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Fatigue and rest of the hamster diaphragm Reid, Wendy Darlene 1988

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FATIGUE AND REST OF THE HAMSTER DIAPHRAGM By WENDY DARLENE REID B . M . R . ( P . T . ) , The Univers i ty of Manitoba, 1979 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF PATHOLOGY We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA February, 1988 (c) Wendy Darlene Reid, 1988 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 The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 DE-6G/81) ABSTRACT Decreased r e s p i r a t o r y muscle strength and/or excessive loads imposed on the r e s p i r a t o r y muscles by disease may r e s u l t in r e s p i r a t o r y muscle fa t igue and v e n t i l a t o r y f a i l u r e . Once the r e s p i r a t o r y muscles f a t i g u e , the only treatment is res t by mechanical v e n t i l a t i o n . However, no one has yet determined the best pro toco l of r e s t . The purpose of these studies was to develop an animal model in the hamster in order to examine the time course of recovery fo l lowing fat igue of the diaphragm and s p e c i f i c a l l y , to tes t whether mechanical v e n t i l a t i o n or spontaneous unloaded breathing was a bet ter mode for f u n c t i o n a l recovery . The studies required the i n i t i a l development of an anesthet ic regimen which produced minimal r e s p i r a t o r y depression in the hamster. A new method of s t imulat ing the diaphragm in small animals was developed by apposing p la te e lectrodes d i r e c t l y against the diaphragm. The v a l i d i t y of t h i s technique was examined and comparison of the mechanical and e l e c t r o p h y s i o l o g i c a l response to that of phrenic nerve s t imula t ion were s i m i l a r at maximal s t i m u l a t i o n . The h i s t o l o g i c a l c h a r a c t e r i s t i c s of the normal hamster diaphragm were determined for f i b r e type proport ions and s i z e s , ox idat ive capac i ty and glycogen l e v e l s in the c o s t a l and c r u r a l regions of t h i s muscle. The examination revealed three d i s t i n c t areas of the diaphragm with d i f f e r e n t h i s t o l o g i c a l features : the abdominal surface of the c r u r a l r e g i o n , the thorac i c surface of the c r u r a l region and the s t e r n a l and c o s t a l r eg ion . Diaphragmatic fa t igue was induced in v ivo by r e p e t i t i v e e l e c t r i c a l s t imula t ion which r e s u l t e d in both high and low frequency f a t i g u e . The fat igue stimulus a lso produced muscle f i b r e damage, p r i m a r i l y along the - i i i -abdominal surface of the diaphragm over the e l ec trodes , and glycogen deple t ion in the type l i b f i b r e s . Rest by continuous mechanical v e n t i l a t i o n re su l t ed in recovery of high frequency fat igue in the hamster diaphragm whereas re s t by spontaneous unloaded breathing r e s u l t e d in no recovery. Sham fat igue groups rested by e i t h e r mechanical v e n t i l a t i o n or spontaneous breathing demonstrated progress ive d e t e r i o r a t i o n in transdiaphragmatic pressure throughout the re s t p e r i o d . Decreased muscle f i b r e damage but increased inflammation and glycogen dep le t ion was demonstrated in a l l four fatigue/sham fat igue and re s t groups compared to that demonstrated by the fatigue/sham fat igue only groups. The r e s u l t s suggest that passive res t by continuous mechanical v e n t i l a t i o n promotes recovery fo l lowing fat igue induced by e l e c t r i c a l s t i m u l a t i o n . A d d i t i o n a l fac tors such as prolonged f a s t i n g , loads imposed on the diaphragm by the p la te e lectrode apparatus, p o s i t i v e pressure v e n t i l a t i o n , and cumulative e f fec t s of i n t r a p e r i t o n e a l urethane l i k e l y contr ibuted to the progress ive d e t e r i o r a t i o n of diaphragmatic funct ion demonstrated in the animals of the two sham groups rested by e i ther spontaneous breathing or mechanical v e n t i l a t i o n , and confounded the r e s u l t s shown by the two fat igue groups rested by e i ther spontaneous breathing or mechanical v e n t i l a t i o n . - i v -TABLE OF CONTENTS Page Abstract i i Table of Contents i v L i s t of Tables v i L i s t of Figures v i i L i s t of Abbreviat ions x i i Acknowledgements x iv Dedicat ion xvi I . Introduct ion The Respiratory Muscles 1 -Respiratory F a i l u r e 2 Neuromuscular Fatigue 9 Recovery from Neuromuscular Fat igue 13 Respiratory Muscle Fatigue 16 Respiratory Muscle Rest 19 An Animal Model of Respiratory Muscle Fatigue and Rest 23 Object ive 24 S p e c i f i c Aims 24 Figures 26 References 30 I I . Development of Anesthet ic Regimen and Parameters of Mechanical V e n t i l a t i o n Introduct ion 39 Methods 40 - V -Results 45 Discuss ion 48 Figures 53 References 62 I I I . Diaphragmatic P late E lec trode St imulat ion of the Hamster Diaphragm Introduct ion 64 Methods 65 Results 75 Discuss ion 78 Figures 83 References 95 IV. H i s t o l o g i c a l Features of the Hamster Diaphragm Introduct ion 97 Methods 100 Results 109 Discuss ion 122 Figures 131 References 152 V . Ef fec t s of Fatigue and Rest on the Hamster Diaphragm Introduct ion 157 Methods 159 Results 168 Discuss ion 182 Figures 206 References 237 V I . Concluding Remarks and Future D i r e c t i o n s 242 - v i -LIST OF TABLES I . Factors c o n t r i b u t i n g to r e s p i r a t o r y f a i l u r e 3 I I . The e f fec t s of r e s t i n g the r e s p i r a t o r y muscles by mechanical v e n t i l a t i o n i n pat ients with r e s p i r a t o r y disease 20 I I I . Anesthet ic agents and doses in the hamster 42 IV. Response to anesthet ic agents 46 V. Morphologica l abnormali t ies 107 V I . Muscle f i b r e diameters in microns in the abdominal and thorac i c surfaces of the diaphragm: comparison of d i f f e r e n t regions 112 V I I . Comparison of muscle f i b r e s i ze i n the abdominal and the thorac ic surface of the c r u r a l region and the r e s t of the diaphragm in 6 hamsters 115 V I I I . Thickness measurements of 12 cryos tat s e r i a l sect ions 116 IX. Summary of h i s t o l o g i c a l features of the three main areas of the hamster diaphragm 120 X. Muscle f i b r e p r o f i l e s across a l l regions of the hamster diaphragm 121 X I . Pdi before and a f ter the fa t igue stimulus in the 3 fa t igue groups: Fat igue on ly , F/SB and F/MV 170 X I I . A r t e r i a l blood measures during fatigue/sham fat igue and r e s t pro toco l 173 X I I I . Number of damaged f i b r e s and scores of inflammation for the fat igue/sham fat igue groups 175 XIV. Summary of r e s u l t s - pos t - fa t igue and during r e s t per iod 181 - v i i -LIST OF FIGURES Figure 1 Diagram of the diaphragm i l l u s t r a t i n g the three major regions of t h i s muscle ( s t e r n a l , c o s t a l , and c r u r a l regions) 26 Figure 2 The primary act ions of the diaphragm 26 Figure 3 The e f fec t of h y p e r i n f l a t i o n on the diaphragm 27 Figure 4 The length- tens ion r e l a t i o n s h i p of s k e l e t a l muscle 27 Figure 5 The pathway involved in muscular contrac t ion 28 Figure 6 Factors which may contr ibute to neuromuscular fa t igue 29 P late I Figure 7 Pressure displacement plethysmograph s p e c i a l l y s i zed for the hamster 53 Figure 8 The e f fec t of d i f f e r e n t anesthet ics on a r t e r i a l blood gas measurements 55 Figure 9 The e f fec t of the combination of anesthet ics on a r t e r i a l blood measurements over time 56 Figure 10 The e f fec t of the combination of anesthet ics on minute v e n t i l a t i o n , t i d a l volume, and frequency of breathing over time 57 Figure 11 The e f fec t of minute v e n t i l a t i o n on PaC02 and Pa02 58 Figure 12 The e f f ec t of h y p e r v e n t i l a t i o n using the mechanical v e n t i l a t o r on apnea time 59 Figure 13 The e f fec t of d e l i v e r e d t i d a l volume on measured t i d a l volume 59 Plate II Figure 14 The diaphragmatic EMG and Pes recording during spontaneous breathing and during various l e v e l s of mechanical v e n t i l a t i o n 60 Figure 15 Animal preparat ion for diaphragmatic p la te e lectrode s t imula t ion 83 - v i i i -Cast of the upper abdominal c a v i t y and the hamster diaphragm S i l i c o n e dome fashioned from the abdominal cast Figure 16 Figure 17 Figure 18 Apparatus for s t imula t ing and electromyographic recording of the hamster diaphragm Figure 19 In terac t ive s i g n a l process ing Figure 20 Technique used to t e s t phase r e l a t i o n s h i p s of Pes and Pga Figure 21 Representative diaphragmatic ac t ion po tent ia l s from inner e lectrode with maximal current Figure 22 Representative diaphragm act ion p o t e n t i a l produced by DPS and recorded from outer e lectrode Figure 23 Representative diaphragmatic act ion p o t e n t i a l s recorded before and a f ter curare P late I I I Figure 24 The e f fec t of increas ing current on P d i , Pga, and Pes in four animals using DPS and PNS Figure 25 The e f fec t of varying external abdominal pressure (Pab) on Pdi during DPS in s ix animals Figure 26 Pdi produced by twitch st imuation (DPS) at varying frequencies of t e tan ic s t imula t ion in four animals - c o n t r o l and two hours Figure 27 Regional s i t e s of b iops ie s cut from the hamster diaphragm Pla te IV Figure 28 Photomicrographs of representat ive cross - sec t ions of p o s t e r i o r c o s t a l and c r u r a l b iops ies Figure 29 The average d i f f erence of the o p t i c a l dens i ty measurements determined from 16 f i b r e s of the c o n t r o l s l i d e P late V Figure 30 Muscle f i b r e type proport ions in abdominal and t h o r a c i c surface of each region of the diaphragm 84 84 85 86 87 88 89 90 91 93 94 131 132 134 135 - ix -P late V Figure 31 Photomicrograph of c r u r a l region surrounding the esophagus 135 Figure 32 Frequency d i s t r i b u t i o n of diameters of type 1 f i b r e s i n the t h o r a c i c surface of the c r u r a l region and the res t of the diaphragm ( s t e r n a l and c o s t a l regions) 137 Figure 33 Frequency d i s t r i b u t i o n of diameters of type 11a f i b r e s in the thorac i c and abdominal surfaces of the c r u r a l region and the re s t of the diaphragm ( s t e r n a l and c o s t a l regions) 138 Figure 34 Frequency d i s t r i b u t i o n s of diameters of type I , I l a and l i b f i b r e s across a l l regions P late VI Figure 35 Scanning e l ec tron photomicrographs of cryos tat sect ions of the hamster diaphragm 139 140 P la te VII Figure 36 Plate VIII Figure 37 Plate IX Figure 38 Frequency d i s t r i b u t i o n of o p t i c a l dens i t i e s of PAS-stained Type I f i b r e s (upper pane l ) , type I l a f i b r e s (middle panel) and type l i b f i b r e s (lower panel) Frequency d i s t r i b u t i o n of o p t i c a l dens i t i e s of NADH-TR sta ined type 1 f i b r e s (upper pane l ) , type I l a f i b r e s (middle panel) and type l i b f i b r e s (lower panel) Photomicrograph of cross - sec t ions of c r u r a l region s ta ined with PAS ( l e f t ) and NADH-TR (r ight ) 142 144 146 Figure 39 Box p lo t s of the o p t i c a l dens i ty of PAS-stained f i b r e s and p values for comparisons between f i b r e types w i t h i n a given area 148 Figure 40 Box p lo t s of o p t i c a l dens i ty for PAS-stained f i b r e s and p values for comparisons between areas wi th in a given f i b r e type 149 Figure 41 Box p lo t s of the o p t i c a l dens i ty for NADH-TR stained f i b r e s and p values for comparisons between f i b r e types w i th in a given area 150 Figure 42 Box p lo t s of o p t i c a l dens i ty for NADH-TR s ta ined f i b r e s and p values for comparisons between areas w i th in a given f i b r e type 151 - X -Figure 43 Experimental design of FATIGUE/SHAM FATIGUE ONLY and FATIGUE/SHAM FATIGUE AND REST experiments 206 Plate X Figure 44 Pga and Pes t r a c i n g at the beginning and at the end of the fa t igue stimulus of r e p e t i t i v e e l e c t r i c a l s t imula t ion appl ied to the hamster diaphragm 207 P la te XI Figure 45 P i c t o r i a l grading scheme used to grade inflammatory response 209 Plate XII Figure 46 Photomicrographs of damaged f i b r e s in the hamster diaphragm i l l u s t r a t e d by c r o s s -sect ions s ta ined with H & E , NADH-TR, and M-ATPase 211 Figure 47 Re la t ionsh ip between the i n i t i a l dynamic Pdi from f a t i g u i n g e l e c t r i c a l s t imula t ion and the endurance time of the f a t i g u i n g s t imula t ion 213 Figure 48 Pdi before and a f t er the fat igue st imulus i n the three fa t igue groups: fa t igue on ly , F / S B , and F/MV 214 Figure 49 Change in Pdi for twi tch and t e tan ic s t imula t ion fo l lowing fatigue/sham fat igue and during four hours of r e s t in the four fatigue/sham fat igue and re s t groups 215 Figure 50 P a r t i a l pressure of a r t e r i a l carbon diox ide during the experiment for the four fatigue/sham fat igue and res t groups 216 Figure 51 P a r t i a l pressure of a r t e r i a l oxygen during the experiment for the four fat igue/sham fat igue and r e s t groups 217 Figure 52 Grade of inflammation and number of damaged c e l l s as ind ica ted by H & E , NADH-TR and M-ATPase s ta ined t i s sue cross - sec t ions in the fa t igue and sham fat igue only groups 218 Plate XIII Figure 53 Photomicrographs of damaged f i b r e s along the abdominal surface of the hamster diaphragm 219 Figure 54 Number of damaged c e l l s over time in the fatigue/sham fat igue and re s t groups 221 - x i -P late XIV Figure 55 Photomicrographs of wisps of cytoplasm (upper pane l ) , empty spaces (middle panel) and e o s i n o p h i l i c f l o c c u l e n t masses (lower panel) observed in muscle f i b r e c r o s s -sect ions of the hamster diaphragm sta ined with H & E Figure 56 Inflammatory response over time i n the fatigue/sham fat igue and res t groups 222 224 Plate XV Figure 57 Photomicrographs of neutrophi l s invading connective t i s sue l a y e r s , surrounding c e l l s and i n f i l t r a t i n g c e l l s i n the hamster diaphragm sta ined with H & E 225 Plate XVI Figure 58 Frequency d i s t r i b u t i o n s of o p t i c a l dens i ty for PAS stained f i b r e s for type I f i b r e s , type I l a f i b r e s and type l i b f i b r e s in fat igue and sham fat igue only groups 227 Plate XVII Figure 59 Frequency d i s t r i b u t i o n s of o p t i c a l dens i ty for PAS sta ined type I f i b r e s in a l l s ix experimental groups 229 Plate XVIII Figure 60 Frequency d i s t r i b u t i o n s of o p t i c a l dens i ty for PAS stained type I l a f i b r e s in a l l s ix experimental groups 231 P la te XIX Figure 61 Frequency d i s t r i b u t i o n s of o p t i c a l dens i ty for PAS sta ined type l i b f i b r e s in a l l s ix experimental groups 233 Figure 62 Box p lo t s of o p t i c a l dens i ty for PAS sta ined f i b r e s for each f i b r e type and three sham fat igue groups: sham fat igue on ly , S/SB, and S/MV 235 Figure 63 Box p lo t s of o p t i c a l dens i ty for PAS s ta ined f igures for each f i b r e type and three fa t igue groups: fa t igue on ly , F / S B , and F/MV 236 - x i i -LIST OF ABBREVIATIONS ANOVA analys i s of variance atm atmospheres ATP adenosine tr iphosphate BW body weight C a ^ + ca lc ium ion APdi change in transdiaphragmatic pressure COPD chronic obs truc t ive pulmonary disease C 0 2 carbon dioxide CV c o e f f i c i e n t of v a r i a t i o n DPS diaphragmatic p la te e lectrode s t imula t ion EMG electromyography FRC f u n c t i o n a l r e s i d u a l capac i ty F/MV group - fat igued rested by mechanical v e n t i l a t i o n F/SB group - fat igued rested by spontaneous breathing f f a frequency of breathing H & E hematoxylin and eosin H + hydrogen ion Hb hemoglobin HCO^ bicarbonate ion Hz hertz IMV in termi t t en t mandatory v e n t i l a t i o n K + potassium ion M-ATPase myosin adenosine tr iphosphatase MW maximum voluntary v e n t i l a t i o n - x i i i -N number in sample Na + sodium ion NADH-TR nicotinomide adenine d i n u c l e o t i d e - t e t r a z o l i u m reductase PaCO^ p a r t i a l pressure of a r t e r i a l carbon d iox ide PaO^ p a r t i a l pressure of a r t e r i a l oxygen PAS p e r i o d i c a c i d - S c h i f f Pab abdominal pressure Pdi transdiaphragmatic pressure Pes esophageal pressure Pga g a s t r i c pressure P . . maximal transdiaphragmatic pressure dimax P„ maximal exp iratory pressure Emax P,. maximal i n s p i r a t o r y pressure Imax PTT peak twitch tension RHF r i g h t - s i d e d heart f a i l u r e rms root mean squared R T 1 / ^ h a l f r e l a x a t i o n time SD standard dev ia t ion SDH s u c c i n i c dehydrogenase S/SB group - sham fat igue and r e s t by mechanical v e n t i l a t i o n S/MV group - sham fat igue and re s t by spontaneous breathing TPT t ime-to-peak tension VC v i t a l capaci ty Vg minute v e n t i l a t i o n t i d a l volume - x iv -ACKNOWLEDGEMENTS Dr. P .D. Pare acted as my supervisor and Dr. R . L . Pardy acted as a major advisor throughout the course of my Masters/PhD program. They provided much time, e f f o r t , and encouragement in o f f e r i n g t h e i r suggestions and advice , and I extend by s incere apprec ia t ion to the both of them. I am g r a t e f u l to Dr. J . C . Hogg, as a member of my committee and also as the D i r e c t o r of the UBC Pulmonary Research Laboratory who provided valuable advice , and a product ive and s t imula t ing atmosphere to work w i t h i n . I thank Dr. W.K. Oval le for h is use fu l comments and suggestions on the h i s t o l o g i c a l aspects of t h i s t h e s i s . I express my s incere apprec ia t ion to Dr. Rodrigo Moreno for in troduc ing me to the t e c h n i c a l aspects of animal research and his innovative suggestions in the ear ly development of t h i s p r o j e c t . I am g r a t e f u l to Mr. Trevor Blogg who provided much expert i se and ass i s tance in the e l e c t r o p h y s i o l o g i c s tud ies . My thanks to Dr. David Walker for his advice and ass is tance in the eva luat ion of the morphological features of the hamster diaphragm and his personal support . I am thankful to Mr. Barry Wiggs for h is ass is tance in s t a t i s t i c a l a n a l y s i s , Mr. Joe Comeau for h is graphics s k i l l s , and Mr. Andre MacKenzie for h is ass is tance in preparat ion of f i g u r e s . I am very thankful to Mr. Stuart Greene whose photographical expert i se contr ibuted a great deal to the imagery of t h i s t h e s i s . I am g r a t e f u l to Ms. Jenn i f er Hards, Ms. Peggy Wright , Ms. E l i z a b e t h Wood, Ms. Carolyn Davies , and Mr. Joe Rolandi for t h e i r ass is tance in the performance of the h i s t o l o g i c a l and p h y s i o l o g i c a l experiments and data c o l l e c t i o n . I am thankful to a l l the l a d i e s i n the front o f f i c e of the UBC Pulmonary Research Laboratory but e s p e c i a l l y to Miss Joan Dixon for her ass i s tance - XV -throughout the years I have spent at the UBC Pulmonary Research Laboratory, and to Ms. Helena Maurice for typing t h i s t h e s i s . I am also indebted to the Canadian Lung A s s o c i a t i o n for f i n a n c i a l support in the form of a fe l lowship for two years and the Medical Research C o u n c i l of Canada for f i n a n c i a l support in the form of a studentship for two years . - xvi -DEDICATION This thes i s i s dedicated to my husband, Danny, who helped me f i n d the t r u t h more than any other . - 1 -I . INTRODUCTION THE RESPIRATORY MUSCLES The r e s p i r a t o r y system cons i s t s of two par t s : a gas-exchanging organ, the lungs , and a pump which moves the a i r in and out of the lungs (30,97). The r e s p i r a t o r y pump cons i s t s of both exp ira tory and i n s p i r a t o r y muscles. The major muscle of i n s p i r a t i o n i s the diaphragm ( F i g . 1 X 7 5 ) . The s t e r n a l region of t h i s muscle ar i ses from the p o s t e r i o r aspect of the x iphoid process , the c o s t a l region ar i ses from the lower s ix r i b s and c o s t a l c a r t i l a g e s , and the c r u r a l region ar i ses from the upper three lumbar vertebrae p o s t e r i o r l y . The f i b r e s r a d i a t e inwards i n s e r t i n g in to the c e n t r a l tendon (72). Other primary muscles of i n s p i r a t i o n are the scalene muscles and the i n s p i r a t o r y i n t e r c o s t a l muscles. Accessory muscles of i n s p i r a t i o n inc lude: the s ternoc le idomasto id , the p e c t o r a l i s major, the abdominals and severa l others of l e s ser importance (75). Contract ion of the diaphragm accounts for most of the i n s p i r e d volume although i t i s not e s s e n t i a l for v e n t i l a t i o n (76). A large por t ion of the diaphragm j u s t d i s t a l to i t s o r i g i n i s d i r e c t l y apposed against the r i b cage and t h i s region i s named the "zone of appos i t ion". The p i s t o n - l i k e ac t ion of t h i s muscle i s due to the shortening of the zone of appos i t ion (75). The diaphragm has three primary act ions ( F i g . 2): i ) diaphragmatic contrac t ion r e s u l t s in an increase in abdominal pressure pushing outwards on the a p p o s i t i o n a l area and the lower r i b cage, and causing outward displacement of the abdominal w a l l ; i i ) force exerted i n the d i r e c t i o n of the f i b r e s on the i n s e r t i o n of the muscle causes r o t a t i o n of the r i b s upwards (pump handle motion) and outwards (bucket handle motion); i i i ) negative p l e u r a l pressure produced by diaphragmatic con trac t ion p u l l s - 2 -inwards on the upper r i b cage when t h i s muscle contracts alone. Therefore , the diaphragm has two primary i n s p i r a t o r y act ions and one exp ira tory act ion (75,97). RESPIRATORY FAILURE Respiratory f a i l u r e can be broadly categorized into two major types: lung f a i l u r e and pump f a i l u r e (95,97). Lung f a i l u r e i s p r i m a r i l y due to abnormali t ies of lung gas exchange which are manifested by hypoxemia. Pump f a i l u r e leads to a l veo l ar hypovent i l a t ion which i s charac ter i zed by CO^ re t en t ion (95,97). There are severa l fac tors which can contr ibute to pump f a i l u r e (Table I ) . C e n t r a l nervous system (CNS) depression can be def ined as decreased neural dr ive from the r e s p i r a t o r y centre in the medulla to the r e s p i r a t o r y pump. This can r e s u l t in a l veo lar hypovent i l a t ion in such condi t ions as drug overdose of r e s p i r a t o r y depressants (barb i tura te s ) (97 ) , "Ondine's Curse" and most f requent ly , by i r r e v e r s i b l e damage to the r e s p i r a t o r y centres of the bra in stem from i n f a r c t i o n , hemmorrhage, anoxia or e x t r i n s i c compression from trauma (66). F a i l u r e of CNS a c t i v a t i o n , neura l conduction, or neuromuscular  transmiss ion can r e s u l t in pump f a i l u r e . Neuromuscular d i sorders include many condit ions with d iverse e t i o l o g i e s and p a t h o l o g i c a l f ea tures . Disease can a f f ec t the upper motor neuron, the lower motor neuron, or neuromuscular t ransmiss ion . An example of an upper motor l e s i o n i s pyramidal t r a c t i n j u r y . Diseases a f f e c t i n g the lower motor neuron are axonal neuropathy and amyotrophic l a t e r a l s c l e r o s i s . Myasthenia gravis i s an autoimmune disease which af fec ts neuromuscular transmiss ion p o s t j u n c t i o n a l l y by - 3 -TABLE I FACTORS CONTRIBUTING TO RESPIRATORY FAILURE CNS DEPRESSION DECREASED CNS ACTIVATION, NEURAL CONDUCTION OR NEUROMUSCULAR TRANSMISSION MYOPATHY MUSCULAR WEAKNESS INCREASED IMPEDANCE MECHANICAL DISADVANTAGE OF RESPIRATORY MUSCLES RESPIRATORY MUSCLE FATIGUE Drug overdose Ondine's Curse Brainstem damage from i n f a r c t i o n , hemmorhage anoxia or ex terna l compression Upper motor neuron Lower motor neuron Neuromuscular junc t ion Dystrophy Myos i t i s Metabol ic muscle disease M a l n u t r i t i o n Disuse atrophy Increased res i s tance Increased elastance Mechanical defect H y p e r i n f l a t i o n Length-tens ion disadvantage +Demand 4-Supply - A -reducing the number of ace ty l cho l ine receptors a v a i l a b l e to ace ty l cho l ine on muscle. Respiratory complicat ions i n c l u d i n g v e n t i l a t o r y f a i l u r e can a r i s e in a large proport ion of pat ients a f f l i c t e d with these d i s o r d e r s . Diseases of the upper motor neuron, neural conduction or neuromuscular transmiss ion r e s u l t in decreased funct ion of the r e s p i r a t o r y pump because of weakness, and/or s p a s t i c i t y (66). In a d d i t i o n , c h r o n i c i t y of the condi t ion can increase loads placed on these muscle due to decreased compliance of the lungs because of m i c r o a t e l e c t a s i s or of the r i b cage due to s t i f f e n i n g of the a r t i c u l a t i o n s in the thorac ic cage (31). F i n a l l y , pat ients with these diseases are at r i s k for the development of r e s p i r a t o r y muscle f a t i g u e . Primary muscle disease can a f fec t the r e s p i r a t o r y muscles l eading to progress ive weakness and v e n t i l a t o r y f a i l u r e . Muscular dystrophies are progress ive hered i tary degenerative d i sorders in which the number of muscle f i b e r s gradua l ly decreases. They are the most common muscle diseases which can r e s u l t in ser ious r e s p i r a t o r y compromise. Disease of muscle a r i s i n g from a metabolic or inflammatory e t io logy a lso leads to muscle weakness. However, r e s p i r a t o r y i n s u f f i c i e n c y i s genera l ly not a primary c l i n i c a l mani fes tat ion in most of these d i sorders (66). Muscular weakness i s common i n n e u r o l o g i c a l and metabolic c o n d i t i o n s , but can a l so a r i s e from poor n u t r i t i o n or as a r e s u l t of c h r o n i c a l l y decreased a c t i v i t y (15). Poor n u t r i t i o n can be due to a de f i c i ency of an e s s e n t i a l nutr i en t and/or inadequate c a l o r i c in take . The major nutr i en t s inc lude: water, carbohydrates , p r o t e i n s , l i p i d s , minerals and v i tamins . Although some can be synthesized by the body, others must be consumed i n the d i e t . Otherwise, a d e f i c i e n c y w i l l r e s u l t and normal - 5 -funct ion of biochemical pathways or formation of c e l l u l a r s tructures w i l l be affected (80). Semi-s tarvat ion leads to pro te in catabol ism and the re su l tant amino acids are used for energy supply (80). The primary source of amino acids during s t a r v a t i o n is s k e l e t a l muscle (80). C l i n i c a l l y , i f increased loads are placed on the r e s p i r a t o r y muscle of the malnourished p a t i e n t , they w i l l be unable to adapt because of decreased substrates a v a i l a b l e for p r o t e i n synthes i s . M a l n u t r i t i o n has been shown to contr ibute to decreased strength of the diaphragm in pat ients with chronic obs truc t ive pulmonary disease (COPD) (3) and rodents (70). Muscular weakness can a lso a r i s e from decreased a c t i v i t y and subsequent muscle f i b r e atrophy, p r i m a r i l y of the type II f i b r e s (15). I n a c t i v i t y because of dyspnea may contr ibute to decreased r e s p i r a t o r y muscle s trength; the i n h i b i t i o n of the performance of strenuous a c t i v i t i e s w i l l minimize v e n t i l a t i o n at high l e v e l s such that the ' c o n d i t i o n i n g ' of the r e s p i r a t o r y musculature i s no longer maintained. Increased impedance of the r e s p i r a t o r y system places a greater load on the r e s p i r a t o r y muscles. Increased loads may a r i s e from wi th in the airways and/or the parenchyma of the lungs , from the chest w a l l , or a combination of these f a c t o r s . Increased loads from wi th in the lungs can a r i s e from e l a s t i c and r e s i s t i v e fac tors (77). In chronic d i sease , the e lastance of the lung can be increased by chronic f i b r o s i s . Resistance i s a f fected by those aspects which decrease the airway lumen s i ze (within the lumen, in the lumen w a l l or outs ide the lumen wal l ) such as edema, mucous p lugging , and bronchospasm. Increased loads on the chest w a l l can occur in extreme obes i ty where added weight on the chest w a l l can increase the work of breathing cons iderably (76). In n e u r o l o g i c a l condi t ions and - 6 -kyphosco l ios i s (76), pat ients have decreased compliance of the thorax and thus increased energy cost for the work of breath ing . This can also occur i n several n e u r o l o g i c a l condit ions such as q u a d r i p l e g i a , p o l i o m y e l i t i s , and s p i n a l muscle a trophies , where muscular weakness leads to s t i f f e n i n g of the thorac i c j o i n t s and r i b cage (31). In k y p h o s c o l i o s i s , the g r e a t l y increased curvature and r o t a t i o n of the spine r e s u l t s in a very immobile r ibcage . Decreased mechanical advantage of the r e s p i r a t o r y muscles can occur in r e s p i r a t o r y disease . Less e f f i c i e n t force product ion of the mechanical ly disadvantaged i n s p i r a t o r y muscle(s) increases the load on the other less af fected muscles. In chronic obs truc t ive pulmonary disease ( F i g . 3 ) , h y p e r i n f l a t i o n places the diaphragm in a more f l a t t ened p o s i t i o n , decreasing i t s p o t e n t i a l for excursion and pumping a c t i o n . I t has been demonstrated in COPD pat ients that the t o t a l length of the i n s p i r a t o r y muscles shorten with the diaphragm being af fected to the greatest extent . Some studies have suggested that the shortened lengths of the i n s p i r a t o r y muscles in COPD pat ients r e s u l t in a l ength- tens ion disadvantage of the s u b - c e l l u l a r s tructures (sarcomeres) of these muscles (76). An apprec ia t ion of t h i s hypothesis i s provided by an examination of the s k e l e t a l muscle l ength- tens ion r e l a t i o n s h i p ( F i g . 4 ) . A l l s k e l e t a l muscle e x h i b i t s a s i m i l a r general r e l a t i o n s h i p in which an optimal length for tension generation occurs r e l a t e d to the most advantageous over lap of a c t i n and myosin f i laments (48,87). H y p e r i n f l a t i o n by shortening the sarcomeres of the i n s p i r a t o r y muscles could r e s u l t i n decreased force output due to excessive overlap of the myosin and a c t i n f i laments . Studies of the diaphragm in emphysematous hamsters (37,107) and of c h r o n i c a l l y shortened - 7 -limb muscles (44,48) in several species s trongly suggest that s k e l e t a l muscle adapts when held in a shortened p o s i t i o n . Sarcomeres are l o s t and i n d i v i d u a l sarcomere length i s maintained. Thus, muscular contrac t ion of shortened muscle occurs with optimal overlap of myosin and a c t i n f i laments so long as s u f f i c i e n t time has passed to enable adaptat ion. Hence, force loss can not be a t t r i b u t e d s o l e l y to l ength- tens ion disadvantage in such chronic condi t ions but rather to other fac tors such as poor mechanical p o s i t i o n or muscular weakness. However, a l ength- tens ion disadvantage may occur in other cases of r e s p i r a t o r y disease where acute changes in the operat ing length of the r e s p i r a t o r y muscles occurs . In an asthmatic attack (97) or the e x e r c i s i n g COPD p a t i e n t , acute h y p e r i n f l a t i o n can place the i n s p i r a t o r y muscles at shortened lengths where less than optimal tension product ion can occur due to increased overlap of the myosin and a c t i n f i laments . Although hypertrophy of the diaphragm has been demonstrated in emphysematous hamsters (38,69) and humans (62,100), others have found atrophy of t h i s muscle i n pat ients (105,110). Arora and Rochester (4) suggest that poor n u t r i t i o n is a primary f a c t o r c o n t r i b u t i n g to the decreased weight of the diaphragm in COPD. Thurlbeck (110) reports that the diaphragm:body weight r a t i o i s a lso decreased in these pat ients implying that other fac tors besides poor n u t r i t i o n contr ibuted to i t s decreased mass. Perhaps when the diaphragm is placed in a completely f l a t t ened and funct ion les s p o s i t i o n , a change in a f ferent output from muscle spindles and G o l g i tendon organs r e s u l t s i n decreased recruitment during i n s p i r a t i o n and subsequent atrophy. With severe h y p e r i n f l a t i o n , not only is the diaphragm at an extreme mechanical disadvantage, but the loads - 8 -of i n s p i r a t i o n are l a r g e l y t rans ferred to the i n s p i r a t o r y accessory muscles for v e n t i l a t o r y needs (33). Respiratory muscle fat igue can occur when the energy demand exceeds the energy supply surpassing any reserve capac i ty of the t i s sue (97). There are , however, s i t u a t i o n s where r e s p i r a t o r y muscle fat igue i s not r e l a t e d to an energy imbalance. In condi t ions l i k e myasthenia grav i s or p a r t i a l c u r a r i z a t i o n , the problem ar i ses at the neuromuscular j u n c t i o n (98). Weakened or decondit ioned muscles have a diminished reserve to draw from during s t r e s s f u l s i t u a t i o n s , and are more suscept ib le to f a t i g u e . Factors c o n t r i b u t i n g to increased muscle demands include increased impedance, mechanical disadvantage, n e u r o l o g i c a l d e f i c i t loading the remaining muscle f i b r e s , or a combination of these f a c t o r s . Decreased energy supply can a r i s e from a diminished blood flow, low oxygen content of a r t e r i a l b lood, or decreased energy stores in the muscles (77). Decreased energy supply due to a low cardiac output during shock has been demonstrated to p r e c i p i t a t e diaphragmatic fa t igue in dogs (5) . Decreased energy stores due to poor n u t r i t i o n alone does not lead to r e s p i r a t o r y muscle f a t i g u e . However, when m a l n u t r i t i o n is combined with r e s p i r a t o r y condi t ions such as COPD, a maladaptive response of the diaphragm may r e s u l t . Although s k e l e t a l muscle i s an extremely adaptable t i s s u e , with the added loads of disease or decreased energy supply, the r e s p i r a t o r y muscles may reach a point where they are no longer able to adapt. At th i s p o i n t , r e s p i r a t o r y fat igue and subsequent pump f a i l u r e is thought to occur. - 9 -NEUROMUSCULAR FATIGUE Fatigue of the neuromuscular system can be defined as the i n a b i l i t y of a muscle or a muscle group to susta in a given load (12). T r a d i t i o n a l l y , fa t igue was thought to occur at the point of force l o s s . More r e c e n t l y , i t i s considered that changes during the f i r s t few muscular contract ions could p o s s i b l y contr ibute to subsequent neuromuscular fat igue (12). Although a number of causes of neuromuscular fa t igue have been hypothesized, few cause and e f fec t r e l a t i o n s h i p s have been c l e a r l y es tab l i shed (12). The pathway which is involved in muscular contrac t ion is shown in F i g . 5. The i n a b i l i t y to sus ta in neuromuscular contrac t ion may be due to one or a combination of a number of a l t e r a t i o n s along the motor pathway. The fac tors can be categorized as: c e n t r a l , neuromuscular transmiss ion or p e r i p h e r a l ( F i g . 6). The cause of fat igue may be c e n t r a l i n o r i g i n due to a decreased output from the CNS. This may be conscious due to lack of mot ivat ion or an i n a b i l i t y to t o l e r a t e exerc ise discomfort (12,34). I t may be unconscious and serve as a p r o t e c t i v e mechanism to minimize i r r e v e r s i b l e damage of the overloaded muscle. During exhaustive loads , s k e l e t a l muscle af ferents may provide important s ignals to the CNS or operate v i a s p i n a l re f lexes r e s u l t i n g in decreased e x c i t a t o r y input to the muscle. Although the maximal force output may decrease, such a mechanism may serve to protec t the muscle from cont inuing to contract u n t i l the point of i r r e v e r s i b l e muscle damage. S k e l e t a l muscle a f ferent input during fat igue may come in the form of propr iocept ive information from the Golg i tendon organ or the muscle s p i n d l e . Group I I I and IV f i b r e s which e x i s t as free nerve endings within the muscle may i n h i b i t motoneuron a c t i v i t y i n response to muscle s t re tch - 10 -(98) or the metabolic state of the f a t i g u i n g muscle. The a f ferent input may be an important feedback l i n k between the c o n t r a c t i l e apparatus and e x c i t a t o r y input from the CNS and/or s p i n a l r e f l e x a c t i v i t y during exhaustive exercise and may provide some explanation for c e n t r a l fat igue demonstrated in the r e s p i r a t o r y muscles (8 ,12) . Fatigue could a r i s e from a loss of neuromuscular transmiss ion . Recent studies cast doubt on previous suggestions that t h i s mechanism may operate in normal s k e l e t a l muscle (12), however, more work is needed to fur ther substant iate th i s hypothesis . Loss of neuromuscular transmiss ion i s the major cause of fat igue in some diseases such as myasthenia g r a v i s , Lambert-Eaton myasthenic syndrome, and botul i sm (34). Fatigue may also be p e r i p h e r a l in o r i g i n . I t may a r i s e from a number of d i srupt ions wi th in the s k e l e t a l muscle f i b r e i n v o l v i n g energy supply, e l e c t r o l y t e balance, or an excess of hydrogen ions and inorganic phosphate (116) from metabolic r e a c t i o n s . Regarding energy supply, glycogen deple t ion (67,82,98,99,100) and supercompensation (replenishment above normal basal l e v e l s ) (82,101) of muscle f i b r e s has been r e l a t e d to endurance time. However, glycogen stores may not be depleted at the point of exhaustion from exerc ise performed at low workloads or very high workloads near or above one's maximal aerobic capac i ty (60). The mechanism by which muscle glycogen l e v e l s a f f ec t sustained performance is not known. Perhaps af ferents from the s k e l e t a l muscle f i b r e provide feedback to the CNS dependent upon the energy state of the muscle f i b r e . S k e l e t a l muscle diseases such as c a r n i t i n e de f i c i ency syndromes pose l i m i t a t i o n s in l i p i d metabolism, and fat igue from sustained a c t i v i t i e s i s a primary complaint . - 11 -However, the p o s s i b i l i t y that l i m i t a t i o n s of l i p i d metabolism in normal s k e l e t a l muscle contr ibute to fat igue during sustained exerc i se has rece ived l i t t l e a t t e n t i o n . Fatigue may also r e s u l t from a reduct ion in the free energy a v a i l a b l e from ATP h y d r o l y s i s . This could a r i s e from a disturbance in the metabolic pathways producing ATP or from decreased hydro lys i s of t h i s high energy phosphate. Decreased ATP could contr ibute to fa t igue by slowing myos in-act in uncoupling (64), the uptake of calc ium (Ca^ + ) by the sarco-plasmic re t i cu lum (64), and/or ac t ive t ransport of sodium (Na + ) and potassium (K + ) by the sarcolemmal pump. An e l e c t r o l y t e imbalance a r i s i n g from slow ac t ive transport of N a + and K + would decrease the a b i l i t y of an ac t ion p o t e n t i a l to be propagated along the sarcolemma or the transverse tubules (34). An increased concentrat ion of hydrogen ions (from excess l a c t i c ac id product ion and/or decreased u t i l i z a t i o n ) can i n h i b i t key regula tory enzymes of the anaerobic catabol ism of carbohydrates i . e . phosphorylase, phosphofructokinase. These two enzymes are almost completely i n h i b i t e d at a pH of 6.4 and th i s l e v e l of a c i d i t y may be reached in s k e l e t a l muscle during exhaustive exerc ise (55). F u r t h e r , excess hydrogen ions can d iminish the a f f i n i t y of troponin for the C a ^ + b inding s i t e and thus i n h i b i t i t s regulatory funct ion on myosin and a c t i n coupl ing (96). I t can a lso increase b inding of C a ^ + by the sarcoplasmic r e t i c u l u m (35,55). P e r i p h e r a l fa t igue has been associated with increased l e v e l s of inorganic  phosphate which may i n h i b i t the myos in-act in uncoupling due to decreased breakdown of ATP because of end product accumulation (84). In add i t i on to a l t e r a t i o n s i n the biochemical funct ions of several c e l l u l a r processes , p e r i p h e r a l fa t igue may occur due to l e t h a l and sub le tha l - 12 -c e l l damage. Several inves t iga tors have documented evidence of muscle f i b r e degeneration and regeneration fo l lowing an acute bout or severa l bouts of exhaustive exercise in both humans (42,56,71,109) and animals (43,54,112, 113,114). Vihko and Salminen (112) described changes in f i b r e s from the quadriceps muscle of mice a f ter exhaustive swimming which were s i m i l a r to those observed post - i schemia . They observed s t r u c t u r a l a l t e r a t i o n s in s u b c e l l u l a r organel les such as the swel l ing of mitochondria and d i s r u p t i o n of the c r i s t a e which were re la t ed to the stages of r e v e r s i b l e and i r r e v e r s i b l e c e l l damage as de l ineated by Trump (111). Poss ib le mechanisms of exerc i se - induced muscle c e l l death could be a decreased energy supply caused by t r a n s i e n t ischemia or the product ion of oxygen r a d i c a l s (29). Mechanical forces may exceed the t e n s i l e strength of the muscle c e l l and d i s r u p t i o n of the z-band (36,55) or co l lagen f ibres (36,80) may t r i g g e r the inflammatory process . Another p o s s i b i l i t y i s that i o n i c imbalances created during exerc ise may increase the i n t r a c e l l u l a r concentrat ion of calc ium leading to a c t i v a t i o n of ca l c ium-ac t iva ted neutra l proteases and subsequent c e l l u l a r degradation (115). I r r e v e r s i b l e and r e v e r s i b l e c e l l damage would decrease the number of muscle f i b r e s a v a i l a b l e for recruitment during sustained a c t i v i t y p o s s i b l y decreasing the force output. In a d d i t i o n , the increased load on the i n t a c t muscle f i b r e populat ion may p r e c i p i t a t e fa t igue of the remaining c e l l s . Neuromuscular fa t igue a r i s i n g d i s t a l to the CNS has been c l a s s i f i e d as low frequency or high frequency dependent upon whether or not force loss occurred in response to e x t e r n a l l y appl ied low or high frequency s t i m u l a t i o n , r e s p e c t i v e l y . High frequency fat igue has u s u a l l y been found to have a quick - 13 -recovery and genera l ly i s a t t r i b u t e d to e l e c t r i c a l f a i l u r e of neuromuscular transmiss ion or reduced e x c i t a b i l i t y of the sarcolemma (58,98). In c o n t r a s t , low frequency fat igue has been demonstrated to have a much longer durat ion and i s hypothesized to be due to f a i l u r e of e x c i t a t i o n / c o n t r a c t i o n c o u p l i n g , or a d i s r u p t i o n of the c o n t r a c t i l e apparatus because of s t r u c t u r a l 2+ damage (98), decreased Ca re lease from the sarcoplasmic re t i cu lum (35) and/or diminished a f f i n i t y of troponin for the C a ^ + b inding s i t e (57,98). I f low frequency i s the r e s u l t of depressed re lease of C a ^ + , high frequency fat igue may not be af fected s ince high e x c i t a t i o n frequency s t imula t ion may compensate for impaired re lease of C a ^ + and maximal a c t i v a t i o n of f ibres w i l l s t i l l occur (35). However, much of the work performed examining the mechanisms of high and low frequency fat igue has been done in a p a r t i c u l a r muscle group (35) or i n i s o l a t e d muscle preparat ions (64) so one should be cautious in ex trapo la t ing the r e s u l t s of these reports to other s i t u a t i o n s . RECOVERY FROM NEUROMUSCULAR FATIGUE Very l i t t l e i s known about the mechanisms and time course of recovery. Because of the m u l t i p l e proposed mechanisms of f a t i g u e , the durat ion of res t and a c t i v i t y l e v e l during re s t which would optimize recovery l i k e l y var i e s cons iderably and depends upon the kind of a c t i v i t y which induced neuromuscular fa t igue . Short bursts of high i n t e n s i t y a c t i v i t y may only requ ire a short recovery per iod (80). I f s u f f i c i e n t oxygen i s present , replenishment of high-energy phosphates (ATP and creat ine phosphate) i s thought to occur during a res t i n t e r v a l of about 10 minutes (116). However, i f exerc ise i s severe and long enough the purine nuc leot ide cyc le may be ac t iva ted with - 14 -increases in IMP and sustained reduct ion in ATP. De novo synthesis of ATP i s very slow. Unsteady-state exercise at high i n t e n s i t i e s r e s u l t s in the bu i ld -up of l a c t i c ac id - the end product of anaerobic metabolism. Excess l a c t i c ac id was formerly impl icated as a cause of neuromuscular f a t i g u e . L a c t i c ac id may not d i r e c t l y i n h i b i t muscular contrac t ion but rather the e f f ec t of i t s accumulation on pH may p r e c i p i t a t e fa t igue (67). The opt imal recovery a c t i v i t y to decrease l a c t i c ac id l e v e l s has been demonstrated to be exercise performed at 60% of maximal oxygen uptake (80) however, l a c t i c ac id and hydrogen ion accumulation may not be r e l a t e d to neuromuscular fa t igue from prolonged exerc i se . The l i t e r a t u r e on s k e l e t a l muscle suggests that fa t igue from exerc ise of prolonged durat ion i s c l o s e l y associated with glycogen deple t ion (82,99). In limb muscles, the r e s t o r a t i o n of muscle glycogen stores takes approximately 48 hours (24). However, i f exerc ise i s prolonged and repeated over severa l days, some i n d i v i d u a l s may require a longer recovery per iod (25), e s p e c i a l l y i f the d i e t i s low in carbohydrate (11,101). In contrast to the ac t ive recovery which promotes l a c t i c ac id removal, passive recovery with minimal a c t i v i t y has been demonstrated to maximize glycogen replenishment in the vastus l a t e r a l i s muscle of humans (13). Several reports have demonstrated signs of muscle f i b r e damage a f ter a s ing le bout or severa l bouts of prolonged exerc i se i n both animal (43,54,112,113,114) and human studies (42,56,71,109). Car l son and Faulkner (21) have stated that contrary to previous b e l i e f , s k e l e t a l muscle has a tremendous capaci ty to regenerate. Unfor tunate ly , very l i t t l e i s known regarding the time course and best p r o t o c o l of res t to f a c i l i t a t e - 15 -regeneration of human s k e l e t a l muscle. A recent report (36) s tated that complete r e p a i r of lower limb muscles in humans fo l lowing an endurance race may take up to 12 weeks. Much of the information on regenerat ion of muscles comes from study of minced or grafted s k e l e t a l muscle. Exerc i se f a c i l i t a t e s regenerat ion of minced muscle by ensuring the i n t e r n a l a r c h i t e c t u r e of the newly formed muscle i s organized in a normal manner. Otherwise, i f minced muscle i s pos i t ioned i n v ivo without tendinous connect ions, the regenerat ing f i b r e s develop very abnormally with the f i b r e s apparently arranged in a random manner (20). In add i t ion to ensuring the most appropriate s t r u c t u r a l i n t e g r i t y , exercise increases mass, p r o t e i n content, ox idat ive capac i ty and glycogen concentrat ion in muscle graf ts of rodents (115). I f muscle regenerat ing a f ter exerc ise - induced damage i s s i m i l a r to graf ted muscle, i t would appear that some a c t i v i t y i s e s s e n t i a l in order to induce normal alignment of f ibres and to res tore the ox idat ive and g l y c o l y t i c capac i ty of the t i s s u e . However, there i s l i t t l e information regarding the best exercise des ign. Regenerating muscle should not be overexercised but no one r e a l l y knows what cons t i tu tes "overexercise". Another fac tor that must be considered during recovery is the prevention of s i g n i f i c a n t muscle f i b r e atrophy. This may only be a subtle point for opt imiz ing recovery in the competit ive a th le te s u f f e r i n g from o v e r t r a i n i n g but i s l i k e l y very important i f r e s t by mechanical v e n t i l a t i o n i s prescr ibed for the pat i ent with r e s p i r a t o r y muscle f a t i g u e . There i s an abundant l i t e r a t u r e examining the e f fec t s of immobi l izat ion on s k e l e t a l muscle, however because of the large number of v a r i a b l e s involved many of the key issues remain unresolved. I t i s known that with atrophy, the tension generating a b i l i t y of s k e l e t a l muscle decreases (106). Numerous - 16 -aspects of muscle metabolism are af fected with general trends towards d e - d i f f e r e n t i a t i o n (106). With the immobilized lower limb some muscle groups such as the quadriceps are p r e f e r e n t i a l l y af fected to a greater degree which may be r e l a t e d to such fac tors as i t s funct ion before immobi l i za t ion , f i b r e t y p i n g , and/or the length at which i t i s immobil ized. Animal s tudies have demonstrated that s k e l e t a l muscle atrophy occurs r e l a t i v e l y q u i c k l y . Goldspink (45) found that cast immobi l izat ion of the limb muscles of rodents for two days produced a s i g n i f i c a n t decrease in mass and rate of prote in synthes i s . Atrophy is described as occurr ing very r a p i d l y during the f i r s t few weeks of immobi l izat ion with a slower rate l a t e r on (106). Studies of cast immobi l izat ion of human limb muscles have examined the e f fec t of e l e c t r i c a l s t i m u l a t i o n , i sometr ic exerc ise and exerc ise through l i m i t e d range (with the use of a mobile cast brace) on maintenance of muscle func t ion . The r e s u l t s from these studies suggest that exerc ise through range which corresponds more c l o s e l y to the funct ion required during d a i l y a c t i v i t i e s i s the best treatment to reduce atrophy and loss of func t ion . Groups of pat ients performing i sometr ic exerc ise or being subjected to e l e c t r i c a l s t imula t ion d id not have be t ter muscle funct ion than that of c o n t r o l groups in some studies (106). RESPIRATORY MUSCLE FATIGUE The r e s p i r a t o r y muscles are s k e l e t a l muscle and they respond to various s t i m u l i in a s i m i l a r manner to that of l imb muscles. In both normal healthy i n d i v i d u a l s and d i f f e r e n t pat i ent populat ions , i t has been demonstrated that the r e s p i r a t o r y muscles can be t r a i n e d and w i l l improve t h e i r funct ion in response to both a s p e c i f i c (1 ,9 ,10 ,22 ,39 ,53 ,73 ,84 ,85 ,88 , - 17 -103) and a non- spec i f i c (68) r e s p i r a t o r y muscle t r a i n i n g program. The r e s p i r a t o r y muscles are capable of impressive feats of endurance. In the case of e l i t e cross -country s k i e r s , v e n t i l a t o r y l e v e l s of 80 to 100 L/min can be maintained for 8 to 10 hours for severa l days in succession ( IA) . S i m i l a r to limb muscles, the r e s p i r a t o r y muscles w i l l fa t igue in response to excessive loads . This has been demonstrated in healthy i n d i v i d u a l s during isocapneic h y p e r v e n t i l a t i o n (76) and i n s p i r a t o r y r e s i s t i v e breathing (63,93). The tension-t ime index (7) , the product of the proport ion of maximal c o n t r a c t i l e force (transdiaphragmatic pressure generated to maximal transdiaphragmatic pressure [Pdi/Pdimax]) and the duty cyc le ( r a t i o of contrac t ion time to contrac t ion and r e l a x a t i o n time) is r e l a t e d to endurance time. S i m i l a r to limb muscles i f t h i s r a t i o exceeds 0.15 neuromuscular fa t igue w i l l occur (12). Roussos et a l (98) found that the c r i t i c a l pressure of a l l the i n s p i r a t o r y muscles i s approximately 50-70% of maximum, and for the diaphragm alone i t i s 40% of the maximum with a t i d a l vo lume: insp iratory time of 0 .6-0.9 (an index of the mean v e l o c i t y of shortening) and a duty cyc le of 0 .3 -0 .4 . Diaphragmatic fa t igue appears to develop at a lower tension-t ime index during exhaustive exerc ise than during i n s p i r a t o r y r e s i s t i v e l o a d i n g . This has been a t t r i b u t e d to competing demand of other e x e r c i s i n g muscles with the i n s p i r a t o r y muscles for card iac output and to the higher energy cost of the higher v e l o c i t y of shortening during exerc ised- induced h y p e r v e n t i l a t i o n compared to i n s p i r a t o r y r e s i s t i v e breath ing . Besides the tension-t ime index, v e l o c i t y of shortening , and type of exerc i s e , diaphragmatic performance can a lso be inf luenced by the l e v e l of oxygen and carbon dioxide breathed. Healthy i n d i v i d u a l s performing i n s p i r a t o r y r e s i s t i v e breathing of hypoxic (63) or hypercapneic (65) - 18 -mixtures have a decreased endurance time compared to normoxic and isocapneic breathing . Observations from c l i n i c a l s i t u a t i o n s provide suggestion that r e s p i r a t o r y muscle fat igue occurs in pat ients i n addi t ion to the above-mentioned examples in healthy i n d i v i d u a l s . COPD pat ients who develop CO^ re tent ion develop i n s p i r a t o r y pressures which are w e l l w i th in the domain of fat igue producing loads . They produce Pdi with each breath of 25 to 30% of maximum Pdi whereas those pat ients with no CO^ re tent ion produce pressure of 10% of Pdi maximum (97). Electromyographic signs of fa t igue have been demonstrated i n pat ients being weaned from mechanical v e n t i l a t o r s (23,52, 94). To date i t i s c o n t r o v e r s i a l whether or not the r e s p i r a t o r y muscles adapt by improving t h e i r funct ion in response to the added loads of lung disease (3,18,68,100,105) or i f there is a l i m i t to t h e i r a d a p t a b i l i t y ; the r e s p i r a t o r y muscles may reach a point where they are no longer able to cope with the increased work of breathing due to high i n s p i r a t o r y loads and mechanical disadvantage. Consequently, they may become exhausted and undergo maladaptat ion. Unl ike fat igued limb muscles, the funct ion of the r e s p i r a t o r y muscles are e s s e n t i a l f or l i f e and they must continue to contract even in an exhausted s ta te . Continued excessive demand on the r e s p i r a t o r y muscles together with i n s u f f i c i e n t recovery may r e s u l t in degenerative changes and a state of "chronic" f a t i g u e . Therapy for r e s p i r a t o r y muscle fa t igue can only be r e s p i r a t o r y muscle r e s t . A very important cons idera t ion i s to determine whether weakness or fat igue i s the cause of low force output (16). A weakened muscle w i l l improve i t s funct ion with proper n u t r i t i o n and a progress ive - 19 -t r a i n i n g program, however, a fat igued muscle w i l l only benef i t from res t and o v e r t r a i n i n g or too much s tress can r e s u l t in permanent damage (16). When v e n t i l a t o r y f a i l u r e occurs because of r e s p i r a t o r y muscle f a t i g u e , r e s t i n g the r e s p i r a t o r y muscles by mechanical v e n t i l a t i o n i s e s s e n t i a l . RESPIRATORY MUSCLE REST There are severa l c l i n i c a l s tudies (most of which are poorly contro l l ed ) that have examined the benef i t of r e s t i n g the r e s p i r a t o r y muscles by mechanical v e n t i l a t i o n (26,27,40,46,57,88,92) . In these s tud ies , the inves t iga tors have examined the e f fec t s of v e n t i l a t o r y muscle r e s t on pat ients with COPD, k y p h o s c o l i o s i s , and neuromuscular disease and the r e s u l t s have shown improved a r t e r i a l blood gases, r e s p i r a t o r y muscle funct ion and f u n c t i o n a l status (Table I I ) . The r e s u l t s of these studies suggest that r e s t i n g the r e s p i r a t o r y muscles by mechanical v e n t i l a t i o n is b e n e f i c i a l . However, to date , no one has determined the mechanism of r e s p i r a t o r y muscle f a t i g u e . Thus i t i s d i f f i c u l t to determine the best p r o t o c o l of mechanical v e n t i l a t i o n which would optimize recovery of the fat igued r e s p i r a t o r y muscles. Glycogen and t r i g l y c e r i d e dep le t ion has been demonstrated i n the diaphragm and i n t e r c o s t a l muscles of sheep exposed to i n s p i r a t o r y flow r e s i s t i v e loads by Bazzy et a l (6) . Several inves t iga tors (47,50) have shown deple t ion of glycogen in the r e s p i r a t o r y muscles of rodents in response to high v e n t i l a t o r y loads induced by whole body exerc i se ( e i ther swimming or t r e a d m i l l running) . - 20 -T A B L E I I THE E F F E C T S OP RESTING THE RESPIRATORY MUSCLES BY  MECHANICAL V E N T I L A T I O N IN P A T I E N T S WITH RESPIRATORY D I S E A S E DURATION OF REST P A T I E N T GROUP (& FOLLOW-UP)  E F F E C T S OF REST CONTROLS AUTHOR(S) n o c t u r n a l ( a v : 10 y r s ) 8 h y p o v e n t i l a t i n g , p a t i e n t s , m i x e d e t i o l o g y : n e u r o m u s c u l a r weakness and k y p h o s c o l i o s i s + P a C 0 2 , + P a © 2 No G a r a y e t a l ( 1 9 8 1 X 4 0 ) n o c t u r n a l D u c h e n n e ' s m u s c u l a r d y s t r o p h y + P a C 0 2 , + Pa02 No C u r r a n ( 1 9 8 1 X 2 7 ) 4 -16 h o u r s (2 -48 mos) 6 COPD h o s p i t a l i z a t i o n days No R o c h e s t e r changed f r o m 141+111 (91) t o 15+12 d a y s / y e a r 4-10 h o u r s (5.2+3.8 mos) 18 l u n g d i s e a s e + 17 n e u r o m u s c u l a r + d i s e a s e PaC02, p I m a x > MW, pEmax No M a r i n o e t a l ( 1 9 8 2 ) ( 7 9 ) 1 hour (one s e s s i o n ) 7 COPD + P d i m a x , + d i a p h r a g m No R a b i n o v i t c h c o n t r a c t i l i t y , + h i g h / e t a l low r a t i o o f EMG (1983) (88) 12 h o u r s 4 k y p h o s c o l i o s i s ( a v : 3 .4 y r s ) + d y s p n e a , + r e s t l e s s No Hoeppner s l e e p , + P a C 0 2 , t Pa02 e t a l + Hb, + FRC, + VC, ( 1 9 8 4 X 5 7 ) r e s o l v e d RHF 4-8 h o u r s 15 s t a b l e COPD (8 t r e a t m e n t & 7 c o n t r o l s ) + P l m a x & + PEmax + s u s t a i n e d v e n t i l a t i o n a t 50 & 70% MVV, 4- P a C 0 2 » + d y s p n e a i n t r e a t m e n t g r o u p o n l y C r o p p e t a l ( 1 9 8 7 X 2 6 ) n o c t u r n a l (8 wks) 5 m i x e d e t i o l o g y + P a C 0 2 , + P a 0 2 , n e u r o m u s c u l a r weakness r e t u r n e d t o work and t h o r a c o p l a s t y No G o l d s t e i n e t a l (1987) (46) A B B R E V I A T I O N S : d e c r e a s e - 4- , e l e c t r o m y o g r a p h y - EMG, f u n c t i o n a l r e s i d u a l c a p a c i t y F R C , h e m o g l o b i n - H b , i n c r e a s e - + , maximum i n s p i r a t o r y p r e s s u r e -P l m a x ' m a x i m u m e x p i r a t o r y p r e s s u r e - Pgniaxj maximum v o l u n t a r y v e n t i l a t i o n - M W , months - m o s , r i g h t - s i d e d h e a r t f a i l u r e - R H F , v i t a l c a p a c i t y - V C , weeks - w k s , y e a r s - y r s - 21 -The benef i t of night- t ime v e n t i l a t i o n may operate through glycogen replenishment s ince the time course of replenishment is s i m i l a r to the length of a n i g h t ' s r e s t . Studies in rodents demonstrated glycogen r e p l e t i o n of the diaphragm to require only 3-4 hours (47,50) so v e n t i l a t i o n overnight may be more than necessary to al low glycogen replenishment of the human diaphragm. From information known to date on other fac tors which a f fec t glycogen replenishment one would hypothesize that the time course of glycogen replenishment of the diaphragm in r e s p i r a t o r y pat ients may be much longer than in healthy rodents . In limb muscles of humans, glycogen replenishment fo l lowing endurance exercise can take up to 48 hours. In a d d i t i o n , a high abdominal pressure (17) such as during ac t ive e x p i r a t i o n (97) and low a r t e r i a l blood pressure (61) impedes diaphragmatic blood flow. These fac tors together with inadequate n u t r i t i o n , chronic loads due to impedance of the lung and chest w a l l and mechanical disadvantage of the r e s p i r a t o r y muscles, may grea t ly prolong glycogen replenishment in the fat igued diaphragm of the mechanical ly v e n t i l a t e d pat ient with chronic r e s p i r a t o r y disease . I f the loss of force generating a b i l i t y i s due to muscle f i b r e damage in the diaphragm, then a much longer recovery per iod i s l i k e l y to be needed. Deplet ion of metabol i tes , and atrophy have been demonstrated in pat ients with COPD although these changes were not found s o l e l y i n the r e s p i r a t o r y muscles (19,59). Such abnormal i t ies are a lso commonly found in aging and d i suse . The c o n t r i b u t i o n of muscle f i b r e damage to r e s p i r a t o r y muscle fat igue i s not known. Prolonged mechanical v e n t i l a t i o n may produce muscle f i b r e atrophy in humans. I t has been recent ly reported that prolonged mechanical - 22 -v e n t i l a t i o n (11 days) in baboons r e s u l t e d in a 46% decrease in r e s p i r a t o r y muscle strength and a 37% decrease in endurance time (2) . Func t iona l exerc ise rather than isometric or e l e c t r i c a l l y s t imulated contract ions have been shown to decrease disuse atrophy in limb muscles of humans (106). I f one can extrapolate these f indings from limb muscles to the r e s p i r a t o r y muscles, there i s strong suggestion that the mechanical ly v e n t i l a t e d pat ient should perform f u n c t i o n a l exercise of h i s r e s p i r a t o r y muscles d a i l y in order to l i m i t or prevent atrophic changes. Intermit tent mandatory v e n t i l a t i o n (IMV) was developed to r e i n f o r c e the normal pat tern of neuromuscular recruitment of the r e s p i r a t o r y muscles and maintain r e s p i r a t o r y muscle condi t ion (32). Further examination of th i s technique of mechanical v e n t i l a t i o n demonstrated that some pat ients complain of extreme dyspnea during IMV (76). In 20 c r i t i c a l l y i l l p a t i e n t s , the workload frequent ly equal led or exceeded the t o t a l workload expected for a spontaneously breathing healthy subject (78). Therefore , i t may be counter-product ive to use IMV because the r e s p i r a t o r y muscle exer t ion required may perpetuate r e s p i r a t o r y muscle f a t i g u e . The optimal r e s p i r a t o r y muscle res t regimen may be a combination of mechanical v e n t i l a t i o n a l t e r n a t i n g with spontaneous breath ing . I f the diaphragm atrophies at a s i m i l a r rate as other s k e l e t a l muscle in response to immobi l i za t ion , t h i s would suggest that the per iod of t o t a l mechanical v e n t i l a t i o n should be kept to a minimum. I f t h i s i s impossible because of compl icat ing f a c t o r s , the weaning process w i l l l i k e l y be very prolonged because of profound atrophy of the r e s p i r a t o r y muscles, and thus they must be r e t r a i n e d before they undertake the i n c r e d i b l e endurance feat of contrac t ing r e p e t i t i v e l y for the remainder of the p a t i e n t ' s l i f e . - 23 -AN ANIMAL MODEL OF RESPIRATORY MUSCLE FATIGUE AND REST Because of the anatomical p o s i t i o n and e s s e n t i a l funct ion of the ' diaphragm i t i s indeed very d i f f i c u l t to study t h i s muscle in humans. We chose to examine the e f fec t s of fa t igue and re s t on the diaphragm using the hamster as our animal model. The hamster appeared to be a s u i t a b l e species because the f i b r e type proport ions of the diaphragm are c l o s e r to humans than are those of the dog which is the species most commonly used to inves t igate diaphragmatic fa t igue . The diaphragm of the dog i s composed e n t i r e l y of type I and I l a f i b r e s which are fa t igue r e s i s t a n t (51). In c o n t r a s t , previous reports described a large percentage of fa t iguab le f ibres in the c o s t a l region of the hamster diaphragm (38,69,70) . The hamster i s often used as a species for examination of diaphragmatic s t ruc ture and funct ion because of the s u s c e p t i b i l i t y of t h i s animal to experimental emphysema (104). Since the hamster had not been used in t h i s l a b o r a t o r y , techniques for both p h y s i o l o g i c a l and h i s t o l o g i c a l examination of the diaphragm in these small animals had to be developed. I developed techniques to perform long-term (s ix to e ight hours) i n v e s t i g a t i o n of the hamster diaphragm in v i v o . Since some animals had to breathe spontaneously during the experimental p r o t o c o l , a new anesthet ic regimen was developed for the hamster which allowed a long-durat ion anesthesia but r e s u l t e d in minimal r e s p i r a t o r y depression Because of the small s i ze and the f r a g i l i t y of the phrenic nerves, i t was necessary to develop a new technique to s t imulate the diaphragm d i r e c t l y using p la te e l ec t rodes . Since the l i t e r a t u r e was very c o n t r o v e r s i a l regarding the homogeneity of the mammalian diaphragm, (28,41,49,74,81,83,89,90,102,108,118) the r e g i o n a l and i n t e r - a n i m a l - 24 -v a r i a b i l i t y of the h i s t o l o g i c a l features of the hamster diaphragm was inves t i ga ted . Repet i t ive e l e c t r i c a l s t imula t ion was used as the fat igue s t imulus . This has been used by severa l other inves t iga tors examining diaphragmatic funct ion and i s advantageous because c e n t r a l input i s bypassed and the parameters of the fat igue stimulus can be very t i g h t l y c o n t r o l l e d in the anesthetized animal. Spontaneous breathing and continuous mechanical v e n t i l a t i o n were used as the re s t protocols and the p h y s i o l o g i c and morphologic consequences of fat igue and r e s t were examined. OBJECTIVE To determine the e f fec t of fat igue and res t on the hamster diaphragm and s p e c i f i c a l l y to tes t whether mechanical v e n t i l a t i o n or spontaneous unloaded r e s p i r a t i o n was the best mode for f u n c t i o n a l recovery of the fat igued diaphragm. SPECIFIC AIMS 1. To develop a s u i t a b l e anesthet ic regimen for long-term (s ix to e ight hours) study of the hamster diaphragm in v i v o . 2. To develop appropriate techniques to perform long-term i n v e s t i g a t i o n of the funct ion of the hamster diaphragm in v i v o . 3. To charac ter i ze the proport ions of muscle f i b r e types , f i b r e s i z e s , glycogen l e v e l s and ox idat ive capac i ty of the hamster diaphragm. 4. To determine the reg iona l v a r i a b i l i t y of the h i s t o l o g i c a l features of the hamster diaphragm. 5. To determine the f u n c t i o n a l changes i n the hamster diaphragm in response to low and high frequency s t imula t ion fo l lowing a fa t igue s t imulus . - 25 -6. To determine from glycogen depletion, the muscle fibre type recruitment pattern produced by fatiguing stimulus of repetitive electrical stimulation. 7. To determine the histological changes in the hamster diaphragm following a fatigue stimulus. 8. To compare recovery of function of the acutely fatigued diaphragm by resting with either spontaneous breathing or mechanical ventilation. 9. To compare recovery of structure in the acutely fatigued diaphragm by resting with either spontaneous breathing or mechanical ventilation. - 26 -Figure 1: Diagram of the diaphragm i l l u s t r a t i n g the three major r e -gions ( s t e r n a l [ s t e r n ] , c o s t a l and c r u r a l ) of t h i s muscle. Figure 2: The primary act ions of the diaphragm. Contrac t ion of the diaphragm r e s u l t s in increased abdominal pressure (Pab) pushing outward on the zone of appos i t ion (Z a p) and lower r ibcage and causing outward movement of the abdomen. Contract ion of the diaphragm also r e s u l t s in force exerted on the i n s e r t i o n of the muscle r o t a t i n g the r i b s upwards and outwards. The generation of negative p l e u r a l pressure (Ppl) r e s u l t s in the inward p u l l on the upper r i b cage. - 27 -— End «xp*r«tory po»rtk>n of diaphragm — End inspir atofy poaltkxi Diaprragmatk; •xcuralon Figure 3: The effect of hyperinflation on the diaphragm. Hyperinflation of the lungs flattens the diaphragm decreasing i t s potential for excursion and pumping a b i l i t y . Figure 4 : The length-tension relationship of skeletal muscle. At shortened lengths less tension i s produced because the myosin and actin filaments overlap excessively. At optimal length, the myosin and actin filaments are able to interact to the greatest degree and thus maximal tension i s produced. At lengths greater than optimal length, the myosin and actin filaments interact to a lesser degree and less tension i s produced. - 28 -Brain Spinal Cord i Peripheral Nerve Neuromuscular Junction I Sarcotemma * Transverse Tubules Ca release by Terminal Cisternae Actin-myosin Coupling FORCE OUTPUT Figure 5: The pathway invo lved i n muscular c o n t r a c t i o n . - 29 -CENTRAL' -Voluntary Involuntary NEUROMUSCULAR TRANSMISSION PERIPHERAL Decreased energy supply E l e c t r o l y t e imbalance Excess H + Excess P i S u b - l e t h a l and l e t h a l c e l l damage decreased glycogen decreased l i p i d ecreased ATP decreased propagation of act ion p o t e n t i a l i n h i b i t i o n of phosphorylase and phosphofructokinase increased b inding of Ca^+ by sarcoplasmic re t i cu lum decreased a f f i n i t y of troponin for C a 2 + i n h i b i t i o n of myosin-a c t i n uncoupling Figure 6: Factors which may contr ibute to neuromuscular f a t i g u e . - 30 -REFERENCES 1. 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J Appl  P h y s i o l 47:43-50, 1979. 114. Vihko V , Salminen A, Rantamaki J : A c i d hydrolase a c t i v i t y in red and white s k e l e t a l muscle of mice during a two-week per iod fo l lowing exhausting exerc i se . Pf lugers Arch 378:99-106, 1978. 115. White TP: Adaptations of s k e l e t a l muscle grafts to chronic changes of p h y s i c a l a c t i v i t y . Fed Proc 45:1470-1473, 1986. 116. W i l k i e D: Shortage of chemical f u e l as a cause of fa t igue : Studies by nuclear magnetic resonance and b i c y c l e ergometry, i n : Human Muscle  Fat igue: P h y s i o l o g i c a l Mechanisms. Porter R, Whelan J (eds) . Pitman M e d i c a l , London, 1981, pp 102-119. 117. Wrogemann K, Pena SDJ: M i t o c h o n d r i a l calc ium overload: a general mechanism for c e l l necros is in muscle d iseases . Lancet V o l 1, 7961:672-677, 1976. 118. Y e l l i n H: Di f ferences in h i s tochemica l a t t r i b u t e s between diaphragm and hindleg muscles of the r a t . Anat Record 173:333-340, 1972. - 39 -I I . DEVELOPMENT OF ANESTHETIC REGIMEN AND PARAMETERS OF MECHANICAL VENTILATION INTRODUCTION Development of Anesthet ic Regimen Although the golden Syrian hamster (mesocricetus auratus) i s frequent ly used for biomedical research , most of the anesthet ic regimens described for t h i s species induce a r e l a t i v e l y short durat ion of anesthesia (5 ,8 ,9 ,11 ,12 ,14 ,17 ,24) . For the performance of long-term p h y s i o l o g i c a l studies examining the e f fec t s of fat igue and r e s t of the diaphragm in v i v o , we required an anesthet ic regimen which would: 1. induce anesthesia adequate to allow neck and abdominal surgery; 2. not produce r e s p i r a t o r y depress ion; and 3. al low an even depth of anesthesia for a s ix -hour per iod . To date, the anesthet ics descr ibed for use in hamsters were su i tab l e for the performance of surgery (5 ,8 ,9 ,12 ,14 ) . However, l o n g -durat ion anesthesia using prev ious ly descr ibed regimens would require frequent supplemental doses, p o s s i b l y making the depth of anesthesia extremely v a r i a b l e . The most commonly employed anesthet ic in the hamster, p e n t o b a r b i t a l , has r e s u l t e d in marked r e s p i r a t o r y depression (2,3,19) and i s d i f f i c u l t to t i t r a t e to a standard depth of anesthesia in other species (22). Af ter examination of the e f fec t s of severa l d i f f e r e n t anes the t i c s , we developed an e f f e c t i v e , l ong-durat ion anesthet ic regimen in the hamster by adminis ter ing a combination of urethane, a lpha-ch lora lose and sodium p e n t o b a r b i t a l . Parameters of Mechanical V e n t i l a t i o n During the long-term studies examining the e f fec t s of fa t igue and re s t on the diaphragm, the hamsters had to be mechanical ly v e n t i l a t e d - 40 -during s u r g i c a l preparat ion and l a t e r on in the experimental protoco l at l e v e l s s u f f i c i e n t to provide diaphragmatic r e s t . They a lso had to be hyper-v e n t i l a t e d to induce apnea for t e s t i n g of diaphragmatic f u n c t i o n . Although l e v e l s of minute v e n t i l a t i o n during spontaneous breathing for the hamster have been reported (17), information regarding response to d i f f e r e n t l e v e l s of mechanical v e n t i l a t i o n i s not known. Recent reports have suggested that mechanical v e n t i l a t i o n does not n e c e s s a r i l y r e s t the r e s p i r a t o r y muscles (1 ,21) . Therefore , we examined the EMG a c t i v i t y of the diaphragm during d i f f e r e n t l e v e l s of mechanical v e n t i l a t i o n in the hamster. This chapter describes the e f fec t s of d i f f e r e n t anesthet ics on anesthesia time, analges ic response and r e s p i r a t o r y c o n t r o l . During some of these experiments, we a lso determined the most e f f e c t i v e l e v e l s of mechanical v e n t i l a t i o n for t i d a l v e n t i l a t i o n and to induce apnea. METHODS  Animals Twenty-one adult male golden Syr ian hamsters (mesocricetus auratus) (Charles R i v e r , La P r a i r i e , Quebec) were used in three sets of experiments. They were housed i n d i v i d u a l l y in wire mesh cages and maintained on a d ie t of Pur ina Laboratory chow, water ad l i b i t u m u n t i l the time of the study. They had a mean body weight (BW) of 128.1+16.1 g (mean+SD). Experimental Protoco ls a. Development of Anesthet ic Regimen In a f i r s t set of animals , s evera l d i f f e r e n t anesthet ics were t r i e d . L i t t l e success was achieved with th iobutabarb i ta l - sod ium ( I n a c t i n - B y k ) , halothane, e ther , and a combination of methohexitone and - 41 -diazepam so fur ther i n v e s t i g a t i o n of these agents was not performed. Eleven hamsters were anesthet ized with an i n t r a p e r i t o n e a l i n j e c t i o n of one of the fo l lowing: 1. urethane (n=3), 2. ch lora lose (n=2), 3. pentobarb i ta l (n=3) or 4. a combination of agents #1-3 (n=3). Doses used are shown in Table I I I . The e f fec t s of the anesthet ics on time to anesthes ia , t o t a l anesthesia t ime, and a r t e r i a l blood measurements were inves t i ga ted . Anesthesia was defined as the state in which the animal was non-responsive to p inching of the paw with s u r g i c a l forceps and then to s u r g i c a l i n c i s i o n of the s k i n . In a second set of animals, the combination of anesthet ic agents (urethane, c h l o r a l o s e , pentobarbi ta l ) was fur ther examined in e ight animals. A r t e r i a l blood measurements and minute v e n t i l a t i o n were monitored in the animals for up to s ix hours. Supplemental doses of urethane and ch lora lose (13.5 and 1.4 mg/lOOg BW, r e s p e c t i v e l y ) were administered every two hours. Since during subsequent experiments, hamsters needed to be mechanical ly v e n t i l a t e d for t i d a l v e n t i l a t i o n and hypervent i la ted to induce apnea in order to measure diaphragmatic force output, these two l e v e l s of mechanical v e n t i l a t i o n were a l so determined. Animals were mechanical ly v e n t i l a t e d (Harvard Rodent V e n t i l a t o r , Model 683, S t . Laurent,Quebec) at v a r i a b l e l e v e l s and a r t e r i a l blood measurements were monitored. The d e l i v e r e d (as i n d i c a t e d by the mechanical v e n t i l a t o r se t t ings ) and measured (as determined by the plethysmograph) t i d a l volumes were compared. In order to determine apnea t ime, three hamsters were hypervent i la ted for three minutes, disconnected from the mechanical v e n t i l a t o r and then apnea time was measured. - 42 -TABLE I I I ANESTHETIC AGENTS AND DOSES IN THE HAMSTER  AGENT DOSE (mR/lOOgm BW) Urethane (50% w/v e t h y l carbamate)* 150 Alpha-ch lora lose (1% w/v)** 8-10 Pentobarb i ta l (6.5 mg/ml)*** 5-9 Combination Urethane, 38 Alpha-choralose and 3.8 Sodium pentobarb i ta l 2.6 * Sigma Chemical C o . , S t . Louis ** A 1% wt/v s o l u t i o n of a lpha-ch lora lose s o l u t i o n was made by heating a lpha-ch lora lose (BDH Chemicals L t d , Poole , England) and a 10% polyethylene g l y c o l (Sigma Chemical Co, MW:6000-7000) to 6 0 ° C . This was administered only up to 4 hours a f ter preparat ion . * * * Somnotol, MTC Pharmaceuticals , Miss i s sauga , Canada - 43 -b. Parameters of Mechanical V e n t i l a t i o n In a t h i r d set of two hamsters, the l e v e l of mechanical v e n t i l a t i o n necessary to minimize e l e c t r i c a l a c t i v i t y of the diaphragm was determined. In these experiments, the animal was anesthet ized with a combination of urethane, ch lora lose and pentobarb i ta l (doses-see Table I I I ) . The esophageal pressure (Pes) and the diaphragmatic electromyogram (EMG) were recorded (see Chapter I I I , pp 70-73 for d e t a i l s of methodology used for measurement of Pes and diaphragmatic EMG) during spontaneous breathing and then during d i f f e r e n t l e v e l s of mechanical v e n t i l a t i o n : h y p o v e n t i l a t i o n , adequate v e n t i l a t i o n and h y p e r v e n t i l a t i o n . Animal Preparat ion Animals were anesthetized with an i n t r a p e r i t o n e a l i n j e c t i o n of the anesthet ic agent(s) i n a quiet room. Fol lowing anesthes ia , they lay supine on a heated table with a r e c t a l probe connected to a thermis tor . Temperature . o was maintained at 37+1 C. Animals were then tracheostomized and allowed to breathe spontaneously through the tracheostomy i n c i s i o n . The l e f t c a r o t i d ar tery was cannulated. Sampling of A r t e r i a l Blood The l e f t c a r o t i d ar tery was d i s sec ted and cannulated using PE10 tubing in order to obta in a r t e r i a l blood samples. In the f i r s t set of experiments, one a r t e r i a l blood sample was taken at one hour. In the second set of experiments, up to f i v e samples of 0.3 ml each were taken during the course of each experiment and the blood volume was replaced with hepar in ized s a l i n e (10 u n i t s / m l ) . Since the hamster's c i r c u l a t i n g blood volume is estimated to be 10 m l , the smallest amount necessary for a r t e r i a l blood gas ana lys i s was obtained. The samples were analysed using an ABL 3 Acid-base - 44 -Laboratory blood gas analyzer (Radiometer, Copenhagen). The analyzer was o c a l i b r a t e d every two hours. PaO^ and PaCO^ were corrected to 37 C. Measurement of minute v e n t i l a t i o n and breathing pattern Minute v e n t i l a t i o n (V^) and breathing pattern were determined during spontaneous breathing and mechanical v e n t i l a t i o n using a body plethysmograph (7) s p e c i a l l y s ized for hamsters (volume was 10.5 L) s i m i l a r to that described by Koo et a l (14) ( F i g . 7) . The animal was pos i t ioned supine in the plethysmograph and the t r a c h e a l cannula was attached to a tapered s t a i n l e s s s t e e l tube (14 gauge). Changes in box pressure were used to measure t i d a l volume. Box pressure was monitored with a +2cm H^O transducer (Model no. MP45 Val idyne C o . , Northr idge , CA) . The volume of the plethysmograph was such that t i d a l breathing produced box pressures which were wel l w i th in the l i n e a r range of s e n s i t i v i t y of the pressure transducer . The box was c a l i b r a t e d at the beginning and the end of the study over the range of 0-10 ml which r e s u l t e d in a maximal change in box pressure of 1 cmH 0. For measurement of V and breathing p a t t e r n , a 2 Ci hamster was placed ins ide the plethysmograph, the plethysmograph was vented several times and the temperature and pressure allowed to e q u i l i b r a t e . The volume s i g n a l was recorded (Hewlett Packard mul t i - channe l recorder , Model no. 7758A, Massachussetts) and frequency of breathing ( f . ) , t i d a l volume b ( V T ) , and minute v e n t i l a t i o n were determined from the t r a c i n g . S t a t i s t i c a l Ana lys i s For the f i r s t set of experiments, a one-way ana lys i s of variance was performed to determine i f there was a d i f f erence between the e f fec t s of the d i f f e r e n t anesthet ic agents on a r t e r i a l blood measurements. Tukeys tes t was used to determine which groups were s i g n i f i c a n t l y d i f f e r e n t . - 45 -RESULTS a. Development of Anesthet ic Regimen The r e s u l t s from the f i r s t set of experiments examining the e f fects of the anesthet ic agents alone and in combination are i l l u s t r a t e d in F i g . 8 and Table IV. A r t e r i a l blood samples were not obtained in the animals anesthet ized with ch lora lose because inadequate s u r g i c a l anesthesia was obtained such that the tracheostomy and c a n n u l i z a t i o n of the c a r o t i d artery could not be performed. Urethane or sodium pentobarb i ta l administered alone in a dose s u f f i c i e n t to produce s u r g i c a l anesthesia produced marked r e s p i r a t o r y depression as manifested by s i g n i f i c a n t hypercapnia and hypoxemia ( F i g . 8) . Compared to animals anesthet ized with the combination, those anesthet ized with pentobarb i ta l and urethane had a higher PaCO^ (p<0.05) and those anesthet ized with pentobarb i ta l had a lower PaO^ (p<0.05). The group anesthet ized with urethane had a lower blood pH in comparison to the group anesthet ized with the combination (p<0.05). In add i t ion to r e s p i r a t o r y a c i d o s i s , a metabolic ac idos i s was assoc iated with the use of urethane. This was demonstrated by a greater base d e f i c i t (-2.2+-1.9) i n the urethane group than in the other two groups (pentobarbital:+2.7++2.5, combination: +0.4+ 0.5) (p=0.066). The d i f f erence may not have reached a s i g n i f i c a n t p value because of the small number of animals i n each group. In c o n t r a s t , the PaCO , PaO , and pH of the animals anesthet ized with the combination 2 2 were w i th in the normal range of values for spontaneously breathing hamsters (17). Animals anesthet ized with p e n t o b a r b i t a l produced large amounts of s a l i v a r e q u i r i n g frequent s u c t i o n i n g . Excess ive s a l i v a t i o n was not apparent fo l lowing admini s tra t ion of the other agents or combination of anes thet i c s . 46 -TABLE IV RESPONSE TO ANESTHETIC AGENTS ANESTHETIC TIME TO ANESTHESIA TOTAL ANESTHESIA TIME (min) (hr) Urethane 15 6.00 Alpha-ch lora lose Pentobarb i ta l 5 0.75 Urethane, a lpha-ch lora lose and pentobarb i ta l 15 1.5-2.0 - 47 -The e f fec t of the combination of anesthet ic agents on PaCO , 2 PaO^, pH and v e n t i l a t i o n over time i s i l l u s t r a t e d in F i g . 9 and F i g . 10 from the second set of experiments. The values were s table over the 6-hour p e r i o d . I f there were any changes, there was a trend towards improvement. The increase in V was p r i m a r i l y due to an increase i n the frequency of cl breathing with no change in the t i d a l volume ( F i g . 10). The f. ranged b between a mean of 63 and 90 breaths/min and the t i d a l volume ranged between a mean of 0.66 and 0.72 ml/lOOg BW. We determined that a f. of 80/min was the most s u i t a b l e frequency b of breathing during mechanical v e n t i l a t i o n for the anesthet ized hamster. When the mechanical v e n t i l a t o r was set at t h i s r a t e , the animals had less of a tendancy to spontaneously breathe in between the v e n t i l a t o r d e l i v e r e d breaths . The e f fec t of minute v e n t i l a t i o n on PaCO„ and PaO„ is shown in 2 2 F i g . 11. As expected, there was c lose correspondence between the PaCO^ achieved by spontaneous breathing and mechanical v e n t i l a t i o n at the same l e v e l of minute v e n t i l a t i o n (as determined by the plethysmograph). The e f f ec t of h y p e r v e n t i l a t i o n (using mechanical v e n t i l a t i o n ) on apnea time is shown in F i g . 12 (n=3). There appeared to be a f a i r l y c lose r e l a t i o n s h i p between minute v e n t i l a t i o n and apnea t ime. However, when the measured t i d a l volume was r e l a t e d to d e l i v e r e d t i d a l volume (as ind ica ted by the mechanical v e n t i l a t o r se t t ings ) the r a t i o v a r i e d between 0.24 and 0.69 ( F i g . 13). Even when a l t e r i n g the bias flow or occ luding the bias flow a l toge ther , the measured t i d a l volume v a r i e d cons iderably for a given d e l i v e r e d t i d a l volume between animals ( F i g . 13). - 48 -Monitoring the diaphragmatic EMG during spontaneous breathing and l e v e l s of mechanical v e n t i l a t i o n demonstrated that an auditory or v i s u a l EMG s i g n a l corresponded c l o s e l y with negative Pes swings ( F i g . 14, panel A & B) . When the l e v e l of mechanical v e n t i l a t i o n was progres s ive ly increased , the diaphragmatic EMG s i g n a l d iminished. No auditory or v i s u a l EMG s i g n a l was detected when only a p o s i t i v e d e f l e c t i o n ( i n synchrony with mechanical v e n t i l a t i o n ) was produced on the Pes t r a c i n g during each V e n t i l a t o r cyc le ( F i g . 14, panel C & D) . DISCUSSION The use of a combination of urethane, ch lora lose and pentobarb i ta l in the hamster r e s u l t e d in anesthesia adequate to permit the performance of surgery and also provided an even depth of anesthesia without r e s p i r a t o r y depress ion. Using t h i s anesthet ic regimen, we were able to determine the l e v e l s of mechanical v e n t i l a t i o n required for adequate t i d a l v e n t i l a t i o n and that necessary to produce apnea for t e s t i n g of diaphragmatic f u n c t i o n . The anesthet ic agents administered alone in the hamster were inadequate. Chlora lose d id not produce s u r g i c a l anesthes ia , and urethane and pento-b a r b i t a l produced marked r e s p i r a t o r y depress ion . The use of ch lora lose ( i n a dose of 8-10 mg/100 gm BW) as an anesthet ic agent alone r e s u l t e d in marked s k e l e t a l muscle h y p e r t o n i c i t y and inadequate s u r g i c a l anesthesia i n hamsters. Chlora lose ( l i k e c h l o r a l hydrate) produces hypnosis and anesthesia and i t s metabolism r e s u l t s in the formation of t r i c h l o r o e t h a n o l . The ac t ion of ch lora lose i s t y p i f i e d by f u n c t i o n a l d i s r u p t i o n or d i s s o c i a t i o n of the CNS through marked CNS s t imula t ion or induct ion of a c a t a l e p t o i d state (2) . Chlora lose increases - 49 -CNS e x c i t a b i l i t y and high doses can r e s u l t in se izures (2) and u l t imate ly r e s p i r a t o r y depression (6 ,10,25) . Since i t only produces stage II anesthesia (2) , i t i s not recommended as an anesthet ic for surgery in other spec ies . Within the l i m i t s of the experimental des ign, t h i s was confirmed in our studies on the hamster s ince the animal demonstrated marked increased muscle tone and f l i n c h i n g upon touch. Supplemental doses of ch lora lose only accentuated signs of increased muscle tone and hyperresponsiveness to touch without any apparent deepening of anesthesia to al low s u r g i c a l i n t e r v e n t i o n . Because of the h y p e r t o n i c i t y of the animal and the i n a b i l i t y to do surgery, we were unable to evaluate the r e s p i r a t o r y response s ince both the monitoring of v e n t i l a t o r y l e v e l s and a r t e r i a l blood measurements required surgery. When urethane alone was administered to the hamster ( i n a dose of 150 mg/100 gm body weight) , profound r e s p i r a t o r y depression r e s u l t e d ( F i g . 8) . In a d d i t i o n , the large decrease in pH was i n d i c a t i v e of both a r e s p i r a t o r y and metabolic a c i d o s i s . The e f f ec t of urethane in the hamster in our study was c e r t a i n l y contrary to the r e s u l t s of some inves t i ga tors (12,20,25) although others (10) have shown comparable v e n t i l a t o r y depress ion. There are severa l poss ib le causes of the metabolic ac idos i s found in the hamster in response to urethane. Th i s anesthet ic has been descr ibed as causing a decrease i n blood pressure (16), d i l a t i o n of the m i c r o -vasculature (15,16), leakage of p e r i t o n e a l f l u i d (18,23), and an increase in permeabi l i ty of the mesenteric microvasculature and other vascu lar beds (4,15) in other spec ies . From our experimental des ign, we were not able to i d e n t i f y whether or not one of these fac tors or other causes may have produced the metabolic a c i d o s i s . - 50 -Urethane had no s t imulatory e f f ec t on muscle l i k e that observed with c h l o r a l o s e . A s ing le dose of urethane had a long (approximately 6-hours in the hamster) durat ion of a c t i o n , s i m i l a r to the durat ion of ac t ion of c h l o r a l o s e . This i s in keeping with the long anesthesia produced by urethane in other species (2 ,4 ,12) . An anesthet ic dose of pentobarb i ta l administered to the hamster r e s u l t e d in marked r e s p i r a t o r y depression s i m i l a r to that descr ibed in other species (2 ,3 ,19) . Pentobarb i ta l i s a r e l a t i v e l y s h o r t - a c t i n g barb i tura te which is a r e s p i r a t o r y depressant in d i r e c t proport ion to the amount given. Induction in the hamster was quick , and the anesthesia deep and of moderate durat ion (45 minutes) making i t a su i tab l e agent for short s u r g i c a l procedures. Besides the marked r e s p i r a t o r y depression induced by pento-b a r b i t a l , recovery in the hamster was quick and unpredictable making th i s agent unsui table for experiments r e q u i r i n g long s u r g i c a l procedures and monitoring of p h y s i o l o g i c a l parameters which necess i ta ted anesthet ic r e s t r a i n t of the animal. Excessive s a l i v a t i o n (which required r e p e t i t i v e suct ioning) was another undes irable e f f e c t of t h i s anes thet i c . Because none of the above-mentioned agents used alone produced adequate anesthesia for our purposes in the hamster, a combination of c h l o r a l o s e , urethane and pentobarb i ta l was t e s ted . Since both urethane and ch lora lose do not peak i n ac t ion u n t i l at l e a s t 15 minutes p o s t - i n j e c t i o n , pentobarb i ta l was included for a qu ick , smooth induc t ion . Pentobarb i ta l would l i k e l y induce the deeper anesthesia necessary for s u r g i c a l preparat ion e a r l y in the experimental des ign. I f the animal was anesthet ized with the combination in a quie t environment with minimal handl ing , loss of - 51 -consciousness occurred wi th in f ive minutes and the animal was allowed to re s t a fur ther 10 minutes before being handled. We were unable to compare the e f fec t s of t h i s combination of anesthet ic agents to other studies s ince the use of t h i s regimen i s unique to our knowledge. An e f f e c t i v e dose of urethane and ch lora lose in the hamster was lower than that used in the r a t (13,19). The lower dose in the hamster could be explained by the supplemental use of p e n t o b a r b i t a l in our experiments and/or that hamsters have a slower rate of degradation or i n a c t i v a t i o n of the anesthet ic agent compared to r a t s . Since we d id not do any experiments with a combination of urethane and ch lora lose we are not able to compare d i r e c t l y to other s tudies . I n t e r e s t i n g l y , an increase in V in the anesthet ized hamster was E achieved by an increase in f^ with no change in except at extremely high l e v e l s of v e n t i l a t i o n . Because the f during t i d a l v e n t i l a t i o n b ranged between 60-90 breaths /min, we se lected a rate of 80/min for mechanical v e n t i l a t i o n and found spontaneous breathing was minimized during mechanical v e n t i l a t i o n at t h i s breathing frequency. The appropriate l e v e l of mechanical v e n t i l a t i o n for t i d a l v e n t i l a t i o n and to induce apnea was determined (Fig .11 and F i g . 12). I n i t i a l l y , i t was hypothesized that adequate mechanical v e n t i l a t i o n could be provided based on the d e l i v e r e d t i d a l volume and f^ of the mechanical v e n t i l a t o r s e t t i n g s . However, there was a very poor r e l a t i o n s h i p between measured and d e l i v e r e d t i d a l volume in d i f f e r e n t animals ( F i g . 13). The same equipment was used, i t was c a r e f u l l y c a l i b r a t e d and determined to be l eak-proof . The discrepancy between d e l i v e r e d and measured t i d a l volume may be a t t r i b u t e d p a r t i a l l y to v a r i a b l e compliances of the r e s p i r a t o r y - 5 2 -system in d i f f e r e n t animals , however some of the d i f ferences may have been due to undetected leaks in the equipment. Because of the poor r e l a t i o n s h i p between the measured and de l ivered t i d a l volume, adequate v e n t i l a t i o n could not be predic ted from the mechanical v e n t i l a t o r set t ings alone. I t was not p r a c t i c a l to perform measurements of t i d a l volume using the plethysmograph in subsequent studies because the hamster was connected to other apparatus. From the studies performed monitoring the diaphragmatic EMG and Pes during d i f f e r e n t l e v e l s of mechanical v e n t i l a t i o n , i t was apparent that the Pes s i g n a l was a reasonable i n d i c a t o r of spontaneous a c t i v i t y of the diaphragm. Thus, adequate t i d a l v e n t i l a t i o n was defined as mechanical v e n t i l a t i o n at l e v e l s s u f f i c i e n t to minimize any negative d e f l e c t i o n of the Pes s i g n a l ( F i g . 14). Pentobarb i ta l in the combination f a c i l i t a t e d a qu ick , smooth induct ion and deep anesthesia to l e v e l I I I such that surgery could be performed. The long durat ion of ac t ion of urethane and ch lora lose described in other species was also observed in the hamster. Although urethane used alone produced marked r e s p i r a t o r y depress ion , i t s use in the combination was at a much lower dose and no such e f f ec t was observed. The h y p e r t o n i c i t y demonstrated by ch lora lose alone seemed to have been countered by the use of the combination by act ion(s ) of e i t h e r pentobarb i ta l and/or urethane, or again perhaps because of the s l i g h t l y lower dose. We concluded that the combination of urethane, choralose and p e n t o b a r b i t a l was a good combination for our long term experiments. Secondly, because of the c lose correspondence between the negative Pes s i g n a l and diaphragmatic EMG, Pes was used in a l l subsequent experiments as the i n d i c a t o r of adequate t i d a l v e n t i l a t i o n and h y p e r v e n t i l a t i o n to induce apnea using mechanical v e n t i l a t i o n . - 53 -PLATE 1 Figure 7: Pressure displacement plethysmograph s p e c i a l l y s ized for the hamster. The photograph (upper) i l l u s t r a t e s the small c y l i n d e r ( in which the animal was placed) connected to the 10 L b o t t l e f i l l e d with copper wool. For the measurement of t i d a l v e n t i l a t i o n , the connection between the two containers was l e f t open such that the t o t a l volume of the c y l i n d e r and bo t t l e was 10.5 L . A s t a i n l e s s s t e e l tracheostomy tube (14 G) (arrow) entered the head box of the small c y l i n d e r (lower diagram). A bias flow (arrow head) of 50-60 ml/min of room a i r was d e l i v e r e d v i a a port w i th in the s t a i n l e s s s t e e l tube to the animal in order to minimize equipment dead space during spontaneous breath ing . The dead space of the apparatus during mechanical v e n t i l a t i o n was c a l c u l a t e d to be 0.15 mL. - 55 -80 - i r 7 . 5 CO 60 -4 0 -2 0 -A A A O Pentobarbital • Urtthan* A Combination PaCO, A A A a a e A A O A O • o - 7 . 4 V) "c 3 - 7 . 3 PaO, PH •7.2 Figure 8: The e f fec t of d i f f e r e n t anesthet ics on a r t e r i a l blood gas measurements. The raw data for each animal i s p l o t t e d . P a r t i a l pressure (mmHg) for PaC02 and Pa02 i s shown on the l e f t y -ax i s and pH uni ts i s shown on the r i g h t y - a x i s . - 5 6 -too-8 0 -E 60-e 3 «n 91 I S. 40-2 0 -I 1 I I • Pa02 • PaCO, 0-7.6-7.5-7.4-X a I 7.3-7.2-—I 1 1 1— 2-3 3-4 4 -5 5-6 Time (hours) Figure 9 : The e f fec t of the combination of anesthetic agents on a r t e r i a l blood measurements over time. The mean+S.D. for each time i n t e r v a l i s i l l u s t r a t e d . - 57 -to-. SO-I s 9 0.7-I > 0.6-0.5-£ 70-50-40- 1 — 2-3 3-4 4-5 Time (hours) 5-« Figure 10: The e f f e c t of the combination of anesthet ics on minute v e n t i l a t i o n , t i d a l volume, and frequency of breathing over time. The mean+SD for each time i n t e r v a l i s shown. - 58 -120 r 100 3 8 0 x E ^ 60 CM 8 & 40 h 20 0 160 r 120 a X E 80 8 s. 40 0 0 0 0 *0 0 MV • SB 100 200 300 400 MINUTE VENTILATION (ml/mln/100gm B.W.) Figure 11: The e f f e c t of minute v e n t i l a t i o n on PaC02 (upper panel) and PaC-2 (lower p a n e l ) . The raw data f o r 8 animals i s shown for spontaneous breathing (SB) and mechanical v e n t i l a t i o n (MV). Animals were para lyzed wi th s u c c i n y l cho l ine for low l e v e l s of v e n t i l a t i o n . - 59 -25 20 h v-^ 16 h v v v v V V V < 10 L U V 0 0 100 200 300 MINUTE VENTILATION (ml/min/100gm) Figure 12: The e f fec t of h y p e r v e n t i l a t i o n using mechanical v e n t i l a t o r on apnea time. The raw data from three animals i s shown. DELIVERED VT (ML) Figure 13: The e f fec t of de l ivered t i d a l volume (as measured on the mechanical v e n t i l a t o r ) on measured t i d a l volume (as determined by the plethysmograph). The raw data from 3 animals i s shown for d i f f e r e n t l e v e l s of bias flow (0,60 and 300 ml /min) . The l i n e of i d e n t i t y i s shown. - 60 -P late II Figure 14: The diaphragmatic EMG ( l e f t ) and Pes ( r ight ) recording during spontaneous breathing (A) and during various l e v e l s of mechanical v e n t i l a t i o n : hypovent i la t ion (B) , adequate v e n t i l a t i o n (C) and h y p e r v e n t i l a t i o n (D). The scale i s the same in a l l cases for the l e f t pane l . For the r i g h t panel , the v e r t i c a l sca le is the same for a l l cases, however, the h o r i z o n t a l scale is e i t h e r 10 sec/5 squares or 2 sec/5 squares as ind ica ted on the f i g u r e . Arrows ind ica te negative esophageal pressure d e f l e c t i o n s on panel B, r i g h t s ide . The e lectrocardiogram was a lso recorded on the EMG t r a c i n g under a l l condi t ions - 62 -REFERENCES 1. Benjamin RG, Chapman GA: D i r e c t electromyographic diaphragmatic a c t i v i t y during mechanical v e n t i l a t i o n ( A b s t r a c t ) . Am Rev Respir Pis 135 (Suppl):A52, 1987. 2. Booth NH, McPonald L E : Ve ter inary Pharmacology and Therapeutics 5th Ed . Ames, Iowa, Iowa State U n i v e r s i t y Press , 1982, Chapter 10, 11 and 13. 3. Borison HL: C e n t r a l nervous depressants , anes the t i c s , hypnot ics , sedatives and other r e s p i r a t o r y depressants. In Respiratory  Pharmacology. JG Widdicombe (ed). N .Y . Pergamon, 1981, pp 65-83. 4. Bree MM, Cohen BJ: Ef fec t s of urethane anesthesia on blood and blood vessels in r a b b i t s . Lab Animal Care 15: 254-259, 1965. 5. C u r l J L , Peters L L : Ketamine hydrochlor ide and xy laz ine hydrochlor ide anaesthesia in the golden hamster (Mesocricetus auratus ) . Lab Animals 17:290-293, 1983. 6. Dripps RP, Oumke PR: The e f fects of narco t i c s on the balance between c e n t r a l and chemoreceptor c o n t r o l of r e s p i r a t i o n . J Pharm Exp Ther 77:290-306, 1943. 7. Dubois AB, Botelho SY, Bede l l GN, M a r s h a l l R, Comroe JH: A r a p i d plethysmographic method for measuring thorac ic gas volume: a comparison with a ni trogen washout method for measuring f u n c t i o n a l r e s i d u a l capac i ty in normal subjects . J C l i n Invest 35:322-326, 1956. 8. Farkas GA, Roussos Ch: Histochemical and biochemical corre la t e s of v e n t i l a t o r y muscle fat igue in emphysematous hamsters. J C l i n Invest 74:1214-1220, 1984. 9. Ferguson JW: Anaesthesia in the hamster using a combination of methohexitone and diazepam. Lab Animals 13:305-308, 1979. 10. F l o r e z J , Borison HL: E f f e c t s of c e n t r a l depressant drugs on r e s p i r a t o r y r e g u l a t i o n in the decerebrate ca t . Resp P h y s i o l 6:318-329, 1969. 11. Green CG: Neuroleptanalges ic drug combinations in the anesthet ic management of small laboratory animals. Lab Animals 9:161-178, 1975. 12. Green CG: Animal Handbook 8 - Animal Anaesthes ia . Laboratory Animals L t d , London, Spottiswoode Bel lantyne L t d , 1979. 13. Hughes EW, Martin-Body RL, S a r e l i u s IH, S i n c l a i r JD: E f f e c t s of urethane-chloralose anesthesia on r e s p i r a t i o n in the r a t . C l i n Exp  Pharm P h y s i o l 9:119-127, 1982. - 63 -14. Koo KW, L e i t h DE, Sherter CB, Snider GL: Respiratory mechanics in normal hamsters. J Appl Phys io l 40:936-942, 1976. 15. Landis EM: M i c r o i n j e c t i o n studies of c a p i l l a r y permeab i l i t y . Am J P h y s i o l 81:124-142, 1927. 16. Longnecker DE, H a r r i s PD: M i c r o c i r c u l a t o r y act ions of general anes thet ics . Fed Proc 39:1580-1583, 1980. 17. Lucey EC, Snider GL, Javaheri S: Pulmonary v e n t i l a t i o n and blood gas values in emphysematous hamsters. Am Rev Respir Pis 125:299-303, 1982. 18. Severs WB, K e i l L C , Klase PA, Oeen KC: Urethane anesthesia in r a t s . A l t e r e d a b i l i t y to regulate hydrat ion . Pharmacology 22:209-226, 1981. 19. Seyde WC, McGowan L , Lund N, Pu l ing B, Longnecker DE: Ef fec t s of anesthet ics on r e g i o n a l hemodynamics in normovolemic and hemorrhaged r a t s . Am J P h y s i o l 249:H164-H173, 1985. 20. Sollman T: Manual of Pharmacology. 7th ed. P h i l a d e l p h i a , Saunders, 1949. 21. Stanescu DC, Rodenstein DO, Scarniere S, Hoeven C: Diaphragmatic electromyography during ass i s t ed v e n t i l a t i o n in a l e r t healthy subjects . Fed Proc 45:36, 1986. 22. Taber R, Irwin S: Anesthesia in the mouse. Fed Proc:1528-1532, 1969. 23. van der Meer C, Vers luys -Broers JAM, Tuynman HARE, Buur VAJ: The e f fec t of e t h y l urethane on hematocri t , blood pressure and plasma glucose. Arch Int Pharmacodyn Ther 217:257-275, 1975. 24. von S t r i t t m a t t e r J : Anaesthesie bein Goldhamster mit Ketamine und Methoxyflurane. Z e i t s c r i f t fur Versuchst ierkunde 14:129-133, 1972. 25. Wang SC, Nims L F : The e f fects of various anesthet ics and decerebrat ion on the CO2 s t imula t ion ac t ion of the r e s p i r a t i o n in ca t s . J Pharm  Exp Ther 92:187-195, 1948. - 64 -I I I . DIAPHRAGMATIC PLATE ELECTRODE STIMULATION OF THE HAMSTER DIAPHRAGM INTRODUCTION Conventional methods of studying the c o n t r a c t i l e force of the diaphragm in animals include measuring the pressure produced by t h i s muscle during e i ther phrenic nerve s t imula t ion (PNS) (1 ,2 ,4 ,7 ,10 ,19 ,22 ,23) or intramuscular s t imulat ion (4 ,15) . The phrenic nerves can be i s o l a t e d in animal preparations and st imulated d i r e c t l y (2 ,4 ,10 ,20 ,22) , or by placement of i n t r a c a v a l e lectrodes (1 ,6 ,18 ,23) . D i r e c t PNS in hamsters has been performed in our laboratory and by others (2) , however, the preparat ion is extremely f r a g i l e . The proposed experiments to examine the e f fects of fa t igue and re s t on the hamster diaphragm required in termi t tent measurements of diaphragmatic force output throughout the durat ion of the s ix to e ight hour experiments. The animal had to be connected and disconnected to a mechanical v e n t i l a t o r , a r t e r i a l blood gases had to be obtained, and movement of the animal was involved in applying external abdominal pressure . Because of the f r a g i l i t y of the PNS preparat ion i t was doubtful that i t could be maintained for these long-term studies examining the e f fec t s of fa t igue and r e s t on the hamster diaphragm. PNS by placement of i n t r a c a v a l e lectrodes would be d i f f i c u l t in hamsters because of t h e i r small s i ze and in the proposed experiments, the need for i n f u s i o n of f l u i d s . Intramuscular s t imula t ion by i n s e r t i o n of hook e lectrodes d i r e c t l y in to the diaphragm has been performed in dogs (4 ,17) . We attempted th i s in three animals. Although i t i s poss ib le to i n s e r t f ine hook e lectrodes d i r e c t l y into the hamster diaphragm, we found that a pneumothorax was - 65 -created e i t h e r during the i n i t i a l i n s e r t i o n or during subsequent t e tan ic s t imula t ion of the muscle. Because of the t e c h n i c a l d i f f i c u l t i e s of using convent ional methods i n small animals and the des ire to do long term studies of diaphragmatic funct ion in the hamster, we developed a new technique of apposing p late e lectrodes d i r e c t l y against the abdominal surface of the diaphragm to apply e l e c t r i c a l s t i m u l a t i o n . The purpose of t h i s chapter i s to descr ibe t h i s technique and to compare both the mechanical and e l e c t r o p h y s i o l o g i c a l responses of the diaphragm during PNS with those obtained with diaphragmatic p la te e lectrode s t imula t ion (DPS) in the hamster. METHODS  Animals Eighteen adult male golden Syr ian hamsters (mean body weight: 129.8+12.3g (SD)) from Charles R i v e r , La P r a i r i e , Quebec, were s tud ied . Experimental Protoco ls The animal preparat ion i s i l l u s t r a t e d in F i g . 15. Animals were i n i t i a l l y anesthet ized using a mixture of urethane (38 mg/100 gm), a lpha-ch lora lose (3.8 gm/100 gm) and sodium pentobarb i ta l (2.6 mg/ 100 gm) . Supplemental increments of the anesthet ic agents were administered as necessary. A tracheostomy was made with an i n c i s i o n between the t h i r d and fourth c a r t i l a g i n o u s r ings of the trachea and a short s t a i n l e s s s t e e l 14-gauge tube was inserted and secured. The hamsters were mechanical ly v e n t i l a t e d with room a i r using a rodent v e n t i l a t o r (Model 683, Harvard, St . Laurent , Quebec) throughout a l l s u r g i c a l preparat ions and the remainder of - 66 -the experiment. Immediately before diaphragmatic s t i m u l a t i o n , animals were hypervent i la ted to apnea. In the f i r s t group of four animals , the mechanism of DPS was examined by comparing the diaphragmatic ac t ion p o t e n t i a l produced using r i g h t DPS and r i g h t PNS. The animals were prepared in a s i m i l a r manner to that described above for b i l a t e r a l s t imula t ion except that only the r i g h t phrenic nerve was i s o l a t e d and s t imulated . For DPS, the dome conta in ing two e lectrodes ( inner and outer) on the r i g h t s ide was used. Once the s u r g i c a l preparat ion was complete, a Pab of 15 cmH^O was appl ied before each s t imulat ion and the diaphragm was s t imulated a l t e r n a t e l y by r i g h t PNS and r i g h t DPS. The diaphragmatic ac t ion p o t e n t i a l was recorded and evaluated for amplitude and latency with increas ing s t imula t ion current . In three animals , the ac t ion p o t e n t i a l s produced by DPS and PNS were observed fo l lowing each of two doses of curare (each dose:0.020 mg/lOOgm). In a second group of four animals , the e f fec t of increas ing current on peak twitch (PTT) and the r e l a t i v e c o n t r i b u t i o n of g a s t r i c (Pga) and esophageal pressure (Pes) with PNS and DPS was compared using b i l a t e r a l PNS and DPS with a constant Pab of 15 cmH^O. S t imulat ion was performed using a twitch pulse and the Pga, Pes and Pdi responses were examined. In a t h i r d group of s ix animals , the e f f ec t of Pab on Pdi using DPS was examined. A f t e r s u r g i c a l p r e p a r a t i o n , a corset with the small water bag was f i t t e d over the lower trunk of the hamster to achieve a base l ine Pga (Pab) of 10 cmHgO. The animal was hypervent i la ted to apnea and during the h y p e r v e n t i l a t i o n , 30 seconds before the st imulus was a p p l i e d , Pab was increased to the prescr ibed amount (up to 30 cmH^O in 5 cmH^O i n t e r v a l s ) , the st imulus appl ied and then Pab re leased to the r e s t i n g l e v e l - 67 -of 10 cmH20 af ter the e l e c t r i c a l s t imula t ion was performed. The corset was loosened to enable measurements of Pdi with Pab lower than 10 cmH^O (Pab of A , 6 and 8 cmH 20). In a fourth group of four animals, the r e p r o d u c i b i l i t y of the Pdi obtained with DPS was measured over 2 hours. Twitch and t e t a n i c pressure measurements with a Pab of 15 cmH^O, were taken every 15 and 30 minutes, r e s p e c t i v e l y . Animals were spontaneously breathing between measurements. Diaphragmatic P late Elec trode Preparat ion From gross d i s s e c t i o n of the hamster, we found that the r i g h t phrenic nerve terminated upon the diaphragm j u s t l a t e r a l to the a n t e r i o r margin of the i n f e r i o r vena cava. The l e f t phrenic nerve entered the diaphragm on the opposite s ide of the c e n t r a l tendon in a s l i g h t l y more a n t e r i o r p o s i t i o n . E a r l y experiments with an open abdomen preparat ion in the hamster demonstrated that Pdi (Pes only) was greatest and more of the diaphragm contracted (as determined by v i s u a l observation) when the s t imula t ing e lec trode was near the s i t e of phrenic nerve i n n e r v a t i o n . In order to make the dome encasing the diaphragmatic p la te e l ec t rodes , we casted the abdominal c a v i t y of a dead ev i scerated 130 gm hamster ( F i g . 16). Using t h i s cas t , we i n i t i a l l y fashioned a hard p l a s t i c dome (using q u i c k - d r y i n g denta l p l a s t i c ) corresponding to the abdominal surface of the diaphragm which encased p la te e l ec trodes . A f t e r t e s t i n g t h i s in three hamsters, i t appeared that a more f l e x i b l e dome would decrease the load and may al low more normal movement of the diaphragm in the spontaneously breathing animal. For a l l subsequent s tud ie s , a p l i a b l e dome corresponding to the abdominal surface of the diaphragm was fashioned ( F i g . 17) by applying a t h i n layer of s i l i c o n e ge l around two "dished" - 68 -plat inum plate e lectrodes (diameter: 1mm) . The e lectrodes were placed on the cast with t h e i r convex surfaces upwards and near the s i t e of phrenic nerve i n n e r v a t i o n . The e lectrodes were each connected to s ing le - s tranded enamelled copper wires , l eading to mul t i s tranded ( f l e x i b l e ) wires in the area of t r a n s i t i o n out of the body c a v i t y . Drying of the ge l secured the e lectrodes in appropriate p o s i t i o n on the s i l i c o n e dome. For one set of experiments, a s p e c i a l dome was fashioned conta in ing two s t imula t ing e lectrodes on the r i g h t side - an inner s t imula t ing and recording e lectrode near the s i t e of phrenic nerve innervat ion and an outer recording e lectrode approximately 2 mm p o s t e r o - l a t e r a l to the inner e l ec trode . Once the s i l i c o n e gel was d r y , the dome was trimmed such that when pos i t ioned ins ide a l i v e animal , i t s i n f e r i o r border lay j u s t below the c o s t a l i n s e r t i o n of the diaphragm. A notch was cut out p o s t e r i o r l y to al low passage of the great vessels and esophagus when placed in s i t u . To prepare an animal for DPS, the animal was anesthet ized , and then a small i n c i s i o n was made in the sk in at the l e v e l of the upper lumbar vertebrae p o s t e r i o r l y . Through the i n c i s i o n , a large (1cm x 5cm) f l a t plat inum i n d i f f e r e n t e lectrode was pos i t ioned lengthwise along the back j u s t beneath the s k i n , and the i n c i s i o n c losed . Through a mid l ine abdominal i n c i s i o n , the l i v e r was gent ly r e t r a c t e d , and the diaphragm was exposed. The s i l i c o n e dome was pos i t ioned under the diaphragm with the s t imula t ing e lectrodes near the s i t e of phrenic nerve i n n e r v a t i o n . The dome was sutured to the abdominal w a l l at severa l points around i t s circumference j u s t below the c o s t a l i n s e r t i o n of the diaphragm. The i n c i s i o n was then c losed t i g h t l y with the f l e x i b l e e lectrode wires e x i t i n g between the suture s i t e s ( F i g . 15). - 69 -Proper e lectrode p o s i t i o n was l a t e r confirmed by autopsy in a l l animals s tud ied . At the end of the experiment the a n t e r i o r r i b cage was removed and the e lectrodes were v i s u a l i z e d d i r e c t l y through the i n t a c t trans lucent diaphragm. Preparat ion for Phrenic Nerve St imulat ion (PNS) Through a mid l ine i n c i s i o n over the neck, the phrenic nerves were i s o l a t e d b i l a t e r a l l y j u s t below t h e i r ex i t s from the f i f t h c e r v i c a l root . Under a d i s s e c t i n g microscope the f a s c i a was gently teased away from the nerve and small b i p o l a r platinum hook e lectrodes ( i n t e r n a l diameter: 2mm) were placed under each nerve. Skin f laps about the neck i n c i s i o n were drawn up and mineral o i l was poured into the depression to cover the nerves . S t imulat ion Technique The apparatus for diaphragmatic s t imula t ion and electromyographic recording i s shown in F i g . 18. P r i o r to s t i m u l a t i o n , the impedances of both the DPS and PNS s t imula t ing e l e c t r o d e - p a i r s were measured in two animals as fo l lows: A 1kHz s i n u s o i d a l s i g n a l of amplitude 1.0 v o l t s rms suppl ied by an o s c i l l a t o r (Hewlett-Packard model 202C, Palo A l t o , CA) and measured with a multimeter (Fluke 8020A, E v e r e t t , WA), was appl ied to the ser ies combination of a 10 kOhm r e s i s t o r and the e l e c t r o d e - p a i r under t e s t , located in s i t u as descr ibed above. The a l t e r n a t i n g p o t e n t i a l r e s u l t i n g across the e l ec t rode -p a i r (V . ) was then measured with the same mult imeter. F i n a l l y , the p a i r impedance (Z . ) of the e l e c t r o d e - p a i r was c a l c u l a t e d from the r p a i r r e l a t i o n s h i p : Zpa ir v p a i r = , - 70 -The impedance of the e lectrode p a i r was assumed to be predominantly r e s i s t i v e . In both cases (DPS and PNS), the impedance of the e lectrode p a i r was in the region of 1 kOhms (+20%). The s t i m u l i employed for both PNS and DPS were suppl ied by the t rans former - i so la t ed s t imulator (model 14E12) i n s t a l l e d in a storage monitor (model 14H05, DISA, Skovlunde, Denmark). The stimulus durat ion was set at 200 us for a l l s t i m u l a t i o n . Knowing the impedances of the e l e c t r o d e - p a i r s , i t was poss ib le to provide c u r r e n t - s t i m u l a t i o n rather than voltage s t imula t ion by simply i n s e r t i n g an accurate r e s i s t o r with a value much greater than the impedance of the e lec trode p a i r in ser ies with the (act ive) cathode of the e l e c t r o d e - p a i r . A value of 100 kOhms +1% was chosen for PNS, and 30 kOhms +1% for DPS. These values r e f l e c t e d the range of stimulus current which i t was f e l t might be needed to obtain supramaximal s t i m u l a t i o n . An accurate s e r i e s - r e s i s t o r and an o s c i l l o s c o p e (model 1740A Hewlett-Packard, Palo A l t o , CA) were used to v e r i f y that square pulses of current of the expected amplitudes were indeed being suppl i ed . Muscle a c t i o n - p o t e n t i a l recording The diaphragmatic muscle a c t i o n - p o t e n t i a l s were recorded in the fo l lowing conf igura t ions : a) s t imula t ion of the phrenic nerve in the neck and recording from the inner p la te e l ec trode , with respect to the large f l a t e lectrode ( i n d i f f e r e n t ) and using a small subcutaneous ground wire inser ted into the animal's r i g h t f o r e l e g . b) with the e lec trode placement as in case a) but prov id ing current s t imula t ion v i a the same inner e l ec t rode . - 71 -c) for DPS prov id ing current s t imula t ion v i a the inner e l ec t rode , but recording from the outer p la te e l ec t rode . Measurements of amplitude and la tency were made in cases , a) and c) using the image storage f a c i l i t i e s of the DISA electromyograph (D1SA, Skovlunde, Denmark). The data was a lso recorded in a l l three of the above mentioned cases ( i n c l u d i n g DPS-st imulat ing and recording from the same e lectrode) by a d i g i t a l EMG apparatus. The d i g i t a l EMG apparatus was developed s p e c i f i c a l l y for recording in case b) to permit removal of the dominant stimulus a r t i f a c t (11). D i g i t a l EMG Recording Apparatus Because of the large s ize of the decaying p o l a r i z i n g voltage of the s t imula t ion e lectrode (200mV) and i t s time course co inc ident with the diaphragmatic ac t ion p o t e n t i a l , the r e l a t i v e l y small diaphragmatic ac t ion p o t e n t i a l (15mV) was d i f f i c u l t to d i s c e r n . Thi s made accurate measurements of amplitude and latency of the diaphragmatic ac t ion p o t e n t i a l very d i f f i c u l t . Therefore , a d i g i t a l recording apparatus was developed to recover an a r t i f a c t - f r e e act ion p o t e n t i a l . The apparatus and technique were developed by a col league in t h i s l aboratory with my ass i s tance . De ta i l s of the apparatus and technique employed are descr ibed i n a paper submitted for p u b l i c a t i o n (6). The technique w i l l be summarized below. A d i g i t a l recording system was developed with the dynamic range required to acquire the combined large st imulus a r t i f a c t and small diaphragmatic ac t ion p o t e n t i a l . In order to record the appropriate data for determining the diaphragmatic ac t ion p o t e n t i a l from the same (inner) e lectrode as that prov id ing current s t imula t ion we d id the f o l l o w i n g : - 72 -a) During the experiment, we recorded a sub-threshold s t imula t ion a r t i f a c t in the hamster preparat ion ( F i g . 19, Panel A ) . This s t imula t ion a r t i f a c t had a s i m i l a r form but smal ler amplitude compared to those recorded at higher current s . b) As stimulus current was increased beyond threshold l e v e l s , both the stimulus a r t i f a c t and diaphragmatic ac t ion p o t e n t i a l was recorded ( F i g . 19, Panel A ) . c) A f t e r the experiment was completed, time synchronizat ion and s ca l ing of the s igna l s were c a r r i e d out in an i n t e r a c t i v e fa sh ion . F i r s t l y , the stimulus a r t i f a c t alone was matched to the same amplitude as the combined stimulus a r t i f a c t and the diaphragmatic ac t ion p o t e n t i a l ( F i g . 19, Panel B ) . Second, the t r a i l i n g edges of the two s igna l s were matched ( F i g . 19, Panel C) and then the d i f f erence of the two s ignals was c a l c u l a t e d prov id ing a s i g n a l equivalent to the diaphragmatic act ion p o t e n t i a l alone ( F i g . 19, Panel D) . L a s t l y , the c a l c u l a t e d diaphragmatic ac t ion p o t e n t i a l was enlarged ( F i g . 19, Panel E ) . Some r e s i d u a l a r t i f a c t remained e a r l y in the time course of the c a l c u l a t e d diaphragmatic ac t ion p o t e n t i a l recorded from the inner e l ec t rodes . Transdiaphragmatic Pressure (Pdi) Transdiaphragmatic pressure (Pdi) was measured as an index of force output of the diaphragm during DPS and PNS. Pdi was def ined as the a lgebra ic sum of g a s t r i c (Pga) and esophageal (Pes) pressure changes from base l ine (Pdi = Pga - Pes) . The passive g a s t r i c and esophageal pressures before diaphragmatic contrac t ion were not part of the measure. Pga and Pes were measured by w a t e r - f i l l e d polyethylene catheters (PE 50) (with several s ide holes cut into the d i s t a l 0.75 cm of each catheter) placed down the - 73 -esophagus. The d i s t a l end of the g a s t r i c catheter was placed in the stomach. The esophageal catheter was inser ted into the stomach, withdrawn u n t i l a negative pressure was produced with i n s p i r a t o r y e f f o r t s and then withdrawn a fur ther 1.5 cm. The proximal end of these catheters were connected to separate d i f f e r e n t i a l transducers (Model 1280 and 267 BC, Hewlett Packard, Waltham, MA). The pressure transducers were c a l i b r a t e d using a water manometer. In a d d i t i o n , the two transducers were determined to be in phase up to 30 Hz by using an o s c i l l a t i n g pressure source. Because of the des i re to examine the time course of the diaphragmatic t w i t c h , the synchrony of the transducers was a lso examined using a short impulse with a s i m i l a r amplitude and a time course h a l f of that expected from the diaphragmatic twitch of the hamster. This was done by p lac ing the Pga and Pes catheters through a rubber stopper into a w a t e r - f i l l e d f l a s k . A 10 ml w a t e r - f i l l e d glass syringe a lso communicated with the f l a s k v i a another opening in the stopper ( F i g . 20). A pressure impulse was produced by applying a sharp blow to the top of the syr inge . Pga and Pes were recorded (Hewlett Packard mult ichannel r e c o r d e r , Model No. 7758A, Waltham, Massachussetts) and Pdi was obtained by e l e c t r o n i c a l l y subtrac t ing Pes from Pga. From c lose examination of the phase response of the pressure transducers , we determined that they were i n phase wi th in at l e a s t h a l f the r e a c t i o n times (time to peak pressure [TPP] and h a l f - r e l a x a t i o n time fRT^/^l ) of the mechanical response of the diaphragm. However, when we t r i e d to determine TPP and RT^/^ of the hamster diaphragm in v i v o , the Pes and Pga were c o n s i s t e n t l y out of phase. We d id not know whether Pes and/or Pga was most representat ive of the response time of the diaphragm in - 74 -the hamster and summing the out-of-phase s igna l s would have been meaningless. Hence, we d id not include the data for TPP and R T ^ / ^ . Twitch and Pressure-Frequency C h a r a c t e r i s t i c s Twitches produced by e l e c t r i c a l s t i m u l i (200 ys pulses) were therefore analyzed for peak twitch pressure (PTP). Pdi was a lso measured during t e t a n i c maximal s t i m u l a t i o n , (200 ys pu l ses , one sec durat ion) and at maximal current and a range of stimulus frequencies (30-70Hz). In previous experiments, we examined the e f fec t of d i f f e r e n t stimulus frequencies (10-100Hz) on P d i . A fused tetanus was not achieved at 10 and 20 Hz and Pdi plateaued at 70 Hz. Therefore , for these experiments and a l l future s tud ies , we chose a range of st imulus frequencies between 30 and 70 Hz, i n c l u s i v e . E x t e r n a l Abdominal Pressure (Pab) In an attempt to maintain constancy of diaphragm geometry, p o s i t i o n and muscle length during c o n t r a c t i o n , constant external abdominal pressure (Pab) was a p p l i e d . During ear ly experiments, we t r i e d cas t ing the abdomen of the hamster, the technique commonly used to maintain increased abdominal pressure in other animal preparat ions . I t was d i f f i c u l t to apply a t i g h t f i t t i n g p l a s t e r to the abdomen alone on t h i s small animal . In a d d i t i o n , once the cast was a p p l i e d , Pab was not constant but rather progres s ive ly f e l l throughout the course of the experiment. A small corset with two wide v e l c r o adhesive straps wrapping around the abdomen and one s trap running from the back across the gro in to the abdomen was much eas i er to apply and readjust on the hamster. In order to to apply in termi t t en t Pab which could be v a r i e d over a wide range of pressures , a small f l u i d - f i l l e d bag was placed over the abdomen and then both the bag and lower trunk of the animal - 75 -was encased in the corset ( F i g . 15). The corset was l e f t in p o s i t i o n throughout the durat ion of the experiment and provided a base l ine Pab of 8 to 10 cmH^O. The small bag was attached to polyethylene tubing ( i n t e r n a l diameter: 5 mm) which in turn lead to a large f l u i d - f i l l e d bag supported on a stand ( F i g . 15). Increased abdominal pressure was appl i ed 30 seconds before s t imula t ion by r a i s i n g the height of the large f l u i d - f i l l e d bag on the stand. Pab was measured as the base l ine Pga before s t i m u l a t i o n . The bag was lowered immediately fo l lowing e l e c t r i c a l s t i m u l a t i o n . S t a t i s t i c a l Ana lys i s An analys i s of variance (ANOVA) was used to determine i f there was a s i g n i f i c a n t d i f f erence in Pdi produced by v a r i a b l e Pab. Secondly, ANOVA with time as a repeated measure and animals as a b lock ing f a c t o r was used to dete rmine the r e p r o d u c i b i l i t y of Pdi from twitch and t e t a n i c s t imula t ion over a two hour p e r i o d . RESULTS Using PNS, the diaphragmatic ac t ion p o t e n t i a l recorded by the DISA electromyograph (EMG) was comparable to the response recorded by the d i g i t a l EMG apparatus. Representative diaphragmatic ac t ion p o t e n t i a l s produced by PNS and recorded by the DISA (upper panel) and the d i g i t a l (middle panel) EMG apparatus are shown for comparison in F i g . 21. The shape, amplitude and la tency of the ac t ion p o t e n t i a l s were s i m i l a r . A representa t ive act ion p o t e n t i a l recorded by the d i g i t a l data a c q u i s i t i o n apparatus with DPS is shown in the lower panel of F i g . 21. By using the d i g i t a l data a c q u i s i t i o n technique, much of the s t imulat ion a r t i f a c t was removed from the compound diaphragmatic ac t ion p o t e n t i a l recorded from the inner e lec trode with DPS. - 76 -However, because of r e s i d u a l s t imulat ion a r t i f a c t , the t a k e - o f f point could not be accurate ly determined ( F i g . 21, lower pane l ) . Therefore the latency for DPS was determined from the compound diaphragmatic ac t ion p o t e n t i a l recorded from the outer e lectrode ( F i g . 22). Although the la tency for DPS was shorter than for PNS, the shape and amplitude of the ac t ion p o t e n t i a l was s i m i l a r . S i m i l a r r e s u l t s were obtained from the other three animals s tud ied . The mean amplitude for PNS was 15.5 + 4.6 mV (mean+SD) and for DPS was 14.4 + 4.0 mV. The latency of s t imula t ion for PNS was 1.9 + 0.1 and for DPS was 0.9 + 0.0 mS. The latency of the ac t ion p o t e n t i a l produced with DPS was cons is tent with that of nerve s t imula t ion and much longer than what would be observed for d i r e c t muscle a c t i v a t i o n . Assuming that a c t i v a t i o n was v i a the small branches of the phrenic nerve, we used the d i f f erence between la t enc i e s for DPS and PNS, and the length of t r a v e l between the two s t imula t ion s i t e s (4 cm) to c a l c u l a t e the v e l o c i t y of phrenic nerve conduction. The conduction v e l o c i t y ranged between 40 and 44.4 m/sec. A f t e r recording ac t ion p o t e n t i a l s for PNS and DPS with supramaximal current , we administered two cumulative doses of curare . With increas ing amounts of curare , the ac t ion p o t e n t i a l s produced with twitch s t imula t ion by both PNS and DPS decreased in amplitude (see F i g . 23) i n a s i m i l a r manner. Peak twitch pressure and the p r o p o r t i o n a l c o n t r i b u t i o n of Pes and Pga produced with increas ing current by DPS and PNS d i f f e r e d i n some respects (N=4) ( F i g . 24). Phrenic nerve s t imula t ion produced progres s ive ly greater Pdi and Pes in response to increas ing current u n t i l a p lateau was reached. Further increases in current r e s u l t e d in no change in Pdi and Pes. Pga contr ibuted very l i t t l e to Pdi produced by PNS. In c o n t r a s t , DPS produced progress ive ly greater Pdi and Pga over the range of stimulus currents examined. No plateau of Pdi or Pga was obvious although an i n f l e c t i o n in the Pdi curve was apparent in three of the four hamsters. Esophageal pressure appeared to plateau at the same point as the i n f l e c t i o n of P d i , and Pdi at t h i s point was equivalent to Pdi produced by maximal current using PNS. Using DPS, Pga contr ibuted minimal ly to Pdi at low stimulus current s , however, once Pes reached i t s plateau va lue , Pga contr ibuted a much greater proport ion to Pdi and i t continued to increase with increas ing c u r r e n t . Subsequent experiments have demonstrated that the point at which Pes plateaus corresponded c l o s e l y with maximal current as ind ica ted by the compound act ion p o t e n t i a l . In s ix animals , increas ing Pab up to 15 cmH^O r e s u l t e d i n a progress ive increase in Pdi ( F i g . 25). However, only the Pdi at 2 and 4 cmH^O of Pab was s t a t i s t i c a l l y d i f f e r e n t (p<.0001) from the Pdi produced at 15 cmH 20. Increas ing Pab beyond 15 cmH^O caused a trend towards decreased Pdi but the decrease was not s i g n i f i c a n t . In the l a s t group of animals, we examined the short - term r e p r o d u c i b i l i t y of repeated measures of the twitch Pdi and Pdi with t e tan ic s t imula t ion (30-70 Hz) using DPS over time. Base l ine and two-hour r e s u l t s are shown in F i g . 26. There was no d i f f erence between the repeated measures of Pdi with twitch and t e tan ic s t imula t ion over a two-hour p e r i o d . Pdi increased cons iderably from 1 to 50 Hz s t i m u l a t i o n , began to plateau at 50 Hz and reached a maximal value at 70 Hz s t i m u l a t i o n . - 78 -DISCUSSIOM Because conventional methods of s t imula t ing the diaphragm were i m p r a c t i c a l to use over a per iod of hours, we developed a new technique for diaphragmatic s t imula t ion in the hamster. We found the p la te e lec trode was a very e f f e c t i v e method of s t imula t ing the diaphragm and was reproducib le over a two-hour p e r i o d . We found that with constant c u r r e n t , the maximal Pdi was produced when the s t imula t ing e lec trode was near the s i t e of phrenic nerve i n n e r v a t i o n . S i m i l a r r e s u l t s have been reported by Nochomovitz et a l in the dog (17). In contrast to intramuscular s t imula t ion of the hamster diaphragm, the incidence of pneumothorax was very low using DPS and only occurred as the r e s u l t of t e c h n i c a l e r r o r when suturing the s i l i c o n e dome in p lace . S i m i l a r to transvenous PNS there was no r i s k of nerve damage due to s u r g i c a l manipulat ion of the phrenic nerve. Unl ike transvenous PNS, DPS d id not r e s u l t in cardiac f i b r i l l a t i o n and the major vesse ls were s t i l l a v a i l a b l e for c a t h e t e r i z a t i o n . In comparison to phrenic nerve s t i m u l a t i o n , DPS was a less f r a g i l e preparat ion and was s impler and quicker to implement in the hamster. Moreover, the e l e c t r o p h y s i o l o g i c a l and mechanical responses at maximal stimulus using DPS and PNS were s i m i l a r . The comparable amplitudes of the compound ac t ion p o t e n t i a l s with DPS and PNS suggest that an equal number of diaphragmatic motor un i t s were r e c r u i t e d by both techniques ( F i g . 21). The estimated phrenic nerve conduction v e l o c i t i e s determined from the d i f f erence in l a t e n c i e s of the compound ac t ion p o t e n t i a l s produced with DPS and PNS suggest that diaphragmatic a c t i v a t i o n using DPS was v i a the small branches of the phrenic nerve. Conduction v e l o c i t i e s of the hamster phrenic nerve ranged between 40 and 44.4 m/sec, which i s w i th in the range reported for small - 79 -myelinated f i b r e s (9) . The threshold e x c i t a t o r y current required for s t imula t ion was only s l i g h t l y higher for DPS (0.5 to 1.0 mA) than for PNS (0.4 to 0.8 mA). Since the threshold for muscle e x c i t a t i o n i s approximately an order of magnitude greater than that required for nerve (15), the comparable current required for DPS and PNS also suggested that the e x c i t a t i o n of the diaphragm with DPS was v i a nerve and not d i r e c t l y v i a the muscle f i b r e s . This was confirmed in three animals by the adminis trat ion of curare . In these experiments, incremental doses of curare r e s u l t e d in a decremental response of the compound act ion p o t e n t i a l produced by both PNS and DPS ( F i g . 5) . The data provide strong evidence that with DPS, a c t i v a t i o n occurred v i a the neuromuscular junc t ion since s u f f i c i e n t amounts of curare o b l i t e r a t e d the compound act ion p o t e n t i a l invoked by both PNS and DPS. Although Pdi with twitch impulses was s i m i l a r using PNS and DPS, the p r o p o r t i o n a l c o n t r i b u t i o n of Pes and Pga d i f f e r e d . For PNS, with increas ing stimulus current , Pdi progres s ive ly increased u n t i l a maximal value was reached; t h e r e a f t e r , fur ther increases in current r e s u l t e d in no fur ther increase in Pdi ( F i g . 24). The plateau of force product ion in response to increas ing l e v e l s of st imulus current i s s i m i l a r to that descr ibed in other nerve-muscle s t imula t ion preparat ions (21). On the other hand, with DPS, Pdi and Pga curves d i d not demonstrate any p e r c e p t i b l e plateau over the range of stimulus currents used although there was an i n f l e c t i o n in the Pdi curve which matched the plateau of the Pes curve. Recruitment of muscles besides the one of in t ere s t i s a we l l - recognized problem of d i r e c t muscle s t imulat ion (15). The recruitment of a d d i t i o n a l t h o r a c i c or abdominal muscles could have increased Pdi by producing a more - 80 -i sometr ic contrac t ion and/or by mainta ining the diaphragm at or near optimal length during c o n t r a c t i o n . This may expla in the progress ive r i s e in Pdi and Pga over the e n t i r e range of stimulus current ( F i g . 24). Laporta and Grassino (12) have shown that the greatest Pdi with maximal e f f o r t is achieved with simultaneous i n s p i r a t o r y and expuls ive maneuvers. The recruitment of abdominal exp iratory muscles during an expuls ive maneuver may have an e f fec t s i m i l a r to the general ized muscle recruitment observed with DPS at higher stimulus current s . The progress ive r i s e in Pga may be a r e f l e c t i o n of the abdominal muscle recrui tment . In contrast to the Pga and Pdi curves , the shape of the Pes curve using DPS c l o s e l y resembled the shape of the Pes curves using PNS and may be a more accurate r e f l e c t i o n of recruitment of the diaphragm alone. Our r e s u l t s suggest that maximal recruitment of the diaphragm using DPS occurs when Pes a t ta ins a plateau va lue . At t h i s p o i n t , Pdi produced by DPS i s of s i m i l a r magnitude to that produced by supramaximal s t imula t ion using PNS. This was confirmed by our observat ion that the plateau on the Pdi curve corresponded to maximal diaphragmatic recruitment assessed by the s i ze of the compound diaphragmatic act ion p o t e n t i a l in subsequent s tud ies . Increas ing Pab resu l t ed in a trend towards a progress ive r i s e in Pdi u n t i l an optimum was reached at a Pab of 15 cmH^O. Beyond t h i s point fur ther increases i n Pab d i d not a f f ec t P d i . Increas ing Pab up to 15 cmH20 could have had a p o s i t i v e e f f ec t on Pdi by increas ing diaphragmatic f i b e r l ength , increas ing the area of appos i t ion of the diaphragm, decreasing the radius of curvature of the diaphragm, and l i m i t i n g diaphragmatic shortening ( r e s u l t i n g in a more i sometr ic contrac t ion during s t i m u l a t i o n ) . Further increases in Pab may have f a i l e d to inf luence Pdi by extending the length - 81 -of diaphragmatic f i b e r s beyond t h e i r opt imal l ength . From our experimental des ign , i t was impossible to determine which of these fac tors were i n f l u e n c i n g Pdi and at what l e v e l of Pab each would have had i t s maximal e f f e c t . Studies in humans (5) and severa l other species have found a greater Pdi production with abdominal b i n d i n g . Maximum trandiaphragmatic pressures ( F i g . 26 - Pdi at 70 Hz s t imulat ion) of the hamster diaphragm was approximately 100 cmH^O which l i e s w i th in the range of maximum Pdi produced in l a r g e r species (1 ,3 ,4 ,8 ,10 , 16,19) . Upon i n i t i a l observat ion , i t may seem s u r p r i s i n g that a small rodent can generate such large P d i . However, according to L o r i n g and DeTroyer (13), at lung volumes where a s i g n i f i c a n t area of the diaphragm is apposed against the r i g cage, trandiaphragmatic pressure i s determined by the fo l lowing equation: Pdi = force of f i b r e s  c r o s s - s e c t i o n a l area of the thorax Force output of s k e l e t a l muscle i s d i r e c t l y r e l a t e d to the c r o s s - s e c t i o n a l area of the muscle f i b r e s (14). The a b i l i t y to generate such large Pdi in the hamster s i m i l a r to l a r g e r species must imply that the r e l a t i o n s h i p between the c r o s s - s e c t i o n a l area of diaphragmatic muscle f i b r e s to the c r o s s - s e c t i o n a l area of the thorax i s s i m i l a r in the hamster compared to l a r g e r spec ies . The frequency of s t imula t ion needed to achieve a plateau in Pdi in the hamster was s i m i l a r to that reported in rabb i t s (1,19) and cats (8) but higher than those found i n dogs (4,10) and humans (3 ,16) . The DPS technique produced s table twitch and t e tan ic pressures over a two-hour p e r i o d . Although transdiaphragmatic pressure i s an index of diaphragmatic tension - 82 -product ion , i t i s a lso dependent on shortening of the diaphragmatic muscle f i b r e s - the l a t t e r of which is r e l a t e d to the compliance of the abdominal and thorac ic c a v i t i e s . Every e f f o r t was made to maintain the corset in the same p o s i t i o n and an external abdominal pressure of 15 cmH^O was appl ied before a l l contract ions so the compliance of the abdominal c a v i t y was cons i s tent during s t imula t ion throughout the two-hour r e s t p e r i o d . I t i s u n l i k e l y that the compliance of the thorac i c c a v i t y changed throughout the two hours. Thus, i t can be assumed that s ince the compliances of the two c a v i t i e s d i d not change, the shortening of the diaphragm during the d i f f e r e n t s t imulat ions at a p a r t i c u l a r frequency was s i m i l a r and the r e p r o d u c i b i l i t y of the Pdi can be s o l e l y a t t r i b u t e d to s i m i l a r tension product ion at the d i f f e r e n t time i n t e r v a l s . This was encouraging since the r e p r o d u c i b i l i t y of DPS was an e s s e n t i a l feature for the technique to be used i n the three to s ix hour experiments examining the e f fec t s of fa t igue and re s t on the diaphragm. We found that the DPS technique i s an e f f e c t i v e r e l a t i v e l y simple and reproduc ib le method of s t imula t ing the diaphragm in the hamster. S i m i l a r to PNS, the mode of transmiss ion was v i a the nerve. Maximal diaphragmatic a c t i v a t i o n can be recognized by the plateau on the curve r e l a t i n g current and esophageal pressure or more p r e c i s e l y by measuring the maximal compound diaphragmatic ac t ion p o t e n t i a l . Pab inf luences diaphragmatic pressure and should be he ld constant near 15 cmH^O. From these experimental r e s u l t s , we concluded that DPS was a u s e f u l technique to evaluate the c o n t r a c t i l e propert ie s of the hamster diaphragm in v i v o . Twitch and t e tan ic transdiaphragmatic pressures were s table over a two-hour per iod suggesting the p o t e n t i a l s u i t a b i l i t y for the s tudies examining the e f f ec t s of fa t igue and r e s t on the hamster diaphragm. - 83 -corset Stimulator n " l ib small bag plate electrodes tracheostomy tube Pes Pga diaphragm silicone dome indifferent electrode Figure 15: Animal preparation for DPS. See text for description. - 84 -Anterior Posterior Figure 16: Cast of the upper abdominal c a v i t y and the hamster diaphragm looking down onto the impression made by the diaphragm. Figure 17: S i l i c o n e dome fashioned from the abdominal cas t . The inner e lectrodes were used for s t imula t ion and recording whereas the outer e lectrodes were used for recording only . - 85 -P#rsoool Compute* Figure 18: Animal p r e p a r a t i o n f o r s t i m u l a t i n g and EMG r e c o r d i n g equipment. Recording technique w i t h the DISA a m p l i f i e r and d i s p l a y i s not shown. The d i g i t a l EMG re c o r d i n g apparatus i s shown as connecting to the s t i m u l a t i n g e l e ctrode p a i r and s t i m u l a t o r synchronizing output. The recorded data i s fed to a d i r e c t coupled d i f f e r e n t i a l a m p l i f i e r whose output feeds a 12 b i t analogue d i g i t a l converter (A.D.C.) which samples synchronously at a rate of 10,000 samples/sec (10 ICS/sec). The r e s u l t i n g data i s stored i n the c e n t r a l processing u n i t (C.P.U.) and i s subsequently d i s p l a y e d and o p t i o n a l l y recorded to mass storage (hard d i s c ) . O s c i l l o s c o p e l i k e commands are issued through the keyboard. The data a c q u i s i t i o n c y c l e i s i n i t i a t e d by the a r r i v a l of the t r i g g e r impulse from the synchronizing output of the DISA s t i m u l a t o r . 86 -C. D. - J ; _ J E. (Optional magnification) Figure 19: I n t e r a c t i v e s i g n a l process ing . See text for d e s c r i p t i o n . - 87 -Figure 20: Technique used to tes t phase r e l a t i o n s h i p s of Pes and Pga. A sharp v e r t i c a l force was appl ied at the large arrowhead. - 88 -Figure 21; Representative diaphragmatic ac t i on po tent ia l s recorded from inner e lec trode with maximal c u r r e n t . Upper: Photograph recorded on DISA electromyograph with PNS. Middle and lower: representat ive recording made with d i g i t a l data a c q u i s i t i o n technique with PNS (middle panel) and DPS (lower pane l ) . - 89 -Figure 22: Representative diaphragmatic action potential produced by DPS v i a the inner electrode and recorded from outer electrode. - 90 -5mV • — » ^ W w w t i i 1ms i i i i i i i il Baseline — 1st Dose — 2nd Dose 7\_A/W v.. II •J.I < I t : J : 1 • i i J : < : Figure 23: Representative diaphragmatic action potentials recorded before and after curare. Upper, PNS; lower; DPS Pre-curare; 10 minutes post .020 mg/100 gms curare; 10 minutes post second dose curare. I - 91 -PLATE I I I Figure 24: The e f fec t of increas ing current on Pdi (%) , Pga (o) , and Pes (•) in k animals (#1-4) using DPS ( l e f t ) and PNS (r ight ) . - 93 -Figure 25: The effect of varying external abdominal pressure (Pab) on Pdi in 6 animals during DPS (mean + SD) * s i g n l f i c a n t l y different than Pdi produced at Pab of 15 cmH20 (p=0.0001>. - 94 -Figure 26: Pdi produced by twitch s t imulat ion (DPS) at vary ing frequencies of t e tan ic s t imulat ion in 4 animals . Values are means+SD. S o l i d bars , c o n t r o l data; s t r i p e d b a r s , data at 2 hours. REFERENCES - 95 -1. A l d r i c h TK, Appel D: Diaphragm fat igue induced by i n s p i r a t o r y r e s i s t i v e loading in spontaneously breathing r a b b i t s . J Appl Phys io l 59:1527-1532, 1985. 2. Arnold JS , Ol iven A, Kelsen SG: E f f e c t of hypoxia on transdiaphragmatic pressure in i n t a c t hamsters ( A b s t r a c t ) . Fed Proc 44(3):1004, 1985. 3. Aubier M, DeTroyer A, Sampson M, Macklem PT, Roussos C: Aminophyll ine improves diaphragmatic c o n t r a c t i l i t y . N Engl J Med 305:249-252, 1981. 4. Aubier M, V i i r e s N, Muriciano D, Medrano G, Lecocquic Y , Pariente R: Ef fec t s and mechanism of ac t ion of t e r b u t a l i n e on diaphragmatic c o n t r a c t i l i t y and fa t igue . J Appl P h y s i o l 56:922-929, 1984. 5. Bellemare F , B i g l a n d - R i t c h i e B: Assessment of human diaphragm strength and a c t i v a t i o n using phrenic nerve s t i m u l a t i o n . Resp P h y s i o l 58:263-277, 1984. 6. Blogg T, Reid WD: The a p p l i c a t i o n of d i g i t a l data recording and analys i s to a problem in electromyography. Submitted to J Appl  P h y s i o l . 7. Daggett WM, P i c c i n i n i J C , Austen WG: In tracava l e l ec t rophren ic s t i m u l a t i o n . I . Experimental a p p l i c a t i o n during barbi tuate i n t o x i c a t i o n , hemorrhage and gangl ion ic blockade. J Thorac Cardiovasc  Surg 51:676-684, 1966. 8. Evanich MJ, Franco MJ , Lourenco R: Force output of the diaphragm as a funct ion of phrenic nerve f i r i n g rate and lung volume. J Appl Phys io l 35:208-212, 1973. 9. Hodgkin AL: The Conduction of Nervous Impulse. L i v e r p o o l U n i v e r s i t y Press , L i v e r p o o l , 1971. 10. Howell S, Roussos C: Isoproterenol and aminophyll ine improves c o n t r a c t i l i t y of fat igued canine diaphragm. Am Rev Respir Pis 129:118-124, 1984. 11. Hultman E , Sjoholm H: Electromyogram, force and r e l a x a t i o n time during and a f ter continuous e l e c t r i c a l s t imula t ion of human s k e l e t a l muscle in s i t u . J P h y s i o l 339:33-40, 1983. 12. Laporta P, Grass ino A: Assessment of transdiaphragmatic pressure in humans. J Appl P h y s i o l 58(5):1469-1476, 1985. 13. L o r i n g SH, PeTroyer A: Act ions of the r e s p i r a t o r y muscles. In The Thorax, Part A , V o l 29, Chapter 10 of Lung Biology i n Health and  P isease . Lenfant C (ed) New York, Marcel Pekker Inc , 1985, pp 327-349. - 96 -14. McArdle WD, Katch F l , Katch VL: Exerc i se Physiology: Energy.  N u t r i t i o n , and Human Performance, 2nd Ed. Lea & Febiger , P h i l a d e l p h i a , 1986. 15. Mortimer J T , Peckham PH: Intramuscular e l e c t r i c a l s t i m u l a t i o n . In: Neural organizat ion and i t s relevance to p r o s t h e t i c s . F i e l d s WF (ed), New York, I n t e r c o n t i n e n t a l Medical Book, 1973, pp 131-146. 16. Moxham J , Wiles CM, Newham D, Edwards RHT: C o n t r a c t i l e funct ion and r e s p i r a t o r y muscles. In: Human, Muscle Fat igue: P h y s i o l o g i c a l  Mechanisms. Porter R, Whelan 3 (eds) , Pitman M e d i c a l , London, 1981, pp 197-212. 17. Nochomovitz ML, Dimarco AF, Mortimer J T , Cherniack NS: Diaphragm a c t i v a t i o n with intramuscular s t imula t ion in dogs. Am Rev Respir Pis 127:325-329, 1983. 18. Planes RF, McBrayer RH, Koen PA: Diaphragmatic pressures: transvenous vs . d i r e c t phrenic nerve s t i m u l a t i o n . J Appl P h y s i o l 59:269-273, 1985. 19. Sant'Ambrogio G, Saibene F : C o n t r a c t i l e propert ie s of the diaphragm in some mammals. Resp P h y s i o l 10:349-357, 1970. 20. Sant'Ambrogio, G, Oecandia M, P r o v i n i L : Piaphragmatic c o n t r i b u t i o n to r e s p i r a t i o n in the r a b b i t . J Appl P h y s i o l 21(3):843-847, 1966. 21. Usherwood P: Nervous Systems. Studies in Biology No. 36. London, Edward Arnold L t d , 1973, p. 48. 22. V i i r e s N, Aubier M, Murciano 0, F l eury B, Talamo C, Par iente R: E f f e c t s of aminophyll ine on diaphragmatic fat igue during acute r e s p i r a t o r y f a i l u r e . Am Rev Respir P i s 129:396-401, 1984. 23. Wanner A, Sackner MA: Transvenous phrenic nerve s t imula t ion in anesthet ized dogs. J Appl P h y s i o l 34:489-494, 1973. - 97 -IV. HISTOLOGICAL FEATURES OF THE HAMSTER DIAPHRAGM INTRODUCTION The diaphragm of severa l mammalian species (8,16,18,61) contains three general types of muscle f i b r e s which have been c l a s s i f i e d by h is tochemical s t a i n i n g for myosin adenosine tr iphosphatase (M-ATPase). In ter - spec ie s v a r i a b i l i t y in proport ions of M-ATPase muscle f i b r e types (18), f i b r e s i ze (8) and f i b r e ox idat ive capac i ty (13,18) have been c l e a r l y demonstrated. In other s k e l e t a l muscles, the proport ions of type I and type II f i b r e s (28,31,43,53) and f i b r e s ize (27,29,30,53) have been demonstrated to vary between reg ions . Regional v a r i a b i l i t y w i th in a muscle has been a t t r i b u t e d to d i f ferences in sex (2 ,27) , s p e c i a l i z a t i o n in r e g i o n a l funct ion (28,29) and d i f f erences i n vascular supply (43). In a d d i t i o n , age (4,29,34, 39,61) , disuse (5,57) and t r a i n i n g (15,48) have been demonstrated to a f fec t the proport ion of muscle f i b r e types and may p r e f e r e n t i a l l y a f f ec t d i f f e r e n t regions of the muscle rather than a f f e c t i n g the muscle homogeneously. Besides intra-muscular v a r i a b i l i t y , cons iderable i n t e r - i n d i v i d u a l v a r i a b i l i t y of proport ions (31,52), s izes and ox idat ive p o t e n t i a l of muscle f i b r e types has been demonstrated i n some limb muscles (52). I t i s commonly held that the ox idat ive and g l y c o l y t i c capac i ty of the three M-ATPase muscle f i b r e types are d i f f e r e n t (6 ,47) . The type I f i b r e i s considered to be more aerobic i n nature possessing higher amounts of ox idat ive enzymes and l i p i d . The type l i b i s more g l y c o l y t i c conta in ing large amounts of glycogen and enzymes involved in anaerobic metabolism. The type 11a f i b r e i s considered to be intermediate between type I and l i b f i b r e s in both ox idat ive and g l y c o l y t i c capac i ty . A c t i v i t y has a profound - 98 -inf luence on the enzymatic l e v e l s of s k e l e t a l muscle (48). Although Halk jaer -Kr i s t ensen and Ingemann-Hansen (24) demonstrated a c l e a r d i s t i n c t i o n in the ox idat ive and g l y c o l y t i c enzyme a c t i v i t y i n type I and type II f i b r e s of human s k e l e t a l muscle, the separat ion of metabolic features of three types of muscle f i b r e s has been questioned by other inves t iga t ions (18,44,49) . I t has been demonstrated i n severa l species that when f i b r e types are categorized according to the M-ATPase c l a s s i f i c a t i o n they do not demonstrate d i s c r e t e d i f f erences in the the amount of succinate dehydrogenase (SDH) (18,49). Rather, there is considerable overlap in the metabolic nature of the d i f f e r e n t f i b r e types. For a p a r t i c u l a r type of f i b r e , there is in terspec ies v a r i a b i l i t y in the ox idat ive capaci ty (18,49). Within a s ing le muscle some descr ibe r e g i o n a l v a r i a b l i t i t y in SDH a c t i v i t y in f i b r e s of the same type (56,61) whereas others do not (16,45). Quant i ta t ive h i s t o l o g i c a l techniques have also been used to examine the r e l a t i v e amounts of glycogen in s k e l e t a l muscle f ibres (19,23,25,26) . However, r e g i o n a l d i f f erences in glycogen content of the diaphragm have not been inves t iga ted . Anatomica l ly , the diaphragm can be d iv ided into s t e r n a l , c o s t a l and c r u r a l regions based on the o r i g i n of the muscle f i b r e s (26). Detroyer et a l (9) demonstrated that p h y s i o l o g i c a l l y , the diaphragm cons i s t s of two muscles - the c o s t a l and c r u r a l - each having a d i f f e r e n t ac t i on on the r i b cage. H i s t o l o g i c a l l y , i t i s c o n t r o v e r s i a l whether or not there i s r eg iona l v a r i a b i l i t y w i th in the diaphragm. Some inves t iga t ions have found no r e g i o n a l d i f f erences (16,20,21,37,38,45,59) , however, s evera l s tudies (14,41,50,51,54,62) have noted r e g i o n a l v a r i a b i l i t y i n f i b r e type proport ions wi th in the diaphragm. Even comparison of s tudies which used - 99 -the same species and s i m i l a r s t a i n i n g methods demonstrated c o n f l i c t i n g r e s u l t s (12,32). The sampling s i t e v a r i e d s l i g h t l y but both studies sampled from the c o s t a l reg ion . In add i t ion to f i b r e type propor t ions , muscle f i b r e s i ze and metabolic c h a r a c t e r i s t i c s r e f l e c t the funct ion and adaptation of a muscle as a whole or wi th in a s p e c i f i c area. Although f i b r e s i ze in a given region of the diaphragm has been described in some species (13,32,39,40) , information concerning r e g i o n a l v a r i a t i o n in f i b r e s i ze (14,16,50,55) and metabolic nature (16,45,55,62) wi th in the diaphragm i s l i m i t e d . To the author's knowledge, t h i s information is unavai lab le in the hamster, an animal which has been used to study the diaphragmatic response to such intervent ions as fat igue (12), exerc i s e , emphysema (12,32) and s tarva t ion (33). For the long-term studies examining the e f fects of fa t igue and res t on the hamster diaphragm, we wanted to obta in diaphragmatic muscle b iops ies for h i s t o l o g i c a l examination at key points in the experimental des ign. Because the l i t e r a t u r e was so c o n t r o v e r s i a l regarding the homo-geneity of the mammalian diaphragm and the information was l a c k i n g in the hamster, i t was necessary to charac ter i ze the h i s t o l o g i c a l features of the normal hamster diaphragm. The purpose of t h i s study was to def ine the i n t e r - a n i m a l and i n t e r - r e g i o n a l v a r i a b i l i t y of f i b r e type propor t ions , s i z e s , ox idat ive capac i ty and glycogen content of the hamster diaphragm. We also examined the diaphragm for any morphological abnormal i t ies at the l i g h t microscopic l e v e l . - 100 -MATERIALS AND METHODS  Animal Care Ten adult male golden Syrian hamsters (Charles R i v e r , La P r a i r i e , Quebec) were housed i n d i v i d u a l l y in wire mesh cages and maintained on a d i e t of Purina Laboratory chow and water. At the time of study, they had a mean body weight (BW) of 127.5+4.9g (mean+S.D.). Six animals were used for the examination of r e g i o n a l v a r i a b i l i t y in the diaphragm and four animals were se lected for examination of the d i s t r i b u t i o n of f i b r e types in the c r u r a l region around the esophagus. Regional Muscle Biops ies Fol lowing anesthesia with an i n t r a p e r i t o n e a l i n j e c t i o n of urethane (Sigma Chemical C o . , S t . Louis)(38mg/100g BW) , ch lora lose (BDH Chemicals L t d . , Poole , England)(3.8mg/100g BW) and sodium pentobarb i ta l (Somnotol) (2.6mg/100g BW), a mid l ine abdominal i n c i s i o n was made. The e n t i r e diaphragm was excised and placed upon phosphate-buffered sa l ine-soaked gauze. The fo l lowing biops ies were cut: s t e r n a l , a n t e r i o r c o s t a l , p o s t e r i o r c o s t a l and c r u r a l ( F i g . 27). Biops ies were mounted on gum tragacanth. A diaphragm sandwich was made from two matching segments from the r i g h t and l e f t s ides of the c o s t a l diaphragm whereas each c r u r a l segment was mounted separate ly . The preparat ions were frozen in isopentane cooled to the temperature of l i q u i d n i trogen and stored at - 7 0 ° C u n t i l s ec t ion ing and subsequent s t a i n i n g . Muscle Histochemistry Transverse sect ions were cut at 10 and 16 um thickness on a cryos ta t (Ames Cryostat I I , E l k h a r t , Indiana) . The 10 um sect ions were s ta ined for hematoxylin and eosin (H & E ) , nicot inamide adenine d inuc leo t ide - 101 -t e t r a z o l i u m reductase (NADH-TR) (46) and myosin adenosine-triphosphatase (M-ATPase) using a modi f i ca t ion of the technique of Dubowitz and Brooke (10). Sixteen um sect ions were s ta ined for the demonstration of glycogen using p e r i o d i c a c i d - S c h i f f (25). A l l sect ions were cut and s ta ined on the same day in the same batch to avoid poss ib le a r t i f a c t s that could a r i s e from interassay v a r i a t i o n . We se lected NADH-TR rather than SDH to demonstrate the ox idat ive capac i ty of the hamster diaphragm because previous inves t iga t ions (12,32) of the hamster diaphragm had used t h i s s t a i n i n g technque. A f t e r t r y i n g various incubat ion times for NADH-TR, i t was found that the usual time of 20 minutes demonstrated considerable f i b r e v a r i a b i l i t y . The modif ied technique for M-ATPase enabled three f i b r e types to be discerned which could be categorized as: Type I - dark, Type I l a - pa l e , and Type l i b - intermediate . The modi f i ca t ions included a pre - incubat ion pH of 4.5 and a pos t - incubat ion pH of 9.5. Proport ions and pat tern of f i b r e type d i s t r i b u t i o n Each biopsy c r o s s - s e c t i o n from the f i r s t s ix animals was examined with a Zeiss U n i v e r s a l research microscope equipped with b r i g h t f i e l d o p t i c s . Using T e c h n i c a l Pan f i l m (32 ASA), enlarged black and white photo-micrographs were taken of each sec t ion at a f i n a l magni f i ca t ion of 77x. A l i n e was drawn on the middle of the photographs of biopsy cros s - sec t ions so that f i b r e proport ions could be determined for the abdominal and thorac i c surface of each specimen ( F i g . 28). The abdominal surface was defined as the area from the mid l ine to the edge of the c r o s s - s e c t i o n fac ing the abdominal c a v i t y . The thorac i c surface was defined as the other h a l f of the c r o s s - s e c t i o n . Any f i b r e s which f e l l onto the mid l ine were not included - 102 -in the a n a l y s i s . Since the f i b r e s izes and proport ions were determined in the abdominal and thorac ic halves of the b i o p s i e s , we r e f e r subsequently to the r e s u l t s in a surface rather than on a surface . F i b r e type proport ions were determined by taking the number of f i b r e s in a p a r t i c u l a r f i b r e type, and d i v i d i n g t h i s by the t o t a l number of f i b r e s in a reg ion . In order to avoid sample bias and to ensure accuracy of the proport ion determinat ion, a l l f i b r e s in a s p e c i f i c region were counted. To determine whether the pat tern of f i b r e d i s t r i b u t i o n was cons is tent along the length of the c r u r a l f i b r e s from o r i g i n to i n s e r t i o n , cross - sec t ions were examined at three d i f f e r e n t l e v e l s : one near the c e n t r a l tendon and then two successive cuts o n e - t h i r d and two-thirds the distance to the o r i g i n of t h i s muscle. Examination of esophageal region in the crus In an a d d i t i o n a l four animals, the c r u r a l region around the esophagus was examined. Biops ies were s tored , cut , and s ta ined in a s i m i l a r manner to that descr ibed above. Only the pat tern of f i b r e type d i s t r i b u t i o n around the esophagus was evaluated in the diaphragmatic b iops ies in these animals. Muscle f i b r e s ize The l e s s e r f i b r e diameter (LFD) was taken as the index of s i ze (2) . Cross - sec t ions of muscle b iops ies were projec ted onto a 36- inch square d i g i t i z i n g surface (GTCO Corporat ion , R o c k v i l l e , MD) and were d i g i t i z e d . The d i g i t i z i n g t a b l e t was connected to a personal microcomputer (Wang, L o w e l l , MA). Before measurements were taken, the d i g i t i z i n g area was c a l i b r a t e d using a g r a t i c u l e to give an accurate i n d i c a t i o n of magni f i cat ion and to ensure that the microscope and m i r r o r were pos i t ioned such that there - 103 -was minimal d i s t o r t i o n of the image over the e n t i r e f i e l d of view. The LFD was determined for 50 f ibres of each type for each surface of each region for the f i r s t s ix animals. Measurement of thickness The thickness of cryos tat sect ions s ta ined for PAS was determined for 12 s e r i a l sect ions using the technique as prev ious ly descr ibed by Lange and Engstrom (35). We used a Zeiss u n i v e r s a l research microscope equipped with a lOOx Planapo o i l immersion objec t ive ( Z e i s s ) . The f ine focus d i a l on t h i s microscope i s c a l i b r a t e d (each d i v i s i o n is the equivalent of 2 um). The thickness of the sec t ion was measured by focusing on the super ior and then the i n f e r i o r surfaces of the cryos ta t sec t ion and not ing the movement of the f ine focus d i a l . An area conta in ing the same 50 f i b r e s was i d e n t i f i e d for each s e r i a l s e c t i o n . Thickness measurements were performed 10 times in each of three locat ions (upper l e f t corner , middle , and lower r i g h t corner) around the area of 50 f i b r e s for each s e r i a l c r o s s - s e c t i o n . In a d d i t i o n , cryos tat sect ions cut at 16 um were prepared for examination under the scanning e l ec tron microscope by t r a n s f e r r i n g them d i r e c t l y to a Balzers 010 Freeze Drying u n i t and d r i e d overnight . Dr ied sect ions were mounted on aluminum stubs with s i l v e r pa int and then sputter coated with gold pal ladium in the same un i t for 200 seconds i n 0.05 atm of argon, at a charge of 45 mA and dis tance of 7.3 cm. Coated sect ions were viewed in a scanning e l ec tron microscope (Model 250T, Cambridge). Quant i ta t ion of NADH-TR and PAS PAS s t a i n i n g i n t e n s i t y (as an i n d i c a t i o n of the amount of glycogen) has been evaluated by c l a s s i f y i n g f i b r e s into three or four ca tegor ie s . However, i t i s d i f f i c u l t to v i s u a l l y d i s c e r n deeply s ta ined f i b r e s which - 104 -may vary grea t ly in t h e i r content of glycogen as assessed b iochemica l ly . Attempts to categorize f i b r e s s tained with NADH-TR as ox idat ive or non-ox idat ive using v i s u a l assessment proved to be very d i f f i c u l t . Because of the problems faced with v i s u a l c l a s s i f i c a t i o n schemes for assessment of PAS and NADH-TR stained muscle f i b r e s , we chose to perform microphotometric q u a n t i t a t i v e assessment (63) on these s ta ined t i s sue sec t ions . The h i s tochemica l r eac t ion for NADH-TR and PAS was q u a n t i f i e d using a computer-based image process ing system (Kontron SEM-IPS, Munich) that consis ted of a video monitor and a computer for the process ing of images. This set-up was connected to a video camera (Model 68, Dage MTI) attached to a l i g h t microscope (Zeiss U n i v e r s a l research) . The wavelength of transmitted l i g h t peaked at 546 nm which corresponded c l o s e l y to the maximal absorbance of PAS and NADH-TR sta ined m a t e r i a l . The video image of the muscle sect ion was composed of a matrix of 480 x 512 p i x e l s . By use of 2 a microscope magni f i ca t ion of x20, each p i x e l had an area of 0.5568 ym . The image process ing system had the a b i l i t y to d i scern 255 grey l e v e l s . At the beginning of each sess ion , the system was c a l i b r a t e d . The background of the s l i d e , mounting medium, and c o v e r s l i p was set to equal a grey l e v e l of zero and an opaque black image ( f l eck of graphite) was set to equal 200. Measurements with and without s tray l i g h t were taken and a transformation tab le was set-up such that the computer automat ica l ly c a l c u l a t e d an o p t i c a l dens i ty measurement without s tray l i g h t for a s ing le f i b r e from the • transmit ted ' and ' inc ident* l i g h t readings . A f t e r the grey l e v e l s were c a l i b r a t e d for a transparent and black image, the upper and lower l i m i t s of the o p t i c a l dens i ty were set at 0 and 2 .2 , r e s p e c t i v e l y . - 105 -O p t i c a l dens i ty measurements were determined for the e n t i r e cross -s e c t i o n a l p r o f i l e of the muscle f i b r e . Since glycogen i s homogeneously d i s t r i b u t e d in the c e l l (25) and the PAS technique a lso s ta ins the po ly -saccharides in the c e l l membrane, an e f f o r t was made to exclude the c e l l borders for o p t i c a l dens i ty measurements of PAS-stained f i b r e s . This was done by d i g i t i z i n g the circumference of each f i b r e and then the o u t l i n e was expanded to a three p i x e l width (by a computer command) such that the t i s sue i d e n t i f i e d wi th in the ou t l i ne was wel l w i th in the c e l l border . The o p t i c a l dens i ty of each p i x e l w i th in the expanded out l ines of muscle f i b r e s was determined and then the mean o p t i c a l dens i ty of a l l p i x e l s w i th in the f i b r e was c a l c u l a t e d . In contrast to the homogeneous d i s t r i b u t i o n of glycogen, mitochondria ( i d e n t i f i e d by NADH-TR) are d i s t i b u t e d non-homogeneously -e s p e c i a l l y w i th in the type I f i b r e which can have a large subsarcolemmal populat ion of mitochondria . Therefore , f or the NADH-TR s ta ined f i b r e s , the o u t l i n e of the muscle f i b r e was narrowed to a s ing le p i x e l width which enabled the maximal i n c l u s i o n of any deeply s ta ined sub-sarcolemmal matter in the o p t i c a l dens i ty measurement. The o p t i c a l dens i ty of each p i x e l w i th in the o u t l i n e d f i b r e was determined and the mean o p t i c a l dens i ty of a l l the p i x e l s w i t h i n the f i b r e was c a l c u l a t e d . Within and between day v a r i a b i l i t y of o p t i c a l dens i ty measurements was examined by taking o p t i c a l dens i ty measurements of a c o n t r o l s l i d e s ta ined for PAS at the beginning and end of every sess ion . Thi s c o n t r o l data was analyzed using a f u l l y crossed three-way ana lys i s of variance examining d i f ferences between time ( f i r s t vs l a s t measure of day) , day (between days) and f i b r e s (between f i b r e v a r i a n c e ) . C o n t r o l o p t i c a l densi ty - 106 -measures between times and days was not d i f f e r e n t . However, there was some time-day i n t e r a c t i o n . Thus, we examined the v a r i a b i l i t y of d i f f e r e n t days by p l o t t i n g the average d i f ference of the f i r s t and l a s t measurement of a l l f i b r e s in the c o n t r o l sample for each day (expressed as a percentage of the average o p t i c a l dens i ty measure) ( F i g . 29). When the average day to day v a r i a b i l i t y was expressed as a percentage of the average o p t i c a l dens i ty , the mean day to day v a r i a b i l i t y was 4.4% with a maximum v a r i a b i l i t y of 11.2% on day 12. In order to determine reg iona l and f i b r e type d i f ferences of glycogen l e v e l s and ox idat ive capac i ty , o p t i c a l dens i ty measures were determined for 50 f i b r e s in both surfaces (abdominal and t h o r a c i c ) of the l e f t c r u r a l r e g i o n , s t e r n a l , and the c o s t a l reg ions . The data for each surface of the c r u r a l region were analyzed separate ly but data for the two surfaces of the s t e r n a l and c o s t a l regions were combined before a n a l y s i s . Assessment of morphological abnormali t ies Muscle biopsy cross - sec t ions s ta ined for H&E, M-ATPase and NADH-TR were s c r u t i n i z e d for the morphological abnormal i t ies l i s t e d in Table V. - 107 -TABLE V  MORPHOLOGICAL ABNORMALITIES Inflammatory c e l l s Increased connective t i s sue Necrot ic f i b r e s B a s o p h i l i c f i b r e s F ibre s p l i t t i n g In t erna l n u c l e i F i b r e atrophy C e n t r a l cores Minicores Motheaten f i b r e s Target f i b r e s Type grouping Type predominance Other - 108 -S t a t i s t i c a l analys i s Dif ferences for proport ions and LFD measurements between sides ( r i g h t vs l e f t ) , surfaces (abdominal vs t h o r a c i c ) , regions ( s t e r n a l , a n t e r i o r c o s t a l , p o s t e r i o r c o s t a l , and c r u r a l ) , and animals (#1-6) were examined. A f u l l y crossed three-way ( s ide , surface , region) ana lys i s of var iance with animals as a b lock ing f a c t o r was u t i l i z e d to determine d i f f e r e n c e s , i f any, in proport ions . Since d i f ferences between sides ( r ight vs l e f t ) were n o n - s i g n i f i c a n t (p>0.2), a new two-way ANOVA was constructed e l i m i n a t i n g a l l s ide terms. Dif ferences were accepted with a s i g n i f i c a n c e l e v e l of p<0.005 because of the m u l t i p l e comparisons being made. The d i f ferences between frequency d i s t r i b u t i o n s of muscle f i b r e s i ze were assessed using a two-sided Kolmogorov-Smirnov tes t (7) . No d i f ferences were observed between sides or surfaces of the s t e r n a l and c o s t a l regions and between the s t e r n a l and c o s t a l reg ions . The data was combined such that d i f ferences were examined for three main areas: the s t e r n a l and c o s t a l r e g i o n , thorac i c surface of c r u r a l r e g i o n , and abdominal surface of the c r u r a l r eg ion . F i b r e s i ze d i f ferences across a l l regions and interanimal d i f ferences wi th in a p a r t i c u l a r f i b r e type and area were also looked f o r . From the analyses examining d i f f erences for s izes and proport ions of muscle f i b r e types between regions and surfaces , three main areas of the hamster diaphragm were i d e n t i f i e d : the s t e r n a l and c o s t a l r e g i o n , the thorac i c surface of the c r u r a l region and the abdominal surface of the c r u r a l r eg ion . Therefore , the data was grouped into these three regions for a l l subsequent examination and analyses of d i f f erences i n the glycogen content and ox idat ive capac i ty between reg ions . - 109 -Dif ferences for o p t i c a l dens i ty measurements of NADH-TR and PAS between three main areas ( s t e r n a l and c o s t a l , abdominal surface of c r u r a l and thorac i c surface of the c r u r a l region) were also determined using a two-sided Kolmogorov-Smirnov tes t (7) . Both reg iona l and f i b r e type d i f ferences in s i z e , and o p t i c a l dens i ty measures for PAS and NADH-TR were determined. Dif ferences were accepted with a s i g n i f i c a n c e l e v e l of p<0.005. RESULTS Proport ions and pat tern of f i b r e type d i s t r i b u t i o n Three f i b r e types could be discerned in a l l regions of the diaphragm in a l l animals ( F i g . 28). There were no side ( r i g h t vs l e f t ) d i f ferences so the data for sides were combined p r i o r to fur ther s t a t i s t i c a l a n a l y s i s . The mean (+S.E.) values for f i b r e type proport ions in the s ix animals for the abdominal and thorac ic surfaces of the s t e r n a l , a n t e r i o r c o s t a l , p o s t e r i o r c o s t a l and c r u r a l regions of the diaphragm are shown in F i g . 30. Henceforth, the data for a given region and a surface w i l l be r e f e r r e d to as a s u r f a c e / r e g i o n . An even mosaic d i s t r i b u t i o n of the three f i b r e types was observed i n the s t e r n a l and c o s t a l regions ( F i g . 28, upper panel) but was not seen i n the c r u r a l region ( F i g . 28, lower p a n e l ) , where there were very few type I f i b r e s in the abdominal surface and a greater number in the thorac i c surface (p<0.005). The proport ion of type I f i b r e s in the abdominal surface of the c r u r a l region was l ess than in a l l other surfaces /reg ions (p<0.005) and the proport ion in the t h o r a c i c surface of the c r u r a l region was greater than a l l other surfaces /reg ions (p<0.005). There were no s i g n i f i c a n t d i f ferences in proport ions between surfaces /reg ions in the type - 110 -I l a f i b r e s . As expected from the manner by which muscle f i b r e type proport ions were c a l c u l a t e d , i f a d i f f erence occurred in the proport ion of type I f i b r e s and no d i f f erence occurred i n type I l a propor t ions , then a d i f f erence must have occurred in the proport ion of type l i b f i b r e s . There was a greater proport ion of type l i b f i b r e s in the abdominal surface of the c r u r a l region than in the thorac i c surface (p<0.005). The proport ion of type l i b f i b r e s in the abdominal and thorac i c surface of the c r u r a l regions were d i f f e r e n t from a l l other surface /reg ions (p<0.005). Although proport ions were only determined for one c r o s s - s e c t i o n of each b iopsy , the pat tern of d i s t r i b u t i o n of f i b r e types in the c r u r a l region was determined from two a d d i t i o n a l c r o s s - s e c t i o n s , o n e - t h i r d and two-thirds the distance to the o r i g i n of t h i s muscle, and from b iops ies taken s p e c i f i c a l l y around the esophagus. A s i m i l a r pattern occurred along the f u l l length of the c r u r a l region from o r i g i n to i n s e r t i o n , and no d i s t i n c t d i f f erences were observed in the region surrounding the esophagus ( F i g . 31). The v a r i a b i l i t y of the proport ions in the three f i b r e type categories could be a t t r i b u t e d to four f a c t o r s : hamster ( i n t e r - a n i m a l ) , r e g i o n a l , surface , or r e g i o n a l - s u r f a c e i n t e r a c t i o n . By ana lys i s of var iance , the proport ion of variance that could be a t t r i b u t e d to i n t e r -animal v a r i a b i l i t y was 4, 23 and 7% for type I , I l a and l i b f i b r e s , r e s p e c t i v e l y . The remainder of the v a r i a b i l i t y was accounted for by r e g i o n a l and surface v a r i a b i l i t y but because of r e g i o n a l - s u r f a c e i n t e r -a c t i o n , the exact proport ion of var iance could not be determined for each of these fac tors alone. - I l l -Muscle f i b r e s ize The mean LFD for each s ide , surface and region i s shown in Table V I . Because of the small number of type I f i b r e s in the abdominal surface of the c r u r a l r e g i o n , the s i ze of these f i b r e s could not be compared in the s t a t i s t i c a l a n a l y s i s . S i m i l a r to proport ion data , no d i f f erence was observed between s ides , so the s i d e - t o - s i d e data was combined. In a d d i t i o n , no d i f ferences were observed between the surfaces and regions for s t e r n a l , a n t e r i o r c o s t a l and p o s t e r i o r c o s t a l regions so t h i s data was combined for comparison to the abdominal and thorac i c surface of the c r u r a l reg ions . - 112 -TABLE VI MUSCLE FIBRE DIAMETERS IN MICRONS IN THE ABDOMINAL AMD THORACIC SURFACES  OF THE DIAPHRAGM: COMPARISON OF DIFFERENT REGIONS REGION TYPE I TYPE IIA TYPE IIB Abdominal Thoracic Abdominal Thoracic Abdominal Thoracic R Sternal 36.5+7.6 37.9+8.0 36.6+7.1 38.3+8.0 47.4+9.9 49.0+9.9 L Sternal 33.6+6.5 37.1+7.5 37.6+7.6 40.4+8.2 45.5+8.8 47.7+9.2 R Ant. Costal 35.8+7.5 35.4+7.6 35.0+7.6 36.8+7.2 45.5+9.1 47.0+8.4 L Ant. Costal 38.5+7.3 38.0+7.1 38.6+6.6 39.9+7.7 50.0+9.6 49.9+9.3 R Post.Costal 37.9+7.9 36.6+7.4 38.6+8.3 37.6+8.1 47.9+9.4 46.3+9.4 L Post.Costal 38.8+7.6 36.6+7.5 38.7+7.5 38.4+7.5 48.3+9.1 48.4+9.3 R Crural 42.2+8.4 35.9+6.6 44.5+8.6 46.0+8.0 51.8+9.5 L Crural 43.1+8.1 33.1+5.9 40.6+7.3 45.5+11.3 46.5+7.9 Values are means+S.D. of 300 fibres (6 animals z 50 fibres) - 113 -The mean diameter of the type I f i b r e s in the t h o r a c i c surface of the c r u r a l region was l arger than in the s t e r n a l and c o s t a l regions (p<0.0001) with a mean of 42.7+8.3 ( ± S . D . ) and 36.9+7.6 ( ± S . D . ) u m , r e s p e c t i v e l y ( F i g . 32). The type ITa f ibres on the t h o r a c i c surface of the c r u r a l region had a mean diameter of 42.6+_8.3 um and were l a r g e r than the type I l a f i b r e s in both the abdominal surface of the c r u r a l reg ion (mean of 3 4 . 5 ± 6 . 4 um), and the s t e r n a l and c o s t a l diaphragm (mean of 3 8 . 0 ± 7 . 8 um [p<0.0001] F i g . 33). There were no d i f ferences in the s i ze of the type l i b f i b r e s between any reg ion . When comparing the diameter of the three f i b r e types across a l l reg ions , there was a large uniform upward s h i f t in the frequency d i s t r i b u t i o n and a l a r g e r mean in the s ize of type l i b f i b r e s ( 4 7 . 7 ± . 9 . 4 um) as compared to both type I (37.7+8.0 um) and I l a (38.2+7.9 um) f i b r e s (p<0.001) ( F i g . 34). In contras t , the frequency d i s t r i b u t i o n s of the type I and type I l a f i b r e s demonstrated cons iderable over lap , however they were d i f f e r e n t (p<0.003) with a s l i g h t upward s h i f t in the mean of the type I l a f i b r e s compared to the type 1 f i b r e s ( F i g . 34). The s i g n i f i c a n t d i f f erence can be l a r g e l y a t t r i b u t e d to the large N (4200 and 4674 f i b r e s r e s p e c t i v e l y for type I and I l a f i b r e s ) . Muscle f i b r e type s izes in the s ix animals were a l so compared. From the comparison of sur face / reg ions , d i f f erences in muscle f i b r e s i ze were found between three main areas: the t h o r a c i c surface of the c r u r a l r e g i o n , the abdominal surface of the c r u r a l region and the r e s t of the diaphragm ( s t e r n a l and c o s t a l ) . Thus, f or the comparison between animals , the data was grouped into these three major areas . The data for the upper (90%) and lower (10%) d e c i l e s , upper (75%) and lower (25%) q u a r t i l e s and - 114 -mean (50%) values are shown for each f i b r e type, animal , and three main areas in table V I I . Ten percent of LFD data is greater than the value ind ica ted in the upper d e c i l e (90%) column, 10% of the LFD i s l e s s than the value ind ica ted in the lower d e c i l e (10%) column, 25% of the LFD data is greater than the value ind ica ted in the upper q u a r t i l e (5%) column, and 25% of the LFD data is l e ss than the value ind ica ted in the lower q u a r t i l e (25%) column. Examination of these numbers revealed a very c lose correspondence of the three areas between the s ix animals. Examination of thickness of cryos tat sect ions Examination of the thickness measurements (Table VIII ) and the scanning e l ec tron micrographs ( F i g . 35) demonstrated that the cryos tat sect ions were r e l a t i v e l y uniform in th ickness . The c o - e f f i c i e n t of v a r i a t i o n of three thickness measurements across a region of 50 f i b r e s for the 12 sect ions ranged between 2 and 19% with a mean value of 12%. The scanning e l ec t ron micrographs also i l l u s t r a t e d the cons is tent thickness of the cryos tat sec t ions . The cryos tat sect ions were ne i ther wedge-shaped, or uneven between or wi th in the muscle f i b r e s ( F i g . 35). Comparison of d i f f e r e n t sect ions revealed a c o e f f i c i e n t of v a r i a t i o n of 9% (equivalent to approximately 1.5 um per sec t ion cut at 16 um). - 115 -TABLE VII COMPARISON OF MUSCLE FIBRE SIZES ACCORDING TO  FIBRE TYPE. MAIN AREA AND EACH EACH ANIMAL RHGIOH HAMSTER 10% 25% 50% 75% 90% TYPE I Sternal & Coatal Thoracic Surface of Crural TYPE I l a Sternal & Costal Abdominal Surface of Crural 2 6 . 1 2 7 . 1 2 9 . 0 2 7 . 1 2 6 . 9 3 1 . 1 3 S . 8 3 3 . 7 2 9 . 4 3 2 . 8 3 3 . 6 31.5 27 .5 2 8 . 2 3 0 . 4 27.4 27.7 3 0 . 4 2 2 . 8 2 9 . 8 2 8 . 8 25 .2 2 8 . 0 28.4 2 9 . 7 3 0 . 4 3 2 . 1 3 1 . 8 31 . 1 3 5 . 9 39. 37. 32. 35. 37. 35. 31 . 1 31 .6 3 4 . 5 31 .9 3 2 . 3 34.4 2 6 . 1 3 3 . 3 31.6 27.4 3 2 . 1 32.4 34 3 4 . 8 35 .7 3 7 . 2 35.7 4 1 . 0 4 4 . 2 4 5 . 1 3 7 . 3 41.2 4 3 . 3 41.4 3 5 . 6 35.4 3 9 . 3 38.2 37 .1 39 . 7 30.5 35 .9 35 .1 2 9 . 9 3 6 . 0 3 6 . 0 3 8 . 2 3 9 . 4 4 0 . 5 4 3 . 0 4 1 . 1 4 7 . 7 4 9 . 3 5 0 . 5 4 3 . 3 4 8 . 0 4 9 . 9 4 6 . 1 3 9 . 9 4 2 . 0 4 4 . 0 4 4 . 6 4 2 . 6 4 5 . 3 3 6 . 8 4 0 . 7 3 8 . 4 33 .7 4 0 . 3 4 0 . 2 4 3 . 6 4 3 . 0 4 4 . 6 4 8 . 7 4 5 . 9 5 2 . 3 53 .2 55 .8 4 8 . 3 52 .2 54.7 5 2 . 2 44 .7 46.4 4 9 . 5 51 .0 47.6 50.6 41 .1 4 4 . 2 4 2 . 8 38.3 43 .9 45 .2 Thoracic Surface of Crural TYPE l i b Sternal & Costal 3 7 . 9 31 .4 32 .5 27 .8 3 3 . 8 34 .7 3 3 . 1 3 5 . 6 37 .4 3 4 . 4 36 .6 4 0 . 6 41 .3 34.5 3 8 . 4 3 0 . 8 3 8 . 3 3 7 . 8 3 8 . 1 4 0 . 9 4 2 . 3 3 9 . 8 41 .8 45.7 44.4 41.6 4 3 . 6 35.4 4 2 . 1 4 3 . 8 43 .6 46.4 4 8 . 1 45.4 4 8 . 2 51.8 4 9 . 0 5 0 . 6 4 9 . 1 4 2 . 1 4 8 . 0 4 9 . 9 4 9 . 4 51 .9 5 4 . 2 5 2 . 7 5 5 . 6 5 7 . 4 53.6 55.6 51.8 47.4 52.4 55 .0 55 .2 57.7 59 .9 58.5 6 2 . 6 62 .4 Abdominal Surface of Crural Thoracic Surface of Crural 27.7 3 9 . 6 3 7 . 2 3 3 . 2 4 1 . 1 3 2 . 4 3 6 . 6 3 8 . 1 3 9 . 9 35 .4 3 9 . 1 3 6 . 2 3 1 . 0 4 4 . 1 4 0 . 8 3 8 . 0 4 5 . 4 3 6 . 3 4 0 . 9 4 2 . 0 4 4 . 0 41 .4 4 4 . 1 4 1 . 2 3 8 . 9 49 .7 4 6 . 3 4 2 . 2 51 .0 41.4 4 6 . 5 4 9 . 9 50.5 4 8 . 1 4 9 . 0 49.4 4 8 . 7 5 5 . 1 51 .9 4 6 . 1 5 8 . 5 4 6 . 7 5 3 . 0 5 7 . 3 5 6 . 1 5 3 . 6 57.7 5 5 . 3 53.7 6 5 . 1 5 8 . 1 51 .0 6 3 . 1 51.4 57 .1 6 2 . 5 6 0 . 6 57.4 6 2 . 8 5 9 . 3 Values are i n microns . - 116 -TABLE VIII THICKNESS MEASUREMENTS OF 12 CRYOSTAT SERIAL SECTIONS UPPER MIDDLE LOWER MEAN OF SD WITHIN SECTION LEFT RIGHT SECTION C . V . (%) 1 16.84 17.54 17.52 14.30 0.40 2.8 2 16.46 18.12 15.98 16.86 1.12 6.6 3 14.38 17.60 14.34 15.44 1.88 12.2 4 14.90 18.86 15.98 16.58 2.04 12.3 5 14.18 17.80 16.70 16.22 1.86 11.5 6 14.88 11.92 13.40 1.82 13.6 7 14.36 14.14 15.80 14.76 0.92 6.2 8 11.70 14.08 12.90 1.46 11.3 9 15.66 15.62 15.06 15.44 0.34 2.2 10 12.30 12.10 14.08 12.82 1.10 8.6 11 16.08 19.68 13.32 16.36 3.20 19.6 12 16.10 16.04 13.54 15.22 1.46 9.6 MEAN SECTION THICKNESS 15.03 SD 1.41 BETWEEN SECTION C . V . 9.4% Note: A l l values are in um with the exception of the c o e f f i c i e n t s of v a r i a t i o n which are expressed as percentages Abbrev ia t ion : C o e f f i c i e n t of V a r i a t i o n - C . V . - 117 -O p t i c a l dens i ty measures of NADH-TR and PAS Frequency d i s t r i b u t i o n curves for the o p t i c a l dens i ty of f ibres s ta ined for PAS and NADH-TR are shown in F i g . 36 and 37. The PAS o p t i c a l dens i ty values were log normally d i s t r i b u t e d while the NADH-TR showed a normal d i s t r i b u t i o n . The data was examined separate ly for each of the three f i b r e types for each of three main areas: abdominal surface of the c r u r a l region ( A b d / C r u r ) , t h o r a c i c surface of the c r u r a l region (Thor /Crur) and the s t e r n a l and c o s t a l regions (S&C). Because of the small number of type I f i b r e s in Abd/Crur , the o p t i c a l dens i ty measures for PAS and NADH-TR from t h i s region could not be compared in the s t a t i s t i c a l ana lys i s and these data are not shown. The o p t i c a l dens i ty of the PAS-stained muscle f ibres demonstrated more f i b r e type ( F i g . 38) and r e g i o n a l v a r i a b i l i t y ( F i g . 36) than the NADH-TR sta ined muscle f i b r e s ( F i g s . 37 & 38). Since i t has been demonstrated that 98.5 % of the PAS-pos i t ive mater ia l ins ide a muscle f i b r e is glycogen (25), when d i scuss ing the PAS s t a i n i t w i l l be assumed that the amount of glycogen i s d i r e c t l y r e l a t e d to the o p t i c a l dens i ty of the PAS-sta ined f i b r e s . PAS Box p lo t s represent ing the d i s t r i b u t i o n s of the o p t i c a l dens i ty of PAS-stained f i b r e s and p values for comparisons between f i b r e type wi th in a given region are shown in F i g . 39. Examination of f i b r e v a r i a b i l i t y wi th in a given area demonstrated that the I l a f i b r e s had the l e a s t amount of glycogen in a l l three of the main areas . The l i b f i b r e s had the most glycogen in Abd/Crur and S&C. The glycogen content was not d i f f e r e n t in type 1 and type l i b f i b r e s of T h o r / C r u r . Both the type I and l i b f ibres had more glycogen than type I l a f i b r e s in T h o r / C r u r . - 118 -Box p lo t s (60) represent ing the d i s t r i b u t i o n s of o p t i c a l densi ty for PAS-stained f ibres and p values for comparisons between regions within a given f i b r e type are shown in Figure 40. Comparison of a s p e c i f i c f i b r e type across the three main areas demonstrated that there was a trend towards the type I f i b r e s of S&C to contain more glycogen than the type I f i b r e s in T h o r / C r u r . The type I l a f i b r e s in the T h o r / C r u r had less glycogen than the type I l a f ibres i n the Abd/Crur and S&C but no d i f f erence was found between the glycogen content of the type I l a f i b r e s in Abd/Crur and the type I l a f i b r e s in the S&C. The type l i b f i b r e s in the Abd/Crur had the most glycogen, the type l i b f i b r e s in S&C were intermediate , the type l i b f ibres in Thor /Crur had the l ea s t glycogen. NADH-TR Box p lo t s of o p t i c a l dens i ty for NADH-TR stained f i b r e s and p values for comparisons between f i b r e types wi th in a given region are shown in Figure 41. Examination of f i b r e v a r i a b i l i t y wi th in a given area demonstrated that the I l a f i b r e s had the highest amount of NADH-TR in Abd/Crur and S&C. Type I had an intermediate amount and the type l i b f ibres had the l ea s t NADH-TR in S&C. There were no d i f ferences between the NADH-TR content of any of the three f i b r e types i n T h o r / C r u r . Box p lo t s of o p t i c a l dens i ty for NADH-TR s ta ined f i b r e s and p values for comparisons between regions wi th in a given f i b r e type are shown in F igure 42. Comparison of a s p e c i f i c f i b r e type across the three main areas demonstrated that the NADH-TR content was not d i f f e r e n t in the type 1 f i b r e s in T h o r / C r u r and S&C. The type I l a f i b r e s had the highest amount of NADH-TR in Abd/Crur compared to the type I l a f i b r e s in S&C and the type I l a f i b r e s in T h o r / C r u r . The NADH-TR content was not d i f f e r e n t in the type 11a - 119 -f i b r e s in S&C and T h o r / C r u r . The type l i b f i b r e s in the T h o r / C r u r tended to have more NADH-TR than the type l i b f i b r e s in A b d / C r u r . The type l i b f i b r e s in S&C had less NADH-TR than the type l i b f i b r e s in both Abd/Crur and T h o r / C r u r . Q u a l i t a t i v e morphological assessment Four biopsy cross - sec t ions s ta ined with NADH-TR demonstrated c e n t r a l dots in f i b r e s (between 3 and 20 f i b r e s per a t o t a l of 1000-2000 f i b r e s per b iopsy ) . A few i n t e r n a l n u c l e i were observed in one cros s -s e c t i o n . One f i b r e in another biopsy c r o s s - s e c t i o n had a motheaten appearance. No inflammatory c e l l s or damaged f i b r e s were noted in any of the biopsy cross s ec t ions . O v e r a l l , there were minimal abnormal i t i e s , and the few observed d id not demonstrate a predominance in one p a r t i c u l a r region of the diaphragm. SUMMARY We examined f i b r e type and r e g i o n a l d i f ferences in propor t ions , s i z e s , glycogen l e v e l s and ox idat ive capac i ty of muscle f i b r e types (based on M-ATPase) in the hamster diaphragm. From examination and analys i s of the h i s t o l o g i c a l f ea tures , we found three d i s t i n c t areas in the hamster diaphragm: abdominal surface of the c r u r a l r e g i o n , t h o r a c i c surface of the c r u r a l r e g i o n , and the s t e r n a l and c o s t a l diaphragm. A summary of the h i s t o l o g i c a l features in the three main areas of the diaphragm i s shown in Table IX. and of the three M-ATPase f i b r e types i s shown in Table X. - 120 -TABLE IX SUMMARY OF HISTOLOGICAL FEATURES OF THE THREE MAIN AREAS  OF THE HAMSTER DIAPHRAGM ABD/CRUR THOR/CRUR S&C PROPORTIONS Type I Type I l a Type l i b smallest no d i f f erence l arges t l a r g e s t no d i f f erence smallest intermediate no d i f f erence intermediate FIBRE SIZE Type 1 Type I l a Type l i b smallest no d i f f erence l a r g e s t l arges t no d i f f erence smallest intermediate no d i f f erence GLYCOGEN LEVELS Type I Type I l a Type l i b more most less (trend) l e s s l e a s t more (trend) more intermediate OXIDATIVE CAPACITY Type I Type I l a l a r g e r Type l i b intermediate no d i f f erence smal ler no d i f f e r e n c e smal ler l arges t ( t rend) smal lest (trend) - 121 -TABLE X MUSCLE FIBRE PROFILES ACROSS ALL REGIONS OF THE HAMSTER DIAPHRAGM TYPE I TYPE I l a TYPE l i b PROPORTIONS 6-38% 22-27% 35-66% FIBRE SIZE (um) 37.7+8.0 38.2+7.9 47.7+9.4 GLYCOGEN LEVELS intermediate l ea s t most OXIDATIVE CAPACITY intermediate greatest smal lest - 122 -DISCUSSION The r e s u l t s of t h i s study c l e a r l y demonstrate the presence of marked reg iona l and sur face - to - sur face v a r i a b i l i t y and some in ter -an imal v a r i a b i l i t y in the h i s t o l o g i c a l features w i th in the hamster diaphragm. The major d i f ferences were apparent w i th in the c r u r a l r e g i o n , and between the c r u r a l region and the re s t of the diaphragm ( s t e r n a l and c o s t a l ) . There was no s i g n i f i c a n t d i f f erence in the s ize and proport ions of f i b r e types between the r i g h t and the l e f t s ide of the diaphragm, and throughout surfaces and regions in the s t e r n a l and c o s t a l reg ions . However, examination of muscle f i b r e type proport ions , s i z e s , ox idat ive capac i ty , and glycogen l e v e l s demonstrated d i f ferences between the abdominal and thorac i c surface of the c r u r a l r eg ion , and these two surfaces of the c r u r a l region d i f f e r e d from the re s t of the diaphragm. We used the M-ATPase based c l a s s i f i c a t i o n scheme of Dubowitz and Brooke (9) which enabled three f i b r e types to be d i scerned . Several inves t iga tors examining f i b r e typing in the diaphragm have s ta ined s e r i a l sect ions using a combination of the technique for m y o f i b r i l l a r adenosine tr iphosphatase of Guth and Samaha (22), and the standard method for nicot inamide adenine d inuc leo t ide t e t r a z o l i u m reductase (46,47). Green et a l (17) demonstrated that the two d i f f e r e n t h i s tochemica l s t a i n i n g techniques for M-ATPase (10,22) showed complete correspondence between 'type I ' f i b r e s in seven species ( i n c l u d i n g three rodents ) . In add i t i on to the M-ATPase c l a s s i f i c a t i o n scheme, we determined muscle f i b r e p r o f i l e s examining the l e v e l s of glycogen and the ox idat ive capac i ty for three major areas of the diaphragm. We were able to demonstrate d i s c r e t e separat ion of f i b r e types using the M-ATPase scheme, however, cons iderable overlap of - 123 -both glycogen l e v e l s and the ox idat ive capac i ty between f i b r e types was demonstrated in a l l regions of the hamster diaphragm. Quant i ta t ion of SDH a c t i v i t y in d i f f e r e n t f i b r e types (based on M-ATPase) have also shown metabolic heterogenity in several other species (18,44,49). Only Ha lk jaer -Kris tensen and Ingemann-Hansen (24) have demonstrated d i s c r e t e separat ion of q u a n t i t a t i v e l y determined enzymatic l e v e l s by determining both the ox idat ive and g l y c o l y t i c capaci ty in M-ATPase based muscle f i b r e types. Several s tudies categorize two or three f i b r e types and sub-categorize the type II M-ATPase f i b r e type based on q u a l i t a t i v e v i s u a l examination of the i n t e n s i t y of s t a i n i n g for another enzyme or substrate . I t would be impossible to r e l a t e our f indings to these s tudies because of the continuous d i s t r i b u t i o n of both the glycogen l e v e l s and ox idat ive capac i ty of the d i f f e r e n t f i b r e types in the hamster diaphragm. G e n e r a l l y , the type I l a f i b r e had the l ea s t glycogen and the highest ox idat ive capaci ty across a l l regions of the hamster diaphragm. The l i b f i b r e contained the most glycogen and had the lowest ox idat ive capaci ty whereas the type I f i b r e was intermediate in these two c h a r a c t e r i s t i c s . The metabolic c h a r a c t e r i s t i c s of the hamster diaphragm contrast with the muscle f i b r e type p r o f i l e most commonly reported in humans (3,58) and other species (19,49) i n which the type I l a f i b r e i s most often intermediate i n nature. There are other species such as the mouse where the intermediate f i b r e i s not the type I l a f i b r e type (49). Because of in ter spec i e s and intramuscular v a r i a b i l i t y of metabolic c h a r a c t e r i s t i c s in the M-ATPase f i b r e types i t i s d i f f i c u l t to compare our r e s u l t s to other i n v e s t i g a t i o n s . Thus, much of the d i s p a r i t y between the r e s u l t s of our i n v e s t i g a t i o n and others may be explained by the i n c o m p a t i b i l i t y of f i b r e type s t a i n i n g and c l a s s i f i c a t i o n schemes used in d i f f e r e n t s tud ies . - 124 -Muscle f i b r e p a t t e r n , propor t ions , and s izes wi th in the s t e r n a l and c o s t a l diaphragm were found to be r e l a t i v e l y homogeneous between surfaces and the three s t e r n a l / c o s t a l regions examined. The r e l a t i v e d i s t r i b u t i o n of the three f i b r e types suggested that the diaphragm is wel l -equipped to perform l o w - i n t e n s i t y , r e p e t i t i v e contract ions and may be r e c r u i t e d during more f o r c e f u l maneuvers. There was considerable v a r i a b i l i t y of both glycogen l e v e l s and the ox idat ive capaci ty between a l l three M-ATPase based f i b r e types in the s t e r n a l and c o s t a l region of the hamster diaphragm. This provides fur ther support to the hypothesis that the s t e r n a l and c o s t a l diaphragm i s r e c r u i t e d during d i f f e r e n t kinds of a c t i v i t i e s . We d id not observe any large d i f ferences in muscle f i b r e type proport ions and s izes wi th in the s t e r n a l and c o s t a l diaphragm. Dif ferences in muscle f i b r e type proport ions wi th in the c o s t a l and s t e r n a l regions have been reported by others (51,55,56,62) . L o c a l proport ions as determined from a c l a s s i f i c a t i o n scheme based on M-ATPase and SDH s t a i n i n g i n t e n s i t y var i ed from 20-40% e s p e c i a l l y around a f i b r o v a s c u l a r septum in the r a t diaphragm (62). Sieck et a l (55) reported a greater number of s low-twi tch , ox idat ive (SO) f i b r e s on the abdominal surface with a predominance of f a s t tw i t ch , o x i d a t i v e , g l y c o l y t i c (FOG) and fas t t w i t c h , g l y c o l y t i c (FG) f i b r e s on the t h o r a c i c surface of the cat diaphragm. A more recent study by Sieck et a l (56) demonstrated l a r g e r and more ox ida t ive type I and type II f i b r e s on the abdominal compared to the thorac i c s ide of the c o s t a l diaphragm. A predominance of SO and FOG f i b r e s was found in the s t e r n a l reg ion of the cat diaphragm (51). We d i d not observe d i f ferences in proport ions and s izes of the c o s t a l and s t e r n a l reg ion (51,55,56,62) . The d i f f erence between our own r e s u l t s and those of previous studies may be r e l a t e d to i n c o m p a t i b i l i t y of - 125 -f i b r e type c l a s s i f i c a t i o n schemes used, i n t e r - s p e c i e s v a r i a b i l i t y , and/or d i f f e r e n c e s in biopsy sampling. Because of the homogeneity of muscle f i b r e proport ions and s i zes in the s t e r n a l and c o s t a l diaphragm, we d id not look f o r d i f f e rences in the ox ida t ive capac i ty and glycogen l e v e l s between s u r f a c e s . The propor t ion of type I to type II f i b r e s in the c o s t a l diaphragm found in our study appears to be in agreement with other s tudies (12,32) examining the hamster diaphragm although both of these i n v e s t i g a t i o n s found a s l i g h t l y smal ler propor t ion of type I f i b r e s (Farkas et al :23.2% [12] and Kelsen et al:25-26% [32]). With respect to three f i b r e type d i f f e r e n t i a t i o n , both Kelsen et a l (32) and Farkas et a l (12) used a d i f f e r e n t c l a s s i f i c a t i o n scheme (47). These two studies presented gross d i s s i m i l a r i t i e s in regards to the f a s t - t w i t c h or type II f i b r e s u b - c l a s s i f i c a t i o n in to FOG and FG. Because of the i n c o m p a t i b i l i t y (1,3,18) of the two schemes f o r three f i b r e type c l a s s i f i c a t i o n (10,47) , i t i s d i f f i c u l t to compare propor t ions from our study to t h e i r s (12,32) although our r e s u l t s appear to compare more c l o s e l y to those by Kelsen et a l (32). We demonstrated considerable v a r i a b i l i t y in the ox idat ive capac i ty of the type II f i b r e s ( F i g . 37, middle and lower panels) in the s t e r n a l and c o s t a l diaphragm which is in d i r e c t contrast to the the f ind ings of Farkas et a l (12). They found the hamster diaphragm to conta in 100% ox ida t ive f i b r e s . There were severa l cont ras t ing features between the c r u r a l region and the r e s t of the diaphragm. The crus d i f f e r s in embryologica l o r i g i n (36), p h y s i o l o g i c a l ac t ion (9) and gross appearance. The th ickness of th is muscle from the abdominal to the t h o r a c i c surface suggests that i t funct ions to provide a greater force output than the r e s t of the diaphragm. We found - 126 -that the M-ATPase f i b r e type proport ions , f i b r e s i z e s , glycogen l e v e l s and ox ida t ive capaci ty i n the c r u r a l regions contrasted in severa l ways to the re s t of the diaphragm. In a d d i t i o n , there was a sharp d e l i n e a t i o n in pat tern and proport ions of M-ATPase f i b r e types between the abdominal and t h o r a c i c surface of t h i s region of the diaphragm; the abdominal surface had a pauci ty of type I f i b r e s whereas the t h o r a c i c surface had s i g n i f i c a n t l y more type I f i b r e s than the re s t of the diaphragm. This i s the converse of what Sieck et a l (54) found in the cat diaphragm but in agreement with what Metzger et a l (41) and R i l e y and Berger (51) found in the r i g h t crus of the r a t and cat diaphragm, r e s p e c t i v e l y . Caution must be used in a d i r e c t comparison of these studies with ours because of the d i f f e r e n t species and/or f i b r e type c l a s s i f i c a t i o n schemes used. In our study, there was a much l a r g e r proport ion of l i b f i b r e s in the abdominal surface and fewer type l i b f ibres in the thorac i c surface of the c r u r a l r e g i o n . Since a l l changes in type l i b f i b r e proport ions were associated with the opposite change in type I f i b r e proport ions (with small f l u c t u a t i o n s in the type I l a category) , one may speculate that the type I and l i b f i b r e s i n t e r a c t in a complementary fa sh ion . We cannot o f f e r a p h y s i o l o g i c a l explanat ion for such i n t e r a c t i o n . With respect to s i ze of muscle f i b r e s , both the type I and type I l a f i b r e s were larger i n the thorac i c surface of the c r u r a l reg ion than the re s t of the diaphragm. This region a l so had the l ea s t amount of glycogen i n a l l f i b r e types and the most NADH-TR i n the type l i b f i b r e s compared to the re s t of the diaphragm. These changes together with the greater proport ion of type I f i b r e s in the thorac i c surface of the crus would suggest more frequent recruitment during long durat ion a c t i v i t i e s . E l e c t r o -- 127 -myographic studies have shown reg iona l s p e c i a l i z a t i o n in funct ion of the crus around the esophagus during a c t i v i t i e s such as e r u c t a t i o n , vomiting and defecat ion (42). We found no s p e c i a l s t ruc ture of the c r u r a l diaphragm around the esophagus with respect to f i b r e type pa t t ern . The authors are unaware of s tudies examining surface to surface d i f f e r e n t i a t i o n in reg iona l funct ion of the crus . The scant number of type I f i b r e s and smaller type I l a f ibres in the abdominal surface of the c r u r a l region may r e f l e c t minimal recruitment of f i b r e s with high endurance c h a r a c t e r i s t i c s . The smaller type I l a f ibes in the abdominal surface of the c r u r a l region may be the r e s u l t of r e l a t i v e d i suse , however in some cases chronic use ( e l e c t r i c a l s t imula t ion) reduces f i b r e s i z e . Another p o s s i b i l i t y may be that l i m i t a t i o n in d i f f u s i o n to t h i s surface during contrac t ion may have r e s u l t e d in the adaptive response of smaller f i b r e s to f a c i l i t a t e d i f f u s i o n during recovery. The large proport ion of type l i b f i b r e s and high l eve l s of glycogen in the abdominal surface of the c r u r a l region would suggest that t h i s area i s involved in quick f o r c e f u l a c t i v i t i e s such as recruitment in the performance of a V a l s a l v a maneuver during defecat ion and other funct ions which require a f i r m abdominal c a v i t y . We d id not f i n d as many d i f ferences between f i b r e types and the three main areas with NADH-TR sta ined f i b r e s compared to PAS-sta ined f i b r e s . This may be i n d i c a t i v e of a much more homogeneous d i s t r i b u t i o n of the ox idat ive capac i ty compared to the glycogen l e v e l s or perhaps the non-s p e c i f i c i t y of NADH-TR may have created extra noise in the s i g n a l . Nicotinamide adenine d inuc leo t ide t e t r a z o l i u m reductase i d e n t i f i e s mito-chondria which contain ox idat ive enzymes but a lso has a l e ss s p e c i f i c r e a c t i o n with the sarcoplasmic tubules (58). The n o n - s p e c i f i c r eac t ion may - 1 2 8 -account for 10 to 12% of the formazan deposited in the t i s sue sec t ion (24). F i b r e type and incubat ion times between 10 and 30 minutes does not appear to a f f ec t the amount of formazan deposi t due to the n o n - s p e c i f i c reac t ion (24). The r a t i o of mean absorbance of formazan in type I : type I I f i b r e s has been reported to be 1.41:1 i n human s k e l e t a l muscle. This i s not un l ike the r a t i o of SDH a c t i v i t y reported in type I:type II f i b r e s of 1.53:1 from microd i s sec t ion of i n d i v i d u a l f ibres and subsequent biochemical analyses (11). The c lose correspondence of the r a t i o of mean absorbance of NADH-TR s ta ined f ibres to the SDH a c t i v i t y of type I : type II f i b r e s would suggest that NADH-TR i s a good representat ion of the ox idat ive capac i ty of s k e l e t a l muscle f i b r e s . Dif ferences in cryos ta t t i s sue thickness may have contr ibuted to the v a r i a b i l i t y in the o p t i c a l dens i ty va lues . We found the cryos ta t sect ions examined to be f a i r l y homogeneous, however some i n t e r - and i n t r a -sec t ion d i f ferences d id e x i s t . In order to minimize d i f ferences we cut sect ions for PAS s t a i n i n g at 16 uM. We had planned to categor ize NADH-TR s ta ined f i b r e s by v i s u a l examination and hence, sect ions for t h i s s t a i n were cut at 10 ym. When t h i s method of c l a s s i f i c a t i o n appeared quest ionable the same 10 ym sect ions were evaluated using microphotometric techniques in order to quant i ta te the s t a i n i n g i n t e n s i t y . The th inner sect ions used for the eva luat ion of the NADH-TR sta ined f i b r e s may have contr ibuted to the lack of s t a t i s t i c a l s i g n i f i c a n c e in the examination of d i f f erences between f i b r e types and/or reg ions . Sieck et a l (54) found a cross -over of f i b r e s from one surface to the other in the c r u r a l region from gross d i s s e c t i o n of t h i s muscle fo l lowing the course of i n d i v i d u a l f i b r e s from o r i g i n to i n s e r t i o n . We - 129 -attempted to examine the cross-over of f i b r e s by taking three cuts of cross - sec t ions vary ing in l e v e l between the o r i g i n and i n s e r t i o n of the c r u r a l diaphragm. In a d d i t i o n , we examined the area around the esophagus. We found a cons is tent pattern in the c r u r a l region independent of the l e v e l in which the c r o s s - s e c t i o n was cut . In other words, the abdominal surface always had very few type I f ibres and the thorac i c surface demonstrated more of a mosaic pat tern (with cons iderably more type I f i b r e s than the abdominal surface) throughout the length of the crus . Our methodology may be less s ens i t i ve for detect ion of f i b r e s cross ing over from the abdominal to the thorac ic surface compared to the technique of Sieck et a l (54). A l t e r n a t i v e l y , the cross -over of f i b r e s between the abdominal and thorac ic surface of the diaphragm may not occur in the hamster. I f cross -over of f ibres does occur, i t would appear that an equal number of the same f i b r e type crosses over from the abdominal to thorac i c surface as from the thorac ic to abdominal surface . Comparison of muscle f i b r e s i ze between animals demonstrated a very small amount of v a r i a b i l i t y in proport ions and s izes of muscle f i b r e types. This i s un l ike the large i n d i v i d u a l v a r i a b i l i t y of muscle f i b r e s i ze found i n human limb muscles which has been a t t r i b u t e d to sex, age, a c t i v i t y l e v e l and biopsy s i t e (28). Three of the fac tors c o n t r i b u t i n g to i n d i v i d u a l v a r i a b i l i t y were c a r e f u l l y c o n t r o l l e d for in our study. We used a l l male hamsters of the same age. The small s i z e of the animal enabled us to examine the t o t a l region of i n t e r e s t ra ther than sampling a small representat ive area . A c t i v i t y l e v e l was not d i r e c t l y c o n t r o l l e d f o r . However, the small in teranimal v a r i a b i l i t y we found suggests s i m i l a r a c t i v i t y (and v e n t i l a t o r y ) l e v e l s in the animals we s tud ied . - 130 -From examination of muscle f i b r e type s ize and propor t ions , t h i s study c l e a r l y demonstrated three d i s t i n c t areas of the diaphragm: the thorac i c surface of the c r u r a l r eg ion , the abdominal surface of the c r u r a l r eg ion , and the s t e r n a l and c o s t a l r eg ion . This would suggest that the hamster diaphragm i s r e c r u i t e d to perform at l e a s t three d i f f e r e n t patterns of a c t i v i t y . - 131 -Figure 27: Regional sites of biopsies cut from hamster diaphragm. Sternal, anterior c o s t a l , and posterior costal were cut from the central tendon to just beyond the midpoint between the o r i g i n and insertion. In contrast, crural segments were cut from the o r i g i n (upper three lumbar vertebrae) almost to the central tendon. - 132 -PLATE IV Figure 28: Photomicrograph of representat ive cross - sec t ions of p o s t e r i o r c o s t a l (upper panel) and c r u r a l (lower panel) b iops ies s ta ined for the demonstration of M-ATPase a f t er ac id (pH=4.5) pre incubat ion . Note that the r i g h t and l e f t p o s t e r i o r c o s t a l b iops ies are sandwiched together . The abdominal surface (AS) of these b iops ies are ins ide and the thorac i c surface (TS) of each i s on the outs ide . Scale=200 um. The c r u r a l biopsy was cut midway between o r i g i n and i n s e r t i o n . The t h o r a c i c surface i s uppermost and the abdominal surface i s lowermost. Scale=200 um. 133 - 134 -Figure 29: The average difference of the optical density measurements determined from 16 fibres of the control s l i d e on 15 days. The average difference was derived by subtracting the control measures obtained at the beginning of the day from those obtained at the end of the day. The average difference i s expressed as a percentage of the mean o p t i c a l density measure. - 135 -PLATE V Figure 30: Muscle f i b r e type proport ions in abdominal ( r i gh t ) and thorac ic ( l e f t ) surface of each region of the diaphragm. In each r e g i o n a l wedge, the inner segment = Type I ; middle = Type I l a ; outer = Type l i b . Values are mean + SE. Figure 31: Photomicrograph of c r u r a l region surrounding esophagus stained for the demonstration of M-ATPase a f t er a c i d (pH=4.5) pre incubat ion . The thorac i c surface i s uppermost and the abdominal surface i s lowermost. The esophageal opening (ES) through the diaphragm is i n d i c a t e d . Scale=200um. l i t . Costal . Costal - 137 -15-i Diameter (jiM) Figure 32: Frequency d i s t r i b u t i o n of diameters of type I fibres in the thoracic surface of c r u r a l regions and the rest of the diaphragm (sternal and costal regions). - 138 -15-i 1 ' Diameter QLIM) Figure 33: Frequency d i s t r i b u t i o n of diameters of type I l a f i b r e s in thorac ic and abdominal surfaces of the c r u r a l reg ion (upper pane l ) ; and in the lower panel , thorac i c surface of the c r u r a l region and the res t of the diaphragm ( s t erna l and c o s t a l r e g i o n s ) . - 139 -15-| O Type I IB Type Ila Z 3 Type lib 10 II 0 o 20 30 40 50 60 Diameter (/xM) 70 so 90 Figure 34: Frequency d i s t r i b u t i o n s of diameters of type I , I l a , and l i b f i b r e s across a l l reg ions . - 140 -PLATE VI Figure 35: Scanning e l ec t ron photomicrographs of cryos ta t sect ions of the hamster diaphragm. Note the uniform thickness of the cryos tat sect ion along the sec t ion edge, w i th in an i n d i v i d u a l muscle f i b r e , and along the o u t l i n e of each muscle f i b r e . C a l i b r a t i o n bar = 20 um on upper panel and 25 um on lower panel . - 142 -PLATE VII Figure 36: "Frequency d i s t r i b u t i o n of o p t i c a l dens i t i e s of PAS stained type 1 f ibres (upper pane l ) , type I l a f i b r e s (middle pane l ) , and type l i b f i b r e s (lower pane l ) . Percent of Fibres Percent of Fibres — — ro o u> o i V \ M V X \ \ , 3T~ I V V V p O o" g_ O (5 o o c 3 o ZSZS V V 1 A A \ 1 A~~VI IH o in i p GO' HOI aT -i O > or CD * 5 o o (A O o Ul _1_ — — ro o in o ' i ' to in _1_ IH IM o m _ J 1_ CD o _i Percent of Fibres i n o u i o i n o i n o u i —I 1 1 1 1 1 I i i pi v v v v v r  i \ \ \ V i 3 0 - 144 -PLATE VII I F igure 37: Frequency d i s t r i b u t i o n of NADH-TR sta ined type I f i b r e s (upper pane l ) , type I l a f i b r e s (middle pane l ) , and type l i b f i b r e s (lower pane l ) . 0.4 0.6 Optical Density (OD units) - 146 -PLATE IX Figure 38: Photomicrograph of s e r i a l cross - sec t ions of c r u r a l regions s ta ined with PAS ( l e f t ) and NADH-TR ( r i g h t ) . The thorac ic surface i s uppermost ( inner margin of page) and the abdominal surface i s lowermost (outer margin of page) in both photomicrographs. Scale = 100 um. - 148 -Ila lib 0.0001 psO.000 0.001 0.01 OPTICAL DENSITY 0.1 1.0 THOR/CRUR I Ila lib 0.0001 p=0.000 p=0.001 poO.018 0.001 0.01 OPTICAL DENSITY 0.1 1.0 STERNAL ft COSTAL l Ila l ib paO.000 p ==0.000 ' psO.000 0.0001 0.001 0.01 OPTICAL DENSITY 0.1 1 to Figure 39: Box p l o t s of the o p t i c a l dens i ty of PAS-s ta ined f i b r e s and p values for comparisons between f i b r e types w i t h i n a given area . The m i d l i n e of the box i s the median, the l e f t border i s the lower q u a r t i l e , the r i g h t border is the upper q u a r t i l e , the l e f t end po in t of the l i n e i s the minimum and the r i g h t end po int of the l i n e i s the maximum. - 149 -TYPE I Thor/Crur Stamal * Costal 0.0001 HELM Abd/Crur Thor/Crur _ Stomal it Costal 0.0001 TYPE Hb Abd/Crur Thor/Crur Stamal A Costal 0.0001 0.001 0.01 OPTICAL DENSITY 0.1 0.001 0.01 OPTICAL DENSITY 0.1 0.001 0.01 OPTICAL DENSITY 0.1 p-0.041 1.0 paO.OOO p=0.001 } p=0.0S6 1.0 p*0.000 p*0.000 ' psO.000 1.0 Figure 40: Box p l o t s of o p t i c a l d e n s i t y f o r PAS-s ta ined f i b r e s and p values for comparisons between areas w i t h i n a g iven f i b r e type . See legend of f i g u r e 39 f o r e x p l a n a t i o n of a box p l o t . - 150 -ABD/CRUR Ila lib 0.2 THOR/CRUR I Ila lib o aa STERNAL * C0STA1 I Ila lib 0.4 0.6 OPTICAL DENSITY 0.4 0.6 OPTICAL DENSITY 0.8 0.8 p«0 .000 p=0.267 poO.282 ) p=0.475 p=0.002 psO.000 > p=0.000 0.4 0.6 OPTICAL DENSITY 0.8 41: Box p l o t s of o p t i c a l dens i ty for NADH-TR s ta ined f i b r e s and p values for comparisons between f i b r e types w i t h i n a given area . 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Metzger JM, Scheidt KB, F i t t s RH: Histochemical and p h y s i o l o g i c a l c h a r a c t e r i s t i c s of the ra t diaphragm. J Appl P h y s i o l 58(4)-.1085-1091, 1985. 42. Monges H, Sa lducc i J , Naudy B: P i s s o c i a t i o n between the e l e c t r i c a l a c t i v i t y of the diaphragmatic dome and crura and muscular f ibres during esophageal d i s t e n s i o n , vomiting and e r u c t a t i o n . J Phys io l (Par i s ) 74:541-554, 1978. - 155 -43. Mortimer J T , Peckham PH: Intramuscular e l e c t r i c a l s t i m u l a t i o n . In: Neural organizat ion and i t s relevance to p r o s t h e t i c s . New York, I n t e r c o n t i n e n t a l Medical Books, 1973, pp 131-146. 44. Nemeth P, Pette D: Succinate dehydrogenase a c t i v i t y in f i b r e s c l a s s i f i e d by Myosin ATPase in three hind limb muscles of r a t . J P h y s i o l 320:73-80, 1981. 45. Nishiyama A: Histochemical studies on r e d , white, and intermediate muscle f i b e r s of some s k e l e t a l muscles I I I . Histochemical demonstration of ox idat ive enzymes, phosphorylase and glycogen in r e s p i r a t o r y muscle f i b e r s . Acta Med Okayama 20:137-146, 1966. 46. Novikoff AB, Shin W, Drucker J : M i t o c h o n d r i a l l o c a l i z a t i o n of ox idat ive enzymes: s t a i n i n g r e s u l t s with two-tetrazol ium s a l t s . J Biophys Biochem C y t o l 9:47-61, 1961. 47. Peter JB , Barnard R J , Edgerton VR, G i l l e s p i e s CA, Stempel KE: Metabol ic p r o f i l e s of three f i b r e types of s k e l e t a l muscle in guinea pigs and r a b b i t s . Biochemistry 11:2627-2633, 1972. 48. Pette D: A c t i v i t y - i n d u c e d fas t to slow t r a n s i t i o n s in mammalian muscle. Med Sc i Spo Ex 16:517-528, 1984. 49. Reichmann H, Pette D: A comparative microphotometric study of succinate dehydrogenase l e v e l s in type I , IIA and IIB f i b r e s in mammalian and human muscles. Histochemistry 74:27-41, 1982. 50. Reid MB, E r i s c o n GC, Feldman HA, Johnson RL: F i b r e types and f i b r e diameters in canine r e s p i r a t o r y muscles. J Appl P h y s i o l 62(4):1705-1712, 1987. 51. R i l e y D, Berger A J : A reg iona l h i s tochemica l and electromyographic ana lys i s of the cat r e s p i r a t o r y diaphragm. Exp Neurology 66:636-649, 1979. 52. S a l t i n B, Henriksson J , Nygaard E , Andersen P, Jansson E : F i b r e types and metabolic p o t e n t i a l s of s k e l e t a l muscles in sedentary man and endurance runners . Ann NY Sciences 301:2-29, 1977. 53. Sandstedt PER: Representativeness of a muscle biopsy specimen for the whole muscle. Acta Neurol Scand 64:427-437, 1981. 54. S ieck GC, Roy RR, Powell P, Blanco C, Edgerton VR, Harper RM: Muscle f i b r e type d i s t r i b u t i o n and a r c h i t e c t u r e of the cat diaphragm. J Appl  P h y s i o l 55:1386-1392, 1983. 55. Sieck GC, Sacks RD, Blanco DE, Edgerton VR: SDH a c t i v i t y and c r o s s - s e c t i o n a l area of muscle f i b r e s in cat diaphragm. J Appl  P h y s i o l 60:1284-1992, 1986. - 156 -56. Sieck GC, Sacks RD, Blanco C: Absence of r eg iona l d i f f erences in the s i ze and ox idat ive capac i ty of diaphragm muscle f i b r e s . J Appl  P h y s i o l 63:1076-1082, 1987. 57. S t . P i e r r e D, Gardiner PF: The e f f ec t of immobi l izat ion and exercise on muscle func t ion: a review. Physiotherapy Canada 39:24-36, 1987. 58. Swash M, Schwartz MS: Biopsy Pathology of Muscle , Chapman & H a l l , London, 1985, pp 19. 59. Tamaki N: E f f e c t of growth on muscle c a p i l l a r i t y and f i b r e type composition in r a t diaphragm. Eur J Appl P h y s i o l 54:24-29, 1985. 60. Tukey JW: Exploratory Data A n a l y s i s . Addison, Wesley Pub. C o . , London, 1977. 61. Vandervoort AA, Hayes KC, Belanger AY: Strength and endurance of s k e l e t a l muscle in the e l d e r l y . Physiotherapy Canada 38:167-173, 1986. 62. Y e l l i n H: Dif ferences in h is tochemical a t t r i b u t e s between diaphragm and hindleg muscles of the r a t . Anat Record 173:33-341, 1972. 63. Zimmer GH: Microphotometry, in Molecular b io logy , b iochemistry and  b iophys i c s . Springer P u b l , 1973, V o l 14: 297-328. - 157 -V. THE EFFECTS OF FATIGUE AND REST ON THE HAMSTER DIAPHRAGM INTRODUCTION Once I had developed a su i tab le anesthet ic regimen, and methods to perform p h y s i o l o g i c a l and h i s t o l o g i c a l examination of the hamster diaphragm, I combined these techniques in order to inves t iga te the e f fec t s of fat igue and res t on the hamster diaphragm. The author i s unaware of any study examining the time course of recovery of both s t r u c t u r a l and p h y s i o l o g i c a l changes in the fat igued diaphragm in e i ther animal models or humans. Numerous studies performed in animals and humans have examined the e f fec t s of fat igue on the diaphragm and how factors such as low cardiac output (4) , hypoxia (3,31) and hypercapnea (33) may p r e c i p i t a t e or compound fat igue but no publ ished i n v e s t i g a t i o n has examined recovery. The e f fec t s of r e s t i n g the r e s p i r a t o r y muscles by mechanical v e n t i l a t i o n has been inves t igated in a number of c l i n i c a l s tudies (12,13,17,21,41,52,53) . Several of these studies in pat ients with chronic neuromuscular d iseases , chest wa l l d iseases , or chronic lung diseases assoc iated with r e s p i r a t o r y muscle weakness and or hypercapneic r e s p i r a t o r y f a i l u r e have demonstrated improvement in r e s p i r a t o r y muscle func t ion af ter vary ing periods of r e s t produced by mechanical v e n t i l a t i o n . In c l i n i c a l s tudies examining the benef i t of mechanical v e n t i l a t i o n on r e s p i r a t o r y muscle f u n c t i o n , i t i s impossible to obta in muscle b iops ie s of the diaphragm, so no i n d i c a t i o n of the s t r u c t u r a l changes assoc iated with diaphragmatic fa t igue and recovery can be determined. Furthermore the information der ived from c l i n i c a l s tudies i s l i m i t e d by v a r i a b i l i t y in the nature and s e v e r i t y of disease between p a t i e n t s . C l i n i c a l and phys io log ic - 158 -abnormali t ies other than r e s p i r a t o r y muscle fat igue may be present in i n d i v i d u a l pat ients and these may require intervent ions in add i t i on to r e s p i r a t o r y muscle r e s t and so may inf luence the r e s u l t s obtained. I chose to fa t igue the diaphragm by r e p e t i t i v e e l e c t r i c a l s t imula t ion because the parameters of the fa t igue stimulus a f f e c t i n g diaphragmatic muscle f i b r e recruitment could be more t i g h t l y c o n t r o l l e d than by using a more p h y s i o l o g i c a l st imulus such as t r a c h e a l banding or exhaustive exerc i se . Since we were performing experiments in anesthet ized animals who may have had a l t ered r e s p i r a t o r y d r i v e , a p h y s i o l o g i c a l overload may have simply r e s u l t e d i n hypovent i la t ion and thus hindered our a b i l i t y to produce r e s p i r a t o r y muscle f a t i g u e . On the other hand, by using e l e c t r i c a l s t i m u l a t i o n , the duty cyc le and r e p e t i t i o n s could be f ixed to produce standardized contrac t ion and r e l a x a t i o n t imes. In a d d i t i o n , diaphragmatic muscle a c t i v a t i o n could be c o n t r o l l e d both by the magnitude of the current which inf luences the proport ion of f i b r e s r e c r u i t e d and by the frequency of s t imula t ion which would c o n t r o l the f i r i n g frequency of the phrenic motoneurons. The end point of fa t igue could be c l e a r l y def ined in terms of a s p e c i f i c decrease in force output (%Pdi/Pdimax). Consequently, by f a t i g u i n g the diaphragm by r e p e t i t i v e e l e c t r i c a l s t i m u l a t i o n , the durat ion and magnitude of the overload could be more c l o s e l y c o n t r o l l e d than by imposing more p h y s i o l o g i c a l over loads . Rest ing the diaphragm by e i t h e r continuous mechanical v e n t i l a t i o n or spontaneous unloaded breathing was performed in order to contras t two d i f f e r e n t kinds of recovery a c t i v i t i e s . Mechanical v e n t i l a t i o n exemplif ied a pass ive recovery , whereas spontaneous unloaded breathing represented a more ac t ive recovery . No supplemental oxygen was given dur ing any part of - 159 -the experiment because of the p o s s i b i l i t y that supplemental oxygen may prolong endurance time. Pardy and Bye (50) demonstrated that the breathing of a hyperoxic mixture during i n s p i r a t o r y r e s i s t i v e breathing in man delayed the onset of diaphragmatic fa t igue . The o v e r a l l object ives of the work presented in t h i s chapter were to examine the time course of recovery fo l lowing fat igue of the hamster diaphragm and s p e c i f i c a l l y , to tes t whether mechanical v e n t i l a t i o n or spontaneous unloaded breathing was a bet ter mode for f u n c t i o n a l recovery. 1 wanted to examine both f u n c t i o n a l and s t r u c t u r a l changes of the hamster diaphragm. In v ivo f u n c t i o n a l responses of the diaphragm to low and high frequency s t imulat ion were examined fo l lowing a f a t i g u i n g stimulus and during the subsequenct re s t p e r i o d . H i s t o l o g i c a l signs of diaphragmatic muscle f i b r e damage were looked for fo l lowing the fa t igue st imulus and during recovery. L a s t l y , glycogen deple t ion and r e p l e t i o n patterns of the diaphragm were examined in order to determine f i b r e type recruitment during the f a t i g u i n g e l e c t r i c a l s t imula t ion and the changes in glycogen l e v e l s induced by the fa t igue stimulus and the r e s t p e r i o d . METHODS Animals and experimental groups Seventy-one adul t male golden Syr ian hamsters obtained from Charles R i v e r , La P r a i r i e , Quebec, were used for the s tud ie s . T h e i r mean body weight was 127.4+19.4 g . (SD) . Hamsters were assigned e i t h e r to a fat igue/sham only pro toco l or a fat igue/sham and re s t pro toco l ( F i g . 43). Of the 71 animals , 14 hamsters were studied i n order to obtain muscle b iops ies immediately fo l lowing the fa t igue or sham p e r i o d : seven fa t igue only and seven sham fat igue only . - 160 -The remaining hamsters were randomly assigned to four groups for the fat igue and re s t s tud ies . Nine animals were subjected to a sham fat igue st imulus and then were allowed to breathe spontaneously during the rest per iod (S/SB); nine animals were subjected to sham fat igue but were mechanical ly v e n t i l a t e d during the re s t per iod (S/MV). In the remaining two groups, diaphragmatic fat igue was induced with r e p e t i t i v e e l e c t r i c a l s t imula t ion and nine of the animals were res ted by spontaneous breathing (F/SB) and eight animals were rested with mechanical v e n t i l a t i o n (F/MV). Twenty-two animals were excluded from the study because of t e c h n i c a l e r r o r (N=5), hypotension (N=12), inadequate a r t e r i a l blood gases (N=2) or other problems (N=3). Most of the 22 animals were excluded because of d i f f i c u l t i e s which began during the s u r g i c a l preparat ion before they were assigned to one of the s ix experimental groups. Experimental protoco l s (Fig .43) A. Fatigue/sham fat igue only Fourteen hamsters were studied to obtain diaphragmatic muscle b iops ies for h i s t o l o g i c examination immediately post fa t igue or sham f a t i g u e . A l l animals were fasted overnight (16 hours) , weighed j u s t before admin i s tra t ion of the anes thet ics , then anesthet ized and s u r g i c a l l y prepared. Measurements of transdiaphragmatic pressure during twitch and t e t a n i c s t imula t ion were obtained. In the sham fat igue only group, measurements were obtained at the same time points as for the fat igue only group but during the per iod corresponding to the f a t i g u i n g e l e c t r i c a l s t imula t ion of the diaphragm, these animals breathed spontaneously through the tracheostomy tube with no a d d i t i o n a l load imposed. A bias flow of 60 mL/min room a i r was infused - 161 -into the tracheostomy tube at a point very near to the trachea in order to minimize the deadspace of the tracheostomy tube. For the fa t igue only group, fa t igue of the diaphragm was induced by e l e c t r i c a l s t imulat ion of the diaphragm using the diaphragmatic p late e lectrodes at a r e p e t i t i o n rate of 80 per minute, frequency of 30 Hz, maximal current , and duty cyc le of 0.30 u n t i l there was a 50% decrease in the dynamic Pdi ( F i g . 44). Fol lowing the fat igue or sham s t imulus , measurement of twitch and te tan ic Pdi was repeated and then the animals were k i l l e d and diaphragmatic muscle b iops ies were obtained. Muscle b iops ies were obtained by opening the laporotomy i n c i s i o n , r e t r a c t i n g the abdominal contents and exc i s ing the l e f t s t e r n a l and a n t e r i o r c o s t a l regions of the diaphragm. Biops ies were cut from these two reg ions , quick frozen and stored in a manner s i m i l a r to that described in Chapter IV, p 100. B. Fatigue/sham fat igue and re s t ( F i g . 43). In the 35 animals who had a sham or f a t i g u i n g stimulus fol lowed by a per iod of r e s t , the s u r g i c a l preparat ion and recording of twitch and t e tan ic Pdi were performed as described above for animals in the F a t i g u e /  sham fat igue only p r o t o c o l . The hamsters i n the groups who were fa t igued (F/SB and F/MV) were subjected to a s i m i l a r f a t i g u i n g stimulus and the sham groups (S/SB and S/MV) underwent a 10-minute per iod of spontaneous breathing through the tracheostomy tube. P h y s i o l o g i c a l parameters were monitored during these experiments in a s i m i l a r manner to that j u s t descr ibed for the fat igue/sham fat igue only experiments in order to ensure that the fa t igue s t i m u l i were s i m i l a r . The r e s p i r a t o r y muscles were res ted by e i ther spontaneous breathing (S/SB and F/SB groups) or mechanical v e n t i l a t i o n (S/MV and F/MV groups) . Previous experiments (Chapter I I , p. 52) had - 162 -demonstrated that when mechanical v e n t i l a t i o n was s u f f i c i e n t to produce an absent diaphragmatic EMG there were no negative de f l ec t ions of esophageal pressure . Therefore , during the surgery for a l l animals and during the res t per iod in the mechanical v e n t i l a t e d groups (S/MV and F /MV) , the l e v e l of mechanical v e n t i l a t i o n was adjusted u n t i l negative Pes d e f l e c t i o n s were abol ished ensuring passive recovery with minimal diaphragmatic a c t i v i t y . Twitch and t e tan ic pressure measurements were repeated every hour for up to s ix hours of res t for a l l four groups. Blood pressure was monitored c o n t i n u a l l y and three or four a r t e r i a l blood gas samples were obtained throughout the course of the experiment. Muscle b iops ies were obtained from the diaphragm, quick frozen and stored in a fashion s i m i l a r to that described prev ious ly under the Fatigue/sham fat igue only p r o t o c o l . S u r g i c a l preparat ion Hamsters were anesthet ized with a combination of pentobarb i ta l (2.6mg/100g BW), ch lora lose (3.8 mg/lOOg BW) and urethane (38 mg/lOOg BW) . Supplemental doses (Chapter I I , p. 41) were administered as necessary. They were tracheostomized and mechanical ly v e n t i l a t e d on room a i r throughout the s u r g i c a l prepara t ion . The l e f t c a r o t i d a r t e r y was d i s sec ted and cannulated (PE 10 tubing) in order to obtain a r t e r i a l blood samples and to monitor blood pressure . Through a mid l ine laparotomy i n c i s i o n , the s i l i c o n e dome conta in ing the diaphragmatic p la te e lectrodes was inser ted and sutured in place as descr ibed prev ious ly (Chapter I I I , p. 67-68). Two water f i l l e d catheters were placed down the esophagus to measure esophageal (Pes) and g a s t r i c (Pga) pressure and Pdi was determined as descr ibed prev ious ly (Chapter I I I , p. 72). Transdiaphragmatic pressure (Pdi) was c a l c u l a t e d as the a lgebra ic sum of Pga and Pes (Pdi=Pga-Pes) changes during e l e c t r i c a l - 163 -s t i m u l a t i o n . S i m i l a r to previous studies using DPS, the corset encasing the small water bag was appl ied (p. 74). This provided a r e s t i n g Pab of 8 to 10 cmH^O. T h i r t y seconds before e l e c t r i c a l s t imulat ion Pab was increased to 15 mcH^O and then the Pab was decreased to r e s t i n g l e v e l s immediately fo l lowing e l e c t r i c a l s t i m u l a t i o n . The corset was kept in p o s i t i o n throughout the durat ion of the experiment. Monitor ing and c o r r e c t i o n of blood pressure Blood pressure was measured by a d i f f e r e n t i a l pressure transducer (Bentley Trantec , Model #800) throughout the laparotomy surgery and the remainder of the experiment. I f the animal became hypotensive ( s y s t o l i c pressure <100 mmHg), 5% human serum albumin was administered in 1.0 mL a l i q u o t s . Albumin was administered to 20 of 35 animals used in the f a t i g u e /  sham fat igue and re s t s tud ies . On average 4.7+2.3 mL was administered (range:2.0-11.0 mL). R e t r o s p e c t i v e l y , p h y s i o l o g i c a l data from the l a t e r course of the experiment was excluded from the analyses i f the s y s t o l i c blood pressure could not be maintained above 100 mmHg. Monitor ing and c o r r e c t i o n of a r t e r i a l blood gases A r t e r i a l blood samples of 0.3 mL were taken during the f i r s t hour of r e s t and every one to two hours t h e r e a f t e r . They were analyzed using an a r t e r i a l blood gas analyzer (Radiometer Copenhagen ABL 3) . A r t e r i a l blood measurements were expressed as values corrected to 37oC. Resp iratory and metabolic disturbances were treated i n the fo l lowing manner: 1. i f an abnormal pCO^ (<30mmHg or >45mmHg) was noted, the mechanical v e n t i l a t o r se t t ings or anesthet ic regime was a l t ered accord ing ly ; 2. i f a metabol ic ac idos i s occurred (any base d e f i c i t ) , i t was correc ted by in fus ion of sodium bicarbonate (0.892 mEq/mL) based on the fo l lowing equation: - 164 -(0.4 x body weight) x base d e f i c i t = mEq sodium bicarbonate requ ired to correc t the base d e f i c i t (29). The f i r s t part of the equation (0.4 x body weight) i s used to estimate i n t r a c e l l u l a r and e x t r a c e l l u l a r f l u i d . I t i s assumed that h a l f the a l k a l i (HC0~) w i l l be accepted by i n t r a c e l l u l a r buffers and the other h a l f w i l l e levate plasma [HCO^l (29). Sodium bicarbonate was administered in 17 of 35 animals used in the fat igue/sham fat igue and re s t s tud ies . An average of 0.24+0.16 mL per animal was administered (range:0.10-0.70mL). Three of the 35 experiments were discont inued before the s ix hour res t per iod was completed because the r e s p i r a t o r y or metabolic disturbances could not be c o r r e c t e d . Fatigue stimulus Fat igue of the diaphragm was induced by r e p e t i t i v e e l e c t r i c a l s t i m u l a t i o n . We chose to s t imulate with short bursts (duty c y c l e : 0.30, 30 Hz, 200 us pulse width) appl ied r e p e t i t i v e l y at 80 reps/min u n t i l fa t igue was induced. Repe t i t i ve s t imula t ion at t h i s low duty cyc le was chosen in order to induce fat igue with an endurance load rather than one of higher i n t e n s i t y and shorter d u r a t i o n . We chose a s t imulat ion frequency of 30 Hz because i t was the lowest frequency at which a fused tetanus could be c o n s i s t e n t l y achieved in the hamster. The airway was open during the r e p e t i t i v e e l e c t r i c a l s t imula t ion and each s t imula t ion was assoc iated with a transdiaphragmatic pressure swing which was of much greater amplitude than that produced during spontaneous breath ing . A bias flow of 60 mL/min room a i r was infused into the tracheostomy tube at a point very near to the trachea i n order to minimize the deadspace of the tracheostomy tube during the f a t i g u i n g r e p e t i t i v e e l e c t r i c a l s t i m u l a t i o n . The corset remained in p o s i t i o n which provided a r e s t i n g Pab of 8-10 cmH 0. The r e p e t i t i v e - 165 -e l e c t r i c a l s t imula t ion was continued u n t i l there was a 50% decrease in the dynamic Pdi ( F i g . 44). The dynamic Pdi was defined as the change in Pdi produced with the r e p e t i t i v e e l e c t r i c a l s t imula t ion with an open airway and Pab of about 8-10 cmH^O. An average of three contract ions was taken for the dynamic Pdi measurement. Measurement of twitch and t e tan ic Pdi Maximal current was determined by gradua l ly increas ing the current a p p l i c a t i o n for r e p e t i t i v e twitches u n t i l a maximal compound diaphragmatic ac t ion p o t e n t i a l was obtained. Once maximal current was determined, base-l i n e Pdi measurements were made for twitch (200 uS pulses) and t e tan ic s t i m u l i (pulse width: 200 uS, one sec d u r a t i o n , frequencies: 30, 50, and 70 Hz) . Twitch and t e tan ic s t imula t ion measures were repeated immediately fo l lowing the fa t igue per iod and once every hour thereaf ter during the res t p e r i o d . Muscle h is tochemistry S e r i a l sect ions of muscle b iops ies were s ta ined for H & E , M-ATPase, NADH-TR, and PAS using s i m i l a r techniques to those descr ibed in Chapter IV, p. 100-101. Sections were evaluated for muscle f i b r e abnormal i t ies and damage using H & E , NADH-TR, and M-ATPase (Chapter IV, p. 106). F i b r e types were assessed using the M-ATPase s t a i n . Glycogen content was quant i ta ted using microdensi tometr ic techniques on s e r i a l cros s - sec t ions s ta ined for PAS in a fashion s i m i l a r to that descr ibed in Chapter IV, p. 103-106. Because of the large number of b i o p s i e s , c r o s s -sect ions of these specimens were s ta ined on two d i f f e r e n t days. S e r i a l sect ions were taken from c o n t r o l b iops ies and sta ined on both days - 166 -Q u a l i t a t i v e H i s t o l o g i c a l Analys i s We quant i tated inflammation by examining the i n f l u x of neutrophi l s (polymorphonuclear leucocytes) using a p i c t o r i a l grading scheme i l l u s t r a t e d i n F i g . 45. A grade of 0, 1, 2, or 3 was given to each of 6 f i e l d s (magnif icat ion 20x) observed using a research microscope ( Z e i s s ) . A grade of '0* was i n d i c a t i v e of a normal c r o s s - s e c t i o n of hamster diaphragm with l e ss than three neutrophi l s observed. A grade of '1 ' was assigned to the f i e l d i f neutrophi l s were observed p r i m a r i l y contained w i th in the blood ves se l s . A grade of '2' was given i f neutrophi l s were observed wi th in the blood vesse ls and along the main connective t i s sue layers of epimysium and perimysium. L a s t l y , a grade '3' was i n d i c a t i v e of the most extreme response with neutrophi l s around i n d i v i d u a l f i b r e s and/or wi th in muscle f i b r e s . To fur ther d i f f e r e n t i a t e the grade a '+' or ' - * was given in add i t i on to the numerical va lue . This was equated to e i t h e r +0.25 or -0.25 i n the t a l l y of the inflammatory response of the s ix f i e l d s . The highest poss ib le l e v e l of inflammation for one diaphragmatic muscle c r o s s - s e c t i o n was 19.5 ( s ix f i e l d s x a grade of 3+). The number of damaged f i b r e s was counted in H & E , NADH-TR, and M-ATPase s ta ined sec t ions . Damaged f i b r e s were defined as those demonstrating d i s rupt ions in the normal morphology and s t a i n i n g c h a r a c t e r i s t i c s . Examples for each of the three s ta ins are i l l u s t r a t e d in F i g . 46. S i m i l a r to the grading of inflammation, counting of damaged f i b r e s was performed on s ix f i e l d s at a magni f i ca t ion of 20x for each biopsy using a research microscope. Six f i e l d s at a magni f i ca t ion of 20x inc luded the viewing of approximately 600 f i b r e s which comprised about 50-75% of the c r o s s - s e c t i o n of the i n d i v i d u a l b i o p s i e s . - 167 -S t a t i s t i c a l Ana lys i s A repeated measures ANOVA was used to assess any d i f ferences between pre - and pos t - fa t igue Pdi measurements in the three fat igue groups: fat igue on ly , F / S B , and F/MV. The repeated measure was frequency of s t imula t ion and d i f ferences between two grouping fac tors were determined: group ( fat igue on ly , F / S B , or F/MV) and time (preor p o s t - f a t i g u e ) . In order to c o n t r o l for d i f ferences in base l ine P d i , the e f fects of fa t igue were expressed as changes in Pdi from base l ine values (APdi ) . Base l ine was defined as the f i r s t measurement before e i t h e r the sham or fat igue s t imulus . The APdi was also used for examination of d i f ferences at each frequency of s t imula t ion and time i n t e r v a l in the four fatigue/sham fat igue and res t groups (see below). As described above, abnormal blood pressure and a r t e r i a l blood measures re su l t ed in the exc lus ion of a number of animals l a t e in the course of some experiments such that the N was decreased at f i v e and s ix hours of r e s t . For t h i s reason the p h y s i o l o g i c a l data was only analyzed for up to four hours of r e s t . Because there were severa l miss ing values even during the four hours of res t (due to t e c h n i c a l d i f f i c u l t y in obta in ing a good Pdi measurement or drop out of animals at the l a t e r time p e r i o d s ) , a three-way analys i s of var iance with time as repeated measure could not be performed. Instead, a two-way ana lys i s of var iance was performed examining for d i f f erences between stimulus (sham vs fat igue) and re s t (spontaneously breathing vs mechanical v e n t i l a t i o n ) at each time i n t e r v a l : one hour -N=35, two hours - N=31, three hours - N=25, four hours - N=17. Because a two-way ANOVA was performed at f i v e time i n t e r v a l s , a more conservat ive l e v e l of p<0.01 was chosen for s t a t i s t i c a l s i g n i f i c a n c e . - 168 -Dif ferences in a r t e r i a l blood gas measures between the four fa t igue and re s t groups were examined for using a one-way ana lys i s of var iance . In order to examine for d i f ferences of inflammation and damaged f i b r e s , a pa ired t - t e s t was performed on the absolute numbers of inflammation grade and the number of damaged f i b r e s for the fat igue and sham fat igue only groups. In a d d i t i o n , a Wilcoxon paired-sample t e s t was performed on the ranked order of the inflammation scores and number of damaged f i b r e s for the fat igue and sham fat igue only groups to examine for d i f f e r e n c e s . Di f ferences in the amount of glycogen in cros s - sec t ions of diaphragmatic muscle b iops ies were analyzed using an ANOVA on the logged values for PAS o p t i c a l dens i ty . The fo l lowing comparisons of the PAS o p t i c a l dens i ty in cross - sec t ions of diaphragmatic muscle b iops ie s were made: 1. sham fat igue only and fat igue only; 2. fa t igue on ly , F/SB and F/MV groups; 3. sham fat igue only , S/SB, and S/MV groups; 4. four fa t igue and res t groups - S/SB, S/MV, F / S B , F/MV. These comparisons were performed by separat ing o p t i c a l dens i ty data according to f i b r e type and making comparisons w i th in a given f i b r e type. The data was a lso grouped according to t ime. Comparisons were made with the data groups for 3-4 hours r e s t , 5-6 hours re s t and 3-6 hours of r e s t . RESULTS Response to fat igue st imulus Although a s i m i l a r fat igue stimulus was appl ied to the diaphragm of a l l hamsters, t h e i r response v a r i e d in both the dynamic transdiaphragmatic - 169 -pressures produced and the time per iod the stimulus was appl ied u n t i l there was a 50% decrease in the dynamic Pdi ( F i g . 47). The mean i n i t i a l dynamic Pdi of the three fa t igue groups was 24.7+6.6 cmH^O with a range of 12.5-44.0 cmH 20. The mean time per iod of f a t i g u i n g e l e c t r i c a l s t imula t ion r e s u l t i n g in a 50% decrease in the dynamic Pdi of the three fa t igue groups was 13:53+8:29 min with a range of 4:30-40:00 mins. There was no obvious r e l a t i o n s h i p between the i n i t i a l Pdi and endurance time to fa t igue ( F i g . 47). Twitch and t e tan ic Pdi Pos t - fa t igue Pdi measures were decreased from p r e - f a t i g u e Pdi for both twitch impulses and for a l l frequencies of t e tan ic s t imula t ion in the three fa t igue groups ( fat igue only , F / S B , and F/MV) as shown in F i g . 48 and Table XI (p<0.0005). The decrease in Pdi pos t - fa t igue was s i m i l a r in a l l three groups. Although a 50% decrease in the dynamic Pdi was produced from 30 Hz s t i m u l a t i o n , a s i m i l a r decrease was not demonstrated by the q u a s i -i sometr ic Pdi measures three (twitch) and s ix minutes ( t e tan ic pressures) fo l lowing the cessat ion of the fa t igue st imulus ( F i g . 48 and Table X I ) . There was no percept ib l e trend towards recovery in Pdi at low frequencies of s t imula t ion (twitch and 30 Hz) during the four-hour res t per iod in the two fat igue and res t groups (F/SB and F/MV) and there was a trend towards a progress ive decrease in Pdi in the two sham fat igue and res t groups which was apparent by one hour post fa t igue ( F i g . 49) . Consequently, there was no d i f f erence in Pdi between the groups with twi tch and 30 Hz s t i m u l a t i o n at any of the hourly i n t e r v a l s during the re s t p e r i o d . - 170 -TABLE XI Pdi BEFORE AND AFTER THE FATIGUE STIMULUS  IN THE 3 FATIGUE GROUPS: FATIGUE ONLY, F/SB AND F/MV FATIGUE ONLY TWITCH 30 Hz 50 Hz 70 Hz HAMSTER PRE- POST- PRE- POST- PRE- POST- PRE- POST-1 20.0 21.7 39.0 25.2 53.0 40.0 69.0 52.0 2 12.2 9.2 30.3 19.0 51.5 33.0 68.4 46.6 3 14.0 9.8 22.0 17.0 47.4 23.5 62.0 51.5 4 9.3 2.4 30.5 12.7 47.0 27.0 56.6 46.0 5 9.0 3.0 36.5 8.6 50.6 20.0 60.4 34.0 6 13.0 4.0 22.0 10.0 42.0 23.0 48.0 64.5 7 13.3 8.0 35.0 25.0 48.1 38.4 63.5 53.0 MEAN 13.0 8.3 30.8 16.8 48.5 29.3 61.1 49.7 S.D. 3.7 6.6 6.7 6.7 3.6 7.9 7.2 9.2 F/SB TWITCH 30 Hz 50 Hz 70 Hz HAMSTER PRE- POST- PRE- POST- PRE- POST- PRE- POST-1 10.1 5.3 21.1 7.0 42.0 19.8 51.4 36.0 2 11.0 5.0 15.0 13.0 46.0 31.0 63.0 52.5 3 21.4 16.5 22.5 18.5 64.0 32.0 85.5 63.0 4 14.5 9.6 23.5 17.0 49.0 36.0 65.0 57.5 5 15.8 12.5 23.5 19.0 51.0 39.4 81.0 60.2 6 23.5 19.7 28.0 20.0 58.0 42.0 66.5 60.0 7 21.0 11.8 26.0 17.0 56.0 34.5 70.0 48.0 8 22.8 17.5 34.0 21.5 65.1 40.4 75.3 60.3 9 10.0 10.6 27.0 22.5 46.0 23.5 51.0 45.0 MEANS 16.7 12.1 24.5 17.3 53.0 33.2 67.6 53.6 S.D. 5.6 5.1 5.2 4.8 8.2 7.6 11.9 9.0 F/MV TWITCH 30 Hz 50 Hz 70 Hz HAMSTER PRE- POST- PRE- POST- PRE- POST- PRE- POST-1 18.0 11.0 20.0 12.0 39.5 21.0 61.0 29.0 2 23.3 10.3 26.0 19.5 45.5 36.5 54.0 41.7 3 15.5 10.0 22.0 14.0 49.0 30.5 64.0 42.0 4 11.2 6.4 27.0 13.8 49.3 33.0 67.5 45.0 5 11.6 8.0 19.2 15.5 51.0 34.0 70.5 67.0 6 24.0 10.0 21.0 12.0 40.8 28.0 56.4 41.0 7 14.0 11.0 29.0 16.9 61.9 37.6 77.1 60.2 8 15.5 13.0 34.5 17.0 47.0 34.0 55.5 54.0 MEANS 16.6 10.0 24.8 15.1 48.0 31.8 63.3 47.5 S.D. 4.9 2.0 5.3 2.6 6.9 5.3 8.1 12.1 Note: A l l pressures are expressed in cnu^O - 171 -A d i f f e r e n t pattern of APdi was demonstrated during the res t per iod at high frequencies (50 and 70 Hz) of t e tan ic s t imula t ion compared to low frequencies of s t imula t ion ( F i g . 49). For 50 Hz at one hour of r e s t , APdi was less in hamsters of both mechanical ly v e n t i l a t e d groups (F/MV and S/MV) than in the spontaneously breathing groups (F/SB and S/SB) (p<0.01). At two hours of r e s t , the APdi was l ess i n hamsters of F/MV than APdi of the animals in F/SB (p<0.01) however both sham groups (S/MV and S/SB) demonstrated a s i m i l a r increase in APdi . At three hours of r e s t , there was a trend towards the APdi in the hamsters of both mechanical ly v e n t i l a t e d groups (F/MV and S/MV) to be less than the APdi in the animals of the spontaneously breathing groups (F/SB and S/SB) (p<0.03). The mean APdi of hamsters in F/MV was very c lose to zero at three hours of r e s t . At four hours of r e s t , there were no d i f f erences in the change i n Pdi from base l ine between the four groups at 50 Hz t e tan ic s t i m u l a t i o n . For 70 Hz s t i m u l a t i o n , there was no d i f f erence in the APdi between any of the four groups at one hour of r e s t . At two hours of r e s t , there was a trend for the APdi in hamsters of the mechanical ly v e n t i l a t e d groups (F/MV and S/MV) to be less than the APdi of animals in the spontaneously breathing groups (S/MV and S/SB) (p<0.03). At three and four hours of r e s t , there were no s i g n i f i c a n t d i f f erences of the APdi between any of the hamsters in the four groups at 70 Hz s t i m u l a t i o n . There was a d e f i n i t e trend towards recovery of Pdi in hamsters of F/MV at three hours of r e s t , however, the small N i n the groups at t h i s po int of the experiment may have contr ibuted to the lack of s t a t i s t i c a l s i g n i f i c a n c e . Because of the l i m i t a t i o n s of the s t a t i s t i c a l a n a l y s i s , s i g n i f i c a n c e could only be examined between APdi of animals in each group - 172 -at each time i n t e r v a l rather than comparison of APdi between time i n t e r v a l s , pos t - fa t igue and the hourly periods during r e s t . By looking at the graph ica l d i s p l a y of the APdi in the hamsters of the four groups, some trends over time were obvious ( F i g . 49). At low and high frequencies of s t i m u l a t i o n , the two sham fat igue and re s t groups (S/SB and S/MV) demonstrated a tendency towards progress ive d e t e r i o r a t i o n during the res t p e r i o d . No obvious recovery of Pdi from low frequency s t imula t ion fat igue was demonstrated in the animals of the two fat igue groups (F/SB and F/MV). The animals in the F/MV group demonstrated a trend towards recovery of Pdi towards base l ine l e v e l s up to three hours of res t at high frequencies of s t i m u l a t i o n , e s p e c i a l l y 50 Hz s t i m u l a t i o n . A r t e r i a l blood measures The raw data for the PaCO and PaO over the durat ion of the 2 2 experiment for the four groups is p l o t t e d in F i g . 50 and 51, r e s p e c t i v e l y . The mean data for the a r t e r i a l blood measures are shown in Table X I I . The PaC0 2 was lower in S/MV than the PaCO^ in the S/SB and F/SB groups (p<0.01). There was a trend towards lower values of PaCO^ in the animals of F/MV than those in the S/SB and F/SB groups. There was no re t en t ion of carbon dioxide in the spontaneously breathing animals , even in the l a t e r hours of the experiment. There was a wide s ca t t er of values for PaO . However, there were no s i g n i f i c a n t d i f ferences or obvious trends 2 towards d i f ferences between any of the groups or over t ime. There was a s i g n i f i c a n t l y lower concentrat ion of hydrogen ions ( [H + ] ) i n the a r t e r i a l blood of hamsters in S/MV than those i n the S/SB and F/SB groups (p<0.01). - 1 7 3 -TABLE XII  ARTERIAL BLOOD MEASURES DURING FATIGUE/SHAM FATIGUE AND REST PROTOCOL GROUP PaC0 2 (mmHg) S/SB 3 7 . 7 + 7 . 8 Pa0 2 (mmHg) PH 5 8 . 1 + 1 4 . 1 7 . 4 0 F/SB 3 6 . 9 + 8 . 1 J 6 9 . 1 + 1 6 . 3 7 . 4 0 [H+] (nmol/L) BASE EXCESS (mEq/L) 4 0 . 1 + 4 . I T - 0 . 7 6 + 3 . 2 1 S/MV 2 9 . 3 + 8 . 2 * 6 4 . 8 + 1 5 . 8 7 . 4 6 3 4 . 4 + 6 . 9 * - 0 . 6 6 + 3 . 4 4 0 . 0 + 5 . 2 J - 0 . 5 4 + 3 . 2 4 F/MV 3 1 . 8 + 8 . 7 6 6 . 7 + 1 7 . 8 7 . 4 5 3 5 . 8 + 7 . 1 - 0 . 2 6 + 3 . 4 1 * p < 0 . 0 1 The number of animals in each group were the same as for the Pdi measurements. - 174 -The [H + ] in the a r t e r i a l blood of animals in F/MV demonstrated a trend towards lower values compared to the [H + ] i n the S/SB and F / S B . Muscle f i b r e damage In the diaphragmatic cross - sec t ions s ta ined with H & E and M-ATPase, there were s i g n i f i c a n t l y more damaged f i b r e s in the fa t igue only group than those of the sham fat igue only group (p<0.01) as shown in F i g . 52 and Table X I I I . The counts of damaged f i b r e s determined from cross - sec t ions s ta ined with H & E , M-ATPase and NADH-TR ( F i g . 52) were very s i m i l a r in the fa t igue only group. However, for the sham fat igue only group, more damaged f i b r e s were counted in the NADH-TR sta ined t i s sue cross - sec t ions than in the M-ATPase and H & E stained c r o s s - s e c t i o n s . However, the number of damaged c e l l s was a very small proport ion of the number of f i b r e s observed in the t i s sue c r o s s - s e c t i o n . In the fat igue only group, an average of 26 damaged f i b r e s were counted from a t o t a l of approximately 600 f i b r e s observed in each t i s sue c r o s s - s e c t i o n . The damaged c e l l s appeared to be located predominantly along the abdominal surface of the diaphragmatic c r o s s -sect ions ( F i g . 53). - 175 -TABLE XIII NUMBER OF DAMAGED FIBRES AND SCORES OF INFLAMMATION  FOR THE FATIGUE/SHAM FATIGUE GROUPS GROUP ANIMAL Sham 1 Fatigue 2 3 4 5 6 7 NUMBER OF DAMAGED FIBRES H & E NADH-TR M-ATPase 0 0 0 0 9 0 24 1 21 22 1 32 2 34 0 6 13 0 20 0 20 INFLAMMATION SCORE 6.50 0.00 0.00 2.25 7.25 2.00 5.25 MEANS & S.D. Fatigue 1 2 3 4 5 6 7 8+11* 29 47 24 26 22 28 2 13+16 28 31 23 40 30 29 3 8+10* 24 47 31 37 34 20 0 3.32+3.23 3.00 5.00 5.00 7.00 3.25 3.25 5.50 MEANS & S.D. 25+13 26+11 28+15 4.57+1.00 * p<0.01 Sham fat igue group d i f f e r e n t than fat igue group. - 176 -There was a trend towards a decreased number of damaged f ibres during the ear ly hours of the re s t period ( F i g . 54). This trend was apparent from counts of diaphragmatic cross - sec t ions s ta ined for H & E , M-ATPase and NADH-TR. There were trends towards decreasing counts of damaged f ibres in a l l four fatigue/sham fat igue and res t groups over time but increases were noted at four and f i v e hours in diaphragmatic c r o s s -sect ions from the S/SB group and at s ix hours in cros s - sec t ions s ta ined from F/SB and F/MV groups. Counting of damaged c e l l s in the diaphragmatic cross - sec t ions of the fatigue/sham fat igue and res t groups was more d i f f i c u l t than in the t i s sue cross - sec t ions of fat igue/sham fat igue only groups. In severa l biopsy samples, there appeared to be wisps of cytoplasm, empty spaces, and e o s i n o p h i l i c f l o c c u l e n t masses ( F i g . 55) which may have been the only remains of prev ious ly damaged muscle f i b r e s such as those i l l u s t r a t e d in F i g . 46. Such h i s t o l o g i c a l features were not counted as damaged f i b r e s . I f they indeed d id represent damaged f i b r e s then the trend towards a decrease in the detectable number of damaged f i b r e s could have been an a r t i f a c t r e l a t e d to the complete or near complete d i s s o l u t i o n of some f i b r e s . Inflammatory response Inflammation as ind ica ted by an i n f l u x of neutrophi l s was apparent in both the diaphragmatic cross - sec t ions of sham fat igue and fa t igue only groups compared to the lack of such signs in the diaphragm of hamsters without the extensive s u r g i c a l preparat ions and p h y s i o l o g i c a l measures of Pdi (Chapter IV, p. 119). The neutrophi l s were located p r i m a r i l y in the blood vessels of the diaphragmatic muscle cross - sec t ions of both the sham fat igue and the fa t igue only groups although t h i s phenomenon was more - 177 -obvious in the t i s sue of the l a t t e r group. In about h a l f of the samples, neutrophi l s were observed in the connective t i s sue layers surrounding the f i b r e s and wi th in the muscle f i b r e s although t h i s observat ion was much less frequent than that of neutrophi l s in the blood vesse l s . The mean grade of inflammation was higher for the fat igue only than the sham fat igue only group however the d i f f erence d id not reach s t a t i s t i c a l s i g n i f i c a n c e ( F i g . 52). The inflammatory response demonstrated a progress ive increase in cross - sec t ions of diaphragmatic b iops ies i n a l l four fat igue/sham fat igue and res t groups up to two and three hours of res t ( F i g . 56). The c e l l u l a r i n f i l t r a t e appeared to plateau in t i s sue cross - sec t ions of three of the fat igue/sham fat igue groups and decreased in those of the F/MV group af ter three hours of r e s t . The inflammatory response was increased three - and f o u r - f o l d in the diaphragmatic cross - sec t ions of the fat igue/sham fat igue and res t groups compared to the b iops ies of the fatigue/sham fat igue only groups. Numerous neutrophi l s invading the t i s sue in the connective t i s sue l a y e r s , surrounding i n d i v i d u a l f i b r e s and i n f i l t r a t i n g muscle c e l l s were observed ( F i g . 57). Glycogen l e v e l s Frequency d i s t r i b u t i o n curves for the o p t i c a l dens i ty of f ibres s ta ined for PAS are shown in F i g s . 58-60. As in the normal c o n t r o l animals (Chapter IV, p. 117), the frequency d i s t r i b u t i o n s of s t a i n i n g i n t e n s i t y were skewed to the r i g h t but demonstrated log normal d i s t r i b u t i o n s . In the type l i b f i b r e s , there was a s i g n i f i c a n t d i f f erence in the mean between the o p t i c a l dens i t i e s of the fa t igue only group (mean:0.167; mean+SD in log : -1.79+-0.71) and the sham fat igue only group (mean:0.292; e mean+SD in log : -1.23+-0.61) (p<0.05). Examimation of the frequency - 178 -d i s t r i b u t i o n s of the type l i b f ibres in these two groups ( F i g . 58) revealed a d i s t i n c t and s i g n i f i c a n t s h i f t to lower o p t i c a l dens i ty values in the frequency d i s t r i b u t i o n of fat igue only group compared to the d i s t r i b u t i o n of the sham fat igue only group (p<0.05). Although there was a decreased amount of glycogen in the type l i b f i b r e s of the fat igue only group, there was s t i l l a measurable amount remaining a f t er the fa t igue s t imulus . In a d d i t i o n , there was no d i f f erence in the frequency d i s t r i b u t i o n s of the glycogen s t a i n i n g i n t e n s i t y in the type I and I l a f i b r e s of the fa t igue and sham fat igue groups. Thus, although the fa t igue stimulus decreased the l e v e l of glycogen s tores , they were c e r t a i n l y not o b l i t e r a t e d . A l l muscle b iops ies for a l l groups were not s ta ined for glycogen on the same day. Biops ies of fatigue/sham fat igue were s ta ined on day 1 and biops ies of fat igue/sham fat igue and re s t were s ta ined on day 2. Comparison of c o n t r o l s e r i a l sect ions stained on the f i r s t s t a i n i n g day to those stained on the second s t a i n i n g day demonstrated an increase in s t a i n i n g i n t e n s i t y on the second day. The average d i f f erence for o p t i c a l dens i ty between muscle f i b r e s s ta ined on day 1 and day 2 was +.052 uni t s or a log e value of -0 .19 . The s t a t i s t i c a l ana lys i s was done on uncorrected data because the most appropriate method to transform the data for such a s h i f t i n s t a i n i n g i n t e n s i t y was not known. However, i t order to examine poss ib le trends which may have been undetected by the s t a t i s t i c a l a n a l y s i s , the data shown i n F i g . 59-63 was p l o t t e d with the o p t i c a l dens i ty values for fat igue and sham fat igue only groups corrected by a uniform upward s h i f t . Without the c o r r e c t i o n , there were no other s i g n i f i c a n t d i f ferences of PAS s t a i n i n g i n t e n s i t y found for any of the f i b r e types between: 1. fa t igue on ly , F/SB and F/MV groups; 2. sham fat igue on ly , S/SB, and S/MV - 179 -groups; 3. four fat igue and res t groups - S/SB, S/MV, F / S B , F/MV. However with the c o r r e c t i o n of the o p t i c a l dens i ty , some trends were apparent. For the type I f i b r e s , there appeared to be lower values in the o p t i c a l densi ty of diaphragmatic cross - sec t ions from fatigue/sham fat igue and re s t compared to sham fat igue and fat igue only groups ( F i g . 59, 62 & 63). No obvious trends were observed between the o p t i c a l dens i ty of the type I l a f i b r e s between the s ix experimental groups ( F i g . 60). I n t e r e s t i n g l y , the frequency d i s t r i b u t i o n s of the type I l a f i b r e s in the four fatigue/sham fat igue and re s t groups appeared to be more bimodal in nature compared to those of the fat igue/sham fat igue groups ( F i g . 60). There appeared to be lower values for o p t i c a l dens i ty values of type l i b f i b r e s in diaphragmatic c r o s s -sect ions from sham fat igue and res t groups (S/SB and S/MV) compared to the sham only group ( F i g . 61 & 62). Summary of r e s u l t s Both low and high frequency fa t igue were produced in the three fa t igue groups. Transdiaphragmatic pressure demonstrated a trend towards progress ive d e t e r i o r a t i o n during the re s t per iod in the animals of the S/SB and S/MV groups. Recovery from high frequency fat igue was demonstrated in the diaphragm of animals in F/MV although no recovery was demonstrated in the diaphragm of animals in F / S B . The PaCO^ and [H +] was s i g n i f i c a n t l y lower in the animals of S/MV and there was a trend towards lower values in the animals of F/MV compared to the a r t e r i a l blood measures in S/SB and F / S B . Muscle f i b r e damage was produced in the diaphragms of animals in both the fa t igue only and sham fat igue only groups but was s i g n i f i c a n t l y greater in the fa t igue only group. There were trends towards a decrease in the number of damaged c e l l s i n the diaphragm of the four fat igue/sham - 180 -fa t igue and re s t groups compared to the fa t igue only and sham/fatigue only groups, however some c e l l damage may have been underestimated. The grade of inflammation was greater in the diaphragm of both the sham fat igue only and fat igue only groups than i n the normal hamster diaphragm . Trends towards a marked increase in inflammation were c l e a r l y obvious in the biopsy specimens of the four fatigue/sham fat igue and re s t groups. Glycogen l eve l s were decreased i n the type l i b f i b r e s i n the diaphragms of the fa t igue only groups compared to the glycogen l e v e l s in the type l i b f i b r e s in the diaphragm of the sham fat igue only group. There were trends towards a decrease i n glycogen l e v e l s i n the diaphragms of the four sham f a t i g u e / fa t igue and res t groups compared to the glycogen l e v e l s in the diaphragms of the sham fat igue group however d i f ferences were not s i g n i f i c a n t when uncorrected for the systematic d i f ferences in s t a i n i n g i n t e n s i t y between days. - 181 -TABLE X I V SUMMARY OF RESULTS - P O S T - F A T I G U E AND DURING REST PERIOD MEASURES SHAM F A T . ONLY S / S B EXPERIMENTAL GROUPS F A T I G U E S/MV ONLY F / S B F / M V P d i P O S T - F A T I G U E No c h a n g e i m m e d i a t e l y p o s t - s h a m f a t i g u e s t i m u l u s s i m i l a r d e c r e a s e o f t w i t c h & t e t a n i c P d i f o r 3 f a t i g u i g r o u p s Low f r e q u e n c y fi h i g h f r e q u e n c y f a t i g u e REST Low F r e q . H i g h F r e q . n / a n / a A r t e r i a l b l o o d m e a s u r e s n / a PaC02 Pa02 [H>] Base E x c e s s C e l l Damage P O S T - F A T I G U E n / a n / a n / a n o r m a l 81. + n o r m a l n o r m a l n o r m a l n o r m a l M O / b i o p s y n / a n / a n / a n / a n / a n / a n / a n o r m a l ^ 2 6 / b i o p s y no c h a n g e no c h a n g e n o r m a l n o r m a l n o r m a l n o r m a l no change r e c o v e r y • normal n o r m a l n / a n / a REST 1-3 h o u r s n / a 4 - 6 h o u r s n / a I n f l a m m a t i o n P O S T - F A T I G U E + v s n o r m a l REST 1-3 H o u r s 4 -6 H o u r s n / a n / a G l y c o g e n l e v e l s P O S T - F A T I G U E B a s e l i n e REST n / a * 4 & 5 h r n / a p r o g . no c h a n g e n / a no c h a n g e n / a p r o g . no c h a n g e n / a t r e n d s - +type I & t y p e l i b n / a n / a + v s n o r m a l & sham f a t . n / a n / a T 6 h r n / a P r o g . no c h a n g e + 6 h r n / a t u n t i l 2 h r d e c r e a s e + - T y p e l i b n / a n / a n / a t r e n d - +type I A B B R E V I A T I O N S : d e c r e a s e - + , f a t i g u e - f a t . , f r e q u e n c y -n / a - n o t a p p l i c a b l e , p r o g r e s s i v e - p r o g . f r e q . , h o u r s - h r , i n c r e a s e • T - 182 -DISCUSSION FATIGUE/SHAM FATIGUE ONLY Pdi Low and high frequency fat igue of the hamster diaphragm was produced by r e p e t i t i v e maximal s t imula t ion v i a diaphragmatic p late e lectrodes at a frequency of 30 Hz ( F i g . 48). Other inves t i ga tors have suggested that the presence of low frequency fat igue ind icates impaired e x c i t a t i o n - c o n t r a c t i o n coupl ing or an a l t e r a t i o n of the c o n t r a c t i l e apparatus whereas the presence of high frequency fat igue ind icates f a i l u r e of e l e c t r i c a l propagation v i a the neuromuscular junc t ion or loss of funct ion of the muscle c e l l membrane system (15,22,39,55) . However, i t i s not known whether or not high frequency fat igue can be a t t r i b u t e d to s i m i l a r p h y s i o l o g i c a l mechanisms i n the presence of low frequency fa t igue (24). Fat igu ing e l e c t r i c a l s t imula t ion was appl ied using a duty cyc le of 0.30 at a mean pressure of 38% of Pdi (mean dynamic Pdi/mean Pdi at 70 max Hz s t imula t ion pre - fa t igue ) . The product of the mean dynamic P d i / P d i max and duty cyc le r e s u l t s in a pressure-t ime product of 0.12. Inves t igat ions of the human diaphragm demonstrated that fat igue occurred when the pressure-t ime product exceeded 0.15 whereas contract ions which were below t h i s product could be sustained i n d e f i n i t e l y (55). In p r e l i m i n a r y s tud ie s , I had used a duty cyc le of 0.50 to fa t igue the hamster diaphragm which was reported to be an appropriate duty cyc le to fa t igue the diaphragm in the dog (28). However, fa t igue (as defined by a 50% decrease in the dynamic Pdi) occurred w i t h i n minutes of a p p l i c a t i o n of the e l e c t r i c a l s t imula t ion to the hamster diaphragm so a shorter duty cyc le was chosen such that the endurance time would be longer . The d i f f erences i n the pressure-t ime - 183 -product that produced fat igue in the hamster to that of other species may be a t t r i b u t e d to in t er - spec i e s d i f ferences in the ox idat ive capac i ty of the diaphragm and a varying recruitment pat tern of the diaphragm in the human studies where vo luntary contrac t ion was used to achieve P d i . During phrenic nerve or diaphragmatic p la te e lectrode s t i m u l a t i o n , the order of recruitment i s f i xed and a higher frequency of s t imula t ion is necessary for a fused tetanus. Thus fat igue i s l i k e l y to occur e a r l i e r during e l e c t r i c a l s t imula t ion than vo luntary contrac t ion (47). Muscle f i b r e damage Diaphragmatic p late e lectrode s t imula t ion produced muscle f i b r e damage which was p r i m a r i l y on the abdominal surface in the a n t e r i o r c o s t a l region of the diaphragm located over the s t imula t ing e lec trode ( F i g . 53). The number of damaged f i b r e s was greater in the fa t igue only group compared to the sham fat igue only group ( F i g . 52). The presence of damaged f i b r e s in the sham fat igue group provides evidence that the r e l a t i v e l y t r i v i a l i n s u l t of p o s i t i o n i n g the s i l i c o n e dome beneath the diaphragm and b r i e f s t imula t ion for measurement of Pdi without the product ion of fa t igue caused some c e l l damage. More damaged c e l l s were detected in the NADH-TR sta ined cross - sec t ions of the sham fat igue group than were detected i n the H & E and M-ATPase s ta ined cros s - sec t ions of the sham fat igue group which suggested that t h i s s t a i n may be more s e n s i t i v e in de tec t ing c e l l damage. Th i s i s not unexpected s ince the NADH-TR s t a i n i d e n t i f i e s the sarcoplasmic re t i cu lum and mitochondria (61) which are the f i r s t c e l l u l a r organel les to be a l t e r e d i n the r e v e r s i b l e stages of c e l l death . Trump et a l (62) described seven stages of c e l l death; stages 1 to 4a are r e v e r s i b l e i f the i n j u r i o u s st imulus i s removed. In stage 2, the - 184 -endoplasmic re t i cu lum becomes d i l a t e d and in stages 3 and 4, the inner and outer compartments of the mitochondria undergo various volume changes of c o n s t r i c t i o n and d i l a t a t i o n . Revers ib le damage of rat muscle mitochondria and plasma membrane has been demonstrated fo l lowing e l e c t r i c a l s t imula t ion of the s c i a t i c nerve (59). Walter et a l (66) also demonstrated r e v e r s i b l e mi tochondr ia l changes in ra t s k e l e t a l muscle fo l lowing e l e c t r i c a l s t imula t ion of the s c i a t i c nerve. In our study, the increased number of abnormal c e l l s detected by NADH-TR compared to the H & E and M-ATPase s ta ins may have been a r e f l e c t i o n of changes in the volumes of the s u b c e l l u l a r compartments of the endoplasmic re t i cu lum and mitochondria in diaphragmatic muscle f i b r e s of the hamster. Damaged c e l l s were observed in the diaphragmatic cros s - sec t ions of the fa t igue group suggesting that the fa t igue stimulus of r e p e t i t i v e e l e c t r i c a l s t imula t ion provided a damaging i n s u l t to the diaphragmatic muscle f i b r e s . Damage at the l e v e l of the muscle f i b r e may have been one of the causes of the low frequency fat igue observed in the fa t igue only group. Revers ib le muscle f i b r e damage fo l lowing nerve e l e c t r i c a l s t imula t ion has been demonstrated by others (59,66). However, the damage i n f l i c t e d in these studies appeared to be much less severe than that observed in the hamster diaphragm which may be a r e f l e c t i o n of the d i f f e r e n t fa t igue stimulus parameters used or the endurance capac i ty of the muscle. A l t e r n a t i v e l y , perhaps the e l e c t r i c a l s t imula t ion v i a the p la te e lectrodes had some kind of d i r e c t e f f ec t on the muscle f i b r e s which was not produced i n the two studies (59,66) us ing nerve s t i m u l a t i o n . Muscle f i b r e damage and c e l l death fo l lowing exerc ise overload has been reported in humans (19,27,34) and in animals (20,26,64) . Greater muscle f i b r e damage (42,48) - 185 -and low frequency fat igue (14) was found in the quadriceps fo l lowing eccentr i c versus concentr ic exerc i se . To my knowlege, t h i s i s the f i r s t documentation of the production of diaphragmatic muscle f i b r e damage by r e p e t i t i v e e l e c t r i c a l s t i m u l a t i o n . According to Trump et a l (62) there are two primary i n t e r - r e l a t e d mechanisms of c e l l i n j u r y : an a l t e r a t i o n of the systems for ATP product ion , and d i srupt ions of the c e l l membrane so that i t f a i l s to funct ion as a permeabi l i ty b a r r i e r between the c e l l i n t e r i o r and the e x t r a c e l l u l a r space. The fat igue stimulus of r e p e t i t i v e e l e c t r i c a l s t imula t ion may have placed extreme demands on ATP production and could have re su l t ed in decreased oxygen suppl ies r e s u l t i n g in a m i l i e u s i m i l a r to ischemia. Such a hypothesis was f i r s t r e f e r r e d to by Vihko et a l (64) who r e l a t e d the mechanisms of c e l l death as described by Trump to the changes induced during exhaustive exercise in t h e i r animal model. Vihko et a l found u l t r a s t r u c t u r a l changes in the mouse quadriceps muscle f i b r e s fo l lowing exhaustive swimming s i m i l a r to the morphological a l t e r a t i o n s observed in the stages of c e l l death in other c e l l types . They stated that the main d i f ferences between ischemia and in tens ive p h y s i c a l exer t ion are that during e x e r c i s e , high l a c t a t e and low oxygen tens ion are reached r e l a t i v e l y q u i c k l y and recovery i s r a p i d , whereas during ischemia such changes in l a c t a t e and oxygen tens ion are r e l a t i v e l y slow and may not re turn to normal. The number of damaged f i b r e s detected by our methods formed a small f r a c t i o n of the t o t a l muscle f i b r e populat ion in the reg ion of the diaphragmatic b i o p s i e s . However, damaged f i b r e s were observed i n every biopsy sample of the fa t igue group. The incidence of damaged f i b r e s appeared to be s i m i l a r to the f indings of others who examined muscle biopsy - 186 -samples from the humans fo l lowing overexert ion from m i l i t a r y (19) and marathon t r a i n i n g (27). G e l l e r (19) examined muscle b iops ies from m i l i t a r y r e c r u i t s who presented with signs and symptoms of rhabdomyolysis wi th in the f i r s t week of t r a i n i n g . Usua l ly muscles of the upper extremit ies and shoulder g i r d l e were affected with minimal involvement of the lower l imbs . He found the d i s t r i b u t i o n of necro t i c f i b r e s to be patchy and i r r e g u l a r often with large regions of normal muscle separating abnormal f i b r e s . Hik ida et a l (27) performed u l t r a s t r u c t u r a l examination of the gastrocnemius muscle from volunteer human marathon runners. They found signs of muscle f i b r e damage such as Z-band streaming, e x t r a c e l l u l a r l o c a l i z a t i o n of mitochondria , d i s r u p t i o n of the sarcolemma, and i n t r a c e l l u l a r ery throcytes , however, most of the muscle was reported to be normal. H i k i d a a lso descr ibed an i n f l u x of inflammatory c e l l s inc lud ing n e u t r o p h i l s , lymphocytes, macrophages and mast c e l l s . Inflammation The grade of inflammation was greater in the fa t igue only group than the sham fat igue only group ( F i g . 52). The accumulation of neutrophi l s in the blood vessels of the diaphragmatic muscle cross - sec t ions of both the sham fat igue and fat igue only groups was l i k e l y an i n d i c a t i o n of margination of these c e l l s in response to a chemotactic s t imulus . Muscle f i b r e damage or t e a r i n g of connective t i s sue layers were poss ib le s t i m u l i to the c e l l u l a r response of inflammation. Another poss ib le explanat ion may be that a n t i -dromic s t imula t ion of the sensory nerves of the diaphragm produced "neurogenic inf lammation." St imulat ion of a f ferent nerves by e l e c t r i c a l s t imula t ion or i r r i t a t i n g substances has been reported to be an important mediator of inflammation in other t i s sues besides muscle such as the eye - 187 -(8) , sk in (30,36) and the airways of the lungs (43). Neurogenic inflammation i s thought to be caused by neuropeptides such as substance P re leased from sensory nerve endings (16,36). Inflammation was more marked in the diaphragmatic muscle c r o s s -sect ions of the fa t igue only group suggesting that damage was more prevalent in t h i s group. The observat ion that the primary l o c a t i o n of the polymorpho-nuclear leukocytes was wi th in the blood vesse ls rather than the connective t i s sue layers or muscle f i b r e s themselves was probably a r e f l e c t i o n of the time course of c e l l u l a r i n f i l t r a t i o n during inflammation rather than a measure of the s e v e r i t y of the r e a c t i o n . Glycogen l e v e l s The type l i b f i b r e s of the diaphragmatic b iops ies in the fat igue only group contained l ess glycogen than the type l i b f i b r e s of the muscle b iops ies in the sham fat igue only group ( F i g . 58). Several inves t iga tors (10,22,23,47,65) have used glycogen dep le t ion studies to i n d i c a t e the pattern of neuromuscular recrui tment . To the extent that glycogen deple t ion ind ica tes which f i b r e types are p r i m a r i l y r e c r u i t e d , the data suggest that diaphragmatic p la te e lectrode s t imula t ion causes predominantly type l i b a c t i v a t i o n . The r e s u l t s are cons is tent with the r e s u l t s of previous inves t i ga tors us ing e l e c t r i c a l s t imula t ion to i n i t i a t e muscle c o n t r a c t i o n . Mortimer and Peckham (47) s tated that the s i ze order of recruitment of muscle f i b r e types i s reversed with e l e c t r i c a l s t imula t ion of motoneurons when compared to vo luntary c o n t r a c t i o n . With vo luntary a c t i v a t i o n the smal lest f i b r e s (type I f i b r e s ) are r e c r u i t e d f i r s t fol lowed by the type I l a and type l i b f i b r e s with higher i n t e n s i t y loads . In c o n t r a s t , e x t e r n a l l y app l i ed e l e c t r i c a l s t imula t ion i s descr ibed as i n i t i a l l y r e c r u i t i n g the - 188 -motoneurons which innervate the larger type l ib fibres and higher levels of stimulation recruit the type I and type Ila fibres. Although the pattern of glycogen depletion suggested the preferential recruitment of type l ib fibres, recording of the diaphragmatic compound action potential during earlier experiments, indicated that a l l of the diaphragmatic fibres should have been recruited with the stimulus parameters used in this study. Previous work comparing phrenic nerve stimulation to diaphragmatic plate electrical stimulation (Chap III, p. 79) demonstrated a similar Pdi production at maximal stimulation. With phrenic nerve stimulation at maximal current one is assured of maximal recruitment of a l l fibre types of the diaphragm and since similar Pdi was produced with phrenic nerve stimulation and diaphragmatic plate electrode stimulation, this is additional evidence that a l l fibres types and a l l of the diaphragm was being recruited with the latter technique. If, as the above discussion suggests, there was maximal recruitment of diaphragmatic muscle fibres then the significant depletion of glycogen in only the type l ib fibres following the fatiguing electrical stimulation must be interpreted in other ways. Perhaps the glycogen depletion patterns indicates recruitment of those muscle fibres more dependent on carbohydrate for energy production. Examination of the depletion of l ipid stores in the diaphragmatic muscle fibre types in response to a similar fatigue stimulus would provide additional information regarding the recruitment pattern of the diaphragm during diaphragmatic plate electrode stimulation. At the level of fatigue defined in this study, there was s t i l l a considerable amount of glycogen remaining in the sternal and costal diaphragm. There was no detectable decrease in the glycogen levels of type - 189 -I and type I l a f i b r e s and many of the type l i b f ibres s t i l l contained a considerable amount of glycogen. The presence of glycogen in the fat igued hamster diaphragm does not preclude glycogen deple t ion as a causat ive fac tor of force l o s s . In the e l e c t r i c a l l y s t imulated diaphragm, the muscle f ibres are r e c r u i t e d in a very f ixed manner. S i g n i f i c a n t glycogen loss in a large proport ion of the l a r g e r , more f o r c e f u l type l i b f ibres of the e l e c t r i c a l l y s t imulated f ibres may have contr ibuted to l i m i t a t i o n of energy supply in t h i s f i b r e populat ion and hence, been a causat ive fac tor c o n t r i b u t i n g to f a t i g u e . C r i t i q u e of fa t igue stimulus Diaphragmatic fa t igue was induced in t h i s study by r e p e t i t i v e e l e c t r i c a l s t imula t ion at maximal c u r r e n t , duty cyc le of 0.30, pulse width of 200 ys and frequency of 30 Hz u n t i l there was a 50% decrease i n the dynamic Pdi ( F i g . 44). This pro toco l was chosen over more p h y s i o l o g i c a l fa t igue s t i m u l i such as t r a c h e a l banding to permit the r e l a t i v e l y r a p i d development of fa t igue and because an end-point (a 50% decrease in dynamic Pdi) to ind ica te fa t igue could be c l e a r l y def ined in a l l animals. The chosen pro toco l had c e r t a i n l i m i t a t i o n s . The f ixed and reversed order of recruitment of muscle f i b r e types with diaphragmatic p la te e lectrode s t imula t ion i s very d i f f e r e n t than vo luntary recruitment of s k e l e t a l muscle. In a d d i t i o n , fa t igue was defined as a 50% drop i n the dynamic Pdi produced by e l e c t r i c a l s t imula t ion which may be very d i f f e r e n t from the point of fa t igue induced by exhaustive vo luntary exerc i se in the unanesthetized animal . Thus, fa t igue produced by r e p e t i t i v e e l e c t r i c a l s t imula t ion i s c e r t a i n l y not d i r e c t l y comparable to fa t igue induced by exhaustive voluntary exerc i s e . - 190 -In contrast to our r e s u l t s , others (35,AA) have reported that t ransmiss ion f a i l u r e at the neuromuscular junc t ion occurs at s t imula t ion f requencies above 20 Hz. Transmission f a i l u r e can occur p r e s y n a p t i c a l l y at the nerve terminal branches, p o s t s y n a p t i c a l l y from a decrease in endplate e x c i t a b i l i t y or l e s s f requent ly from a dep le t ion of synapt ic t ransmi t ter substance (5) . However, dur ing the s tudies in which t ransmiss ion f a i l u r e of the neuromuscular junct ion occurred , continuous s t imu la t ion was appl ied fo r at l e a s t 30 seconds. McNeal (AA.A5) compared the e f f e c t s of d i f f e r e n t s t imu la t ion f requencies on force output using supramaximal s t imula t ion (pulse width: 100 us) v i a the s c i a t i c nerve in the cat gastrocnemius muscle. Continuous supramaximal s t imu la t ion at a s t imula t ion frequency of 30 Hz showed that there was maintenance of normalized force f o r the f i r s t 30 seconds fol lowed by a f a l l to about 75% of maximum in two minutes. The use of short bursts (0.225 sec) of t e tan ic s t imula t ion combined with a longer res t i n t e r v a l (0.525 sec) in our study may have allowed s u f f i c i e n t recovery time to prevent s i g n i f i c a n t t ransmiss ion f a i l u r e . The demonstration of s i g n i f i c a n t glycogen dep le t ion p o s t - f a t i g u e and p e r s i s t e n t low frequency fa t igue during the r e s t per iod in our study does not support the hypothesis that t ransmiss ion f a i l u r e was the major cause of fa t igue in s p i t e of the s t imu la t ion frequency chosen. FATIGUE AND REST GROUPS: F/SB AND F/MV Pdi Transdiaphragmatic pressure measurements demonstrated high and low frequency fa t igue immediately fo l lowing the fa t igue st imulus in animals of both fa t igue and r e s t groups ( F i g . A8) but recovery from high frequency - 191 -fa t igue was demonstrated up to three hours of res t in the animals of the F/MV group ( F i g . 49). The changes in Pdi produced by low and high frequency s t imula t ion immediately fo l lowing the fa t igue stimulus and during the res t per iod provided fur ther suggestion regarding the mechanism of fa t igue . According to severa l inves t iga tors (28,54) the recovery of high frequency fat igue may i n d i c a t e re turn of normal funct ion of neuromuscular transmiss ion or membrane funct ion of e i t h e r the sarcolemma or t - tubu le system. A trend towards recovery was demonstrated in the F/MV group by the time of the f i r s t measurements during the re s t p e r i o d . Recovery may be a t t r i b u t e d to a r e a d i l y r e v e r s i b l e process such as the c o r r e c t i o n of an i o n i c imbalance. However, such a suggestion is speculat ive s ince t h i s study d id not d i r e c t l y address poss ib le causat ive mechanisms at the c e l l u l a r l e v e l . The experimental condi t ions in other studies which have inves t iga ted high and low frequency fat igue (32) were very d i f f e r e n t than those in t h i s study so i t i s d i f f i c u l t to extrapolate from previous r e s u l t s . The pers i s tence of low frequency fat igue rather than high frequency fat igue in t h i s study is s i m i l a r to what others have found and t h i s may r e f l e c t a problem in e x c i t a t i o n contrac t ion coupl ing where l ess tens ion is generated for a given membrane e x c i t a t i o n . There are severa l pos s ib l e a l t e r a t i o n s in the s k e l e t a l muscle f i b r e which may lead to t h i s i n c l u d i n g s t r u c t u r a l damage (28), 2+ decreased Ca re lease from the sarcoplasmic re t i cu lum and/or diminished 2+ a f f i n i t y of troponin for the Ca b inding s i t e (28,54). The pers i s tence of low and high frequency fat igue in hamsters of the F/SB group would i n d i c a t e that ' a c t i v e ' r e s t was not adequate to enable recovery; in c o n t r a s t , the t o t a l r e s t imposed by continuous mechanical v e n t i l a t i o n promoted optimal recovery. - 192 -The animals in the F/SB group d id not demonstrate any CO^ r e t e n t i o n , however, the animals in the F/MV group demonstrated a strong trend towards lower values of PaCO^ and H + compared to the a r t e r i a l blood measures of the animals in the 2 spontaneously breathing groups (F/SB and S /SB) . Although a l k a l o s i s has been shown to have a p o s i t i v e i n o t r o p i c e f f ec t on the myocardium (67), i t has not been demonstrated to have a p o s i t i v e e f f e c t on c o n t r a c t i l i t y of the diaphragm in dogs (56) and humans (33). I t i s doubtful that the r e s p i r a t o r y a l k a l o s i s induced in the animals of F/MV had any e f f e c t on the force output of the hamster diaphragm. Diaphragmatic b iops ies were only obtained from animals of the F/SB and F/MV groups in the re s t per iod while those in the hamsters of the fa t igue only group (discussed e a r l i e r ) were taken j u s t a f t er fa t igue had been induced. The end-point for de f in ing fa t igue was i d e n t i c a l in these three groups of animals and s i m i l a r decreases in the Pdi produced by twitch and t e tan ic s t imula t ion were observed fo l lowing the fa t igue stimulus in the two fat igue and re s t groups (F/SB and F/MV) compared to the fa t igue only group ( F i g . 48). Since p h y s i o l o g i c a l changes were comparable in the three fa t igue groups immediately fo l lowing the fa t igue s t imulus , one would expect s i m i l a r morphological changes in a l l three groups. Hence, the e f fec t s of r e s t on the s t ruc ture of the diaphragm can be made by comparison of b iops ies from the F/SB and F/MV groups to those of fa t igue only group. Muscle f i b r e damage Damage of the diaphragm was observed in the hamsters of the two fat igue and res t groups and may have ar i s en from the i n i t i a l i n s e r t i o n of the dome, r e p e t i t i v e rubbing of the dome against the diaphragm during mechanical v e n t i l a t i o n or spontaneous brea th ing , or from the e l e c t r i c a l - 193 -s t imula t ion performed for the product ion of fat igue and measurements of P d i . Somewhat s u r p r i s i n g l y , the count of damaged muscle f i b r e s was lower i n the b iops ies of the fa t igue and re s t group throughout the r e s t per iod than in the fa t igue only and sham fat igue only groups ( F i g . 54). However, the extent of t i s sue damage of the diaphragm in the fa t igue and re s t groups may have been underestimated in t h i s study because of the l i m i t a t i o n of our h i s t o l o g i c a l techniques i n detect ing severe ly damaged c e l l s . Some of the damaged c e l l s in the diaphragmatic muscle b iops ies may have been undetected because the count of damaged c e l l s was l i m i t e d to those c e l l s which s t i l l had enough form to be recognized as muscle f i b r e s . This e l iminated severe ly damaged c e l l s which may have been the e o s i n o p h i l i c f l o c c u l e n t masses observed i n the b iops ies of the fa t igue and re s t groups. Damaged c e l l s may have a lso completely d i s so lved by the time the b iops ie s were obtained in the l a t e r course of the experiment. The methods used to i d e n t i f y damaged c e l l s could have been l i m i t e d by other fac tors which may have af fected experimental groups to various degrees. Although the h is tochemical techniques d i d i d e n t i f y some sub-c e l l u l a r s t r u c t u r e s , they were not the most s e n s i t i v e method to detect a l t e r a t i o n s in organe l l e s . I t i s therefore poss ib le that our methods f a i l e d to detect e a r l y stages of c e l l i n j u r y . The study was a lso l i m i t e d by the small sample s i ze and thus i t i s d i f f i c u l t to make strong conclus ions regarding the r e s u l t s obtained. Although the number of damaged f i b r e s was lower during the e a r l y hours of the re s t p e r i o d , the count of damaged f i b r e s increased i n animals of both fat igue and r e s t groups at s ix hours of r e s t . Th i s may have been i n d i c a t i v e of c e l l s with i n j u r y undetected e a r l y in the experiment. Over - 194 -the s ix hour res t p e r i o d , as the c e l l s proceeded through stages of c e l l death, morphological changes became more apparent. Inflammation Inflammation scores or grades were 2.5 to 4 f o l d higher during the re s t per iod in the b iops ies of the fa t igue and res t groups (F/SB and F/MV) compared to those of the fat igue only group ( F i g . 56). Tissue damage was the most l i k e l y f a c t o r i n c i t i n g an inflammatory response although antidromic s t imula t ion of the sensory nerves (30,36,43) and the presence of an in fec t ious agent cannot be ru led out s ince a port of entry v i a the laporotomy i n c i s i o n was a v a i l a b l e and our s u r g i c a l technique was clean at best . The i n f l u x of neutrophi l s into cutaneous l e s ions of r a b b i t s has been demonstrated to peak between two and four hours (9 ,46,51) . The extent and k i n e t i c s of n e u t r o p h i l accumulation was found to be r e l a t e d to the dose and kind of chemotactic agent (9) . Intracutaneous i n j e c t i o n of zymosan induced a s l i g h t l y delayed response which was cons i s tent with the requirement for the chemotaxinigen to ac t iva te complement which in turn acted as a chemotaxin (9) . In t h i s study, the inflammatory response d i d not demonstrate a sharp peak but rather p e r s i s t e d for severa l hours in the diaphragm of three of the four fat igue and r e s t groups: S/SB, S/MV, and F/MV. I f the diaphragm of the hamster responds in a s i m i l a r manner as cutaneous l e s ions in r a b b i t s , the prolonged response would imply the presence of a p e r s i s t e n t chemotaxin or an e a r l i e r st imulus may have t r i g g e r e d a second chemotactic agent which r e s u l t e d in a p e r s i s t e n t inflammatory response. • The grade for the inflammatory response of the diaphragm biops ies from the F/MV group decreased progres s ive ly from two to f i v e hours of the - 1 9 5 -res t p e r i o d . The recovery of high frequency fat igue occurred for up to three hours of r e s t , so i t i s d i f f i c u l t to know whether the small decrease i n inflammation up to three hours af fected Pdi product ion . A decrease in inflammation may have been r e l a t e d to the increase in P d i , however, an explanat ion of cause or e f f ec t cannot be substant ia ted . Glycogen l eve l s Trends towards lower values in the o p t i c a l dens i ty of the type 1 f i b r e s in diaphragmatic cross - sec t ions from hamsters in the two fat igue and re s t groups were observed compared to the o p t i c a l dens i ty of the type I f i b r e s in b iops ies of the fat igue only groups ( F i g . 5 9 , 61) , although these d i f ferences were not s i g n i f i c a n t . No s i g n i f i c a n t d i f f erences or trends were noted in the o p t i c a l dens i ty values of the type I l a and type l i b of the diaphragm in the fat igue and re s t groups compared to those of hamsters in the fat igue on ly , and sham fat igue and re s t groups. There were trends towards more of a bimodal d i s t r i b u t i o n in the o p t i c a l dens i ty values of the type I l a f i b r e s in the fa t igue and r e s t groups. Recovery of the diaphragm in response to high-frequency s t imula t ion was demonstrated in F/MV whereas recovery d i d not occur in the F/SB group. This r e s u l t suggests that the l e s s e r loads imposed on the diaphragm of animals in the F/MV group provided bet ter re s t to promote recovery compared to the exerc ise imposed on the diaphragm of animals i n F / S B . Ahlborg (1,2) has demonstrated that fo l lowing muscle con trac t ion glycogen r e p l e t i o n was augmented during recovery in man. A bout of exhaustive exerc i se fol lowed by carbohydrate loading has been demonstrated to be more e f f e c t i v e in promoting glycogen supercompensation than carbohydrate loading alone during endurance t r a i n i n g (60). Bonen et a l (7) demonstrated greater glycogen - 196 -replenishment when exerc ise was followed by passive rather than act ive recovery. I f passive recovery promotes glycogen r e p l e t i o n t h i s could serve as one explanation for recovery i n the fat igued hamster diaphragm of the F/MV group. However, there was no s i g n i f i c a n t increase in the o p t i c a l dens i ty for glycogen in the F/MV group and in f a c t , there was a trend towards lower l e v e l s in the type I f i b r e s of t h i s group compared to the o p t i c a l dens i ty of the type I f i b r e s in the fa t igue only and sham fat igue only groups. The reasons we d id not f i n d glycogen r e p l e t i o n with continuous mechanical v e n t i l a t i o n may have been the small sample s i ze or a number of the many compl icat ing fac tors inherent in the experimental des ign . Most l i k e l y , s i g n i f i c a n t glycogen replenishment d id not occur because animals had been fasted for 18-24 hours and no supplemental glucose was administered. An experimental design which optimized pre-experiment glycogen stores may have provided a be t ter base l ine to study the e f fec t s of fa t igue and re s t on glycogen l e v e l s . The data do allow the conclus ion that recovery from high frequency fat igue in the F/MV groups was not accounted for by glycogen r e p l e t i o n during the re s t p e r i o d . SHAM FATIGUE AND REST GROUPS: S/SB AND S/MV Pdi In contrast to e a r l i e r studies demonstrating r e p r o d u c i b i l i t y of Pdi over a two-hour p e r i o d , (Chap I I I , p. 77) the two sham fat igue and res t groups (S/SB and S/MV) demonstrated progress ive d e t e r i o r a t i o n in t r a n s -diaphragmatic pressure during the four-hour re s t per iod ( F i g . 49). The d i f f erence between the r e p r o d u c i b i l i t y of Pdi in the e a r l i e r experiments compared to d e t e r i o r a t i o n in Pdi during t h i s experiment may be explained by - 197 -either the more extensive surgery (insertion of carotid cannula in addition to insertion of silicone dome) involved in the sham fatigue and rest experiments or by spurious results in earlier studies resulting from an inadequate sample size. There was deterioration of Pdi from both high and low frequency stimulation in the sham fatigue and rest studies. This would indicate that uncontrolled factors contributed to progressive diaphragmatic fatigue. The factors potentially responsible for the deterioration can be divided into those which increase demands on the muscle, muscle fibre damage, and those factors which decrease energy stores and supplies to the muscle. Factors contributing to diaphragmatic fatigue could have affected the two sham fatigue and rest groups to different degrees. In the S/SB group, a number of factors could have increased the load on the diaphragm. They include: the resistance of the tracheostomy tube, and the impedance of the silicone dome containing the plate electrodes, the corset and water bag. If mechanical ventilation was adequate in the S/MV group, these factors increasing the load on the diaphragm would not have been operative. Adequate levels of ventilation were given in the majority of the hamsters in the S/MV group as indicated by minimal negative deflection of the Pes during the rest period. For both S/SB and S/MV, muscle fibre damage and inflammation may have decreased the number of functional muscle fibres and thus, decreased the force generating ability of the diaphragm and increased the load on the remaining muscle fibre pool. In both groups, factors contributing to decreased energy stores may have been the prolonged fasting, the circadian rhythm of glycogen stores and increased levels of epinephrine. Decreased energy supply may have occurred from a decreased blood pressure - 198 -from the cumulative e f fects of i n t r a p e r i t o n e a l urethane, vasospasm of the diaphragmatic vascu la ture , and p o s i t i v e pressure mechanical v e n t i l a t i o n ( in S/MV o n l y ) , and hypoxemia. a. Factors increas ing energy demands Assuming that flow i s laminar , the res i s tance of the tracheostomy tube can be estimated based on the fo l lowing equation: R = 8 1 u irr* (38) where 1 i s the length of the tube, u i s the v i s c o s i t y of a i r and r i s the radius of the tube. Using t h i s equat ion, the c a l c u l a t e d res i s tance of the tracheostomy tube was approximately equivalent to the res i s tance of the lower r e s p i r a t o r y t r a c t of hamsters as measured by Niewoehner et a l (49). In man, res i s tance of the lower r e s p i r a t o r y t r a c t i s approximately equal to the res i s tance of the upper r e s p i r a t o r y t r a c t . Assuming a s i m i l a r r e l a t i o n s h i p ex i s t s in the hamster, the res i s tance of the tracheostomy tube may have been equivalent to the res i s tance of the upper r e s p i r a t o r y t r a c t i t bypassed and hence, the tube d id not impose any a d d i t i o n a l loads above those normally found during spontaneous breathing in the hamster. The s i l i c o n e dome encasing the p la te e lectrodes and the p o s i t i o n i n g of the small water bag and corset over the lower trunk of the hamster may have provided extra impedance for the diaphragm to contract against . Although the s i l i c o n e dome, water bag, and corset may have increased diaphragmatic l o a d , the increase in Pab for the major i ty of the experiment was minimal . In f a c t , even i f there was an increased l o a d , t h i s could have operated in a p o s i t i v e manner by increas ing the area of diaphragmatic appos i t i on , improving the l ength- tens ion r e l a t i o n s h i p , decreasing the radius - 199 -of diaphragmatic curvature , enabl ing a more i sometric con trac t ion and thus, improving diaphragmatic force output. Examination of the Pes and Pga t rac ings during spontaneous breathing at the very beginning of the experiment (when no apparatus was present) and during the sham fat igue per iod (when a l l apparatus was present) demonstrated an increase in Pdi of between 5-20% and increased frequency of breathing in some animals a f ter the apparatus was in p lace . Although there was an increase in Pdi generated by some animals, there was no information regarding diaphragmatic c o n t r a c t i l i t y (Pdi/EMGdi) recorded. I t i s d i f f i c u l t to assess whether or not the s i l i c o n e dome, water bag, and corset was a s i g n i f i c a n t load to the diaphragm c o n t r i b u t i n g to i t s progress ive fa t igue because the experiment was not s p e c i f i c a l l y designed to examine t h i s quest ion , b. Muscle f i b r e damage The degree of muscle f i b r e damage in the two sham fat igue and res t groups (S/SB and S/MV) was small r e l a t i v e to the fat igue and sham fat igue only groups ( F i g . 54). However, s i m i l a r to the b iops ies of the two fat igue and re s t groups, the ac tua l t i s sue damage of the diaphragm in the sham fat igue and res t groups may have been underestimated because of l i m i t a t i o n s of the h i s t o l o g i c a l techniques used to detect severe and minor c e l l damage, and the small sample s i ze (as discussed e a r l i e r ) . Damage of the diaphragm may have ar i sen in the two sham fat igue and re s t groups from the i n i t i a l i n s e r t i o n of the dome, r e p e t i t i v e rubbing of the dome against the diaphragm during mechanical v e n t i l a t i o n or spontaneous breath ing , or from the e l e c t r i c a l s t imula t ion performed for measurements of P d i . The inflammatory response was marked in the b iops ies of the sham fat igue and res t groups ( F i g . 56). I t was greater than that observed in - 200 -diaphragmatic cross - sec t ions of the sham fat igue only group and s i m i l a r to inflammation observed in the b iops ies of the fa t igue and re s t groups (F/SB and F/MV) . S i m i l a r to the fat igue and re s t groups, t i s sue damage was the most l i k e l y fac tor i n c i t i n g an inflammatory response although antidromic s t imula t ion of sensory f i b r e s of the phrenic nerve and the presence of an in fec t ious agent were other p o t e n t i a l f a c t o r s . A decrease in funct ion of the hamster diaphragm was r e f l e c t e d by a progessive drop in Pdi throughout the re s t p e r i o d . Regardless of the poss ib le causes of c e l l damage and the inflammatory response, both of these fac tors l i k e l y contr ibuted to a decrease in funct ion of the diaphragmatic muscle f i b r e s . c. Factors decreasing energy stores The progress ive d e t e r i o r a t i o n in diaphragmatic strength which was apparent in both sham fat igue and re s t groups could have been p a r t i a l l y a t t r i b u t e d to by glycogen d e p l e t i o n . Glycogen stores must have been markedly depleted in these animals because of the length of f a s t i n g , the c ircadium rhythm of glycogen s tores , and increased sympathetic a c t i v i t y due to s u r g i c a l s t r e s s . By the time b iops ies were obtained, the hamsters had fasted 18 to 24 hours. Fas t ing was necessary i n order to empty the stomach. An empty stomach was e s s e n t i a l for the measurement of Pdi by use of water-f i l l e d catheters pos i t ioned down the esophagus - one in the lower esophagus and the other in the stomach. I f the animal was not fasted overnight , the catheters q u i c k l y became clogged making measurements of Pdi imposs ible . Because hamsters s leep during the day and are most p h y s i c a l l y ac t ive at n i g h t , i t i s poss ib le that the f a s t i n g per iod for t h i s study was longer than the ac tua l time of food removal. I t p o s s i b l y inc luded the previous - 201 -day as w e l l . Fas t ing for 36 to 44 hours has been demonstrated to reduce hepat ic glycogen to 10 to 15% of normal basal concentrat ions in man (6). We may have produced comparable hepatic glycogen deple t ion in the hamster by simply f a s t i n g the animal overnight . Glycogen l e v e l s i n rats have been demonstrated to f luc tua te during a 24-hour p e r i o d . Glycogen l e v e l s peak i n the diaphragm at 0400 hours and may be approximately 50% of those values during the e a r l y afternoon hours when the animals are maintained on a 12-hour day/night cyc le with the day beginning at 0600 hours (18,25). I f the c i r c a d i a n rhythm of glycogen l e v e l s i n rats i s s i m i l a r to hamsters, the point at which the diaphragmatic muscle b iops ies were obtained in most animals was w e l l into the time of day when glycogen l e v e l s were below 50% of maximal va lues . Although sympathetic a c t i v i t y has been reported to decrease in response to s t a r v a t i o n , i t increases in response to s u r g i c a l s tress (68). This can r e s u l t in an increase in basa l metabolic rate and m o b i l i z a t i o n of glycogen stores and free f a t t y a c i d s . Epinephrine can act d i r e c t l y on s k e l e t a l muscle convert ing s k e l e t a l muscle glycogen to l a c t i c a c i d which is transported to the l i v e r and converted to new glucose (68). I t i s h ighly l i k e l y that sympathetic a c t i v i t y was s t imulated during the experiments o u t l i n e d in t h i s chapter s ince surgery was extensive and prolonged (between 1.5 and 3.0 hours) . The extent to which t h i s mechanism was operat ive in dep le t ing glycogen stores already af fec ted by f a s t i n g and normal c i r c a d i a n rhythm is d i f f i c u l t to p r e d i c t but c e r t a i n l y increased sympathetic a c t i v i t y from s u r g i c a l s tress would not have had a p o s i t i v e e f f ec t on glycogen stores i n the hamster diaphragm. - 202 -We d i d not detect any d i f ferences in glycogen stores between the S/SB and S/MV groups, and between these two groups and the fa t igue and res t groups even though glycogen deple t ion was demonstrated by the fat igue stimulus ( F i g . 59-61). Examination of the frequency d i s t r i b u t i o n s d id demonstrate a s h i f t in a l l four fat igue/sham fat igue and re s t groups to lower values of s t a i n i n g i n t e n s i t y in the type I f i b r e s . In add i t ion the d i s t r i b u t i o n of type I l a f i b r e s appeared to become more bimodal in nature suggesting that type I l a f i b r e s are composed of 2 d i f f e r e n t populations with d i f f e r e n t recruitment pat terns . The small sample s i z e , and many uncontro l l ed fac tors l i k e l y contr ibuted to the i n a b i l i t y to detect any fur ther dep le t ion during the res t p e r i o d . Factors decreasing energy suppl ies Low blood pressure may have contr ibuted to a decreased energy supply to the diaphragm. Hypotension was the primary cause of m o r t a l i t y and exc lus ion of p h y s i o l o g i c a l data . Although the p h y s i o l o g i c a l data analyzed was only from hamsters who met the c r i t e r i a o u t l i n e d in the methodology, t h e i r blood pressure i n e v i t a b l y de ter iora ted throughout the course of the experiment neces s i ta t ing in fus ion of 5% human serum albumin for i t s maintenance. Progress ive d e t e r i o r a t i o n in blood pressure may have been due to the cumulative e f fec t s of urethane and the e f fec t s of p o s i t i v e pressure v e n t i l a t i o n . Urethane was used in r e l a t i v e l y small amounts i n combination with other agents to produce anesthes ia , however, l a r g e r proport ions were given l a t e r in the course of the experiment. Urethane alone has been demonstrated to cause increased c a p i l l a r y permeabi l i ty (37), d i l a t a t i o n of a r t e r i o l e s (39), increased p e r i t o n e a l f l u i d accumulation (57,63), decreased cardiac - 203 -output (58) and blood pressure (39) in other animals. I n t e r e s t i n g l y , the e f fec t s of urethane on p e r i t o n e a l f l u i d accumulation was decreased by i n t r a -a r t e r i a l (63) or intravenous (57) rather than i n t r a p e r i t o n e a l i n j e c t i o n . I f urethane has a s i m i l a r ac t ion in hamsters as described i n other animals, the use of i n t r a p e r i t o n e a l adminis trat ion of cumulative doses of urethane i n t h i s study may have contr ibuted to increased vascu lar permeab i l i t y , p e r i t o n e a l f l u i d accumulation, and d e t e r i o r a t i o n of blood pressure . Continuous p o s i t i v e pressure v e n t i l a t i o n decreases venous re turn in man (11,40) and t h i s mechanism may have contr ibuted to a decreased cardiac output and hypotension in the hamsters of F/MV and S/MV. During continuous p o s i t i v e pressure v e n t i l a t i o n , the d r i v i n g pressure for venous re turn may be decreased by an increased r i g h t a t r i a l i n t r a c a v i t a r y pressure during lung expansion. I f the mean systemic pressure does not increase s u f f i c i e n t l y to compensate, the pressure d i f f erence d r i v i n g venous re turn w i l l decrease (40). I t i s h igh ly l i k e l y that other fac tors such as the cumulative e f fec t s of urethane af fected the a b i l i t y of the systemic vasculature to compensate in t h i s study, such that the loads imposed by the continuous p o s i t i v e pressure v e n t i l a t i o n could have contr ibuted to a decrease i n card iac output and hypotension. Although blood pressure , was monitored i t was not evaluated in a systematic way and hence no s t a t i s t i c a l ana lys i s could be performed in order to determine whether or not blood pressure was lower i n the two mechanical ly v e n t i l a t e d groups compared to the two groups who performed spontaneous breathing during the r e s t p e r i o d . Besides low blood pressure , decreased energy suppl ies to the diaphragm may have ar i s en from vasospasm of the diaphragmatic vasculature and hypoxemia. - 204 -P o s i t i o n i n g of the s i l i c o n e dome against the hamster diaphragm may have induced vasospasm of the vascu la ture . This in turn would have resu l t ed in decreased substrate flow to the diaphragm and contr ibuted to f a t i g u e . There was a trend towards lower values of PaO^ in the animals of S/SB. Jardim et a l (31) has demonstrated that breathing a hypoxic mixture during i n s p i r a t o r y r e s i s t i v e loading r e s u l t e d in a shorter endurance time to f a t i g u e . The animals in the S/SB d i d not demonstrate any CO^ r e t e n t i o n , however, they had a s i g n i f i c a n t l y lower PaCO^ and FH +] compared to the a r t e r i a l blood measures of the animals in the two spontaneously breathing groups (S/SB and F / S B ) . Although i t i s doubtful that the r e s p i r a t o r y a l k a l o s i s induced in the animals of S/MV had any e f f ec t on the force output of the hamster diaphragm, the lower PaO^ shown in the animals of S/SB may have contr ibuted to the d e t e r i o r a t i o n of Pdi in these animals. Because of the long durat ion of the experiment, the necess i ty to fas t the animal , and the apparatus involved for Pdi measurements, many of the above fac tors could have placed greater demands or decreased energy suppl ies to the diaphragm of the sham fat igue and re s t hamsters. Loads caused by the tracheostomy tube, s i l i c o n e dome and c o r s e t , and hypoxemia may have been more of a f a c t o r in the S/SB group whereas p o s i t i v e pressure v e n t i l a t i o n l i k e l y af fected the S/MV group to a much greater degree. The e f fec t s of c e l l damage, inflammation, urethane and changes i n glycogen l e v e l s (due to f a s t i n g , c i r c a d i a n rhythm and increased epinephrine) l i k e l y af fected both groups to a s i m i l a r degree. Our methodology does not allow q u a n t i t a t i o n of the c o n t r i b u t i o n of these fac tors and one can only speculate on t h e i r r e l a t i v e importance. C e r t a i n l y s evera l p o t e n t i a l f ac tors were o u t l i n e d which could have contr ibuted to the progress ive d e t e r i o r a t i o n of Pdi in the sham fat igue and re s t groups (S/SB and S/MV). - 205 -A l l of the fac tors discussed which may have contr ibuted to increased energy demands and decreased energy stores in the diaphragm of the S/SB and S/MV groups could have operated in the F/SB and F/MV groups. However, other fac tors induced by the fa t igue stimulus may have negated or a l t e r e d the e f fec t s of some of these f a c t o r s . Exhaustive exerc ise i s a strong stimulus to hormonal s ecre t ion and glycogen r e p l e t i o n (7) . The e f fec t s of various hormones may have had a p o s i t i v e e f fec t on Pdi production in the fa t igue groups. However, the changing l e v e l s of hormones preand pos t - fa t igue is a very complex issue not w e l l understood, and speculat ion of poss ib le mechanisms of ac t ion is d i f f i c u l t . Perhaps the recovery of Pdi in animals of F/MV was due to glycogen r e p l e t i o n which was not detected by our methods and sampling t imes. This i s doubt fu l , however, because of the many other fac tors which l i k e l y contr ibuted to glycogen d e p l e t i o n . L a s t l y , there was a drop-out of animals i n the l a t e r times of the experimental des ign. The recovery of Pdi in animals of F/MV may represent only the strong animals that were able to survive the experimental pro toco l ra ther than r e f l e c t the benef i t of mechanical v e n t i l a t i o n versus spontaneous breath ing . - 206 -EXPERIMENTAL DESIGN MEASUREMENTS PREP. A MTERVFi^iONft Pre-fatigue Pdi Post-fatigue Pdi Biopsies obtained —* from FATIGUE ONLY & S H A M ONLY Post-fatigue Pdi repeated hourly \ In addition: ABQ & B P monitored Muscle b i o p s y - * obtained at end of experiment r o Anesthetized Surgical Preparation - 1 -.EITHER: J Fatigue OR Sham stimulus REST BY EITHER: Spontaneous Breathing 8 ( S / S B & F / S B ) OR Mechanical ventilation ( S / M V & F / M V ) TME (Hours) Figure 43 Experimental design of fatigue/sham fatigue only and fatigue/sham fatigue and rest experiments. The central v e r t i c a l axis indicates the time scale in hours. Animal preparations (prep) and interventions are indicated in the right hand column and the measurements and monitoring performed are outlined in the l e f t column. - 207 -Plate X Figure 44 Pga and Pes trac ings at the beginning and at the end of the fa t igue stimulus of r e p e t i t i v e e l e c t r i c a l s t imula t ion appl ied to the hamster diaphragm. The upper Pga ( l e f t ) and Pes ( r igh t ) t rac ings (pre - fa t igue) were recorded during the f i r s t contract ions produced by the f a t i g u i n g e l e c t r i c a l s t i m u l a t i o n . The lower trac ings (post - fa t igue) were recorded during the l a s t contract ions produced by the f a t i g u i n g e l e c t r i c a l s t i m u l a t i o n . Pdi was c a l c u l a t e d from Pes and Pga as shown in the table below the pressure t r a c i n g s . zoe '9 a P r e -F a t i g u e 10 c m r H2O L Ii ! i ! i ! i e s Tr I i j I! 1 i TT; : f i Ii! 11 i i r n j!H ' ' '• i iMI i:: !• .1 ill i i . i H i 4 i 1 ' i | 'Ii! iiii ] I j i ill. jijl V i : jlli [iii ; j Iiii j it i -I -'j JH! i 1 i ii 1; ' i i i i • ; • i 11 hf i i i i : i i: i i II! mi !it| '' i! i; i i ! !' | ill! Sili .: i; i ! t I 11: i iiii • ! • i i j j 11 i ill! J I : { II' I1!! iiii iiii iii i . i ilJ. U i 111! j i • i i i; i i ' lijl iji; T l T f : : 11 *' i • i i: i; i ; i ; < t M M • ; i ; iii: ill! I i i i . i>i * • 11 i i: i P o s t -F a t i g u e l O c m f H 2 o [ m .Hi! Ijli iiii i-iii ill; iii iiii ii'! ilil ll'i iiii ijli iiii iiii i d iii !• i 1 • I i :Ti I; iiii iiii :i i j- •<- jiji . ; • ! • ! Iiii iiii iiii <* n ii +11:: i i i i : I iiii M i : j ; •'; ; i liiii * 'iii m r ii — f : : t-* | i I!.! iiii -i iiii P ilil iiii Iiii iiii iiii iii ' ' M iiii lji! iii I i I! !; i ; i i • • j mi i i i ifc ii L I : i I ; t i h y !! 1 i i«f 1! ( i iiii iiii m m 'jii i TT TT ii Iiii i; i; i • • i iiii WW ill; i!:; iiii i i Ii !ili iiii !!: i i i i ' iiii iiii i'ii iiii 1 ii I il iiii ! • 11 Iii! iiii 1 • ' • 1 I |! ii : ! j * iiii i:!: ! ; ! ! I s I s P g a ~ p e s = p d i P r e . P o s t . 2 7 . 5 - 1 0 3 7 . 5 15 .5 - 6 2 1 . 5 - 209 -P late XI Figure 45 P i c t o r i a l grading scheme used to grade inflammatory response. Grade 0 (panel A) represented normal hamster diaphragmatic muscle with l ess than three neutrophi l s observed. A grade 1 (panel B) was assigned to a f i e l d i f there were neutrophi l s observed p r i m a r i l y in the blood vesse l s . A grade 2 (panel C) was given i f an i n f l u x of neutrophi l s into the muscle t i s sue p r i m a r i l y located wi th in the t h i c k e r connective t i s sue layers (epimysium and perimysium) was observed. A grade 3 (panel D) represented the most marked inflammatory response with the presence of neutrophi l s surrounding severa l muscle f i b r e s and/or i n f i l t r a t i n g in to the c e l l s . A l l t i s sue cross - sec t ions of the hamster diaphragm were s ta ined with H & E and the magni f i cat ion is the same for a l l photomicrographs. C a l i b r a t i o n bar = 100 uM. 2 I O - 211 -P late XII Figure 46 Photomicrographs of damaged f i b r e s in the hamster diaphragm i l l u s t r a t e d by cross - sec t ions s ta ined with H & E (upper pane l ) , NADH-TR (middle pane l ) , and M-ATPase (lower pane l ) . The magni f i cat ion i s the same for a l l photomicrographs. C a l i b r a t i o n bar = 25 uM. - 213 -60 r c E 40 30 20 10 • x • x * • x 0 0 o x> *° ox x° 10 20 30 40 Dynamic Pdi (cmH20) o F/MV X F/SB j • Fatigue only 60 Figure 47: Re la t ionsh ip between the i n i t i a l dynamic Pdi from the f a t i g u i n g e l e c t r i c a l s t imula t ion and the endurance time of the f a t i g u i n g stimulus (necessary to produce a 50% decrease in the dynamic P d i ) . The data for each animal i s shown in for the 3 fa t igue groups: fat igue on ly , F / S B , and F / M V . - 214 -FATIGUE ONLY i i 1 . i F/SB I I I 1 I I I 1 F/MV I I 1 I T • Post-fatigue • ± 1 • Pre-fatigue 1 i i i Twitch 30 Hz 50 Hz 70 Hz FREQUENCY OF STIMULATION Figure 48 Pdi (means + SD) before and a f t e r the fa t igue st imulus in the three fa t igue groups: fa t igue only (upper p a n e l ) , F/SB (middle panel) and F/MV (lower panel) - 215 TWITCH 30 Hz 50 Hz 70 Hz POSI-fAJIOUE (HOUR 2 HOUR SHOOK 4 HOUR POSt-fAHOUE 1 MOJR 2 HOUR 9 HOUR 4 HOUR REST REST GROUP REST POST-FATIGUE 1 HOUR 2 HOUR 3 HOUR 4 HOUR S/SB 9 9 8 7 5 S/MV 9 9 9 8 5 F/SB 9 9 8 6 4 F/MV 8 8 6 4 3 4 9 Change i n Pdi for twitch and t e t a n i c s t imula t ion fo l lowing fa t igue and during four hours of r e s t for the four fatigue/sham fat igue and re s t groups. The mean value+SD i s shown. N for each time i n t e r v a l i s shown table below the p l o t s . - 216 -100 r f 80r S/SB <T <or a. TIME (hra) 80 • 80 -S / M V 20 r * X TIME (hrs) F / S B TIME (hra) 100 F/MV TIME (hra) Figure 50 P a r t i a l pressure of a r t e r i a l carbon d iox ide dur ing the experiment for the four fat igue/sham fa t igue and re s t groups. - 217 -S/SB » » 0» 2 4 a TIME (hrs) tso tX3 S O eo 40 0 a o S/MV • » TIME (hrs) F/SB TIME (hrs) tjo -W O • ao -ao -40 to - 1 o a o . F/MV TIME (hrs) Figure 51 P a r t i a l pressure of a r t e r i a l oxygen during the experiment for the four fatigue/sham fat igue and r e s t groups. - 218 -i Figure 52 Grade of inflamination and number of damaged c e l l s as indicated by H & E, NADH-TR, and M-ATPase stained tissue cross-sections in the fatigue and sham fatigue only groups. The mean score of seven animals for the grade of inflammation and number of damaged c e l l s i s shown. - 219 -PLATE XIII Figure 53 Photomicrograph of damaged f ibres along the abdominal surface of the hamster diaphragm. Fibres were s ta ined with M-ATPase at a pre incubat ion of pH 4.5 . Upper panel -c a l i b r a t i o n bar= 50 uM. Lower panel - c a l i b r a t i o n bar= 25 uM. - 221 -30 r 0 2 4 6 8 T I M E ( H O U R S ) Figure 54 Number of damaged cells over time in the fatigue/sham fatigue and rest groups. Note: Points at zero were values of Fatigue only for F/SB (••*-•) and F/MV (—'—) lines and Sham fatigue only for S/MV (—±—) and S/SB (-e-) lines. - 222 -PLATE XIV Figure 55 Photomicrographs of wisps of cytoplasm (upper p a n e l ) , empty spaces (middle panel) and e o s i n o p h i l i c f l o c c u l e n t masses (lower panel) observed in muscle f i b r e cross - sec t ions of the hamster diaphragm sta ined with H & E . Upper pane l - c a l i b r a t i o n bars = 10 uM, middle panel - c a l i b r a t i o n bars = 25 um, lower panel - c a l i b r a t i o n bars = 50 uM. 2 * 3 55 - 224 -Figure 56 Inflammatory response over time in the fat igue/sham fat igue and res t groups. Note: Points at zero were values of Fatigue only for F/SB (-•*••) and F/MV (——) l i n e s and Sham fat igue only for S/MV (—+—) and S/SB (--S-7 l i n e s . - 225 -PLATE XV Figure 57 Photomicrographs of neutrophi l s invading connective t i s sue layers (upper pane l ) , surrounding c e l l s (middle panel) and i n f i l t r a t i n g c e l l s (lower panel) of the hamster diaphragm stained with H & E . Upper panel -c a l i b r a t i o n bar= 100 uM. Middle and lower panel -c a l i b r a t i o n bar= 25 uM. - 227 -Plate XVI Figure 58 Frequency d i s t r i b u t i o n of o p t i c a l dens i ty for PAS sta ined f i b r e s for type I f ibres (upper pane l ) , type I l a f i b r e s (middle pane l ) , and type l i b f i b r e s (lower panel) in fat igue and sham fat igue only groups. 30-25 -£ 20-_Q O 15-c a> o i_ a> 10-5-0-30-25-20-Type I in fl) c a> o i_ a> C L o 15-10-0-30-25-vt O 15-1 0.25 0.50 0.75 1.25 Type Ila 0 C L 10-0.25 0.50 0.75 1.25 type lib 4-• I Fatigue d Sham Fatigue J 0 0.25 0.50 0.75 1 Optical Density (OD units) -r-.25 - 229 -P la te XVII F igure 59 Frequency d i s t r i b u t i o n of o p t i c a l dens i ty for PAS s ta ined type I f i b r e s in a l l 6 experimental groups. The o p t i c a l dens i ty for the sham fat igue and fat igue only groups have been corrected by an upward uniform s h i f t of O.052 o p t i c a l dens i ty u n i t s . Q 40 35-30-Opticd Density (OD units) Fatigue 0.2 0.4 0.6 0.8 1 Corrected Optical Density (OD units) 0.2 0.4 0.6 0.8 1 Optical Density (OD units) - 231 -P late XVIII Figure 60 Frequency d i s t r i b u t i o n of o p t i c a l densi ty for PAS sta ined type I l a f i b r e s in a l l 6 experimental groups. The o p t i c a l dens i ty for the sham fat igue and fat igue only groups have been corrected by an upward uniform s h i f t of 0.052 o p t i c a l dens i ty u n i t s . to 0 0.2 0.4 0.6 0.8 1 Optical Density (OD units) - 233 -P late XIX Figure 61 Frequency d i s t r i b u t i o n of o p t i c a l dens i ty for PAS sta ined type l i b f ibres in 6 experimental groups. The o p t i c a l dens i ty for the sham fat igue and fat igue only groups have been corrected by an upward uniform s h i f t of 0.052 o p t i c a l dens i ty u n i t s . 35-30-O 20- S h a m £ 15-10-•llllBaiI.mil.. -• 0.2 0.4 0.6 0.8 1 Corrected Optical Density (OD units) 40-35-30-O 20-E2 S/SB Optical Density (0D units) £2 l / S B 0.2 0.4 0.6 Optical Density (OD units) - 235 -TYPE I Sham fatigue S/SB S/MV TYPE I l a Sham fatigue S/SB S/MV 0.0001 _ J •4.0 ID-0.001 I -3 .0 0.01 _ _ l •2.0 •1.0 O Z _ > C O 0,0 2JL 1X> log^ 0.0001 —I 0.001 - J 0.01 — J -4.0 •3.0 -2 .0 -1.0 0.0 1.0 log« TYPE l i b Sham fatigue L Z 3 S/SB S/MV 0.0001 I 0.001 - J 0.01 1 0 -4.0 - 3 .0 -2 .0 -1.0 OPTICAL DINSITY 0,0 1.0 log, Figure 62 Box plots of o p t i c a l density for PAS stained fi b r e s for each f i b r e type and three sham fatigue group: sham fatigue only, S/SB and S/MV. Optical density has been corrected for the sham fatigue group by an upward uniform s h i f t of l o g e -0.19 o p t i c a l density units. The midline of the box i s the median, the l e f t border i s the lower q u a r t i l e , the right border i s the upper q u a r t i l e , the l e f t end point of the l i n e i s the minimum and the ri g h t end point of the l i n e i s the maximum. - 236 -TYPE I Fatigue only F/SB F/MV O.OOOl _ J 4.0 0 .001 -3.0 0.01 _J ill. -2.0 •1.0 0.0 1.0 l o g a TYPE I l a Fatigue only F/SB F/MV 0 . 0 0 0 1 -4.0 TYPE l i b Fatigue only F/SB F/MV 0 .0001 _ J 0 .001 _ J 0.01 _ l — ill -3.0 -2.0 -1.0 T r 0.001 — J 0.01 —J ill 4.0 -3.0 -2.0 -1.0 O P T I C A L DENSITY 0.0 0.0 1 0 1.0 l og . 1.0 log. Figure 63 Box p lo t s of o p t i c a l dens i ty for PAS stained f i b r e s for each f i b r e type and three fat igue group: fa t igue o n l y , F/SB and F/MV. 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Mortimer J T , Peckham PH: Intramuscular e l e c t r i c a l s t i m u l a t i o n . In: Neural Organizat ion and i t s Relevance to P r o s t h e t i c s , F i e l d s WF (ed). I n t e r c o n t i n e n t a l Medical Books, New York, 1973, pp 131-146 48. Newham DJ , M i l l s KR, McPhail G, Edwards RHT: Muscle damage in response to exerc i s e . Eur J C l i n Invest 12:29- ,1982 49. Niewoehner DE, Kleinerman J : E f f e c t s of experimental emphysema and b r o n c h i o l i t i s on lung mechanics and morphometry. J Appl P h y s i o l 35:25-31, 1973 50. Pardy RL, Bye A J : The e f fec t s of hyperoxia on r e s p i r a t o r y muscle f u n c t i o n . Am Rev Respir Pis 130:27-40, 1985 51. Paterson DS, Twose TM, McCormick ME, Cunni A E , Foster S J : Polymorphomuclear leukocyte accumulation in inflammatory dermal s i t e s measured by a novel n e u t r o p h i l s p e c i f i c marker p r o t e i n . Agents  A c t i o n 16 (1/2) ,1985. 52. Rabinovi tch B, Pardy RL, Hussain SNA, Macklem PT: The acute e f fec t s of r e s t on v e n t i l a t r o y muscles in pat ients with severe chronic a i r f l o w l i m i t a t i o n ( A b s t r a c t ) . The P h y s i o l o g i s t 26:A21, 1983 53. Rochester DF, Mart in L L : Resp iratory muscle r e s t . In: The Thorax, Part B, V o l 29, Chapter 43 of Lung Biology i n Hea l th . Lenfant C. (ed) Marcel Dekker Inc , New York, 1985, pp 1303-1328 54. Roussos Ch, Moxham J : Respiratory muscle f a t i g u e . In: The Thorax, Part B, V o l 29, Chapter 27 of Lung Bio logy i n Health and Disease. Lenfant C (ed) Marcel Dekker Inc , New York, 1985, pp 829-870 - 241 -•55. Roussos Ch, Macklem PT: Insp ira tory muscle fa t igue . In: Handbook  of Phys io logy. Sect ion 3: The Respiratory System, V o l I I I , Chap 29. Wil l iams & W i l k i n s , Bal t imore , MD, 1986, pp 511-527. 56. Schnader JY, Juan G, Howell S, F i t z g e r a l d R, Roussos Ch: A r t e r i a l CO2 p a r t i a l pressure a f fec ts diaphragmatic f u n c t i o n . J Appl P h y s i o l 58:823-829, 1985. 57. Severs WB, K e i l L C , Klase PA, Deen KC: Urethane anesthesia in r a t s . A l t e r e d a b i l i t y to regulate hydrat ion . Pharmacology 22:209-226, 1981 58. Seyde WC, McGowan L , Lund N, Dul ing B, Longnecker D: E f f e c t s of anesthet ics on reg iona l hemodynamics in normovolemic and hemorrhaged r a t s . Am J P h y s i o l 249:H164-H173, 1985 59. Shah A, Nagao F , Sahgal V , Singh H: E f f e c t of nerve s t imula t ion on ra t s k e l e t a l muscle. A study of plasma membrane. E x p e r i e n t i a 41:1396-1398, 1985. 60. Sherman WM, C o s t i l l DL: The marathon: d i e t a r y manipulat ion to optimize performance. Am' J Spor Med 12:44-51, 1984 61. Swash M, Schwartz MS: Biopsy Pathology of Muscle . Chapman & H a l l , London, 1985, p 19. 62. Trump BF, Laiho KA, Mergner WJ, A r s t i l a AU: Studies on the s u b c e l l u l a r pathophysiology of acute l e t h a l c e l l i n j u r y . B e i t r  Pathol 152:243-271, 1974 63. van der Meer C, Vers luys JAM, Tuynman HARE, Buur VAJ: The e f fec t s of ethylurethane on hematocrit , blood pressure and plasma glucose. Arch  in t Pharmacodyn Ther 217:2570275, 1975 64. Vihko V , Rantamaki J , Saliminen A: Exhaustive p h y s i c a l exerc ise and ac id hydrolase a c t i v i t y in s k e l e t a l muscle. Histochem 57:237-249, 1978 65. V o l l e s t a d NK, Vaage 0, Hermansen L : Muscle glycogen deple t ion patterns in Type I and subgroups of type f i b r e s during prolonged severe exerc i se in man. Acta P h y s i o l Scand 122:433-441, 1984 66. Walters GF, Brucher J , Tass in S, and Bergmans J : Experimental changes in muscle mitochondria induced by e l e c t r i c a l s t imula t ion and i n h i b i t i o n of energy metabolism. In: Mitochondria and Muscular  Diseases . Busch HFM, Jennekens FGI , Scholte HR (Eds) Mefar b . v . Beetsterzwaag, The Netherlands, 1981. 67. Wang H and Katz RL: E f f e c t s of changes in coronary blood pH on the heart . C i r c Res 17:114-122, 1965. 68. Wilmore DW, A u l i c k LH, Becker RA: Hormones and the c o n t r o l of metabolism. In: S u r g i c a l N u t r i t i o n . F i s cher JE (ed). L i t t l e , Brown, and C o . , Boston, 1983. Chapter 3, pp 65-95 - 242 -V I . CONCLUDING REMARKS AND FUTURE DIRECTIONS I developed an animal model in the hamster in order to examine the e f fec t s of fat igue and re s t on the diaphragm. This included the development of techniques for both p h y s i o l o g i c a l and h i s t o l o g i c a l examination of the diaphragm. The most appropriate anesthet ic regimen was found to be a combination of sodium p e n t o b a r b i t a l , c h l o r a l o s e , and urethane which allowed a l o n g - d u r a t i o n , even anesthesia and minimal r e s p i r a t o r y depress ion . The most appropriate l e v e l of mechanical v e n t i l a t i o n for t i d a l breathing and to induce apnea in the hamster was a lso determined in these s tud ie s . To ensure t o t a l diaphragmatic res t fo l lowing f a t i g u e , the l e v e l of v e n t i l a t i o n s u f f i c i e n t to abo l i sh diaphragm contrac t ion was determined. I t was found that when mechanical v e n t i l a t i o n was s u f f i c i e n t to produce an absent diaphragmatic EMG, there were no negative de f l ec t ions of esophageal pressure . Therefore , the negative d e f l e c t i o n s of esophageal pressure were used as a gu ide l ine for adjustment of mechanical v e n t i l a t i o n to achieve t o t a l r e s t in a l l subsequent s tudies . A new method to st imulate the hamster diaphragm was developed by apposing p la te e lectrodes encased in a s i l i c o n e dome d i r e c t l y against t h i s muscle. Diaphragmatic muscle e x c i t a t i o n was shown to be v i a the nerve using t h i s method of diaphragmatic s t i m u l a t i o n . This was shown by comparison to phrenic nerve s t i m u l a t i o n . Compared to phrenic nerve s t imula t ion there was: a s i m i l a r amplitude and s l i g h t l y shorter la tency of the ac t ion p o t e n t i a l , a s i m i l a r decrease in the amplitude of the ac t ion p o t e n t i a l in response to c u r a r e , and a comparable force output (Pdi) at maximal c u r r e n t . During - 243 -t h i s ser ies of experiments, the external abdominal pressure which produced maximal Pdi was determined to be 15 cmH^O. In a d d i t i o n , the DPS technique gave reproducib le transdiaphragmatic pressures over a 2-hour p e r i o d . The in t er -an imal and i n t e r - r e g i o n a l v a r i a b i l i t i e s of the proport ions of f i b r e types (M-ATPase), s i zes of f i b r e types (M-ATPase), ox idat ive capaci ty (NADH-TR) and glycogen l e v e l s (PAS) in the hamster diaphragm were determined. There was very l i t t l e v a r i a b i l i t y between animals in the proport ions and s izes of the muscle f i b r e types of the hamster diaphragm. Previous h i s t o l o g i c a l work on the hamster diaphragm (1,3) focused s o l e l y on evaluat ion of the c o s t a l region as being representat ive of the hamster diaphragm. There was disagreement about the proport ions of the three f i b r e types w i th in the hamster diaphragm even though s i m i l a r s t a i n i n g methods were used in the two s tud ie s . By c a r e f u l examination of r e g i o n a l d i f ferences in h i s t o l o g i c a l features of the hamster diaphragm I found three major areas: the abdominal surface of the c r u r a l r e g i o n , the thorac i c surface of the c r u r a l r e g i o n , and the s t e r n a l and c o s t a l diaphragm (see Table IX, p. 120 for d e t a i l s ) . The three major areas have very d i f f e r e n t proport ions and s izes of muscle f i b r e types and the three f i b r e types have very d i f f e r e n t p r o f i l e s of ox idat ive capac i ty and glycogen content in the three main areas . Since q u a l i t a t i v e c a t e g o r i z a t i o n of the i n t e n s i t y of NADH-TR and PAS s ta ined muscle f i b r e s of the hamster diaphragm was d i f f i c u l t , I chose to perform q u a n t i t a t i v e ana lys i s which revealed over lapping d i s t r i b u t i o n s of the three muscle f i b r e s . Because of the d i f f e r e n t techniques used, i t i s d i f f i c u l t to compare the NADH-TR data from t h i s study to previous work (1,3) however, my r e s u l t s would appear to compare more c l o s e l y to those by Kelsen et a l (3) and contrast sharply to - 244 -those reported by Farkas et a l (1) . This i s the f i r s t report of glycogen l e v e l s in the d i f f e r e n t f i b r e types and regions of the hamster diaphragm and i t i s i n t e r e s t i n g to note the reg iona l v a r i a b i l i t y of t h i s feature and the of ten complementary r e l a t i o n s h i p of glycogen l e v e l s to the ox ida t ive capaci ty in the muscle f i b r e s types and regions of the hamster diaphragm. The d i f f e r e n t h i s t o l o g i c a l features of the three main areas of the hamster diaphragm probably r e f l e c t t h e i r recruitment dur ing very d i f f e r e n t kinds of a c t i v i t i e s . I propose to inves t iga te th is hypothesis by examining the reg iona l response of the diaphragm to var ious overloads such as experimental emphysema, t rachea l banding, and exerc ise - induced hyperven t i l a t ion (from swimming). The l a t t e r two overloads w i l l be induced at both submaximal and exhaustive l e v e l s . The d i f f e r e n t regions of the diaphragm w i l l be evaluated fo r substrate (glycogen and l i p i d ) dep le t ion patterns in response to acute r e s p i r a t o r y muscle loads and muscle f i b r e hypertrophy in response to chronic over loads . These studies should provide u s e f u l information regarding the diaphragmatic muscle f i b r e recruitment pat tern during d i f f e r e n t r e s p i r a t o r y overloads which w i l l be h e l p f u l to determine the most appropr iate phys io -l o g i c a l overload fo r future studies to examine the e f f e c t s of res t on the fa t igued hamster diaphragm. Such informat ion should a lso be h e l p f u l in the design of the most appropriate exerc ise program fo r the r e s p i r a t o r y muscles of pa t ien ts with COPD. These studies w i l l provide suggest ion regarding whether or not whole body and/or s p e c i f i c r e s p i r a t o r y exerc ise st imulates the diaphragm to s u f f i c i e n t degree to provide a t r a i n i n g e f f e c t or r e s u l t in exhaustive over load , and secondly , which form of exerc ise provides the - 245 -most su i tab le reg iona l recruitment pat tern of the diaphragm fo r cond i t ion ing of the r e s p i r a t o r y muscles in these p a t i e n t s . Once the p h y s i o l o g i c a l and h i s t o l o g i c a l methods were developed, the long-dura t ion studies (5-7 hours) examining the e f f e c t s of res t on the fa t igued hamster diaphragm were performed. Both high and low frequency fa t igue of the diaphragm were produced by r e p e t i t i v e e l e c t r i c a l s t imula t ion of the hamster diaphragm. Type l i b f i b r e s were depleted of glycogen to the greatest degree which p o s s i b l y r e f l e c t s a greater recrui tment or greater dependence on glycogen metabolism of these f i b r e s dur ing e l e c t r i c a l s t i m u l a t i o n . Future work examining both glycogen and l i p i d dep le t ion pat terns in response to an acute exhaustive load may provide a bet ter representa t ion of the recruitment pat tern of the diaphragm. Recovery of the diaphragm in response to h igh- f requency s t imu la t ion was demonstrated in the animals of the F/MV group whereas recovery d id not occur in the diaphragm of animals in the F/SB group. Progress ive d e t e r i o r a t i o n of Pdi throughout the four -hour res t per iod was demonstrated in the animals of the S/SB and S/MV groups. Many f a c t o r s may have contr ibuted to the progress ive decrease in diaphragmatic func t ion in animals of the S/SB and S/MV groups in the four hours post-sham f a t i g u e , and complicated the i n t e r p r e t a t i o n of the r e s u l t s demonstrated in the F/SB and F/MV groups in the recovery p e r i o d . I conclude that the l e s s e r loads imposed on the diaphragm of animals in the F/MV group provided more e f f e c t i v e recovery than the exerc ise imposed on the diaphragm of animals in the F/SB group. However, the conc lus ion that passive r e s t was be t te r than ac t ive recovery must be accepted with caut ion because of the small sample s i ze and the many extraneous fac to rs which could have cont r ibuted to the - 2 4 6 -outcome of t h i s study. Recovery of the F/MV group at three hours of rest may have been r e l a t e d to the progress ive decrease in inflammation noted in t h i s group between two and f i v e hours of res t or these may have been two unre la ted events. This decrease in inflammation grade was a trend only and was not submitted to s t a t i s t i c a l a n a l y s i s . The p e r s i s t e n t low frequency fa t igue may be a t t r i b u t e d p a r t i a l l y to the muscle f i b r e damage. From th is experimental design and the subsequent l o s s of animals throughout the p r o t o c o l , i t i s indeed d i f f i c u l t to make any strong conclus ions regarding the r e s u l t s and mechanisms. Mod i f i ca t ions of the experimental design for future s tudies may provide a more s tab le preparat ion such that d e t e r i o r a t i o n of the animals in the sham fa t igue and r e s t groups i s minimized and the r e s u l t s of fa t igue and r e s t groups may al low a more l u c i d i n t e r p r e t a t i o n . I t would be i n t e r e s t i n g to inves t iga te the ac tua l load imposed on the diaphragm from the s i l i c o n e dome conta in ing the diaphragmatic p l a t e s , water bag and c o r s e t . I f the load imposed i s s i g n i f i c a n t , an a l t e r n a t i v e technique of diaphragmatic s t imula t ion i s v i a the phrenic nerves, however, the author s i n c e r e l y doubts the f e a s i b i l i t y of t h i s technique fo r long term studies because of the f r a g i l i t y of the p repara t ion . Blood pressure i s determined by the product of blood f low and p e r i p h e r a l vascu la r r e s i s t a n c e . There are many fac to rs which can a f f e c t these two parameters and thus contr ibuted to progress ive d e t e r i o r a t i o n of blood pressure in some of the hamsters in t h i s study. M o d i f i c a t i o n of the anesthet ic regime may prevent the development of hypotension seen in some hamsters during the prolonged time of anes thes ia . The n e c e s s i t y for minimal r e s p i r a t o r y depression l i m i t s the s e l e c t i o n of anesthet ics which may be - 247 -appropr ia te . However, previous work in the l i t e r a t u r e does suggest that i n t r a - a r t e r i a l (5) or intra-venous (4) rather than i n t r a - p e r i t o n e a l i n j e c t i o n of urethane decreases accumulation of per i tonea l f l u i d (which may contr ibute to the development of hypotension) . Another anesthet ic combination descr ibed as having minimal e f f e c t s on the r e s p i r a t o r y and card iovascu la r systems is f luan isone and fen tany l c i t r a t e (2) but t h i s has not been t r i e d in long term small animal experiments. The mean PaO^ of animals in the fat igue/sham fa t igue and res t groups was only s l i g h t l y lower than other reports of the PaO^ of spontaneously breathing animals. However, supplemental oxygen may produce a more stable preparat ion when one considers the other i n s u l t s imposed from t h i s experimental p r o t o c o l . Supplementation of 5% dextrose dur ing the f a s t i n g per iod and the course of the experiment may minimize the e f f e c t s of f a s l i n g . The f e a s i b i l i t y of mainta in ing animals on 5% dextrose overnight has been examined in a pre l iminary t r i a l by mainta in ing 7 animals on 5% dextrose and 7 animals on water overn ight . Those animals maintained on dextrose drank more f l u i d s and l o s t l e s s weight during the f a s t i n g p e r i o d . Hamsters are nocturnal animals which are most ac t ive and feed at n i g h t . The animals were maintained on a 12-hour day /n ight cyc le with l i g h t hours beginning at 0700. Changing of the day /n ight c y c l e such that experiments began towards the middle of t h e i r ac t ive per iod may prove to be l e s s s t r e s s f u l and a lso provide higher basa l l e v e l s of glycogen stores in the animals. Maintainance of animals on dextrose combined with bet ter t iming of the experiments with the c i r c a d i a n rhythm of glycogen stores are two f a c t o r s which may r e s u l t in the experimental in tervent ions c o i n c i d i n g - 248 -with the peaking of glycogen s t o r e s . This may enable a more c l e a r demonstration of the e f f e c t s of only the experimental in tervent ions uncomplicated by the e f f e c t s of f a s t i n g . Of i n t e r e s t , marked inflammation was found in the hamster diaphragm in response to diaphragmatic p la te e lect rode s t i m u l a t i o n . The greater inflammatory response in the diaphragm of the fa t igue only group compared to the sham fa t igue only groups ind ica ted that the fa t igue st imulus accentuated th is response. However, dur ing the res t p e r i o d , equa l ly severe inflammation was noted in the diaphragm of a l l four fa t igue and r e s t groups which may be a r e f l e c t i o n of the i n a b i l i t y to d iscern d i f f e r e n c e s because of the small sample s i ze or the ac t ion of f ac to r (s ) which a f fec ted the diaphragm of animals in d i f f e r e n t groups to a s i m i l a r degree. T issue damage was the most l i k e l y f a c t o r i n c i t i n g an inflammatory response although a n t i -dromic s t imula t ion of the sensory f i b r e s of the phrenic nerve and the presence of an i n f e c t i o u s agent were other p o s s i b i l i t i e s . I propose to inves t iga te the con t r ibu t ion of an i n f e c t i o u s agent and antidromic s t imula t ion of sensory f i b r e s as i n c i t i n g agents by comparison of the inflammatory response of the diaphragm to phrenic nerve s t imu la t ion (by placement of the e lec t rodes in the neck region) and diaphragmatic p la te e lect rode s t i m u l a t i o n . The r e s u l t s of the proposed studies may not only provide an explanat ion f o r the inflammation observed in these experiments but may ind ica te the operat ion of neurogenic inflammation in the hamster diaphragm. Although neurogenic inflammation has been descr ibed in other t i s s u e s , i t has never been documented in s k e l e t a l muscle and may serve as a mechanism i n i t i a t i n g inflammation in muscle exposed to an exhaustive over load . - 2 4 9 -The muscle f i b r e type composit ion of the diaphragm and the s u s c e p t i b i l i t y to experimental emphysema of the hamster lends i t s e l f as an appropr iate animal f o r a model of r e s p i r a t o r y muscle f a t i g u e . However, i t s smal l s i ze l i m i t s the a b i l i t y to monitor and maintain the hamster during p h y s i o l o g i c a l experiments which requi re long-term anesthes ia . Through my t h e s i s work, I was able to develop techniques in order to charac te r i ze the h i s t o l o g i c a l and p h y s i o l o g i c a l features of the hamster diaphragm. Within the l i m i t a t i o n s of the experimental des ign , r e s t by mechanical v e n t i l a t i o n provided bet ter recovery of the hamster diaphragm. Further work examining the response of the hamster diaphragm to more p h y s i o l o g i c a l exhaustive loads and the con t ro l of f ac to rs which cont r ibute to d e t e r i o r a t i o n of the animal preparat ion during recovery may provide more l u c i d r e s u l t s to determine the res t p ro toco l which would optimize recovery of the fa t igued diaphragm. - 250 -REFERENCES 1. Farkas GA, Roussos Ch: Histochemical and biochemical c o r r e l a t e s of v e n t i l a t o r y muscle fa t igue in emphysematous hamsters. J C l i n Invest 74:1214-1220, 1984. 2. Green CG: Neuroleptanalgesic drug combinations in the anesthet ic management of smal l laboratory animals. Lab Animals 9:161-178, 1975. 3. Kelsen SG, Wolanski , T , Supinski GS, Roessmann U: The e f f e c t of e las tase - induced emphysema on diaphragmatic muscle s t ruc ture in hamsters. Am Rev Respir P is 127:330-334, 1983. 4. Severs WB, K e i l LC , Klase PA, Oeen KC: Urethane anesthesia in r a t s . A l te red a b i l i t y to regulate hydra t ion . Pharmacology 22:209-226, 1981 5. van der Meer C, Vers luys -Broers .7AM, Tuynman HARE, Buur VAJ: The e f f e c t of e thy l urethane on hematocr i t , blood pressure and plasma g lucose . Arch Int Pharmacodyn Ther 217:257-275, 1975. PUBLICATIONS Papers 1. Reid WD, Loveridge BM: Review paper - Physiotherapy management of p a t i e n t s with chron ic o b s t r u c t i v e airways d i s e a s e . Physiotherapy Canada 35(4) : 183-195, 1983 2. Reid WD, Loveridge BM: Review paper - V e n t i l a t o r y muscle endurance t r a i n i n g i n p a t i e n t s with chron ic o b s t r u c t i v e airways d i s e a s e . Physiotherapy Canada, 35(4):197-205, 1983 3. Reid WD, Warren CPW: V e n t i l a t r o y muscle strength and endurance t r a i n i n g i n e l d e r l y subjects and p a t i e n t s with chron ic a i r f l o w l i m i t a t i o n . Physiotherapy Canada 36:305-311, 1984 4. Taylor RD, Reid WD, Fleetham J A , Pare PD: C i g a r e t t e smoke i n h a l a t i o n pat terns and b r o n c h i a l r e a c t i v i t y . Thorax 43:65-70, 1988 5. Richardson J A , Reid WD, Dunn L , Pardy RL: R e p r o d u c i b i l i t y of t e s t s of r e s p i r a t o r y muscle performance i n chron ic o b s t r u c t i v e pulmonary d isease (COPD). Physiotherapy Canada 40:34-39, 1988 6. Pardy RL, Reid WD, Belman MJ: Respiratory muscle t r a i n i n g . C l i n i c s i n Chest Medicine 9(2) June 1988 7. Pardy RL, Reid WD: R e h a b i l i t a t i o n i n COPD. Current Therapy i n Respiratory D iseases , 1989 Papers submitted 1. Reid WD, Blogg T , Wiggs B J , Pare PD, Pardy RL: Diaphragmatic p l a t e e lec t rode s t i m u l a t i o n i n the hamster. Submitted to J Appl P h y s i o l 2. Reid WD, Hards JM, Wiggs BR, Wood EN, Wright PV, Pardy RL: Muscle f i b r e type propor t ions and s i z e s i n the hamster diaphragm. Submitted to Muscle & Nerve 3. Reid WD, Davies C , Pare PD, Pardy RL: Anesthe t ics for 6-hour experimentat ion i n the hamster. Submitted to Laboratory Animals 4. Blogg T , Reid WD: The a p p l i c a t i o n of d i g i t a l data recording and a n a l y s i s to a problem i n electromyography. Submitted to J Appl P h y s i o l Abst rac ts 1. Reid WD, Loveridge BM: T r a d i t i o n a l chest physiotherapy i n COPD p a t i e n t s - a review. Proceedings of the Canadian Lung A s s o c i a t i o n (CLA) 1982, p . 34 2. Reid WD, Loveridge BM: Secre t ion removal techniques i n s tab le c h r o n i c o b s t r u c t i v e airways d isease (COAD) p a t i e n t s . Proceedings of Canadian Physiotherapy A s s o c i a t i o n (CPA) 1983, p. 40 3. Reid WD, Loveridge BM: T r a d i t i o n a l breathing exerc ises i n s t a b l e , ch r on ic o b s t r u c t i v e airways d isease (COAD) p a t i e n t s . Proc CPA 1983, p . 41 4. Reid WD, Loveridge BM: V e n t i l a t o r y muscle t r a i n i n g i n chron ic o b s t r u c t i v e airways d isease p a t i e n t s . Proc CPA 1983, p . 44 5. Reid WD Taylor DR, Fleetham JA, Pare PD: The r e l a t i o n s h i p between c i g a r e t t e smoke i n h a l a t i o n pat terns and b r o n c h i a l r e a c t i v i t y . Proceedings of Canadian Physiotherapy A s s o c i a t i o n (CPA) 1983, p. 16 6. Taylor DR, Reid WD, Pare PD, Fleetham JA: C i g a r e t t e smoke i n h a l a t i o n pat terns and b r o n c h i a l r e a c t i v i t y . Am Rev Respir D i s 129(4) (Suppl):A249, A p r i l 1984 7. Reid WD, Moreno RH, Pare PD, Pardy RL: Diaphragmatic s t i m u l a t i o n by p l a t e e lec t rodes i n the hamster. Fed Prod 44(3):839, 1985 8. Reid WD, Moreno RH, Pare PD, Pardy RL: D i r e c t muscle s t imu la t ion using p l a t e e lec t rodes against the diaphragm i n the hamster. C l i n Invest Med 8:A203, 1985 9. Reid WD, Pare Pd, Pardy RL: The e f f e c t s of r e s t on the fa t igued hamster diaphragm. Fed Proc 46(3):818, 1987 10. Reid WD: Respi ra tory muscle fa t igue and r e s t . Good, bad, or i n d i f f e r e n t . Presented a t Advances i n Pulmonary Medicine Course . American Co l lege of P h y s i c i a n s . June 1987 11. Reid WD, Hards JM, Pare PD, Pardy RL: Propor t ions and s i z e s of muscle f i b r e types i n the hamster diaphragm. Fed Proc 1988 12. Reid WD, Hards JM, Pare PD, Pardy RL: F i b r e type and r e g i o n a l v a r i a b i l i t y of g lycogen and ox ida t i ve capac i ty i n the hamster diaphragm. Fed Proc 1988 

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