyethylene tubing (2.15mm 1D, 3.25 mm OD) (Baxter Scientific Products, McGraw Park, IL) with 6 sideholes beginning a distance of 8 catheter diameters from the sealed tip.9 Tracheal and pharyngeal catheters were constructed so as to be phase matched to 10 Hz.77 Tracheal and pharyngeal pressures were measured using differential pressure transducers (DP-45-22, Validyne Engineering Sales, Northridge, Calif.) and were recorded on a respiratory function computer (Buxco LS-14, Buxco Electronics, Inc., Sharon, CT). The differential pressure transducers used to measure tracheal and pharyngeal pressures measured pressure ranges of j; 56 cm of H20 and :1; 22.5 cm of H20, respectively. The differential pressure transducers were calibrated with a water manometer before each experiment. Peak inspiratory and expiratory tracheal and pharyngeal pressure and respiratory frequency were determined from the pressure tracings. Ten consecutive breaths were averaged to determine each data point. 53 Experimental design Horses were exercised on the treadmill for three minutes at four m/s and one min at HRmmsu. A videoendoscope was then placed through the right naris and secured to the halter with Velcro” strips. Horses were then exercised at HR,,,,,,50, HRW75, and HR“m for two min at each speed. Pressure measurements were recorded during the second 60 seconds at each speed and measurements were made during the last 30 seconds. Catheters were flushed with air during the experiment to avoid fluid accumulation and dampening of pressure values. Horses performed the exercise trial twice: once after bilateral hypoglossal and glossopharyngeal nerve block, and once without nerve block. The Videoendoscopic image was correlated with the respiratory cycle by observing the pressure tracings on the respiratory function computer. Each protocol was recorded on videotape. The sequence of exercise trials was randomized so that three horses performed the trial with motor blockade first, and two horses performed the trial first without nerve blockade. Hypoglossal and glossopharyngeal nerve block A lip twitch was applied to the horse’s nose for restraint. The videoendoscope was passed through the right naris. An injection apparatus was made using a 12-cc syringe attached to a 30—cm length of polyethylene tubing (1.19 mm ID, 1.7 mm OD) (Baxter Scientific Products, McGraw Park, IL). A 22 gauge needle, with the hub removed, was attached to the opposite end of the polyethylene tubing. The tubing was passed through a hollow plastic rod, 45.5 cm in length ( 1D. 4.0 mm, 0D. 5.0 mm). This rod was passed through the right naris and used to elevate the cartilaginous flap of 54 the nasopharyngeal opening of the guttural pouch, allowing the endoscope to be passed into the guttural pouch, followed by the plastic rod, which contained the injection apparatus. The hypoglossal and glossopharyngeal nerves were identified coursing from caudodorsal to cranioventral in the medial compartment of the guttural pouch. The needle was inserted between the hypoglossal and glossopharyngeal nerves, directly over the hypoglossal nerve, at a point approximately 2/3 of the distance along the course of the nerves through the guttural pouch (Figure 12). One to two ml of 2% mepivicaine hydrochloride (The UpJohn Company, Kalamazoo, M1) were injected directly over the hypoglossal nerve. Due to the proximity of the two nerves, glossopharyngeal nerve block was also achieved. The procedure was repeated on the left side. In all five horses, blockade of glossopharyngeal nerves was demonstrated by lack of gagging or swallowing when the dorsal pharyngeal recess and the lateral pharyngeal walls were probed with a biopsy instrument passed through the videoendosc0pe. Successful nerve block was indicated by protrusion of the horse’s tongue from the mouth. Horses were able to swallow with the nerve block. Horses wore a muzzle for two hours after hypoglossal nerve block to prevent eating and chewing the tongue. Each horse received phenylbutazone (4 mg/kg, orally) and potassium penicillin (22,000 iu/ kg, IV) once at the end of the hypoglossal nerve block trial. The muzzle was removed when the horse was able to retract its tongue. The horse was then observed while eating for any signs of dysphagia. 55 Data analysis Data were analyzed by two-way analysis of variance with a 2 X 5 repeated measures design and post hoc comparisons were made using the Student-Newman-Keuls’ test. A significance level of P < 0.05 was chosen. Results Bilateral hypoglossal and glossopharyngeal nerve block was easily performed in standing horses with no lasting side effects. All horses regained normal tongue function and were able to eat normally by two hours after the procedure. The main factors in the two-way analysis of variance were treadmill speed and nerve block. As has been shown in multiple previous studies, there was a significant effect of speed on peak tracheal and pharyngeal inspiratory and expiratory pressures and respiratory frequency.”"""99 Increasing the treadmill speed caused significantly (P < 0.0001) more negative peak tracheal inspiratory pressures and more positive peak tracheal and pharyngeal expiratory pressures in both control and nerve block groups, but only caused more negative peak pharyngeal inspiratory pressure in the control group (Table 3). Peak tracheal inspiratory pressure was significantly (P = 0.027) more negative after bilateral hypoglossal and glossopharyngeal nerve block compared to unblocked values (Table 3). There was a significant (P = 0.002) interaction between speed and nerve block such that increasing treadmill speed produced a greater change in peak tracheal inspiratory pressure after bilateral hypoglossal nerve block. 56 Peak pharyngeal inspiratory pressure was significantly (P = 0.005) less negative after bilateral hypoglossal and glossopharyngeal nerve block compared to unblocked values. Again, there was a significant (P = 0.005) interaction between speed and nerve block, so that as treadmill speed increased, the change in peak inspiratory pharyngeal pressure was less after nerve block. No significant effect of nerve block was detected for peak tracheal or pharyngeal expiratory pressures. Respiratory frequency was significantly (P = 0.024) lower after nerve block. During Videoendoscopic examination, epiglottic retroflexion was consistently observed during inhalation, based on negative tracheal and pharyngeal pressure tracings, in all horses exercising at HRWSO, HRW75, and HRW after bilateral hypoglossal and glossopharyngeal nerve block (Figure 13). Table 3. Mean tracheal and pharyngeal pressures and respiratory frequency during 57 exercise in five horses before and after bilateral hypoglossal nerve block. FREQ Unblk Rest -2.1 -I.9 1.7 1.4 -1.8 -I.5 1.3 0.9 20 26 1; 0.7 1; 0.6 1' 0.6 i 0.7 :1; 0.8 1; 0.6 : 0.6 i 0.5 1: 0 i 13 50 -13.3 -I3.8 7.8 9.7 -10.5 -7.5 4.9 4.9 102 65 i 2.3 i 2.8 i (1.7 t 0.9 :t 2.8 i 2.5 : 1.0 i 1.2 i 28 i 21. 75 —18.8 -24.7 10.2 ".6 -10.5 -7.6 5.4 6.9 112 68 :1; 3.4 i 3.0’ t 1.6 i 1.8 :1; 3.5 :t 2.5‘ : 1.6 i 2.5 i 8 j: 8‘ 100 -26.4 -35.7 14.3 13.3 -13.6 -7.5 8.6 8.3 122 58 i; 2.7 i 3.8 i 2.5 i 2.4 :1; 3.8 1: 3.0‘ 3 2.7 i 2.3 t 15 j; 5‘ Pressure in cm of H20 : STD; 5 %HR,,,a,; * significantly different from unblocked value; Unblk = pressures without bilateral hypoglossal nerve block; Blk = pressures with bilateral hypoglossal nerve block; Bold values are bilateral hypoglossal nerve block values; TIP = peak tracheal inspiratory pressure; TEP = peak tracheal expiratory pressure; PIP = peak pharyngeal inspiratory pressure; PEP = peak pharyngeal expiratory pressure; FREQ = respiratory frequency, breaths/minute The epiglottis rapidly changed position, as it retroflexed during inhalation and returned to a normal position during exhalation. As treadmill speed increased and peak tracheal inspiratory pressure became more negative, the epiglottis displaced further away from the soft palate toward the rima glottis during each breath. At HR,,,,,50, the epiglottis was slightly elevated above the soft palate during inspiration, but at HR,,,,,,, the epiglottis formed almost a 90’ angle with the soft palate and the tip of the epiglottis was in contact with the corniculate processes of the arytenoid cartilages or actually prolapsed through the rima glottis during peak inspiration. No abnormal respiratory noise was heard during epiglottic retroflexion. Despite complete loss of contact between the epiglottis and the soft palate, the soft palate maintained its normal position. 58 Discussion An important observation in this study was that bilateral hypoglossal and glossopharyngeal nerve block caused epiglottic retroflexion in exercising horses. This condition has been observed clinically in horses.59 Retroflexion occurred during inspiration and became more severe as exercise intensity increased, suggesting that it is a dynamic obstruction resulting from the airflow and negative intraluminal pressure that occurs during inhalation. As a result of this dynamic inspiratory obstruction peak, tracheal inspiratory pressure became more negative as the horse worked to maintain airflow. Simultaneously, peak inspiratory pharyngeal pressure became less negative because obstruction of the rima glottis by the epiglottis probably reduced airflow. Bilateral hypoglossal and gloss0pharyngeal nerve block had no effect on expiratory pressures because the obstruction occurred only during inhalation. Horses altered their breathing strategy because of the inspiratory obstruction by decreasing respiratory frequency by approximately 50% after nerve block. Work of breathing is made more efficient by increasing tidal volume and decreasing respiratory frequency, which compensates for the added resistive work imposed by the inspiratory obstruction.‘6 Loss of motor function of the hyoepiglotticus muscle was most likely responsible for the epiglottic retroflexion, and may even be involved in the pathogenesis of a similar clinical syndromes9 Contraction of the geniohyoideus muscle also alters epiglottic position by pulling the hyoid arch rostrally which increases tension on the hyoepiglottic ligament, pulling the epiglottis toward the basihyoid bone.8° However, gross anatomical dissection and muscle stimulation, suggest that the hyoepiglotticus muscle is responsible 59 for maintaining the position of the epiglottis and that the range of motion of the basihyoid bone may be insufficient to explain epiglottic retroflexion. Epiglottic hypoplasia or epiglottic dysfunction has been suggested as an etiologic factor in the pathogenesis of DDSP”'”*”"5 because an anatomically abnormal epiglottis may be mechanically incapable of holding the soft palate in its normal position.” The results of this study question this assumption. The intrinsic soft plate muscles responsible for stabilizing the caudal half of the soft palate are innervated by the pharyngeal branch of the vagus nerve, which is anatomically very separate from the hypoglossal and glosso- pharyngeal nerves in the area where the nerves were blocked and was therefore not blocked.” Consequently, in the present study, the intrinsic neuromuscular control of the soft palate was presumably normal so that the caudal half of the soft palate was tensed and stabilized during exercise. Under these conditions, the epiglottis apparently served no support function. Despite complete loss of contact between the epiglottis and the soft palate during inspiration, the soft palate maintained its normal position. Horses used in this study exercised at HRH“. The speed that results in HR,” is lower than maximum racing speed, and peak inspiratory pressures measured in the horses in this study were less negative than peak inspiratory pressures measured in fit horses exercising at 14 m/s. ‘9'“ If horses with bilateral hypoglossal and glossopharyngeal nerve blocks had exercised at racing speed, perhaps DDSP would have occurred. However, Rehder et al. showed that DDSP is not caused by excess negative inspiratory pressures in the nasopharynx."4 It is important to realize that the results of this study pertain only to horses with normal soft palate function. When soft palate function is abnormal, the epiglottis may 60 provide support by holding the soft palate down. In such cases, epiglottic augmentation may provide a thicker and more rigid structure that helps to maintain the soft palate ventral to the epiglottis.“ In conclusion, removing epiglottic contact from the caudal free margin of the soft palate did not affect soft palate position, and therefore, epiglottic dysfunction or malformation is unlikely to be the cause of DDSP in exercising horses. Bilateral hypoglossal nerve block did cause epiglottic retroflexion, and therefore a neuromuscular dysfunction involving the hypoglossal nerve or hyoepiglotticus muscle may be involved in the pathogenesis of clinical epiglottic retroflexion in horses. Chapter 4 BILATERAL NERVE BLOCKADE OF THE PHARYNGEAL BRANCH OF THE VAGUS NERVE PRODUCES PERSISTENT SOFT PALATE DYSFUNCTION IN HORSES Abstract Objective The objective of this research was to determine the effect of bilateral blockade of the pharyngeal branch of the vagus nerve on soft palate function in horses. Animals Five Standardbred horses were used. Procedure Peak tracheal inspiratory and expiratory pressure and airflow were measured while horses exercised at the speeds corresponding to the speed that resulted in HRmam and HR,m with and without pharyngeal branch of the vagus nerve blockade. Respiratory frequency:stride frequency coupling (Rf:Sf) was measured by attaching a catheter connected to a pressure transducer to the right forelimb of the horse and correlating the foot-fall measurements with respiratory frequency. By means of a videoendoscope and 61 62 injection apparatus, the pharyngeal branch of the vagus nerve was blocked bilaterally as the nerve coursed through the guttural pouch across the longus capitus muscle. Results Persistent dorsal displacement of the soft palate occurred in all horses after nerve blockade. Peak expiratory tracheal pressure increased (14.1 to 36.2 cm H20) (P = 0.001), expiratory impedance increased (0.25 to 0.88 cm HID-14s") (P = 0.007), and minute ventilation decreased (1090.2 to 875.2 l/min) (P = 0.04) compared to control values. Respiratory frequency:stride frequency coupling ratio decreased from 1 to 0.63 (P = 0.009) such that horses took one breath per stride without the nerve block and, approximately, one breath per two strides with the block. Conclusions The results of this study show that DDSP creates a flow limiting expiratory obstruction and may be caused by neuromuscular dysfunction involving the pharyngeal branch of the vagus nerve. Dorsal displacement of the soft palate may alter performance by causing an expiratory obstruction and by altering breathing strategy in horses. Clinical relevance Young horses are commonly infected with upper respiratory viruses, accompanied at times by secondary bacterial infections. If the pharyngeal branch of the vagus nerve is damaged during these infections, improved prevention and treatment of airway infections in young horses may make DDSP a preventable and rare disease. 63 Introduction Dorsal displacement of the soft palate is an intermittent obstructive upper airway condition that occurs in athletic horses during high-intensity exercise.” The epiglottis is positioned dorsal to the soft palate and the caudal free margin of the soft palate closely approximates the larynx at the base of the epiglottis, so that there is no communicating space between the nasopharynx and the oropharynx in horses.” Dorsal displacement of the soft palate occurs when the caudal free margin of the soft palate is positioned dorsal to the epiglottis, creating a velopharyngeal obstruction.29 The pathogenesis of this condition remains obscure and only fragmentary knowledge exists concerning the physiology of veIOpharyngeal patency in horses. Primary neuropathy and muscle dysfunction have been implicated as a cause of DDSP in horses.”13 Indeed, if the efferent innervation to the muscles supporting the caudal half of the soft palate was damaged, instability of this portion of the soft palate may occur, leading to DDSP. The position of the soft palate is determined by the coordinated activity of groups of antagonistic muscles, which include the levator veli palatini, tensor veli palatini, palatinus, and palatopharyngeus muscles.‘3'5‘ The levator veli palatini muscle elevates the soft palate during swallowing, vocalization, and eructation, and facilitates oral ventilation.‘3'5' The tensor veli palatini muscle expands the nasopharynx during inspiration by tensing the palatine aponeurosis, depressing the rostral half of the soft palate toward the tongue."7"5‘ Impairing the function of the tensor veli palatini muscles by transecting the tendon of the muscle prior to ramification in the palatine aponeurosis causes the rostral half of the soft palate to become flaccid, but DDSP did not occur.“ 64 The palatinus and palatopharyngeus muscles control the position of the caudal half of the soft palate, which is the portion of the soft palate that displaces dorsally and obstructs airflow in affected horses."3'5"’” The palatinus muscle consists of two fusiform muscles, which lie on either side of the midline of the soft palate, caudal to the hard 5 The muscle attaches to the palate, and just ventral to the nasopharyngeal mucosa.2 caudal aspect of the palatine aponeurosis and terminates near the caudal free margin of the soft palate. A small muscle bundle arising from the lateral aspect of each muscle continues a short distance caudodorsally into the palatopharyngeal arch.” The palatopharyngeus muscle originates from the palatine aponeurosis and the lateral border of the palatinus muscle. It travels caudally along the lateral wall of the nasopharynx to the pharyngeal raphe, forming part of the superior constrictor muscle group.” Contraction of the palatinus and palatopharyngeus muscles shortens the soft palate and depresses the caudal portion toward the tongue.‘3'5"3‘ Both muscles are innervated by the pharyngeal branch of the vagus nerve.” This nerve branches from the parent vagus nerve at the level of the cranial cervical ganglion and courses cranioventrally along the medial wall of the guttural pouch to the dorsal wall of the pharynx, where it ramifies in the pharyngeal plexus.” Because of this innervation, we hypothesized that bilateral nerve block of the pharyngeal branch of the vagus nerve would cause DDSP in horses. We developed a unique, non-invasive technique to block these nerves using endoscopic guidance. 65 Materials and methods This study was divided into three experiments. Experiment One was a study performed to investigate the effect of the bilateral pharyngeal branch of the vagus nerve block on soft palate function. Based on the results of Experiment One, Experiment Two was performed to measure upper airway mechanics in horses with DDSP, produced by bilateral pharyngeal branch of the vagus nerve block, and Experiment Three was performed to measure the effect of DDSP on Rf:Sf in horses. Five Standardbred horses (three geldings and two mares), two to seven years old, weighing between 400 and 427 Kg were used in all three experiments, which were approved by the All-University Committee for Animal Use and Care at Michigan State University. All horses were vaccinated against tetanus, equine influenza, rhinopneumonitis, Eastern and Western equine encephalitis, and Streptococcus equi. Physical examinations of the horses and endoscopic examinations of the larynx and nasopharynx at rest and during high-speed treadmill exercise failed to reveal any abnormalities. All horses were trained to run on the treadmill prior to the experiment. Three horses paced and 2 horses trotted. Each horse maintained the same gait throughout the experiment. Maximum heart rate was determined using a telemetry system (Digital VHF Telemetry System, MI403A, Hewlett Packard, Palo Alto, CA) and an incremental exercise test."6 The speed corresponding to HR,,,,,,75 was interpolated from these data. A lip twitch was applied to the horse’s nose for restraint. The videoendoscope was passed through the right naris. An injection apparatus was constructed using a Darien tracheal—wash catheter (Mill Rose Laboratories, 7310 Corporate Blvd. Mentor, 66 OH). The inner tubing was removed and replaced with 30 cm of polyethylene tubing (1.19 mm ID, 1.7 mm OD). A 22-gauge needle, with the hub removed, was attached to the end of the tubing. The polyethylene tubing was passed, in a retrograde fashion, through the Darien catheter until the tip of the needle was covered by the outer sleeve of the catheter. The catheter was then passed through the biopsy portal of the endoscope. A 10-ml syringe filled with mepivicaine hydrochloride (The UpJohn Co., Kalamazoo, MI) was attached to the end of the injection apparatus. A hollow plastic rod, 45.4 cm in length (4.0 mm ID, 5.0 mm OD) was also passed through the right naris and was used to elevate the cartilaginous flap of the nasopharyngeal opening of the guttural pouch, allowing the endoscope to be passed into the guttural pouch. The pharyngeal branch of the vagus nerve was identified in the ventral medial compartment of the guttural pouch as it coursed across the longus capitus muscle (rectus capitus ventralis major).” The needle was advanced and inserted just beneath the nerve, under the guttural pouch membrane, and 1 ml of mepivicaine hydrochloride was injected. The left pharyngeal branch of the vagus nerve was blocked using the same procedure. Endoscopic examination was performed after bilateral nerve block of the pharyngeal branch of the vagus nerve to assess the position of the soft palate. Horses wore a muzzle for four hours after the bilateral nerve block to prevent them from eating and aspirating feed. Experiment One Experiment One measured the effect of bilateral pharyngeal branch of the vagus nerve block on soft palate function in horses. 67 Instrumentation The soft palate was observed by means of a videoendosc0pe that was placed in the nasopharynx and secured to the halter with adhesive strips. Observations were made with horses standing and exercising on the treadmill. Experimental protocol Within ten minutes after bilateral pharyngeal branch of the vagus nerve blockade, horses were exercised on a treadmill for three minutes at four Ms“. Horses then exercised at HRW75 for two minutes. The treadmill was stopped and horses rested for one minute. The treadmill speed was increased to HRmax for two minutes. Experiment TWO Experiment Two examined upper airway mechanics in horses with DDSP, produced by bilateral pharyngeal branch of the vagus nerve block. Instrumentation Horses were instrumented to measure tracheal inspiratory and expiratory pressure and inspiratory and expiratory airflow while running on a treadmill. Measurement techniques for upper airway measurements have been described.‘7 Briefly, a tracheal catheter that measured lateral pressure was percutaneously placed at the junction of the proximal and middle thirds of the cervical trachea.”"8 The tracheal catheter was made with 150 cm of polyethylene tubing (2.15 mm ID, 3.25 mm OD) (Baxter Scientific Products, McGraw Park, IL). Tracheal pressure was measured using a differential 68 pressure transducer (DP45-22, Validyne Engineering Sales, Northridge, CA) and was recorded on a respiratory function computer (Buxco LS-14, Buxco Electronics, Inc., Sharon, CT). The differential pressure transducer was calibrated with a water manometer to 10 cm H20 before each experiment. Airflow was measured using a 15.2 cm—diameter pneumotachograph laminar flow element (Laminar flow straightening element, Merriam Instruments, Grand Rapids, MI) mounted on an airtight face mask. The fiberglass face mask covered the mouth and nostrils, was fitted on the horse’s head, and sealed with a rubber shroud and adhesive tape. The pneumotachograph was mounted on the end of the face mask with a protective wire screen positioned between the horse’s muzzle and the pneumotachograph. The resistance of the pneumotachograph was 0.04 cm of H20 L"s‘l measured up to an airflow of 90 L5". The combined resistance of the mask-pneumotachograph system was 0.05 cm of H20 L“s" at 90 L 5". Pressure changes across the pneumotachograph were measured using a differential pressure transducer. The pressure signal was proportional to inspiratory and expiratory airflow. Prior to each protocol the pneumotachograph was calibrated using a rotameter flow meter (Model FP- 2-37-P-10/77, Fisher and Porter Co. , Wanninster, PA) capable of measuring airflow up to 90 Ls". Pressure signals were passed into a pulmonary function computer and then through low-pass filters. Peak inspiratory and expiratory tracheal pressures, peak inspiratory and expiratory airflow, and respiratory frequency were obtained from the physiograph tracings. Tidal volume was obtained by digitally integrating the flow signal with respect to time. Peak tracheal inspiratory and expiratory impedances were calculated as the ratio of peak tracheal pressure and peak airflow. Minute ventilation was 69 calculated as the product of tidal volume and respiratory frequency. Ten consecutive breaths were obtained to determine a data point. Experimental design Horses were exercised on a treadmill for three minutes at four Ms". Horses then exercised at HRmam for two minutes. The treadmill was stopped and horses rested for one minute. The treadmill speed was increased to HR,,,,,, for two minutes. Catheters were flushed with air during the experiment to avoid fluid accumulation and dampening of pressure values. Horses performed the exercise trial two times: once with the nerve block while upper airway function measurements were made and then without the nerve block. The sequence of exercise trials was randomized so that three horses performed the trial with the nerve block first and two horses performed the trial first without the nerve block. Nerve block and control measurements were made 7-10 days apart. After each experiment, horses received phenylbutazone (4 mg/ kg, IV) and potassium penicillin (22,000 iu/kg,1V). Experiment Three Experiment Three measured the effect of DDSP on Rf:Sf in horses. Instrumentation Stride frequency and respiratory frequency were measured while horses exercised on the treadmill. These measurements were made separately from the upper airway measurements because the face mask pneumotachograph system may alter respiratory 70 frequency.” Respiratory frequency was measured using a tracheal catheter described in Experiment Two. Stride frequency was measured using a 2—M polyethylene catheter with a water balloon attached to the end of the catheter. The catheter was attached to the distal right forelimb of the horse and connected to a pressure transducer. The contact phase of the right forelimb produced a positive pressure deflection. Tracheal pressure and the pressure signal from the right forelimb were displayed on the pulmonary function computer so that Rf:Sf could be determined by counting the number of breaths and the number of strides per minute and computing the ratio. Experimental design The same exercise protocol and design used in Experiment Two was used in Experiment Three. Three horses performed the Rf:Sf protocol first and then rested for 45 minutes, after which the face mask was placed on the horse’s head and upper airway measurements were made. The order of the protocol was reversed for the last two horses. Data analysis Quantitative data were analyzed using a 2 X 5 repeated measures analysis of variance and post-hoe comparisons were made using the Student-Newman-Keuls’ test. A significance level of P < 0.05 was chosen. 7 1 Results Experiment One Bilateral pharyngeal branch of the vagus nerve block was easily performed in standing horses with no long-term effects. Persistent DDSP occurred in all horses in this study within two to fifteen minutes after the nerve block. Videoendoscopy during exercise revealed that DDSP was persistent and that the caudal free margin of the soft palate billowed across the rima glottis during exhalation. All horse regained pharyngeal function and were able to swallow within four hours. Experiment 2 The 2 main effects in the analysis of variance were speed and nerve block. As has been reported previously, increasing speed from HRW75 to HRm, resulted in significantly (P < 0.001) more negative peak tracheal inspiratory pressure, more positive peak expiratory pressure, higher inspiratory and expiratory flows, increased tidal volume, and increased minute ventilation‘7'33'3‘-‘°'°‘ (Table 4). Bilateral pharyngeal branch of the vagus nerve block produced persistent DDSP in all horses. This obstruction caused an increase in peak tracheal expiratory pressures (P = 0.0015) and expiratory impedance (P = 0.007), and can therefore be considered an expiratory obstruction. No significant difference was detected in peak expiratory flow between horses with bilateral pharyngeal branch of the vagus nerve blockade and control horses. Tidal volume was not significantly affected by nerve block, but respiratory frequency was significantly lower (P = 0.04), causing minute ventilation (respiratory frequency X tidal volume) to be significantly reduced (P = .048) with the nerve block 72 compared to control values (Table 4). Although both inspiratory and expiratory time increased after nerve block, we were unable to detect a significant difference in either time compared to control values. Interestingly, peak tracheal inspiratory pressure was less negative (P = 0.029), and peak inspiratory flow was not significantly affected compared to control values. Therefore, tracheal impedance decreased (P = 0.018) with nerve block compared to control values. Table 4. Mean tracheal expiratory and inspiratory peak pressures and impedance, expiratory and inspiratory airflows, tidal volume, respiratory frequency, minute ventilation, and Rf:Sf ratios (j; STD) in five horses before and after bilateral pharyngeal branch of the vagus nerve block. SPEED (961m...) TEP 2, EF TIP 2. 1r VT 1' vE Te Ti 75 comm] 10.4 0.26 39.6 21.5 0.62 35 10.3 69 785.8 0.39 0.47 1 2.6 1 0.07 1 3.26 1 3.67 1 0.08 1 4.45 1 1.19 1 11.4 1 90.8 1 0.04 1 .03 75 black 24.7. 0.7. 372 10.3- 0.33. 33.1 12.6 511° 721° 0.49 0.53 1 5.1 1 0.26 1 6.67 1 2.47 1 0.05 1 6.7 1 6.7 1 9.0 1 99.6 1 0.09 1 .03 100 14.1 0.26 54.3 32 0.69 46.6 14.1 79 1090.2 0.38 0.43 control 1 2.2 1 0.07 1 3.21 1 7.9 1 0.16 1 5.0 1 2.22 1 13.4 1 147.7 1 0.06 1 .06 100 block 36.2‘ 0.11. 46.7 18.6‘ 0.41. 44.5 15.0 57‘ 375.2. 0.48 0.44 1 11.3 1 0.33 1 10.4 1 7.61 1 0.11 1 9.0 1 3.31 1 9.11 1 114.5 1 0.11 1 .07 * Significantly different from preoperative values; HRH“, = maximum heart rate; TEP = peak tracheal expiratory pressure in cm of H20; Zc = expiratory impedance in cm of HZO-l"-s"; EF = expiratory flow in Is“; TIP = peak tracheal inspiratory pressure in cm of H2); Z = tracheal inspiratory impedance in cm of H2)-l"-s"; IF = inspiratory flow in Is"; VT = tidal volume in l, f = frequency in breaths-min"; VE minute ventilation in l-min"; Te = expiratory time; block = with bilateral pharyngeal branch of the vagus nerve block; control = without nerve block Experiment 3 Respiratory frequency was significantly lower (P = 0.01) while stride frequency was not affected by nerve block. Therefore, Rf:Sf was significantly lower (P = 0.009). 73 Also, inspiratory and expiratory times were significantly longer, (P = 0.0002 and P = 0.007, respectively), compared to control values (Table 5). Table 5. Respiratory and stride frequency, Rf:Sf ratios, and inspiratory and expiratory times in five horses with and without bilateral pharyngeal branch of the vague nerve block SPEED Resp. freq. Stride (%HRmax) freq“ 75 control 122 120 1.02 0.32 0.28 i 1.1 :l: O :l: 0.09 i 0.02 i 0.02 75 block 68 120 0.57* 0.45* 0.42* i 1.8 i 0 :l: 0.15 :l: 0.01 :l: 0.09 100 control 130 120 1.0 0.29 0.26 :l: 1.0 i 0 :t 0.0 i 0.01 :l: 0.02 100 block 82 132 0.63" 0.44“ 0.50" i 2.8 :l: 8.0 :l: 0.21 i 0.08 i 0.12 * Significantly different from preoperative values; HR”, = maximum heart rate; Resp. freq. = respiratory frequency in breaths min; Stride freq. = stride frequency in strides per min; R:S = respiratoryzstride frequency coupling ratio; Ti = inspiratory time in seconds; Te = expiratory time in seconds; block = bilateral pharyngeal branch of the vagus nerve block; control = without nerve block Discussion This is the first report of a reproducible model of persistent DDSP in horses. This model allowed us to measure upper airway mechanics and breathing strategy in horses while the soft palate was displaced, and it provided information about the potential etiology of DDSP. Bilateral pharyngeal branch of the vagus nerve block produced persistent displacement, implicating dysfunction of the pharyngeal branch of the vagus nerve and the palatinus and palatopharyngeus muscles in the pathogenesis of the clinical disease. 74 An important conclusion of this study was that DDSP. created an expiratory nasopharyngeal obstruction in research horses. This observation has been made in clinical cases by other investigators.“'°8 During exhalation, air flow caused the soft palate to billow, somewhat like a compliant sheet, obstructing expiratory airflow. As a result of this obstruction, expiratory pressure increased in an attempt to maintain expiratory airflow. Peak expiratory airflow did not change, but expiratory impedance increased. Tracheal inspiratory pressure became less negative and inspiratory impedance decreased. Previous studies have reported less negative peak tracheal inspiratory pressures while the soft palate was dorsally displaced in clinical cases.“68 Peak inspiratory airflow did not change, and therefore upper airway caliber must have increased, suggesting that horses were breathing trans-orally while the soft palate was displaced dorsally. Based on clinical observations, it has been suspected that horses might open—mouth breathe during episodes of DDSP.” Trans-oral breathing would be a unique feature of this syndrome, because horses generally are obligate nasal breathers. Horses decreased their respiratory frequency in response to the increased expiratory impedance. Tidal volume did not significantly increase in blocked horses and minute ventilation decreased. Blood gases were not measured in this study, but reduced minute ventilation in blocked horses compared to controls suggests that horses with DDSP may not maintain appropriate ventilation at high speeds. Stride frequency and respiratory frequency were measured simultaneously in control and blocked horses to determine the effect of DDSP on breathing strategy. This portion of the study was performed without the face mask because wearing a face mask 75 pneumotachograph system imposed increased resistance to breathing, altered respiratory frequency, and, presumably, altered breathing strategy in horses.33 Respiratory frequency:stride frequency coupling may occur in order to maximize stride efficiency and efficient energetics of breathing.° In the control horses exercising at the trot and the pace, Rf:Sf was almost always 1:1 (Table 5). In blocked horses, respiratory frequency decreased by almost 50% compared to unblocked horses exercising at the same speed, while stride frequency remained unchanged. Therefore, Rf:Sf became 1:2, suggesting that breathing and stride synchronization was altered by the persistent DDSP. The results of this study have led us to the hypothesis that DDSP is caused by a primary dysfunction of the neuromuscular regulation of the soft palate, involving the pharyngeal branch of the vagus nerve and the palatinus and palatopharyngeus muscles. The pharyngeal branch of the vagus nerve is intimately associated with the retropharyngeal lymph node chain prior to ramifying in the pharyngeal plexus.” Retropharyngeal lymph node inflammation occurs frequently in young horses due to the high prevalence of viral upper respiratory tract diseases and secondary bacterial infections. In clinical cases of DDSP, the pharyngeal branch of the vagus nerve may be damaged by local lymphadenopathy, inflammation, and infection. Since observing that blockade of the pharyngeal branch of the vagus nerve produced DDSP, we have examined the guttural pouches of four horses presented with clinical DDSP. In all four horses, we observed evidence of guttural pouch inflammation and retropharyngeal lymphadenopathy. Although the numbers are small, this lends credence to our hypothesis that DDSP is caused by a primary neuropathy of the pharyngeal branch of the vagus nerve. 76 Bilateral pharyngeal branch of the vagus nerve block induced persistent DDSP and dysphagia at rest in our horses. In the clinical syndrome, the soft palate displaces intermittently during intense exercise, not persistently, as occurred in our horses, and is therefore a less severe obstruction. Also, dysphagia is not reported as a symptom of clinical intermittent DDSP. The pharyngeal branch of the vagus nerve was blocked as it coursed across the longus capitus muscle in the guttural pouch. This nerve is a purely motor nerve that innervates the palatinus, palatopharyngeus, and levator veli palatine muscles, as well as the dorsal pharyngeal constricting muscles, which include the hyopharyngeus, cricopharyngeus, thyropharyngeus, and pterygopharyngeus muscles.” Because the horses with clinical intermittent DDSP can swallow, the levator veli palatini muscle and the dorsal pharyngeal constrictor muscles are seemingly unaffected. Perhaps the nerve is damaged at its distal limits, prior to innervation of the palatinus and palatopharyngeus muscles in horses that exhibit the clinical disease. Neurogenic effects on the palatopharyngeus muscle and inflammatory changes in the musculus uvulae (palatinus muscle) have been identified in humans with sleep-related obstructive breathing disorders.”°”'”'7°'78 Sleep apnea is characterized by repetitive episodes of complete or partial upper airway obstruction, leading to apnea or hypopneas. ”3"” The repetitive upper airway occlusion occurs predominantly at the level of the velopharynx and oropharynx.‘2'” Inspiratory recruitment of soft palate muscles has been demonstrated in laboratory animals by increased electromyographic activity of these muscles during inspiration, negative pressure augmentation, and chemical stimulation.” Inspiratory activity of soft palate muscles has also been shown to be associated with upper airway patency during sleep in patients with obstructive sleep 77 apnea, and electromyographic activity of these muscles was abnormal just prior to the onset of obstructive episodes.” Therefore, although DDSP occurs in horses during exercise, whereas sleep apnea occurs during somnolence, the velopharyngeal obstruction and potential abnormal muscle function involved in both syndromes may be similar. Chapter 5 ELECTROMYOGRAPHIC ACTIVITY OF THE PALATINUS AND PALATOPHARYNGEUS MUSCLES IN EXERCISING HORSES Abstract We studied the respiratory related electromyographic activity of the palatinus and palatopharyngeus muscles in four horses while they exercised on a treadmill. Moving time average EMG activity was measured in the palatinus and the palatopharyngeus muscles using bipolar fine-wire electrodes. Pharyngeal pressure was correlated with muscle activity in one horse and Rf:Sf were correlated with EMG muscle activity in one horse. Measurements were made while the horses completed an incremental exercise protocol on the treadmill. The palatinus and palatopharyngeus muscles displayed synchronous expiratory activity that increased significantly (P = 0.002 and P = 0.013, respectively) with exercise intensity. Phasic expiratory activity of the palatinus muscle increased 150 1 12.3%, whereas phasic expiratory activity of the palatopharyngeus muscle increased by 62.7 i 8.9% as the treadmill speed increased from 6 M/s to 10 M/s. Muscle activity was coordinated with respiration and not with stride frequency. These results suggest that the palatinus and palatopharyngeus muscles may be important in stabilizing the position of the soft palate during exhalation in obligate nasal breathers, such as the horse. 78 79 Introduction The soft palate is recognized as having important respiratory functions in humans, cats, dogs, and horses.3"'””"~""87 Coordinated palatal movements are responsible for oral nasal airflow partitioning, panting, and pursed-lip breathing in humans"7 During intense exercise, oral nasal breathers adopt transoral breathing in order to decrease airflow resistance and nasal work of breathing.”66 Unique to other mammalian athletes, the horse is an obligate nasal breather so that oronasal airflow partitioning does not occur in horses.” The caudal free margin of the soft palate contacts the base of the larynx so that there is no communication between the oropharynx and nasopharynx during breathing.” Rather than switching to oral breathing, the horse employs multiple mechanisms to dilate the nares and nasopharynx in order to minimize airflow resistance, as peak inspiratory airflow increases from 5.09 :l: 0.34 US at rest to 75 :1; 9.35 US while exercising at high intensity.“'°° The position of the soft palate is critical in maintaining retropalatal airway patency.67 If the neuromuscular coordination that controls soft palate position is inappropriate, obstruction of the retropalatal area can occur, exemplified by obstructive sleep apnea in humans and DDSP in horses.“29 The etiologies of both syndromes are currently speculative; however, soft palate collapse causes airway obstruction in both diseases. ‘2'” Dorsal displacement of the soft palate is an obstructive upper respiratory tract disease that occurs during exhalation.”'°"°8 The obstruction causes increased expiratory impedance and peak expiratory pressures and decreased minute ventilation, as well as altering Rf:Sf during intense exercise.” Previously, I blocked the pharyngeal branch of the vagus nerve bilaterally, and induced persistent DDSP.35 The pharyngeal branch of 80 the vagus nerve provides efferent innervation to the levator veli palatini, palatinus, and palatopharyngeus muscles, which control the position of the caudal half of the soft palate.”56 Therefore, I hypothesized that DDSP is caused by a primary dysfunction of the neuromuscular regulation of the soft palate involving the pharyngeal branch of the vagus nerve, the palatinus muscle, and the palatopharyngeus muscle. In dogs, the palatinus and palatopharyngeus muscles exhibit electromyographic activity that is coincident with breathing.7'”'88 Because the palatinus and palatopharyngeus muscles have respiratory activity in other species and because neural blockade of the efferent supply to these muscles caused persistent DDSP, I measured the electromyographic activity of the palatinus and palatopharyngeus muscles in horses exercising on a treadmill. I hypothesized that the palatinus and palatopharyngeus muscles would have synchronous respiratory activity that increases with breathing intensity. Materials and methods Four horses were used in the experiment, which was approved by the All- University Committee for Animal Use and Care at Michigan State University. All horses were vaccinated against tetanus, equine influenza, rhinopneumonitis, Eastern and Western equine encephalitis, and Streptococcus equi infections. Physical examinations of the horses and endoscopic examinations of the larynx and nasopharynx at rest and during high-speed treadmill exercise failed to reveal any abnormalities. All horses were trained to run on the treadmill prior to the experiment. Bipolar fine wire electrodes and a ground wire were constructed using teflon- coated wire Cooner Wire, Chatsworth, PA). A small amount of resin cement (Justi 81 Products, Oxnard, CA) was applied to the end of the electrodes so the electrode could be seated in the soft palate. Electrodes were placed in the palatinus and palatopharyngeus muscles using a transoral approach under general anesthesia. Horses were premedicated with xylazine (0.04 mg/kg, IV) and anesthesia was induced with ketamine (2.2 mg/kg, IV) and diazepam (0.1 mg/kg, IV) and positioned in sternal recumbency. Anesthesia was maintained by use of an IV infusion of 5% guaifenesin containing 0.5 mg of xylazine/ml and 2.0 mg of ketamine/ml, delivered at 2 ml/kg/h. The head was elevated and the mouth opened, using a mouth speculum. A videoendoscope was passed through the left naris and positioned in the nasopharynx. One end of the wire electrode was fed through the pointed end of an l8-gauge needle. The palatinus muscle was palpated midline, caudal to the palatine aponeurosis. The hamulus of the pterygoid bone was used for additional orientation. The needle was passed through the palatinus muscle in the soft palate by driving the needle from the oropharynx to the nasopharynx. Using the videoendoscope, the wire that had been passed through the needle was grasped with a biopsy instrument and was pulled through the nose until the resin cement plug seated against the oral surface of the soft palate. The protocol was repeated and the second wire was placed 5—8 mm away from the first wire, through the palatinus muscle. The electrode was placed in the palatopharyngeus muscle, using the same method. The palatopharyngeus muscle was located by palpating caudal to the hamulus of the pterygoid bone along the lateral wall of the nasopharynx. The wires were passed through the nostril using a l4-gauge needle, and were secured to the horse’s muzzle using elastic tape. A ground wire was placed in the subcutaneous space, just over the left sternocephalicus muscle. Five to six hours after electrode 82 placement, muscle function measurements were made while the horses exercised on a treadmill . Experimental protocol A 150-cm polyethylene sidehole catheter (polyethylene tubing, 2.15 mm ID, 3.25 mm OD, Baxter Scientific Products, McGaw Park, IL) was placed through the right naris and secured to the muzzle with tape in two horses. The catheter was made with six side holes, beginning a distance of eight catheter diameters from the sealed tip.“ The catheter tip was placed at the level of the nasopharyngeal opening of the Eustachian tube in the nasopharynx. A catheter with a balloon attachment was secured to the left forelimb of the horse to synchronize stride and respiratory frequency. Horses completed an incremental exercise protocol that consisted of a three-minute warm-up at 2 M/s followed by one minute at 6 M/s and 30 s at 8, 9, 10 , 11, and 12 M/s or until exhaustion. EMG measurements The EMG signals were processed through a sixth-order Butterworth filter (band pass, 50 to 5000 Hz), amplified, rectified, and moving time-averaged with a constant of 100 milliseconds. Both raw EMG and moving time average signals were recorded. Quantification of the EMG was performed by digitization of the moving time-averaged EMG signal. Nasopharyngeal pressure was measured using a differential pressure transducer. Foot fall was detected using a blood pressure transducer. Nasopharyngeal pressure, footfall, and EMG signals were recorded on a physiologic recorder (Gould Instruments, Valleyview, OH). 83 After the exercise protocol, the electrodes were removed by sedating the horses, opening the mouth with a speculum, and retrieving the electrodes by pulling them through the mouth. Horses wore a muzzle for two hours after electrode removal. Data analysis Mean electrical activity of each EMG was determined by dividing the total area of each moving time average wave form by the duration of the electrical activity. The average of 10 consecutive breaths at each speed was calculated for each horse. In order to standardize the data for each horse, mean electrical activity was reported as a percentage of the activity measured at 6 M/s. Data were analyzed using a one-way analysis of variance. Post hoc comparisons were made using the Student-Newman-Keuls’ test. A significance level of P < 0.05 was selected. Results The surgical procedure used to place the electrodes was easily performed in 10 to 15 minutes. None of the horses exhibited apparent postoperative complications. All horses completed the incremental exercise test up to and including 30 seconds at 10 M/s. One horse completed the exercise protocol and EMG recordings were made at 11 and 12 M/s. To standardize the EMG muscle activity, mean electrical activity was reported as a percentage of the activity recorded at 6 M/s. Specifically, the mean electrical activity of the palatinus muscle at 6 M/s was arbitrarily designated 1 for each horse and as muscle activity increased with treadmill speed, mean electrical activity was reported as 84 145.7% at 8 M/s, 179.7% at 9 M/s, and 250% at 10 M/s of the activity measured at 6 M/s. The main effect on EMG muscle activity was treadmill speed. As treadmill speed increased, phasic expiratory activity increased significantly (P = 0.002 and P = 0.012) for both the palatinus and palatopharyngeus muscles (Table 6) (Figure 14). Tonic inspiratory and expiratory activity also increased over baseline measurements. The phasic activity was in phase with respiration and not with stride (Figure 15). Table 6. Mean electrical activity (1 STD) reported as the percent of activity at 6 M/s for the palatinus and palatopharyngeus muscles in three horses exercising on a treadmill. I Muscle 8 M/s 9 M/s 10 M/s 11 M/s 12 M/s ! Palatinus 145.7% 179.9% 250.7% 391% 526% i 45.0 i 86.6 :1: 123.1 Palatopharyngeus 108.7% 115 % 162.7% 265 % 348% 1 38.7 1 26 :1; 89.4 M/s = meters per second Discussion Palatal movement and position is thought to be determined by coordinated function of four muscles: the levator veli palatini, tensor veli palatini, palatinus and ”'5‘ The function of these muscles has been studied palatopharyngeus muscles. extensively during speech and deglutition; however, the precise action of each muscle, and timing of these muscles during respiration is unclear. Respiratory-related EMG activity has been reported for the soft palate muscles in humans and laboratory animals.2'7-‘2'”'4""“517179-87” However, the reported activities of these muscles are often 85 contradictory, which may reflect differences in species used, anesthetic techniques, and the stimulus used to evoke muscular activity, be it pressure, sleep, or chemical stimulation. The tensor veli palatini muscle is a fusiform muscle that originates from the muscular process of the petrous temporal bone, pterygoid bone, and lateral lamina of the auditory tube. This muscle extends cranioventrally along the lateral wall of the pharynx.” The tendon of the tensor veli palatini courses at a 90' angle around the hamulus of the pterygoid bone and ramifies in the palatine aponeurosis.” Contraction of this muscle tenses this fascia and depresses the soft palate toward the tongue.” In humans and dogs, contraction of the tensor veli palatini muscle is synchronous and coincident with respiration."°8'79'” Specifically, phasic and tonic inspiratory EMG activity, measured in anesthetized dogs, increased when negative pressure was applied to the larynx and pharynx and when the dogs were breathing a hypoxic hypercapnic mixture, suggesting that contraction of tensor veli palatini muscle is stimulated by chemical drive as well as by local reflex mechanism.7“’7'88 In humans, the tensor veli palatini EMG activity was synchronous with inspiration during wakefulness but was dramatically reduced during rapid eye movement sleep.72 In one patient, previously diagnosed with obstructive sleep apnea, marked cessation of tensor veli palatini activity was consistently associated with apnea initiation, and lack of tensor veli palatini EMG activity lasted for the duration of the apneic episode.2 Although EMG measurement of tensor veli palatini have never been made in horses, bilateral tenectomy of the tensor veli palatini muscle caused collapse of the nasopharynx during inspiration in exercising horses and increased peak inspiratory tracheal pressures, suggesting that tensor veli palatini 86 activity may be coincident with inspiration in an attempt to support the nasopharynx during intense inspiratory efforts.” The levator veli palatini muscle arises from the muscular process of the petrous part of the temporal bone and form the lateral lamina of the auditory tube and courses rostroventrally along the lateral pharyngeal wall.” The right and left muscle meet above the glandular layer in the soft palate, forming the levator sling so that contraction of the levator veli palatini muscles elevates the soft palate during swallowing and facilitates oral ventilation.” Contradictory information exists concerning electromyographic activity of levator veli palatini muscle during breathing. In anesthetized dogs, phasic inspiratory and expiratory activity was measured in levator veli palatini.7'7°'” Negative pressure application to the larynx and oropharynx and hypoxic hypercapnic breathing resulted in increased phasic inspiratory and expiratory activity in the levator veli palatini, suggesting that, as was noted with the tensor veli palatini muscle, levator veli palatini muscle activity is stimulated centrally by chemical drive and by local reflexes?”87 In humans, the levator veli palatini muscle is active during swallowing and oral breathing, with reduced activity during nasal breathing.”31 Interestingly, the levator veli palatini muscle has been shown to have phasic inspiratory, phasic expiratory, and also lack of respiratory activity by different investigators.2‘*’-”'72 Sleep state and obstructive sleep apnea appear to affect levator veli palatini activity.”79 Peak inspiratory and end-expiratory EMGs of levator veli palatini decreased significantly during non-rapid-eye-movement sleep in normal men; EMG activity in the levator veli palatini was absent during apneic episodes; and activity increased in levator veli palatini concomitant with apnea resolution.'2'7” Inspiratory activity of levator veli palatini during nasal breathing is difficult to explain. 87 Intuitively, inactivity of levator veli palatini would seem essential for nasal breathing because levator veli palatini contraction elevates the soft palate, essentially closing the nasopharynx and opening the oropharynx, such that nasal breathing would not seem possible. The palatinus and palatopharyngeus muscles control the position of the caudal half of the soft palate."'” The palatinus is a fusiform bilobed muscle that originates from the caudal aspect of the palatine aponeurosis and extends caudally along the midline of the soft palate, just beneath the nasopharyngeal mucosa. The muscle infiltrates the caudal free margin of the soft palate, and muscle fibers course dorsally in the lateral pillars of the soft palate.” The actual function of the palatinus muscle is unknown. Contraction of the palatinus muscle shortens the soft palate and may either depress the caudal soft palate toward the tongue or elevate the caudal soft palate toward the dorsal pharyngeal wall.“"‘2"’3'51 This controversial action may depend on the soft palate conformation, the simultaneous contraction of other muscles, as well as the teleological basis for enhanced respiration in a species. Specifically, if the soft palate terminates with the uvula, as in humans, and does not contact the epiglottis, contraction of the palatinus and palatopharyngeus muscles in concert with levator veli palatini contraction may lift the caudal margin of the soft palate toward the dorsal pharyngeal wall.‘”2 In the horse, because the soft palate extends to the base of the larynx, with the lateral pillars coalescing to form the palatopharyngeal arch, contraction of the palatinus muscle may shorten and depress the caudal soft palate toward the tongue during breathing and aide in soft palate elevation during swallowing. In anesthetized dogs the palatinus muscle exhibited phasic inspiratory and expiratory activity that was enhanced with upper airway 88 pressure augmentation.7'”'” Hypercapnic hypoxic breathing, however, resulted only in increased phasic expiratory activity in the palatinus.87 In the study reported here, the palatinus exhibited phasic expiratory action that increased with exercise intensity. Exercising horses experience both increased negative nasopharyngeal inspiratory pressures as well as chemical stimulation during high-intensity exercise. Therefore, different stimuli may result in variable muscle activity and respiratory timing. These differences may be explained by specific species differences, the effects of anesthesia on muscle activity, or different stimuli used to trigger muscle activity. Horses are obligate nasal breathers, whereas dogs open-mouth breathe during intense exercise. Contraction of the palatinus during inspiration may be important in the dog in order to position that soft palate, thus allowing oral breathing. In the horse, nasal breathing may be more efficient if the soft palate is supported during exhalation, thus minimizing dead space and maintaining contact around the larynx. In this case, the palatinus muscles may act like a support within the soft palate to maintain a rigid palate. The palatopharyngeus muscle originates along the caudal margin of the palatine aponeurosis and along the lateral margins of the palatinus muscles and courses dorsolaterally into the pharyngeal wall. The function of this muscle is not known, but is hypothesized to be important in the ventral motion of the soft palate toward the base of the tongue. The interaction of palatinus and palatopharyngeus muscles, especially as it relates to soft palate position, is unknown. Dorsal displacement of the soft palate is an expiratory obstructive syndrome. It is plausible that loss of function of an expiratory muscle would create an expiratory obstruction. Little information exists as to the onset of DDSP during the respiratory 89 cycle. Rehder et al. reported that seven horses displaced the soft palate during inspiration, five during exhalation, and eight during swallowing.64 Inappropriate palatinus and palatopharyngeus function could result in flaccidity of the caudal soft palate, causing DDSP during exhalation and swallowing. Loss of tonic activity of these muscles might contribute to palate displacement during inspiration, or possibly these muscles have a phasic inspiratory activity that we were unable to measure. Electromyographic recordings of the palatinus and palatopharyngeus muscles should be made in horses during episodes of DDSP in order to conclusively implicate dysfunction of this neuromuscular group in the disease. SUMMARY AND CONCLUSIONS The results of these experiments have provided information about the neuromuscular regulation of the nasopharynx of the horse and have refuted some theories and strengthened others concerning the etiology of DDSP in horses. Evidence suggests that dysfunction of the pharyngeal branch of the vagus nerve, the palatinus and the palatopharyngeus muscles, may be implicated as a potential cause of DDSP because bilateral nerve blockade induced the syndrome and these muscles have synchronous respiratory function that increases as exercise intensity increases. However, we have literally just begun to address the potential cause of this upper respiratory obstruction. The primary expiratory activity of the palatinus and palatopharyngeus muscles is confusing, and thus future work will be directed at confirming the expiratory activity of these muscles and investigating the phase of the respiratory cycle when DDSP occurs in horses. Then, the EMG activity of the palatinus and palatopharyngeus muscles will be measured in horses during episodes of DDSP.This experiment will confirm or refute the role of dysfunction of these muscles in DDSP. Optimistically, if the palatinus and palatopharyngeus muscles have abnormal (decreased) activity during DDSP, this strengthens the hypothesis that this neuromuscular unit may be involved in the syndrome, but certainly does not confirm it. Dorsal displacement of the soft palate may be similar to obstructive sleep apnea in that multiple muscles may 90 91 have decreased activity during the obstructive episode, but not all of these muscles are responsible for initiating obstruction. However, if the palatinus and palatopharyngeus muscles have evidence of denervation, based on histopathologic examination, and if the pharyngeal branch of the vagus nerve has distal axonal degeneration and demyelinization, from horses with DDSP compared to non-affected horses, then neuromuscular dysfunction as a basis for DDSP is a stronger hypothesis. Identification of an abnormality in neuromuscular function does not explain the etiology of DDSP. We have theorized that the pharyngeal branch of the vagus nerve may be damaged by upper airway inflammation. Specifically, because this nerve courses through the retropharyngeal lymph node chain, perhaps lymphadenopathy in this area, common in young horses with upper respiratory tract infection, precipitate nerve damage resulting in abnormal muscle function and DDSP. The questions to be answered include the following: How does a respiratory virus induce inflammation that may result in neural damage? Is there any effect on the surrounding nerves? Is this effect mediated by toxins, inflammatory mediators, or something else? Is there an epidemiological connection between the onset of upper respiratory tract infection in young horses and the development of DDSP? Can DDSP be induced by causing upper respiratory tract infection? In conclusion, we have gained some information concerning the neuromuscular regulation of the nasopharynx in the horse and provided introductory information concerning the pathogenesis of DDSP, a debilitating syndrome that affects the horse. LIST OF REFERENCES 10. 11. LIST OF REFERENCES Ainsworth DM, Ducharme NG, Hackett RP, et al. Regulation of respiratory muscle activities during chemoreceptor stimulation in adult horses. Am J Vet Res l995;56(3):366- 373. Anch AM, Remmers J E, Sauerland EK, et al. Oropharyngeal patency during waking and sleep in the pickwickian syndrome: electromyographic activity of the tensor veli palatini. Electromyogr Clin Neurophysiol 1981;21:317-330. Anderson JD, Tulleners EP, Johnson JK, et al. Sternothyrohyoid myectomy or staphylectomy for treatment of intermittent dorsal displacement of the soft palate in racehorses: 209 cases (1986 - 1991). JAm Vet Med Assoc 1995;206(12):1901-1912. Azzam NA and DP Kuehn. The morphology of musculus uvulae. Cleft Palate J 1977;14(1):78-87. Blythe LL, Cardinet GH, Meagher DM, et al. Palatal myositis in horses with dorsal displacement of the soft palate. J Am Med Assoc 1983;183(7):781-785. Bramble DM, and Carrier DR. Running and breathing in mammals. Science 1983;219:251- 256. Brancatisan A, Van der Touw T, O’neil N, et al. Influence of upper airway pressure oscillations of soft palate muscle electromyographic activity. J Appl Physiol 1996;81(3):1190—1196. Brancatisano TP, Dodd DS, Collett PW, et al. Effect of expiratory loading on glottic dimensions in humans. J Appl Physiol 1985;58:606611. Brouillette RT, and TT Bradley. A neuromuscular mechanism maintaining extrathoracic airway patency. J Appl Physiol: Respirat Environ Exercise Physiol l979;46(4):772-779. Brouillette RT, and TT Bradley. Control of genioglossus muscle inspiratory activity. J Appl Physiol: Respirat Environ Exercise Physiol l980:49(5);801-808. Bruce EN, Mitra J, Cherniack NS. Central and peripheral chemoreceptor inputs to phrenic and hypoglossal motoneurons. J Appl Physiol: Respirat Environ Exercise Physiol l982;53(6)21504-1511. 92 12. l3. 14. 15. l6. l7. 18. 19. 20. 21. 22. 23. 24. 25. 26. 93 Carlson DM, Onal E, Carley DW, et al. Palatal muscle electromyographic activity in obstructive sleep apnea. Am J Respir Crit Care Med 1995; 152: 1022-1027. Cook WR. The diagnosis of respiratory unsoundness in the horse. Vet Rec l965;77(l9):516-528. Cook WR. Observations on the aetiology of epistaxis and cranial nerve paralysis in the horse. Vet Rec 1966;78(12):396-405. Cook WR. Some observations on form and function of the equine upper airway in health and disease: 1.The pharynx. In: Proc 27th Annu Conv Am Assoc Equine Pract, 1981:355. Daubenspeck JA. Influence of small mechanical loads on variability if breathing pattern. J Appl Physiol 1981;50:200-206. Derksen F1, Stick JA, Scott ED, et al. Effect of laryngeal hemiplegia and laryngoplasty on airway flow mechanics in exercising horses. Am J Vet Res 1986;47:16-20. Derksen JF, and NE Robinson. Esophageal and intrapleural pressures in the healthy conscious pony. Am J Vet Res 1980;41:1756-1761. Ducharme NG, Hackett RP, Ainsworth D, et al. Repeatability and normal values for measurement of pharyngeal and tracheal pressured in exercising horses. Am J Vet Res l994;55(3):368—374. Edstrom L, Larsson H, Larsson L. Neurogenic effects on the palatopharyngeal muscle in patients with obstructive sleep apnea: a muscle biopsy study. J Neural Neurosci Psych 1992;55:916-920. Furusawa K, Yamaoka M, Kogo M, et al. The innervation of the levator veli palatini muscle by the glossopharyngeal nerve. Brain Res Bulletin 1990;26:599-604. Hackett RP, and NG Ducharme. Soft palate displacement: pathophysiology and treatment. ZIst Annu Forum Amer Coll Vet Surg 1993; 179-181. Hairston LE, and EK Sauerland. Electromyography of the human palate: discharge patterns of the levator and tensor veli palatini. Electromyogr Clin Neurophysiol 1981;21:287-297. Hairston LE, and EK Sauerland. Electromyography of the human pharynx: discharge patterns of the superior pharyngeal constrictor during respiration. Electromyogr Clin Neurophysiol 1981 ;21 :299-306. Hare WCD. Equine respiratory system. In: Getty R (Ed): Sisson and Grossman’s The Anatomy of Domestic Animals. 5th ed. Philadelphia, W. B. Saunders Company 1975;504- 511. Harrison 1W, Raker CW, Wheat JD. Sternothyrohyoid myectomy in horses: 17 cases (1984-1985). J Am Vet Med Assoc 1988;193:1299-1302. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 94 Haxihu MA Cherniack NS, Mitra J, et al. Nonvagal modulation of hypoglossal neural activity. Respir Physiol 1992;59:65-71. Haynes PF. Persistent dorsal displacement of the soft palate associated with epiglottic shortening in two horses. J Am Vet Med Assoc 1981;179:677-681. Haynes PF. Dorsal displacement of the soft palate and epiglottic entrapment: diagnosis, management, and interrelationship. Compend Cont Educ l983;5: 8379-8388. Hida W, Kurosawa H, Okabe S, et al. Hypoglossal nerve stimulation affects the pressure- volume behavior of the upper airway. Am J Respir Crit Care Med 1995;151:455-460. Hinton VA and DW Warren. Relationships between integrated oral-nasal differential pressure and velopharyngeal closure. Cleft Palate Craniofac J 1995:32(4):306-310. Holcombe SJ, Beard WL, Hinchcliff KW, et al. Effect of Sternothyrohyoid myectomy on upper airway mechanics in normal horses. J Appl Physiol l994;77(6):28l2-2816. Holcombe SJ, Beard WL, Hinchcliff KW. et al. Effect of a mask and pneumotachograph on tracheal and nasopharyngeal pressures, respiratory frequency, and ventilation in horses. Am J Vet Res 1996;57(3):250-254. Holcombe S J, Derksen Fl, Stick JA, et al. Effect of bilateral tenectomy of the tensor veli palatini muscle on soft palate function in horses. Am J Vet Res l997:58(3): 317-321. Holcombe SJ, Derksen FJ, Stick JA, Robinson NE. Bilateral nerve blockade of the pharyngeal branch of the vagus nerve produces persistent soft palate dysfunction in horses. Amer J Respir Crit Care Med; Ab. 155(4):A412,1997. Honnas CM, Schumacher J, Dean PW. Identifying and correcting displacements of the soft palate and pharyngeal tissues. Eq Prac 1990;1unez622-63l. Hornet RL, Innes JA, Holden HB, et al. Afferent pathways for pharyngeal dilator reflex to negative pressure in man: a study using upper airway anaesthesia. J Physiol 1991;436:31-44. Hwang .1, Bartlett D, St. John WM. Characterization of respiratory-modulated activities of hypoglossal motorneurons. J Appl Physiol; Respirat En vir Exercise Physiol 1983 ;55(3):793- 798. Hwand J, St. John WM, Barlett D. Afferent Pathways for hyoglossal and phrenic responses to changes in upper airway pressure. Respirat Physiol 1984;55:341-354. Kogo M, N ishia J, Matsuya T, et al. Coordination of the levator veli palatini and intrinsic laryngeal muscles: an evoked electromyographic study in the dog. Cleft Palate J l987;24(2):1 19-125. Kuehn DP, Moon 18, Folkins 1W. Levator veli palatini muscle activity in relation to intranasal air pressure variation. Cleft Palate Craniofac J 1993:30l41236l-368. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 95 Kuehn DP, Folkins JW, Linville RN. An electromyographic study of the musculus uvulae. Cleft Palate J l988;25(4):348-355. Kuehn DP, Folkins JW, CB Cutting. Relationships between muscle activity and velar position. Cleft Palate] 1982; 19:25-35. Leiter JC, Knuth SL, Bartlett D. Dependence of pharyngeal resistance on genioglossal EMG activity, nasal resistance, and airflow. J Appl Physiol 1992;73(2):584-590. Linford RL, O’Brien TR, Wheat JD, et al. Radiographic assessment of epiglottic length and pharyngeal and laryngeal diameters in Thoroughbreds. Am J Vet Res 1983;44:1660- 1666. Lumsden J M, Derksen FJ, Stick J A, et al.. Use of flow-volume loops in evaluating upper airway obstruction in exercising horses. Am J Vet Res l993;54(5):766-774. Mathew OP, Abu-Osba YK, Thach BT. Genioglossus muscle responses to upper airway pressure changes: afferent pathways. J Appl Physiol: Respirat Environ Exercise Physiol l984;52(2):445-450. Mathew OP, Sant’Ambrogio G, Fisher JT, et al. Laryngeal Pressure Receptors. Respirat Physiol 1984;57:113-122. Mathew OP. Upper airway negative-pressure effects on respiratory activity of upper airway muscles. J Appl Physiol: Respirat Environ Exercise Physiol l984;56(2):500-505. McEnvoy RD, Popovic RM, Saunders NA, et al. Effects of sustained and repetitive isocapnic hypoxia on ventilation and genioglossal and diaphragmatic EMGs. J Appl Physiol l996;81(2):866-875. Moon J B, Smith AE, Folkins JW, et al. Coordination of velopharyngeal muscle activity during positioning of the soft palate. Cleft Pal Craniofac J 1994;31(1):45-55. Morris EA, and HI Seehennan. Evaluation of upper respiratory tract function during strenuous exercise in racehorses. J Am Vet Med Assoc 1990;196(3):431-438. Mortimore IL, Mathur R, Douglas NJ. Effect of posture, route of respiration, and negative pressure on palatal muscle activity in humans. J Appl Physiol l995;79(2):448-454. Nielan GJ, Rehder RS, Ducharme NG, et al. Measurement of tracheal static pressure in exercising horses. Vet Surg 1980;41:1756-1761. Niinimaa V, Cole P, Mintz S. The switching point from nasal to oronasal breathing. Respir Physiol 1980;42:61-71. Nishia J, Matsuya T, Machida J. The motor nerve supply of the velopharyngeal muscles. 28111 annual meeting of Japan Society of Plastic and Reconstructive Surgery, Kyoto, Japan. April, 1975. 57. 58. 59. 60. 61. 62. 63. 65. 66. 67. 68. 69. 70. 71. 96 Odeh M, Schall R, Gavriely N, et al. Effect of upper airway muscle contraction on supraglottic resistance and stability. Respir Physiol 1993;92:139-150. O’leary MJ, and RP Millman. Technical modifications of uvulopharyngoplasty: the role of the palatopharyngeus. Laryngoscope 1992;101:1332-1335. Parente EJ. Diagnosing upper airway obstructive diseases. Proc 6th Annu Symposium of the American College of Veterinary Surgeons 1996: 172-173. Pelletier N, and DE Leith. Ventilation and carbon dioxide exchange in exercising horses: effect of inspired oxygen fraction. J Appl Physiol 1995;78:654-662. Peloso JG, Stick JA, Nickels FA, et al. Epiglottic augmentation by use of polytetrafluoroethylene to correct dorsal displacement of the soft palate in a Standardbred horse. J Am Vet Med Assoc l992;201(9): 1393-1395. Philip-Joet F, Marc 1, Series F. Effects of genioglossal response to negative airway pressure on upper airway collapsibility during sleep. J Appl Physiol 1996;80(5): 1466- 1474. Raphel C. Endoscopic findings in the upper respiratory tract of 479 horses. J Am Vet Med Assoc 1982;181(5):470-473. Rehder RS, Ducharme NG, Hackett RP et al.. Measurement of upper airway pressures in exercising horses with dorsal displacement of the soft palate. Am J Vet Res 1995;56(3):269- 274. Roberts J L, Reed WR, Bradley TT. Pharyngeal airway stabilizing function of sternohyoid and sternothyroid muscles in the rabbit. J Appl Physiol: Respirat Environ Exercise Physiol l984;75(6): 1790-1795. Robinson NE and PR Sorenson. Pathophysiology of airway obstruction in horses: a review. J Am Vet Med Assoc 1978;172(3):299-303. Rodenstein DO and DC Stanescu. Soft palate and oronasal breathing in humans. J Appl Physiol Respirat Environ Exercise Physiol l984;57(3):651-657. Roethlisberger-holm K. Tracheal pressure in horses with signs of dorsal displacement of the soft palate. Equine Vet J Suppl 1995;18:70-75. Rothstein RJ, Narce SL, deBerry-Borowiecke B, et al. Respiratory-related activity of upper airway muscles in anesthetized rabbits. J Appl Physiol: Respirat Environ Exercise Physiol l983;55(6): 1930-1836. Salomone RJ, and E van Lunteren. Effects of hypoxia and hypercapnia on geniohyoid contractility and endurance. J Appl Physiol 1991 ;7l(2):709-715. Sant’ambrogio G, Mathew OP, Fisher 1T, et al. Laryngeal receptors responding to transmural pressure, airflow, and local muscle activity. Respirat Physiol 1983;54:317-330. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83. 84. 85. 86. 97 Sauerland EK, Orr WC, Hairston LE. EMG patterns of oropharyngeal muscles during respiration in wakefulness and sleep. Electromyogr Clin Neurophysiol 1981;21:307-316. Schnall RP, Pillar G, Kelsen SC, et al. Dilatory effects of upper airway muscle contraction induced by electrical stimulation in awake humans. J Appl Physiol l995;78(5): 1950-1956. Schwartz AR, Thut DC, Russ B, et al. Effect of electrical stimulation of the hypoglossal nerve on airflow mechanics in the isolated upper airway. Am Rev Respir Dis 1993;147:1144-1150. Sekosan M, Zakkar M, Wenig BL, et al. Inflammation in the uvula mucosa of patients with obstructive sleep apnea. Laryngoscope 1996;106:1018-1020. Series F, Simoneau J, St. Pierre S, et al. Characteristics of the genioglossus and musculus uvulae in sleep apnea hypopnea syndrome and in snorers. Am J Respir Crit Care Med 1996; 153: 1870-1874. Strohl KP, Wolin AD, van Lunteren E, et al. Assessment of muscle action on upper airway stability in anesthetized dogs. J Lab Clin Med 1987;110:221-230. Swift AC, Goulding H, Elder J, et al. A histopathological comparison of the uvula between snorers and non-snorers. Clin Otolaryngol 1995;20:517-521. Tangel DJ, Mezzanotte WS, White DP. Influences of NREM sleep on activity of palatoglossus and levator veli palatini muscles in normal men. J Appl Physiol 1995;78(2):689-695. Travers JB and Norgren R. Afferent projections to the oral motornuclei in the rat. J Comp Neurol 1983;220:280-298 Trigos I, Ysunza A, Vargas D, et al. The San Venero Roselli pharyngoplasty: an electromyographic study of the palatopharygeus muscle. Cleft Palate J l988;25(4):385-388. Truy E, Cote-Deplus I, Morgon A. Effectiveness of musculus palatopharyngeus resection in uvulopalatopharyngoplasty for snoring. J Otolaryngol 1995;24(2):79-83. Tsubone H, Mathew OP, Sant’Ambrogio G. Respiratory activity in the superior laryngeal nerve of the rabbit. Respirat Physiol 1987;69:195-207. Tulleners EP, Mann P Raker CW. Epiglottic augmentation in the horse. Vet Surg l990;l9(3)2181-l90. Tulleners E, and A Hamir. Evaluation of epiglottic augmentation by use of polytetrafluoroethylene paste in horses. Am J Vet Res l99l;52(l 1): 1908-1915. Van der Graaff WB. Gottgried SB, Mitra J, et al. Respiratory function of hyoid muscles and hyoid arch. J Appl Physiol: Respir Environ Exercise Physiol l984;57( l)l97-204. 87. 88. 89. 90. 91. 92. 93. 94. 95. 96. 97. 98. 99. 100. 98 Van der Touw T, O’neill N, Amis T, et al. Soft palate muscle activity in response to hypoxic hypercapnia. J Appl Physiol l994;77(6):2600-2605. Van der Touw T, O’Neill N, Brancatisan A, et al. Respiratory-related activity of soft palate muscles: augmentation by negative upper airway pressure. J Appl Physiol 1994;76(1):424-432. van Lunteren E, Van de Graaff WB, Parker DM, et al. Activity of upper airway muscles during augmented breaths. Respirat Physiol 1983;53:87-98. van Lunteren E, Van de Graaff WB, Parker DM, et al. Nasal and laryngeal reflex responses to negative upper airway pressure. J Appl Physiol: Respirat Environ Exercise Physiol 1984;56(3)746-752. van Lunteren E, Strohl KP, Parker DM, et al. Phasic volume-related feedback on upper airway muscle activity. J Appl Physiol: Respirat Environ Exercise Physiol 1984;56(3):730- 736. van Lunteren E, Haxiu MA, Cherniack NS. Relation between upper airway volume and hyoid muscle length. J Appl Physiol l987;63(4): 1443-1449. van Lunteren E, Haxhiu MA, Cherniack NS. Mechanical function of hyoid muscles during spontaneous breathing in cats. J Appl Physiol 1987;62(2):582-590. van Lunteren E. Respiratory muscle coordination. J Lab Clin Med 1988;112(3):285-300. van Lunteren E, and TE Dick. Breath to breath variability in hypoglossal motor unit firing. Respir Physiol 1992;89:37-46. Wagner PD, Gillespie JR, Landgren GL, et al. Mechanism of exercise-induced hypoxemia in horses. J Appl Physiol 1989;66(3):1227-1233. Weiner D, Mitra J, Salamone J. Effect of chemical stimuli on nerves supplying upper airway muscles. J Appl Physiol l982;52(2)530—536. Wiegand DA, Latz B, Zwillich CW. Upper airway resistance and geniohyoid muscle activity in normal men during wakefulness and sleep. J Appl Physiol l990;69(4):1252- 1261. Williams JW Pascoe JR, Meagher DM, et al. Effects of left recurrent laryngeal neurectomy, prosthetic laryngoplasty, and subtotal arytenoidectomy on upper airway pressure during maximal exertion. Vet Surg 1990;l9(2): 136-141. Woodall DL, Hokanson J A, Mathew OP. Time of application of negative pressure pulses and upper airway muscle activity. J Appl Physiol l989;67(l):366-370. APPENDIX 99 Figure 1. Videoendoscopic image of a normal larynx and nasopharynx of a horse with the sofi palate (S) positioned ventral to the epiglottis (E). Figure 2. Videoendoscopic image of the nasopharynx of a horse with the soft palate (S) displaced dorsal to the epiglottis. Figure 3. Videoendoscopic image of the nasopharynx of a horse with DDSP during high- intensity treadmill exercise. Notice how the soft palate (S) billows across the airway, obstructing the rima glottis. Hyoglmus A” Genioglossus ' Figure 4. Computer graphic illustration of the anatomical relationship between the hyoid apparatus, the tongue, and the muscles that attach to the basihyoid bone. (SH) stylohyoid bone: (CH) ceratohyoid bone; (BH) basihyoid bone. Figure 5. Computer graphic illustration of the hyoid apparatus illustrating the angle between the stylohyoid and ceratohyoid bones. The diameter of the nasopharynx is determined by the angle (A) between the stylohyoid and ceratohyoid bones. The vertical distance between the base of the cranium and the basihyoid bone determines the vertical diameter of the pharynx. Figure 6. Computer graphic illustration of the change in position of the hyoid apparatus during contraction of the genioglossus muscles. Contraction of the genioglossus muscle or cranial traction on the tongue is causing ventral displacement of the basihyoid which increases the angle formed by the stylohyoid and ceratohyoid bone, expanding the longitudinal dimensions of the nasopharynx. Figure 7. Computer graphic illustration of a transverse section through the larynx and nasal and oral pharynx of a horse. The hamulus of the pterygoid bone, muscles, and tissue layers of the sofi palate are labeled. The m. tensor veli palatini and m. levator veli palatini are emphasized with hatch marks to indicate that theses muscles course beneath the nasopharyngeal mucosa. Figure 8. Computer graphic illustration ofthc ventral aspect ofthc horse’s skull. Notice the muscles ofthc soft palate, the palatine aponcurosis, and the hamulus ofthc pterygoid bone (arrow). Figure 9. Videoendoscopic view of the nasopharynx of a horse following bilateral tensor veli palatini muscle tenectomy during an inspiratory effort with both nostrils occluded. The sofi palate billows dorsally into the nasopharynx. (1) nasal septum, (2) soft palate, and (3) junction of the hard and soft palate. Figure 10. Videoendoscopic view of the nasopharynx of a horse following bilateral tensor veli palatini muscle tenectomy during an expiratory effort with both nostrils occluded. The of palate collapses ventrally, developing a concave conformation. (1) nasal septum, (2) soft palate, and (3) junction of the hard and sofi palate. Figure 11. Computer graphic illustration of a transverse section of a horse head. The basihyoid bone and the muscles attaching to the basihyoid bone are Figure 12. Videoendoscopic image of the ventro-medial aspect of the guttural pouch. A bleb of mepivicaine (arrow) is injected between the hypoglossal nerve (X11) and the glossopharyngeal nerve (XI). Figure 13. Videoendoscopic image of the nasopharynx of a horse exercising on a treadmill at the speed resulting in HRH“. The ventral surface of the epiglottis (E) is seen because the epiglottis is retroflexed into the rima glottis. The caudal free margin of the soft palate (arrow) can also be seen. The insertion of the hyoepiglotticus muscle (H) is seen beneath the mucosal covering the ventral surface of the epiglottis.