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The role of the abdominal muscles in breathing Leevers, Ann Margaret 1991

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THE ROLE OF THE ABDOMINAL MUSCLES IN BREATHING by ANN MARGARET LEEVERS B. Sc. (Biology) Simon Fraser U n i v e r s i t y M. Sc. (Kines io logy) Simon Fraser U n i v e r s i t y A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES EXPERIMENTAL MEDICINE We accept t h i s thes i s as conforming to the requ ired standard THE-UNIVERSITY OF BRITISH COLUMBIA 1991 CcS Ann Margaret Leevers , 1991 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives, it is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of fj^rirtri^6*{~^( The University of British Columbia Vancouver, Canada Date DE-6 (2/88) ii ABSTRACT Nine tracheotomized dogs were c h r o n i c a l l y instrumented wi th sonomicrometer length measurement transducers and f ine wire EMG electrodes i n each of the four abdominal muscles: transversus abdominis (TA), i n t e r n a l obl ique (10), ex terna l obl ique (E0) and rectus abdominis (RA) . To assess the e f f ec t s of anesthesia and chronic implantat ion of transducers on abdominal muscle r e s t i n g length and length changes, muscle length measurements were made i n the anesthet ized dogs and then repeated i n the same dogs when awake, on successive days over per iods ranging from two to e ight weeks. Over the durat ion of the study per iods the awake dogs were a l so exposed to d i f f e r e n t experimental p r o t o c o l s . The experimental pro toco l s were p o s t u r a l changes, e x p i r a t o r y thresho ld loading (ETL) and C0 2 r ebreath ing , and ETL and C0 2 rebreath ing before and a f t er r e v e r s i b l e vaga l blockade. The object ives were to determine i n d i v i d u a l abdominal muscle ton ic and phas ic , exp ira tory shortening i n response to these d i f f e r e n t s t i m u l i . We hypothesized that the i n d i v i d u a l abdominal muscles would e x h i b i t d i f f e r i n g amounts of ton ic and phasic a c t i v i t y and that the i n t e r n a l muscle layer (TA and 10) would be r e c r u i t e d p r e f e r e n t i a l l y compared to the ex terna l muscle l a y e r (E0 and RA) i n response to each of the s t i m u l i . To assess the r e l a t i v e importance of vagal ly -mediated ref lexes i n the c o n t r o l o f abdominal muscle a c t i v a t i o n , the awake dogs were exposed to ETL and progress ive hypercapnia before and a f t e r r e v e r s i b l e vagal blockade. The r e s u l t s of these studies demonstrate the f e a s i b i l i t y of us ing c h r o n i c a l l y implanted sonomicrometer transducers for measurement of abdominal muscle length changes i n awake dogs. Phasic exp ira tory shortening of the TA, 10 and EO was increased by changes i n posture from the l a t e r a l d e c u b i t i s to the s i t t i n g and s tanding p o s i t i o n s , by ETL and by hypercapnia . Tonic shortening o f the TA and/or the 10 was a l so present under a l l three c o n d i t i o n s . There was p r e f e r e n t i a l recruitment of the i n t e r n a l muscles under a l l three condi t ions and t h i s p r e f e r e n t i a l recrui tment p e r s i s t e d dur ing ETL and C0 2 rebreathing a f t er vagal blockade. We conclude that abdominal muscle a c t i v i t y i s mediated by vagal and chemical re f l exes and that segmental re f l exes from muscle propr ioceptors p l a y a r o l e i n modulating the l e v e l o f i n d i v i d u a l abdominal muscle a c t i v i t y . iv TABLE OF CONTENTS A b s t r a c t i i Table of Contents . . . . . i v L i s t of F igures v i L i s t of Tables x L i s t of Abbrev ia t ions x i Acknowledgement x i i I . In troduct ion 1 L i t e r a t u r e R e v i e w . . 1 In troduct ion 1 F u n c t i o n a l Anatomy of the Abdominal Muscles 2 Methods of Study 6 Pos tura l E f f e c t s on and Regional Dif ferences i n Abdominal Muscle A c t i v i t y 8 Abdominal Muscle A c t i v a t i o n by Exp ira tory Threshold Loading . .10 Abdominal Muscle A c t i v a t i o n by Hypercapnia 11 D i f f e r e n t i a l A c t i v a t i o n of the Abdominal Muscles 11 Summary 12 Object ives 13 S i g n i f i c a n c e 14 References 15 I I . Chronic Implantat ion and Length Measurements . .20 In troduct ion 20 Methods 21 S u r g i c a l Preparat ion 22 Tracheostomy . . . 22 Implantat ion of transducers and EMG wires 24 Measurements 27 Pro toco l 32 A n a l y s i s 33 Results ' 34 Discuss ion 39 References 45 I I I . E f f e c t s of Posture on Abdominal Muscle Length 47 In troduct ion 47 Methods , 49 Measurements 49 Protoco l 52 A n a l y s i s 53 Results 54 Discuss ion . . . 60 References 69 V IV. Abdominal Muscle A c t i v a t i o n by Expira tory Threshold Loading .72 In troduct ion .72 Methods 73 Measurements 73 Pro toco l 75 A n a l y s i s 76 Resul ts 76 D i scuss ion 91 References • 97 V. Abdominal Muscle A c t i v i t y During Hypercapnia 99 In troduc t ion 99 Methods 100 Instrumentation 100 Measurements 101 P r o t o c o l 103 A n a l y s i s • • 103 Resul t s 104 Di scuss ion 117 References 126 V I . E f f e c t s of Vagal Blockade on Abdominal Muscle A c t i v a t i o n 129 In troduc t ion 129 Methods 130 Measurements 131 Ex p ira t o ry Threshold Loading 133 C0 2 Rebreathing 133 Pro toco l 134 ETL 135 C0 2 Rebreathing 135 Vagal Blockade 135 A n a l y s i s 136 ETL 136 C0 2 Rebreathing 137 Vagal blockade 138 Resul ts 138 E f f e c t s of Vagal Blockade on Contro l Parameters , 138 ETL 139 C0 2 Rebreathing 151 D i s c u s s i o n 161 E f f e c t s of Vagal Blockade on Contro l Parameters 161 E f f e c t s of Vagal Blockade during ETL 162 E f f e c t s of Vagal Blockade during C0 2 Rebreathing 167 References 170 V I I . Summary and Conclusions 174 Summary of Major Findings 174 Conclusions 176 References 180 vi LIST OF FIGURES Figure 1 Schematic diagram of the canine abdominal muscles 4 Figure 2 Photograph showing a permanent tracheostomy i n a mongrel dog two weeks pos t - surgery 23 Figure 3 Photograph showing the placement of a p a i r of sonomicrometer transducers and f ine wire EMG electrodes i n the ex terna l ob l ique muscle 25 Figure 4 Schematic diagram of the canine abdomen showing p o s i t i o n s and f i b e r o r i e n t a t i o n s o f the four abdominal muscles 28 Figure 5 A representa t ive record ing from an awake dog ( l y i n g i n l e f t l a t e r a l decubitus) showing transversus abdominis (TA) length changes and TA EMG a c t i v i t y 30 F igure 6 Example of the experimental setup with the dog l y i n g i n the l e f t l a t e r a l decubitus p o s i t i o n 33 Figure 7 Two x - sec t ions o f i n t e r n a l obl ique muscle s ta ined with Masson's tr ichome. The muscle biopsy had been f i x e d i n f o r m a l i n wi th the sonomicrometer transducer i n place 38 Figure 8 A representa t ive record ing from an awake dog l y i n g i n l e f t l a t e r a l decubitus p o s i t i o n (top panel) and standing on a l l fours (bottom panel) showing transversus abdominis (TA) length changes and TA EMG a c t i v i t y , 50 Figure 9 Bar graph showing a c t i v e shortening of the TA, 10 and EO i n the l e f t l a t e r a l decubitus (LLD), s i t t i n g (SIT) and standing (STAND) postures 55 Figure 10 Box p l o t s o f the change i n r e s t i n g length (LRL) with s h i f t i n posture from l e f t l a t e r a l decubitus (LLD) to s i t t i n g (SIT) . . .57 Figure 11 Box p l o t s of the change i n r e s t i n g length (LRL) with s h i f t i n posture from l e f t l a t e r a l decubitus (LLD) to s tanding (STAND). 58 vii Figure 12 A representa t ive t r a c i n g from an awake dog ( l e f t l a t e r a l decubitus p o s i t i o n ) showing t i d a l volume (VT) , airway pressure (Pao), i n t e r n a l obl ique (10) length changes and EMG a c t i v i t y 78 F igure 13 Length changes (%LRL) of three abdominal muscles (TA, 10 and E0) at c o n t r o l (Pao=0) and four l eve l s of PEEP (Pao=6, 10, 14 and 18 cmH20) dur ing exp ira tory threshold loading . ; . . . . .80 F igure 14 T o t a l t i d a l length excursions of three abdominal muscles (TA, 10 and E0) expressed as a percentage of the i n i t i a l base l ine r e s t i n g length (%LRL) p l o t t e d against increas ing airway pressure (Pao) dur ing e x p i r a t o r y threshold loading 83 F igure 15 A c t i v e , phas ic exp ira tory shortening of three abdominal muscles (TA, 10 and E0) expressed as a percentage of the i n i t i a l base l ine r e s t i n g length (%LRL) p l o t t e d against i n c r e a s i n g airway pressure (Pao) dur ing exp ira tory threshold loading 84 F igure 16 The change i n lung volume (<5Vol) produced by ETL p l o t t e d against airway pressure (Pao) 88 Figure 17 I n s p i r a t o r y a i r flow durat ion (Ti) (top) and e x p i r a t o r y d u r a t i o n (T E) (bottom) p l o t t e d against airway pressure (Pao) dur ing ETL 89 F igure 18 E x p i r a t o r y durat ion (TE) p l o t t e d against the change i n volume (6vol) dur ing ETL 90 F igure 19 A representa t ive recording from an awake dog ( l e f t l a t e r a l d e c u b i t i s p o s i t i o n ) showing transversus abdominis (TA) length changes, TA EMG a c t i v i t y and t i d a l volume (VT) during C0 2 r e b r e a t h i n g . . . . 1 0 6 Figure 20 V T p l o t t e d as a func t ion of expired minute v e n t i l a t i o n (VE) dur ing C0 2 rebreath ing 109 Figure 21 V T p l o t t e d against T E and Ti during C0 2 rebreathing 110 Figure 22 Length changes of the transversus abdominis (TA), i n t e r n a l obl ique (10) and externa l obl ique (E0) during a i r - b r e a t h i n g c o n t r o l and at three l e v e l s of e n d - t i d a l C0 2 113 Figure 23 T o t a l t i d a l length changes of the TA, 10 and EO (%LRL) p l o t t e d against i n c r e a s i n g minute v e n t i l a t i o n during C0 2 r ebrea th ing . 115 Figure 24 A c t i v e shortening of the TA, 10 and EO (%LRL) p l o t t e d against i n c r e a s i n g minute v e n t i l a t i o n during C O 2 rebreath ing 116 Figure 25 Photograph showing placement of a s i l a s t i c c u f f around the r i g h t c e r v i c a l vagus nerve i n one of the dogs 132 Figure 26 Abdominal muscle a c t i v e phasic shortening versus airway pressure (Pao) dur ing ETL 140 Figure 27 Abdominal muscle a c t i v e phasic shortening versus the change i n volume dur ing ETL 141 Figure 28 Representat ive record ing from an awake dog ( l e f t l a t e r a l decubitus p o s i t i o n ) showing t i d a l volume (V T ) , airway pressure (Pao), i n t e r n a l obl ique (10) length changes and 10 EMG a c t i v i t y dur ing exp ira tory thresho ld loading 142 Figure 29 T i d a l volume versus airway pressure (Pao) during ETL before and a f t e r vagal blockade 146 Figure 30 TXOT versus airway pressure (Pao) during ETL before and a f t e r vagal blockade 147 Figure 31 Tj and T E versus airway pressure (Pao) during ETL before and . a f t er vagal blockade 148 Figure 32 Change i n lung volume (<Svol) versus airway pressure (Pao) dur ing ETL before and a f t e r vagal blockade 149 Figure 33 Representat ive record ing from an awake dog ( l e f t l a t e r a l decubitus p o s i t i o n ) showing V T , TA phasic shortening (%LRL) and TA EMG during moderate hypercapnia before ( l e f t panel) and a f t e r ( r i g h t panel) vaga l blockade 152 Figure 34 Abdominal muscle a c t i v e phasic shortening p l o t t e d against e n d - t i d a l PC0 2 during rebreath ing , before and a f t er vagal b lockade. . . . 154 ix Figure 35 Abdominal muscle ac t ive shortening p l o t t e d against minute v e n t i l a t i o n during rebreathing , before and a f t e r vaga l blockade 155 F igure 36 T i d a l volume ( V T ) p l o t t e d against minute v e n t i l a t i o n dur ing C0 2 rebreath ing , before and a f ter vagal blockade 157 F igure 37 T o t a l a i r flow durat ion ( T T O T ) p l o t t e d against minute v e n t i l a t i o n dur ing C0 2 rebreath ing , before and a f t e r vaga l blockade 158 X LIST OF TABLES Table I Rest ing base l ine lengths (RL) of the abdominal muscles i n anesthet ized compared to awake dogs and ac t ive shortening (%LRL) i n awake dogs ( l e f t l a t e r a l decubitus pos i t i on ) 37 Table II T i d a l volume and t iming parameters i n three postures 59 Table III V e n t i l a t i o n and t iming parameters during ETL 86 Table IV A r t e r i a l b lood gas values during ETL 87 Table V A r t e r i a l b lood gas and pH values during C 0 2 rebreathing 1 0 5 Table VII V e n t i l a t o r y Parameters for r e s t i n g a i r breath ing c o n t r o l and three l e v e l s o f e n d - t i d a l C0 2 I l l Table VII V e n t i l a t i o n and t iming parameters during ETL before and a f t e r vagal blockade 145 Table VIII A r t e r i a l b lood gases during ETL before and a f t er vagal blockade. . 1 5 0 Table IX V e n t i l a t o r y Parameters for r e s t i n g a i r breathing c o n t r o l and three l e v e l s of e n d - t i d a l C 0 2 156 Table X A r t e r i a l Blood Gas and pH values during C 0 2 Rebreathing before and a f t e r vaga l blockade 160 xi LIST OF ABBREVIATIONS 5Vol change i n lung volume EEL end-expiratory length EELV end-expiratory lung volume EILV end-inspiratory lung volume EMG electromyogram EO external oblique F R C f u n c t i o n a l r e s i d u a l capacity 10 i n t e r n a l oblique L L D l e f t l a t e r a l decubitus L A B L a c t i v e baseline length L E E length at end-expiration L E I length at e n d - i n s p i r a t i o n L R L r e s t i n g baseline length % L R L muscle length change as a percent of L R L P aC0 2 a r t e r i a l pressure of carbon dioxide P E T C 0 2 end-tidal pressure of carbon dioxide PC0 2 p a r t i a l pressure of carbon dioxide P O 2 p a r t i a l pressure of oxygen RA rectus abdominis SIT s i t t i n g p o s i t i o n of the dog STAND standing p o s i t i o n of the dog TA transversus abdominis T E duration of e x p i r a t i o n Ti duration of i n s p i r a t i o n T T O T t o t a l breath duration V T t i d a l volume AKNOWLEDGEMENTS I would l i k e to thank my supervisor Dr. Jeremy Road for h i s support and encouragement and my supervisory committee for t h e i r cons truc t ive c r i t i c i s m s and support. Spec ia l thanks and a p p r e c i a t i o n to the t e c h n i c a l s t a f f who made the experiments p o s s i b l e : S a l l y Osborne and Sharon Barwick and a l l the s t a f f of the UBC U n i v e r s i t y H o s p i t a l Animal Resource U n i t , e s p e c i a l l y Michael and Mique l . DEDICATION For my mother. 1 I . INTRODUCTION LITERATURE REVIEW I n t r o d u c t i o n The four muscles of the v e n t r a l abdominal wall are considered to be the major muscles of e x p i r a t i o n (1,12,17). The abdominal muscles have been reported to be p h a s i c a l l y active during C02 rebreathing (42,43,53), exercise (1,35) and expiratory threshold loading (9,34,43) and t o n i c a l l y a c t i v e i n the upright posture (12,16,22). These four muscles: rectus abdominis (RA), external oblique (EO), i n t e r n a l oblique (10) and transversus abdominis (TA), d i f f e r i n t h e i r o r i g i n s , i n s e r t i o n s , f i b e r o r i e n t a t i o n s and surface area. There i s also evidence that they vary i n t h e i r proportions of f i b e r types (11). The presence of such v a r i a t i o n s i n anatomy and structure would suggest functional d i f f e r e n c e s and hence, with respect to breathing, abdominal muscle a c t i o n could be quite complex. The majority of studies however, have generally assumed that the abdominal muscles act as a unit and have not d i f f e r e n t i a t e d among the i n d i v i d u a l muscles (1,12,30,32,58). For the most part, studies done on humans have used surface electrodes and/or measured abdominal displacement, which do hot allow for d i f f e r e n t i a t i o n of i n d i v i d u a l muscle a c t i v i t y . A l t e r n a t i v e l y , a number of i n v e s t i g a t o r s have used intramuscular electrodes i n anesthetized animals but anesthesia i s known to depress muscle a c t i v i t y (51). Despite the l i m i t a t i o n s of the above techniques, a few studies have demonstrated regional differences i n abdominal muscle a c t i v i t y i n the upper and lower abdomen with postural changes (16,22,23) and hypercapnia (54). There i s also some evidence 2 that i n d i v i d u a l abdominal muscles can act independently and that the deeper muscle layer (10 and TA) plays a greater r o l e i n v e n t i l a t i o n (46,54). Recently, the technique of sonomicrometry has been used to measure abdominal muscle length changes i n anesthetized dogs during expiratory threshold loading (ETL) (34,43) and hypercapnia (5,28,43). This technique allows separate measurement of i n d i v i d u a l muscle a c t i v i t y which can not be d i f f e r e n t i a t e d by surface electrodes or volume displacements. The r e s u l t s confirm that i n d i v i d u a l abdominal muscles have d i f f e r e n t degrees of a c t i v i t y . In p a r t i c u l a r , i t has been shown that the i n t e r n a l layer (10 and TA) i s the more important one i n v e n t i l a t i o n during ETL (34) and hypercapnia (41) i n anesthetized dogs. The mechanisms responsible f o r abdominal muscle recruitment are thought to be p r i m a r i l y v a g a l l y mediated during ETL (7,23,48) and p r i m a r i l y due to chemoreceptors during hypercapnia (33,43,56). However, there i s also evidence that segmental reflexes may be involved (31,49), such that passive s t r e t c h i n g of the muscles activates muscle proprioceptors and r e f l e x l y stimulates muscle a c t i v i t y . The patterns of abdominal muscle recruitment may d i f f e r depending on the r e l a t i v e contributions of these mechanisms. Functional Anatomy of the Abdominal Muscles The four muscles of the ve n t r a l abdominal w a l l : rectus abdominis (RA), external oblique (E0), i n t e r n a l oblique (10) and transversus abdominis (TA) d i f f e r i n o r i g i n , i n s e r t i o n , f i b e r o r i e n t a t i o n and surface area. The RA i s most medial and runs a x i a l l y , from the sternum to the pubis on ei t h e r side of the l i n e a alba. The E0 l i e s l a t e r a l l y and s u p e r f i c i a l l y , extending caudiodorsally from the outer surfaces of the 3 lower eight r i b s to i n s e r t into the i l i a c c r e s t and l i n e a alba. The 10 l i e s deep to the E0 but more medial and with i t s f i b e r s running at approximately r i g h t angles to those of the E0, from i t s o r i g i n on the i l i a c c r e s t . At i t s point of i n s e r t i o n into the lower three r i b s , the 10 i n t e r d i g i t a t e s with the i n t e r n a l i n t e r c o s t a l s . The deepest of the abdominal muscles i s the TA. I t runs c i r c u m f e r e n t i a l l y , a r i s i n g from the inner surface of the l a s t s i x c o s t a l c a r t i l a g e s where i t i s continuous with the transversus thoracis and i n t e r d i g i t a t e s with the diaphragm (4). A schematic i l l u s t r a t i o n of the canine abdominal muscles i n d i c a t i n g the i n d i v i d u a l muscle f i b e r o r i e n t a t i o n and p o s i t i o n i n the abdominal wall i s shown i n Figure 1. The anatomy of the canine abdominal muscles appears to be very s i m i l a r to humans. However, the shape of the r i b cage i s quite d i f f e r e n t between the two species. In dogs, the r i b cage anteroposterior diameter i s greater than the transverse diameter, whereas the reverse i s the case i n humans (37). The d i f f e r e n c e i n r i b cage shape may produce some fun c t i o n a l v a r i a t i o n of i n d i v i d u a l abdominal muscles. The v e n t i l a t o r y function of the muscles of the abdominal w a l l has been deduced p r i m a r i l y from anatomical analysis and electromyographical studies. Based on t h e i r anatomy, they were thought to compress the abdomen and p u l l down the lower r i b s , thus having an expiratory action on lung volume. However, when i n d i v i d u a l abdominal muscles were stimulated separately (17,37) they were found to have d i f f e r i n g actions on the r i b cage. In the supine, anesthetized dog, the RA was found to have a d e f l a t i o n a r y e f f e c t on the lower r i b cage, the E0 an i n f l a t i o n a r y e f f e c t and the 10 and TA no noticeable e f f e c t (17). In contrast, separate stimulation of both the RA and E0 i n humans r e s u l t e d i n 4 Figure 1: Schematic diagram of the canine abdominal muscles: transversus abdominis (•) , i n t e r n a l oblique ( O ) , external oblique (A) and rectus abdominis ( A ) , showing the f i b e r o r i e n t a t i o n and p o s i t i o n i n the abdominal wall. 5 6 displacement of the r i b cage and reduction i n lung volume (37) . These actions however were achieved d i f f e r e n t l y : the RA produced a decrease i n anteroposterior diameter of the r i b cage, whereas the EO caused a decrease i n transverse diameter. Despite the d i f f e r e n t actions of the i n d i v i d u a l muscles when stimulated separately, the net e f f e c t i s expiratory and depends on the recruitment pattern and coordinated a c t i o n of the abdominal muscles. The abdominal muscles are supplied by the lower i n t e r c o s t a l and lumbar nerves (4). The motoneurons innervating i n d i v i d u a l muscles are d i s t r i b u t e d from T4-L3. The segmental d i s t r i b u t i o n of motoneurons f o r each abdominal muscle i s T4-L3 for the RA, T6-L3 f o r the EO, T13-L3 f o r the 10 and T9-L3 f o r the TA (38). Central drive i s conveyed to the abdominal motoneurons v i a projections from expiratory neurons located p r i m a r i l y i n the caudal p o r t i o n of the v e n t r a l r e s p i r a t o r y group (VRG) (39). Methods of Study Most of the information regarding the recruitment or a c t i v i t y of the abdominal muscles during breathing i s derived from electromyography (usually v i a surface electrodes). The abdominal muscles have been studied under a v a r i e t y of conditions using these techniques. However, due to the l i m i t a t i o n s imposed by surface electrodes, only the a c t i v i t y of the s u p e r f i c i a l muscles (RA and EO) can be d i f f e r e n t i a t e d . More recent studies have u t i l i z e d intramuscular f i n e wire EMG electrodes, which have enabled detection of i n d i v i d u a l muscle a c t i v i t y (18,57). Nevertheless, there are l i m i t a t i o n s inherent to electromyography: only muscle a c t i v a t i o n i s measured and neither muscle r e s t i n g length nor 7 pass ive a c t i v i t y ( i n s p i r a t o r y lengthening and pass ive shortening) can be determined. In a d d i t i o n , ton ic muscle a c t i v i t y can only be i n f e r r e d from the increase i n background noise . Studies u t i l i z i n g measurements o f abdominal muscle displacement provide some informat ion about the c o n t r i b u t i o n of the abdominal muscles to v e n t i l a t i o n , i n terms of i n s p i r a t o r y lengthening as w e l l as phasic shortening . However, ton ic muscle a c t i v i t y i s even more d i f f i c u l t to assess and pass ive shortening can not be separated from ac t ive shortening. In a d d i t i o n , there i s no means of i d e n t i f y i n g i n d i v i d u a l muscle act ions with these techniques. Sonomicrometry i s a technique which allows measurement of the t r a n s i t time of a sound wave t r a v e l l i n g between two p i e z o e l e c t r i c transducers ( c r y s t a l s ) . The t r a n s i t time i s measured 1543 times per second and i s averaged to give a continuous DC vo l tage . Given the known conduct ion v e l o c i t y of sound i n muscle (1580m/sec), the l i n e a r distance between two c r y s t a l s implanted i n muscle can be obtained from the measured t r a n s i t time. Thus, sonomicrometry provides a d i r e c t , i n v ivo method for measuring muscle length changes. An important advantage of sonomicrometry i n the study of r e s p i r a t o r y mechanics i s that i t provides a means of quant i fy ing the mechanical consequences of muscle a c t i v a t i o n and when used i n conjunct ion with electromyography (EMG), pass ive and a c t i v e shortening can be i d e n t i f i e d . The f i r s t use of sonomicrometry to measure r e s p i r a t o r y muscle length changes was accomplished i n the dog diaphragm (40). Recently i t has been used to measure abdominal muscle length changes i n anesthet ized dogs (5,34,41,43) and found to be a use fu l technique i n the abdominal muscles. 8 Postural E f f e c t s on and Regional Differences i n Abdominal Muscle A c t i v i t y Data from studies using electromyographic techniques have i l l u s t r a t e d the e f f e c t s of body p o s i t i o n on muscle a c t i v i t y of the RA and EO. I t i s generally accepted that the abdominal muscles (RA and EO) are i n a c t i v e i n human subjects when r e s t i n g supine (1,13,22,30,35). C o n f l i c t i n g r e s u l t s have been found with other postures. Several studies i n man using e i t h e r electromyography (12,13,16,22) and/or displacement measurements (22,35) have found tonic abdominal muscle a c t i v i t y , e s p e c i a l l y i n the lower "gravity dependent" p o r t i o n of the abdomen i n upright, quiet breathing. There i s also evidence of phasic abdominal contraction during expiration i n the upright p o s i t i o n , i n some subjects (13,35,52). S i m i l a r r e s u l t s have been reported f o r the seated p o s i t i o n , although with a les s e r degree of a c t i v i t y (13,16,35). In contrast, other i n v e s t i g a t o r s observing abdominal wall displacements suggested that the abdominal muscles were not act i v e i n standing subjects at r e s t (30,32). The cont r o v e r s i a l r e s u l t s may be p a r t i a l l y explained by the d i f f e r e n t methodologies u t i l i z e d . Electromyography most l i k e l y provides a more s e n s i t i v e measure of muscle recruitment, compared to abdominal displacement techniques (11,22). Indeed, more recent studies i n which intramuscular EMG electrodes were placed i n the in t e r n a l abdominal muscles (TA and 10) , c l e a r l y demonstrated phasic a c t i v i t y of those muscles, i n the standing p o s i t i o n during the exp ira tory phase of quiet breathing (18,57). Therefore , i t seems reasonable to conclude that contraction of the abdominal muscles during quiet breathing i n upright man contributes to breathing. The abdominal muscles of dogs appear to be more ac t i v e at r e s t 9 than those i n man. Phasic EMG a c t i v i t y has been found i n the TA (5,23,28) and o c c a s i o n a l l y 10 (34) and EO (28) i n supine anesthetized dogs and i n awake dogs (19). In the small number of studies i n which abdominal muscle length changes were made (sonomicrometry), phasic shortening has been found i n a s s o c i a t i o n with the EMG when present, i n supine anesthetized dogs (5,28,34,41,43). When anesthetized dogs are t i l t e d towards upright, phasic EMG a c t i v i t y appears i n the previously i n a c t i v e abdominal muscles and increases i n those that were active i n the supine p o s i t i o n (20,23,28). Phasic EMG a c t i v i t y has also been shown i n awake standing (19,53) and s i t t i n g dogs (19). In addition, there i s evidence to suggest that tonic a c t i v i t y develops i n the upright postures, p a r t i c u l a r l y i n the TA of the dog (19,26). In upright postures, phasic and tonic a c t i v i t y of the abdominal muscles i s thought to help maintain FRC (23), defend diaphragm end-expiratory length (26) and contribute to t i d a l volume v i a t h e i r r e l a x a t i o n at end-expiration (24,25). The c o n t r i b u t i o n of the abdominal muscles to t i d a l volume has been estimated to be more than 50% i n anesthetized dogs t i l t e d to the headup p o s i t i o n (24). The assumption of. upright posture produces an increase i n FRC (2,23). The increase i n FRC i s considered to be p r i m a r i l y responsible f o r abdominal muscle a c t i v a t i o n v i a a vagal r e f l e x from lung s t r e t c h receptors (15,20,23). Indeed, when vagal r e f l e x e s were eliminated by vagotomy, abdominal muscle a c t i v i t y was reduced i n upright anesthetized (20,23) and awake dogs (19) and anesthetized rabbits (15). However, evidence suggests that segmental reflexes from muscle proprioceptors are also involved i n abdominal muscle a c t i v a t i o n i n upright postures, 10 p a r t i c u l a r l y tonic a c t i v i t y (15,19,50). G r a v i t a t i o n a l e f f e c t s on the abdominal contents would tend to cause the abdominal wall to move outward, s t r e t c h i n g the abdominal muscles (2) and thus, stimulating muscle proprioceptors (29). Regional differences i n tonic abdominal muscle a c t i v i t y were alluded to e a r l i e r i n the discussion of postural e f f e c t s . Several previous investigators found regional differences i n abdominal muscle a c t i v i t y i n man i n the upright posture (35,50) and i n dogs during hypercapnia (28,42). In man the lower region of the abdominal muscles had greater tonic muscle a c t i v i t y than the upper regions i n the upright posture (35,54). This i s thought to be due to the presence of la r g e r hydrostatic forces i n the lower abdomen. There i s also evidence of regional differences i n abdominal muscle a c t i v a t i o n i n dogs during CO2 rebreathing, which are opposite to postural differences (greater a c t i v i t y i n the upper region) (28,42). Abdominal Muscle A c t i v a t i o n by Expiratory Threshold Loading Expiratory threshold loading (ETL) stimulates abdominal muscle a c t i v i t y i n man (1,36,58) and i n anesthetized cats (6,10) and dogs (34,43). Abdominal muscle active shortening during ETL has also been demonstrated i n anesthetized dogs (34,43). Abdominal muscle a c t i v a t i o n i s one of the load compensating mechanisms employed by the r e s p i r a t o r y system (14,36). A c t i v a t i o n of the abdominal muscles during ETL helps to prevent too great an increase i n FRC and hence, defends diaphragm length and t i d a l volume (14,34,45). Expiratory threshold loading produces an increase i n FRC (34,36,45). I t i s the increase i n FRC which i s thought to be the 11 primary mediator of abdominal muscle a c t i v a t i o n v i a s t i m u l a t i o n of vagal r e f l e x e s by lung s t r e t c h receptors (6,8,10,14,48). However, segmental refle x e s may also be involved (7,49). The increase i n lung volume produced by ETL could s t r e t c h the abdominal muscles and thus, stimulate muscle proprioceptors. Indeed, passive lung i n f l a t i o n i n anesthetized, paralyzed dogs r e s u l t e d i n lengthening of the abdominal muscles, p a r t i c u l a r l y the TA and 10 (34). Abdominal Muscle A c t i v a t i o n by Hypercapnia Abdominal muscle phasic EMG a c t i v i t y i n humans (55,57) and anesthetized (5,43,56) and awake (3,53) dogs i s stimulated by hypercapnia. Abdominal muscle phasic shortening has also been demonstrated i n anesthetized dogs (5,28,43) during hypercapnia. A c t i v a t i o n of the abdominal muscles by C O 2 increases expiratory flow, allowing breathing frequency to increase. In addition, abdominal muscle a c t i v a t i o n enables t i d a l volume to increase by encroaching on both i n s p i r a t o r y and expiratory reserve volumes. Thus, the work of breathing may be r e d i s t r i b u t e d between the expiratory and i n s p i r a t o r y muscles when v e n t i l a t i o n i s increased by hypercapnia. The abdominal muscle response to hypercapnia appears to be mediated p r i m a r i l y v i a peripheral and c e n t r a l chemoreflexes (33) and i s a function of an increased drive to breathe. However, a vagal component also appears to be involved, since abdominal muscle EMG a c t i v i t y i s reduced i n vagotomized, anesthetized dogs during hypercapnia (28,42). D i f f e r e n t i a l A c t i v a t i o n of the Abdominal Muscles In addition to regional v a r i a t i o n i n recruitment and a c t i v a t i o n 12 with changes i n posture , d i f f e r e n t i a l a c t i v a t i o n of the i n d i v i d u a l abdominal muscles has been found during exp ira tory load ing (34,43) and hypercapnia (28,42,43). Robertson et a l . . (46) reported increased b lood flow predominantly to the TA and 10, dur ing e x p i r a t o r y r e s i s t a n c e l oad ing . I t has a l so r e c e n t l y been shown that , i n supine anes thet ized dogs, ETL produced much greater a c t i v a t i o n and shortening of the i n t e r n a l abdominal muscles (43), p a r t i c u l a r l y the 10 (34). D i f ferences a l so e x i s t i n the p a t t e r n of contrac t ion of the abdominal muscles with s t i m u l a t i o n by CO2. There i s evidence of d i f f erences of both EMG a c t i v i t y (28,43) and shortening (41,43) among the muscles; the TA appearing to be the most a c t i v a t e d . In a d d i t i o n , Robertson et a l . , (47) found the TA to have the greatest blood flow dur ing CO2 r ebrea th ing , i n anesthet ized dogs. With a few exceptions (3,19,53), the major i ty o f s tudies of abdominal muscle response to ETL and hypercapnia have used supine anesthet ized animals . However, anesthesia may enhance v a g a l re f l exes dur ing ETL and a f f e c t the v e n t i l a t o r y (44) and breath ing p a t t e r n (27) response to CO2. These e f fects of anesthes ia cou ld a f f e c t the recruitment p a t t e r n o f the abdominal muscles. Summary The abdominal muscles are st imulated to contrac t wi th phas ic exp ira tory a c t i v i t y dur ing both expiratory thresho ld load ing (9,34,58) and hypercapnia- induced hyperpnea (35,43,57). The mechanisms respons ib le for the recruitment are l i k e l y to be d i f f e r e n t depending on the s t imulus . Therefore , the degree of a c t i v a t i o n of i n d i v i d u a l abdominal muscles and the pat tern of recruitment may vary under 13 d i f f e r e n t condi t ions and w i l l depend on the r e l a t i v e c o n t r i b u t i o n of l o c a l p r o p r i o c e p t i v e inputs (29,31,49), vagal a f ferents (7,9,19,48) and chemoreceptor input (5,33,43,56). OBJECTIVES A chronic dog model was used i n the experiments p r i m a r i l y because the abdominal muscles of the dog have been s tud ied p r e v i o u s l y i n acute preparat ions and t h e i r large s i ze allows the techniques to be performed. In a d d i t i o n , although there are some d i f f e r e n c e s , the dog's abdominal muscles are s i m i l a r i n anatomy and funct ion to humans (21). The canine model has been used extens ive ly and there i s a large amount of in format ion a v a i l a b l e on the r e s p i r a t o r y muscles. The objec t ives of t h i s study were to assess the v e n t i l a t o r y funct ions of the i n d i v i d u a l abdominal muscles i n the awake dog under a v a r i e t y of s t i m u l i which produce hyperpnea, increased load and g r a v i t a t i o n a l e f f e c t s . In a d d i t i o n , muscle recruitment p a t t e r n was examined with the aim of determining the primary c o n t r o l mechanisms. Thi s informat ion may a l so further understanding of the i n t e r a c t i o n between the abdominal muscles and the other muscles of r e s p i r a t i o n . Therefore , the major object ives of t h i s study were to tes t the fo l l owing hypotheses: 1. That the a c t i v i t y during quiet breathing of a l l of the abdominal muscles w i l l be greater i n the awake state than i n the anesthet ized dog. 2. That t on i c and phasic a c t i v i t y w i l l be greater i n the upr ight (standing on a l l fours and s i t t i n g ) p o s i t i o n compared to the l y i n g p o s i t i o n ( l a t e r a l decubitus) due to the h y d r o s t a t i c pressure gradient and that p o s t u r a l e f fec t s w i l l be more pronounced on the i n t e r n a l 14 abdominal muscles (TA and 10) compared to the ex terna l muscles (RA and E0) . 3. That a c t i v i t y of the i n t e r n a l muscle layer (TA and 10) w i l l be greater than the e x t e r n a l l ayer (E0 and RA) during e x p i r a t o r y thresho ld load ing i n the awake dog, as was found i n the anesthet ized dog (34). 4. That hypercapnia w i l l produce a greater e f f e c t on the abdominal muscles i n the awake animal than the anesthet ized one and that the i n d i v i d u a l muscles may e x h i b i t d i f ferences i n recruitment p a t t e r n . 5. That both vagal inputs and segmental re f lexes are invo lved i n abdominal muscle recrui tment and may exp la in d i f ferences i n recrui tment among the i n d i v i d u a l abdominal muscles. The objec t ives were met by determining the c o n t r i b u t i o n o f the abdominal muscles to v e n t i l a t i o n i n the awake dog under cond i t ions of l oad ing , C0 2 - induced hyperpnea and pos tura l changes and comparing the r e s u l t s to those found for the anesthet ized dog and the awake dog post-vagotomy. S i g n i f i c a n c e These experiments w i l l provide i n s i g h t in to the i n v ivo c o n t r a c t i l e p r o p e r t i e s of the abdominal muscles and the c o n t r o l o f e x p i r a t i o n . Since the r e s u l t s from th i s study w i l l be d e r i v e d i n the awake s ta te , they may be more appl i cab le than those from studies performed on anesthet ized animals. In a d d i t i o n , the use o f var ious s t i m u l i to r e c r u i t the abdominal muscles w i l l s imulate to some degree the s i t u a t i o n s found i n a number of disease c o n d i t i o n s . For example, exp ira tory thresho ld load ing produces an increase i n FRC s i m i l a r to chronic obs truc t ive lung disease (52). C o n t r i b u t i o n to the understanding 15 of the complex coordination of these muscles during breathing and t h e i r i n t e r a c t i o n with the other muscles of r e s p i r a t i o n under various conditions may be valuable i n some c l i n i c a l s i t u a t i o n s . REFERENCES 1. Agostoni, E., and E.J.M. Campbell. The abdominal muscles. In: The  Respiratory Muscles: Mechanics and Neural Control. E.J.M. Campbell, E. Agostoni, and J . Newsom-Davis (eds). Saunders, Philadelphia, PA, 1970, 175-180. 2. Agostoni, E., and R.E. Hyatt. S t a t i c behavior of the r e s p i r a t o r y system. 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Robertson, C.H., W.L. Eschenbacher and R.L. Johnson. Respiratory muscle blood flow d i s t r i b u t i o n during expiratory r e s i s t a n c e . J .  C l i n . Invest. 60: 473-480, 1977. 47. Robertson, C H . , M.A. Pagel and R.L. Johnson. The d i s t r i b u t i o n of blood flow, oxygen consumption and work output among the r e s p i r a t o r y muscles during unobstructed hyperventilation. J . C l i n . Invest. 59: 43-50, 1977. 48. R u s s e l l , J.A. and B. Bishop. Vagal afferents e s s e n t i a l f o r abdominal muscle a c t i v i t y during lung i n f l a t i o n i n cats. J . Appl. P h y s i o l . 41(3): 310-315, 1976. 49. R u s s e l l , J.A., B. Bishop and R.E. Hyatt. Discharge of abdominal muscle alpha and gamma motorneurons during expiratory loading i n cats. Exper. Neurol. 97: 179-192, 1987. 50. Shannon, R. I n t e r c o s t a l and abdominal muscle a f f e r e n t influence on medullary dorsal r e s p i r a t o r y group neurons. Respir. P h y s i o l . 39: 73-94, 1980. 51. Shannon, R. Reflexes from respiratory muscles and costovertebral j o i n t s . In: Handbook of Physiology. Section 3: The Respiratory  System. Vol.11. A.P.Fishman. (ed). Williams & Wilkins, Baltimore, MD, 1986, 431-447.' 52. Skarvan, K. The v e n t i l a t o r y function of the abdominal muscles i n normal subjects and i n patients with chronic obstructive lung disease. R e s p i r a t i o n 28: 347-359, 1971. 53. Smith, CA., D.M. Ainsworth, K.S. Henderson and J.A. Dempsey. 19 D i f f e r e n t i a l responses of expiratory muscles to chemical s t i m u l i i n awake dogs. J . Appl. Physiol. 66(1): 384-391, 1989. 54. S t r o h l , K.P., J . Mead, R.B. Banzett, S.H. Loring and P.C. Kosch. Regional differences i n abdominal muscle a c t i v i t y during various maneuvers i n humans. J. Appl. Physiol. 51: 1471-1476, 1981. 55. Takasaki, Y., D. Orr, J . Popkin, A. Xie and T.D. Bradley. E f f e c t of hypercapnia and hypoxia on re s p i r a t o r y muscle a c t i v a t i o n i n humans. J . Appl. Phvsiol. 67(5): 1776-1784, 1989. 56. van Lunteren, E., M.A. Haxhiu, N.S. Cherniack and J.S. Arnold. Rib cage and abdominal expiratory muscle responses to C O 2 and esophagel distension. J . Appl. Physiol. 64(2): 846-853, 1988. 57. Wakai, Y., M.M. Welsh, A.M. Leevers and J.D. Road. The e f f e c t of continuous p o s i t i v e airway pressure and hypercapnia on expiratory muscle a c t i v i t y during wakefulness and sleep. Am. Rev. Respir. P i s . 141(4): A125, 1990.(Abstract) 58. Wolfson, D.A., K.P. Strohl, A.F. Dimarco and M.D. Altose. E f f e c t s of an increase i n end-expiratory lung volume on pattern of thoracoabdominal movement. Respir. Physiol. 53: 273-283, 1983. 20 I I . CHRONIC IMPLANTATION AND LENGTH MEASUREMENTS INTRODUCTION The technique of sonomicrometry was o r i g i n a l l y used to measure v e n t r i c u l a r dimensions (18) and was adapted to measure diaphragm muscle length changes by Newman et a l . (10) . Since then, sonomicrometry has been used to measure length changes i n various other r e s p i r a t o r y muscles, i n c l u d i n g the diaphragm, i n anesthetized animals (2,7,11,13). Recently, diaphragm length measurements (sonomicrometry) have also been made i n awake dogs (1,4) and sheep (20). We previously placed sonomicrometer transducers i n each of the four muscles of the ven t r a l abdominal wall: rectus abdominis (RA), external oblique (EO), i n t e r n a l oblique (10) and transversus abdominis (TA) i n anesthetized dogs (8), and were able to measure t i d a l muscle length changes and acti v e shortening during expiratory threshold loading (ETL). However, that study and most studies employing sonomicrometry have predominantly used anesthetized animals and anesthesia a f f e c t s v e n t i l a t i o n (14) and respiratory muscle a c t i v i t y (15). For example, evidence suggests that anesthesia reduces the r e s t i n g tone of the diaphragm (6). In addition, animals have generally been studied i n the supine p o s i t i o n , a p o s i t i o n which may be less natural f o r dogs compared to the prone or l a t e r a l decubitus p o s i t i o n . F i n a l l y , implantation of the c r y s t a l s per se may a f f e c t muscle shortening by such f a c t o r s as sca r r i n g or l o c a l i n h i b i t i o n . Consequently, i t was hypothesized that abdominal muscle tonic a c t i v i t y ( r e f l e c t e d by r e s t i n g length) and activ e , expiratory shortening would be d i f f e r e n t i n the awake compared to the anesthetized animal. 21 The objectives of t h i s study were to assess the e f f e c t s of anesthesia on abdominal muscle r e s t i n g length and active shortening by comparing i n d i v i d u a l abdominal muscle length changes measured i n anesthetized dogs to those measured i n the same dogs when awake. A further objective was to determine the f e a s i b i l i t y of long-term use of sonomicrometry f o r abdominal muscle length measurements. Since many of the methods for subsequent chapters are s i m i l a r , the d e s c r i p t i o n of methods f o r a l l the experimental procedures are d e t a i l e d i n the present chapter and only the protocols w i l l be o u t l i n e d i n l a t e r chapters. The r e s u l t s from experiments on the e f f e c t s of posture, ETL and hypercapnia are discussed i n Chapters I I I , IV and V. METHODS Nine female mongrel dogs between 18-30 kg were selected. The c r i t e r i a f o r s e l e c t i o n were a f r i e n d l y , even temperament and dogs of s i m i l a r s i z e and shape (.ie. a breed such as a labrador with a chest b u i l d which i s neither deep nor f l a t i n the anteroposterior diameter). Sur g i c a l and experimental protocols were approved by the Animal Care Committee of the U n i v e r s i t y of B r i t i s h Columbia. D a i l y care and maintenance was provided by trai n e d personnel i n the Animal Resource Unit, following the guidelines of the Canadian Counsel on Animal Care. A permanent tracheostomy was prepared and followed by a two week recovery period. During the recovery period, each dog was f a m i l i a r i z e d with the personnel and experimental setups and trai n e d to follow simple commands. A f t e r the sutures were removed from the tracheostomy, the dog underwent t r i a l sessions with a tracheostomy tube i n place. 22 S u r g i c a l P r e p a r a t i o n A l l the s u r g i c a l procedures were performed under s t e r i l e c o n d i t i o n s , i n the Animal Resource Uni t of the UBC acute care h o s p i t a l . The dogs were premedicated 30 minutes p r i o r to surgery with acepromazine ( l .Omg/kg IM) and atropine (0.5mg/kg SQ). Anesthes ia was produced with sodium pentotha l (20mg/kg IV) . The dogs were then intubated with a cu f fed #9 endotracheal tube and placed on an anesthet ic machine which d e l i v e r e d a combination i n h a l a t i o n agent, halothane and n i t r o u s oxide (halothane 1.5% and supplemental O 2 , F 1 0 2 1 0 at 2 1 /min . ) . A l l appropriate areas were shaved and prepped. Body temperature was maintained at 37°C by a heat ing pad. F l u i d balance was maintained in travenous ly wi th l a c t a t e d Ringer ' s s o l u t i o n . A n t i b i o t i c s were given i n the morning before and for f i v e to seven days a f t e r surgery ( e i t h e r 1 tab co- tr imoxazole (NOVO/DS) o r a l l y or 1 cc p e n i c i l l i n - s t r e p t o m y c i n mix IM) . Tracheostomy A m i d l i n e , 2-4 cm v e r t i c a l s k i n i n c i s i o n was made approximately two cm i n f e r i o r to the c r i c o i d c a r t i l a g e . The muscle and f a s c i a o v e r l y i n g the trachea were c a r e f u l l y d i s sec ted and the connect ive t i s sue removed from the trachea . A U-shaped i n c i s i o n , approximately one t h i r d the circumference of the trachea, was made through three or four of the c a r t i l a g i n o u s r ings and the r e s u l t i n g f lap was removed. The s k i n was then sutured (4.0 monofilament nylon) to the t r a c h e a l mucosa and the wound was l e f t uncovered to h e a l . The tracheostomy was c leaned d a i l y and the sutures were removed a f t er 10 to 14 days. An example of a permanent tracheostomy i s shown i n Figure 2. Figure 2: Photograph showing a permanent tracheostomy i n a mongrel dog two weeks post-surgery. The dog i s pictured i n the s i t t i n g p o s i t i o n with i t s head held up. 24 Implantation of transducers and EMG wires One p a i r of 2.5mm double-lensed, p i e z o e l e c t r i c transducers ( c r y s t a l s ) was s u r g i c a l l y implanted (10-15 mm apart) i n each of the four abdominal muscles. Implantation of the c r y s t a l s was accomplished i n two stages. The dog was placed i n the r i g h t l a t e r a l decubitus p o s i t i o n and a h o r i z o n t a l i n c i s i o n was made i n the sk i n on the l e f t side of the abdomen, j u s t below the umbilicus and then extended about 10 cm l a t e r a l l y from the midline. F i r s t the rectus abdominis, and then the external oblique muscles were exposed by blunt d i s s e c t i o n of the f a s c i a . For each muscle, p a r a l l e l muscle f i b e r s were separated and a pocket formed between the f i b e r s . A c r y s t a l was placed i n the pocket, oriented i n the d i r e c t i o n i n which the f i b r e s ran and anchored with a purse-s t r i n g suture (4.0 s i l k ) . The second c r y s t a l of the p a i r was s i m i l a r l y placed i n another pocket 10-15 mm away, between the same muscle f i b e r s . The c r y s t a l s were oriented perpendicular to the muscle f i b e r s so that the lenses were fa c i n g each other. The placement of a p a i r of c r y s t a l s i n the external oblique muscle i s shown i n Figure 3. Bipo l a r , f i n e wire electromyogram (EMG) electrodes were then sewn into d i f f e r e n t f i b e r s 2-3 cm away from the c r y s t a l s . The EMG electrodes consisted of FEP-teflon coated, 75-strand s t a i n l e s s s t e e l wire (Cooner Wire Co. #AS637). The end of each wire was stripped of i n s u l a t i o n and then sewn d i r e c t l y into the muscle with a s u r g i c a l needle. A knot was made, the free end cut short and the wire was anchored i n place by covering the knot with dental cement. A small loop was formed i n the c r y s t a l wires to allow some slack f o r muscle movement and the f a s c i a over the two exposed muscle areas was sutured closed (3.0 v i c r y l ) leaving the loop of wires under the f a s c i a . Figure 3: Photograph showing the placement of a p a i r of sonomicrometer transducers and fine wire EMG electrodes i n the external oblique muscle. The top of the photograph i s towards the c r a n i a l end of the dog. 26 A smal l mid-scapular sk in i n c i s i o n was made and the wires from the RA and EO were tunne l led under the sk in to e x i t d o r s a l l y through the mid-scapular i n c i s i o n . The s k i n i n c i s i o n s over the muscles were then sutured c lo sed (2.0 pro lene ) . For the second stage of the procedure, the dog was p laced i n the l e f t l a t e r a l decubitus p o s i t i o n . A 6-10 cm diagonal i n c i s i o n was made i n the d i r e c t i o n of the i n t e r n a l obl ique muscle f i b r e s , s t a r t i n g 2 cm below the umbi l icus approximately 10 cm from the mid l ine and extending d o r s o - l a t e r a l l y and cauda l ly . The external obl ique muscle f i b e r s were separated and the f a s c i a b lunt d issected to expose the i n t e r n a l obl ique muscle. A p a i r of c r y s t a l s and EMG wires were p laced i n the muscle us ing the technique described e a r l i e r . The f a s c i a l l a y e r and the o v e r l y i n g EO muscle layer were sutured c losed (3.0 v i c r y l ) l e a v i n g a loop of the c r y s t a l wires under the f a s c i a . A f i n a l 6-10 cm diagonal i n c i s i o n was made 2-5 cm above the umbi l i cus , running i n the d i r e c t i o n o f the ex terna l obl ique f i b r e s ; d o r s o - l a t e r a l l y and cephalad. The area between the RA and 10 muscles where only the EO muscle and the 10 f a s c i a o v e r l i e the transversus abdominis was loca ted . At t h i s p o i n t , the f a s c i a was b l u n t d i s sec ted to expose the TA and c r y s t a l s and EMG wires p laced as o u t l i n e d above. The f a s c i a and muscle layers were c l o s e d (3.0 v i c r y l ) l e a v i n g a loop of the wires underneath. The wires from the TA and 10 were tunne l l ed under the sk in to e x i t through the d o r s a l mid-scapular i n c i s i o n . Both s k i n i n c i s i o n s were then sutured c losed (2.0 p r o l e n e ) . A l l the wounds were dusted with powdered c e f a z o l i n sodium (Ancef) a n t i b i o t i c and bandaged and a dog torso s u i t ( A l i c e Chatham A r t s ) was l o o s e l y f i t t e d over the dog to r e t a i n and protec t the wires . The s i t e of 27 placement of c r y s t a l s and EMG wires for each muscle i s shown schemat ica l ly i n Figure 4. Measurements Abdominal muscle lengths were measured with the p i e z o e l e c t r i c transducers ( c r y s t a l s ) . The c r y s t a l s were connected to i s o l a t e d f ine wires . The bared ends of the e x t e r i o r i z e d wires were, i n t u r n , connected by minigrabbers and sh ie lded cable to a sonomicrometer (model 120, T r i t o n Technology, San Diego, C a l . ) . Abdominal muscle electromyograms were measured v i a the f ine wire EMG e l ec trodes . The bared ends of the EMG e lectrode wires were connected to a m p l i f i e r s by minigrabbers and sh ie lded c o a x i a l cab le . The electromyographic s igna l s were ampl i f i ed (Grass, model P511), low and h igh pass f i l t e r e d (30Hz and lKHz) and recorded i n tandem with the sonomicrometer s i g n a l s . The technique of sonomicrometry has been adapted to a l low accurate measurement of diaphragm length changes, as descr ibed p r e v i o u s l y (10) and has a l so been shown to be an e f f e c t i v e method of measuring abdominal muscle length changes i n an acute preparat ion (8). In that e a r l i e r acute study (8), the c r y s t a l s were p laced i n approximately the same area of each abdominal muscle as they were i n the present study. These placements were found to give good representat ive measurements of abdominal muscle length changes. To evaluate p o s s i b l e r e g i o n a l v a r i a t i o n i n length changes, c r y s t a l s were p laced i n d i f f e r e n t regions of each abdominal muscle and the measurements between regions were compared, i n the previous study (8). No s i g n i f i c a n t r e g i o n a l v a r i a t i o n i n abdominal muscle length changes was found. Therefore , the c r y s t a l s Figure 4: Schematic diagram of the canine abdomen showing p o s i t i o n s and f i b e r or i enta t ions of the four abdominal muscles: rectus abdominis ( A ) , ex terna l obl ique ( A ) , i n t e r n a l obl ique ( O ) and transversus abdominis (•) . The symbols i n d i c a t e the approximate placement of the p i e z o e l e c t r i c transducers and EMG wires i n each of the muscles. 29 were placed i n approximately the same area of each abdominal muscle i n a l l of the dogs i n the present study (Figure 4). The r e s t i n g length of the abdominal muscles was defined as e i t h e r the end-expiratory length, i f no active, expiratory (phasic) shortening was present, or the length immediately preceding a c t i v e , phasic shortening and was termed LRL The s l i g h t plateau of the TA length t r a c i n g indicated by the arrows i n Figure 5 i s LRL. The LRL was not meant to represent the true r e s t i n g length of the muscle, since tonic muscle a c t i v i t y may have been present, but was the baseline length preceding a c t i v e shortening c o i n c i d i n g with phasic EMG a c t i v i t y , regardless of the dog's p o s i t i o n . Changes i n length from the r e s t i n g length, e i t h e r lengthening or shortening, during t i d a l breathing, were expressed as a percentage change from the i n i t i a l r e s t i n g length (%LRL) . Muscle shortening was further defined as tonic or ac t i v e . A decrease i n the baseline from the i n i t i a l r e s t i n g length was termed tonic shortening. The length measurements were recorded i n tandem with the EMGs. Length changes were matched to the phasic raw EMG by using a st r a i g h t edge to l i n e up the beginning of the phasic EMG to the length measurement. Active shortening was determined to be phasic shortening which coincided with the raw expiratory EMG a c t i v i t y and which u s u a l l y corresponded with the beginning of the downward d e f l e c t i o n of the length tracing, a f t e r the r e s t i n g length plateau (Figure 5). Airfl o w was measured with a pneumotachograph ( F l e i s c h #1) and pressure transducer (Validyne MP45, Medfield, Mass.) attached to the d i s t a l end of the tracheostomy tube and integrated to give t i d a l volume. Volume was c a l i b r a t e d with a 3 l i t e r c a l i b r a t i o n syringe p r i o r to each experiment. Inspiratory (Ti) and expiratory duration (T E) were 30 Figure 5: A representative recording from an awake dog ( l y i n g i n l e f t l a t e r a l decubitus) showing transversus abdominis (TA) length changes and TA EMG a c t i v i t y . The arrows in d i c a t e the r e s t i n g length ( L R L ) of the muscle and the downward d e f l e c t i o n of the length trace a f t e r the arrow (c o i n c i d i n g with i n i t i a t i o n of EMG a c t i v i t y ) i s a c t i v e , phasic shortening ( % L R L ) . 31 LLD 32 determined from the a i r f l o w t r a c i n g on the s t r i p chart recorder (Gould, Model 8000S) us ing a d i g i t i z i n g device (Sigma-Scan, Jande l S c i e n t i f i c , Corte Madera, CA). P r o t o c o l The study p e r i o d began immediately a f t e r the implanta t ion surgery, whi le the dog was s t i l l anesthetized (before f i t t i n g of the j a c k e t ) . At that t ime, r e s t i n g lengths of a l l the abdominal muscles were recorded with the dog l y i n g i n the l e f t l a t e r a l decubitus p o s i t i o n (N=8). Two to three days fo l l owing instrumentation, the awake pro toco l s began. A d i f f e r e n t , randomly assigned protoco l was fol lowed on each study day. The var ious protoco l s are described i n d e t a i l i n the f o l l o w i n g chapters . They inc luded p o s t u r a l changes, expiratory thresho ld l oad ing and C O 2 rebrea th ing . The t o t a l durat ion of study for each dog v a r i e d from two to 8 weeks. At the end of the awake pro toco l s , the dogs were euthanized and b i o p s i e s of muscle containing the c r y s t a l s were performed. The muscle samples were sect ioned, s ta ined by Masson's trichome method and examined under a l i g h t microscope. At the beginning of each experiment, the j a c k e t was removed and a f t e r a p p l i c a t i o n of a t o p i c a l anesthet ic (2% viscous x y l o c a i n e ) , a cuf fed tracheostomy tube (#7) was inser ted through the tracheotomy. The dog was p laced on a p lat form, pos i t ioned i n the l a t e r a l decubitus and c r y s t a l and EMG wires were connected to the sonomicrometer and a m p l i f i e r s , r e s p e c t i v e l y (Figure 6). When the dog was re laxed and breath ing s t a b i l i z e d , c o n t r o l measurements of abdominal muscle r e s t i n g l ength , length changes and EMG a c t i v i t y and t i d a l volume were made.. A l l Figure 6: Example of the experimental setup with the dog l y i n g i n the l e f t l a t e r a l decubitus p o s i t i o n . 34 measurements were recorded on an eight channel, thermal chart recorder (Gould, Model 8000S). A n a l y s i s Paired T-tests were used to t e s t f o r differences i n r e s t i n g lengths of i n d i v i d u a l muscles i n the anesthetized dogs compared to the awake dogs. When comparisons of the amounts of t i d a l shortening among the d i f f e r e n t abdominal muscles were made, a one-way ANOVA was used. A s i g n i f i c a n c e l e v e l of P<0.05 was accepted. Post-hoc multiple comparisons were made using a Tukey multiple comparison t e s t and a s i g n i f i c a n c e l e v e l of P<0.05 accepted. When standard deviations d i f f e r e d s i g n i f i c a n t l y , log transformations of the data were made and the ANOVAS repeated. RESULTS Immediately following implantation, with the dogs anesthetized, r e s t i n g muscle lengths (LRL) were recorded i n the l e f t l a t e r a l decubitus p o s i t i o n (LLD). To compare awake to anesthetized, muscle lengths were measured i n the awake dogs two to four days a f t e r surgery. There were no consistent d i f f e r e n c e s i n the LLD TA, 10 and E0 LRL compared to the LLD lengths of the anesthetized dogs (N=8). However, the RA was approximately 12% longer at r e s t (P<0.05) i n the anesthetized dog (Table I ) . To explore the e f f e c t s of chronic implantation of c r y s t a l s on muscle length, measurements of r e s t i n g muscle lengths were repeated i n the awake dogs (LLD position) at regular i n t e r v a l s , over periods ranging from two to eight weeks. Length measurements obtained within two to four days of implantation (awake dogs) compared c l o s e l y to those 35 made over a per iod of two weeks (Table I) and even a f t e r e ight weeks i n one dog. The c o e f f i c i e n t of v a r i a t i o n of each r e s t i n g length measurement (over a two week p e r i o d ) , w i th in each dog, ranged from 0.05 to 0.12, 0.01 to 0.15, 0.03 to 0.13 and 0.04 to 0.21 for the TA, 10, EO and RA, r e s p e c t i v e l y . The amount of ac t ive shortening (%LRL) of the TA, 10 and EO i n the awake dogs ( L L D p o s i t i o n ) at day 2-4 increased s i g n i f i c a n t l y from e s s e n t i a l l y zero i n the anesthet ized dogs (Table I ) . The smal l amount of mean ac t ive shortening r e f l e c t e d the fac t that ac t ive shortening d i d not occur i n a l l the muscles or i n a l l the dogs. None of the dogs exh ib i t ed ac t ive shortening of the RA. Act ive shortening of the TA, 10 and EO was present on at l ea s t one study day i n 9 out of 9, 5 out of 7 and 2 out of 9 dogs, r e s p e c t i v e l y . Since the standard d e v i a t i o n i n t i d a l shortening d i f f e r e d s i g n i f i c a n t l y among the d i f f e r e n t muscles and was p r o p o r t i o n a l to the mean, l og transformations of the data were performed. The transformed data demonstrated a s i g n i f i c a n t d i f f erence i n shortening among the muscles: the TA and 10 shortened more than the EO or RA (P<0.05) (Table I ) . The amount of t i d a l shortening of each muscle w i t h i n each dog v a r i e d from one recording sess ion to the next over the whole study p e r i o d . A c t i v e shortening was not always present i n those muscles which exh ib i t ed i t . However, there was no cons i s tent trend of e i t h e r an increase or a decrease i n t i d a l shortening over the study p e r i o d . The c o e f f i c i e n t of v a r i a t i o n of each measurement (over a two week p e r i o d ) , w i t h i n each dog, ranged from 0 to 2.4, 0 to 2.0 and 0 to 2.6 for the TA, 10 and EO, r e s p e c t i v e l y . At terminat ion of the experiments, b iops i e s were taken from the muscle around the transducers and f i x e d i n formal in . These were then 36 sectioned, stained by Masson's trichome method and examined h i s t o l o g i c a l l y . Two x-sections from an 10 muscle biopsy, f i x e d with the sonomicrometer transducers i n place, are shown i n Figure 7. The x-sections shown i n Figure 7 are perpendicular to the plane of the muscle f i b e r s and reveal that the 10 i n t h i s example was approximately 5 cm th i c k . The technique of b l u n t l y separating the muscle f i b e r s to place the transducers i n the muscle caused some l o c a l i z e d damage, as shown by the purple coloured area of f i b r o s i s j u s t below the transducer pocket i n Figure 7. Further muscle damage due to the transducers was l i m i t e d to small capsules of f i b r o s i s immediately surrounding the transducers. The muscle t i s s u e between the f i b r o t i c pockets appeared to be normal, healthy t i s s u e l i k e that stained red i n Figure 7. Since the transducers were placed with t h e i r lenses perpendicular to the muscle f i b e r s , f a c i n g i n the d i r e c t i o n i n which the f i b e r s ran, the t h i n capsule of f i b r o s i s around them d i d not i n t e r f e r e with muscle f i b e r length changes between pa i r s of transducers. 37 Table I : Res t ing base l ine lengths (RL) of the abdominal muscles i n anesthet ized compared to awake dogs and a c t i v e shortening (%LRL) i n awake dogs ( l e f t l a t e r a l decubitus p o s i t i o n ) . TA 10 EO RA RL (mm) Anesthet ized 9.71 ± 0 . 9 2 10.3 ± 0 . 9 6 8.89 ± 0 . 6 1 9.92 ± 0 . 6 0 RL (mm) Awake (d2-4) 9.24 ± 0 . 9 5 10.0 ± 0 . 7 9 8.21 ± 0 . 3 1 * 8.72 ± 0 . 8 3 RL (mm) Awake (2 wks) 9.38 ± 0 . 8 2 8.80 ± 1 . 1 7 9.50 ± 0 . 6 0 8.49 ± 0 . 8 2 %LRL LLD Awake * 0.71% ± 0 . 1 6 * * 0.38%* ± 0 . 1 5 * * 0.04% ± 0 . 0 3 0% d2-4 = f i r s t measurements of r e s t i n g length (RL) i n awake dogs 2-4 days a f t e r surgery 2 wks = measurements of RL i n awake dogs 2 weeks a f t e r ^surgery Values are means ± SE A s t e r i s k s (*) i n d i c a t e s i g n i f i c a n t l y d i f f e r e n t from anesthet ized (P<0.05). * * ind ica te s l og transformed data s i g n i f i c a n t l y greater than EO and RA (P<0.05). 38 Figure 7: Two x-sections of inte r n a l oblique muscle stained with Masson's trichome. The muscle biopsy had been f i x e d i n formalin with the sonomicrometer transducer i n place. Eight weeks post implantation. Diameter of pocket i s equal to that of transducer ( 2 . 5 m m ) . The purple area of f i b r o s i s below each capsule which had contained the transducer i s a r e s u l t of the separation of the muscle f i b e r s to form the pocket f or the transducer. The transducers were placed with t h e i r lenses perpendicular to the muscle f i b e r s , i e . facing i n the d i r e c t i o n i n which the f i b e r s ran. The red stained tissue i s healthy muscle. 39 DISCUSSION This study examined the e f f e c t s of anesthesia and chronic implantation of sonomicrometer transducers on abdominal muscle r e s t i n g length and shortening. The r e s u l t s suggest that anesthesia does not e f f e c t the r e s t i n g tone of the TA, 10 or EO muscles i n the l a t e r a l decubitus p o s i t i o n , since the r e s t i n g lengths of these muscles was not d i f f e r e n t i n the awake dog. However, anesthesia d i d r e s u l t i n lengthening of the RA therefore, a change i n muscle tone can not be conc l u s i v e l y discounted. In contrast, anesthesia appears to reduce act i v e shortening i n a l l four abdominal muscles. Neither r e s t i n g length nor shortening changed s i g n i f i c a n t l y over a f i f t e e n day period, i n d i c a t i n g that sonomicrometry i s a useful technique to measure i n vivo abdominal muscle length changes i n chronic animal models. Anesthesia e f f e c t s r e s p i r a t o r y muscle tone but the response depends on the type of anesthetic agent and may also be species s p e c i f i c (15). In the present study, the i n h a l a t i o n anesthetic, halothane, was used. Anesthetic agents such as halothane are thought to induce limb muscle r e l a x a t i o n v i a depression of s p i n a l reflexes (9). There i s also evidence that halothane causes loss of tone of chest wall (which could include r i b cage, abdomen and diaphragm) muscles, i n humans (15) and dogs (17,19). For example, chest wall compliance, which i s determined p a r t l y by muscle tone, increased during halothane anesthesia i n humans (15) and dogs (17,19). In f a c t , diaphragm end-expiratory length was the same i n anesthetized man compared to diaphragm end-expiratory length i n paralysed-anesthetized man, suggesting that diaphragm muscle tone may be abolished by anesthesia alone (6). Furthermore, diaphragm tonic EMG a c t i v i t y was reduced with induction of halothane anesthesia i n man (9). 40 Consistent with loss of chest wall muscle tone during halothane anesthesia, there was a decrease i n the anteroposterior and an increase i n the l a t e r a l diameters of both the r i b cage and abdomen, i n supine subjects (21,22) and, i n the l a t e r a l decubitus p o s i t i o n , an increase i n anteroposterior diameters but no change i n l a t e r a l diameters (21,22). I f anesthesia reduced abdominal muscle tone, or reduced tone and hence lengthened the diaphragm i n the present study, one might p r e d i c t a change i n abdominal muscle r e s t i n g length. The change i n r e s t i n g length would be dependent to some degree on body p o s i t i o n . In the present study, halothane anesthesia had no consistent e f f e c t on TA, 10 or EO muscle r e s t i n g lengths but r e s u l t e d i n a lengthening of the RA. Therefore, these r e s u l t s suggest e i t h e r that halothane has no e f f e c t on TA, 10 and EO abdominal muscle tone or that r e s t i n g tone i s not present i n those muscles i n the q u i e t l y breathing awake dog, at l e a s t not i n the l e f t l a t e r a l decubitus p o s i t i o n . I t i s not p o s s i b l e to determine from the present r e s u l t s whether the lengthening of the RA r e f l e c t s an actual d i f f e r e n c e i n the e f f e c t of anesthetic on the d i f f e r e n t muscles. I t may be that i n dogs, abdominal muscle lengths are not as t i g h t l y coupled to lung volume change below FRC as they are above FRC (8) or there may have been a loss of abdominal muscle tone i n the anesthetized dog which was not apparent because of the dog's p o s i t i o n . In the l a t e r a l decubitus p o s i t i o n , l o s s of abdominal muscle tone might be expected to r e s u l t i n an increase i n the antero-p o s t e r i o r diameter of the abdomen (21,22) and thus, lengthening of a l l the abdominal muscles, but p a r t i c u l a r l y the more v e r t i c a l l y oriented muscles (EO and RA) . The RA however, due to i t s f i b e r o r i e n t a t i o n and p o s i t i o n i n the midline of the anterior abdominal wall would be most 41 a f f e c t e d by outward displacement of the anterior wall and would lengthen most, as was found. The f i n d i n g that the RA was s i g n i f i c a n t l y longer during anesthesia compared to the awake state, suggests there was. a decrease i n RA muscle tone during anesthesia. I t follows that i f tonic a c t i v i t y was present i n the RA, i t would l i k e l y be present i n the TA, 10 and EO as w e l l . O v e r a l l , there may simply be more tone i n the RA during wakefulness, or the passive r e l a x a t i o n of tone i n a l l the abdominal muscles was manifest most i n the RA. There does not appear to be any other studies i n which abdominal muscle r e s t i n g length i n anesthetized dogs i s compared to awake dogs. However, a study of the e f f e c t s of pentobarbital sodium anesthesia on diaphragm r e s t i n g length found the r e s t i n g length of the c r u r a l diaphragm ( l a t e r a l decubitus position) to be longer i n the anesthetized dog compared to the awake dog (4). I t was concluded that a reduction i n muscle tone during anesthesia was responsible f o r lengthening of the diaphragm. Furthermore, during halothane anesthesia, supine dogs showed a decrease i n FRC (19) presumably i n conjunction with a lengthening of the diaphragm, perhaps as a r e s u l t of a loss i n tone. In the l a t e r a l p o s i t i o n (as i n the present study), lengthening of the c r u r a l diaphragm might lead to a s l i g h t passive shortening of the abdominal muscles on the nondependent side. Conversely, the abdominal muscles on the dependent side would be more l i k e l y to lengthen as a r e s u l t of g r a v i t a t i o n a l forces. In th i s study, r e s t i n g length measurements were made on the nondependent side f o r the TA and 10 and the dependent side f o r the EO and RA and except f o r the RA, there were no s i g n i f i c a n t differences between the anesthetized lengths compared to the awake lengths (Table I ) . Therefore, the present study does not show 42 convincing evidence of a decrease i n FRC or diaphragm tone with anesthesia i n the l a t e r a l decubitus p o s i t i o n . The p o s i t i o n may be a fa c t o r , since although there was a decrease i n FRC i n supine dogs (19), no change i n FRC was found i n prone dogs (17). In addition, lengthening of the diaphragm ( a t t r i b u t e d to loss of diaphragm tone) occurred during pentobarbital sodium anesthesia (4), which may have d i f f e r e n t e f f e c t s than halothane. A l l the abdominal muscles showed a greater amount of active shortening i n the awake compared to the anesthetized dog, i n which shortening was n e g l i g i b l e (LLD p o s i t i o n ) . The TA and 10 shortened more than the EO or RA, i n agreement with previous reports from supine, anesthetized dogs (8,12) and thus, supporting the hypothesis that the i n t e r n a l abdominal muscles are more active i n breathing than the external muscles. Although the TA and 10 had s i g n i f i c a n t l y greater a c t i v i t y at r e s t than the EO or RA, the amount of shortening was less than one percent of the r e s t i n g length. The amount of shortening i n the TA and EO i s less than that reported i n some studies of supine, anesthetized dogs (12,13), but greater than that observed by Leevers and Road (8). V a r i a t i o n i n degree of active shortening reported among these previous studies could be due to factors such as dif f e r e n c e s i n measurement technique or l e v e l of anesthesia. The methods f o r measuring shortening d i f f e r e d between the studies, i n that both passive (due to e l a s t i c r e c o i l ) and active (EMG present) shortening were included as acti v e shortening i n the studies reporting more shortening i n anesthetized dogs (12,13). This measurement technique would lead to reports of a greater amount of shortening than one i n which j u s t a c t i v e (EMG) shortening was measured (8). In addition, barbituates, such as 43 pentobarbital sodium, appear to reduce phasic, abdominal muscle a c t i v a t i o n i n a dose r e l a t e d manner (15). For example, pentobarbital sodium anesthesia r e s u l t e d i n a dose dependent reduction i n phasic, expiratory abdominal nerve a c t i v i t y i n cats (5). Thus, reports of " l i g h t l y anesthetized" supine dogs with phasic EMG a c t i v i t y and shortening of the TA at r e s t (12), i n contrast to reports that no r e s t i n g abdominal muscle shortening was found i n a s i m i l a r preparation (8), perhaps r e f l e c t s a d i f f e r e n c e i n the l e v e l of anesthesia. The d i s p a r i t y between the amount of shortening i n the anesthetized dogs (12,13) and the awake dogs i n the present study may also have been due a differ e n c e i n p o s i t i o n . Unlike the l a t e r a l decubitus posture, the supine posture i s not a natural one for the dog and may therefore, r e s u l t i n d i f f e r e n t abdominal muscle recruitment l e v e l s or patterns. Abdominal muscle a c t i v i t y i n the present study may have been underestimated i n comparison to awake non-tracheostomized dogs. The upper airway i s thought to produce expiratory braking and hence contribute to abdominal muscle a c t i v a t i o n (16). Since the dogs breathed through a tracheostomy, the upper airway was bypassed and expiratory braking may have been reduced. At two weeks, the e f f e c t s of chronic implantation were studied (Table 1). There could have been sca r r i n g (as a foreign body reaction) which would have changed muscle r e s t i n g length and shortening. A l t e r n a t i v e l y , shortening might have increased from the immediate post-operative period because of r e f l e x i n h i b i t o r y e f f e c t s consequent to surgery. However, chronic implantation of sonomicrometer transducers i n the abdominal muscles d i d not e f f e c t abdominal muscle r e s t i n g length or shortening over the two week experimental period. These r e s u l t s d i f f e r 44 from previous reports of chronic sonomicrometer implantat ion i n the diaphragm (1,20). In sheep (20), both diaphragm r e s t i n g length and t i d a l shortening were reduced on postoperat ive day one compared to postoperat ive day 28 and i n dogs (1), diaphragm t i d a l shortening gradua l ly increased and s t a b i l i z e d three weeks a f t e r implanta t ion . However, the diaphragm dysfunct ion which occurred i n both these s tudies was probably not due to a d i r e c t e f f ec t of implanta t ion of the transducers on the diaphragm. I t was more l i k e l y a r e f l e x i n h i b i t i o n of the diaphragm r e s u l t i n g from the s u r g i c a l procedure: a laparotomy (1) and a thoracotomy (20), s ince both procedures have been shown to e f f e c t diaphragm a c t i v i t y (3). Laparotomy has a l so been shown to e f f e c t abdominal muscle a c t i v a t i o n i n anesthet ized dogs, r e s u l t i n g i n a decrease i n TA but no change or an increase i n EO EMG a c t i v i t y (3). Since i n the present study, implantat ion of the transducers i n the muscles was v i a a s u p e r f i c i a l approach which d i d not r e q u i r e a laparotomy, s i m i l a r e f fec t s on abdominal muscle a c t i v i t y would not be expected, but minor t i s sue damage could r e s u l t . However, acute implantat ion of transducers i n the diaphragm d i d not impair diaphragm shortening despi te some l o c a l muscle f i b e r damage (10). Therefore , the r e s u l t s suggest that implantat ion per se does not e f f e c t abdominal muscle r e s t i n g length or shortening. Examination of muscle sect ions which had contained the transducers, revealed t h i n f i b r o t i c capsules between areas of heal thy muscle t i s sue . S i m i l a r f ind ings were reported for the diaphragm (1). In conc lus ion , the r e s u l t s i n d i c a t e that sonomicrometry i s a use fu l method for measuring i n v ivo muscle length changes i n long-term, chronic animal models. 45 REFERENCES 1. Easton, P . A . , J .W. F i t t i n g , R. Arnoux, A . Gueraty and A. Grass ino . Recovery o f diaphragm funct ion a f t er laparotomy and chronic sonomicrometer implantat ion . J . A p p l . P h v s i o l . 66(2): 613-621, 1989. 2. Farkas , G . A . , R . E . Baer, M. Estenne and A. De Troyer . Mechanical r o l e of e x p i r a t o r y muscles during breathing i n upr ight dogs. J .  A p p l . P h v s i o l . 64(3): 1060-1067, 1988. 3. Farkas , G .A . and A. De Troyer . Ef fec t s of midl ine laparotomy on e x p i r a t o r y muscle a c t i v a t i o n i n anesthet ized dogs. J . A p p l . P h v s i o l . 67(2): 599-605, 1989. 4. F i t t i n g , J . W . , P . A . Easton, R. Arnoux, A. Gueraty and A. Grass ino . E f f e c t of anesthes ia on canine diaphragm length. Anesthes io logy . 66: 531-536, 1987. 5. F r e g o s i , R . F . , S . L . Knuth, D .K. Ward and D. B a r t l e t t . J r . Hypoxia i n h i b i t s abdominal exp ira tory nerve a c t i v i t y . J . A p p l . P h v s i o l . 63(2): 211-220, 1987. 6. Froese, A . B . and A . C . Bryan. Ef fec t s of anesthesia and p a r a l y s i s on diaphragmatic mechanics i n man. Anesthesiology. 41: 242-255, 1974. 7. Greer , J . J . and R . B . S t e i n . Length changes of i n t e r c o s t a l muscles dur ing r e s p i r a t i o n i n the ca t . Resp ir . P h v s i o l . 78: 309-322, 1989. 8. Leevers , A . M . and J . D . Road. Mechanical response to h y p e r i n f l a t i o n of the two abdominal muscle l a y e r s . J . A p p l . P h y s i o l . 66(5): 2189-2195, 1989. 9. M u l l e r , N . , G. V o l g y e s i , L . Becker, M.H. Bryan and A . C . Bryan. Diaphragmatic muscle tone. J . A p p l . P h y s i o l . 47: 279-284, 1979. 10. Newman, S . , J . Road, F . Bellemare, J . P . C l o z e l , C M . Lavigne and A. Grass ino . Resp ira tory muscle length measured by sonomicrometry. J .  A p p l . P h v s i o l . 56: 753-764, 1984. 11. Ninane, V . and A. De Troyer . Mechanics of paras terna l s and t r i a n g u l a r i s s t e r n i i n upr ight vs . supine dogs. J . A p p l . P h y s i o l . 65(1): 452-459, 1988. 12. Ninane, V . , J . J . G i l m a r t i n and A. De Troyer . Changes i n abdominal muscle length during breath ing i n supine dogs. R e s p i r . P h v s i o l . 73: 31-42, 1988. 13. O l i v e n , A. and S . G . Kelsen . E f f e c t of hypercapnia and PEEP on exp ira tory muscle EMG and shortening. J . A p p l . P h y s i o l . 66(3): 1408-1413, 1989. 14. P a v l i n , E . G . , and T . F . Hornbein. Anesthesia and the c o n t r o l of 46 v e n t i l a t i o n . In: Handbook of Physiology Sect ion 3 : The Resp iratory  System. V o l . I I I . P . T . Macklem, and J . Mead (eds) . 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EFFECTS OF POSTURE ON ABDOMINAL MUSCLE LENGTHS INTRODUCTION The four muscles of the v e n t r a l abdominal wall (TA, 10, EO and RA) are thought to be the primary contributors to e x p i r a t i o n and the defence of lung volume. I t has been suggested that increasing abdominal muscle a c t i v a t i o n i n upright postures helps to maintain FRC (13), optimize diaphragm length (17) and also a s s i s t s i n s p i r a t i o n by producing an increase i n passive outward r e c o i l of the chest wall at end-expiration (14,16). In addition, there i s i n d i r e c t evidence that abdominal muscle c o n t r i b u t i o n to t i d a l volume increases to more than 50% when anesthetized dogs are moved from supine to upright (14). Increased abdominal muscle a c t i v a t i o n following assumption of upright postures i s thought to be mediated by vagal reflexes (9,13) and abdominal muscle segmental r e f l e x e s (6) due to g r a v i t a t i o n a l e f f e c t s on the chest w a l l . The abdominal muscles exhibit both increased tonic and phasic, expiratory a c t i v i t y i n upright postures i n man (7,11) and anesthetized (9,18) and awake dogs (8,17). However, both tonic and phasic, expiratory abdominal muscle a c t i v i t y were generally assessed by electromyography (8,9,18) or i n d i r e c t l y v i a measurements of g a s t r i c pressure (5,17) or abdominal diameters (11). A l i m i t a t i o n of these methods i s that they do not allow i n t e r p r e t a t i o n of mechanical events i n terms of abdominal muscle r e s t i n g length and shortening. In p a r t i c u l a r , abdominal muscle tonic a c t i v i t y i s poorly described by ton i c EMG a c t i v i t y , since tonic EMG a c t i v i t y i s measured by inference from the degree of background noise. In addition, although phasic a c t i v i t y may be described by the EMG, increases i n phasic EMG a c t i v i t y w i l l not 48 n e c e s s a r i l y produce increased t i d a l length changes because the amount of shortening w i l l be in f luenced by the muscle's i n i t i a l length and the a f t e r l o a d on the muscle. Indeed, g r a v i t a t i o n a l forces would tend to increase abdominal muscle a f t e r l o a d and i n i t i a l length i n u p r i g h t postures . Furthermore, abdominal muscle ton ic a c t i v i t y i s in f luenced by o r i e n t a t i o n i n the g r a v i t a t i o n a l f i e l d and hence posture . An increase i n t on i c a c t i v i t y might be r e f l e c t e d by a decrease i n i n i t i a l muscle length as a r e s u l t of a c t i v a t i o n of muscle propr ioceptors . However, the e f f ec t s of changing body p o s i t i o n on abdominal muscle r e s t i n g ( i n i t i a l ) l ength and shortening has not been prev ious ly inves t iga ted i n awake dogs. The use of sonomicrometry provides a method to measure i n v i v o abdominal muscle r e s t i n g length and t i d a l length changes (3,19,23). A n a l y s i s of EMG data reveals that the abdominal muscles are d i f f e r e n t i a l l y r e c r u i t e d i n upr ight postures (8,18). This d i f f e r e n t i a l recruitment may be r e l a t e d to the d i s t i n c t r e l a t i o n s h i p s and attachments to the r i b cage, as w e l l as the d i f f e r e n t or i enta t ions to the abdominal compartment of each muscle. However, the mechanical consequences of d i f f e r e n t i a l recrui tment i n terms of abdominal muscle r e s t i n g length and a c t i v e , exp ira tory shortening have not been prev ious ly reported . We hypothes ized that the abdominal muscles would show more phasic exp ira tory a c t i v i t y i n the s i t t i n g (SIT) and standing (on a l l fours) (STAND) p o s i t i o n s compared to the l a t e r a l decubitus p o s i t i o n and that the r e s t i n g lengths i n the d i f f e r e n t pos i t i ons would be dependent on i n d i v i d u a l abdominal muscle f i b e r o r i e n t a t i o n , p o s i t i o n i n the abdominal w a l l and r e l a t i v e c o n t r i b u t i o n to breath ing . We fur ther hypothesized that the i n t e r n a l abdominal muscle layer (TA and 10) would be more ac t ive than the ex terna l muscle layer (EO and RA). Therefore , 49 the objec t ives of t h i s study were to assess the e f f ec t s of posture on phas ic and ton ic muscle a c t i v i t y of i n d i v i d u a l abdominal muscles by measuring shortening and r e s t i n g length i n d i f f e r e n t postures i n awake dogs. METHODS Five tracheotomized, female mongrel dogs were s u r g i c a l l y implanted with sonomicrometer transducers and f ine wire EMG e lec trodes i n each of the four abdominal muscles as described i n Chapter II (Figure 4 ) . Mea surement s Abdominal muscle r e s t i n g lengths and shortening were measured with the sonomicrometer transducers (see Chapter I I ) . As descr ibed i n Chapter I I , the r e s t i n g length of the abdominal muscles was def ined as e i t h e r the end-expiratory length, i f no a c t i v e , e x p i r a t o r y (phasic) shortening was present , or the length immediately preceding a c t i v e , phasic shortening (plateau ind ica ted by arrows i n Figure 8 ) , regardless of the dog's p o s i t i o n , and was termed LRL- The three p o s i t i o n s s tud ied were the l e f t l a t e r a l decubitus (LLD), s i t t i n g on the rear haunches with the f r o n t legs and chest v e r t i c a l (SIT) and standing on a l l four legs (STAND). The change i n LRL between the LLD p o s i t i o n and the SIT or STAND p o s i t i o n s was expressed as a percent change from LLD L R L . Change from the r e s t i n g l ength , lengthening or shortening, dur ing t i d a l b r e a t h i n g , was expressed as a percentage change from the i n i t i a l r e s t i n g length (%LRL) i n that p o s i t i o n . Ac t ive shortening was determined to be that c o i n c i d i n g with raw exp ira tory EMG a c t i v i t y . 50 Figure 8: A representa t ive recording from an awake dog l y i n g i n l e f t l a t e r a l decubitus p o s i t i o n (top panel) and standing on a l l fours (bottom panel) showing transversus abdominis (TA) length changes and TA EMG a c t i v i t y . The arrows i n d i c a t e the r e s t i n g length (LRL) o f the muscle and the downward d e f l e c t i o n of the length trace a f t e r the arrow ( co inc id ing with i n i t i a t i o n o f EMG a c t i v i t y ) i s a c t i v e , phasic shortening (%LRL) . The LRL i n the standing p o s i t i o n i s 26% shorter than LRL i n the L L D p o s i t i o n . Note the background EMG a c t i v i t y i n the standing p o s i t i o n , perhaps r e f l e c t i n g ton ic EMG a c t i v i t y (bottom p a n e l ) . 5 s e c . 52 The electromyographic s igna l s from the implanted f ine wire e lectrodes were a m p l i f i e d and f i l t e r e d below 30Hz and above lKHz (Grass, model P511). The EMG s igna l s were recorded i n p a i r s with abdominal muscle l ength . A i r f l o w was measured with a pneumotachograph ( F l e i s c h #1) and pressure transducer ( ± l c m H 2 0 , Val idyne MP45, M e d f i e l d , Mass.) at tached to the d i s t a l end of the tracheostomy tube and in tegrated to give t i d a l volume. I n s p i r a t o r y (Ti) and exp ira tory durat ion (TE) were determined from the flow t r a c i n g on the chart recorder us ing a d i g i t i z i n g device (Sigma-Scan, Jandel S c i e n t i f i c , Corte Madera, CA). Protoco l Each dog was s tud ied a number of times and the t o t a l d u r a t i o n of t e s t i n g for each dog v a r i e d from two to e ight weeks. A d i f f e r e n t , randomly assigned p r o t o c o l was fol lowed on each study day. The var ious protoco l s inc luded p o s t u r a l changes, exp ira tory thresho ld load ing and C0 2 rebreath ing . E x p i r a t o r y threshold loading and C0 2 rebreath ing are descr ibed i n d e t a i l i n the fo l lowing chapters . The p r o t o c o l for p o s t u r a l changes i s descr ibed i n the present chapter. At the beginning of each awake p r o t o c o l , a cuf fed tracheostomy tube (#7) was i n s e r t e d through the tracheotomy. The dog was p laced on a p la t form, p o s i t i o n e d i n the l a t e r a l decubitus and c r y s t a l and EMG wires were connected to the sonomicrometer and a m p l i f i e r s , r e s p e c t i v e l y . When the dog was re laxed and breath ing s t a b i l i z e d , c o n t r o l measurements of abdominal muscle r e s t i n g length , length changes and EMG a c t i v i t y and t i d a l volume were made. The dog was then moved to the SIT or STAND p o s i t i o n and once breath ing s t a b i l i z e d , measurements were repeated. 53 T i d a l volume and t iming parameters i n a l l three p o s i t i o n s were obtained from only three dogs, due to d i s lodg ing of the pneumotachograph dur ing p o s i t i o n changes. A n a l y s i s Measurements of abdominal muscle t i d a l shortening were averaged over f i v e breaths for each p o s i t i o n , for each t r i a l of p o s t u r a l changes. I n d i v i d u a l muscle shortening from a l l the t r i a l s for each dog was averaged and then the mean t i d a l shortening per abdominal muscle, i n each p o s i t i o n , was der ived . To tes t for d i f ferences i n the amounts of abdominal muscle t i d a l shortening i n the three p o s i t i o n s , a one-way ANOVA was performed. A one-way ANOVA was a l so performed to t e s t for d i f f erences i n the amount of t i d a l shortening among the d i f f e r e n t abdominal muscles, i n each p o s i t i o n . A s i g n i f i c a n c e l e v e l o f P<0.05 was accepted. Post hoc mul t ip l e comparisons were made us ing a Tukey m u l t i p l e comparison t e s t . When the standard dev ia t ions i n the measurements were s i g n i f i c a n t l y d i f f e r e n t and p r o p o r t i o n a l to the mean, log transformations of the data were made and the ANOVAs repeated. The r e s t i n g lengths i n the LLD p o s i t i o n were used as the reference lengths . A s h i f t i n the dog's p o s i t i o n to SIT or STAND was made randomly, but the change i n r e s t i n g length was always c a l c u l a t e d as the change when moving from LLD to SIT or LLD to STAND. The change i n r e s t i n g length with s h i f t i n p o s i t i o n from LLD to SIT or LLD to STAND was determined for each t r i a l , and the average change i n LRL wi th s h i f t i n p o s i t i o n , for a l l the t r i a l s , was c a l c u l a t e d for each dog and then the mean change i n LRL per p o s i t i o n change was determined. T i d a l volume and t iming parameters were also compared between LLD and SIT, and LLD 54 and STAND i n three dogs. Comparisons between LLD and SIT, and LLD and STAND were made us ing the Student's T - t e s t and a s i g n i f i c a n c e l e v e l of P<0.05. RESULTS Both phas ic and tonic r e s p i r a t o r y a c t i v i t y of the abdominal muscles, r e f l e c t e d by degree of ac t ive shortening (%LRL) and changes i n r e s t i n g length (LRL) r e s p e c t i v e l y , were a f fec ted by movement and changes i n posture . The e f f ec t s of moving from LLD to STAND on TA %LRL and LRL are demonstrated by a t y p i c a l t r a c i n g shown i n Figure 8. The amount of t i d a l shortening of each muscle v a r i e d between t r i a l s . However, there was no cons i s tent change over the durat ion of each study p e r i o d and the abdominal muscles a l l showed the same trend from one t r i a l to the next: that i s , shortening e i t h e r increased or decreased i n a l l the muscles ( i n which shortening was present ) . A c t i v e , exp iratory shortening i n each of the abdominal muscles except the RA was s i g n i f i c a n t l y greater i n the SIT and STAND p o s i t i o n s compared to the LLD (Figure 9) . The c o e f f i c i e n t of v a r i a t i o n i n shortening per muscle, w i th in each dog, ranged from 0.4 to 2.4, 0.07 to 2.0 and 0 to 2.5 i n the LLD, from 0 to 1.4, 0 to 1.1 and 0 to 2.0 i n SIT and from 0 to 0.6, 0 to 1.1 and 0 to 2.0 i n STAND for the TA, 10 and E0, r e s p e c t i v e l y . The RA showed no phasic a c t i v i t y i n any p o s i t i o n , and the TA and 10 had greater ac t ive shortening i n a l l three postures than the E0 (Figure 9) . There were changes i n the r e s t i n g length of each muscle when moving from the LLD p o s i t i o n to e i ther the SIT or STAND p o s i t i o n s (Figures 10 and 11) . The TA was most c o n s i s t e n t l y e f fec ted by p o s i t i o n changes; a longer r e s t i n g length when moving from LLD to the s i t t i n g Figure 9: Bar graph showing ac t ive shortening of the TA, 10 and EO i n the l e f t l a t e r a l decubitus (LLD), s i t t i n g (SIT) and standing (STAND) postures . The shortening i s expressed as a percentage of the r e s t i n g length i n each p o s i t i o n . As ter i sks (*) i n d i c a t e s i g n i f i c a n t l y l e ss shortening of EO than TA and 10 i n a l l three p o s i t i o n s (P<0.05). N=5. 56 p o s i t i o n and a shorter r e s t i n g length when moving from LLD to s tanding . The other three muscles were more v a r i a b l e i n t h e i r responses to p o s t u r a l change however, the changes were cons i s tent w i t h i n each dog. Moving from LLD to SIT, the 1 0 had a shorter r e s t i n g length i n 3 out of 5 dogs ( 1 5 to 25%) and a longer r e s t i n g length i n 2 / 5 ( 3 to 5%); the EO had a shorter r e s t i n g length 3 / 5 ( . 9 to 8.6%) and a longer r e s t i n g length i n 2 / 5 ( 1 4 to 25%); and the RA had a shorter r e s t i n g length 3 / 5 ( 4 . 7 to 12.3%) and a longer r e s t i n g length i n one dog ( 1 2 . 6 % ) . Moving from LLD to STAND, the 1 0 a longer r e s t i n g length i n 3 out of 5 dogs ( 4 to 22.5%) and a shorter r e s t i n g length i n two (6 to 7 . 6 % ) ; the EO a longer r e s t i n g length i n 4 of 5 ( 4 to 24.6%) and shorter r e s t i n g length i n one (3%); and the RA a longer r e s t i n g length i n 4 of 5 ( 1 . 7 to 25.8%) and shorter r e s t i n g length i n one ( 1 5 . 7 % ) . The mean change i n EO LRL was to a s i g n i f i c a n t l y longer length i n the standing p o s i t i o n compared to LLD (lengthened by 9 . 2 ± 4 . 6 % (mean ± SE) ) . T i d a l volume and t iming parameters measured i n the SIT and STAND p o s i t i o n s were compared to measurements i n the LLD p o s i t i o n , i n three dogs (Table I I ) . There was no d i f ference i n exp ira tory d u r a t i o n (T E ) i n the three p o s i t i o n s but VT was s i g n i f i c a n t l y greater i n STAND and i n s p i r a t o r y d u r a t i o n (Ti) and the i n s p i r a t o r y f r a c t i o n of the t o t a l breath durat ion ( T I / T T O T ) were s i g n i f i c a n t l y greater i n SIT and STAND compared to LLD. I n s p i r a t o r y dr ive as r e f l e c t e d by V T / T i was not s i g n i f i c a n t l y d i f f e r e n t i n the three pos i t i ons ( 2 5 6 ± 1 7 i n LLD, 2 5 4 ± 1 0 i n SIT and 2 2 1 ± 1 2 i n STAND). 30 r 20 10 TA Q io RA SIT -10 -20 -30 Figure 10: Box p l o t s of the change i n r e s t i n g length (LRL) wi th s h i f t i n posture from l e f t l a t e r a l decubitus ( L L D ) to s i t t i n g (SIT) . The length i s expressed as a percent of the L L D length (100%). The boxes are the 95% confidence i n t e r v a l , v e r t i c a l l ine s ind ica te the range and h o r i z o n t a l l i n e s are the mean. A s t e r i s k s (*) i n d i c a t e s i g n i f i c a n t l y d i f f e r e n t from L L D LRL (P<0.05). N=5. 58 Figure 11: Box p l o t s of the change i n r e s t i n g length (LRL) wi th s h i f t i n posture from l e f t l a t e r a l decubitus (LLD) to standing (STAND). The length i s expressed as a percent of the LLD length (100%). The boxes are the 95% confidence i n t e r v a l , v e r t i c a l l i n e s ind icate the range and h o r i z o n t a l l i n e s are the mean. A s t e r i s k s (*) i n d i c a t e s i g n i f i c a n t l y d i f f e r e n t from LLD LRL ( P < 0 . 0 5 ) . N=5 . 59 Table I I : T i d a l volume and t iming parameters i n three postures . P o s i t i o n LLD SIT STAND Ti(secs) 1.251.02 1.4711.0* 1 .671.06* T E (secs) 3.331.35 2.751.04 2.751.15 T I / T I O T 0.281.02 0.351.02* 0.391.01* V T (mls) 297125 348126 374114* A s t e r i s k s (*) i n d i c a t e s i g n i f i c a n t l y d i f f e r e n t from LLD (P<0.05). N-3. 60 DISCUSSION The r e s u l t s of t h i s study show that abdominal muscle ac t ive e x p i r a t o r y shortening i s greater i n the s i t t i n g (SIT) and standing (STAND) p o s i t i o n s , compared to the l a t e r a l decubitus, i n awake dogs. In a d d i t i o n , the r e s u l t s support the hypothesis that the i n t e r n a l abdominal muscle l a y e r ( transversus abdominis and i n t e r n a l obl ique) i s more ac t ive i n breath ing than the ex terna l muscle layer (external obl ique and rectus abdominis) . Furthermore, the cons is tent change i n r e s t i n g length of the transversus abdominis (TA) to a shorter length when moving from LLD to STAND suggests an increase i n tonic a c t i v i t y and i n d i c a t e s , the importance of the TA i n defending end-expiratory lung volume and mainta in ing diaphragm length . The three p o s i t i o n s examined i n the present study are n a t u r a l postures for awake dogs. Other studies of pos tura l e f fec t s i n awake dogs have used s i m i l a r SIT and STAND p o s i t i o n s , whereas the reference p o s i t i o n was prone i n one study (8) and r i g h t l a t e r a l decubitus i n the other (17), compared to LLD i n th i s study. In contras t , p o s t u r a l changes i n s tudies of anesthet ized dogs general ly involve t i l t i n g from the supine p o s i t i o n to the headup (14,20,24) or headdown p o s i t i o n s (24) or s h i f t i n g from the supine to prone-cradled (21,24) or prone-suspended (16). The v a r i e t y of postures u t i l i z e d i n studies of anesthet ized and awake dogs, and man makes comparisons d i f f i c u l t . However, i t has been shown i n anesthet ized dogs that diaphragm length i n the l a t e r a l decubitus p o s i t i o n i s c lose to supine FRC diaphragm length (21,24) and thus, LLD i s a reasonable reference p o s i t i o n . The s i t t i n g p o s i t i o n i n awake dogs i s s i m i l a r to the t i l t e d headup p o s i t i o n i n anesthet ized dogs, and the s i t t i n g and standing pos i t ions i n man. The standing 61 p o s i t i o n of awake dogs approximates the prone p o s i t i o n s of anesthet ized dogs and man, to some degree, but the g r a v i t a t i o n a l e f f ec t s on the chest w a l l and abdominal contents would most l i k e l y be grea ter . In a d d i t i o n , c o n t r a c t i o n of the s t a b i l i z i n g muscles of the p e l v i s and shoulder g i r d l e may e f f e c t abdominal muscle length and a c t i v a t i o n . Although d i f f erences i n posture among the var ious studies makes comparisons d i f f i c u l t , i t i s c l e a r that both the s i t t i n g (SIT) and standing (STAND) p o s i t i o n s of awake dogs would increase the g r a v i t a t i o n a l e f f ec t s on the abdominal contents and hence, e f f e c t abdominal muscle length and a c t i v i t y compared to the LLD p o s i t i o n . The increase i n abdominal muscle a c t i v e , e x p i r a t o r y shortening i n the s i t t i n g and standing pos i t i ons found i n the present study i s cons i s tent with previous reports of increased phas ic EMG a c t i v i t y i n u p r i g h t anesthet ized dogs (9,13) and awake, s i t t i n g and s tanding dogs (8). More s i g n i f i c a n t l y however, the present r e s u l t s conv inc ing ly demonstrate that the increased phasic EMG a c t i v i t y has a measurable mechanical e f f e c t i n terms of abdominal muscle shortening . A c t i v e e x p i r a t o r y shortening of the abdominal muscles would be an important compensatory mechanism to counter the detr imenta l e f f ec t s that upr ight and prone-crad led postures have on diaphragm length (20,21,24). Indeed, i t was r e c e n t l y reported that diaphragm r e s t i n g end-expiratory length was unchanged from l a t e r a l decubitus end-expiratory length i n awake standing dogs and s l i g h t l y reduced i n awake s i t t i n g dogs (17). In c o n t r a s t , prone and upr ight anesthet ized dogs had much greater reduct ions i n diaphragm end-expiratory length (compared to supine) (13,24). The r e l a t i v e preservat ion of diaphragm end-expiratory length i n the awake dogs was a t t r i b u t e d to phasic exp ira tory c o n t r a c t i o n of the 62 abdominal muscles, assessed from abdominal pressure swings (17). In a d d i t i o n to mainta in ing diaphragm length at a s i m i l a r operat ing length and hence, p r e s e r v i n g i t s pressure generating a b i l i t y (21,24), the abdominal muscles a l so a s s i s t i n i n s p i r a t i o n by r e l a x i n g at end-e x p i r a t i o n . Indeed, i t has been suggested that t h i s mechanism o f the abdominal muscles contr ibuted more than 50% of the t i d a l volume i n anesthet ized dogs t i l t e d towards upright (14). Further support for the important p o s t u r a l r o l e of the abdominal muscles i s prov ided by the t i d a l volume and t iming r e s u l t s . The greater V T i n the s tanding p o s i t i o n i s cons i s tent with previous reports i n awake dogs (17). D i f f e r e n t l i n e s of evidence suggest that the increase i n V T when moving to u p r i g h t postures i s a r e s u l t of an increase i n e x p i r a t o r y muscle a c t i v i t y . F i r s t l y , when abdominal muscle a c t i v i t y was reduced by vagotomy i n anesthet ized dogs, V T decreased with t i l t i n g to u p r i g h t , whereas i n the i n t a c t dogs there was a trend to an increase i n VT (14). Secondly, i n the present study there was no apparent increase i n i n s p i r a t o r y d r i v e , s ince V J / T J was the same i n the three p o s i t i o n s and i n another s i m i l a r study, diaphragm EMG d i d not change s i g n i f i c a n t l y i n the s i t t i n g and standing p o s i t i o n s compared to l a t e r a l decubitus (17). Abdominal muscle a c t i v i t y may not only be p a r t i a l l y respons ib le for the l a r g e r V T , but may a lso provide a greater c o n t r i b u t i o n to V T i n the standing p o s i t i o n compared to the l a t e r a l decubitus . Ti was greater i n the s i t t i n g and standing p o s i t i o n s compared to the LLD but t h i s was i n s p i r a t o r y flow d u r a t i o n not neural Ti (EMG a c t i v i t y ) . I t has been shown i n awake standing dogs that a s i g n i f i c a n t p o r t i o n of i n s p i r a t o r y flow (19%) i s pass ive i . e . precedes the onset of diaphragm EMG (25). Thus r e l a x a t i o n of phasic exp ira tory muscle a c t i v i t y can c o n t r i b u t e to 63 V T by producing pass ive outward r e c o i l of the chest w a l l before ac t ive i n s p i r a t i o n . The greater amount of a c t i v e , exp ira tory shortening of a l l the abdominal muscles i s a mechanical consequence that might be expected from the increased phasic abdominal EMG a c t i v i t y reported for upr ight man (7,12,27) and awake (8) and anesthet ized dogs (9,13). However, both abdominal muscle i n i t i a l length and a f t e r l o a d w i l l be e f f ec ted by changes i n posture (1). When the i n i t i a l length of a muscle or the a f t e r l o a d on a muscle i s changed, increased EMG a c t i v i t y i s not n e c e s s a r i l y assoc ia ted with increased shortening . For example, when anes thet ized dogs were t i l t e d towards upr ight there was an increase i n a f t e r l o a d , a decrease i n diaphragm i n i t i a l length and a corresponding decrease i n shortening , but no change i n diaphragm EMG a c t i v i t y (20). Converse ly , i n another study of anesthet ized dogs, t i l t i n g towards u p r i g h t r e s u l t e d i n a progress ive increase i n p a r a s t e r n a l i n t e r c o s t a l EMG a c t i v i t y but a decrease i n phasic shortening (22). In that study, the decrease i n shortening was assoc iated with an increase i n i n i t i a l l ength . In the present study, abdominal muscle i n i t i a l l ength v a r i e d cons iderab ly . Indeed, TA i n i t i a l length decreased by approximately 12% i n the standing p o s i t i o n . That amount of decrease i n i n i t i a l length cou ld inf luence shortening, depending on the length at which tens ion generat ion would be maximal (L 0 ) for the TA. Unfortunate ly , the a v a i l a b l e data on abdominal muscle c o n t r a c t i l e proper t i e s i s l i m i t e d . In supine, anesthet ized dogs, the i n v ivo r e s t i n g length at FRC i n r e l a t i o n to L 0 was reported to be 83% for the EO and 105% for the RA (15) and although there are no publ i shed data on TA Lo, TA supine FRC length was found to be approximately 74% of L 0 (G. Farkas, personal communication). 64 The EO and TA would there fore , be on a more optimal p o r t i o n of t h e i r l ength- tens ion curves i f LRL were longer i n the s i t t i n g or s tanding p o s i t i o n s . As was found, i n 4 of 5 dogs EO LRL lengthened when moving from LLD to STAND (Figure 11) and TA LRL lengthened when moving from LLD to SIT (Figure 10). I f i t i s assumed that the increase i n EO and TA LRL would optimize muscle l ength , then the inference i s that t h e i r LRL i n the l a t e r a l decubitus p o s i t i o n i s at a shorter length i n r e l a t i o n to L Q , s i m i l a r to supine EO (15) and TA length. However, i n the s tanding p o s i t i o n , TA LRL was cons iderably reduced. Such shortening o f TA LRL i n the standing p o s i t i o n i s not cons is tent with opt imiz ing TA l ength , s ince i t would then be at an even shorter length than LLD TA LRL and there fore , TA tens ion generating capaci ty would presumably be compromised. Based on these wide f luc tuat ions i n L R L , i t i s c l e a r that EMG data may not be an accurate r e f l e c t i o n of mechanical events. I f the abdominal w a l l was re laxed, the standing p o s i t i o n should have produced outward movement of the abdominal compartment and hence lengthening of a l l the abdominal muscles. The obl ique muscles (10 and EO) would a l so presumably be lengthened when moving to the s tanding p o s i t i o n due to t h e i r o r i e n t a t i o n to the abdominal w a l l and t h e i r i n s e r t i o n s on the p e l v i s . As was found, the EO d i d lengthen i n the standing p o s i t i o n and although not s i g n i f i c a n t , there appeared to be a trend to lengthening of the 10 and RA (Figure 11). In c o n t r a s t , TA length decreased i n the standing p o s i t i o n . The decrease i n TA LRL may be explained by cons ider ing i t as tonic shortening r e f l e c t i n g an increase i n ton ic EMG a c t i v i t y . This r e s u l t provides d i r e c t evidence of ton ic abdominal muscle a c t i v i t y which prev ious ly has only been i n f e r r e d from i n d i r e c t measurements. The tonic shortening of the TA found i n the 65 present study i s cons i s tent with reports of ton ic TA and RA EMG a c t i v i t y i n awake s i t t i n g dogs (8). That same study does, not spec i fy whether ton i c EMG a c t i v i t y was found i n the standing p o s i t i o n . In a d d i t i o n , abdominal muscle a c t i v i t y i n the s i t t i n g and s tanding p o s i t i o n s was compared to a c t i v i t y i n the prone p o s i t i o n . I t i s p o s s i b l e that there was abdominal muscle a c t i v i t y present i n the prone p o s i t i o n which would make i t d i f f i c u l t to determine the presence of t on i c a c t i v i t y i n the s i t t i n g or standing p o s i t i o n s when compared to prone. Tonic shortening of the TA i n the standing p o s i t i o n would serve to defend end-expiratory lung volume and diaphragm l ength . In prone, anes thet ized dogs, FRC was increased (16) and diaphragm i n i t i a l length was decreased (21) compared to the supine p o s i t i o n . However, abdominal muscle a c t i v a t i o n was found to prevent a greater increase i n FRC i n the prone, anesthet ized dogs (16) and was a l so concluded to be respons ib le f o r the p r e s e r v a t i o n of diaphragm length i n standing awake dogs (17). Since diaphragm i n i t i a l length was less than L 0 i n prone, anesthet ized dogs (21,24), i t i s probable that diaphragm i n i t i a l l ength would a l so be l e ss than optimal i n the standing awake dog, i f there were no mechanism to counteract the g r a v i t a t i o n a l e f fec t s on the diaphragm i n that p o s i t i o n . Therefore , TA tonic shortening (decrease i n L R L ) , by prevent ing a decrease i n diaphragm end-expiratory l ength , would help to optimize diaphragm length . Increased a c t i v a t i o n , phasic and/or t o n i c , o f the abdominal muscles with change i n posture from supine to upr ight i s most l i k e l y due to g r a v i t a t i o n a l e f fec t s on the chest w a l l (abdomen-diaphragm and r i b cage) . The primary mechanism c o n t r o l l i n g abdominal muscle response to changes i n posture appears to be a vagal r e f l e x from lung s t r e t c h 66 receptors . I t i s thought that pulmonary af ferents are s t imulated by the increased FRC assoc ia ted with assumption of upr ight posture to produce a v a g a l l y mediated r e f l e x a c t i v a t i o n of the abdominal muscles (6). Although FRC was not measured i n the present study, i t was found to increase when l i g h t l y anesthet ized dogs were t i l t e d to u p r i g h t and increased more when exp ira tory muscle a c t i v i t y decreased fo l l owing vagotomy (13). I t i s a l so w e l l e s tab l i shed that FRC i s increased i n the u p r i g h t posture i n man (2). Indeed, the f i n d i n g that abdominal muscle e x p i r a t o r y a c t i v i t y i s decreased a f ter vagotomy i n upr ight anesthet ized dogs (9,13) and r a b b i t s (6) would support a v a g a l l y mediated response. Abdominal muscle a c t i v a t i o n i s most l i k e l y a lso augmented by segmental r e f l e x e s . In r a b b i t s , when lung volume was increased by s i m i l a r amounts with p o s i t i v e pressure breathing and t i l t i n g , abdominal muscle a c t i v i t y was greater during upright t i l t i n g (6). In the upr ight posture , g r a v i t y would tend to cause the abdominal w a l l to move outward, s t r e t c h i n g the abdominal muscles and thus, s t i m u l a t i n g muscle p r o p r i o c e p t o r s . This hypothesis i s supported by the observat ion that i n upr ight man, the ton ic a c t i v i t y of the muscles i n the lower abdomen i s greater than those of the upper abdomen. Presumably, t h i s r e f l e c t s a greater h y d r o s t a t i c pressure being exerted on the lower abdomen causing greater d i s t e n s i o n of the lower abdomen (26). I t has been suggested that the expiratory a c t i v i t y of the abdominal muscles has been genera l ly underestimated due to measurement techniques which were e i t h e r l i m i t e d to the s u p e r f i c i a l muscles or measured abdominal muscle movement as a s ing le u n i t (7). Studies that have measured i n d i v i d u a l abdominal muscle shortening i n supine anesthet ized dogs dur ing expiratory threshold loading (19) and 67 hypercapnic hyperpnea (3) have shown that the i n t e r n a l abdominal muscles (TA and 10) tend to be r e c r u i t e d e a r l i e r and shorten more than the ex terna l l ayer (EO and RA). The present r e s u l t s extend previous f ind ings of v a r i a b l e amounts of abdominal muscle shortening to inc lude p o s t u r a l d i f f erences . Evidence of d i f f e r e n t i a l abdominal muscle recrui tment with changes i n posture has p r e v i o u s l y been demonstrated by EMG a c t i v i t y . Anesthet ized dogs moved from supine to prone (16) and supine to headup (18) had greater amounts of EMG a c t i v i t y i n the TA than the EO. Greater TA EMG a c t i v i t y compared to EO has a l so been shown i n awake s tanding , s i t t i n g and prone dogs (8). S i m i l a r r e s u l t s of TA versus EO and RA EMG a c t i v i t y were found for s i t t i n g man (7). There does not appear to be any p o s t u r a l data a v a i l a b l e for 10 EMG, but the 10 was r e c r u i t e d e a r l i e r and at lower l e v e l s of v e n t i l a t i o n than the EO i n man dur ing hypercapnic hyperpnea (27). Therefore , the greater amount of shortening of the i n t e r n a l abdominal muscles shown i n t h i s study i s cons i s t ent with the d i f f e r e n t i a l a c t i v a t i o n reported i n other s tud ie s . What fac tors could account for the p r e f e r e n t i a l recruitment of the i n t e r n a l abdominal muscle layer? 1. There may be d i f f e r e n t i a l a c t i v a t i o n . The TA and 10 are lengthened more by pass ive lung i n f l a t i o n than the EO or RA (19). Greater lengthening of the i n t e r n a l muscles could a c t i v a t e muscle spindles and l ead to r e f l e x f a c i l i t a t i o n of a c t i v i t y . Conversely, descending output from c e n t r a l c o n t r o l l e r s may p r e f e r e n t i a l l y a c t i v a t e the i n t e r n a l muscles. 2. The pass ive length-tens ion c h a r a c t e r i s t i c s of the i n d i v i d u a l abdominal muscles may be important i n terms of abdominal muscle c o n t r i b u t i o n to V T . In hamsters, the pass ive tens ion generated when the muscles were s t re tched beyond Lo was s i g n i f i c a n t l y greater for the TA than the EO (4). The greater 68 pass ive tens ion of the TA for a given change i n f i b e r length compared to the EO suggests that i t may contr ibute more to the r e c o i l pressure of the abdominal w a l l and hence shorten more than the EO. 3_. The range i n operat ing length of the i n d i v i d u a l muscles may a l so be important i n terms o f abdominal muscle c o n t r i b u t i o n to Vx. In hamsters the range i n operat ing length of the TA was smaller than the EO, that i s the t ens ion generated by a given change i n length was greater for the TA than the EO (4). Thus, the i n t e r n a l muscles may be more e f f e c t i v e generators of abdominal pressure . This would enable the i n t e r n a l muscles to contr ibute more to V T by decreasing end-expiratory lung volume and by i n c r e a s i n g the a p p o s i t i o n a l component of diaphragm c o n t r a c t i o n , a s s i s t the diaphragm during i n s p i r a t i o n (10). What are the advantages of p r e f e r e n t i a l recrui tment of the i n t e r n a l abdominal muscle layer? When st imulated separate ly , the TA and 10 decrease lung volume and increase abdominal pressure , whereas the EO, although i t decreases lung volume and increases abdominal pressure , i n f l a t e s the r i b cage (10). Thus, contrac t ion of the EO has opposing act ions which may make i t l ess e f f e c t i v e at defending diaphragm length . Furthermore, the o r i e n t a t i o n and p o s i t i o n of the i n t e r n a l muscles i n the abdominal w a l l would make them e f f e c t i v e at compressing the abdomen i n upr ight p o s i t i o n s . These r e s u l t s emphasize the r o l e of the i n t e r n a l muscle l ayer of the abdominal wa l l i n breath ing . 69 REFERENCES 1 . Agos ton i , E . , and E . J . M . Campbell. The abdominal muscles. In: The  Resp ira tory Muscles: Mechanics and Neural C o n t r o l . E . J . M . Campbell , E Agos ton i , and J . 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A p p l . P h y s i o l . 5 4 : 4 6 5 -4 6 9 , 1 9 8 3 . 1 1 . Druz, W.S. and J . T . Sharp. A c t i v i t y of r e s p i r a t o r y muscles i n upr ight and recumbent humans. J . A p p l . P h y s i o l . 5 1 ( 6 ) : 1 5 5 2 - 1 5 6 1 , 1 9 8 1 . 1 2 . Estenne, M . , V. Ninane and A. De Troyer . T r i a n g u l a r i s s t e r n i muscle use during eupnea i n humans: e f fec t of posture . R e s p i r . P h y s i o l . 7 4 : 1 5 1 - 1 6 2 , 1 9 8 8 . 1 3 . Farkas , G . A . , R . E . Baer, M. Estenne and A. De Troyer . Mechanical r o l e of exp ira tory muscles during breathing i n upr ight dogs. J__ 70 A p p l . P h v s i o l . 64(3): 1060-1067, 1988. 14. Farkas , G . A . , M. Estenne and A. De Troyer . Exp ira tory muscle c o n t r i b u t i o n to t i d a l volume i n head-up dogs. J . A p p l . P h v s i o l . 67(4): 1438-1442, 1989. 15. Farkas , G .A . and D . F . Rochester. C h a r a c t e r i s t i c s and f u n c t i o n a l s i g n i f i c a n c e of canine abdominal muscles. J . A p p l . P h y s i o l . 65(6): 2427-2433, 1988. 16. Farkas , G . A . and M.A. Schroeder. Mechanical r o l e of e x p i r a t o r y muscles dur ing breath ing i n prone anesthet ized dogs. J . A p p l .  P h v s i o l . 69(6): 2137-2142, 1990. 17. F i t t i n g , J . W . , P . A . Easton, R. Arnoux, A. Guerraty and A. Grass ino . Diaphragm length adjustments with body p o s i t i o n changes i n the awake dog. J . A p p l . P h v s i o l . 66(2): 870-875, 1989. 18. G i l m a r t i n , J . J . , V. Ninane and A. De Troyer . Abdominal muscle use during breath ing i n the anesthet ized dog. Resp ir . P h y s i o l . 70: 159-171, 1987. 19. Leevers , A . M . and J . D . Road. Mechanical response to h y p e r i n f l a t i o n of the two abdominal muscle l a y e r s . J . A p p l . P h y s i o l . 66(5): 2189-2195, 1989. 20. Leevers , A . M . and J . D . Road. E f f e c t of lung i n f l a t i o n and u p r i g h t posture on diaphragmatic shortening i n dogs. R e s p i r . P h y s i o l . 85: 29-40, 1991. 21. Newman, S . L . , J . D . Road and A. Grass ino. In v ivo length and shortening of canine diaphragm with body p o s t u r a l change. J . A p p l .  P h v s i o l . 60(2): 661-669, 1986. 22. Ninane, V. and A. De Troyer . Mechanics of paras terna l s and t r i a n g u l a r i s s t e r n i i n upr ight vs . supine dogs. J . A p p l . P h v s i o l . 65(1): 452-459, 1988. 23. Ninane, V . , J . J . G i l m a r t i n and A. De Troyer . Changes i n abdominal muscle length dur ing breath ing i n supine dogs. R e s p i r . P h y s i o l . 73: 31-42, 1988. 24. Road, J . , S. Newman, J . P . Derenne and A. Grass ino . In v ivo l ength-force r e l a t i o n s h i p of canine diaphragm. J . A p p l . P h v s i o l . 60(1): 63-70, 1986. 25. Smith, C . A . , D.M. Ainsworth, K . S . Henderson and J . A . Dempsey. D i f f e r e n t i a l t iming of r e s p i r a t o r y muscles i n response to chemical s t i m u l i i n awake dogs. J . A p p l . P h y s i o l . 66(1): 392-399, 1989. 26. S t r o h l , K . P . , J . Mead, R . B . Banzett, S .H. L o r i n g and P . C . Kosch. Regional d i f f erences i n abdominal muscle a c t i v i t y dur ing var ious maneuvers i n humans. J . A p p l . P h y s i o l . 51: 1471-1476, 1981. 71 27. Wakai, Y . , M.M. Welsh, A . M . Leevers and J . D . Road. The e f f e c t of continuous p o s i t i v e airway pressure and hypercapnia on exp ira tory muscle a c t i v i t y dur ing wakefulness and s leep . Am. Rev. R e s p i r . P i s . 141(4): A125, 1990.(Abstract) 72 I V . ABDOMINAL MUSCLE ACTIVATION BY EXPIRATORY THRESHOLD LOADING INTRODUCTION The four muscles of the v e n t r a l abdominal w a l l : rectus abdominis (RA) , e x t e r n a l ob l ique (EO) , i n t e r n a l obl ique (10) and transversus abdominis (TA), are known to be s trong ly r e c r u i t e d dur ing forced e x p i r a t i o n (1) or e x p i r a t o r y loading (2,8,20,21). The a p p l i c a t i o n of p o s i t i v e end-exp ira tory pressure (PEEP) presents an exp ira tory thresho ld load (ETL) to the r e s p i r a t o r y system and r e s u l t s i n an increase i n f u n c t i o n a l r e s i d u a l capac i ty (FRC) (8). However, the increase i n FRC i s l e s s than would be expected from passive pressure-volume c h a r a c t e r i s t i c s (23), due to recrui tment of the expiratory muscles, p a r t i c u l a r l y the abdominal muscles (18). This recruitment of the abdominal muscles i s thought to be due p r i m a r i l y to vaga l ly mediated re f lexes (2-4) but l o c a l segmental re f l exes may a lso be involved (3,24). Previous s tudies of abdominal muscle response to ETL have g e n e r a l l y employed anesthet ized animals (6,18,21). Since anesthes ia i s known to inf luence re f l exe s , p a r t i c u l a r l y vagal re f lexes (25,26), the responses i n the anesthet ized animal can not be extrapolated to the awake animal . In a d d i t i o n , studies i n v o l v i n g anesthet ized animals of ten have the animal i n the supine p o s i t i o n , one that i s not n a t u r a l f or the dog and may a l so e f f e c t abdominal muscle response independently o f other f a c t o r s . Indeed, as discussed i n Chapter I I I , abdominal muscle a c t i v a t i o n i s dependent on changes i n posture . We have p r e v i o u s l y shown i n anesthet ized dogs, that the abdominal muscles are r e c r u i t e d non-uni formly and have d i f f e r e n t r e l a t i v e contr ibut ions to e x p i r a t i o n when a c t i v a t e d by e x p i r a t o r y threshold loading (ETL) (18). Therefore , 73 a c t i v a t i o n of the i n d i v i d u a l abdominal muscles may not involve the same r e f l e x pathways. We hypothesized that exp ira tory thresho ld l oad ing , i n the l e f t l a t e r a l decubitus p o s i t i o n ( L L D ) , would produce a s trong recruitment o f the abdominal muscles of the awake dog r e s u l t i n g i n defence o f end-exp ira tory lung volume. We a lso hypothesized that there would be non-uniform a c t i v a t i o n of the abdominal muscles, with greater a c t i v a t i o n of the i n t e r n a l muscle l ayer (TA and 10) than the ex terna l muscle l a y e r (EO and RA) i n the awake dog, as was found i n the anesthet ized dog. Therefore , the objec t ives of t h i s study were to i d e n t i f y i n d i v i d u a l abdominal muscle recrui tment , as measured by the d i f ferences i n r e s t i n g length and shortening, dur ing exp ira tory loading i n awake dogs. METHODS Eight tracheotomized, female mongrel dogs were s u r g i c a l l y implanted with sonomicrometer transducers and f ine wire EMG e lec trodes i n each of the four abdominal muscles as descr ibed i n Chapter I I . Three dogs were s u r g i c a l l y implanted with femoral a r t e r y catheters (vascular access p o r t , model GPV, Access Technologies , I L ) which al lowed a r t e r i a l b lood sampling from a subcutaneous port located i n the d o r s a l lumbar reg ion . Measurements Abdominal muscle end-expiratory lengths and length changes were measured with the sonomicrometer transducers (see Chapter I I ) . The r e s t i n g base l ine length of the abdominal muscles, with the dog i n the l e f t l a t e r a l decubitus p o s i t i o n , was termed L R L . Changes i n muscle 74 l ength dur ing t i d a l breath ing , e i t h e r i n s p i r a t o r y lengthening or e x p i r a t o r y shorten ing , were expressed as a percentage of the i n i t i a l r e s t i n g length (%LRL) . A c t i v e shortening was determined to be that c o i n c i d i n g wi th raw exp ira tory EMG a c t i v i t y . A change i n base l ine length from the i n i t i a l r e s t i n g base l ine length was a l so expressed as a percentage o f the i n i t i a l r e s t i n g length. The new b a s e l i n e length dur ing ETL was termed the ac t ive base l ine length ( L A B L ) • LABL was u s u a l l y shorter than LRL and therefore , was considered to be a r e f l e c t i o n of t o n i c a c t i v i t y . The electromyographic s ignals from the implanted f i n e wire e lec trodes were a m p l i f i e d (Grass, model P511) and f i l t e r e d (band width 100-10,000 Hz) and recorded i n p a i r s with the sonomicrometer s i g n a l s . A i r f l o w was measured with a pneumotachograph ( F l e i s c h #1) and pressure transducer (Validyne MP45, Medf ie ld , Mass.) at tached to the d i s t a l end of the tracheostomy tube and integrated to give t i d a l volume. The opposite end of the pneumotachograph was connected to a two-way, non-rebreath ing va lve (Hans Rudolph, model 2600). E x p i r a t o r y thresho ld loads of 6,10, 14 and 18 C111H2O were produced by app ly ing p o s i t i v e end-e x p i r a t o r y pressure (PEEP) v i a a PEEP valve (Medigas, Model BE-142) attached to the exp ira tory side of the two-way v a l v e . End-exp ira tory pressure was measured v i a a porthole i n the two-way va lve and recorded as airway pressure (Pao) ( d i f f e r e n t i a l pressure transducer , Va l idyne MP-45, M e d f i e l d , M a s s . ) . During the a p p l i c a t i o n of e x p i r a t o r y thresho ld load ( E T L ) , the change i n lung volume ( 6 V 0 I ) was measured i n a d d i t i o n to the c o n t r o l parameters. The 6 V 0 I was determined from the volume expired when the exp ira tory load was re leased . I n s p i r a t o r y (Ti) and exp ira tory ( T E ) durat ion were determined from the flow t r a c i n g on the 75 chart recorder us ing a d i g i t i z i n g device (Sigma-Scan, Jandel S c i e n t i f i c , Corte Madera, CA) . One ml a r t e r i a l b lood samples were obtained, v i a the v a s c u l a r access p o r t , dur ing r e s t i n g breath ing , before and a f t e r the ETL p r o t o c o l , and at three l e v e l s of PEEP (6,10 and 14 cmH20) dur ing ETL. Blood samples were analyzed (Corning, 168 pH/Blood gas analyzer) for PC0 2 , pH, and P0 2 , and H C O 3 " determined by e x t r a p o l a t i o n . Protocol The e ight dogs used i n t h i s study a lso underwent one or more of the other protoco l s on d i f f e r e n t days: p o s t u r a l changes (Chapter I I I ) , C0 2 rebreath ing (Chapter V) and vagal blockade (Chapter VI) . Th i s chapter descr ibes the p r o t o c o l fol lowed for exp ira tory thresho ld load ing (ETL) . Repeat s tudies of ETL were performed on d i f f e r e n t days to assess the r e p r o d u c i b i l i t y o f the r e s u l t s . A l l the studies f o r each dog were performed over per iods ranging from two to e ight weeks. At the beginning of each ETL t r i a l , a cuf fed tracheostomy tube (#7) was i n s e r t e d through the tracheotomy. The dog was p laced on a p la t form, p o s i t i o n e d i n the l e f t l a t e r a l decubitus and the c r y s t a l and EMG wires were connected to the sonomicrometer and a m p l i f i e r s , r e s p e c t i v e l y . The two-way valve and PEEP valve were connected to the d i s t a l end of the pneumotachograph and when the dog was re laxed and breath ing s t a b i l i z e d , c o n t r o l measurements of abdominal muscle r e s t i n g length , length changes and EMG a c t i v i t y , Pao and t i d a l volume were made. An exp ira tory load was then appl i ed and a short time p e r i o d (1-2 minutes) al lowed for a regular breathing pat tern to be r e e s t a b l i s h e d . A f t e r ETL, breath ing was allowed to re turn to c o n t r o l values before the 76 next l oad . The awake dogs were exposed to three or four randomly a p p l i e d exp ira tory thresho ld loads (6,10,14 and 18 cmH 20). (Four dogs were not able to t o l e r a t e a PEEP of 18 cmH20) . A n a l y s i s Measurements were averaged over f i v e breaths for each l e v e l of PEEP, for each study day. Since there were no cons i s tent d i f f erences between s tud ies , the means from each PEEP study were then averaged for each dog and the means per dog, at each l e v e l of PEEP were obtained. The means ± SE were then c a l c u l a t e d for each v a r i a b l e . Whenever comparisons were made among the d i f f e r e n t abdominal muscles, a one-way ANOVA was used and a s i g n i f i c a n c e l e v e l of P<0.05 accepted. Post hoc m u l t i p l e comparisons were made us ing a Tukey's mul t ip l e comparison t e s t . When s i g n i f i c a n t heterogeneity of standard d e v i a t i o n ex i s t ed , l og transformations of the data were made and the ANOVAs repeated. To compare the r e s u l t s at d i f f e r e n t l e v e l s of PEEP, a one-way ANOVA was used and a s i g n i f i c a n c e l e v e l of P<0.05 accepted. A Dunnett's mul t ip l e comparison t e s t was used to tes t for d i f ferences from c o n t r o l measures. RESULTS A l l e ight dogs were e a s i l y able to t o l e r a t e exp ira tory thresho ld loads up to 14 cmH20 but only four of the e ight dogs could adjust to an airway pressure of 18 cmH20. The other four dogs became r e s t l e s s and ag i ta ted at the higher Pao. Exp ira tory threshold load ing produced recruitment of three of the abdominal muscles (TA, 10 and EO) but had no measurable e f f e c t on RA a c t i v i t y . The e f f e c t of a PEEP of 10 cmH20 on V T , the change i n volume ( S V o l ) , abdominal muscle (10) ac t ive base l ine 77 length (LABL) and t i d a l shortening (%LRL) i s shown by a representative t r a c i n g i n Figure 1 2 . The r e s t i n g baseline length of the muscle (LRL) i s the length preceding the downward d e f l e c t i o n of the t r a c i n g before the a p p l i c a t i o n of PEEP (when Pao=0). The LABL i s the length immediately preceding the onset of active shortening. The downward d e f l e c t i o n of the t r a c i n g immediately following the LABL i s a c t i v e , phasic expiratory shortening (%LRL) and coincides with the i n i t i a t i o n of the phasic EMG. The upward d e f l e c t i o n of the t r a c i n g above the r e s t i n g baseline i s i n s p i r a t o r y lengthening. The end-expiratory length and the end-inspiratory length comprise the t o t a l t i d a l length excursion of the muscle. The difference between the r e s t i n g baseline length (LRL) and the a c t i v e baseline length (LABL) was assumed to r e f l e c t tonic muscle a c t i v i t y . An active baseline length shorter than LRL implied tonic muscle shortening. Total t i d a l length excursions, a c t i v e phasic, expiratory shortening and changes i n baseline length -(tonic a c t i v i t y ) of three abdominal muscles during ETL are shown i n Figure 1 3 . T o t a l t i d a l length excursions ( i n s p i r a t o r y lengthening plus expiratory shortening) of the TA and 1 0 increased with increasing Pao but the EO d i d not change s i g n i f i c a n t l y (Figures 13 and 1 4 ) . 78 Figure 12: A representat ive t r a c i n g from an awake dog ( l e f t l a t e r a l decubitus p o s i t i o n ) showing (from top to bottom) t i d a l volume (VT) , airway pressure (Pao), i n t e r n a l obl ique (10) length changes and EMG a c t i v i t y . The l e f t s ide of the t r a c i n g i s with a PEEP of 10 cmH20. The r i g h t s ide i s a f t er re lease of the load . The 10 r e s t i n g length (LRL) i s the length immediately preceding the downward d e f l e c t i o n of the length trace when Pao=0. The downward d e f l e c t i o n of the length trace c o i n c i d i n g with i n i t i a t i o n of EMG a c t i v i t y i s ac t ive phasic shortening expressed as %LRL. 10 Sec 80 Figure 13: Length changes (%LRL) of three abdominal muscles (TA, 10 and EO) at control (Pao=0) and four levels of PEEP (Pao=6, 10, 14 and 18 CH1H2O) during expiratory threshold loading. The 0 line is the i n i t i a l , resting baseline length of the muscles ( L R L ) , the positive length changes are inspiratory lengthening and the negative changes are shortening below the resting baseline length. Active shortening is represented by the black part of the bars and tonic shortening by the unshaded portion of the negative length changes. Values are means ± SE. 81 82 The amount of a c t i v e exp ira tory shortening (%LRL) f or each muscle increased as the l e v e l o f PEEP increased and was s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l at a PEEP o f 10 cmH20 (Figures 13 and 15). %LRL increased from 1 . 0 ± 0 . 1 6 to 4 . 9 ± 0 . 9 6 % for the TA, 0 . 5 ± 0 . 1 8 to 6 . 4 ± 1 . 2 9 % for the 10 and 0 . 0 5 ± 0 . 0 3 to 1 . 8 ± 0 . 3 1 % for the EO (means ± SE) when Pao was increased from c o n t r o l to 14 cmH20. Since shortening was expressed as a percentage of the r e s t i n g base l ine length ( f r a c t i o n a l shortening) , changes i n the base l ine length that occurred with i n c r e a s i n g Pao could e f f e c t the c a l c u l a t i o n of shortening . A decrease i n the base l ine length would l ead to an underest imation of f r a c t i o n a l shortening, whereas an increase i n base l ine length would lead to an overest imat ion. However, shortening was expressed i n terms of the r e s t i n g base l ine length for two reasons: (1) the present study was concerned with shortening r e l a t i v e to shortening at r e s t and (2) the change i n base l ine was not considered to be s u f f i c i e n t to e f f e c t the o v e r a l l r e s u l t s . ETL a l so e f fec ted the LABL of the 10 and EO (Figure 13). The LABL of the 10 was shorter than LRL at each l e v e l of PEEP up to a Pao of 14 cmH20. At 18 cmH20 Pao, measured i n four dogs, 10 LABL was longer than L R L . The E0 LABL was longer than LRL at each l e v e l of PEEP (Figure 13). As was found p r e v i o u s l y i n anesthet ized dogs (18), the i n t e r n a l muscles (TA and 10) p h a s i c a l l y shortened more than the ex terna l muscles (RA and E0) at a l l l e v e l s of Pao (Figures 13 and 15). In a d d i t i o n , there was a d i f f erence i n tonic a c t i v i t y (change i n LABL) between the i n t e r n a l and externa l muscles (Figure 13). 83 12 15 18 Pao (cmhLO) Figure 14: T o t a l t i d a l length excursions of three abdominal muscles (TA, 10 and EO) expressed as a percentage of the i n i t i a l baseline r e s t i n g length (%LRL) p l o t t e d against increasing airway pressure (Pao) during expiratory threshold loading. The asterisks (*) i n d i c a t e s i g n i f i c a n t d i f f e r e n c e i n t o t a l t i d a l length changes between the TA and 10 compared to the EO (P<0.05). 84 Pao (cmH 0) Figure 15: Active, phasic expiratory shortening of three abdominal muscles (TA, 10 and EO) expressed as a percentage of the i n i t i a l baseline r e s t i n g length (%LRL) p l o t t e d against increasing airway pressure (Pao) during expiratory threshold loading. The a s t e r i s k s (*) i n d i c a t e s i g n i f i c a n t d i f f e r e n c e i n active shortening between the TA and 10 compared to the EO (P<0.05). 85 As the l e v e l of PEEP increased, there was a s i g n i f i c a n t increase i n lung volume, i n d i c a t i n g an increase i n FRC (Figure 16). T i d a l volume and b r e a t h i n g frequency ( 1 / T T O T ) were unchanged dur ing ETL. However, there was a h y p o v e n t i l a t i o n at the highest load of 18 cmH20 (Table III ) . Although TTOT d i d not change during ETL, T i decreased ( s i g n i f i c a n t at Pao 10 cmH20) and T E increased at 14 cmH20 (Figure 17). Ti decreased from 1 . 1 5 ± 0 . 0 7 sec to 0.93+0.05 sec and T E increased from 2 . 5 6 ± 0 . 2 3 sec to 3 . 3 5 ± 0 . 3 3 sec at Pao=0 and 14 cmH20, r e s p e c t i v e l y . The increase i n T E was l i n e a r l y r e l a t e d to the increase i n lung volume (Figure 18). The e f f ec t s of ETL on a r t e r i a l blood gases are shown i n Table IV. The mean values for three dogs at each l e v e l of PEEP i n d i c a t e that hypercapnia d i d not develop during ETL up to a PEEP of 14 cmH20. Table III: V e n t i l a t i o n and t iming parameters during E T L . Pao v E VT T T O T T E (cm H 20) (ml "min"1) (ml) (sec) (sec) 0 5981 292 3.61 2.56 ± 8 4 2 ± 2 3 ± . 2 9 ± . 2 3 6 4960 283 3.99 3.05 ± 6 6 2 ± 2 0 ± . 3 1 ± . 2 7 10 4648 266 4.11 3.26 ± 6 5 1 ± 2 3 ± . 3 2 ± . 2 8 14 4627 258 4.18 3.35 * ± 8 3 7 ± 2 1 ± . 3 7 ± . 3 3 18 3529 * 240 4.59 3.70 * ± 5 8 5 ± 1 2 ± . 4 4 ± . 4 0 Pao=airway pressure ( contro l = 0 cmH20) Values are means ± S E N=8 (N=4 at Pao=18 cmH20) A s t e r i s k (*) ind ica tes s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l , Pao=0 cmH20 (P<0.05). Table IV: A r t e r i a l blood gas values during ETL. Pao PC0 2 P0 2 pH (cm H20) mmHg mmHg 0 (pre) 37.6 104.7 7.36 ±2.6 ±12.2 ±.005 6 38.2 97.9 7.36 ±2.4 ±6.1 ±.011 10 37.1 92.9 7.37 ±1.6 ±8.2 ±.011 14 39.5 83.9 7.35 ±0.8 ±5.0 ±.004 0 (post) 35.0 86.8 7.38 ±1.2 ±7.0 ±.007 Pao=airway pressure (control = 0cm H20; pre and post are co n t r o l before and a f t e r ETL) N=3 0 3 6 9 12 15 18 Pao (cm H 2 0) Figure 16: The change i n lung volume (6Vol) produced by ETL p l o t t e d against airway pressure (Pao). Values are means ± SE. As t e r i s k s (*) indicate s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l (P<0.05). 0.00 ^ 1 1 1 1 1 4.0 -3.5 O (0 UJ 3.0 2.5 6 9 12 Pao (cm H.O) 18 Figure 17: I n s p i r a t o r y a i r flow durat ion (Ti) (top) and exp ira tory durat ion (T E) (bottom) p l o t t e d against airway pressure (Pao) during ETL. Values are means ± SE. T E i s s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l at Pao=18cmH20 (P<0.05). 90 4.0 Figure 18: Expiratory duration (T E) p l o t t e d against the change i n volume (6"vol) during ETL. Values are means ± SE 91 DISCUSSION This study shows that awake dogs a c t i v e l y r e c r u i t t h e i r abdominal muscles i n response to expiratory threshold loading (ETL). As was found pr e v i o u s l y i n anesthetized dogs (18), the i n t e r n a l layer of muscles (TA and 10) i s r e c r u i t e d p r e f e r e n t i a l l y and appears to be more important i n the defence of lung volume that r e s u l t s from abdominal muscle a c t i v a t i o n . Abdominal muscle a c t i v a t i o n i s one of the load compensating mechanisms employed by the respiratory system when anesthetized animals are exposed to ETL (2-4,20). Phasic, expiratory shortening of the abdominal muscles helps prevent the increase i n fu n c t i o n a l r e s i d u a l capacity (FRC) and hence, the r e s u l t i n g decrease i n diaphragm i n i t i a l length which would compromise the mechanical e f f i c i e n c y of the diaphragm (22). In anesthetized animals the abdominal muscle response to ETL i s dependent p r i m a r i l y on vagall y mediated reflexes (2-4), since vagotomy e f f e c t i v e l y eliminates phasic abdominal muscle a c t i v i t y (6,24). Other mechanisms i n the abdominal muscle response to ETL may be involved i n awake animals. The responses of awake animals (other than humans) have not been well investigated. However, i t might be expected that the abdominal muscles would be more active during ETL i n awake animals compared to anesthetized animals. Greater abdominal muscle a c t i v a t i o n might be expected i n the awake animal i n spite of the increased a r t e r i a l PC0 2 found during ETL i n anesthetized animals (8), and thus stimulation of expiratory neurons (17), because chemoreceptor responsiveness i s decreased by anesthesia (12). Therefore, load compensation i n anesthetized animals i s not considered to be very dependent on chemoreflexes (8) but chemoreflexes may be more relevant i n the awake 92 dog. In addition, pentobarbital anesthesia has been shown to i n h i b i t abdominal motoneuron phasic discharge i n cats (11) thus presumably r a i s i n g the threshold f o r a c t i v a t i o n . Moreover, CNS mechanisms that may be involved i n the r e s p i r a t o r y response to v e n t i l a t o r y loading i n conscious goats appeared to be affe c t e d by anesthesia (15). Therefore, i t would appear that more p o t e n t i a l , neural pathways to abdominal muscle a c t i v a t i o n are present i n awake animals. On the other hand, vagal reflexes may be so enhanced i n anesthetized animals (7,10,25), that these i n h i b i t i n g e f f e c t s of anesthesia on chemoreceptor mediation of abdominal muscle a c t i v a t i o n may be counteracted. In the present study, the amount of active, phasic expiratory shortening of the TA and EO abdominal muscles e l i c i t e d by ETL was qu a n t i t a t i v e l y s i m i l a r to that found i n anesthetized dogs i n an e a r l i e r study (18), but the phasic shortening of the 10 was less i n the awake dogs. The differe n c e i n 10 shortening could be a r t i f a c t u a l due to differences i n the analysis of active shortening between the two studies. In the e a r l i e r study, t o t a l shortening was measured, which included both tonic (change i n the baseline length from LRL) and act i v e phasic shortening. I f t o t a l shortening i s ca l c u l a t e d i n the present study (tonic plus a c t i v e ) , 10 shortening increases to 9.0±1.3% (Pao = 14 cmH20) which i s c l o s e r to the amount found i n the anesthetized dogs (12.4±0.5%). However, there could be less 10 shortening due to postural e f f e c t s (see Chapter I I I ) : the anesthetized dogs were studied i n the supine p o s i t i o n (18) and the awake dogs were studied i n the l e f t l a t e r a l decubitus p o s i t i o n . A l t e r n a t i v e l y a stronger vagal r e f l e x could be operating i n the anesthetized dog (7,10,25) generating greater a c t i v a t i o n of the abdominal muscles. Since the 10 had a r e l a t i v e l y 93 greater a c t i v a t i o n during ETL i n the anesthet ized dog (18), i t i s reasonable to expect i t to be r e l a t i v e l y l e s s a c t i v a t e d i f vagal re f l exes are reduced i n the awake dog. L a s t l y , although s e n s i t i v i t y to C0 2 i s reduced i n anesthet ized animals (12), ETL genera l l y leads to C0 2 r e t e n t i o n (8) and therefore provides an a d d i t i o n a l st imulus for abdominal muscle a c t i v a t i o n (17). A r t e r i a l PC0 2 may w e l l have increased dur ing the e a r l y phases of l o a d i n g , however i t d i d not increase dur ing ETL (up to a PEEP of 14 cmH20) i n the three dogs i n which i t was measured (Table I V ) . Therefore , i t s quest ionable whether s t i m u l a t i o n of the abdominal muscles by chemoreflexes was a f a c t o r i n the awake dogs. An i n t e r e s t i n g aspect of the present study, and one that has not been p r e v i o u s l y reported , was the presence of ton ic a c t i v i t y i n the 10, as r e f l e c t e d by the shorter ac t ive base l ine length (LABL) compared to LRL, which occurred with increas ing Pao. An LABL shorter than the LRL was cons idered to i n d i c a t e an increase i n ton ic a c t i v i t y . Indeed, i t i s most l i k e l y that ton ic a c t i v i t y was a c t u a l l y underestimated, s ince ETL increases FRC (8) and would be expected to lengthen the abdominal muscles (18). Thus, j u s t maintaining the LABL at the r e s t i n g base l ine length would require an increase i n ton ic a c t i v i t y . I t fo l lows there fore , that the 10 had a s i g n i f i c a n t increase i n ton i c muscle a c t i v i t y because i t s ' LABL was shorter than L R L , and i n a d d i t i o n , the TA probably a l so had an increase i n tonic a c t i v i t y s ince i t maintained i t s ' LABL equal to LRL (Figure 13). Tonic shortening of the abdominal muscles would, l i k e phas ic e x p i r a t o r y shortening, help prevent an increase i n FRC and thus optimize diaphragm length . Furthermore, tonic abdominal muscle c o n t r a c t i o n 94 during i n s p i r a t i o n would reduce the compliance of the abdominal compartment and hence, increase the i n f l a t i o n a r y a c t i o n of the diaphragm on the r i b cage (19). There i s both d i r e c t and i n d i r e c t evidence that suggests expiratory loading can increase abdominal muscle tonic a c t i v i t y . In a study of anesthetized cats, 20 percent of the cats exhibited continuous EO EMG a c t i v i t y during p o s i t i v e pressure breathing (2). I n d i r e c t evidence i s provided by the observation that abdominal muscle e f f e r e n t , gamma motoneurons discharged t o n i c a l l y during quiet breathing and the frequency of the tonic discharge increased with increasing Pao, during p o s i t i v e pressure breathing (24). An increase i n tonic gamma motoneuron discharge would increase the discharge rate and s e n s i t i v i t y of the abdominal muscle spindle afferents (14) and consequently, r e f l e x l y shorten the muscle. In addition, the increase i n end-inspiratory lung volume produced by ETL (8) would s t r e t c h the abdominal w a l l and excite passive s t r e t c h responses i n the abdominal muscle spindles, thus p o s s i b l y a c t i v a t i n g the abdominal muscles during i n s p i r a t i o n (24). Such a p o s s i b i l i t y i s supported by the demonstration of an abdominal muscle s t r e t c h r e f l e x i n humans, when the e x c i t a b i l i t y of the motoneuron pool was f a c i l i t a t e d by sustained s t r e t c h of the abdominals induced by standing (16). I t i s c l e a r from the preceding discussion that, although c o n t r o l of abdominal muscle tonic a c t i v i t y i s most l i k e l y l a r g e l y mediated by. vagal r e f l e x e s , supraspinal and segmental proprioceptive r e f l e x e s could f a c i l i t a t e the response.to ETL. The observation that vagotomy d i d not abolish tonic gamma motoneuron discharge during expiratory loading suggests that supraspinal proprioceptor pathways can be involved (24). Two a d d i t i o n a l pieces of evidence suggest supraspinal and segmental 95 r e f l e x involvement i n the abdominal muscle response to ETL: (1) thoracic s p i n a l transection eliminated the response and (2) b i l a t e r a l lower thoracic and upper lumbar rhizotomy with s p i n a l cord i n t a c t also eliminated the response (3). The p r e f e r e n t i a l a c t i v a t i o n of the i n t e r n a l abdominal muscle layer ( T A and 10) by ETL i s consistent with previous reports from anesthetized dogs (13,18). As was concluded i n these e a r l i e r studies, differences i n t i d a l length changes between the i n t e r n a l and external abdominal muscles i s l i k e l y r e l a t e d to differences i n o r i g i n and i n s e r t i o n , f i b e r o r i e n t a t i o n and compliance of the i n d i v i d u a l muscles. Such anatomical differences could have mechanical e f f e c t s on the load on the muscle and i t s p o s i t i o n on i t s length-tension curve, leading to diff e r e n c e s i n shortening. In addition, the fact that the i n t e r n a l muscles are lengthened more by passive lung i n f l a t i o n (18), might r e s u l t i n greater a c t i v a t i o n v i a proprioceptor afferents. Expiratory threshold loading i n awake dogs produced an increase i n FRC but the increase was less than would be expected i f load compensating mechanisms ( p a r t i c u l a r l y abdominal muscle a c t i v a t i o n ) were not i n i t i a t e d (18,23). Despite the increased FRC, V T was maintained, as was V E u n t i l a PEEP of 18 cmH20, at which point V E decreased. T i d a l volume i s generally also maintained i n anesthetized dogs during ETL, however there tends to be a greater reduction i n V E, p r i m a r i l y as a r e s u l t of a greater reduction i n breathing frequency (Bf) (21,23), compared to what was found i n the awake dogs. The slower Bf may be mediated by stronger vagal a c t i v i t y under anesthesia (26). V e n t i l a t i o n was decreased at a Pao of 18 cmH20 because of a n o n s i g n i f i c a n t decrease i n V T and B f (increase i n T T O T ) at PEEP 18 cmH20. 96 The trend towards an increased T T O T (and thus a decreased Bf) was i n turn due to an increased TE ( s i g n i f i c a n t at Pao = 14 and 18 C111H2O). Expiratory prolongation i s an expected r e s u l t of ETL (8) and has been shown i n anesthetized dogs (21) and cats (5,10) exposed to ETL. Again, the response i s p r i m a r i l y mediated by vagal reflexes (5,6). In f a c t , T E was l i n e a r l y r e l a t e d to the increase i n FRC (Figure 18). In contrast to the increased T E, Ti was s i g n i f i c a n t l y reduced during ETL. A i n i t i a l decrease i n Ti would be consistent with vagal i n h i b i t i o n of i n s p i r a t i o n due to the Hering-Breuer i n f l a t i o n r e f l e x (9). Indeed, an i n h i b i t i o n of i n s p i r a t i o n was observed i n the awake dogs with the onset of ETL, which would c e r t a i n l y support the presence of an Hering-Breuer r e f l e x i n the awake dogs. However, the Ti i n the present study was measured from the flow t r a c i n g a f t e r breathing s t a b i l i z e d and therefore, i s probably not representative of neural T i . Because ETL loads the i n s p i r a t o r y muscles as well as the expiratory muscles, before a i r f l o w can begin, the in s p i r a t o r y muscles must contract to return airway pressure to zero. As a re s u l t , neural Ti would have to e i t h e r remain constant or increase to maintain Vj. Since VT was maintained i n the present study, the measured Ti most l i k e l y considerably underestimated i n s p i r a t o r y drive. In summary, expiratory threshold loading i n awake dogs a c t i v a t e s the abdominal muscles. Both tonic and phasic shortening of the abdominal muscles helps prevent an increase i n FRC, thus preserving diaphragm i n i t i a l length and maintaining V T. In addition, the i n t e r n a l abdominal muscle layer (TA and 10) appears to be p r e f e r e n t i a l l y r e c r u i t e d and hence, more e f f e c t i v e i n load compensation than the external layer (EO and RA). 97 REFERENCES 1. Agos ton i , E . , and E . J . M . Campbell. The abdominal muscles. In: The  R e s p i r a t o r y Muscles: Mechanics and Neural C o n t r o l . E . J . M . Campbell , E Agos ton i , and J . Newsom-Davis (eds) . Saunders, P h i l a d e l p h i a , PA, 1970, 175-180. 2. Bishop, B. Abdominal muscle and diaphragm a c t i v i t i e s and c a v i t y pressures i n pressure breath ing . J . A p p l . P h y s i o l . 18(1): 37-42, 1963. 3. Bishop, B. Ref lex c o n t r o l of abdominal muscles dur ing p o s i t i v e pressure brea th ing . J . A p p l . P h v s i o l . 19(2): 224-232, 1964. 4. Bishop, B. Diaphragm and abdominal muscles response to e levated airway pressures i n the cat . J . A p p l . P h y s i o l . 22: 959-965, 1967. 5. Bishop, B. Vagal c o n t r o l of diaphragm t iming i n cat while breath ing at e levated lung volumes. Respir . P h y s i o l . 30: 169-184, 1977. 6. Bishop, B. and H. Bachofen. Vagal c o n t r o l of v e n t i l a t i o n and r e s p i r a t o r y muscles during e levated pressures i n the c a t . J . A p p l .  P h v s i o l . 32(1): 103-112, 1972. 7. B j u r s t e d t , H. Influence of the abdominal muscle tone on the c i r c u l a t o r y response to p o s i t i v e pressure breath ing i n anesthet ized dogs. Acta Phys. Scandinav. 29: 145-162, 1953. 8. Cherniack, N . S . , and M.D. A l t o s e . Resp iratory Responses to V e n t i l a t o r y Loading. In: The Regulat ion of Breath ing . Marcel D e k k e r . I n c , New York, 1981, 905-964. 9. C l a r k , F . J . and C. von E u l e r . On the r e g u l a t i o n of depth and rate of b r e a t h i n g . J . P h y s i o l . 222: 267-295, 1972. 10. F i n k l e r , J . and S. Iscoe. Contro l of breath ing at e levated ],ung volumes i n anesthet ized cat s . J . A p p l . P h y s i o l . 56(4): 839-844, 1984. 11. F r e g o s i , R . F . , S . L . Knuth, D.K. Ward and D. B a r t l e t t . J r . . Hypoxia i n h i b i t s abdominal exp ira tory nerve a c t i v i t y . J . A p p l . P h y s i o l . 63(2): 211-220, 1987. 12. G a u t i e r , H. Pat tern of breathing during hypoxia or hypercapnia of the awake or anesthet ized cat . Resp ir . P h y s i o l . 27: 193-206, 1976. 13. G i l m a r t i n , J . J . , V. Ninane and A. De Troyer . Abdominal muscle use dur ing breath ing i n the anesthet ized dog. R e s p i r . P h y s i o l . 70: 159-171, 1987. 14. Greer , J . J . and R . B . S t e i n . Fusimotor c o n t r o l of muscle sp indle s e n s i t i v i t y during r e s p i r a t i o n i n the ca t . J . P h y s i o l . 422: 245-264, 1990. 98 15. Isaza, G . D . , J . D . Posner, M.D. A l to se , S .G . Kelsen and N.S . Cherniack. Airway o c c l u s i o n pressures i n awake and anesthet ized goats. R e s p i r . P h y s i o l . 27: 87-98, 1976. 16. Kondo, T . , B. Bishop and C. Shaw. Phasic s t r e t c h r e f l e x o f the abdominal muscles. Exper. Neurol . 94: 120-140, 1986. 17. L e d l i e , J . F . , A . I . Pack and A . P . Fishman. Ef fec t s of hypercapnia and hypoxia on abdominal exp ira tory nerve a c t i v i t y . J . A p p l . P h y s i o l . 55(5): 1614-1622, 1983. 18. Leevers , A . M . and J . D . Road. Mechanical response to h y p e r i n f l a t i o n of the two abdominal muscle l a y e r s . J . A p p l . P h v s i o l . 66(5): 2189-2195, 1989. 19. Macklem, P . T . , D.M. Macklem and A. De Troyer . A model of i n s p i r a t o r y muscle mechanics. J . A p p l . P h y s i o l . : Resp ira t . E n v i r o n .  Exerc ise P h y s i o l . 55: 547-557, 1983. 20. Mead, J . Responses to loaded breath ing . A c r i t i q u e and a synthes i s . B u l l . Eur . Phys iopatho l . Resp ir . 15: 61-71, 1979. 21. O l i v e n , A . and S . G . Kelsen . E f f e c t of hypercapnia and PEEP on exp ira tory muscle EMG and shortening. J . A p p l . P h y s i o l . 66(3): 1408-1413, 1989. 22. Road, J . D . and A . M . Leevers . E f f ec t of lung i n f l a t i o n on diaphragmatic shortening . J . A p p l . P h y s i o l . 65(6): 2383-2389, 1988. 23. Road, J . D . , A . M . Leevers , E . Goldman and A. Grass ino . Resp ira tory muscle c o o r d i n a t i o n dur ing expiratory threshold l oad ing . J . A p p l .  P h v s i o l . 70(4): 1554-1562, 1991. 24. R u s s e l l , J . A . , B. Bishop and R . E . Hyatt . Discharge of abdominal muscle alpha and gamma motorneurons during exp ira tory load ing i n ca t s . Exper. Neuro l . 97: 179-192, 1987. 25. Sant'Ambrogio, G. and J . G . Widdicombe. Resp iratory re f lexes a c t i n g on the diaphragm and i n s p i r a t o r y i n t e r c o s t a l muscles of the r a b b i t . J . P h v s i o l . 180: 766-779, 1965. 26. Younes, M . K . , and J . E . Remmers. Contro l of T i d a l Volume and Resp iratory Frequency. In: The Regulat ion of Breathing . Marce l Dekker, I n c . , New York, 1981, 621-671. 99 V. ABDOMINAL MUSCLE ACTIVITY DURING HYPERCAPNIA INTRODUCTION The abdominal muscles of dogs and humans are r e c r u i t e d by hyperpnea produced by hypercapnia (3,15,19,32). Both increased a c t i v a t i o n (EMG) and increased shortening (sonomicrometry) of the abdominal muscles have been demonstrated i n anesthetized dogs during hypercapnia (3,18,20). There do not appear to be any reports of the simultaneous response of the four muscles of the v e n t r a l abdominal wall: rectus abdominis (RA), external oblique (EO), i n t e r n a l oblique (10) and transversus abdominis (TA) to hypercapnia i n awake dogs. However, as has been previously shown i n anesthetized (16,20) and awake (see Chapter IV) dogs, the four abdominal muscles are d i f f e r e n t i a l l y r e c r u i t e d and have d i f f e r e n t r e l a t i v e contributions to expiration, when activa t e d by expiratory threshold loading (ETL); the i n t e r n a l l a y e r (TA and 10) i s p r e f e r e n t i a l l y r e c r u i t e d . Whether differences e x i s t i n the pattern and degree of recruitment of these four abdominal muscles with stimulation by C O 2 i s unclear. Recent studies i n anesthetized dogs (3,18,20) examined both EMG a c t i v i t y and length changes of the abdominal muscles during C O 2 rebreathing. The re s u l t s from the EMG data suggested that the TA was the l e a s t activated by hypercapnia (20). However, analysis of abdominal muscle length changes indicated that the TA shortened more than the EO and RA during hypercapnia (3,18). In addition, Robertson et al., (28) found the greatest blood flow was to the TA during C02 rebreathing i n anesthetized dogs. Shortening of the 10 was not analyzed i n these studies, but evidence from ETL (16) suggests that the 10 would have a response s i m i l a r to the TA. 100 Hypercapnia produces abdominal muscle a c t i v a t i o n v i a p e r i p h e r a l and c e n t r a l chemoreflexes (21) with p o s s i b l y some involvement of vaga l re f lexes (10). In c o n t r a s t , abdominal muscle a c t i v a t i o n dur ing ETL i s thought to be mediated p r i m a r i l y v i a vagal re f l exes and p o s s i b l y modif ied by s p i n a l and supraspinal ref lexes (14). We hypothes ized that d i f f erences i n the mechanisms of a c t i v a t i o n could produce d i f f erences i n degree of a c t i v a t i o n and p a t t e r n of recruitment of the abdominal muscles during hypercapnia , compared to ETL. The major i ty of s tudies ^ of abdominal muscle response to hypercapnia have used supine anesthet ized animals. However, anesthes ia may a l t e r the e f f ec t s of C O 2 on v e n t i l a t i o n (21), brea th ing p a t t e r n (10) and r e s p i r a t o r y muscle a c t i v i t y (14). Furthermore, posture can inf luence abdominal muscle recruitment (see Chapter III ) and c l e a r l y , the supine p o s i t i o n i s not p h y s i o l o g i c a l for the dog. These confounding fac tors may e f f e c t the recruitment pa t t ern of the abdominal muscles. Therefore , to assess the response of the major exp ira tory muscles (the abdominal muscles) to hypercapnia, abdominal muscle length changes and a c t i v a t i o n were measured during C0 2 rebreathing i n awake dogs i n the l e f t l a t e r a l decubitus p o s i t i o n . METHODS Five tracheotomized female, mongrel dogs (weight 23-29 kg) were s tud ied . The dogs were t r a i n e d to l i e q u i e t l y , while brea th ing through a cuffed tracheostomy tube, during the experimental p r o t o c o l s . Instrumentation One p a i r of 2.5mm double- lensed, p i e z o e l e c t r i c transducers 101 ( c r y s t a l s ) (10-15mm apart) and b i p o l a r , f i n e wire EMG electrodes were s u r g i c a l l y implanted i n each of the four abdominal muscles as described i n Chapter I I . Two dogs were s u r g i c a l l y implanted with femoral a r t e r y catheters (vascular access port, model GPV, Access Technologies, I L ) which allowed a r t e r i a l blood sampling from a subcutaneous port located i n the dorsal lumbar region. Measurements Abdominal muscle lengths were measured with the p i e z o e l e c t r i c c r y s t a l s . The c r y s t a l s were connected to i s o l a t e d f i n e wires. The bared ends of the e x t e r i o r i z e d wires were i n turn connected by minigrabbers and shielded cable to a sonomicrometer (model 120, T r i t o n Technology, San Diego, C a l . ) . The r e s t i n g length of the muscle, with «• the dog l y i n g i n the l e f t l a t e r a l decubitus p o s i t i o n , was termed L R L (as described i n Chapter I I ) . Change from the r e s t i n g length, e i t h e r lengthening or shortening, during t i d a l breathing, was expressed as a percentage of the i n i t i a l FRC r e s t i n g length ( % L R L ) . Muscle shortening was further defined as active phasic or t o n i c . Active shortening was determined to be that c o i n c i d i n g with phasic raw EMG a c t i v i t y . During C0 2 rebreathing, there was often a decrease i n the baseline length from the i n i t i a l r e s t i n g length ( L R L ) . A new baseline length during hypercapnia was termed the active baseline length ( L A B L ) and was considered to be tonic shortening. The bared ends of the EMG electrode wires were connected by minigrabbers and shielded coaxial cable to a m p l i f i e r s . The electromyographic signals were amplified and f i l t e r e d (Grass model P511, 102 band width 100-10,000 Hz) and recorded i n p a i r s with the sonomicrometer s i g n a l s . V e n t i l a t i o n was measured with a pneumotachograph ( F l e i s c h #1) attached to the d i s t a l end of the tracheostomy tube and a i r f l o w was integrated to give t i d a l volume. Inspiratory (Ti) and expiratory (T E) duration were determined from the flow t r a c i n g on the chart recorder. A two-way valve (#2600 Hans-Rudolph Inc., Kansas Cit y , Mo.) was attached to the pneumotachograph, and the i n s p i r a t o r y and expiratory a i r f l o w was set up as a rebreathing c i r c u i t . Progressive hypercapnia was attained by having the dogs rebreathe from a 10 l i t e r bag containing a f i v e l i t e r , gas mixture of 7%C02, 50%02 and balance N2. End-tidal C0 2 was sampled at the tracheostomy, v i a a needle inserted through the attachment to the pneumotachograph, and concentration (%) determined with a C0 2 analyzer (model IL-200, Fisher S c i e n t i f i c L td.). One m i l l i l i t e r blood samples were obtained during r e s t i n g breathing, before and a f t e r the rebreathing protocol, and twice during the rebreathing period. PC02, pH and P0 2 were measured with a blood gas analyzer (Corning, 168 pH/Blood gas analyzer) and corrected to the dog's r e c t a l temperature. 103 Protocol The f i v e dogs used i n th i s study also underwent one or more of the other protocols on d i f f e r e n t days: postural changes (Chapter I I I ) , expiratory threshold loading (Chapter IV) and vagal blockade (Chapter VI) . This chapter describes the protocol followed f o r C0 2 rebreathing. Repeat studies of C0 2 rebreathing were performed on d i f f e r e n t days to assess the r e p r o d u c i b i l i t y of the r e s u l t s . A l l the studies f o r each dog were performed over periods ranging from two to eight weeks. The rebreathing protocols began with the dog l y i n g on a platform i n the l e f t l a t e r a l decubitus p o s i t i o n . A f t e r a l l the wires were connected and the tracheostomy tube was inserted, a f i v e to ten minute co n t r o l period of quiet breathing followed, to allow the dog to relax and breathing to s t a b i l i z e . The awake dog then rebreathed from a bag containing a mixture of 7% C0 2 and 50% 0 2, f o r approximately f i v e minutes. A l l signals were recorded on an eight channel recorder (Gould, model 8000S). Analysis For each experimental t r i a l , a mean of s i x breaths was analyzed fo r the co n t r o l values. During the rebreathing, e n d - t i d a l C0 2 increased progressively, therefore breathing d i d not s t a b i l i z e and the mean of three breaths around each l e v e l of C0 2 was used. To a i d i n data analysis, end-tidal C0 2 was grouped into three l e v e l s . Low C0 2 ranged from 5.8-6.2%, mid C0 2 ranged from 6.8-7.0% and high C0 2 ranged from 7.4 to 7.6%. For each dog, the data from one or more t r i a l s were averaged and the o v e r a l l mean of these i n d i v i d u a l mean values was c a l c u l a t e d . Data are presented as mean ± SE. whenever comparisons were made among 104 the d i f f e r e n t abdominal muscles, a one-way ANOVA was used and a s i g n i f i c a n c e l e v e l of P<0.05 accepted. Post hoc multiple comparisons were made using a Tukey's multiple comparison t e s t . When s i g n i f i c a n t heterogeneity of standard deviation existed, l og transformations of the data were made and the ANOVAs repeated. To compare a l l the r e s u l t s at d i f f e r e n t l e v e l s of end-tidal C02, a one-way ANOVA was used and a s i g n i f i c a n c e l e v e l of P<0.05 accepted. A Dunnett's multiple comparison tes t was used to t e s t f o r differences from c o n t r o l measures. RESULTS The a r t e r i a l blood gases and pH obtained from the two dogs implanted with the vascular access ports confirms that progressive hypercapnia was attained during the C0 2 rebreathing protocol (Table V) . A r t e r i a l PC0 2 increased and pH decreased as end-tidal C0 2 concentration increased. V e n t i l a t i o n and abdominal muscle a c t i v i t y were stimulated by rebreathing C0 2. A representative t r a c i n g of t i d a l volume (V T) , transversus abdominis (TA) EMG a c t i v i t y and TA length changes during co n t r o l and towards the end of the rebreathing period i s shown i n Figure 19. The TA lengthened with i n s p i r a t i o n (upward d e f l e c t i o n of the volume tracing) and shortened both t o n i c a l l y (change i n baseline length) and p h a s i c a l l y (corresponding to TA EMG a c t i v i t y ) with e x p i r a t i o n during rebreathing. / Table V: A r t e r i a l b lood gas and pH values during CO2 rebreath ing . PETC02 PaC02(mmHg) Pa02(mmHg) pH a C o n t r o l 3 7 . 5 ± 0 . 5 9 5 ± 2 7 . 3 6 ± 0 . 0 3 Low C0 2 4 3 . 8 ± 0 . 5 8 8 ± 2 7 . 3 4 ± 0 . 0 1 Mid C0 2 4 4 . 4 ± 0 . 5 1 3 0 ± 2 7 . 2 9 ± 0 . 0 1 106 Figure 19: A representa t ive recording from an awake dog ( l e f t l a t e r a l decubitus p o s i t i o n ) showing transversus abdominis (TA) length changes, TA EMG a c t i v i t y and t i d a l volume (VT) dur ing C O 2 rebreath ing . The l e f t panel i s c o n t r o l qu ie t breath ing (normocapnia) and the r i g h t panel i s during C O 2 r ebrea th ing . The downward d e f l e c t i o n of the length trace c o i n c i d i n g with i n i t i a t i o n of EMG a c t i v i t y i s ac t ive shortening. 108 The e f f ec t s of hypercapnia on V T , minute v e n t i l a t i o n (V E) and brea th ing p a t t e r n are shown i n Figure 20 and Table V I . At c o n t r o l l e v e l s , V T was 2 9 0 ± 1 1 mis , T T O T was 3 . 6 3 ± 0 . 1 9 sec and V E was 5 . 0 ± 0 . 4 4 1/min (mean ± SE) . With increas ing e n d - t i d a l C 0 2 , V T increased to 8 0 9 ± 6 6 mis, TTOT decreased to 3 . 0 8 ± . 2 1 sec and V E increased to 1 6 . 5 ± 2 . 3 7 1/min. The increase i n v e n t i l a t i o n was due p r i m a r i l y to an increase i n V T ra ther than breathing frequency (Bf) s ince TTOT d i d not change s i g n i f i c a n t l y . However, although Bf d i d not change, T E decreased s i g n i f i c a n t l y at the highest l e v e l of e n d - t i d a l C 0 2 . I n s p i r a t o r y d u r a t i o n (Ti) d i d not change s i g n i f i c a n t l y (Figure 21 and Table VI) . 109 Figure 20: Vj p l o t t e d as a funct ion of expired minute v e n t i l a t i o n (VE) dur ing C 0 2 rebreath ing . V T and V E increased with increas ing e n d - t i d a l C 0 2 concentrat ions . N=5. Values are means ± SE. A s t e r i s k s (*) i n d i c a t e s i g n i f i c a n t l y -d i f f e r e n t from c o n t r o l ( P < 0 . 0 5 ) . 110 Figure 21: V T p l o t t e d against T E and T i during C0 2 r e b r e a t h i n g . N=5. Values are means ± SE. As ter i sks (*) i n d i c a t e s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l (P<0.05). Table VI V e n t i l a t o r y Parameters for r e s t i n g a i r breathing c o n t r o l and three l e v e l s of e n d - t i d a l C O 2 . P E T C 0 2 V T (mls ) TTOT (sec) T E ( sec) Ti (sec) C o n t r o l 2 9 0 ± 1 1 3.631.19 2 . 6 0 ± . 2 1 1 . 0 3 ± . 1 5 Low C0 2 4 4 7 ± 5 7 3 . 4 3 ± . 2 8 2 . 2 6 ± . 2 8 1 . 1 7 ± . 2 3 Mid C0 2 6 6 0 ± 7 1 * 3 . 1 8 ± . 2 3 1 . 8 5 ± . 1 3 1 . 3 3 ± . 1 8 High C0 2 8 0 9 ± 6 6 * 3.081.21 1 . 6 9 ± . l l * 1 . 3 9 ± . 1 5 N=5. Values are means ± SE. A s t e r i s k s (*) i n d i c a t e s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l (P<0.05) 112 The length changes (%LRL) of three abdominal muscles dur ing r e s t i n g c o n t r o l breath ing and at three l e v e l s of e n d - t i d a l C0 2 are shown i n F igure 22. The rectus abdominis was not inc luded because i t had n e g l i g i b l e changes i n l ength . The 0 l i n e i s the r e s t i n g b a s e l i n e length (LRL) , the p o s i t i v e length changes are i n s p i r a t o r y lengthening and the negative changes are shortening below the r e s t i n g b a s e l i n e l ength . A c t i v e shortening i s represented by the shaded p o r t i o n and ton ic shortening by the unshaded p o r t i o n of the negative l ength changes. I n s p i r a t o r y lengthening d i d not change s i g n i f i c a n t l y from c o n t r o l values for any of the abdominal muscles, during the s t imulated b r e a t h i n g . Both ton ic and a c t i v e shortening were increased from c o n t r o l values at the h igh C0 2 l e v e l , i n the three abdominal muscles shown. Since v e n t i l a t i o n was s t imulated by the hypercapnia, t o t a l t i d a l l ength changes ( i n s p i r a t o r y lengthening and expiratory shortening) and a c t i v e phas i c , e x p i r a t o r y shortening were p l o t t e d against minute v e n t i l a t i o n (V E) (Figures 23 and 24). As can be seen from these two f i g u r e s , the increase i n t o t a l t i d a l length change of each of the abdominal muscles as V E increased was due p r i m a r i l y to the increase i n a c t i v e phas ic shortening (Figure 24). For example, the t o t a l t i d a l l ength change o f the TA increased to 1 2 . 8 ± 2 . 4 % L R L of which approximately 70% was a c t i v e exp ira tory shortening . Both the i n t e r n a l obl ique (10) and TA a c t i v e l y shortened to a greater extent than the external obl ique (EO) as V E increased wi th i n c r e a s i n g l e v e l s of e n d - t i d a l C0 2 (Figure 24). 113 Figure 22: Length changes of the transversus abdominis (TA), i n t e r n a l obl ique (10) and ex terna l obl ique (EO) during a i r - b r e a t h i n g c o n t r o l and at three l e v e l s of e n d - t i d a l C0 2 . The 0 l i n e i s the r e s t i n g length of the muscles during normocapnia, the p o s i t i v e length changes are i n s p i r a t o r y lengthening and the negative changes are shortening below the r e s t i n g base l ine l ength . A c t i v e shortening i s represented by the blackened p o r t i o n and ton ic shortening by the unshaded p o r t i o n of the negative length changes. Values are means ± SE. 115 5 7 9 11 13 15 17 Ventilation ( l /min) Figure 23: T o t a l t i d a l length changes of the TA, 10 and EO (%LRL) p l o t t e d against increas ing minute v e n t i l a t i o n dur ing C O 2 rebreath ing . N=5. Values are means ± SE. A s t e r i s k (*) ind ica te s s i g n i f i c a n t d i f f erence between EO compared to TA and 10 (P<0.05). Figure 24: A c t i v e shortening of the TA, 1 0 and EO (%LRL) p l o t t e d against increas ing minute v e n t i l a t i o n dur ing C 0 2 rebrea th ing . N=5. Values are means ± SE. A s t e r i s k s (*) i n d i c a t e s s i g n i f i c a n t d i f ference between EO and TA and 1 0 ( P < 0 . 0 5 ) . 117 DISCUSSION This study shows that the awake dog responds to progress ive hypercapnia with an increase i n minute v e n t i l a t i o n ( V E ) , p r i m a r i l y by means of an increase i n t i d a l volume ( V T ) . The abdominal muscles are a c t i v a t e d and e x h i b i t both ton ic and phasic exp ira tory shortening during C0 2 r ebreath ing . In a d d i t i o n , the i n t e r n a l abdominal muscle l ayer (TA and 10) i s p r e f e r e n t i a l l y r e c r u i t e d and shortens to a greater extent than the ex terna l l ayer (RA and EO). Hypercapnia can generate a v a r i e t y of d i f f e r e n t breath ing p a t t e r n s . Breathing patterns observed i n var ious s tudies inc lude: increased V T and breath ing frequency (Bf) (23); increased V T and decreased Ti and T E (22,29); and increased V T , decreased T E and no change i n T i (6,10). Di f ferences i n breathing patterns could r e s u l t from a number of fac tors such as the range of PC0 2 used, the method of measuring t iming parameters, species d i f f erences , anesthes ia and the method of producing hypercapnia . In the present study, rebreath ing C0 2 increased v e n t i l a t i o n by increas ing VT alone. Although Bf (TTOT) d i d not change, T E decreased ( s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l at the higher PC0 2 l e v e l s ) and the T i / T E r a t i o increased during hypercapnia. S i m i l a r r e s u l t s have been reported for the v e n t i l a t o r y response o f awake cats (10) and dogs (1) to s teady-state hypercapnia. In those two s tud ies , T E decreased but Ti was unchanged, and thus the T i / T E r a t i o increased as PC0 2 increased . However, i n contrast to what was found i n the present study, T T OT a l so decreased. This divergence i n r e s u l t s could be a t t r i b u t e d to the e f fec t s of rebreathing versus s teady-state methods. I t has been shown i n awake human subjects that i n the f i r s t 2-3 minutes of t r a n s i t i o n to a new l e v e l of PC0 2 , Ti and T E tend to change i n 118 opposite d i r e c t i o n s with consequently, l i t t l e or no change i n TTOT (9) . Such was the case i n the present study; T i a c t u a l l y increased s l i g h t l y dur ing rebreath ing , whereas TE decreased (Table V I ) . Breathing patterns i n which V T increased and Tj and T E both decreased dur ing hypercapnia were reported from two a d d i t i o n a l s tudies of awake dogs, a l so us ing steady-state methods (22,29). Aga in , methodological d i f f erences could exp la in some o f the d i screpanc ie s i n r e s u l t s among the s tud ie s . Smith et al. (29), used a flow through hood apparatus and Ti and T E were measured from flow record ings . The c o n t r o l Ti reported i n that study was greater than that found i n the present study and i n other s tudies from the same labora tory , i n which awake dogs breathed through masks (1). Therefore, i t may be that the t iming parameters were a l t e r e d by the hood apparatus. P h i l l i p s o n et al. (22), reported r e s u l t s from four awake, tracheotomized dogs but gave no s t a t i s t i c s for the hypercapnia data. However, upon c lose examination of the data , i t appears that c o n t r o l Bf and Ti had a wide range of v a r i a b i l i t y and two of the dogs had a much slower Bf and greater Ti at r e s t and there fore , a r e l a t i v e l y greater increase i n Bf and decrease i n Ti as PC02 increased . Regardless of whether or not hypercapnia r e s u l t e d i n a decrease i n T i , the T i / T E r a t i o increased with i n c r e a s i n g P C O 2 i n both these s tud ie s . Therefore , based on the present r e s u l t s and those from previous s tud ie s , i t appears that awake dogs respond to hypercapnia much l i k e humans (6,10); p r i m a r i l y by increas ing V T and any increases i n Bf r e s u l t from a decrease i n T E , wi th l i t t l e or no change i n T i . The d i f f erence i n breathing pat tern response to hypercapnia i n awake dogs (present r e s u l t s and (1)) and cats (10) compared to the anesthet ized animal supports the conclus ion that anesthes ia e f f e c t s the 119 breath ing p a t t e r n response to hypercapnia , as w e l l as the v e n t i l a t i o n response. Un l ike awake animals , anesthet ized dogs (19,27) and cats (10,35) responded to hypercapnia by i n c r e a s i n g both V T and Bf; the increase i n Bf r e s u l t i n g from a p r o p o r t i o n a l decrease i n both T i and TE ( T I / T e r a t i o d i d not change). I t i s thought that the decrease i n T i , as VT increases during hypercapnia , r e f l e c t s a p o t e n t i a t i o n of vaga l i n h i b i t o r y re f l exes by anesthes ia (24,36). Previous s tudies i n anesthet ized (19,34) and awake dogs (29) and humans (32) demonstrated that abdominal muscle exp ira tory a c t i v i t y i s s t imulated by hypercapnia . In a d d i t i o n , i t has been shown i n anesthet ized dogs that abdominal muscle a c t i v a t i o n (EMG) i s as soc ia ted with phas i c , exp ira tory shortening (sonomicrometry) dur ing hypercapnia (3,20). The present study extends these f indings to awake dogs and shows that abdominal muscle a c t i v a t i o n by hypercapnia r e s u l t s i n phas i c , exp ira tory shortening and i n a d d i t i o n , ton ic shortening of the abdominal muscles. I t i s d i f f i c u l t to compare the a c t i v e , phasic abdominal muscle shortening found i n the present studies with that reported i n s tudies of anesthet ized dogs because of the d i f ferences i n v e n t i l a t i o n and breath ing p a t t e r n response to hypercapnia and the d i f ferences i n methods of ana lyz ing ac t ive shortening . Two studies r e p o r t i n g abdominal muscle length changes dur ing hypercapnia (3,20) analyzed phasic shortening as the decrease i n abdominal muscle length below the re laxed FRC length . This method would inc lude ton ic shortening, which i f present , would r e s u l t i n an overest imat ion of a c t i v e , phasic shortening as def ined i n the present study. In a d d i t i o n , only one study d e t a i l e d v e n t i l a t o r y parameters (20). Despite these d i f f i c u l t i e s , when EO t o t a l t i d a l length 120 changes were compared at a s i m i l a r v e n t i l a t i o n (12.41/min (20) and 12.81/min (Figure 23)) , they were both approximately 4 . 0 % L R L . Furthermore, i f i t i s assumed that a s i m i l a r e n d - t i d a l PCO2 would produce comparable V E i n s i m i l a r preparat ions of anes thet ized dogs (3,20), then the r e s u l t s of TA length changes during CO2 rebreath ing (3) can be compared to those found i n the present study. When TA shortening below FRC was c a l c u l a t e d ( tonic shortening and ac t ive phas ic shortening) i t was found to be qui te comparable to that reported i n the e a r l i e r study (3), approximately 8 .0%LRL and 10 .5%LRL , r e s p e c t i v e l y . The present r e s u l t s of TA % L R L a l so compare favourably to those reported at a V T o f 750 ml (5.1%), for awake dogs l y i n g i n the r i g h t l a t e r a l decubitus p o s i t i o n (12). No other studies describe 10 length changes but i t i s l i k e l y that i t fo l lows the same trend. The general conc lus ion there fore , i s that abdominal muscle exp iratory shortening i s s i m i l a r i n the awake compared to the anesthetized dog (at l e a s t i n the l y i n g p o s i t i o n s ) when i t i s normalized to V E . Phas ic , abdominal muscle shortening has severa l important e f fec t s dur ing hypercapnic hyperpnea. C l e a r l y , a d i r e c t e f f e c t i s an increase i n e x p i r a t o r y flow r e s u l t i n g i n a greater ac t ive e x p i r a t o r y c o n t r i b u t i o n to V T . But i n a d d i t i o n , there are a number of i n d i r e c t e f f e c t s as a consequence of the increase i n ac t ive , exp ira tory shortening which occurs below the muscle's i n i t i a l r e s t i n g length ( L R L ) . That i s , hypercapnic s t i m u l a t i o n of the abdominal muscles r e s u l t e d i n a decrease i n abdominal muscle end-expiratory length ( L E E ) (Figure 22). One r e s u l t would be an increase i n outward r e c o i l of the chest w a l l when the muscles re laxed at end-exp ira t ion . This r e c o i l may produce a s i g n i f i c a n t pass ive component of i n s p i r a t o r y mechanical flow, despite 121 increas ing i n s p i r a t o r y muscle a c t i v a t i o n and i n s p i r a t o r y flow. Evidence to support t h i s proposal i s provided by observations from two studies of awake dogs (1,30). I t was shown that there was a delay between onset of i n s p i r a t o r y mechanical flow (Ti) and the onset of diaphragm EMG a c t i v i t y that was approximately 19% of T i at r e s t and 23% of T i during hypercapnic (6 .5% C 0 2 ) hyperpnea (30). Thus, assuming that diaphragm EMG a c t i v i t y r e f l e c t s ac t ive i n s p i r a t i o n , a s i g n i f i c a n t p o r t i o n of i n s p i r a t o r y flow was pass ive . Indeed, i t was r e c e n t l y reported that p a r a s t e r n a l i n t e r c o s t a l i n s p i r a t o r y muscles had t iming and a c t i v i t y patterns s i m i l a r to the c o s t a l diaphragm during hypercapnia i n awake dogs and thus, would not be respons ib le for any i n s p i r a t i o n preceding diaphragm a c t i v a t i o n (12). Another e f f ec t of a decrease i n abdominal muscle L E E would be to al low VT to increase p a r t i a l l y by u t i l i z i n g the e x p i r a t o r y reserve volume (ERV). In f a c t , Smith et al., (30) estimated from the increase i n end-expiratory p l e u r a l pressure i n awake standing dogs, that hypercapnic s t i m u l a t i o n r e s u l t e d i n a decrease i n end-expiratory lung volume (EELV) of approximately 0 . 1 6 l i t e r s . In contras t , during hypocapnic hypoxia , when there was no recruitment of the exp ira tory muscles ( t r i a n g u l a r i s s t e r n i and TA) , EELV increased by an estimated 0 . 2 7 l i t e r s . F i n a l l y , s ince the c r u r a l diaphragm i s more e f fec ted by abdominal excurs ion than the c o s t a l diaphragm (7), a decrease i n abdominal muscle L E E would presumably lengthen the c r u r a l diaphragm. In supine anesthet ized dogs c r u r a l diaphragm r e s t i n g length i s estimated to be 93% of LQ (optimal pressure generating length) (26). Thus, increas ing c r u r a l diaphragm L E E would place i t on a more opt imal part of i t s l eng th - t ens ion curve and increase i t s pressure generat ing c a p a b i l i t y (26). Therefore , the o v e r a l l e f fec t s of abdominal muscle 122 phasic shortening would be to d i s t r i b u t e the work of brea th ing between the i n s p i r a t o r y and exp ira tory muscles and to increase the mechanical e f f i c i e n c y o f the diaphragm. The present study appears to be the f i r s t to demonstrate ton ic abdominal muscle shortening during hypercapnic hyperpnea. There i s some s l i g h t i n d i r e c t evidence of tonic abdominal muscle a c t i v i t y from two s tudies o f awake dogs (1,30). A trend towards an increase i n base l ine g a s t r i c pressure reported by Ainsworth et a l . , (1) was a t t r i b u t e d to ton i c abdominal muscle a c t i v i t y . However, s ince the awake dogs were s tud ied i n the standing p o s i t i o n , any change i n ton i c a c t i v i t y due to hypercapnic s t i m u l a t i o n may have been obscured by the e f f ec t s of posture on abdominal muscle ton ic a c t i v i t y (see Chapter I I I ) . Another study from the same labora tory , a lso us ing standing dogs, showed post-e x p i r a t o r y e x p i r a t o r y EMG a c t i v i t y (PEEA) o f the TA extending into i n s p i r a t i o n , dur ing hypercapnia (30). I t i s p o s s i b l e that PEEA would not a l low the muscle enough time to re lax back to i t s r e s t i n g base l ine length and would therefore manifest as ton ic shortening . Aga in , any change i n ton i c EMG a c t i v i t y would be d i f f i c u l t to determine, s ince ton i c a c t i v i t y may have already been present due to the standing p o s i t i o n (see Chapter I I I ) . What would be the p o t e n t i a l e f fects of t on i c abdominal muscle shortening? 1. I t i s poss ib le that by a l lowing the diaphragm to begin shortening from a more optimal length (26), the mechanical advantage of the diaphragm would be increased. 2. Tonic abdominal muscle a c t i v i t y would decrease abdominal w a l l compliance. Reduced abdominal compliance enables the diaphragm to expand the r i b cage more e f f e c t i v e l y through both i n s e r t i o n a l and a p p o s i t i o n a l components (17). 3. I t may be that 123 ton i c abdominal muscle a c t i v i t y serves to "track" or brake i n s p i r a t i o n i n much the same manner that p o s t - i n s p i r a t o r y i n s p i r a t o r y a c t i v i t y (PIIA) i s thought to "track" e x p i r a t i o n (25). That i s , simultaneous c o n t r a c t i o n o f an tagon i s t i c exp ira tory and i n s p i r a t o r y muscles would act to r e t a r d i n s p i r a t o r y flow. This i n turn would help to prevent a decrease i n Ti and al low V T to increase during progress ive hypercapnia (6). Indeed, T i d i d not change as PCO2 increased i n the present study. The observat ion that i n c r e a s i n g CO2 dr ive reduces PIIA (30), which enables TE to decrease, tends to i n d i r e c t l y support t h i s i n s p i r a t o r y t r a c k i n g hypothes is . Even a l lowing for a decrease i n EELV, there would c l e a r l y a l so be an increase i n e n d - i n s p i r a t o r y lung volume (EILV), s ince VT almost t r i p l e d during rebrea th ing . In a d d i t i o n , i t has prev ious ly been shown that both diaphragm (27) and paras terna l i n t e r c o s t a l muscle (33) shortening increases dur ing hypercapnia. Considering that pass ive lung i n f l a t i o n produced abdominal muscle lengthening i n anesthet ized dogs (16), such an increase i n V T might be expected to increase abdominal muscle e n d - i n s p i r a t o r y length ( L E I ) . However, the LEI of the muscles d i d not change s i g n i f i c a n t l y from c o n t r o l during hypercapnia and was l e ss than might be p r e d i c t e d from the passive c h a r a c t e r i s t i c s of the abdominal muscles (16). These r e s u l t s are i n agreement with those found i n two s tudies of supine, anesthet ized dogs (3,20). The f a c t that the abdominal muscles d i d not lengthen i n sp i t e of an increase i n EILV, impl ies that an increase i n r i b cage c o n t r i b u t i o n to i n s p i r a t i o n was respons ib le for the increased EILV during hypercapnia. Mechanisms that could be invo lved i n a greater r i b cage c o n t r i b u t i o n are increased p a r a s t e r n a l i n t e r c o s t a l muscle shortening or a progress ive increase i n 124 diaphragmatic shortening , i n a s s o c i a t i o n with reduced abdominal compliance (produced by ton ic abdominal muscle a c t i v i t y ) . Thus, although diaphragmatic shortening progres s ive ly increases (8,27), i t s mechanical a c t i o n i s t r a n s f e r r e d to displacement o f the more compliant r i b cage (17) . The l ack of increase i n abdominal muscle e n d - i n s p i r a t o r y (EI) l ength , despi te an increase i n EILV, a lso suggests the presence of ton ic a c t i v i t y mediated by segmental re f l exes . Increas ing EILV would s t r e t c h the abdominal muscles and ac t iva te muscle s t r e t c h r e f l e x e s , r e s u l t i n g i n shortening of the muscle. This hypothesis i s cons i s t ent with the observat ion that the muscles which are the most s t re tched dur ing pass ive lung i n f l a t i o n (TA and 10) (16) are the ones which showed the greatest ton i c shortening during hypercapnia (Figure 22). Abdominal muscle recruitment by hypercapnia i n awake dogs appears to be a f u n c t i o n of the hyperpnea which i s produced. Although p e r i p h e r a l chemoreflexes might be involved i n d i r e c t l y , v i a inf luences on V E (13), c e n t r a l chemoreflexes are thought to be the primary mediators of e x p i r a t o r y muscle recruitment (31). In the absence o f propr ioceptor feedback, C0 2 has an e x c i t a t o r y e f f ec t on exp ira tory neurons (4) and abdominal motoneurons (15). However, there i s a l so a l arge body of evidence to support the conc lus ion that abdominal muscle a c t i v i t y during hypercapnia i s , at l e a s t p a r t i a l l y , mediated by vaga l a f ferents (2,5,11,19). In a d d i t i o n , the re su l t s from the present study suggest that segmental re f l exes may also play a r o l e . Although the c o n t r o l mechanisms invo lved i n abdominal muscle recruitment are d i f f e r e n t during hypercapnia compared to e x p i r a t o r y thresho ld load ing (ETL-see Chapter IV) , the recruitment p a t t e r n which 125 was observed, o f predominantly i n t e r n a l muscle l ayer a c t i v a t i o n , does not d i f f e r from the p a t t e r n seen during ETL. I t cannot be concluded there fore , that vaga l a f ferents a l t e r the general recruitment p a t t e r n of the abdominal muscles i n comparison to CO2 s t i m u l a t i o n a lone . There were some d i f f e r e n c e s , however. ETL produced a greater increase i n abdominal muscle e n d - i n s p i r a t o r y length (Figure 13) compared to hypercapnia (Figure 22) yet there appeared to be less TA ton ic shortening , despi te the greater TA e n d - i n s p i r a t o r y l ength . There a l so appeared to be l e ss phasic TA shortening during ETL compared to hypercapnia but 10 and EO phasic shortening were s i m i l a r . What mechanisms cou ld be respons ib le for these apparent d i f f erences i n abdominal muscle length changes during ETL compared to hypercapnia? Speculat ion leads to two p o s s i b l e explanat ions: (1) i t may be that the TA i s l e ss in f luenced by vagal re f lexes than the other abdominal muscles, which cou ld account for i t s smal ler amount of shortening dur ing ETL; (2) or hypercapnia may have a d i r e c t e f f e c t on abdominal muscle motoneurons making them more exc i tab le and thus, i n c r e a s i n g t h e i r s u s c e p t i b i l i t y to a c t i v a t i o n by muscle sp indle a f f e r e n t s . In summary, progress ive hypercapnia produces ton ic and phasic shortening of the abdominal muscles i n the awake dog, of which the o v e r a l l e f f ec t s would be to r e d i s t r i b u t e some of the work of breath ing to the exp ira tory muscles and to increase the mechanical e f f i c i e n c y o f the diaphragm. 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De Goede and C N . O l i e v i e r . 127 Rela t ive c o n t r i b u t i o n of c e n t r a l and p e r i p h e r a l chemoreceptors to the v e n t i l a t o r y response to C02 during hyperoxia . R e s p i r . P h v s i o l . 37: 365-379, 1979. 14. Isaza, G . D . , J . D . Posner, M.D. A l t o s e , S .G. Kelsen and N . S . Cherniack. Airway o c c l u s i o n pressures i n awake and anesthet ized goats. R e s p i r . P h y s i o l . 27: 87-98, 1976. 15. L e d l i e , J . F . , A . I . Pack and A . P . Fishman. E f f e c t s of hypercapnia and hypoxia on abdominal exp ira tory nerve a c t i v i t y . J . A p p l . P h y s i o l . 55(5): 1614-1622, 1983. 16. Leevers , A . M . and J . D . Road. Mechanical response to h y p e r i n f l a t i o n o f the two abdominal muscle l a y e r s . J . A p p l . P h y s i o l . 66(5): 2189-2195, 1989. 17. Macklem, P . T . , D.M. Macklem and A. De Troyer . A model of i n s p i r a t o r y muscle mechanics. J . A p p l . P h v s i o l . : R e s p i r a t . Env iron .  Exerc i se P h v s i o l . 55: 547-557, 1983. 18. Ninane, V . , J . J . G i l m a r t i n and A. De Troyer . Changes i n abdominal muscle length dur ing breath ing i n supine dogs. R e s p i r . P h v s i o l . 73: 31-42, 1988. 19. O l i v e n , A . , E . C . D e a l , J r . , S .G . Kelsen and N.S . Cherniack. E f f e c t s of hypercapnia on i n s p i r a t o r y and exp ira tory muscle a c t i v i t y dur ing e x p i r a t i o n . J . A p p l . P h y s i o l . 59(5): 1560-1565, 1985. 20. O l i v e n , A. and S . G . Ke l sen . E f f e c t of hypercapnia and PEEP on exp ira tory muscle EMG and shortening . J . A p p l . P h y s i o l . 66(3): 1408-1413, 1989. 21. P a v l i n , E . G . , and T . F . Hornbein. Anesthesia and the c o n t r o l o f v e n t i l a t i o n . In: Handbook of Physiology Sect ion 3: The R e s p i r a t o r y  System. V o l . I I I . P . T . Macklem, and J . Mead (eds) . Wi l l iams & W i l k i n s , Ba l t imore , MD, 1986, 22. P h i l l i p s o n , E . A . Vagal c o n t r o l of breathing pa t t ern independent of lung i n f l a t i o n i n concious dogs. J . A p p l . P h y s i o l . 37(2): 183-189, 1974. 23. P h i l l i p s o n , E . A . , R . F . Hickey, C R . Bainton and J . A . Nadel . E f f e c t of vagal blockade on r e g u l a t i o n of breathing i n concious dogs. J .  A p p l . P h v s i o l . 29(4): 475-479, 1970. 24. P h i l l i p s o n , E . A . , R . F . Hickey, P .D. Graf and J . A . Nadel . Her ing -Breuer i n f l a t i o n r e f l e x and r e g u l a t i o n of breath ing i n concious dogs. J . A p p l . P h v s i o l . 31(5): 746-750, 1971. 25. Remmers, J . E . and D\ B a r t l e t t , J r . . Ref lex c o n t r o l of e x p i r a t o r y a i r f l o w and d u r a t i o n . J . A p p l . P h v s i o l . 42(1): 80-87, 1977. 26. Road, J . , S. Newman, J . P . Derenne and A. Grass ino . In v i v o l ength-force r e l a t i o n s h i p of canine diaphragm. J . A p p l . P h y s i o l . 60(1): 63-128 70, 1986. 27. Road, J . D . , S . L . Newman and A. Grass ino . Diaphragm length and brea th ing p a t t e r n changes during hypoxia and hypercapnia . R e s p i r .  P h v s i o l . 65: 39-53, 1986. 28. Robertson, C . H . , M.A. Pagel and R . L . Johnson. The d i s t r i b u t i o n of b lood flow, oxygen consumption and work output among the r e s p i r a t o r y muscles dur ing unobstructed h y p e r v e n t i l a t i o n . J . C l i n . Invest . 59: 43-50, 1977. 29. Smith, C . A . , D.M. Ainsworth, K . S . Henderson and J . A . Dempsey. D i f f e r e n t i a l responses of exp iratory muscles to chemical s t i m u l i i n awake dogs. J . A p p l . P h y s i o l . 66(1): 384-391, 1989. 30. Smith, C . A . , D.M. Ainsworth, K . S . Henderson and J . A . Dempsey. D i f f e r e n t i a l t iming of r e s p i r a t o r y muscles i n response to chemical s t i m u l i i n awake dogs. J . A p p l . P h y s i o l . 66(1): 392-399, 1989. 31. S t . J o h n , W.M. Resp ira tory neuron responses to hypercapnia and c a r o t i d chemoreceptor s t i m u l a t i o n . J . A p p l . P h y s i o l . 51(4): 816-822, 1981. 32. Takasak i , Y . , D. O r r , J . Popkin, A. X i e and T . D . Brad ley . E f f e c t of hypercapnia and hypoxia on r e s p i r a t o r y muscle a c t i v a t i o n i n humans. J . A p p l . P h v s i o l . 67(5): 1776-1784, 1989. 33. van Lunteren, E . and N.S . Cherniack. E l e c t r i c a l and mechanical a c t i v i t y of r e s p i r a t o r y muscles during hypercapnia . J . A p p l .  P h v s i o l . 61(2): 719-727, 1986. 34. van Lunteren, E . , M.A. Haxhiu, N.S. Cherniack and J . S . A r n o l d . Rib cage and abdominal exp ira tory muscle responses to C02 and esophagel d i s t e n s i o n . J . A p p l . P h v s i o l . 64(2): 846-853, 1988. 35. Widdicombe, J . G . and A. Winning. Ef fec t s of hypoxia , hypercapnia and changes i n body temperature on the p a t t e r n of brea th ing i n c a t s . R e s p i r . P h v s i o l . 21: 203-221, 1974. 36. Younes, M . K . , and J . E . Remmers. Contro l of T i d a l Volume and Resp ira tory Frequency. In: The Regulat ion of Breath ing . Marce l Dekker, I n c . , New York, 1981, 621-671. 129 V I . EFFECTS OF VAGAL BLOCKADE ON ABDOMINAL MUSCLE ACTIVATION INTRODUCTION The abdominal muscles are r e c r u i t e d during e x p i r a t o r y thresho ld loading (ETL) and hypercapnia as has been shown i n Chapters IV and V. The mechanisms o f a c t i v a t i o n are thought to be p r i m a r i l y v i a a v a g a l l y -mediated lung i n f l a t i o n r e f l e x during ETL and v i a vaga l ly -mediated lung i n f l a t i o n re f l exes and c e n t r a l and p e r i p h e r a l chemoreflexes dur ing hypercapnia. However, as has been shown i n Chapters IV and V, and prev ious ly i n anesthet ized dogs, these muscles are r e c r u i t e d non-uniformly and have d i f f e r e n t r e l a t i v e c o n t r i b u t i o n s ' t o e x p i r a t i o n when ac t iva ted by ETL or hypercapnia (13,18,19,21). Such d i f f erences may r e f l e c t d i f f e r i n g contr ibut ions of the p o t e n t i a l mechanisms of a c t i v a t i o n of the abdominal muscles; for example v a r i a b l e degrees of s p i n a l or suprasp ina l r e f l e x a c t i v a t i o n v i a abdominal muscle propr ioceptors . Indeed, i t has recent ly been shown i n unanesthet ized dogs that transversus abdominis (TA) EMG a c t i v i t y was not s i g n i f i c a n t l y reduced fo l l owing vagotomy (9), which suggests that the TA i s under extravagal in f luences . In a d d i t i o n , removal of the v a g a l l y mediated i n s p i r a t o r y i n h i b i t o r y and exp ira tory e x c i t a t o r y e f fec t s on v e n t i l a t i o n and t iming parameters (8) may i n d i r e c t l y e f f ec t abdominal muscle recruitment pa t t erns . Previous s tudies have predominantly u t i l i z e d anesthet ized animals (13,18,19). Since anesthes ia i s known to inf luence re f l exes (12,29) the e f fects of vagotomy i n anesthet ized animals may be confounded by the anesthesia and d i f f e r from the e f fec t s of vagal blockade i n awake animals. Therefore , the object ives of t h i s study were to i d e n t i f y 130 i n d i v i d u a l abdominal muscle recruitment during ETL and hypercapnia i n awake dogs before and a f t e r r e v e r s i b l e vagal blockade to determine to what degree the i n d i v i d u a l muscles are under vagal in f luence . METHODS Six tracheotomized, female mongrel dogs (22-29kg) were c h r o n i c a l l y implanted wi th sonomicrometer transducers and f ine wire EMG e lec trodes i n each of the four abdominal muscles as descr ibed i n Chapter I I . In a d d i t i o n , the dogs had cuffs p laced around both vagus nerves i n the c e r v i c a l reg ion us ing a method modif ied from one developed for r e v e r s i b l e blockade o f p e r i p h e r a l motoneurons (14) (Figure 25). The cuffs cons i s t ed of a 2.5cm long piece of s i l a s t i c tubing , 3/16 i n . I . D . x 5/16 i n . O.D. (Dow Corning C o r p . , Midland, MI) with two 40cm long s i l a s t i c catheters (.04 i n . I . D . x .085 i n . O.D.) penetrat ing two holes punched through the c u f f near the ends. The catheters acted as i n l e t and o u t l e t ports to f a c i l i t a t e the a p p l i c a t i o n and c i r c u l a t i o n of a t o p i c a l anesthet ic around the enclosed nerve. A s t e r i l e s u r g i c a l procedure was fo l lowed as descr ibed prev ious ly (see Chapter II) . Three of the dogs had the cu f f s p laced during the same surgery i n which the sonomicrometer transducers and EMG wires were implanted. The other three dogs had the cuf f s p laced i n a separate surgery, a week a f t e r the implantat ion surgery . The cuffs were placed around the nerve i n s i d e the c a r o t i d sheath, with the catheters d i r e c t e d l a t e r a l l y . The nerves were 2-3mm i n diameter and occupied approximately 50% of the c u f f volume. The c a r o t i d sheath was sutured c losed around the cuf f s to s e a l the c u f f and nerve. The inner f a s c i a l and muscle layers were then sutured separate ly . The catheters were tunnel led under s k i n and e x i t e d through 131 the s k i n of the d o r s a l neck reg ion . To prevent p lugg ing , the catheters were f lushed every 2-3 days with 1 ml of s t e r i l e s o l u t i o n of sodium hepar in (50 u n i t s / m l ) i n l a c t a t e d Ringers . Three of the dogs were a l so s u r g i c a l l y implanted with femoral a r t e r y catheters (vascular access p o r t , model GPV, Access Technologies , I L ) which al lowed a r t e r i a l b lood sampling from a subcutaneous por t l o c a t e d i n the d o r s a l lumbar reg ion . The a r t e r i a l catheters were implanted dur ing the separate surgery i n which the vaga l cuf f s were p laced i n three of the dogs. Measurements Abdominal muscle end-expiratory lengths and length changes were measured wi th the sonomicrometer transducers (see Chapter I I ) . The r e s t i n g base l ine length of the abdominal muscles, with the dog i n the l e f t l a t e r a l decubitus p o s i t i o n , was termed L R L . Changes i n muscle l ength dur ing t i d a l breath ing , e i t h e r i n s p i r a t o r y lengthening or e x p i r a t o r y shortening , were expressed as a percentage o f the i n i t i a l r e s t i n g length (%LRL) . Ac t ive shortening was determined to be that c o i n c i d i n g with raw exp ira tory EMG a c t i v i t y . A change i n base l ine length from the i n i t i a l r e s t i n g base l ine length was a l so expressed as a percentage of the i n i t i a l r e s t i n g l eng th . The new base l ine length was termed the ac t ive base l ine length (LABL)• LABL was u s u a l l y shorter than LRL and there fore , was considered to be a r e f l e c t i o n of ton ic a c t i v i t y . The electromyographic s igna l s from the implanted f ine wire e lec trodes were a m p l i f i e d and f i l t e r e d (Grass, model P511) and recorded i n p a i r s with the sonomicrometer s i g n a l s . Figure 25: Photograph showing placement of a s i l a s t i c c u f f around the r i g h t c e r v i c a l vagus nerve i n one of the dogs. The l e f t side of the picture i s the head end of the dog. 133 Expiratory Threshold Loading A i r f l o w was measured with a pneumotachograph ( F l e i s c h #1) and pressure transducer (Validyne MP45, Medf i e ld , M a s s . ) , attached to the d i s t a l end of the tracheostomy tube, and in tegra ted to give t i d a l volume. The opposite end of the pneumotachograph was connected to a two-way, non-rebreathing valve (Hans Rudolph, model 2600). E x p i r a t o r y thresho ld loads of 6, 10 and 14 cmH20 were produced by apply ing p o s i t i v e end-expiratory pressure (PEEP) v i a a PEEP va lve (Medigas, Model BE-142) attached to exp ira tory s ide of the two-way v a l v e . End-expiratory pressure was measured v i a a porthole i n the two-way va lve and recorded as airway pressure (Pao) ( d i f f e r e n t i a l pressure transducer , Val idyne MP-45, M e d f i e l d , M a s s . ) . During the exp ira tory thresho ld load ( E T L ) , the change i n lung volume (<SVol) was measured i n a d d i t i o n to the c o n t r o l parameters. The SVol was determined from the volume expired when the e x p i r a t o r y load was re leased . I n s p i r a t o r y (Ti) and exp ira tory (T E ) durat ion were determined from the flow t r a c i n g on the chart recorder . One ml a r t e r i a l b lood samples were obtained, v i a the vascu lar access p o r t , during r e s t i n g breathing before and a f t e r the ETL p r o t o c o l , and each l e v e l of PEEP (6,10 and 14 cmH20) dur ing ETL. Blood samples were analyzed (Corning, 168 pH/Blood gas analyzer) f o r PC0 2 , pH and P0 2 . The H C O 3 " was extrapolated from known equations. CO2 Rebreathing V e n t i l a t i o n was measured with a pneumotachograph ( F l e i s c h #1) attached to the d i s t a l end of the tracheostomy tube and a i r f l o w was in tegrated to give t i d a l volume. I n s p i r a t o r y and exp ira tory durat ion were determined from the flow t r a c i n g on the chart recorder . A two-way 134 valve (#2600 Hans-Rudolph I n c . , Kansas C i t y , Mo.) was attached to the pneumotachograph, and the i n s p i r a t o r y and exp ira tory a i r f l o w was setup as a rebreath ing c i r c u i t . Progress ive hypercapnia was a t t a i n e d by having the dogs rebreathe from a 10 l i t e r bag conta in ing a f i v e l i t e r , gas mixture o f 7% C 0 2 , 50% 0 2 and balance N 2 . E n d - t i d a l C0 2 was sampled at the tracheostomy, v i a a needle inser ted through the attachment to the pneumotachograph, and concentrat ion (%) determined wi th a C0 2 analyzer (model IL-200, F i s h e r S c i e n t i f i c L t d . ) . One m i l l i l i t e r b lood samples were obtained dur ing r e s t i n g brea th ing , before and a f t e r the rebreathing p r o t o c o l , and two times during the rebreath ing p e r i o d . Blood samples were analyzed (Corning, 168 pH/Blood gas analyzer) for PC0 2 , pH, and P0 2 , and HC0 3" was determined by e x t r a p o l a t i o n . Protocol A t o t a l o f s i x dogs were used i n the pre and post vaga l blockade experiments: s i x were exposed to expiratory thresho ld load ing (ETL) and three to C0 2 r ebrea th ing . A l l the dogs p r e v i o u s l y underwent one or a l l of the p r o t o c o l s descr ibed i n the preceding chapters . For the vaga l blockade experiments the two d i f f e r e n t experimental pro toco l s (ETL and C0 2 rebreathing) were fo l lowed on d i f f e r e n t days. Every experimental p r o t o c o l began wi th the dog l y i n g i n the l e f t l a t e r a l decubitus p o s i t i o n , on a p l a t f o r m . A f t e r a l l the wires were connected and the tracheostomy tube was i n s e r t e d , a f i v e to ten minute c o n t r o l p e r i o d o f quie t breath ing fo l lowed to al low the dog to re lax and brea th ing to s t a b i l i z e . The awake dog was then exposed to e i t h e r e x p i r a t o r y thresho ld load ing or C0 2 rebreath ing . 135 ETL The two-way non-rebreathing valve and PEEP va lve were connected to the d i s t a l end of the pneumotachograph and when the dog was re laxed and breath ing s t a b i l i z e d , c o n t r o l measurements of abdominal muscle r e s t i n g l ength , length changes and EMG a c t i v i t y , Pao and t i d a l volume were made. An e x p i r a t o r y load was then app l i ed and a short time p e r i o d (1-2 minutes) allowed for a regular breathing p a t t e r n , on the l o a d , to be re -e s t a b l i s h e d . The awake dogs were exposed to three randomly a p p l i e d exp ira tory thresho ld loads (6,10 and 14 CI11H2O) . Between each load , breath ing was al lowed to r e t u r n to quiet base l ine va lues . CO2 Rebreathing Rest ing c o n t r o l measurements of abdominal muscle r e s t i n g l ength , length changes and EMG a c t i v i t y , t i d a l volume and P C O 2 were recorded. The rebreathing c i r c u i t was then attached and the dog rebreathed from a bag conta in ing a mixture of 7% C O 2 and 50% O 2 , f or approximately f i v e minutes. Measurements were made cont inuously throughout the rebreath ing p r o t o c o l . Vagal Blockade Another c o n t r o l p e r i o d fol lowed the ETL or rebreathing p r o t o c o l s , to al low the dog to r e t u r n to previous r e s t i n g l e v e l s . When breath ing had s t a b i l i z e d , the vagus nerves were r e v e r s i b l y blocked b i l a t e r a l l y by i n j e c t i o n of t o p i c a l anesthet ic (4% xylocaine) in to the vagal cuf fs v i a the c u f f catheters . The c o r r e c t dosage of anesthet ic r e q u i r e d and the optimum procedure to fo l low to achieve blockage of the nerve was determined i n 136 three p i l o t s tudies conducted p r i o r to the chronic implanta t ion of the vagal c u f f s . Two a p p l i c a t i o n s (5 minutes apart) o f 2 .5cc of 4% xy loca ine in to each c u f f r e s u l t e d i n an increase i n t i d a l volume and a decrease i n b r e a t h i n g frequency. The r e s u l t s d i d not change when the vagus nerves were cut a f t e r the xylocaine blockade, i n the p i l o t s tud ie s . The anes thet i c was appl i ed by i n f u s i n g 2.5cc in to each c u f f s imultaneously , v i a one of each c u f f s ' catheter and then s lowly withdrawing l - 2 c c , v i a the other catheter from each c u f f , approximately 5 minutes l a t e r . Fresh anesthet ic was then r e a p p l i e d and the procedure repeated. Thi s procedure allowed the anesthet ic to c i r c u l a t e around the nerve and was found to be the most e f f e c t i v e to produce blockage o f the nerve. Vagal blockade was assessed by the fo l lowing c r i t e r i a : (1) reduct ion or e l i m i n a t i o n of the Hering-Breuer i n f l a t i o n r e f l e x fo l lowing e n d - i n s p i r a t o r y occ lus ion of the tracheostomy tube; (2) b i l a t e r a l Horner 's syndrome; (3) increased t i d a l volume; and (4) decreased breath ing frequency. The vagus nerves were cons idered to be e f f e c t i v e l y b locked i f at l e a s t three out of four c r i t e r i a were present . The experimental p r o t o c o l was then repeated with the vagus nerves b locked . A l l s i g n a l s were recorded on an e ight channel recorder (Gould, model 8000S). A n a l y s i s ETL Measurements were averaged over f i v e breaths for each l e v e l o f PEEP, for each study day. Since there were no cons i s tent d i f f erences 137 between s tud ies , the means from each PEEP study were then averaged for each dog and the means per dog, at each l e v e l o f PEEP, were obtained. The means ± SE were then c a l c u l a t e d for each v a r i a b l e . To assess the e f f ec t s of ETL on each v a r i a b l e , a one-way ana lys i s of var iance (ANOVA) was performed and a Dunnett's mul t ip l e comparison t e s t used to determine at what l e v e l of PEEP any d i f f erence may have occurred . A s i g n i f i c a n c e l e v e l o f P<0.05 was accepted. Whenever comparisons were made among the d i f f e r e n t abdominal muscles, a one-way ANOVA was used and m u l t i p l e comparisons were made us ing a Tukey's m u l t i p l e comparison t e s t . When s i g n i f i c a n t heterogenei ty of standard d e v i a t i o n ex i s t ed , l o g transformations of the data were made and the ANOVAs repeated. CO2 Rebreathing For each experimental t r i a l , a mean of s i x breaths was analyzed for the c o n t r o l va lues . During the rebreath ing , e n d - t i d a l CO2 increased p r o g r e s s i v e l y , therefore breath ing d i d not s t a b i l i z e and the mean of three breaths around each l e v e l of C0 2 was used. For each dog, the data from one or more t r i a l s were meaned and the o v e r a l l mean of these i n d i v i d u a l mean values ± SE was then c a l c u l a t e d . A n a l y s i s of var iance was used to determine s i g n i f i c a n t d i f f erences among the v a r i a b l e s and at d i f f e r e n t l e v e l s of PC0 2 . M u l t i p l e comparisons were made us ing e i t h e r a Tukey or Dunnett's m u l t i p l e comparison t e s t (whichever was a p p r o p r i a t e ) . When s i g n i f i c a n t heterogeneity of standard d e v i a t i o n ex i s t ed , log transformations o f the data were made and,, the ANOVAs repeated. Di f ferences were considered to be s i g n i f i c a n t at P<0.05. 138 Vagal blockade A l l the analyses were performed separate ly on the pre and post vaga l blockade data . To assess the e f fects of vagal blockade, p a i r e d T-tes ts were used to compare r e s u l t s pre and post blockade, f o r each v a r i a b l e . D i f ferences were considered to be s i g n i f i c a n t at P<0.05. RESULTS E f f e c t s of Vagal Blockade on Contro l Parameters A l l the dogs exh ib i t ed at l eas t three of the c r i t e r i a used to assess the e f fec t iveness of the vagal b lock: decreased Hering-Breuer r e f l e x , increased t i d a l volume (VT) , decreased breath ing frequency (Bf) or b i l a t e r a l Horner 's syndrome. Completeness of the b l o c k was a l so determined i n one dog, anesthet ized at the end of the experimental p r o t o c o l s , by comparing the re su l t s of c u t t i n g the v a g i to anes thet i c blockade. There was no change i n V T or t iming parameters a f t e r vagotomy compared to blockade. B i l a t e r a l vaga l blockade increased V T by approximately 30% (range of 20 to 73%) and TTOT (decreased breathing frequency) by a mean of 20% (range 5 to 123%) dur ing r e s t i n g breathing (Table V I I and Figures 29 and 30) . Although there was no s i g n i f i c a n t d i f ference between m'ean VE before and a f t e r vaga l blockade (Table V I I ) , i n d i v i d u a l dogs e x h i b i t e d changes; two dogs decreased V E and four dogs increased V E a f t e r blockade. C o n t r o l a r t e r i a l b lood gases were a lso e f fec ted by b l o c k i n g the nerve. When the c o n t r o l values from the ETL and C0 2 rebreath ing 139 experiments were combined, the P aC0 2 a f t e r blockade was 33.5±1.4mmHg (mean±SE); s i g n i f i c a n t l y lower (P<0.05) than the P aC0 2 before blockade (37.6±1.7mmHg) (Tables VIII and X). In addition, e n d - t i d a l C0 2 measurements were made i n two dogs, at the same time as P aC0 2. For these two dogs, end-tidal C0 2 also decreased a f t e r vagal blockade, from 4.5 to 4.1 and 4.4 to 3.8%. ETL Six dogs were exposed to expiratory threshold loading before and a f t e r blockade of the vagus nerves. A l l s i x dogs were included i n the r e s u l t s of the eight dogs exposed to ETL discussed i n Chapter IV, however only the r e s u l t s from these s i x dogs are presented i n t h i s chapter. Before vagal blockade, ETL produced recruitment of the TA, 10 and EO as was discussed i n Chapter IV. Following vagal blockade, a c t i v e , phasic expiratory shortening ( % L R L ) of the 10 and EO was reduced but % L R L of the TA was unchanged i n response to ETL, at comparable l e v e l s of PEEP (Figure 26). Since the change i n lung volume (increase i n FRC) produced by ETL was greater a f t e r vagal blockade (Figure 32) and i t i s the increase i n lung volume which i s thought to stimulate abdominal muscle a c t i v i t y , abdominal muscle shortening was normalized to the change i n lung volume. When % L R L was compared at the same change i n lung volume, a l l three muscles (TA, 10 and EO) shortened s i g n i f i c a n t l y l e s s a f t e r vagal blockade (Figure 27) . The e f f e c t s of vagal blockade on 10 a c t i v i t y and V T are shown by the representative t r a c i n g i n Figure 28. 140 0 I i l i l i J i l i l i I i I 0 2 4 6 8 10 12 14 Pao (cmH 2 0) Figure 26: Abdominal muscle ac t ive phasic shortening versus airway pressure (Pao) during ETL. A s t e r i s k s (*) ind ica te s i g n i f i c a n t d i f f e r e n c e between pre and post vagal blockade shortening (P<0.05). I . I . I I i I i L _ 0 50 100 150 200 250 300 350 Volume Change (ml) Figure 27: Abdominal muscle active phasic shortening versus the change i n volume during ETL. Asterisks (*) indicate s i g n i f i c a n t difference between pre and post vagal blockade shortening at the same change i n volume (P<0.05). 142 Figure 28: Representat ive recording from an awake dog ( l e f t l a t e r a l decubitus p o s i t i o n ) showing t i d a l volume (V T) , airway pressure (Pao), i n t e r n a l obl ique (10) length changes and 10 EMG a c t i v i t y during expiratory threshold l o a d i n g . The l e f t panel (Pre) i s before vagal blockade and the r i g h t panel (Post) i s a f t e r vagal blockade. The downward d e f l e c t i o n of the l ength trace c o i n c i d i n g with i n i t i a t i o n of EMG a c t i v i t y i s a c t i v e , phasic shortening. 10 EMG a c t i v i t y and shortening are e f f e c t i v e l y e l iminated fo l lowing vagal blockade. 144 V e n t i l a t o r y parameters are given i n Table VII. T i d a l volume was maintained during ETL both before and a f t e r vagal blockade but was s i g n i f i c a n t l y greater at a l l le v e l s of PEEP when the vagus nerves were blocked compared to i n t a c t (Figure 29). In contrast, T T O T was unchanged by ETL before vagal blockade but increased with increasing Pao a f t e r blockade and was greater at a l l lev e l s of PEEP compared to i n t a c t (Figure 30). As a r e s u l t , there was a s i g n i f i c a n t h y p o v e n t i l a t i o n during ETL when the vagus nerves were blocked. The increase i n T T O T a f t e r vagal blockade was due pr i m a r i l y to a greater Ti at r e s t but during ETL, the increase i n T T O T was due to an increase i n T E (Figure 31). Both Ti and T E were s i g n i f i c a n t l y d i f f e r e n t a f t e r vagal blockade at a l l l e v e l s of PEEP compared to i n t a c t . As the l e v e l of PEEP increased, there was an increase i n end-expiratory lung volume, i n d i c a t i n g an increase i n FRC (Figure 32) . There was a greater increase i n FRC a f t e r vagal blockade at a l l l e v e l s of PEEP (Figure 32). A r t e r i a l blood gases were obtained during ETL before and a f t e r vagal blockade. There were no s i g n i f i c a n t differences between PC0 2 or pH at a l l l e v e l s of PEEP pre and post blockade (Table V I I I ) . However, the dogs had a lower P0 2 during ETL a f t e r vagal blockade compared to before (Table VIII) . There was also a trend to an increase i n P aC0 2 with increasing Pao a f t e r vagal blockade (P=.08) Table V I I : V e n t i l a t i o n and t iming parameters during E T L before and a f t er vagal blockade. Pre Vagal Blockade Pao v E V T T T O T T E ..-(cm H 20) (ml "min"1) (ml) (sec) (sec) 0 6486 288 3.21 2.23 ± 1 0 3 3 ± 2 8 ± . 5 3 ± . 4 1 6 5669 287 3.68 2.77 ± 9 4 6 ± 2 5 ± . 5 8 ± . 5 0 10 5333 257 3.50 2.72 ± 8 6 6 ± 2 9 ± . 6 3 ± . 5 6 14 5503 251 3.53 2.76 ± 1 1 7 5 ± 2 8 ± . 6 7 ± . 5 9 Post Vagal Blockade Pao v E vT T T O T T E (cm H 20) (ml 'min"1) (ml) (sec) (sec) 0 6441 * 371 * 3.84 2.60 ± 7 8 9 ± 2 8 ± . 4 4 ± . 3 3 6 5255 * 355 * 4.45 3.33 ± 6 3 6 ± 2 5 ± . 4 1 10 4618 * 336 * 4.68 * 3.61 ± 5 5 8 ± 2 4 ± . 4 2 ± . 3 8 14 ** 4099 321* 4.97* 3.90* ± 4 4 8 ** ± 2 5 ** ± . 4 2 ± . 3 8 * * Pao=airway pressure ( contro l = 0cm H20) Values are means ± S E N=6 A s t e r i s k (*) ind ica tes s i g n i f i c a n t d i f f erence between before and a f t e r vagal blockade (P<0.05). * * ind ica tes s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l (P<0.05). 146 400 - F 375 350 o Pre-vagotomy • Post—vagotomy co vZ> 325 E - § 300 > "5 5- 275 250 _l 14 6 8 10 Pao (cm hLO) 12 Figure 29: T i d a l volume versus airway pressure (Pao) during ETL before and a f t e r vagal blockade. A s t e r i s k s (*) in d i c a t e s i g n i f i c a n t difference between pre and post vagal blockade (P<0.05). 147 F i g u r e 3 0 : T T OT versus airway pressure (Pao) during E T L before and a f t e r vagal blockade. Asterisks (*) i n d i c a t e s i g n i f i c a n t difference between pre and post vagal blockade (P<0.05). v Pre—vagotomy • Post—vagotomy 6 8 10 Pao (cm H.O) Figure 31: Ti and T E versus airway pressure (Pao) during ETL before and after vagal blockade. Solid symbols are post and open symbols are pre vagal blockade. Asterisks (*) indicate significant difference between pre and post vagal blockade (P<0.05). 149 o Pre—vagotomy • Post—vagotomy 0 2 4 6 8 10 12 14 Pao (cm H20) Figure 32: Change i n lung volume (fivol) versus airway pressure (Pao) during ETL before and a f t e r vagal blockade. As t e r i s k s (*) indicate s i g n i f i c a n t difference between pre and post vagal blockade (P<0.05). 150 Table VIII: A r t e r i a l blood gases during ETL before and af t e r vagal blockade. Pre Vagal Blockade Pao PC0 2 P0 2 pH a (cm H20) mmHg mmHg 0 37.6 104.7 7.36 ±2.6 ±12.2 ±.005 6 38.2 .97.9 7.36 ±2.4 ±6.1 ±.011 10 37.1 92.9 7.37 ±1.6 ±8.2 ±.011 14 39.5 83.9 7.35 ±0.8 ±5.0 ±.004 Post Vagal Blockade Pao PC02 P0 2 pH a (cm H20) mmHg mmHg 0 * 33.5 95.8 7.39 ±1.6 ±5.0 ±.013 6 38.1 87.9 7.37 ±2.6 ±15.5 ±.010 10 37.0 84.5 7.37 ±3.1 ±7.9 ±.010 14 39.3 76.3 7.36 ±2.1 ±7.0 ±.006 N=3 Pao=airway pressure A s t e r i s k (*) indicates s i g n i f i c a n t l y d i f f e r e n t from pre-vagal blockade c o n t r o l (P<0.05). 151 CC>2 Rebreathing Three dogs followed the C0 2 rebreathing protocol before and a f t e r vagal blockade. A representative t r a c i n g of TA length changes and EMG and V T during C0 2 rebreathing, before and a f t e r vagal blockade, i s shown i n Figure 33. As was shown previously i n Chapter V, abdominal muscle act i v e , expiratory shortening ( % L R L ) increased with progressive hypercapnia produced by rebreathing C0 2. When abdominal muscle % L R L was compared at s i m i l a r end-tidal PC0 2 a f t e r vagal blockade, shortening of the 10 and EO was s i g n i f i c a n t l y reduced at the highest e n d - t i d a l C0 2 l e v e l compared to pre blockade, but TA % L R L was the same (Figure 34). Since blocking the vagus nerves a f f e c t e d v e n t i l a t o r y parameters and v e n t i l a t i o n increased l i n e a r l y with progressive hypercapnia (Table IX), abdominal muscle % L R L was normalized to minute v e n t i l a t i o n . This was p a r t i c u l a r l y important as the v e n t i l a t o r y response to C0 2 increased post vagal blockade (see below). At i s o v e n t i l a t i o n , % L R L was s i g n i f i c a n t l y reduced (P<0.05) i n a l l three muscles (TA, 10 and EO) a f t e r vagal blockade compared to i n t a c t (Figure 35). The three dogs involved i n the C0 2 rebreathing were also exposed to ETL and as demonstrated i n those s i x dogs, these three dogs had an increase i n r e s t i n g V T and T TOT a f t e r blocking the vagus (Table IX and Figures 36 and 37). V T and V E increased and T E decreased during progressive hypercapnia both pre and post vagal blockade. However, a f t e r vagal blockade, VT, V E, T T O T , T I and T E were a l l s i g n i f i c a n t l y greater (P<0.05) compared to before blockade at any l e v e l of PC0 2 (Table IX and Figures 36 and 37). The increase i n V E may indic a t e that the v e n t i l a t o r y response to C0 2 increased when the vagi were blocked. 152 Figure 33: Representative recording from an awake dog ( l e f t l a t e r a l decubitus position) showing V T , TA phasic shortening (%L R L) and TA EMG during moderate hypercapnia before ( l e f t panel) and a f t e r ( r i g h t panel) vagal blockade. 153 Pre Post 7.2% P E T C 0 2 7.2% P E T C 0 2 154 Figure 34: Abdominal muscle active phasic shortening p l o t t e d against end-tidal PC02 during rebreathing, before and a f t e r vagal blockade. 155 _ l cn c *c CD -+-» o CO CD > o < 4 2 0 12 10 8 6 4 2 0 14 12 10 8 6 v EO pre-vagotomy • EO post-vagotomy • 10 pre-vagotomy • 10 post—vagotomy O TA pre-vagotomy • TA post—vagotomy 8 10 12 14 16 18 Ventilation ( l /min) 20 22 Figure 35: Abdominal muscle ac t ive phasic shortening p l o t t e d against minute v e n t i l a t i o n during rebreath ing , before and a f t e r vaga l blockade. A s t e r i s k s (*) i n d i c a t e s i g n i f i c a n t d i f f erence i n shortening at the same l e v e l of v e n t i l a t i o n before and a f t e r vagal blockade (P<0.05). 156 Table IX: V e n t i l a t o r y Parameters f or r e s t i n g a i r breathing c o n t r o l and three l e v e l s of end-tidal C0 2. Pre Vagal Blockade  PETC02 V T(mls) T T O T (sec) T E(sec) Ti(sec) Control 288±21 4.271.72 3.051.69 1.221.15 Low C0 2 359115 4.121.72 2.211.74 1.911.23 Mid C0 2 6 3 1 + 9 1 3.511.34 2.031.21 1.481.18 High C0 2 767±93 3.481.16 1.891.18 1.581.15 Post Vagal Blockade PETC02 Vi(mls) T T O T (sec) T E(sec) Tj(sec) Control 455186* 5.001.93 3.341.69 1.661.15* Low C0 2 7791203* 4.271.51 2.351,32 2.921.23 Mid C0 2 10661210* 4.131.28* 2.331.39* 1.801.18 High C0 2 13291229* 3.931.41 2.151.48* 1.781.15 N=5. Values are means 1 SE. Asterisks (*) indic a t e s i g n i f i c a n t difference between before and a f t e r vagal blockade (P<0.05) 157 Figure 36: T i d a l volume (V T) pl o t t e d against minute v e n t i l a t i o n during C0 2 rebreathing, before and a f t e r vagal blockade. Asterisks (*) i n d i c a t e . s i g n i f i c a n t d i f f e r e n c e between V T pre and post vagal blockade. Figure 37: T o t a l a i r flow duration (T T O T ) p l o t t e d against minute v e n t i l a t i o n during C 0 2 rebreathing, before and a f t e r vagal blockade. Asterisks (*) indicate s i g n i f i c a n t d i f f e r e n c e between T T O T pre and post vagal blockade. 159 A r t e r i a l blood gases were obtained from two dogs during C O 2 rebreathing before and a f t e r vagal blockade. There were no apparent dif f e r e n c e s between PC02, P0 2 or pH at a l l l e v e l s of PC0 2 pre and post blockade (Table X). Table X: A r t e r i a l Blood Gas and pH values Pre Vagal Blockade PETC02 PaC02(mmHg) Pa02(mmHg) pH a C o n t r o l 3 7 . 5 ± 0 . 4 9 5 ± 2 7 3 6 ± 0 . 0 3 Low C0 2 4 3 . 8 ± 0 . 5 8 8 ± 2 7 . 3 4 ± 0 . 0 1 Mid C0 2 4 4 . 4 ± 0 . 5 1 3 0 ± 2 7. 2 9 ± 0 . 0 1 Post Vagal Blockade PETC02 PaC02(mmHg) Pa02(mmHg) pH a C o n t r o l 3 3 . 5 ± 3 . 5 8 6 ± 1 1 7 . 3 7 ± 0 . 0 2 Low C0 2 4 2 . 8 ± 2 . 5 7 8 ± 6 7 . 3 3 ± 0 . 0 1 Mid C0 2 4 7 . 7 ± 2 . 3 150+16 7. 2 9 ± 0 . 0 1 (N-2) 161 DISCUSSION E f f e c t s of Vagal Blockade on Control Parameters The approximately 30% increase i n V? and the 20% decrease i n Bf, which occurred a f t e r a p p l i c a t i o n of a t o p i c a l anesthetic to the vagus nerves, are consistent with the attainment of vagal blockade. Anesthetic block has been shown to e f f e c t the large myelinated (lung i n f l a t i o n afferents) and small myelinated (lung i r r i t a n t a f f e rents) f i b e r s equally, with no si z e d i f f e r e n t i a l (10). Therefore, complete block of the vagus nerves would remove affer e n t information from both the i n s p i r a t o r y i n h i b i t o r y and expiratory excita t o r y lung i n f l a t i o n receptors and from the i n s p i r a t o r y f a c i l i t a t o r y lung i r r i t a n t receptors (8). The r e s u l t s of t h i s study are consistent with reduction of both i n h i b i t o r y and ex c i t a t o r y influences (8). Although V T increased and Bf decreased, there was no change i n V E, unlike r e s u l t s from other studies i n which V E increased a f t e r vagal blockade i n awake dogs (22,23). However, several of these e a r l i e r studies were from the same laboratory and appeared to be conducted on the same three dogs (22,23). Results s i m i l a r to those of the present study were reported by Kelsen et a l . , (16). They found considerable v a r i a b i l i t y i n the change i n V E produced by vagal blockade: V E increased i n three and decreased i n two dogs but the mean V E was unchanged. In the present study, V E increased i n four and decreased i n two dogs but the mean V E was unchanged. The control pre vagal blockade a r t e r i a l blood gas values are i n agreement with a previous study which s p e c i f i c a l l y i n v e s t i g a t e d the "normal" blood gases f o r awake dogs (36). For example, r e s t i n g a r t e r i a l 162 PC0 2 ( P a C 0 2 ) was 37.6 mmHg i n the present study compared to the "normal" P a C 0 2 o f 35.9mmHg (36). Fol lowing vaga l b lockade, c o n t r o l P a C 0 2 decreased by a small but s i g n i f i c a n t amount (Table V I I I ) . In a d d i t i o n , i n two dogs i n which i t was measured, e n d - t i d a l C0 2 a l so decreased. Few reports are a v a i l a b l e on the e f fects of vagal blockade on e i t h e r P a C 0 2 or a l v e o l a r PC0 2 ( P A C 0 2 ) i n awake animals and those that are , present c o n f l i c t i n g r e s u l t s . An e a r l y study of three awake dogs, whose v a g i were b locked wi th t o p i c a l anesthet ic ( t e t r a c a i n e ) , found no change i n P a C 0 2 (23). In contras t , a l a t e r study of f i v e awake dogs whose v a g i were b locked by c o o l i n g to 0°C reported a mean decrease i n P A C 0 2 (16). Furthermore, P a C 0 2 was found to decrease i n anes thet ized r a b b i t s a f t e r vaga l blockade by c o o l i n g (17). A decrease i n P a C 0 2 would not be s u r p r i s i n g given that the increase i n V T produced by vaga l blockade cou ld r e s u l t i n greater a l v e o l a r v e n t i l a t i o n . E f f e c t s of Vagal Blockade during ETL The e f f ec t s of exp ira tory threshold load ing (ETL) on abdominal muscle phas ic shortening (%LRL) , .V? and FRC pre vaga l blockade were the same as d iscussed i n Chapter IV. The recruitment o f the abdominal muscles helps prevent the increase i n FRC produced by ETL and hence, defends diaphragm length and Vf. Indeed, the change i n volume (6"Vol) that occurred during ETL was much less than would be p r e d i c t e d from pass ive lung i n f l a t i o n curves (26), i n d i c a t i n g the e f fec t iveness of abdominal muscle recrui tment . Abdominal muscle recruitment by ETL i s thought to be p r i m a r i l y v a g a l l y mediated v i a lung i n f l a t i o n re f l exes (4-6). Therefore , e l i m i n a t i o n of pulmonary s t r e t c h receptor a f f erent in format ion by 163 blocking the vagus nerves would be expected to abolish or s i g n i f i c a n t l y reduce abdominal muscle phasic %LRL during ETL. Evidence from studies of anesthetized animals would support t h i s reasoning. That i s , vagotomy e f f e c t i v e l y eliminated abdominal EMG a c t i v i t y i n anesthetized cats (27,28) and dogs (21) during pressure breathing. However, another study of anesthetized dogs found that vagotomy abolished RA and EO EMG a c t i v i t y but only reduced TA a c t i v i t y during ETL (13). S i m i l a r l y , the r e s u l t s a f t e r vagal blockade i n the present study demonstrate that, although abdominal muscle phasic %LRL i s reduced at a s i m i l a r 6Vol (Figure 28) , there i s s t i l l s i g n i f i c a n t recruitment of the abdominal muscles with i n c r e a s i n g PEEP i n awake dogs. This suggests that extravagal mechanisms may be responsible f o r at l e a s t some of the abdominal muscle recruitment. One possible mechanism i s a c t i v a t i o n of abdominal muscle proprioceptors by increased lung volume, r e s u l t i n g i n s p i n a l (segmental) and supraspinal r e f l e x a c t i v a t i o n of the abdominal muscles. The f i n d i n g that the TA was the l e a s t e f f e c t e d by the vagal blockade i s consistent with t h i s .mechanism, since the TA i s the most stretched by passive lung i n f l a t i o n (18). I t has been proposed that the TA has a d i f f e r e n t innervation from the other abdominal muscles, one closer to that of the t r i a n g u l a r i s s t e r n i , and as such, i s not governed by vagal reflexes (9,20). At f i r s t glance, i t might appear that the present r e s u l t s would support t h i s proposal, since the TA i n p a r t i c u l a r did not show a reduction i n phasic expiratory shortening (%LRL) when %LRL was compared at a s i m i l a r Pao, before and a f t e r . v a g a l blockade (Figure 27). However, i n the previous studies apparently no VT or V E data were analyzed and thus, there was no means of normalizing the abdominal muscle a c t i v a t i o n data (which was 164 l i m i t e d to EMG a c t i v i t y ) . Since V T i s increased by vagotomy, the abdominal muscles may be more stretched at end-inspiration, which might a c t i v a t e muscle proprioceptors, thus r e f l e x l y i ncreasing abdominal muscle a c t i v a t i o n . Due to the greater e f f e c t of passive lung i n f l a t i o n on the TA (18), i t would be more l i k e l y to be e f f e c t e d . Therefore, any reduction i n abdominal muscle a c t i v a t i o n r e s u l t i n g from e l i m i n a t i o n of vagal reflexes would be obscured by increases i n segmental r e f l e x e s . Furthermore, one study involved awake dogs whose vagi had been cut 24 hours p r i o r to the study (9). Cutting the vagus nerves would most l i k e l y have profound e f f e c t s on the dogs o v e r a l l state and i n p a r t i c u l a r on v e n t i l a t o r y parameters. In agreement with the present study, recent evidence from awake standing dogs with the vagi blocked by cooling showed a decrease i n TA EMG a c t i v i t y (2). I t i s possible that abdominal muscle recruitment during ETL post vagal blockade was due i n part to an increase i n drive, since there was a trend to an increase i n P aC0 2 as Pao increased (Table V I I I ) . In addition, although mean P aC0 2 may not have changed s i g n i f i c a n t l y , i t has been shown that vagotomy induces within-breath o s c i l l a t i o n s i n a r t e r i a l pH without changing the mean P aC0 2 (34). Therefore, there may have been an increase i n chemical drive to breathe, not r e f l e c t e d by mean P aC0 2. However, despite a possible increase i n drive, abdominal muscle active shortening was s t i l l reduced compared to pre vagal blockade. F i n a l l y , complete vagal blockade may not have been achieved, i n which case lung i n f l a t i o n reflexes could s t i l l have been operative and responsible f o r abdominal muscle recruitment, a l b e i t reduced. However, three out of four of the c r i t e r i a f o r accepting block (increased V T, decreased Bf, decreased Hering-Breuer and b i l a t e r a l Horner's syndrome) 165 were found i n a l l the dogs. These c r i t e r i a are i n d i c a t i v e of e l i m i n a t i o n of vagal r e f l e x e s (23) and would suggest that vagal blockade d i d occur. Although there was a greater change i n lung volume (6"Vol) a f t e r vagal blockade, i t was le s s than would be predicted from passive lung i n f l a t i o n curves and l e s s than that found i n anesthetized dogs a f t e r vagotomy (26). Hence, V T was defended and d i d not decrease with ETL u n t i l a PEEP of 14cmH20 (Figure 33). Some of t h i s load compensation could be a t t r i b u t e d to abdominal muscle phasic shortening, but i t i s apparent that other f a c t o r s are involved. In other words, f o r the same 6"Vol pre and post vagal blockade, abdominal muscle % L R L was reduced. Therefore, other expiratory muscles must have increased t h e i r a c t i v a t i o n to compensate f o r the decrease i n abdominal muscle a c t i v a t i o n . Indeed, i t has been shown that t r i a n g u l a r i s s t e r n i (TS) , an expiratory r i b cage muscle, EMG a c t i v i t y increases during eupneic breathing i n awake dogs, following vagal c o o l i n g (2). Furthermore, i n anesthetized dogs, TS recruitment during ETL was not effected by vagotomy (26). There was a smaller difference between pre and post vagal blockade 6"Vol i n the awake dogs compared to anesthetized dogs pre and post vagotomy (26). A number of factors may be involved to explain t h i s d i f f e r e n c e . (1) Vagal reflexes could be enhanced during anesthesia (11,29). Enhancement of vagal reflexes would be consistent with the previous findings i n Chapter IV. (2) There may be an a f f e c t of state (awake versus anesthetized) such that the importance of vagal mechanisms i s reduced i n the awake state (15). For example, segmental r e f l e x e s may assume a higher p r i o r i t y i n the awake state or c e n t r a l expiratory motoneurons may be more s e n s i t i v e to chemical stimulation i n t h i s s tate. 166 Despite defending V T u n t i l PEEP 14cmH20, the s i g n i f i c a n t h y p o v e n t i l a t i o n dur ing ETL, post vagal blockade (Table V I I ) , ind icates that l oad compensating mechanisms were compromised. The h y p o v e n t i l a t i o n was due to both a decrease i n V T and Bf, whereas before vaga l blockade, n e i t h e r V T or Bf decreased s i g n i f i c a n t l y with load ing (Table V I I ) . This h y p o v e n t i l a t i o n post vaga l blockade i s s i m i l a r , but l e s s marked, than that found i n anesthet ized dogs post vagotomy (26) and again , emphasizes the e f f e c t of anesthes ia to increase vagal tone. In that study, there was a greater increase i n volume during load ing , a f t e r vagotomy, than that found i n the present study. I t was the r e s u l t i n g decrease i n diaphragm length and displacement to a l e ss favourable p o r t i o n of i t s l eng th - t ens ion curve (25), which was considered to be respons ib le for the decrease i n V T that occurred with ETL. As d iscussed i n Chapter IV, abdominal muscle a c t i v a t i o n during ETL helps to maintain optimal diaphragm length and thus defend V T during ETL. Although reduced abdominal muscle a c t i v a t i o n a f t er vagotomy can e x p l a i n the reduct ion i n V T that occurred during ETL, i t does not e x p l a i n the decrease i n Bf. The decrease i n Bf a f t e r vagal blockade was due to a pro longat ion of T E . Such a pro longat ion of T E during ETL, a f t e r vagal blockade, i s unexpected, as T E i s prolonged by a c t i v a t i o n of vagal a f ferents during lung i n f l a t i o n (8). Therefore , non-vagal a f ferents a r i s i n g from the chest w a l l or abdominal muscles may have prolonged T E a f t er vagal b lockade. Support for t h i s hypothesis i s provided by the observat ion that i n t e r c o s t a l ( r i b cage) and lumbar nerve (abdominal muscle) s t i m u l a t i o n i n h i b i t s d o r s a l r e s p i r a t o r y group (DRG) i n s p i r a t o r y neurons i n a manner s i m i l a r to lung i n f l a t i o n (30). The i n h i b i t o r y a c t i o n of the muscle a f ferents on i n s p i r a t o r y neuron a c t i v i t y appears to have a 167 secondary e f f e c t of prolonging the duration of expiratory a c t i v i t y when the muscle afferents are stimulated during e x p i r a t i o n (31). The r e s u l t s i l l u s t r a t e the importance of the vagus nerve and abdominal muscle recruitment f o r load compensation. E f f e c t s of Vagal Blockade during CO2 Rebreathing The r e s u l t s of abdominal muscle phasic shortening (%LRL) during hypercapnia i n the i n t a c t dogs were the same as those discussed i n Chapter V . That i s , the abdominal muscles were r e c r u i t e d by hypercapnia, %LRL increased as end-tidal PCO2 increased and the i n t e r n a l muscle layer (TA and 1 0 ) was more active. Following vagal blockade, there was a s i g n i f i c a n t reduction i n %LRL of a l l the abdominal muscles when compared at a s i m i l a r VE (Figure 3 5 ) . There are apparently no other studies which have investigated the e f f e c t s of e l i m i n a t i n g vagal reflexes on abdominal muscle %LRL during hypercapnia. However, TA %LRL has been shown to be l i n e a r l y r e l a t e d to EMG a c t i v i t y (3) and thus, i t might be expected that abdominal muscle %LRL would be reduced i f EMG a c t i v i t y were decreased. Therefore, these r e s u l t s would seem to be i n agreement with the few others that have investigated the e f f e c t s of vagal blockade or vagotomy on abdominal muscle a c t i v a t i o n (EMG) during hypercapnia (13,20). However, these studies were performed i n anesthetized dogs and anesthesia c l e a r l y enhances vagal a c t i v i t y . One of these studies found that vagotomy abolished EO a c t i v i t y but only reduced TA a c t i v i t y (13), whereas another study reported a reduction i n both EO and TA EMG a c t i v i t y , but not e l i m i n a t i o n of a c t i v i t y during hypercapnia (20). 1 0 a c t i v i t y does not appear to have been i n v e s t i g a t e d i n previous studies. 168 The reduct ion i n abdominal muscle % L R L i n the awake dog, f o l l owing vaga l blockade, impl ies that there i s indeed a s i g n i f i c a n t v a g a l l y mediated component invo lved i n the response to hypercapnia . However, despi te the reduct ion i n abdominal muscle a c t i v i t y , T E was s t i l l able to decrease i n response to C 0 2 , although i t was greater at a l l l e v e l s of PC0 2 ( s i g n i f i c a n t at the highest l e v e l , P<0.05) a f t e r vaga l blockade compared to pre blockade (Table I X ) . T E decreased and hence, Bf increased ( T T O T decreased) dur ing progress ive hypercapnia . Rib cage e x p i r a t o r y muscle a c t i v i t y a f t e r vaga l blockade may have increased. There i s evidence that the t r i a n g u l a r i s s t e r n i (TS) , a r i b cage exp ira tory muscle, i s i n h i b i t e d by pulmonary s t r e t c h receptor feedback, l i k e the diaphragm (33,35). In a d d i t i o n , TS EMG a c t i v i t y was s t imulated by hypercapnia i n awake dogs (32). Therefore , wi th e l i m i n a t i o n of vagal i n h i b i t o r y r e f l e x e s , TS a c t i v i t y might be expected to increase more f o r the same degree of s t i m u l a t i o n , during hypercapnia and counteract the e f f ec t s o f reduced abdominal exp ira tory a c t i v i t y . Accord ing ly , s ince end-expiratory lung volume ( E E L V ) would be defended by t h i s a c t i o n , i t may al low reduct ions i n T E during hypercapnia . I t i s speculated that a predominance o f r i b cage exp ira tory a c t i v i t y w i l l produce a shorter diaphragm at any lung volume and there fore , increase the i n s p i r a t o r y work of b r e a t h i n g . As was found p r e v i o u s l y i n Chapter V , V E increased dur ing progress ive hypercapnia and the increase i n V E was due p r i m a r i l y to an increase i n V T and a decrease i n T E and no change i n T i . A f t e r vaga l blockade, a s i m i l a r pa t t ern of response was found (Table IX) : V E increased due to an increase i n V T and a decrease i n T E (no change i n T i ) . These r e s u l t s would seem to c o n t r a d i c t the e a r l i e r conc lus ion that 169 there i s a s i g n i f i c a n t vagal component to the response to hypercapnia . I f vaga l re f l exes were invo lved i n the hypercapnic response, T i might be expected to decrease dur ing hypercapnia when the v a g i were i n t a c t (7) and fo l l owing vaga l blockade, Ti would not be expected to change. E a r l i e r s tudies o f the e f f ec t s of vagal blockade on hypercapnic response i n awake dogs found these expected r e s u l t s (22,23). However, as d i scussed e a r l i e r , both these studies appeared to be conducted on the same three dogs. In a d d i t i o n , i n the e a r l i e r study (23), two of the dogs were walking on a t r e a d m i l l throughout the hypercapnia p r o t o c o l s . I t i s c l e a r that locomotion would have confounding e f f ec t s on the response to hypercapnia (1). In the second study (22), the v a g i were b locked d i f f e r e n t i a l l y by coo l ing to d i f f e r e n t temperatures. The r e s u l t s are d i f f i c u l t to i n t e r p r e t s ince no s t a t i s t i c s are given and c o o l i n g to 8°C apparent ly increased the v e n t i l a t o r y response to CO2, whereas f u r t h e r c o o l i n g decreased the v e n t i l a t o r y response. In agreement wi th the present r e s u l t s are those from a study i n i n t a c t awake ca t s , i n which Ti d i d not decrease during hypercapnia (12) and those from a study i n awake dogs, i n which the frequency response to hypercapnia was not abo l i shed by vagal blockade (33). Therefore , i t i s concluded that although breath ing during c o n t r o l condi t ions i s markedly a l t e r e d by vaga l blockade, the breathing pa t t ern i n response to hypercapnia i s not . An i n t e r e s t i n g f i n d i n g i n the present study was that f o r the same e n d - t i d a l C 0 2 there was a greater V E a f t er vagal blockade. Th i s increase i n V E was due to an almost doubling of V T . Thi s f i n d i n g appears to be i n contras t to previous reports i n awake dogs, which have reported a r e d u c t i o n i n v e n t i l a t o r y response a f t e r vaga l blockade 170 (22,23). However, i n awake r a b b i t s , vagal blockade d i d not change the s lope or p o s i t i o n of the v e n t i l a t o r y response curve (V E /PC0 2 ) (24) suggesting that there was no reduct ion i n C0 2 response. Furthermore, i n anesthet ized r a b b i t s , v e n t i l a t i o n was increased at low and moderate l e v e l s of PC0 2 f o l lowing vagotomy (17). I t was a l so suggested that vaga l blockade i n awake dogs "may not reduce the response of V E to m i l d degrees of hypercapnia" (23). The l e v e l s o f hypercapnia a t t a i n e d i n the present study could be considered moderate, based on the l e v e l s reported i n other s tudies (17,22,23). In t h i s study, the increase i n v e n t i l a t o r y response to C0 2 post vagal blockade, was marked. I t i s b e l i e v e d there fore , that a pos s ib l e conclus ion to be drawn from these r e s u l t s i s that at low and moderate l eve l s of C0 2 , vaga l re f l exes may be i n h i b i t o r y to the v e n t i l a t o r y response. REFERENCES 1. Ainsworth, D.M. , C A . Smith, S.W. E i c k e r , K . S . Henderson and J . A . Dempsey. The e f f ec t s of locomotion on r e s p i r a t o r y muscle a c t i v i t y i n the awake dog. R e s p i r . P h y s i o l . 78: 145-162, 1989. 2. Ainsworth, D.M. , C A . Smith, B.D. Johnson, S.W. E i c k e r , K . S . Henderson and J . A . Dempsey. Resp iratory muscle a c t i v i t y dur ing vagal blockade i n awake dogs. Am. Rev. R e s p i r . P i s . 141(4): A168, 1990.(Abstract) 3. A r n o l d , J . S . , M.A. Haxhiu, N .S . Cherniack and E . van Lunteren. Transverse abdominis length changes during eupnea, hypercapnia and airway o c c l u s i o n . J . A p p l . P h y s i o l . 64(2): 658-665, 1988. 4. Bishop, B. 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De Troyer , A . , J . J . G i l m a r t i n and V. Ninane. Abdominal muscle use dur ing brea th ing i n unanesthetized dogs. J . A p p l . P h y s i o l . 66(1): 20-27, 1989. 10. F i n k , B .R. and A . M . C a i r n s . D i f f e r e n t i a l slowing and b l o c k of conduct ion by l i d o c a i n e i n i n d i v i d u a l a f f erent myel inated and unmyelinated axons. Anesthesiology 60: 111-120, 1984. 11. F i n k l e r , J . and S. Tscoe . Contro l of breath ing at e l evated lung volumes i n anes thet ized ca t s . J . A p p l . P h y s i o l . 56(4): 839-844, 1984. 12. G a u t i e r , H. P a t t e r n of breathing during hypoxia or hypercapnia o f the awake or anes thet ized cat . Resp ir . P h y s i o l . 27: 193-206, 1976. 13. G i l m a r t i n , J . J . , V . Ninane and A. De Troyer . Abdominal muscle use during brea th ing i n the anesthet ized dog. R e s p i r . P h y s i o l . 70: 159-171, 1987. 14. Hof fer , J . A . and G . E . Loeb. 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P h i l l i p s o n , E . A . , R . F . Hickey, C R . Bainton and J . A . Nadel . E f f e c t of vagal blockade on r e g u l a t i o n of breath ing i n concious dogs. J .  A p p l . P h v s i o l . 2 9 ( 4 ) : 4 7 5 - 4 7 9 , 1 9 7 0 . 2 4 . Richardson, P . S . and J . G . Widdicombe. The r o l e of the vagus nerves i n the v e n t i l a t o r y responses to hypercapnia and hypoxia i n anaesthet ized and unanaesthetized r a b b i t s . R e s p i r . P h v s i o l . 7 : 1 2 2 -1 3 5 , 1 9 6 9 . 2 5 . Road, J . , S. Newman, J . P . Derenne and A. Grass ino . In v ivo length-force r e l a t i o n s h i p o f canine diaphragm. J . A p p l . P h y s i o l . 6 0 ( 1 ) : 6 3 -7 0 , 1 9 8 6 . 2 6 . Road, J . D . , ' A . M . Leevers , E . Goldman and A. Grass ino . Resp iratory muscle c o o r d i n a t i o n during exp ira tory thresho ld load ing . J . A p p l .  P h v s i o l . 7 0 ( 4 ) : 1 5 5 4 - 1 5 6 2 , 1 9 9 1 . 2 7 . R u s s e l l , J . A . and B. Bishop. Vagal a f ferents e s s e n t i a l for abdominal muscle a c t i v i t y dur ing lung i n f l a t i o n i n ca t s . J . A p p l . P h y s i o l . 4 1 ( 3 ) : 3 1 0 - 3 1 5 , 1 9 7 6 . 2 8 . R u s s e l l , J . A . , B. Bishop and R . E . Hyat t . Discharge of abdominal muscle a lpha and gamma motorneurons dur ing exp ira tory loading i n c a t s . Exper. Neuro l . 9 7 : 1 7 9 - 1 9 2 , 1 9 8 7 . 2 9 . Sant'Ambrogio, G. and J . G . Widdicombe. Resp iratory re f lexes a c t i n g on the diaphragm and i n s p i r a t o r y i n t e r c o s t a l muscles of the r a b b i t . J . P h v s i o l . 1 8 0 : 7 6 6 - 7 7 9 , 1 9 6 5 . 3 0 . Shannon, R. I n t e r c o s t a l and abdominal muscle a f ferent inf luence on medullary d o r s a l r e s p i r a t o r y group neurons. R e s p i r . P h y s i o l . 3 9 : 7 3 -9 4 , 1 9 8 0 . 3 1 . Shannon, R. Reflexes from r e s p i r a t o r y muscles and cos tover tebra l j o i n t s . In: Handbook of Physiology. Sect ion 3 : The Resp iratory  System. V o l . 1 1 . A .P .Fishman. (ed). Wi l l iams & W i l k i n s , Bal t imore , MD, 1 9 8 6 , 4 3 1 - 4 4 7 . 3 2 . Smith, C . A . , D.M. Ainsworth, K . S . Henderson and J . A . Dempsey. D i f f e r e n t i a l responses of exp iratory muscles to chemical s t i m u l i i n awake dogs. J . A p p l . P h y s i o l . 6 6 ( 1 ) : 3 8 4 - 3 9 1 , 1 9 8 9 . 173 33. Smith, C . A . , D.M. Ainsworth, K . S . Henderson and J . A . Dempsey. The inf luence o f c a r o t i d body chemoreceptors on exp ira tory muscle a c t i v i t y . R e s p i r . P h v s i o l . 82: 123-136, 1990. 34. Takahashi , E . , A . S . Menon, H. Kato, A . S . S lutsky and E . A . P h i l l i p s o n . C o n t r o l of exp iratory durat ion by a r t e r i a l C02 o s c i l l a t i o n s i n vagotomized dogs. Resp ir . P h y s i o l . 79: 45-56, 1990. 35. van Lunteren, E . , M.A. Haxhiu, N.S . Cherniack and J . S . A r n o l d . Rib cage and abdominal exp ira tory muscle responses to C02 and esophagel d i s t e n s i o n . J . A p p l . P h y s i o l . 64(2): 846-853, 1988. 36. Wise, W.C. Normal a r t e r i a l blood gases and chemical components i n the unanesthet ized dog. J . A p p l . P h y s i o l . 35(3): 427-429, 1973. 174 VII. SUMMARY AND CONCLUSIONS SUMMARY OF MAJOR FINDINGS i (1) The f i r s t ob jec t ive of t h i s study, addressed i n Chapter I I , was to determine whether the abdominal muscles were more a c t i v e i n the awake dog compared to the anesthet ized dog. In a d d i t i o n , the f e a s i b i l i t y of measuring abdominal muscle length changes i n awake dogs, from c h r o n i c a l l y implanted sonomicrometer transducers , was assessed. I t was found that cons i s tent measurements of muscle r e s t i n g length and length changes could be obtained over periods of two weeks and more, without any i n h i b i t i o n of muscle movement or muscle damage (except l o c a l s c a r r i n g conf ined to the area immediately surrounding the implanted transducer) . When abdominal muscle shortening i n the awake dog was compared to that i n the anesthet ized dog, there was s i g n i f i c a n t l y more shortening . I t was not pos s ib l e to determine c o n c l u s i v e l y whether there was greater abdominal muscle tone i n the awake dog, but i t can not be discounted. (2) The second major objec t ive was to assess the e f fec t s of posture on abdominal muscle ton ic and phasic shortening (Chapter I I I ) . I t was hypothesized that the abdominal muscles would p h a s i c a l l y shorten more i n the upr ight postures compared to the l a t e r a l decubitus and that ton ic shortening might be present i n the upr ight postures , due to the h y d r o s t a t i c pressure gradient . A l l of the abdominal muscles shortened more i n the standing and s i t t i n g p o s i t i o n s compared to the l e f t l a t e r a l decubitus . Furthermore, the TA had a cons i s tent s h i f t i n i t s r e s t i n g base l ine length to a shorter length i n the standing p o s i t i o n , which was cons idered to be an i n d i c a t i o n of tonic shortening. (3) Based on r e s u l t s from studies i n anesthet ized dogs (13,14,20), i t was hypothes ized that the abdominal muscles would be p r o g r e s s i v e l y a c t i v a t e d by e x p i r a t o r y threshold loading (ETL) and that the i n t e r n a l muscle l a y e r (TA and 10) would be p r e f e r e n t i a l l y r e c r u i t e d , i n the awake dog. Th i s was indeed what was found (Chapter IV) . In a d d i t i o n , the i n t e r n a l l a y e r was found to exh ib i t tonic shortening dur ing ETL. The recruitment of the abdominal muscles helped to defend t i d a l volume by prevent ing the increase i n lung volume produced by ETL. (4) Since hypercapnia had been shown to ac t iva te the abdominal muscles i n anesthet ized dogs (3,19-21) and since anesthesia i s known to a f f e c t the v e n t i l a t o r y (22) and breathing pa t t ern (12) response to hypercapnia , i t was hypothes ized that the abdominal muscles would be more a c t i v a t e d by CO2 i n the awake dog (Chapter V ) . Furthermore, i t was thought that a c t i v a t i o n of the abdominal muscles by hypercapnia would involve d i f f e r e n t c o n t r o l pathways than ETL and therefore , that the recruitment p a t t e r n of the i n d i v i d u a l muscles might vary and be d i f f e r e n t from the p a t t e r n found dur ing ETL. For the two abdominal muscles (TA and EO) i n which shortening dur ing hypercapnia had been measured i n anes thet ized dogs (3,21), comparable amounts of shortening were found i n the present study. However, t on i c shortening of the muscles, which has not been p r e v i o u s l y reported , was a lso found during progress ive hypercapnia . The abdominal muscles e x h i b i t e d the same pat tern of response dur ing hypercapnia as that found during ETL; p r e f e r e n t i a l recrui tment o f the 176 i n t e r n a l muscle layer (TA and 10). (5) The f i n a l objec t ive of t h i s study was to t r y to assess the c o n t r i b u t i o n of vagal re f lexes to the a c t i v a t i o n of the abdominal muscles, by comparing abdominal muscle a c t i v a t i o n dur ing ETL and hypercapnia , before and a f t er b l o c k i n g the vagus nerves (Chapter V I ) . Although, the abdominal muscles are thought to be a c t i v a t e d v i a d i f f e r e n t mechanisms dur ing ETL and hypercapnia , vagal blockade had a s i m i l a r e f f e c t of reducing the a c t i v a t i o n during both ETL and hypercapnia. In a d d i t i o n , the pa t t ern of recruitment was not changed by e l i m i n a t i o n of the vagal r e f l e x e s . CONCLUSIONS There are severa l conclusions which can be drawn from t h i s study and they w i l l be d iscussed separate ly . (1) The r e s u l t s from t h i s study confirm that the abdominal muscles are r e c r u i t e d at a lower l e v e l of v e n t i l a t i o n than p r e v i o u s l y proposed (5,9,15) and emphasizes the importance of measuring the deeper muscle l a y e r , i n order to obta in an accurate r e f l e c t i o n of abdominal muscle exp ira tory a c t i v i t y . Indeed, recent s tudies i n man, u t i l i z i n g intramuscular e l ec trodes , have demonstrated a c t i v i t y i n the 10 and TA ( i n t e r n a l muscle layer ) at much lower l e v e l s of V E than found p r e v i o u s l y (7,25). The demonstration of abdominal muscle a c t i v e shortening i n the awake dog l y i n g i n the l e f t l a t e r a l decubitus p o s i t i o n (LID), suggests that e x p i r a t i o n i s an ac t ive process even at r e s t . Furthermore, these 177 r e s u l t s i n d i c a t e that measurements of abdominal muscle e x p i r a t o r y a c t i v i t y from surface EMG or abdominal displacement techniques are not s e n s i t i v e to phas ic exp ira tory a c t i v i t y and length changes o f the deeper abdominal muscles (TA and 10). In a d d i t i o n , measurement o f abdominal muscle l ength changes with sonomicrometry provides a means of q u a n t i f y i n g t o n i c shortening as w e l l as ac t ive e x p i r a t o r y shor ten ing . (2) The greater amount of ac t ive exp ira tory shortening found i n the u p r i g h t postures i s not s u r p r i s i n g , g iven the increase i n FRC that occurs and the attendant e x c i t a t i o n of vagal a f f erent s (10). The increase i n phas ic shortening may a l so , i n p a r t , be due to an increase i n e x c i t a b i l i t y o f the s p i n a l motoneurons v i a a c t i v a t i o n o f muscle propr ioceptors (6). Phasic shortening can produce pass ive i n s p i r a t i o n and thus, VT i s shared by i n s p i r a t o r y and exp ira tory muscles. Abdominal muscle proprioceptors may be a c t i v a t e d i n response to the increase i n the h y d r o s t a t i c pressure gradient wi th assumption o f u p r i g h t postures (2). This a c t i v a t i o n i s thought to be re spons ib l e for the appearance of t on i c a c t i v i t y i n the abdominal muscles i n u p r i g h t postures (1,9,15). This study appears to be the f i r s t to c l e a r l y demonstrate ton ic abdominal muscle shortening. In previous s tud ie s , t on i c abdominal muscle a c t i v i t y has been i n f e r r e d from an increase i n EMG background noise (8), g a s t r i c pressure measurements (4) or abdominal diameters (9). The demonstration of ton ic shortening al lows assessment o f the mechanical consequences of tonic a c t i v i t y . I t i s suggested that the ton i c shortening of the TA, found i n the standing p o s i t i o n , was due to greater a c t i v a t i o n of TA muscle proprioceptors compared to the other abdominal muscles. I t was reasoned that the p o s i t i o n of the TA i n the 178 abdominal w a l l and the transverse o r i e n t a t i o n of i t s muscle f i b e r s would make i t more suscept ib l e to s t r e t c h i n g i n the standing p o s i t i o n . Tonic shortening o f the abdominal muscles, p a r t i c u l a r l y the TA ( for the reasons j u s t o u t l i n e d ) , would serve to maintain diaphragm length (11) and prevent the increase i n FRC assoc iated with assumption of upr ight postures (10), and by reducing abdominal compliance, improve r i b cage expansion. Indeed, the diaphragmatic c o n t r i b u t i o n to V T has probably been underestimated by previous measurements o f abdominal volume. (3) I t i s concluded that the a c t i v a t i o n o f the abdominal muscles by ETL helps to prevent the increase i n FRC produced by ETL and hence, defends diaphragm length and V T (24). In the awake dog, abdominal muscle a c t i v a t i o n appears to be inf luenced to a large degree by vaga l r e f l exe s , but i t i s suggested that segmental re f l exes have a greater modulating e f f e c t , than i n the anesthet ized dog. Evidence to support t h i s conc lus ion i s provided by the presence of tonic abdominal muscle shortening dur ing ETL. In anesthet ized dogs, vagal re f l exes are enhanced, as evidenced by the greater amount o f abdominal muscle phasic shortening found dur ing ETL (14,21) compared to that found i n the awake dogs. Furthermore, anesthesia has a depressing e f f ec t on segmental re f l exes (18). (4) I t i s concluded t h a t . t h e a c t i v a t i o n of the abdominal muscles by CO2 funct ions to optimize diaphragm length and, i n response to an increase i n V E , d i s t r i b u t e s the work of breathing between the i n s p i r a t o r y and e x p i r a t o r y muscles. The a c t i v a t i o n of the abdominal muscles during progress ive hypercapnia appears to be p r i m a r i l y v i a chemoreflexes i n the 179 awake dog. However, the tonic shortening e x h i b i t e d by the muscles aga in , suggests a c o n t r i b u t i o n from segmental r e f l e x e s . The ton ic a c t i v i t y may serve to "track" i n s p i r a t i o n (23) and enable shar ing of the work o f b r e a t h i n g . The smaller amount of phasic shortening found i n the awake dogs compared to anesthet ized (3,21) dur ing hypercapnia , and reports that anesthes ia depresses r e s p i r a t o r y neuron response to CO2 (22), would suggest that vagal ref lexes are more invo lved i n abdominal muscle a c t i v a t i o n i n anesthet ized dogs than i n awake dogs. (5) There i s p r e f e r e n t i a l recruitment of the i n t e r n a l abdominal muscle l ayer (TA and 10) dur ing changes i n posture, ETL and hypercapnia; that i s , under what are considered d i f f e r e n t mechanisms o f a c t i v a t i o n . Furthermore, even a f t e r vagal ref lexes are e l iminated by vaga l b lockade, the p a t t e r n of recrui tment i s the same. P r e f e r e n t i a l recrui tment of the i n t e r n a l l a y e r occurred i n anesthet ized dogs (13,14) and awake dogs, during ETL and dur ing hypercapnia and t h i s recruitment p a t t e r n p e r s i s t e d a f t e r vaga l b lockade. I t i s i n f e r r e d that p r e f e r e n t i a l s t r e t c h i n g o f the i n t e r n a l muscles dur ing increases i n lung volume (14) or abdominal volume, leads to a c t i v a t i o n of segmental r e f l exe s , which accounts for e a r l i e r a c t i v a t i o n and greater phasic and ton ic shortening of the i n t e r n a l muscle l a y e r . In a d d i t i o n , there i s no evidence to support separate d i f f e r e n t i a l innervat ion of the i n d i v i d u a l abdominal muscles and the e x p i r a t o r y neurons pro jec t to a l l of the abdominal motoneurons (16,17). Therefore , the p r e f e r e n t i a l recruitment of the i n t e r n a l l a y e r i s probably not due to c e n t r a l mechanisms but , ra ther to d i f f e r e n t pat terns of a c t i v a t i o n of segmental r e f l e x e s . Although vaga l re f lexes may not be respons ib le for d i f f e r e n t i a l 180 a c t i v a t i o n of the abdominal muscles, the vagus nerve i s c l e a r l y important for the load compensating func t ion of the muscles dur ing ETL. Thi s i s p a r t i c u l a r l y true i n the anesthet ized dog, s ince a f t e r vagotomy, lung volume and hence, diaphragm length and V T , are not defended (24). However, i n the awake dog, the pers i s tence o f defence o f end-expiratory lung volume, even a f t e r vaga l blockade, i s evidence o f the r o l e p layed by non-vagal mechanisms. Although vaga l re f l exes do not appear to be r e q u i r e d for the v e n t i l a t o r y and breath ing p a t t e r n response to hypercapnia , they may p lay a r o l e i n abdominal muscle a c t i v a t i o n . During C0 2 s t imulated brea th ing , v a g a l l y mediated augmentation of abdominal muscle a c t i v i t y may help prevent gas t rapping and an increase i n dynamic lung volume thus, defending diaphragm length and a l lowing sharing o f the work of breath ing . REFERENCES 1. Agostoni , E . , and E . J . M . Campbell. The abdominal muscles. In: The  Resp iratory Muscles: Mechanics and Neural C o n t r o l . E . J . M . Campbell , E . Agostoni , and J . Newsom-Davis (eds) . Saunders, P h i l a d e l p h i a , PA, 1970, 175-180. 2. Agostoni , E . , and R . E . Hyatt . S t a t i c behavior of the r e s p i r a t o r y system. In: Handbook of Physiology Sect ion 3: The Resp ira tory  System. V o l . I I I . Wil l iams & W i l k i n s , Bal t imore , MD, 1986, 113-130. 3. A r n o l d , J . S . , M.A. Haxhiu, N .S . Cherniack and E . van Lunteren. Transverse abdominis length changes during eupnea, hypercapnia and airway o c c l u s i o n . J . A p p l . P h y s i o l . 64(2): 658-665, 1988. 4. Campbell, E . J . M . and J . H . Green. The v a r i a t i o n s i n intra-abdominal pressure and the a c t i v i t y of the abdominal muscles dur ing breath ing;a study i n man. J . P h y s i o l . 122: 282-290, 1953. 5. Campbell, E . J . M . and J . H . Green. The exp ira tory func t ion o f the abdominal muscles i n man. An electromyographic study. J . P h y s i o l . 120: 409-418, 1953. 181 6. Davies , A . , F . B . Sant'Ambrogio and G. Sant'Arabrogio. C o n t r o l o f p o s t u r a l changes of end expiratory volume (FRC) by airways s lowly adapting mechanoreceptors. Resp ir . P h y s i o l . 41: 211-216, 1980. 7. De T r o y e r , A . , M. Estenne, V. Ninane, D. van Gansbeke and M. G o r i n i . Transversus abdominis muscle funct ion i n humans. J . A p p l . P h v s i o l . 68(3): 1010-1016, 1990. 8. De T r o y e r , A . , J . J . G i l m a r t i n and V. Ninane. Abdominal muscle use dur ing b r e a t h i n g i n unanesthetized dogs. J . A p p l . P h y s i o l . 66(1): 20-27, 1989. 9. Druz, W.S. and J . T . Sharp. A c t i v i t y of r e s p i r a t o r y muscles i n u p r i g h t and recumbent humans. J . A p p l . P h y s i o l . 51(6): 1552-1561, 1981. 10. Farkas , G . A . , R . E . Baer, M. Estenne and A. De Troyer . Mechanical r o l e of e x p i r a t o r y muscles during breathing i n upr ight dogs. J .  A p p l . P h v s i o l . 64(3): 1060-1067, 1988. 11. F i t t i n g , J . W . , P . A . Easton, R. Arnoux, A. Guerraty and A. Grass ino . Diaphragm length adjustments with body p o s i t i o n changes i n the awake dog. J . A p p l . P h v s i o l . 66(2): 870-875, 1989. 12. G a u t i e r , H. Pa t t ern of breathing during hypoxia or hypercapnia o f the awake or anesthet ized cat . Resp ir . P h v s i o l . 27: 193-206, 1976. 13. G i l m a r t i n , J . J . , V . Ninane and A. De Troyer . Abdominal muscle use dur ing b r e a t h i n g i n the anesthet ized dog. R e s p i r . P h v s i o l . 70: 159-171, 1987. 14. Leevers , A . M . and J . D . Road. Mechanical response to h y p e r i n f l a t i o n of the two abdominal muscle l a y e r s . J . A p p l . P h y s i o l . 66(5): 2189-2195, 1989. 15. L o r i n g , S . H . and J . Mead. Abdominal muscle use during q u i e t , brea th ing and hyperpnea i n uninformed subjects . J . A p p l . P h y s i o l . 52: 700-704, 1982. 16. M i l l e r , A . D . L o c a l i z a t i o n of motorneurons innervat ing i n d i v i d u a l abdominal muscles of the cat . J . Comp. Neurol . 256: 600-606, 1987. 17. M i l l e r , A . D . , K. Ezure and I . Suzuki . Contro l of abdominal muscles by b r a i n stem r e s p i r a t o r y neurones i n the ca t . J . Neurophys io l . 54(1): 155-167, 1985. 18. M u l l e r , N . , G. V o l g y e s i , L . Becker, M.H. Bryan and A . C . Bryan. Diaphragmatic muscle tone. J . A p p l . P h y s i o l . 47: 279-284, 1979. 19. Ninane, V . , J . J . G i l m a r t i n and A. De Troyer . Changes i n abdominal muscle l ength dur ing breathing i n supine dogs. R e s p i r . P h y s i o l . 73: 31-42, 1988. 182 20. O l i v e n , A . , E . C . D e a l . J r . , S . G . Kelsen and N.S . Cherniack. E f f e c t s of hypercapnia on i n s p i r a t o r y and exp ira tory muscle a c t i v i t y dur ing e x p i r a t i o n . J . A p p l . P h y s i o l . 59(5): 1560-1565, 1985. 21. O l i v e n , A . and S . G . Ke l sen . E f f e c t of hypercapnia and PEEP on exp ira tory muscle EMG and shortening. J . A p p l . P h v s i o l . 66(3): 1408-1413, 1989. 22. P a v l i n , E . G . , and T . F . Hornbein. Anesthesia and the c o n t r o l Of v e n t i l a t i o n . In: Handbook of Physiology Sect ion 3: The Resp ira tory  System. V o l . I I I . P . T . Macklem, and J . Mead (eds) . Wi l l iams & W i l k i n s , Bal t imore , MD, 1986, 23. Remmers, J . E . and D. B a r t l e t t . J r . . Ref lex c o n t r o l of e x p i r a t o r y a i r f l o w and d u r a t i o n . J . A p p l . P h v s i o l . 42(1): 80-87, 1977. 24. Road, J . D . , A . M . Leevers , E . Goldman and A. Grass ino . Resp ira tory muscle c o o r d i n a t i o n dur ing exp ira tory thresho ld l oad ing . J . A p p l .  P h v s i o l . 70(4): 1554-1562, 1991. 25. Wakai, Y . , M.M. Welsh, A . M . Leevers and J . D . Road. The e f f e c t of continuous p o s i t i v e airway pressure and hypercapnia on e x p i r a t o r y muscle a c t i v i t y dur ing wakefulness and s leep. Am. Rev. R e s p i r . P i s . 141(4): A125, 1990.(Abstract) 

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