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The role of the diaphragm in task failure during inspiratory resistive loading in the rabbit Osborne, Salma 1994

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THE ROLE OF THE DIAPHRAGM IN TASK FAILURE DURINGINSPIRATORY RESISTIVE LOADING IN THE RABBIT.bySALLY SALMA OSBORNEB.Sc., University of British Columbia (1983)M.Sc., University of British Columbia (1988)A THESIS SUBMITTED IN PARTIAL FULFILMENT OFTHE REQUIREMENTS FOR THE DEGREE OFDOCTOR OF PHILOSOPHYinTHE FACULTY OF GRADUATE STUDIES(Department of Experimental Medicine)We accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIAMay 1994(c) Sally Salma OsborneIn presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature)Department of £peri eiIal iied c Ii LThe University of British ColumbiaVancouver, CanadaDate j4zc3 I- -. 9 VDE.6 (2)88)11ABSTRACTIn experimental animal models, fatigue of the diaphragm has been implicated asthe predominant determinant of hypercapnic ventilatory failure and ultimatelyas the cause of task failure of inspiratory muscles during inspiratory resistivebreathing. The purpose of this study was to examine the effects of increasedinspiratory resistive loads on diaphragm function in the anesthetized rabbitmodel to test three hypotheses: first, that task failure results from a decrease inneural activation; second, that task failure results from a decrease inneuromuscular transmission to the diaphragm; and third, that the developmentof hypoventilation and hypercapnia precede task failure. We assessed centralmotor output and neuromuscular transmission to the diaphragm by continuousmonitoring of phrenic nerve activity and electromyogram activity of the costaldiaphragm during both sustainable and exhaustive inspiratory resistive loads. Wefound a linear relationship between the severity of the target inspiratory airwaypressure achieved with resistive loading and the indices of motor output to thediaphragm and activity of this muscle. Central motor output to the diaphragmremained elevated throughout resistive loading even at task failure.Neuromuscular transmission, as assessed by evoked compound potentials of thediaphragm, remained intact throughout inspiratory resistive loading including attask failure. The activity of the diaphragm remained elevated and coupled tocentral motor output throughout resistive loading, including at task failure.111Hence, task failure did not result from either a decrease in neural activation norfrom a decrease in neuromuscular transmission to the diaphragm. We foundthat despite substantial increases in inspiratory effort, rabbits hypoventilatedduring both sustainable and exhaustive loads. Therefore, hypercapnia typicallyaccompanied inspiratory resistive loading. Furthermore, we found that theelevated levels of arterial Pc02 associated with prolonged loading alone,suppressed central drive to the diaphragm through a time-dependent reductionin breathing frequency. We observed task failure only during intense loading attarget pressure close to the maximum strength of the rabbit diaphragm. Theactivity of the inspiratory muscles (parasternal intercostal and diaphragm)remained elevated and coupled despite severe arterial hypoxemia andhypercapnia during task failure. In contrast, a susbstantial decay in expiratorymuscle activity and in abdominal pressure swings preceded task failure. Inconclusion, neural activation and impulse propagation to the diaphragm weremaintained during inspiratory resistive loading even at task failure. Task failurefollowed a loss in abdominal muscle assist to the diaphragm.ivTABLE OF CONTENTSAbstract iiTable of Contents ivList of Figures viList of Tables iixList of Abbreviations ixAcknowledgements xiiDedication xiiiChapter I: General Introduction 1Skeletal Muscle Fatigue: Definitions 3Etiology of Fatigue 4Respiratory Muscle Fatigue: A Definition 7Diaphragm Function and Fatigue in Response to Inspiratory FlowResistive Loads: Human studies 8Diaphragm Function and Fatigue in Response to Inspiratory FlowResistive Loads: Animal Studies 12Objectives 16Specific Aims 17Significance 18References 26Chapter II: Diaphragm Activation and Phrenic Nerve ImpulsePropagation During Prolonged Inspiratory ResistiveLoading 33Introduction 33Methods 35Results 43The effect of prolonged inspiratory resistive loads onactivation of the diaphragm 43The effect of severe inspiratory resistive loadson activation and neuromuscular transmission to theVdiaphragm.52Discussion 64Evaluation of Methods 64Neuromuscular Transmission to the Diaphragm 66Constant Diaphragm Activation during ProlongedLoading 72Prolonged Inspiratory Resistive Loading and Indices ofDiaphragm Fatigue 73Task Failure of the Respiratory Muscles during SevereInspiratory Resistive Loading 77References 80Appendix I 85Chapter III: Ventilation During Prolonged Hypercapnia in theAnesthetized Rabbit 86Introduction 86Methods 89Results 91Steady state response to hypercapnia: 30 minute exposureto hypercarbia 91Ventilatory response to prolonged hypercapnia: 3 hourexposure to hypercarbia 95Discussion 108References 114Chapter IV: Respiratory Muscle Activity and Task Failure During SevereInspiratory Resistive Loading 117Introduction 117Methods 118Results 123Discussion 133References 140Chapter V: Summary and Conclusions 146References 154viLIST OF FIGURESFigure 1: Mechanical characteristics of skeletal muscle 19Figure 2: Schematic diagrams of the human and the rabbit diaphragm. . 21Figure 3: A schematic diagram of the chest wall illustrating theprimary actions of the diaphragm 23Figure 4: Pathway involved in skeletal muscle tension development . . . 25Figure 5: A schematic diagram of the experimental setup 39Figure 6: Peak electrical activity of the phrenic nerve, the costa!diaphragm and transdiaphragmatic pressure swings duringprolonged inspiratory resistive loads 45Figure 7: Peak inspiratory airway opening pressure andtransdiaphragmatic pressure swings during prolongedinspiratory resistive loads 47Figure 8: Changes in arterial pH, pCO2 and ventilation duringprolonged inspiratory resistive loads 49Figure 9: Peak electrical activity of the phrenic nerve and thecosta! diaphragm during severe loads 53Figure 10: The relationship between peak inspiratory pressure andindices of activity and drive to the diaphragm 56Figure 11: Changes in arterial blood gases and negative inspiratorypressures recorded at the airway opening during severeinspiratory resistive loads 58Figure 12: The change in minute ventilation during severe inspiratoryresistive loading 61Figure 13: Representative evoked diaphragm compound actionpotentials during severe inspiratory resistive loading 70viiFigure 14: The relationship between peak inspiratory pressureand transdiaphragmatic pressure swings during briefinspiratory resistive loading 75Figure 15: Minute ventilation and arterial PC02 levels duringprolonged exposure to FICO2= 0.10 in hyperoxic rabbits 97Figure 16: Comparison of arterial Pco2 levels and minute activityof the diaphragm in rabbits exposed to prolongedhypercarbia and rabbits exposed to prolonged inspiratoryresistive loading 102Figure 17: Comparison of activity of the diaphragm and breathingfrequency in rabbits exposed to prolonged hypercarbia andrabbits exposed to prolonged inspiratory resistive loading . . . 104Figure 18: Comparison of arterial Pc02 levels and the minuteventilation in rabbits exposed to prolonged hypercarbiaand rabbits exposed to prolonged inspiratory resistiveloading 106Figure 19: Electromyographic activity of the costal diaphragm,parasternal intercostal and transversus abdominis duringsevere inspiratory resistive loading 126Figure 20: Sample tracing of arterial blood pressure, airway openingpressure, abdominal pressure, airflow and phasic-movingaverage of costal diaphragm, parasternal intercostal andtransversus abdominis during baseline and severeinspiratory resistive loading 129Figure 21: Sample tracing of the activity of inspiratory musclesduring the final minutes leading to respiratory arrest 131vii’LIST OF TABLESTable I: Respiratory variables at baseline and duringprolonged inspiratory resistive loading in theanesthetised rabbit 51Table II: Respiratory variables at baseline and during severeinspiratory resistive loading in the anesthetisedrabbit 63Table III: Arterial blood gases and ventilatory variables duringthe initial 30 minutes of exposure to 10% inspiredC02 in hyperoxic anesthetized rabbits 93Table IV: Mean values for respiratory times, mean inspiratoryflow rate (VT/TI) and duty cycle (TI/TrOT) inanesthetized rabbits exposed to 10% inspired C02for the initial 30 minutes 94Table V: Arterial blood gases and changes in ventilatoiyvariables produced by 3 hours of exposure to 10%inspired C02 in anesthetized rabbits 99Table VI: Mean values for respiratory times, mean inspiratoryflow rate (VT/TI) and duty cycle (TI/TrOT) inanesthetized rabbits exposed to 10% inspired C02for 3 hours 100Table VII: Respiratory variables, arterial blood gases and pH atbaseline and during severe inspiratory resistive loadingin the anesthetized rabbit 125ixLIST OF ABBREVIATIONS AND SYMBOLSAapp Area of appositionBf Breathing frequencyCSF Cerebrospinal fluidCNS Central nervous systemcm H20 Centimeters of waterE ExpiratoryEELV End expiratory lung volumeEMGdi The moving average of the diaphragm electromyogramEMGps The moving average of the parasternal electromyogramEMGta The moving average of the transversus abdominis electromyogramENGdi The moving average of the diaphragm electroneurogramFc Centroid frequency of the EMG power spectrumFRC Functional residual capacityH Hydrogen cationHC03 Bicarbonate anionI InspiratoryLM. IntramuscularI.V. IntravenousMg2 Magnesium cationmm Minutesxml Millilitersmm Hg Millimeters of mercurymV MillivoltsM wave Evoked compound muscle action potentialPhosphate ionPab Abdominal pressurePACO2 Alveolar partial pressure of carbon dioxidePaCO2 Arterial partial pressure of carbon dioxidePao Airway pressurePdi Transdiaphragmatic pressurePa02 Arterial partial pressure of oxygenPes Esophageal pressurepH Negative logarithm of hydrogen ion concentrationPpl pleural pressureptp Peak to peakQB Quiet breathingsec secondsS.E.M. Standard error of the meanTE Expiratory durationTI Inspiratory durationTI/TrOT Duty cycle‘PrOT Duration of a breath or total respiratory timeAirflowVE Minute ventilationVT Tidal volumeVT/TI Mean inspiratory flow ratexixl’ACKNOWLEDGEMENTSI am indebted to my supervisor Dr. Jeremy Road for his support and patiencethroughout the course of my studies. I sincerely appreciate Dr. John Ledsome’svaluable advice as my interim advisor during Dr. Road’s absence and grateful forhis wisdom and integrity. I am equally grateful to Dr. Bill Milsom whoseenthusiasm for the study of control of breathing encouraged me to continuefurther with my graduate studies. He has invested much time in my postgraduate work and his unconditional support is most appreciated. Many thanksto Dr. Darlene Reid for her advice, useful resources and unendingencouragement. Thanks to Dr. Angelo Belcastro for introducing me to thetechniques in his laboratory and biochemical correlates of muscle fatigue.Special thanks and appreciation for the expert technical assistance of MichaelBoyd, Miguel Pachenko and the enthusiastic assistance of the animal care unitstaff at the University Hospital, U.B.C.; their high professional standards madethe protocols feasible.xliiDEDICATIONTo my children, Simone Kimberly and Corbin Elliot Osborne whose sunnypersonalities were a source of strength and inspiration.To my husband, David Nelson Osborne who surpassed the standards for equalpartnership.In memory of Rene Theophile Hyacinth Laennec and his prescient wisdom:“Nothing hurts the progress of science more than to divert terms from theircustomary meaning without sufficient reason and to create bad new ones”.1I: General IntroductionLife depends on the ability of respiratory muscles to overcome resistive andelastic forces and to produce phasic contractions continuously. Normal breathingis accomplished easily due in good part to the enormous reserve capacity of therespiratory muscles. Functionally, respiratory muscles are skeletal muscles andas such share common mechanical characteristics with other skeletal muscles(Figure 1) and can adapt to long-term functional alterations (Farkas andRoussos, 1983; Farkas, 1991).In the mammalian species, the diaphragm is considered to be the principalmuscle for inspiration during quiet breathing. This dome-shaped structureseparates the thoracic and abdominal cavities. The muscle fibres of thediaphragm originate from three different regions: 1) the sternal region arisingfrom the posterior aspect of the xiphoid process; 2) the costal region arising fromthe lower six ribs and the costal cartilages and 3) the crural region arising fromthe upper three lumbar vertebrae. These fibres ascend and radiate inwardsinserting into the central tendon, a thin but strong aponeurosis (Leak, 1979;Figure 2).During quiet inspiration contraction of the diaphragm decreases pleural pressure,expands lung volume and increases abdominal pressure pushing the ribcage2upward and outwards and leading to protrusion of the anterior abdominal wall(Figure 3). Other primary muscles of inspiration include the scalene andparasternal intercostal muscles. Quiet expiration is thought to be passive andmainly due to the elastic recoil of the lungs.During augmented breathing activity, more of the respiratory muscle mass isrecruited to achieve greater ventilation. The accessory muscles of inspirationsuch as the sternocleidomastoid, external intercostals, serratus posterior superiorand pectoralis major may be activated. Furthermore, expiration becomes activewith phasic contractions of the primary expiratory muscles in the ventralabdominal wall as well as the ribcage internal intercostals.In theory, the respiratory muscles could fail to generate adequate forcecontracting against respiratory loads that require too great an effort for too longa period of time. In 1977, Macklem and Roussos proposed that respiratorymuscle fatigue might underlie respiratory failure observed with several clinicalconditions such as atrophy of respiratory muscles with prolonged mechanicalventilation, neuromuscular weakness, chronic obstructive pulmonary disease orwith increased inspiratory loads. To date, it has been difficult to confirmrespiratory muscle fatigue in patients progressing slowly to chronic respiratoryfailure (Begin and Grassino, 1991; Grassino and Clanton, 1991; Rochester,1991). It has long been accepted that acute inspiratory resistive loading can3induce fatigue of the diaphragm both in man (Roussos and Macklem, 1977;Aubier Ct aL, 1981; Moxham et aL 1981, 1982; Cohen et al. 1982; Grassino andMacklem, 1984; Bellemare and Bigland-Ritchie, 1987 and Yan et aL 1992) andin experimental animal models (Aldrich, 1985, 1987, 1988, 1991; Alexandrovnaand Isaev, 1990; Bazzy and Haddad, 1984; Bazzy and Donnelly, 1993; Mayocket aL 1987, 1991; Oliven et aL 1988) although confirmation for this hypothesis hasbeen elusive.Skeletal Muscle Fatigue: DefinitionsIn 1979, Edwards defined skeletal muscle fatigue in mechanistic terms as adecrease in force generation due to either a decrease in neural impulsepropagation known as “neural fatigue” or due to the failure of the contractileapparatus known as “muscle fatigue” (Edwards, 1979). Since then, variousadditional factors have been considered and the definition of muscle fatigue hasbeen simplified by Hultman and Sjoholm (1986) as “the failure to maintain anexpected force or power output”.Figure 4 shows the pathway involved in muscular contraction. In theory, fatiguecan be brought about by impaired function of any one or combination ofalterations in steps involved in the pathway from the CNS to the contractileapparatus. Therefore, depending on its origin, skeletal muscle fatigue can be4broadly categorized in three groups: 1) Central fatigue, 2) failure ofneuromuscular transmission and 3) peripheral fatigue.Etiology of FatigueFirst, the cause of fatigue may be “central” due to a decrease in the frequencyor intensity of neural output to the skeletal muscle from the central nervoussystem. This may be conscious due to a lack of motivation or an inability totolerate the discomfort associated with the fatiguing stimulus. It may beunconscious due to protective feedback mechanisms operating through inhibitoryafferent input to the central nervous system (for a review see Enoka and Stuart,1992). Skeletal muscle afferent input during increased work may come in formof the proprioceptive information from the Golgi tendon organs or the musclespindles. Alternatively, Group Ill and IV afferent fibres may decreasemotoneuron output in response to stretch or the metabolic state of the muscle.Hence, afferent input from the muscle may serve as an important feedback linkbetween the working muscle and excitatory motor output from the CNS and/orspinal reflex activity during exhaustive work.Secondly, fatigue of skeletal muscle might also result from a decrement inneuromuscular transmission (Naess and Storm-Mathesen, 1955). Whereasneuromuscular transmission failure is undoubtedly the major cause of muscle5fatigue in some pathologic conditions such as botulism, Lambert-Eatonmyasthenic syndrome and myasthenia gravis, its role in the response of skeletalmuscle to voluntary contractions against exhaustive loads remains controversial.Recently this issue has been reexamined in animal models of respiratory musclefatigue. Two studies have considered the importance of declining neuromusculartransmission to the diaphragm under severe inspiratory resistive loads (Aldrich,1987, 1991; Bazzy and Donnelly, 1993).Finally, fatigue of skeletal muscle may be peripheral (myogenic) in origin. Theloci for this fatigue are distal to the neuromuscular junction including thesarcolemma, the transverse-tubule, the sarcoplasmic reticulum and the regulatoryand contractile proteins. There is little evidence that propagation of actionpotentials across the sarcolemma or t-tubules is blocked except in the artificialsituation of continuous high-frequency electrical stimulation wheretransmembrane flux of water and local electrolytes, specifically potassium acrossthe t-tubule, result in decreased contractile force (Westerbiad et aL 1991). Themajor focus of current research on skeletal muscle fatigue looks beyondexcitability of the cell membrane to the various processes involved in couplingof sarcolemmal action potential and cross bridge interaction resulting in musclecontraction (excitation-contraction coupling).Metabolic consequences of sustained muscular work have long been considered6as correlates of peripheral fatigue by virtue of their effect(s) on excitationcontraction coupling and/or cross bridge interaction. In the past 25 years, twohypotheses [the exhaustion (or depletion) hypothesis and the accumulation (buildup of metabolites) hypothesis] have been proposed as metabolic causes offatigue. Depleted metabolites include ATP, PCr and glycogen. Accumulatedmetabolites include H, P, lactate, free intracellular Ca2 and Mg2 (forreviews see Green, 1987; Kirdendall, 1990; Vollestad and Sejersted, 1988; Enokaand Stuart, 1992 and Westerblad et aL, 1991). The extent to which any of thesemetabolites contribute to fatigue of the muscle will depend on the intensity ofwork, the amount of time elapsed since the onset of activity, and the specificmetabolic profile of the muscle or muscle fibre. Owing to the specificity of themetabolic profile of muscle fibres, the current approach in determining the roleof myogenic factors in fatigue is a reductionist one. Typically, studies considerthe levels, compartmental content, rates of production/utilization, andmechanism of action of these metabolites, in vitro, in single intact or skinnedfibre preparations. Typically, these studies employ artificial electrical stimulationregimens to induce fatigue and to assess responsivity (Westerblad et aL, 1991).A coherent interpretation of the literature on the etiology of skeletal musclefatigue is complicated by the diversity of definitions of muscle fatigue, the varietyof indices used to define muscle fatigue and the numerous paradigms used tostudy it. There is general acceptance that the specific mechanism causing fatigue7of a skeletal muscle will depend on the nature of the fatiguing stimulus (Enokaand Stuart, 1992). This point is of considerable importance when examining thevarious breathing maneuvers employed to trigger respiratory muscle fatigue.Respirato,y Muscle Fatigue: A DefinitionThe definition of respiratory muscle fatigue was recently considered at aNational Heart, Lung and Blood Institute Workshop (1990). The currentdefinition of respiratory muscle fatigue is based on a consensus arrived at thisworkshop which involved primarily chest physicians. This new definitiondescribes respiratory muscle fatigue in functional terms as a condition in whichthere is a loss in the capacity for developing force and or velocity of a muscle inresponse to a load and which is reversible by rest. Furthermore, the traditionaldefinition of skeletal muscle fatigue as a failure to generate an expected forcewas redefined as “task failure”.Introducing the concept of reversibility in this new definition of fatigue permitsone to contrast fatigue from muscle weakness. Further, this new definition hasbroadened the concept of respiratory muscle fatigue significantly. An implicationof this definition is that fatigue may be observed with respect to a task eventhough the force required for its completion can still be generated. For example,the capacity of the diaphragm in vivo to develop force is estimated by the8pressure generated across the diaphragm (Pdi). Animal models of diaphragmfatigue have demonstrated a reversible loss in the capacity to develop Pdi inresponse to tetanic electrical stimulation following inspiratory resistive loadedbreathing. However, the capability of the diaphragm to generate pressure duringspontaneous breaths against these loads known as tidal Pdi swings, is maintainedfor periods up to five hours after one observes a decrement in response totetanic electrical stimulation (Mayock, et aL, 1991; see Chapter II as well).According to the current definition of respiratory muscle fatigue proposed at theNational Heart Lung and Blood Institute workshop (1990), these preparations(Mayock et at., 1987, 1991; Aldrich 1990), qualify as models of diaphragmfatigue.Diaphragm Function and Fatigue in Response to Inspiratoiy Flow Resistive Loads:Human studiesThe diaphragm is the primary muscle of inspiration. Accordingly, there has beenmuch focus on its response to artificial external loading. Specifically, theresponse to flow resistive loads which are defined by the resistance of porousdiscs placed externally at the inspiratory inlet of a breathing circuit have beenstudied. The human ability to tolerate high inspiratory flow resistive loads iswell documented (Freedman and Campell, 1970). It has been shown that theincrease in oxygen consumption associated with inspiratory resistive loading is9proportional to the integral of mouth pressure. In turn, oxygen consumption canbe modified through pattern of breathing (Jones et aL, 1985). Although theimmediate response of awake human subjects to inspiratory resistive loading isextremely variable (Axen and Haas, 1979), the steady state response tosustainable inspiratory loads is typically characterized by an increase ininspiratory time, a decrease in breathing frequency and a reduced meaninspiratory flow rate (Axen et aL, 1983; Tm Hof et aL 1986; Jones et aL, 1985).Furthermore, relatively large inspiratory loads can result in an increase inexpiratory time, elevated arterial Pco2 levels (Tm Hof et aL, 1986) and reducedarterial P02 levels (Eastwood et aL, 1994). The specific pattern of breathingemployed by conscious humans may delay fatigue. Load perception and otherfactors may influence this phenomenon (Clague et aL, 1992 and Eastwood et aL,1994). Therefore, it is necessary that the complex nature of the potentialinterplay between load perception and behavioral responses be emphasized wheninterpreting ventilatory responses to loaded breathing in conscious humans.Inspiratory pressure swings across the diaphragm (Pdi) decrease against relativelysevere target inspiratory flow resistive loads (60-80% Pdimax) in conscioushumans. This loss in target pressure generation is thought to reflect a decrementin diaphragm force generation and hence diaphragm fatigue in the conscioushuman. Although breathing against such high inspiratory resistance is hard workand not particularly pleasant (Mead, 1979), it has been argued that fatigue under10these circumstances is not purely motivational. Other indices of fatigue are alsopresent. These indices include a drop in the frequency-Pdi curve (commonlyreferred to as the force-frequency curve), alterations in diaphragmelectromyogram power spectrum (high-low ratio or centroid frequency), andtwitch occlusion (Aubier et aL, 1981, 1985; Moxham et aL, 1981, 1982; Bellemareand Bigland-Ritchie, 1987).Any conclusions arising from the above findings must be tentative as theunderlying mechanisms involved in changes in these indices are unclear and theirpresence precedes rather than being causative of task (pressure) failure. Adecrease in the frequency-pressure curve of the diaphragm after inspiratoryresistive loading suggests alteration of those processes involved in forcegeneration which are distal to the CNS. For example, a decrease in pressureoutput of the diaphragm in response to high frequency electrical stimulation ofthe phrenic nerve is thought to reflect either a decrease in phrenic nerve impulsepropagation, neuromuscular junction failure, or loss of action potentialpropagation across the sarcolemma or t-tubule (high frequency fatigue see above:skeletal muscle etiology). It is regarded as uncertain whether severe inspiratoryresistive loading leads to high frequency fatigue in humans (Moxham, et aL, 1981cf Aubier, et aL, 1981).There is some agreement that a decrease in pressure output of the diaphragm11occurs in response to low frequency stimulation (Moxham, et aL, 1981, 1982;Aubier, 1981). Although low frequency fatigue is thought to reflect fatigue ofmyogenic origin, the cellular mechanisms remain to be elucidated.Similarly, the mechanisms underlying shifts in the diaphragm EMG powerspectrum are unknown. A shift in the diaphragm EMG power spectrum isevaluated by a change in it’s centroid frequency (Fc) or a change in the ratio ofhigh frequency power to low frequency power (H/L ratio). It has been shownthat Fc is more sensitive to shifts in the diaphragmatic EMG power spectrum.Additionally, changes in the Fc have been detected during levels of voluntaryhyperpnea that can be readily sustained (Sieck et aL, 1985). Consequently, thespecificity of using power spectral changes of diaphragm EMG in predictingfatigue has been questioned (Sieck et aL, 1985).The degree to which the diaphragm is activated by the CNS during inspiratoryresistive loading has been assessed by the twitch occlusion technique. Thistechnique suggests that half of the reduction in diaphragm pressure generationis due to central fatigue in awake human subjects (Bellemare and BiglandRitchie, 1987). Recent data show that this technique could be insensitive to thecontribution of central mechanisms of fatigue to pressure failure due to thephenomenon of twitch potentiation (Mador et aL, 1994; Wragg et aL, 1993).12Diaphragm Function and Fatigue in Response to Inspiratoiy Flow Resistive Loads:Animal StudiesSeveral studies in animals breathing against inspiratory resistive loads attemptto define the role of diaphragm fatigue in respiratory failure. Bazzy and Haddad(1984) were the first to show a decrease in diaphragm activity (EMG) andpressure output (Pdi) in response to intense inspiratory resistive loads in a studyon three chronically instrumented awake sheep. These loads were sustainablefor prolonged periods (up to 3 hours) but eventually resulted in a decrease indiaphragm activity (EMG) followed by a drop in diaphragm pressure output andrespiratory acidosis (Bazzy and Haddad, 1984). In a subsequent study using thesame preparation, Bazzy and Donnelly (1992) show that phrenic activity (ENGdi)increased at a time when diaphragm muscle activity (EMGdi) and pressure (Pdi)were maintained. Based on these observations and the decrease seen in theevoked compound potentials of the diaphragm (M-waves) in two sheep, theauthors concluded that neuromuscular transmission fatigue contributed to failureof the diaphragm to generate inspiratory pressure (Bazzy and Donnelly, 1992).In anesthetized rabbits exposed to approximately one hour of inspiratory resistiveloading, Aldrich (1987) arrived at a similar conclusion based on twoobservations. Firstly, there was a substantial decrease in diaphragm M-waveamplitude and area with inspiratory resistive loading. Secondly, inspiratory13resistive loading reduced the frequency-pressure response elicited by stimulationof the phrenic nerve but did not affect the frequency-pressure response elicitedby direct stimulation of the diaphragm. In contrast, prolonged (6 hours)inspiratory resistive loads of similar magnitude in the anesthetized piglet did notaffect the M-wave despite a reduction in the frequency-pressure response of thediaphragm to phrenic nerve stimulation (Mayock et aL, 1987, 1991). Anexamination of neuromuscular response to inspiratory muscle challenge undera variety of loads has not been done.In other species, in both awake and anesthetized preparations, there is evidencethat central fatigue can play a significant role in respiratory failure associatedwith inspiratory resistive loaded breathing. In the unanesthetized infant monkey,intense inspiratory resistive loading resulted in respiratory acidosis andmaintained levels of peak tidal airway pressure and diaphragm EMG (Watchkoet aL, 1988). Although the activity of each diaphragm contraction (EMG) wasnot reduced, there was a decrease in diaphragm activity per minute due toreduced frequency of breathing. These findings are consistent with the notionthat an optimization of inspiratory work occurs in response to loaded breathingand is achieved through a decrease in central rhythm.Central modulation of respiratory activity during inspiratory resistive loading isnot limited to the processes involved in rhythm generation. For example,14Scardella and associates found no change in frequency of breathing in responseto prolonged (2.5 hour) intense inspiratory resistive loading (Scardella et aL,1986) in awake goats. Instead they observed a decrease in tidal volumeassociated with an increase in immunoreactive beta endorphin in thecerebrospinal fluid (Scardella, Santiago and Edelman, 1989). This increase inendogenous opioids in the CNS produced differential inhibition of respiratorymuscle electrical activity (Scardella et aL, 1990). Furthermore, lactic acidosis ofrespiratory muscles is another feature of intense inspiratory resistive loading inthis model (Petrozinno et aL, 1992). To account for these findings in the awakegoat, Scardella and Petrozzino have developed a conceptual model involvingboth facilitatory and inhibitory pathways activated by lactic acid stimulation ofGroup III and IV afferents (Petrozzino et aL, 1993). These findings are alsoconsistent with the notion that central fatigue may indeed play a protective oradaptive role in delaying the onset of myogenic fatigue in response to intenseinspiratory resistive loads.The possibility that peripheral (myogenic) fatigue of the diaphragm mayeventually occur if the inspiratory resistive loads are sufficiently intense and/orprolonged has been examined in a handful of studies. In the anesthetized piglet,submaximal contractions of the diaphragm under moderate inspiratory resistiveloads causes a decrease in the maximal force generating capacity of thediaphragm (Mayock, et aL 1991). However, there is no change in the levels of15ATP, phosphocreatine, lactate or glycogen in the diaphragm after prolongedloading (6 hours) in this preparation. Similarly, the anesthetized rabbit breathingagainst incremental increases in inspiratory threshold load fails to show anyevidence of diaphragm glycogen depletion or lactate accumulation even atmaximal loads that result in respiratory arrest (Ferguson et at, 1990). Otherindices of myogenic fatigue have not been measured during inspiratory resistiveloading in animal models.It is clear that breathing against an inspiratory resistive load reduces thoseindices for which a reduction would imply that central and neuromusculartransmission fatigue of the diaphragm contribute to respiratory failure. Theeffects of inspiratory resistive loading appear to depend on the intensity of theload and its duration. What remains to be determined from the studiesconducted to date is the extent to which central and neuromuscular transmissionfatigue of the diaphragm contributes to the failure of the diaphragm to generatean expected inspiratory pressure (task failure). Furthermore, in all animalspecies where inspiratory resistive loading has been employed to trigger fatigueof the diaphragm, the target inspiratory load has been sufficiently intense toresult in hypoventilation and hypercapnic ventilatory failure. The relationshipbetween hypercapnic ventilatory failure, diaphragm fatigue and task failureduring inspiratory resistive loading remains unclear.16ObjectivesIn the studies discussed below, the anesthetized rabbit model of inspiratoryresistive loading was chosen to examine the effects of increased inspiratoryresistive loads on diaphragm function. The rabbit is a suitable model to assessdiaphragm function since the rabbit diaphragm has a similar proportion offatiguable fibres as man (Green et aL, 1984) and therefore has the potential todemonstrate fatigue. The diaphragm of other species, for example the dog, iscomposed entirely of type I and ha fibres which are fatigue resistant (Green etaL, 1984).Additionally, it has been shown that anesthetized rabbits will breathespontaneously against loads that result in a decrease in the maximal force-generating capacity of the diaphragm (Aldrich, 1985, 1987). Since fatigue canresult from failure of one or more elements of a closed-loop system, examiningdiaphragm function in a preparation that breathes spontaneously underanesthesia offers the opportunity to distinguish the extent to which each of theseelements contributes to diaphragm failure independent of the conscious factorswhich might contribute to fatigue.Accordingly, the major objective of these studies was to test the followinghypotheses:171. The failure of the respiratory system to maintain a target inspiratory pressure(task failure) results from a decrease in central motor output to thediaphragm.2. Task failure results from a decrease in neuromuscular transmission to thediaphragm.3. The development of hypoventilation and hypercapnia precede taskfailure.Specific AimsThe specific aims of the studies discussed below were to:1. Assess central motor output and neuromuscular transmission to the diaphragmduring periods of prolonged sustainable and exhaustive inspiratory resistiveloading.2. Determine the changes in breathing pattern (volume, frequency, and timing)in response to inspiratory resistive loading of different intensities anddurations, ranging from sustainable to exhaustive loads.3. Determine whether the respiratory muscles could be loaded to produce taskfailure.184. Determine the relationship between task failure and hypercapnic ventilatoryfailure during inspiratory resistive loading.SignificanceThese experiments will provide insight into the in vivo function of the normaldiaphragm under inspiratory resistive loading and the control of ventilationunder these conditions. Since the results of these studies are derived in theanesthetized state, they will provide information on mechanisms involved inrespiratory failure during inspiratory resistive loading independent of consciousfactors.19Figure 1: Mechanical characteristics of skeletal muscle:Panel A: The active length-tension relationship of skeletal muscle (adapted fromLieber, 1992). The tension generated in skeletal muscle is a direct function ofthe magnitude of overlap between actin (insert solid lines) and myosin (insertcrossed line) filaments. Maximal tension is produced at optimal length. Lengthsshorter or greater than the optimal result in less filament overlap and lowertension. Optimal length of the diaphragm is reported at a lung volume belowend expiratory lung volume (EELV) at the end of a normal quiet breath (FRC).Panel B: An increase in EELV can place the diaphragm in a position ofmechanical disadvantage. In contrast, a decrease in EELV can be mechanicallyadvantageous in increasing the potential for diaphragm excursion.20125 -100 -75 -50 -25 -01.0ASarcomere Length (urn)BDiaphragmposition at:End ExpirationEnd Inspiration< >.DiaphragmExcursionC0CI—C)C0U,IEEx0CU,C,U,0I I I I1 .5 2.0 2.5 3.0 3.51 EELV21Figure 2: Schematic diagrams of the human diaphragm in situ (top panel) andthe peritoneal surface of the diaphragm of the rabbit (bottom panel). Adaptedfrom Farkas, 1991 and Leak, 1979.a23Figure 3. A schematic diagram of the chest wall showing the rib cage andabdominal compartments. The area of apposition (Aapp) is the region of thediaphragm apposed against the lower ribcage. Contraction and shortening of thediaphragm leads to progressive “peeling away” of this muscle from the ribcageat the Aapp. This down ward “piston-like” action of the diaphragm increasesabdominal pressure and decreases pleural pressure. The increase in abdominalpressure in turn acts as a fulcrum for movement of the rib cage by twomechanisms: 1) Insertional action: the force applied at the origin of diaphragmon the ribcage in the direction of muscle fibre insertion (central tendon) rotatingthe ribs upward (pump handle motion) and outward (bucket handle motion) 2)Appositional action: a distending force on the lower rib cage at the Aapp movingthe ribs outward. The decrease in pleural pressure displaces the upper ribcageinward. [Adapted from De Troyer & Loring, 1986]ZLIRib CageDome-----,.AbdomenA app.25BrainCentral fatigue Spinal CordINeuromuscular Peripheral NerveFatigue Neuromuscular JunctionISarcolemmaTransverse TubulesPeripheral Calcium Release(Myogenic) Crossbridge FormationFatigue ContractionTensionFigure 4. Pathway involved in skeletal muscle tension (force) development(adapted from Kirdendall, 1990).26References1. Aldrich, T.K. (1987). Transmission fatigue of the rabbit diaphragm. RespirPhysioL 69: 307-3 19.2. Aldrich, T. K. (1991). Central and transmission fatigue. Seminars inRespiratoiy Medicine 12: 322-330.3. Aldrich, T. K. (1988). Central fatigue of the rabbit diaphragm. Lung 166:233-241.4. Aleksandrovna, N.P. and G.G. Isaev (1990). Central and peripheralcomponents of the fatigue of respiratory muscles in inspiratory resistive loadin cats. Sechenov Physiological Journal of the US.S.R. 76: 658-666.5. Aubier, M., D., Murciano, Y. Lecocguic, N. Virres and R. Pariente (1985).Bilateral phrenic stimulation; a simple technique to assess diaphragmaticfatigue in humans. J. AppL PhysioL 58: 58-64.6. Aubier, M., G. Farkas, A. de Troyer, R. Mozes and C. Roussos (1981).Detection of diaphragmatic fatigue in man by phrenic stimulation. J AppLPhysiol. 50: 538-544.7. Axen, K. and S.S. Haas (1979). Range of first-breath ventilatory responsesto added mechanical loads in naive men. J AppL PhysioL 46: 743-75 1.8. Axen, K., S.S. Hass, R. Hass, D. Guadino and A. Haas (1983).Ventilatory adjustments during sustained mechanical loading in conscioushumans. J. AppL PhysioL 55: 1211-1218.279. Bazzy A. R. and D. F. Donnelly (1993). Diaphragmatic failure during loadedbreathing: role of neuromuscular transmission. J AppL PhysioL 74: 1679-1683.10. Bazzy A.R. and 0.0. Haddad (1984). Diaphragmatic fatigue inunanesthetized adult sheep. J AppL PhysioL 57: 182-190.11. Bellemare F. and B. Bigland-Ritchie (1987). Central components ofdiaphragmatic fatigue assessed by phrenic nerve stimulation. J AppLPhysioL 62: 1307-1316.12. Begin, P. and A. Grassino (1991). Inspiratory muscle dysfunction andchronic hypercapnia in chronic obstructive pulmonary disease. Am. Rev.Respir Dis. 143: 950-9 12.13. Clague, J.E., J. Carter, M.G. Pearson and P.M.A. Calverley (1992). Effortsensation, chemoresponsiveness, and breathing pattern during inspiratoryresistive loading. J. AppL PhysioL 73: 440-445.14. Cohen, C.A., 0. Zagelbaum, D. Gross, C. Roussos and P.T. Macklem(1982). Clinical manifestations of inspiratory muscle fatigue. Am. J Med.73: 308-316.15. Daubenspeck, J.A. (1994). Invited editorial on “Discrimination of transientlyapplied mechanical loads: breathing versus pulling. J AppL PhysioL 76: 3-4.16. De Troyer, A. and S.H. Loring (1986). Actions of the respiratory muscles.In: Mead J, Macklem P.T. (eds.). Handbook of Physiology, Accessorymuscles. Washington, D.C., American Physiological Society, p. 447.2817. Eastwood, P.R., D. Hiliman and K.E. Finucane (1994). Ventilatoryresponses to inspiratory threshold loading and role of muscle fatigue in taskfailure. .L AppL PhysioL 76: 185-195.18. Enoka, R.M. and D.G. Stuart (1992). Neurobiology of muscle fatigue. JAppL PhysioL 72: 1631-1648.19. Farkas, G.A., and C. Roussos (1983). Diaphragm in emphysematoushamsters: Sarcomere adaptability. J AppL PhysioL 54: 1635-40.20. Farkas, G. A. (1991). Functional characteristics of the respiratory muscles.Semmars in Respir. Med. 12: 247-257.21. Ferguson G.T., C.G. Irvin, and R.M. Cherniack (1990). Relationship ofdiaphragm glycogen, lactate, and function to respiratory failure. Am. Rev.Resp. Dis. 141: 926-932.22. Freedman, S. and E.J.M. Campbell (1970). The ability of normal subjectsto tolerate added inspiratory loads. Respir. PhysioL 10: 213-235.23. Grassino, A. and T. Clanton (1991). Respiratory muscle fatigue. Sem.Respir. Med. 12: 305-321.24. Green, H.J. Neuromuscular aspects of fatigue (1987). Can. 1. Spt. Sci, 12:7S-18S.25. Green, H.J., Rechman, H. and D. Pette (1984). Inter- and intra-speciescomparisons of fibre type distribution and of succinate dehydrogenaseactivity in type I, ha and JIb fibres of mammalian diaphragms.Histochemistiy 81: 67-73.2926. Huitman, E. and H. Sjoholm (1986). Biochemical causes of fatigue. In:Human Muscle Power, N.L. Jones, N. M. Cortney, and A. J. McComas (Eds.)Champaign, IL: Human Kinetics pp. 215-235.27. Im Hof, V., P. West and M. Younes (1986). Steady state response ofnormal subjects to inspiratory resistive load. .1 AppL PhysioL 60: 1471-81.28. Jones G.L., K.J. Killian, E. Summers and N.L. Jones (1985). Inspiratorymuscle forces and endurance in maximum resistive loading. J AppLPhysioL 58: 1608-1615.29. Kirkendall, D. T. (1990). Mechanisms of peripheral fatigue. Med. Sci.Sports Exerc. 22: 444-449.30. Leak, L.V. (1979). Gross and ultrastructural morphologic features of thediaphragm. Am. Rev. Respir Dis. 119:3-21.31. Lieber, R. L. Skeletal Muscle Structure and Function. Williams &Wilkins, Baltimore, MD, p.55, 1992.32. Loring, S.H. and A. De Troyer (1985). Actions of the respiratorymuscles. In Lung Biology in Health and Disease. The Thorax, Part A, Vol.29 (Ed: C. Lenfant), Marcel Dekker Inc., N.Y., pp. 327-349.33. Macklem, P.T. and C. Roussos (1977). Respiratory muscle fatigue: a caseof respiratory failure? Clin. ScL MoL Med. 53: 419-22, 1977.34. Mador, J.M., U.J. Maglang and T.J. Kufel (1994). Twitch followingvoluntary diaphragmatic contraction. Am. J Resp. Crit. Care Med. 149:739-743.3035. Mayock, D.E., R. J. Badura, J.F. Watchko, T.A. Standaert and D.E.Woodrum (1987). Response to resistive loading in the newborn piglet.Pediatr. Res. 21:121-125.36. Mayock, D.E., T.A. Standaert, T.D. Murphy and D.E. Woodrum (1991).Diaphragmatic force and substrate response to resistive loaded breathing inthe piglet. J AppL PhysioL 70: 70-76.37. Mead, J. (1979). Responses to loaded breathing . A critique and a synthesis.BuLL Eur. PhysiopathoL Respii 15: 61-71.38. Moxham, J., A.J.R. Morris, S.G., Spiro, R.H.T., Edwards and M. Green(1981). Contractile properties and fatigue of the diaphragm in man. Thorax36: 164-168.39. Moxham, J., R.H.T. Edwards, M. Aubier, A. Dc Troyer, G. Farkas, P.T.Macklem and C. Roussos (1982). Changes in EMG power spectrum(high-low ratio) with force fatigue in humans. J AppL PhysioL 53: 1094-1099.40. National Heart Lung and Blood Institute Workshop Summary. RespiratoryMuscle Fatigue (1990). Am. Rev. Respir Dis. 142: 474-480.41. Naess, K. and A. Storm-Mathesen (1955). Fatigue and sustained tetaniccontractions. Acta PhysioL Scand. 34: 35 1-366.42. Oliven, A., S. Lohda, M.A. Adams, B Simhjai and S.G. Kelsen (1988).Effect of fatiguing resistive loads on the level and pattern of respiratoryactivity in awake goats. Respir PhysioL 73: 3 11-324.3143. Petrozzino, J.J., A.T. Scardella, T.V. Santiago and N. H. Edelman (1992).Dichioroacetate blocks endogenous opioid effects during inspiratory flow-resistive loading. J AppL PhysioL 72: 590-596.44. Petrozzino, J.J., A.T. Scardella, N.H. Edelman and T.V. Santiago (1993).Respiratory muscle acidosis stimulates endogenous opioids during inspiratoryloading. Am. Rev. Respu Dis. 147: 607-6 15.45. Rochester, D.F. (1991). Respiratory muscle weakness, pattern of breathing,and CO2 retention in chronic obstructive pulmonary disease. Am. Rev.Respu Dis. 143: 901-903.46. Scardella, A.T., R.A. Parisi, D.K. Phair, T.V. Santiago and N.H. Edelman(1986). The role of endogenous opioids in the ventilator)’ response to acuteflow resistive loads. Am. Rev. Respir Dis. 133: 26-31.47. Scardella, A.T., T.V. Santiago and N. H. Edelman (1989). Naloxone altersthe early response to an inspiratory flow-resistive load. J AppL PhysioL 67:1747-1753.48. Scardella, A.T., JJ. Petrozinno, M. Mandel, N.H. Edelman and T.V.Santiago (1990). Endogenous opioid effects on abdominal muscle activityduring inspiratory loading. J AppL PhysioL 69: 1104-1109.49. Sieck, G.C., A. Mazar and M. J. Belman (1985). Changes in diaphragmaticEMG spectra during hyperpneic loads. Respii PhysioL 61: 137-152.50. Vollestad N.K. and O.M. Sejersted (1988). Biochemical correlates offatigue. Europ. J. AppL PhysioL 57: 336-347.3251. Westerbiad, H.J.A., 3. Lee, J. Lannnergren and D.G. Allen (1991). Cellularmechanisms of fatigue in skeletal muscle. Am. J PhysioL 261: C195-C209.52. Wragg, S., C. Hamnegard, J. Road, J. Goidsone, M. Green and 3. Moxham(1993). Twitch Pdi depends on contractile history. Am. Rev. Respu Dis. 147:A699 (Abstract).53. Yan, S., T. Similowski, A.P. Gauthier, P.T. Macklem, and F. Bellemare(1992). Effect of fatigue on diaphragmatic function at different lungvolumes. J AppL PhysioL 72: 1064-1067.33II: Diaphragm Activation and Phrenic Nerve Impulse Propagation DuringProlonged Inspiratory Resistive LoadingIntroductionExperimental animals subjected to inspiratory flow resistive loads breathespontaneously against loads sufficiently intense to cause a decrease in alveolarventilation and result in hypercapnic ventilatory failure. Several investigatorshave suggested that diaphragm fatigue is an important, if not predominant,determinant of hypercapnic ventilatory failure in these studies (Aldrich andAppel, 1985; Aldrich 1987, 1988, 1991; Alexandrovna and Isaev, 1990; Bazzy andHaddad, 1984; Bazzy and Donnelly, 1993; Mayock et aL 1987, 1991; Oliven etaL 1988).Failure of processes proximal to the diaphragm during inspiratory resistiveloading has been hypothesized in studies which show that indices of fatigue atmore distal sites [such as diaphragm pressure response to supramaximal tetanicstimulation of the phrenic nerve (frequency-pressure curve), diaphragmelectromyogram centroid frequency and the concentrations of glycogen andlactate in the diaphragm] remain unchanged (Aldrich, 1988; Watchko et aL, 1988;Ferguson et aL, 1990). In the rabbit and sheep models of inspiratory resistiveloading, electromyographic evidence for neuromuscular transmission failure has34been documented (Aldrich, 1987; Bazzy and Donnelly, 1993). However, Mayocket aL found no electromyographic evidence for neuromuscular transmissionfailure (Mayock et aL, 1991; 1992) in the piglet with a similar fatiguing load.More proximally, the activity of the phrenic nerve (ENGdi), an index of centraldrive to the diaphragm during inspiratory resistive loading, has recently beenexamined in the cat and sheep. According to these studies, there is no decreasein neural drive to the diaphragm at a time when a drop in pressure generationof the diaphragm (Pdi) is observed (task failure). However, decreases in ENGdiare observed in the period following task failure or when higher loads areapplied (Bazzy and Donnelly, 1993; Aleksandrovna and Isaev, 1990).The role of central and neuromuscular fatigue during inspiratory resistive loadingtherefore remains controversial. We set out to answer two questions in a seriesof studies on rabbits subjected to inspiratory resistive loads of varying intensitiesand durations 1) Does neural activation of the diaphragm decline withinspiratory resistive loading? 2) Does neuromuscular transmission to thediaphragm decrease during inspiratory resistive loading? We describe the effectsof four inspiratory resistive loads on ventilation and diaphragm function in theanesthetized rabbit. Loads 1 and 2 were applied for a four hour period. Load1 was less and Load 2 was equivalent to loads previously reported to besufficient to cause diaphragm fatigue in the rabbit (Aldrich, 1985, 1987). Loads353 and 4 were greater in intensity and they are applied in a stepwise fashion untiltask failure. Our studies show that both neural activation and neuromusculartransmission to the diaphragm remain intact even under extreme loadingconditions when the respiratory muscles failed as pressure generator during tidalventilation.MethodsAnimals and groups. Thirty three, male, New Zealand White rabbits wereobtained from Geo-Bat Rabbitries (Abbotsford, B.C.) and cared for accordingto the principles outlined by the Canadian Council for Animal Care at theAnimal Resource Unit facility at the University Hospital (U.B.C). Experimentalprotocols received ethical approval from the University of British ColumbiaAnimal Care Committee.Preparation. The rabbits were anesthetized with intramuscular injection ofKetamine (Ketavet, Parke-Davis, 30 mg/kg) and a sedative, Xylazine (Rompun,Bayer, 7 mg/kg) in the hind limb. Anesthesia was maintained throughout thestudies by supplementing half the initial dose every 30-40 minutes. Rectaltemperature was continuously monitored and maintained between 37.5-39°C witha heating pad. Saline was infused via a marginal ear vein to maintain bloodpressure.36A schematic diagram of the experimental setup is shown in Figure 5. Rabbitswere placed in the supine position and the trachea was cannulated (I.D. = 4 mmstainless steel L shaped tubing) and connected to a heated pneumotachograph(Fleisch # 00) in series with a miniature two way non-rebreathing valve (HansRudolph no. 2814; 2.5 ml dead space). Pressure across the pneumotachographwas measured with a differential pressure transducer (± 2 cm H20, Validyne MP45) and a carrier preamplifier (Gould model 13-44615-35). The carrier outputwas electronically integrated (Gould integrator amplifier 13-4615-70) to recordtidal volume (calibrated for a range of 5-30 ml). An adjustable needle valve wasplaced at the inspiratory port of the non-rebreathing valve to apply flow resistiveloads. Supplemental oxygen was provided at the inspired port throughout theexperiments and adjusted to prevent hypoxia when possible. The left carotidartery was cannulated with polyethylene tubing (PE 150, Clay Adams,Parsippany, N.J.) to measure blood pressure and to sample blood for blood gasand pH analysis (Model 168 pH/blood gas analyzer, Corning Medical, Medfield,MA).The left phrenic rootlet (C4) was exposed at the neck. The nerve was cleanedthen secured in contact with bipolar platinum electrodes and immersed in a poolof mineral oil. Whole phrenic nerve discharge activity recorded from the intactnerve was amplified (Grass P5 series AC preampliers; Grass Instruments Co.,Quincy, MA), filtered (100 and 10,000 Hz low and high frequency cutoffs) and37whole wave rectified. The moving average (time constant = 0.1 sec) wascomputed using a filter with Paynter response (EMG signal processor, RaytechInstruments, Vancouver, Canada). The phrenic nerve discharge activity wasmonitored on an oscilloscope (Gould DST 1421) and the moving average(ENGdi) amplified (Gould medium gain DC preamplifier model 13-4615-10) andrecorded on an 8 channel chart recorder (Gould model 8188-812, Cleveland,OH).To record the diaphragm electromyogram and evoked compound diaphragmpotentials (M-waves), a midline upper abdominal incision was made and theuninsulated tips of two multi-stranded stainless steel fine wires (Cooner wire#AS 631) were sutured 1 cm apart into the left costal hemi-diaphragm midwaybetween the costal margin and central tendon. The moving average of theelectromyogram (EMGdi) was processed as described above for the phrenicnerve signal and continuously recorded on the chart paper. M- waves wereelicited during expiration at zero flow. Single shocks (0.2 msec duration) weredelivered to the left phrenic nerve via a Grass constant current stimulator(model S48 equipped with a PSIU6E stimulus isolation unit) and the M-waveswere displayed on the oscilloscope. The current stimulus was increased untilmaximum M-wave amplitude was observed and then set at five times thisthreshold during the experiment.38Transdiaphragmatic pressure (Pdi) was measured using two air filled balloon-catheters assembled from polyethylene tubing (PE 200 Clay Adams, Parsippany,N.J.) and 5 cm long latex balloons (A&E Corp., Farmingdale, NJ.). Oneballoon catheter was placed through the abdominal incision underneath thediaphragm (Pab) and a second balloon-catheter was placed transorally into theesophagus (Pes). The catheters were placed across a differential pressuretransducer (Validyne, Northridge, CA) to measure Pdi as Pab-Pes. Theesophageal catheter was positioned where the greatest negative peak pressurecould be obtained during tidal inspiration. Airway pressure (Pao) was measuredat the tracheal tube using a differential pressure transducer (Validyne,Northridge, CA). To ensure an adequate translation of pleural pressure toesophageal pressure, both airway pressure and esophageal pressure wererecorded during an occluded inspiratory effort at end expiratory lung volume.A pressure difference of less than 10% in the two pressures was deemedacceptable.39Figure 5. A Schematic Diagram of the Experimental Setup.LIDPhrenic Ne,ve StimulationPhrenw Neive Recording (ENGdI)I.V SalineArterial Blood Gases & pHArterial Blood PressureclosedduringEMGdiBody Temperature41Protocol A: The effect of prolonged mspiratoiy resistive loads on activation of thediaphragm.After a 20 minute period of stabilization, baseline measurements were made.Resistive loading was applied at end expiration in two groups of seven rabbits.This was accomplished by adjusting the inspiratory needle valve to increaseswings in Pdi to approximately 1.5 (Load 1) or 5 times baseline values (Load 2).Inspiratory resistive loads were maintained for a period of 4 hours. Thefollowing variables were measured hourly. Arterial blood (0.3 ml) was sampledin heparinized syringes and arterial blood gases and pH measured within oneminute of sampling, corrected for rectal temperature. Arterial bicarbonateconcentration was calculated based on predicted equations. Pressures generatedby the respiratory system (Pao, Pdi), ENGd1, EMGdi, airflow, tidal volume,arterial blood pressure were recorded continuously. The mean value for tidalvolume, breathing frequency, peak inspiratory pressures (Pao, Pdi) and peakENGdi and EMGdi were calculated hourly during a representative 60 secondinterval. Duration of inspiration (TI) and expiration (TE) were determined fromthe electromyographic recordings. TI was defined from the EMGdi as the timefrom initial rise to the point where a rapid decline was first observed. TEspanned the time from the rapid decline in the EMGdi to its next initial rise. Ina separate sham control group (n = 6), rabbits were allowed to breathespontaneously for 4 hours, without any resistive load, under the same anesthetic,surgical and variable measurement regimen as the loaded group.42Protocol B: The effect of severe incremental inspiratoiy resistive loads on activationand neuromuscular transmission to the diaphragm.Another group of rabbits (n =13) were subjected, in a stepwise fashion, to twointense inspiratory resistive loads sufficient to produce additional 10-15 cm H20increments in airway inspiratory pressure (Loads 3 and 4). Following baselinemeasurements, arterial blood samples, peak ENGdi, EMGdi and respiratoryvariables were analyzed every 10 minutes. The integrity of neuromusculartransmission to the diaphragm was determined by evoked diaphragm potentials(M-waves) obtained in duplicate every 10 minutes. Baseline measures of Mwave amplitude (peak to peak, ptp) were made both before and immediatelyfollowing the imposition of inspiratory resistive loads. The baseline M wave wasstored on the oscilloscope and compared to the M waves monitored during theloaded period. To ensure that the stimulus was maximal, M-wave thresholdswere confirmed after each set of M-wave recordings.Data Analysis.All variables were statistically compared before and after inspiratory loadingusing a paired Student’s t-test. A Friedman’s ANOVA (repeated measures) wasused to detect any significant changes during the loaded period, followed by aWilcoxon signed rank test to determine which values were significantly different43from each other (Systat 5.02). Statistical significance was defined as P <0.05. Allvalues represent mean ± S.E.M.ResultsThe inspiratory resistive loads are defined in this study in terms of mean targetpeak airway inspiratory pressures (Pao). These target loads and the equivalentresistances are tabulated in Appendix I along with values for loads reported inthe literature in other animal studies of inspiratory resistive loading.Protocol A: The effect of prolonged uzspiratoiy resistive loads on activation of thediaphragmFollowing application of inspiratory resistive loads, ENGdi increased graduallyand reached a steady state level within 10-15 minutes. This level of diaphragmactivation was maintained throughout the 4 hour loading period and was directlyproportional to the severity of the load. Parallel changes in transdiaphragmaticpressure swings (Pdi) and diaphragm phasic inspiratory activity (EMGdi) wereobserved during the 4 hour period (Figure 6). The ENGdi:EMGd1 ratio did notchange during the loaded period with either load (Table I). Figure 7 shows thechange in transdiaphragmatic pressure swings (Pdi) for the target inspiratorypressures (Pao) achieved in the two groups of rabbits exposed to prolonged44inspiratory resistive loads and in the sham control group.Within the first hour breathing against Loads 1 and 2, ventilation (<rE) was foundto drop 15% and 40% respectively relative to baseline (Figure 8). For theremainder of the loaded period, ventilation (‘TE) was maintained (Figure 8).Breathing against Load 1, the initial drop in VE resulted entirely from a decreasein breathing frequency (44± 5 versus 34± 4 min.’, p <0.05). The frequency ofbreathing (Bf) decreased due to an increase in Ti (0.69± 0.08 versus 0.84± 0.05sec. p < 0.05). There was no significant change in tidal volume or expiratory time.In contrast, breathing against Load 2, the initial drop in ‘CTE resulted from adecrease in Bf (44± 5 versus 31± 3, p <0.05) and a decrease in VT (Table I). Thefrequency of breathing decreased due to an increase in both inspiratory andexpiratory times (Table I).During loaded breathing there was a gradual decrease in both pH and a gradualincrease in PaCO2 (Figure 8). Pa02 was maintained above 100 mm Hg and nosignificant change in arterial bicarbonate levels was detected throughout the 4hours of loaded breathing (Table I). In the sham control group, no significantchanges in respiratory variables were observed (Figures 6 and 7). Blood gasanalysis showed only a decrease in pH after 3 hours of anesthesia (Figure 8).45Figure 6: Peak electrical activity of the phrenic nerve (ENGdi), the costaldiaphragm (EMGdI) and transdiaphragmatic pressure swings (Pdi) at baseline (B)and during prolonged inspiratory resistive loads (. load 1, n=7; • load 2, n=7)in anesthetized rabbits. Six sham control rabbits (0) breathing spontaneouslywithout imposition of a resistive load under the same anesthetic regime.*= Significantly different from baseline (B), p < 0.05.0 0 ci CD ci 0 0 D(-0 C (I)PdI(cmH20)IIIIIIIEMG(%baseline)ENC(%baseline)—iN.)C’4-01—iN)C.J-01--iN)NJCJ00000000000(31001001001000000000000uJ*III*QJ—-*1”)-*IIII**cJ*****47Figure 7. Peak inspiratory airway pressure (Pao) and transdiaphragmaticpressure swings (Pdi) at baseline (B) and during prolonged inspiratory resistiveloads (.load 1, n=7; • load 2, n=7) in anesthetized rabbits. Six sham controlrabbits (0) breathing spontaneously without imposition of a resistive load underthe same anesthetic regimen.*= Significantly different from baseline (B), p <0.05.0EC)-o0—35—s —300IE—20C-)0Ca10—50***35302520151050* *B 1 2 3 4Loaded period (hours)* * **** * *B 1 2 3 4Loaded period (hours)49Figure 8: Changes in arterial pH, pCO2 and ventilation ((rE) during prolongedinspiratory resistive loads (. Load 1, n = 7; • Load 2, n = 7) in anesthetizedrabbits. Six control rabbits (0) breathing spontaneously without imposition of aresistive load.*= Significantly different from baseline (B), p < 0.05.t = Significantly different from the first hour of loaded breathing, p < 0.05.VE(%baseline)0 U U-CD U--c CD 0 ci0 C 0,-i4o1o)-JcYJcD000000000IIIIIIIIIPOCO2(mmHg)O(D0-N)0000000000IIIIIIII-IV-F•i*I1*\_-___-I-_LCJ00pHbU,r\J-IIU,r’J (,1**51Table I: Mean (± S.E.M.) values for respiratory variables at baseline and duringLoad 2 (n=7).Duration of Loading (hours)Baseline 1 2 4VT (ml) 18(2) 15(1)* 15(1)* 13(1)*t 13(1)*tBf 44(5) 31(3)* 32(3)* 31(3)* 31(3)*(min’)ENGdi/ 1 0.8 0.9 0.9 0.9EMGdi (0.0) (0.3) (0.4) (0.4) (0.4)VT/TI 25(5) 16(3)* 16(2)* 15(2)* 14(2)*t(mi/sec)TI/TrOT 0.50 0.47 0.47 0.44 0.45(0.02) (0.03) (0.03) (0.03) (0.03)Pa02 258 239 214 206 197(mm Hg) (42) (27) (31) (33) (29)[HCO3] 24 24 27 28 26.4(mM/L) (1) (1) (1) (1) (1.2)TI (sec) 0.7 1.0* 1.0* 0.9* 1.0*(0.1) (0.1) (0.2) (0.1) (0.1)TE (sec) 0.7 1.0* 1.1* 1.2* 1.2*(0.1) (0.1) (0.2) (0.2) (0.2)*p <0.05 compared to baseline.tp <0.05 compared to value at other loaded periods.52Protocol B: The effect of severe inspiratoiy resistive loads on activation andneuromuscular transmission to the diaphragm.Loads 3 and 4 resulted in a sustained four to six fold increases in both ENGd1and EMGdi (Figure 9). There was no significant change in the ENGdi to EMGdIratio and no significant reduction in the amplitude of the evoked compounddiaphragm potentials observed during loading (Table II). Nor was there anysignificant change in the duration of M-waves. In fact, the compound actionpotentials were superimposable and no significant change in peak to peakamplitude was observed during loaded breathing (Figure 13, panel A).Application of Loads 3 and 4 produced target inspiratory pressures exceeding-30 cm H20. The increase in resistance required to achieve these extreme targetpressures was associated with arousal and swallowing. Arousal due to thesudden imposition of extreme inspiratory resistive loads was circumvented by agradual increase of inspiratory resistance via the needle valve until the desiredtarget peak Pao was obtained. Target loads were established within 10 minutes.Rabbits swallowed throughout load 3 and load 4. Swallowing was reflected aspositive pressure swings in the esophageal pressure recordings. Accordingly, acontinuous estimate of pleural pressure by recording esophageal pressure couldnot be obtained. For this reason, the peak inspiratory pressure generated by therespiratory system at tracheal opening (Pao) is shown as an index of mechanical53Figure 9: Peak electrical activity of the phrenic nerve (ENGdi) and the costaldiaphragm (EMGd1) at baseline (B) and during severe loads. Failure to sustaininspiratory pressure, designated “F” on the abscissa, occurs 40-60 minutes afterinitiation of Load 4.*= Significantly different from baseline, p < 0.05.(1)C>C541 20011001000a) 900800Load4Load3V•ENGI IEMG* ****6005004003002001000B 10 20 30 40 50 10 20 30 FLoaded period (minutes)55failure of the respiratory muscles. Pao increased with graded loading. There wasa linear relationship between Pao and EMGdi and between Pao and ENGdi inboth individual rabbits and among the groups of rabbits studied at differentinspiratory resistive loads (Figure 10). The highest Pao was obtained with Load4 and could be sustained for 30 minutes (Figure 11). Thereafter, Pao droppedsignificantly within 10 to 30 minutes (represented as time “F” on the abscissa,Figure 11). Pao decreased from 52± 1 to 42± 2 during this period indicatingfailure of the respiratory muscles to maintain a target force (task failure).56Figure 10: The relationship between peak inspiratory pressure (Pao) and indicesof activity (top panel) and drive to (bottom panel) the diaphragm. Symbolsrepresent the mean ± S.E.M. for the following groups: 0 baseline (n=7);• Load 1 (n=7); • Load 2 (n=7); v Load 3 (n=8); Load 4 (n=8).Solid lines represent first order regression, r = 0.99. Dashed lines correspond to95% confidence limits for the regression.ciCciC’,QciCa)C’,C-QzLU80070010030 40 50 60800700600500400300/-o02001000 I...._i I I I I I I0 10 20 30 40 50 60Pao (cm H20)////600500400300200//////0/II /I j • I I I •0 10 20Pao (cm H20)58Figure 11: Changes in arterial blood gases (Pa02, PaCO2) and negativeinspiratory pressures recorded at the airway (Pao) at baseline (B) and duringsevere inspiratory resistive loads in anesthetized rabbits (n=8). Failure to sustaininspiratory pressure designated “F” on the abscissa occurs 40-60 minutes afterinitiation of Load 4.*= Significantly different from baseline, p <0.05.t = Significantly different from the previous loaded period, p < 0.05.S90IE‘30ci0.4—65—60—55—50—45—40—35—30—25—20—15—10—50Load311IAILoad 4V*/1*Y*tI*fE1p.m0)=EE0C)02001801 601 401 201 00806040200B 10J__L_L_ jL L20 30 40 50 10 20Loaded period (minutes)30 F60Severe loading resulted in a 40-45% decrease in minute ventilation (Figure 12)along with a decrease in mean inspiratory flow rate (VT/TI) and an increase inrespiratory times (Table II). No change in inspiratory duration was seen withdecreased inspiratory pressure. However, a significant increase in expiratoryduration resulted in a decreased duty cycle (TI/Pr0T) (Table II). In addition,Load 4 led to significant acidemia (Table II), hypercapnia, and hypoxemia(Figure 11).Introduction of the severe loads resulted in a pneumothorax in five rabbits.These animals were withdrawn from the study. A sudden change in breathingpattern and a large reduction in M-wave amplitude was observed in all rabbitsthat developed a pneumothorax. Post mortem examination was carried on allrabbits whose data was included in the study (n = 8) to confirm an intact pleura.61Figure 12: The average decrease in minute ventilation (‘7E) during severeinspiratory resistive loading as percent of baseline (B) value. Failure to sustaininspiratory pressure designated “F” on the abscissa occurs 40-60 minutes afterinitiation of Load 4.*= Significantly different from baseline (B), p <0.05.= Significantly different from the previous loaded period, p <0.05.2100 d3 d480- V* * *C T * * T60 I *IIINTI I I I Ij I I I I I0 10 20 30 40 50 10 20 30 FLoaded period (minutes)63Table II: Mean (i S.E.M.) values for respiratory variables at baseline andduring Load 4 (n = 8).Duration of Loading (minutes)Baseline 10 20 30 FVT (ml) 18(2) 15(2)* 13(1)* 12(1)*Bf 56(5) 38(6)* 38(5)* 39(4)* 31(4)*t(1/mm)ENGd1/ 1.0 1.2 1.2 1.4 1.7EMGdi (0.0) (0.2) (0.2) (0.4) (0.5)VT/Ti 31(3) 21(2)* 21(2)* 19(2)*(mi/sec)0.50 0.43* 0.43* 0.41*TI/TrOT (0.02) (0.02) (0.02) (0.02) (0.02)pH 7.36 7.14* 7.10* 7.06*(0.01) (0.03) (0.03) (0.03) (0.04)[HC03] 27 28 29 28 28(mM/L) (0.4) (0.6) (0.7) (0.7) (0.7)M wave 3.9 3.9 3.5 3.4 3.2(ptp-mV) (0.4) (0.5) (0.4) (0.4) (0.4)Ti (sec) 0.6 0.7* 0.7* 0.7* 0.7*(0.0) (0.1) (0.1) (0.1) (0.1)TE (sec) 0.6 1.2* 1.1* 1.1* 1.6*4(0.1) (0.2) (0.2) (0.2) (0.4)*p <0.05 compared to baseline.<0.05 compared to value at other loaded periods.tp <0.05 compared to value at 30 minutes.64DiscussionThis study demonstrates that despite hypoventilation and significant changes inblood gases, both activation of and neuromuscular transmission to the diaphragmare maintained throughout prolonged and severe inspiratory resistive loadingeven when the target inspiratory pressure is no longer sustained and a criticallevel of Pa02 is reached (Figure 11, load 4). Additionally, we find no significantchange in the ratio between the activity of the phrenic nerve and that of thediaphragm (ENGdi:EMGdi) throughout loaded breathing at all intensities (Loads1-4). These results provide additional support to the hypothesis that there is nosignificant change in neuromuscular transmission during both sustainable andexhaustive inspiratory resistive loads.Evaluation of MethodsThe study of anesthetized animals breathing spontaneously against inspiratoryloads offers two distinct advantages in determining the locus (loci) of failure inexperimental fatigue of the diaphragm. In the awake subject, apparent failureof the diaphragm as a pressure generator may, in fact, be due to loss ofmotivation of the subject unwilling to breathe against intolerable inspiratoryloads. This behavioral response is predictable and has been documented inuntrained subjects asked to breathe against inspiratory resistive loads. The same65loss of motivation may underlie performance decrements in the awake animalwithout the need to resort to the notion of fatigue as an explanation. Therefore,the first benefit of this protocol is that in the anesthetized preparation, it isassumed conscious factors such as motivational fatigue do not play a role indiaphragm performance. The second advantage is that spontaneous breathingagainst an inspiratory resistive load allows fatigue to develop without the needto impose a non-physiological pattern of diaphragm activation such as electricalstimulation.Ketamine-xylazine anesthesia was chosen for several reasons. First, it has a widemargin of safety when administered intramuscularly. Secondly, it is one of thefew anesthetics which does not depress spontaneous breathing. Finally, its usepermits comparison of our study with results obtained in previous studies ofinspiratory loading in the rabbit all of which have used the same anesthetic agent(Aldrich and Appel, 1985; Aldrich 1987, 1988; Ferguson Ct aL, 1990).Ketamine alone produces a so-called “dissociative” anesthetic state that has beendescribed as a functional and electrophysiological dissociation between thethalamo-neocortical and limbic systems. This unique state is characterized bycatalepsy involving unconsciousness and somatic analgesia without muscularrelaxation (White et aL, 1982). In combination with xylazine, a potent hypnoticwith central muscular relaxant properties, ketamine provides adequate analgesia66and muscle relaxation for surgical procedures (Borkowksi et aL, 1990).Hypotension, hypercapnia and respiratory acidosis and hypoxia are characteristiccardiopulmonary effects of nonvolatile anesthetics in rabbits including ketaminexylazine anesthesia (Borkowksi et aL, 1990). We documented the effects of ouranesthetic regime on ventilation, PaCO2 and arterial pH over a 4 hour period onrabbits (see control group, Figure 8). Prolonged anesthesia alone did not resultin any significant change in ventilation or PaCO2 from baseline. Resting valuesof PaCO2 in the awake rabbits range from 25 to 37 mm Hg (Honda, 1968;Gauthier, 1973) . The range of baseline values of PaCO2 in our anesthetizedpreparation are considerably higher (37-48 mm Hg) owing to the effects ofhyperoxia and anesthesia (Honda, 1968; Borkowski et aL, 1990). Hypoxia andhypotension were purposely avoided (by design), in order to assess diaphragmfatigue independent of these factors. After 3 hours of anesthesia a slightdecrease in arterial pH was observed. This change is likely due to the repetitiveketamine [hydrochloride] administration employed in our protocol.Neuromuscular Transmission to the DiaphragmThe occurrence of neuromuscular transmission failure during maximal voluntarycontractions in skeletal muscle remains open to debate. It has been suggestedthat conflicting findings may be due to methodological differences (BiglandRitchie, 1987). Merton (1954) was first to show the integrity of neuromuscular67transmission during voluntary contractions in the adductor pollicis. He reportedno decrease in the size of muscle compound action potentials (M-waves) evokedby single maximal shocks to the ulnar nerve during sustained maximalcontraction of the adductor pollicis muscle despite a force decay to almost zero.Stephens and Taylor (1972) presented conflicting results using the firstinterosseous muscle. In a subsequent series of experiments, Bigland-Ritchie andcoworkers reexamined the adductor pollicis and first interosseous muscles of thehand as well as other limb muscles and demonstrated that neuromusculartransmission is preserved during maximal voluntary contractions (see Thomas etaL, 1985) not withstanding force decay in the muscle.In the diaphragm, neuromuscular transmission, as assessed by the M-wave,remains intact in humans following both severe inspiratory resistive breathing(Aubier et aL, 1981, 1985, Yan et aL, 1992) and maximal expulsive manoeuvres(McKenzie et aL, 1992) which result in a decrease in Pdi. Similarly, duringprolonged inspiratory resistive breathing in the anaesthetized piglet (Mayock etaL 1991) the M-wave does not change despite a drop in the frequency-pressurecurve of the diaphragm. In contrast to these results, Aldrich (1987) has showna reversible 44% decrease in diaphragm M-wave amplitude in the anesthetizedrabbit after a period of 58± 14 minutes of inspiratory resistive loading underrelatively moderate loads (adjusted to an average Pao = 27 cm H20). Bazzy andDonnelly (1993) have reported similar changes in the diaphragm M-wave68amplitude of two awake sheep breathing against severe inspiratory resistiveloads. These changes were accompanied by an increase in the ENGdi:EMGd1ratio of the diaphragm prior to failure of the diaphragm to generate inspiratorypressure in the awake sheep (Bazzy and Donnely, 1993).In our studies, we have demonstrated that anesthetized rabbits can generatesimilar inspiratory pressures (Load 2) as the pressures observed in the Aldrich(1987) study for 4 hours without a significant change in the ENGdi:EMGd1 ratio.Furthermore, we observed no significant change in the ENGdi:EMGd1 or Mwave, two indices of the integrity of neuromuscular transmission to thediaphragm during two successive periods of sustained inspiratory pressure at evengreater loads (Load 3 and Load 4).Methodological differences may explain the conflict between our findings andthose of Aldrich (1987) and Bazzy and Donnelly (1993). It has been shown thatinspiratory resistive breathing causes a decrease in end expiratory lung volume(Mayock et aL, 1987; Oliven et aL, 1988). We were particularly wary of thepossibility that the M-wave could be altered by such an artifact. Indeed, in ourpilot studies, the M-wave amplitude decreased immediately upon loading butremained constant throughout loading. Sudden removal of the load resulted inan immediate return of the M wave amplitude to baseline levels (Figure 13,panel B). In our view, the shift in baseline M-wave amplitude upon loaded69breathing makes the M-wave obtained immediately upon loading a bettermeasure of baseline M-wave. The shift in M-wave with loading probably resultsfrom changes in muscle length (Kim et aL, 1985) and/or a change in the volumeconductivity of the surrounding tissues (see Brancatisano et aL, 1989). It isunlikely that changes in volume conductivity affected our recordings because thediaphragm electrodes were placed away from the area of apposition whererecordings are vulnerable to such influences (Grassino, Whitelaw and MilicEmili, 1976). The most likely explanation for the immediate shift in the M-waveupon loading is a change in diaphragm length in response to severe inspiratoryresistive loading.In addition to a decrease in M-wave amplitude due to mechanical artifact suchas a change in muscle length, other distinct limitations in the use of M-waveamplitude have been documented. For example, the M-wave amplitude ofshocks delivered during contractions produced by tetanic simulation or sustainedvoluntarily can increase transiently early in tetany [pseudo-facilitation] (Hicks etaL, 1989). It is important to note, however, that changes associated with M-waveamplitude during sustained contractions do not preclude the comparison of Mwave amplitude generated during periods of rest in an intermittently activemuscle. In our study, we observed no significant change in the amplitude or areaof diaphragm M-wave generated during expiration throughout loaded breathing.70Figure 13: Panel A: Representative evoked diaphragm compoundpotentials (M-waves) at baseline and at time intervals during thesevere inspiratory resistive loads. 13 = Load3; LA = Load 4;“F” = at time of pressure failure.Panel B: An example of shift in baseline evoked diaphragmcompound potential (M wave) upon loaded breathing.0.5 m V2 msec.9.,L4, 10 mm 50 mm loadedLoad removed72Constant Diaphragm Activation during Prolonged LoadingHypercapnia inevitably followed inspiratory resistive loading during breathingagainst all loads examined in our study. It is interesting that with prolongedloading (Loads 1 and 2), neural activation of the diaphragm (ENGdi) ismaintained at a constant level despite rising PaCO2 (Figure 6). Clearly, maximalneural activation of the diaphragm is not reached with these loads since applyinga greater load (Load 3) results in higher electrical activity of the phrenic nerve(ENGdi) and electromyographic activity of the diaphragm (EMGd1) (Figures 6and 9). A reduction in responsiveness to increasing Pco2 may be due entirelyto central effects of prolonged (greater than one hour) exposure to high levelsof PaCO2 alone. Alternatively, it has been hypothesized that the augmentedactivity of group III and IV muscle afferents during inspiratory resistive loadingproduce an inhibition of drive to respiratory muscles, possibly by the release ofendogenous opioids (Petrozzino et aL 1992). Such feedback inhibition mayaccount for the constant activation of the diaphragm seen during prolongedinspiratory resistive loading.The effect of prolonged exposure to hypercapnia on activity of the diaphragmand ventilation is examined and described using this rabbit preparation inChapter III.73Prolonged Inspiratoiy Resistive Loading and Indices of Diaphragm FatigueCurrently, there are no universally accepted indices of diaphragm fatigue.Studies examining inspiratory muscle function in anesthetized animals duringinspiratory resistive loading define fatigue as a decline in the frequency-pressurecurve of the diaphragm in response to supramaximal tetanic stimulation of thephrenic nerves. However, it has been shown that the pressure generated by thediaphragm (Pdi) and its activity (EMGdI) are maintained during six hours ofinspiratory resistive loading despite a decline in the frequency-pressure curveobserved within the first hour of loaded breathing (Mayock et aL 1991).Consequently, a drop in the frequency-pressure relationship of the diaphragmdoes not correlate with the activity (EMGd1) or the pressure it generates duringspontaneous breathing. Similarly, in our study, target Pdi is maintained for 4hours under target pressures (Load 2) which have been shown to result in asignificant decrease in the frequency-pressure curve within one hour using thispreparation (Aldrich, 1987). Such a functional loss in maximal pressuregenerating capacity of the diaphragm in response to electrical stimulation reflectsfailure of all motor units to respond to supramaximal stimuli. However, unlikeepiphrenic electrical stimulation, phrenic motor unit recruitment is not uniformduring spontaneous loaded breathing (Cairns and Road, 1993). Therefore,pressure loss during electrical stimulation does not necessarily require the74conclusion that force loss will arise in response to spontaneous loaded breathing.In support of this view, we have demonstrated that inspiratory pressures higherthan those previously reported in the literature (e.g. Aldrich, 1985, 1987, 1988;Ferguson et aL, 1990) can be maintained in the anethetized rabbit for periods upto or greater than 30 minutes. Furthermore, failure to maintain target pressureis associated only with the most severe load (Figure 12, load 4). Targetinspiratory pressures (Pao) achieved under this extreme load are within thereported range of maximum Pdi obtained during supramaximal stimulation of thephrenic nerves in rabbits with bound abdomens (55 ± 5 cm H20; Aldrich 1985,1987, 1988, Ferguson et aL, 1990) and well above those previously achieved inthe spontaneously breathing loaded animals prior to task failure (35± 2.6 cmH20; Ferguson et aL, 1990). Figure 14 shows the relationship between Pao andPdi during brief loads in this preparation. It is clear that there is a good 1:1relationship between Pao and Pdi at pressures between 10-35 cm H20. At higherloads, Pao overestimates Pdi by approximately 5 cm H20 indicating thatperformance seen under Load 4 reflects the extreme sustainable pressure of thediaphragm. In support of this view, Cairns and Road (1993) have shown that allphrenic motoneurons examined under inspiratory resistive loading are recruitedby Pdi = 40 cm H20 and many fire at very high rates of activation (approaching80 Hz).75Figure 14: The relationship between peak inspiratory pressure (Pao) andtransdiaphragmatic pressure (Pdi) swings in two rabbits during brief inspiratoryresistive loading. Airway pressures of -55 cm H20 measured during maximalload (Load 4) compare to Pdi swings of 50 cm H20. Symbols represent peakvalues during single inspirations.70c\J00CPdi (cm H20)EC)0C060-5040-30.20-100-60-50400cpl, Nov180 10 20PdiI I30 40 50 60(cm H20)Q8cp6, Nov18-20-100 0 i I I I I0 10 20 30 40 50 6077Task Failure of the Respirato,y Muscles during Severe Inspiratoiy Resistive LoadingSeveral factors may contribute to our subjects’ failure to maintain the targetinspiratory pressure observed under Load 4. First, hypercapnia has beenreported to attenuate diaphragm contractility in humans, dogs and piglets (Juanet aL, 1984; Schnader et aL, 1985; Watchko et aL, 1987). It is important to notehowever that the effect of high levels of carbon dioxide on diaphragmcontractility are assessed by the frequency-pressure technique. As mentionedabove, this technique measures the maximal contractile response (pressureoutput) of the diaphragm to a supramaximal stimulus which generates anartificial pattern of muscle fibre recruitment. Whether the same mechnismsleading to loss of maximal contractility contribute to diaphragm task failure isopen to debate. In the rabbit, in particular, it has been shown that short termC02 rebreathing does not affect diaphragm contractility (Aldrich and Appel,1985). During each of the loads applied in this study, hypercapnia is severe andlong term. Consequently, any change in diaphragm contractility that wouldcontribute to the loss in pressure generation by this muscle should be manifestunder all the loads examined and relatively early on. Since the activity of thediaphragm (EMGdi) and its ability to maintain pressure were maintainedthroughout loading for prolonged periods it seems unlikely that any change inthe contractility of the diaphragm due to hypercapnia contributed to the loss inpressure generation.78Hypoxemia is a second factor that could result in respiratory muscle task failureor central nervous system depression, or both. As we were unable to measurePdi during the extreme load at the time when inspiratory pressure (Pao)decreased, it is possible that the diaphragm or other respiratory musclesactivated during the load, or both, displayed peripheral (myogenic) fatigue. Onestudy has examined biochemical correlates of diaphragm fatigue duringinspiratory resistive loading in the rabbit (Ferguson, Irvin and Cherniack, 1990).In this study, respiratory arrest induced by incremental inspiratory thresholdloading resulted in the same Pa02 as measured in our study at task failure withno evidence of contractile fatigue of the diaphragm or alterations in diaphragmglycogen or lactate concentrations. Furthermore, the role of hypoxia relateddepression of central output to the respiratory muscles other than the diaphragmcannot be excluded since there seem to be differences in susceptibility of theseoutputs to hypoxia (Fregosi et aL, 1987; Neubauer, Ct aL, 1990). Hence, the lossof inspiratory pressure observed in our study could have been due to peripheralfatigue of the respiratory muscles or to hypoxic depression of output to therespiratory muscles.In conclusion, although it would be difficult to predict to what extent theprogressive and severe perturbations in arterial Pc02, P02 and pH alone orinteractively contribute to respiratory muscle task failure during extreme loads,the data in our studies clearly show that activation and neuromuscular79transmission to the diaphragm are maintained throughout both prolonged andsevere inspiratory resistive loads in the anesthetized rabbit.80References1. Aldrich T.K. and D. Appel (1985). Diaphragm fatigue induced by inspiratoryresistive loading in spontaneously breathing rabbits. J AppL PhysioL 59:1527-1532.2. Aldrich, T.K. (1987). Transmission fatigue of the rabbit diaphragm. RespirPhysioL 69: 307-319.3. Aldrich T.K. (1988). Central fatigue of the rabbit diaphragm. Lung 166: 233-241.4. Aleksandrovna, N.P. and G.G. Isaev (1990). Central and peripheralcomponents of the fatigue of respiratory muscles in inspiratory resistiveload in cats. Sechenov Physiological Journal of the US.S.R. 76: 658-666.5. Aubier, M., D. Murciano, Y. Lecoeguic N. Viires and R. Pariente (1985).Bilateral phrenic stimulations: A simple technique to assess diaphragmaticfatigue in humans. J AppL Physiol. 58: 58-64.6. Aubier M., G. Farkas, S. De Troyer, R. Mozes and C. Roussos (1981).Detection of diaphragmatic fatigue in man by phrenic stimulation. J AppLPhysiol. 50: 538-544.7. Bazzy, A.R. and D.F. Donnelly (1993). Diaphragmatic failure during loadedbreathing: role of neuromuscular transmission. J AppL Physiol. 74: 1679-1683.8. Bazzy A.R. and G.G. Haddad (1984). Diaphragmatic fatigue in81unanesthetized adult sheep. J AppL PhysioL 57: 182-190.9. Bigland-Ritchie, B. (1987). Respiratory Muscles and their NeuromotorControl. New York, Liss, 1987, pp. 379-390.10. Borkowksi, G.L., PJ. Danneman, G.B. Russell, and C.M. Lang (1990). Anevaluation of three intravenous anesthetic regimens in New Zealand Rabbits.Lab. Animal ScL 40: 270-276.11. Brancatisano, A., Kelly, S.M., Tully, A., Loring, S.H. and L.A. Engel (1989).Postural changes in spontaneous and evoked regional dipahragmatic activityin dogs. J. AppL PhysioL 66: 1699-1705.12. Cairns A.C. and R.D. Road (1993). Phrenic motor axon firing rates duringbrief inspiratory resistive loads. Am. Rev. Resp. Dis. 147: A702.13. Ferguson G.T., C.G. Irvin, and R.M. Cherniack (1990). Relationship ofdiaphragm glycogen, lactate, and function to respiratory failure. Am. Rev.Resp. Dis. 141: 926-932.14. Fregosi, R.F., S.L. Knuth, D.K. Ward and D. Bartlett, Jr. (1987). Hypoxiainhibits abdominal expiratory nerve activity. J AppL PhysioL 63: 221-220.15. Gauthier, H. (1973). Respiratory responses of the anesthetized rabbit tovagotomy and thoracic dorsal rhizotomy. Respir PhysioL 17: 238-247.16. Grassino, A.E., Whitelaw, W.A. and J. Milic-Emili (1976). Influence of lungvolume on electrode position on electromyography of the diaphragm. JAppL PhysioL 40: 97 1-975.17. Hicks, A., J. Fenton, Garner, S. and A. J. McComas (1989). M wave82potentiation during and after muscle activity. J. Appi. Physiol. 66: 2606-1610.18. Honda, Y. (1968). Ventilatory response to C02 during hypoxia andhyperoxia in awake and anesthetized rabbits. Respir PhysioL 5:279-287.19. Juan, G., P. Calverley, C. Talamo, J. Schnader and C. Roussos (1984).Effect of carbon dioxide on diaphragmatic function in human beings. N.Eng. J Med. 310: 874-9.20. Kim, M.J. S. Druz and J.T. Sharp (1985). Effect of musce length onelectromyogram in a canine diaphragm strip prearation. J. App. PhysioL 58:1602-1607.21. Mayock, D.E., T.A. Standaert and D.E. Woodrum (1992). Effect ofinspiratory resistive loaded breathing and hypoxemia on diaphragmaticfunction in the piglet. J AppL PhysioL 73: 1888-1893.22. Mayock, D.E., R.J. Badura, J.F. Watchko, T.A. Standaert and D.E. Woodrum(1987). 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Twiggs and D.E. Woodrum(1988). Ventilatory failure during loaded breathing: the role of central neuraldrive. J AppL Physiol. 65: 249-255.35. White, P.F., Way, W.L., and A.J. Trevor (1982). Ketamine-Its pharmacologyand therapeutic uses. Anesthesiology 56: 119-136.36. Yan, S, T. Similowski, A.P. Gauthier, P.T. Macklem, and F. Bellemare(1992). Effect of fatigue on diaphragmatic function at different lungvolumes. J. AppL Physiot 72: 1064-1067.85APPENDIX IFlow Resistances Reported in Animal Studies of Inspiratory Resistive Loading.This thesis: Rabbit (measuredLoad 1: 900 cm H20/L/secLoad 2: 3780 cm H20/L/secLoad 3: 6900 cm H20/L/secLoad 4: 7500 cm H20/L/secAldrich (1985, 1987):Mayock et aL (1987, 1991):Watchko et aL (1988):Bazzy and Haddad (1984)at flow rate of 1 Litre/minute)Target Pao = 8± 2 cm H20 (1.5 X Pdi QITarget Pao = 27± 3 cm H20 (5.5 X Pdi QITarget Pao = 39± 3 cm H20 (8 X Pdi QITarget Pao = 53± 4 cm H20 (11 X Pdi QiRabbit Target Pao = 27 cm H20Piglet 0.65 cm H20/ml/secInfant monkey 0.24-0.42 cm H20/ml/secSheep > 150 cm H20/L/sec86III: Ventilation During Prolonged Hypercapnia in the Anesthetized RabbitIntroductionAs indicated in the preceding chapter, when a flow-resistive inspiratory load isadded to the airway of the anesthetized rabbit, minute ventilation decreasesimmediately. If the applied resistance is maintained, inspiratory efforts willprogressively increase. This response is reflected in the gradual rise in peakinspiratory pressures and tidal volume which result in increased ventilation overthe next few minutes. In its final steady state, minute ventilation is lower thanthe level prior to loaded breathing in the anesthetized animal.Mechanisms that offset added mechanical loads placed on the respiratory systemare termed load compensation. In the anesthetized cat (Shannon and Zechman,1972) and rabbit (Sant’Ambrogio and Widdicombe, 1965), neural reflexes whichincrease inspiratory alpha motoneuron activity are evoked during the firstinspiratory effort after inspiratory resistive loading. These immediate loadcompensatory reflexes are mediated via thoracic dorsal roots and vagal afferentsand are responsible for the first breath response to inspiratory resistive loads.Studies in which blood gases are kept constant by cross corporeal orextracorporeal gas exchange demonstrate that the transient progressive increasein inspiratory effort after the first inspiratory resistive loaded breath is due to the87early changes in blood gas tensions alone (Orthner and Yamamoto, 1974; Bruceet aL, 1974). Thus, initially, load compensation is neurally mediated andsubsequently, load compensation involves the response of all respiratory musclegroups to changes in blood gas tensions.Few studies have examined the effect of prolonged (i.e. longer than one hour)inspiratory resistive loading on ventilation in anesthetized animals. In theprevious chapter, we have shown that during prolonged inspiratory resistiveloading, a constant level of minute ventilation is established within 10-15 minutesof the onset of the load. During the following 3 hours, despite rising PaCO2,minute ventilation is maintained, as is the frequency (Bf) and intensity ofmotoneuron output (ENGdi) to the diaphragm under both Load 1 and Load 2.Hence, load compensation is incomplete and the respiratory muscles do notincrease their work load in spite of progressive hypercapnic ventilatory failure.The constant level of diaphragm activation in our preparation despite a risingC02 stimulus could result from either competing inhibitory and excitatoryfeedback mechanisms to the respiratory centre intrinsic to loaded breathing orfrom the central effects of prolonged exposure to severe hypercapnia or both.There are few published reports on central effects of prolonged exposure tohypercapnia without hypoxia or disease. A handful of studies have examined theeffects on ventilation of prolonged (i.e. greater than 30 minutes) inhalation of88C02 (for a review see Dempsey and Forster, 1982). Most of these reportsconsidered exposure of humans to relatively low levels of C02 (up to 5%inspired C02) for several days. In these studies, a slight reduction in ventilationbetween 3-10 days of exposure to increased levels of carbon dioxide is observedas there is a slightly reduced ventilatory response to inspired C02. Few reportshave examined the effect of the first few hours of exposure to the very highlevels of C02 that would produce elevated levels of arterial PC02 of themagnitude observed during the prolonged inspiratory resistive loading used inour preparation.Bleich, Berkman and Schwartz (1964) measured the CSF bicarbonate responseto sustained hypercapnia in awake dogs exposed to 12% inspired C02. In thefirst 30 minutes of exposure to elevated FICO2, PaCO2 rose from 36 to 80 mmHg and continued to increase for 3 hours. Furthermore, both CSF and plasmabicarbonate responses to severe hypercapnia were rapid. For example, CSFbicarbonate increased by 2 mEq/L within the first 30 minutes of hypercapnicexposure and continued to increase a further 5 mEq/L over the next three hours.Although ventilation was not monitored in their study, it is likely that thedecrease in hydrogen ion drive to central chemoreceptors due to bicarbonatebuffering would cause a drop in ventilation during this period. Whether suchrapid ventilatory acclimation to high levels of C02 could account for theprogressive hypercapnic ventilatory failure in our preliminary study (Chapter II)89is unknown. Accordingly, the following study was designed to determine whetherany significant time-dependent changes in ventilation would occur in ourpreparation solely as a result of prolonged hypercapnia. If so, then the constantlevel of drive to the diaphragm observed with prolonged inspiratory resistiveloading, could be explained at least partially, by the effects of prolongedhypercapnia alone on central respiratory control.MethodsAnimals. Six male, New Zealand White rabbits weighing 3-3.5 kg were studied.The source, care, surgical preparation and anesthetic regime utilized wereidentical to those described in Chapter II except for the following modifications.Following tracheostomy and carotid artery cannulation, the phrenic nerve wasleft intact in four rabbits. No recording of the whole nerve potentials was madein these animals. In two rabbits, the whole phrenic nerve (rather than therootlet) was exposed, secured to bipolar platinum fine wire stimulating electrodesand bathed in mineral oil to record M-wave amplitudes, as described previously.ProtocolAfter a 20 minute period of stabilization following surgery, baselinemeasurements of respiratory variables and blood gases were made in rabbits90breathing room air with supplemental oxygen. The rabbits were then exposedto an inspired gas mixture of 10% C02, 50% 02 balanced N2 (precision gas,Linde-Union Carbide) via a meterological bag in series with the inspiratory portfor a period of 3 hours. The expiratory port of the two way non-rebreathingvalve was exposed to room air. All measurements were obtained at 10, 30, 60,120 and 180 minutes following hyperoxic hypercarbia and after 30 minutes ofrecovery from this gas mixture. Arterial blood (0.3 ml) was sampled inheparinized syringes and gases and pH measurements made within one minuteof sampling. Pressures generated by the respiratory system (Pao, Pdi), peakmoving average of the costal diaphragm electromyogram (EMGdi), airflow, tidalvolume and arterial blood pressure were recorded continuously. The mean valuefor tidal volume, breathing frequency, peak negative inspiratory pressures (Pao,Pdi), TI, TE, VT/TI, TI/rrOT, EMGdi, and minute activity were calculated hourlyduring a representative 60 second interval. To compare the relative activation ofthe diaphragm during hypercarbia and inspiratory resistive breathing, peakEMGdi multiplied by breathing frequency (minute activity) was calculated. Thedurations of inspiration (TI) and expiration (TE) were determined from theEMOdi recordings by the procedure described in Chapter II.Data Analysis. The initial response to hypercapnia was analyzed by comparingmeasures taken during baseline and after 30 minutes from the onset ofhypercarbia. A paired student’s t-test analysis was adopted. Comparison of91measurements taken during C02 exposure was made by Friedman ANOVA(repeated measures). A Wilcoxon signed rank test was used to determine whichvalues differed with time during the high C02 exposure (Systat 5.02). Statisticalsignificance was defined as P <0.05. All values in the text represent mean ±S.E.M.ResultsThe results are described separately for the initial 30 minute exposure tohypercarbia and the prolonged exposure to 3 hours of hypercarbia.Steady state response to hypercapnia: 30 minutes of exposure to hypercarbia.Arterial blood gases and ventilatory variables at baseline and during the first 30minutes of exposure to 10% inspired C02 are summarized in Table Ill. Therewas a two fold increase in minute ventilation in response to FICO2=0.10. Thisventilatory response is within the range previously reported for anesthetizedrabbits (Richardson and Widdcombe, 1969; Gautier, 1973). The rise in minuteventilation was due to an approximate two fold increase in VT and a 20%increase in breathing frequency (Bf). Respiratory times and related variables aresummarized in Table IV. The increase in Bf was due to a reduction in TE in allthe rabbits exposed to 10% inspired CO2. In three of the six rabbits tested, Ti92decreased in response to hypercapnia as well. The increase in mean inspiratoryflow rate (VT/Ti) with C02 breathing was mainly as a result of the increase inVT. On average, there was no significant change in duty cycle (TI/TroT) withhypercapnia. Hypercapnia resulted in increased central drive to the diaphragmas reflected by a two fold rise in the activity of the diaphragm (EMGdi) and atwo fold rise in pressure swings generated by this muscle (Pdi) (Table III).93Table III: Mean values (± S.E.M.) for arterial blood gases and ventilatoryvariables during the initial 30 minutes of exposure to 10% inspired C02 inhyperoxic anesthetized rabbits (n = 6).Duration (minutes)Baseline 10 30PaCO2 47 65* 67*(1) (2) (3)Pa02 158 227 236(mm Hg) (17) (14) (14)pH 7.35 7.24* 7.24*(0.02) (0.01) (0.01)HC03 26 28* 28*(mM/L) (1) (1) (1)VE (%) 100 219* 236*(0) (20) (23)VT (ml) 19 34* 37*(1) (3) (3)Bf 44 53* 53*(min’) (4) (3) (3)EMGdi (%) 100 201* 212*(0) (29) (21)Pdi 5.2 8.8* 8.5*(cm H20) (0.4) (1.0) (0.8)*p <0.05 compared to baseline value.94Table W: Mean values (± S.E.M.) for respiratory times, mean inspiratory flowrate (VT/TI) and duty cycle (TI/Vr0T) in anesthetized rabbits (n = 6)exposed to 10% inspired C02 for the initial 30 minutes.Duration (minutes)Baseline 10 30Ti (sec) 0.6 0.5 0.5(0.1) (0.1) (0.1)TE (sec) 0.9 0.7*t(0.1) (0.1) (0.1)TTOT (sec) 1.4 1.1* 1.1*(0.2) (0.1) (0.1)TI/TroT 0.40 0.42 0.42(0.02) (0.01) (0.02)VT/TI 36 75* 80*(mi/sec) (3) (8) (8)*p< 0.05 compared to baseline value.t p < 0.05 compared to other values obtained during 10% C02 exposure.95Ventilatoiy response to prolonged hypercapnia: 3 hours of exposure to hypercarbia.Figure 15 shows ventilation in response to prolonged inhalation of 10% inspiredC02 in anesthetized hyperoxic rabbits and the corresponding changes in PaCO2.PaCO2 rose relative to the steady state established within the first half hour ofexposure to high C02. By 120 minutes of exposure to hypercarbia, minuteventilation fell as a result of a drop in Bf from 53 breaths/mm at 30 minutes to44 breaths/mm at 120 minutes (see Table V). This decrease in Bf resulted inan average frequency of breathing equivalent to the baseline prior to hypercarbicexposure. In four of the six rabbits tested there was a decrease in VT from 40± 4ml to 30± 3 ml during this period as well. In contrast, the elevated VT observedearly in hypercapnia was either maintained or increased in the other two rabbits.Therefore, the average VT response of the group to prolonged hypercapnia wasnot significantly different over time (Table V).Respiratory times increased variably by 120 minutes of hypercarbia. In tworabbits both TI and TE increased. In the remaining four rabbits, there was onlyan increase in TE with sustained hypercarbia. On average, duty cycle remainedconstant throughout sustained hypercarbia (Table VI). A drop in ventilatorydrive late in hypercarbia was reflected by decreased mean inspiratory flow rate(VT/TI). The average activity (EMGdi) and pressure generated by thediaphragm (Pdi) did not change significantly during the the first 2 hours of96exposure to severe hypercarbia (Table V). However, by 3 hours of CO2exposure, there was a significant drop in both EMGdi and Pdi.We measured no change in the evoked compound action potential of thediaphragm during prolonged exposure to 10% inspired CO2 in the two rabbitstested. The profile of M-waves were superimposable and the mean peak to peakamplitudes of the diaphragm M waves ranged from 5.2 ± 0.3 mV to 5.4 i 0.6 mVduring prolonged hypercarbia.97Figure 15: Top Panel: Arterial PCO2 levels during prolonged exposure toFICO2=O.1O in hyperoxic anethetized rabbits (n=6).Bottom Panel: Minute ventilation during prolonged exposure toFICO2=0.10 in hyperoxic anesthetized rabbits (n=6).Arrows represent the onset of CO2 exposure.* Significantly different from baseline (B), p < 0.5.t = Significantly different from 30 minute value, p <0.05.c8100*1•9080IE/E60-o II40 /0I//I I I I I I I IB 1030 60 120 180Time (minutes)*600 *500400C I’. 300 IIIlit2009>iooØ_ _ _B 1030 60 120 180Time (minutes)99Table V: Arterial blood gases and changes in ventilatory variables produced by3 hours of exposure to 10% inspired C02 in anesthetized rabbits (n = 6).Duration (minutes)Baseline 10 30 60 120PaCO2 47 65* 67* 77*t 80*t 89*t(1) (2) (3) (1) (2) (3.0)Pa02 158 227 236 245 238 243(mm Hg) (17) (14) (14) (17) (21) (21)pH 7.35 7.24* 7.24* 7.21*t 7.17*t 7.14*t(0.02) (0.01) (0.01) (0.01) (0.01) (0.01)HC03 26 28* 28* 30* 29* 30*(mM/L) (1) (1) (1) (1) (1) (0.5)VE (%) 100 219* 236* 206* 164*t 151*t(0) (20) (23) (23) (14) (16)VT (ml) 19 34* 37* 36* 31* 29*(1) (3) (3) (3) (2) (2)Bf 44 53* 53* 47* 44*j. 42*t(min1) (4) (3) (3) (4) (4) (4)EMGdi (%) 100 201* 212* 193* 168* 155*t(0) (29) (21) (21) (15) (15)Pdi 5.2 8.8* 8.5* 8.3* 75* 6.9*t(cm H20) (0.4) (1.0) (0.8) (0.8) (0.8) (0.7)*p <0.05 compared to baseline value.tp <0.05 compared to other values obtained during 10% C02 exposure.100Table VI: Mean values (± S.E.) for respiratory times, mean inspiratory flow rate(VT/TI) and duty cycle (TI/TroT) in anesthetized rabbits (n = 6) exposedto 10% inspired C02 for 3 hours.Duration (minutesBaseline 10 30 60 120 180Ti 0.6 0.5 0.5 0.5 0.6 0.6(sec) (0.1) (0.0) (0.0) (0.0) (0.0) (0.1)TE 0.9 0.7*t 0.7*t 0.8 0.8 0.8(sec) (0.1) (0.1) (0.1) (0.1) (0.1) (0.1)rrOT 1.4 1.1* 1.1* 1.3 1.44 1.4(sec) (0.2) (0.1) (0.1) (0.1) (0.1) (0.1)TI/TroT 0.40 0.42 0.42 0.39 0.41 0.43(0.02) (0.01) (0.02) (0.02) (0.01) (0.02)VT/Ti 36 75* 80* 72* 55*j. 50*t(mi/sec) (3) (8) (8) (6) (5) (5)*p <0.05 compared to baseline value.<0.05 compared to other values obtained during 10% C02 exposure.<0.05 compared to values obtained during the first hour of 10%C02 exposure.101The data obtained during prolonged exposure to 10% C02 alone is comparedto the degree of hypercapnia associated with prolonged inspiratory resistiveloading (load 2, Chapter II) in Figures 16-18. Both stimuli result in similar levelsof PaCO2, however there are distinct differences in breathing pattern in responseto the two conditions. During loaded breathing the rabbits breathe slowly andshallowly whereas during inhalation of C02, breathing is deep and rapid.102Figure 16: Comparison of arterial PCO2 levels (top panel) and thecorresponding levels of minute activity (EMGdi X Bf) of thediaphragm (bottom panel) in rabbits exposed to prolongedhypercarbia (n = 6) and rabbits exposed to prolonged inspiratoryresistive loading (n=7; data from Load 2, Chapter II).*= Significantly different between the two groups, p < 0.05).jO3110- I ILoad 2100- K\\NFICO2=0.10 }Q) 90IE80 +E0C0°60504Q-___B 1 2 3Time (hours)120>*I+± +.EE 60D<40 +Lra.9 20C0 ———B 1 2 3Time (hours)104Figure 17. Comparison of the peak electromyographic activity of thediaphragm (EMGdi) (top panel) and breathing frequency (Bf)(bottom panel) in rabbits exposed to prolonged hypercarbia (n = 6)and rabbits exposed to prolonged inspiratory resistive loading(n = 7; data from Load 2, Chapter II).*= Significantly different between the two groups, p < 0.05).‘cc400 I ILoad 2RNxNFICO2=0.10300 + +a)cria200-DCDw100 -0-B 1 2 3Time (hours)6050a)D40ECl)-c 30oa)204-QD100_----IB 1 2 3Time (hours)106Figure 18. Comparison of arterial Pco2 levels (top panel) and thecorresponding levels of minute ventilation (bottom panel) inrabbits exposed to prolonged hypercarbia (n = 6) and rabbitsexposed to prolonged inspiratory resistive loading (n=7; data fromLoad 2, Chapter II).*= Significantly different between the two groups, p <0.05).D’T110 -___I ILoad 2k\NFICO2=O.l 0100-++90E 80-E(N 700C—)60I5040- -B 1 2 3Time (hours)650600550500-Th450- 400Iic 350300250200 +150501000 -_ _— — — — —B 1 2 3Time (hours)108DiscussionThis study demonstrates a significant time-dependent change in the regulationof ventilation during prolonged exposure to severe hypercapnia in theanesthetized hyperoxic rabbit. Specifically, there is a drop in breathing frequency(Bf) within two hours of exposure to severe hypercapnia followed by a reductionin activation of the diaphragm (EMGdi) by the third hour of C02 exposure(Table V). Further work needs to be done to ascertain the mechanism(s)underlying this phenomenon. We have shown that this effect is mediated initiallythrough its action on central rhythm and subsequently by a decrease in thecentral motor output to the diaphragm per breath (EMGdi).The top panels in figures 16 and 18 show the degree of hypercapnia associatedwith exposure to C02 alone compared with the degree of hypercapnia associatedwith prolonged inspiratory resistive loading. Although both stimuli result insimilar levels of PaCO2, there are distinct differences in breathing pattern inresponse to the two conditions within the first hour of hypercapnia. Whereas Bfis elevated during C02 breathing, it is decreased during loaded breathing as aresult of neurally mediated load compensatory mechanisms (Figure 17).Therefore, there is a greater level of diaphragmatic minute activity during C02breathing compared to loaded breathing at 1 hour (Figure 16). In contrast, thereis no significant difference between activation of the diaphragm per breath109(EMGdi) under the two conditions at similar levels of PaCO2 at 1 hour (Figure17). Consequently, the VT generated during C02 breathing (37± 3 ml) issubstantially greater compared to the volume generated against the flowresistance imposed by the inspiratory load (15± 1 ml). Hence, the increase in themechanical time constant of the respiratory system during external inspiratoryloading and the load compensating mechanisms operating during the first hourof loaded breathing result in a level of minute ventilation that is significantly lessthan predicted by the C02 stimulus alone (Figure 18).While there are differences in breathing pattern during the first hour of exposureto C02 alone compared to loaded breathing, the drop in Bf with prolongedexposure (2 and 3 hours) to C02 is analogous to the initial load compensatoryresponse to loaded breathing (Figures 16-17). It can be hypothesized that thereduction in Bf functions as an effective strategy to reduce the energetic cost ofbreathing and avert or forestall the development of task failure during conditionsof increased ventilatory demand. Evoked compound action potentials of thediaphragm did not change significantly during prolonged exposure to C02suggesting that there is no failure in neuromuscular transmission during this task.However, the fact that activation of the diaphragm (EMGdi) and hence theability to generate pressure (Pdi) was found to have decreased significantly by thethird hour of C02 breathing shows that prolonged hyperpnea associated with thisventilatory stimulus can result in a decrease in central motor output to the110diaphragm.Reduction in Bf within two hours of C02 exposure may be due to 1) the effectsof prolonged anesthesia; 2) a generalized depression of the CNS due to severehypercapnia; or 3) the central effects of prolonged hypercapnia onchemoreceptor activity. The data obtained in the control group during our initialstudies (Chapter II) show that there is no change in Bf as a result of prolongedanesthesia (40± 5 at baseline versus 37± 4 after three hours of anesthesia).Therefore, it is unlikely that the reduction in Bf during prolonged C02 exposureis due to prolonged anesthesia.Rising concentrations of C02 in the arterial blood have different effects onvarious anatomical structures within the brain. According to Wyke andWoodbury (see Wyke, 1963), there are three phases associated with the sequenceof neurophysiological responses to increasing amounts of C02. The initial phaseinvolves direct depression of cortical synaptic activity only and is achieved byinhalation of C02 in concentrations between 3.5 and 7 percent. The next phaseinvolves a generalized reticulo-cortical activation which overcomes the initialdirect cortical depression. This stimulatory phase is increasingly evoked byinhalation of C02 in concentrations between 5-20% . The final phase isassociated with narcosis or anesthesia, which leads to generalized depression ofreticulo-cortical activity where inhaled C02 concentrations exceed 25 percent.111Therefore, it is unlikely that the decrease in breathing frequency observed aftertwo hours of inhalation of 10% C02 is due to a generalized depressive effect ofC02 on the CNS. Furthermore, it has been shown in the past that centralinspiratory activity as indicated by phrenic motor discharge is tolerant ofextensive hypercapnia in rabbits (up to PACO2 of 200 mm Hg; Kobayasi andMurata, 1979).It has been demonstrated that in mammals central chemoreceptors situatedbeneath the ventrolateral medullary surface (Mitchell et aL, 1963; Schlaefke etaL, 1970) stimulate or inhibit ventilation in response to changes in extracellularfluid pCO2/[H9 or [WI alone (Bledsoe and Hornbein, 1981; Dempsey andForster, 1982). Recent reports suggest that other discrete areas within thebrainstem (in the vicinity of the nucleus tractus solitarius and locus coeruleus)may be involved in driving ventilation in response to this chemical stimulus aswell (Coates, Li and Nattie, 1993; see Severinghaus, 1993 as well). Themechanisms by which extracellular pCO2/[W] is sensed by centralchemoreceptors is not known.It seems likely that the decrease in Bf following two hours of sustainedhypercapnia is due to a depressed H stimulus at the central chemoreceptorsites. Rapid increases in cerebrospinal fluid bicarbonate concentration and braintissue bicarbonate concentration are known to occur in response to sustained112hypercarbia, in several species including in the ketamine-xylazine anesthetizedrabbit (Bleich et aL, 1964, Ponten, 1966; Messeter and Siesjo, 1971; Hasan andKazemi, 1976; Ahmad, Berndt and Loeschke, 1976; Nattie and Giddings, 1988).The mechanism which permits bicarbonate ion [HCO3] to accumulate in theCSF during hypercapnia is not known. However, since the CSF is an ionicsolution low in protein and poorly buffered this increase must stem from eitherblood or brain tissue origin. Ionic regulation of [HCO;] in the cerebralextracellular fluids (CSF and brain interstitial fluid) during prolonged andespecially severe hypercapnia is poorly understood (for a review see Fend, 1986).According to Nattie (1980), the time course of the continuous increase in CSF[HC03] in response to severe sustained hypercapnia suggests 3 phases ofregulation: 1) an immediate 5-20 minute response, 2) a slower response thatplateaus over 1-3 hours and 3) an increase over hours to days which isconcomitant with the increase in blood [HCO3]. It is assumed the third phasereflects the slow exchange of ions between blood and CSF.Exposure to high levels of C02 during the second phase, as described by Nattie(1980), can also result in alterations in brain metabolic byproducts which affectchemoreceptor activity directly or indirectly via other central respiratory neurons.These changes include a down regulation in brain excitatory amino acids,glutamate and aspartate, and increases in the inhibitory amino acid, GABA(Kazemi and Hoop, 1991). Irrespective of mechanisms that could potentially113mediate a reduction in chemoreception during the first few hours of severehypercapnia, the question remains whether such change(s) are sufficient toproduce a significant drop in ventilation. In a study on rats, Nattie (1980)suggests that alteration in central chemoreceptor activity during this period mayresult in decreased ventilation.In conclusion, prolonged (greater than 1 hour) and severe hypercapnia alone canresult in a decrease in minute ventilation in the anesthetized hyperoxic rabbitpreparation. It seems that the levels of hypercapnia associated with prolongedmoderate inspiratory resistive loading are sufficient to provide an inhibitory inputto the central respiratory controller resulting in a suppressed level of drive to thediaphragm. Although activation of the diaphragm (EMGdi) is maintained withinthe first two hours of C02 exposure, Bf is reduced [rroT increases] and dutycycle decreases. Consequently, the breathing pattern associated with prolongedhypercapnia can potentially protect the respiratory muscles from overload. Thismechanism may prevent excessive activation of the respiratory muscles whenloading is severe. Therefore, we infer that although load compensatingmechanisms are initiated at the onset and early in loaded breathing, theycompete with changes in breathing pattern that result from the effects ofprolonged hypercapnia that are load decompensating. Surprisingly, thisdecompensation develops within 1-2 hours of loading and is progressive.114References1. Ahmad, H.R., J. Berndth and H. H. Loeschcke (1976). Bicarbonate exchangebetween blood, brain extracellualar fluid and brain cells at maintained PCO2.In: Acid Base Homoeostasis of the Brain Extracellular Fluid and TheRespirator,’ Control System. H.H. Loeschcke (Ed.). Georg Thieme, Stuttgart,pp. 19-27.2. Bledsoe, W.W. and T. Hornbein (1981). Central chemosensors and theregulation of their chemical environment. In: Regulation of Breathing, T.Hornbien (ed.). Lung Biology in Health and Disesase. New York, Dekker,pp. 347-428.3. Bleich, H.L., P.M. Berkman and W.B. Schwartz (1964). The response ofcerebrospinal fluid composition to sustained hypercapnia. J Clin. Invest. 43:11-16.4. Bruce, E.N., J.D. Smith and F.S. Grodins (1974). Chemical and reflex drivesto breathing during resistance loading in cats. .. AppL PhysioL 37: 176-182.5. Coates, E.L., H. Li and E.E. Nattie (1993). Widespread sites of brain stemventilatory chemoreceptors. J AppL PhysioL 75: 5-14.6. Dempsey J.A. and H.V. Forster (1982). Mediation of ventilatory adaptations.Physiological Reviews 62: 262-346.7. Fend, V. (1986). Acid-base balance in cerebral fluids. In: Handbook ofPhysiology- The Respirator,’ System. Williams & Wilkins, Baltimore, MD, Vol115II, Part 1. Cherniack, N.S. and Widdicombe, J.G. pp. 115-139.8. Hasan, F.M. and H. Kazemi (1976). Dual contribution theory of regulationof CSF HCO3 in respiratory acidosis. J AppL PhysioL 40: 559-567.9. Kazemi, H. and B. Hoop (1991). Glutamic acid and alpha-amino butyric acidneurotransmitters in central control of breathing. J AppL PhysioL 70 1-7.10. Kobayasi, S. and K. Murata (1979). Phrenic activity during severehypercapnia in vagotomized rabbits. J AppL PhysioL 47: 9 1-97.11. Messter K. and B.K. Siesjo (1971). Regulation of the CSF pH in acute andsustained respiratory acidosis. Ada. PhysioL Scand. 83: 21-30.12. Mitchell, R.A., H.H. Loeschcke, J.W. Severinghaus, B.W. Richardson and W.H. Maission (1963). Regions of respiratory chemosensitivity on the surfaceof the medulla. Ann. NY Acad. Sci. 109: 661-681.13. Nattie. E.E. (1980). Brain and cerebrospinal fluid ionic composition andventilation in acute hypercpania. Respiz PhysioL 40: 309-322.14. Nattie, E.E. and B. Giddings (1988). Effects of amiloride and diethylpyrocarbonate on CSF HC03 and ventilation in hypercapnia. J. AppLPhysiol. 65: 242-248.15. Orthner F.H. and W.S. Yamamoto (1974). Transient respiratory response tomechanical loads at fixed blood gas levels in rats. J AppL PhysioL 36: 280-287.16. Ponten, U. (1966). Consecutive acid-base changes in blood, brain tissue andcerebrospinal fluid during respiratory acidosis and alkalosis. Acta. NeuroL116Scand. 42: 455-471.17. Sant’Ambrogio, G. and J.G. Widdicombe (1965). Respiratory reflexes actingon the diaphragm and inspiratory intercostal muscle of the rabbit. J PhysioL180: 776-779.18. Severinghaus, J.W. (1993). Invited editorial on “widespread sites of brainstem ventilatory chemoreceptors”. J AppL PhysioL 75: 3-4.19. Schlefke, M. E., W.R. See and H.H Loeschcke (1970). Ventilatory responseto alterations of hydrogen ion concentration in small areas of the ventralmedullary surface. Respir. PhysioL 10: 198-212.20. Shannon, R. and F.W. Zechman (1972). The reflex and mechanical responseof the inspiratory muscles to an increased airflow resistance.Respir. PhysioL 16: 5 1-79.21. Wyke, B. (1963). The neurological basis of pH effects on brain function. In:Brain Function and Metabolic Disorders. Butterworth & Co., London, pp.158-165.117W: Respiratory Muscle Activity and Task Failure During Severe InspiratoryResistive Loading.IntroductionFatigue of the diaphragm has been implicated as a contributing factor inventilatory pump failure induced by severe inspiratory resistive loading (Aldrich1985, 1987, 1988; Alexadrovna and Isaev, 1990; Bazzy and Haddad, 1984;Mayock, 1987, 1991). We have shown that neural activation, neuromusculartransmission and activity of the diaphragm are maintained in anesthetized rabbitsunder severe inspiratory resistive loads even when inspiratory pressuregeneration is reduced i.e. task failure occurs (Chapter II). Since we used peakinspiratory airway pressure (Pao), which reflects total respiratory muscle outputas an index of muscle force, the specific contribution of respiratory muscles otherthan the diaphragm to force failure during extreme inspiratory resistive loading(Load 4) could not be assessed. Therefore, task failure may be a consequenceof reduced function of extradiaphragmatic muscles.In this study, we monitored the electromyographic activity of the other majorinspiratory muscle, the parasternal intercostal (EMGps), because loss ofinspiratory pressure may be due to failure of activation of this muscle.Alternatively, the expiratory muscles are thought to provide an important assist118to inspiration and hence failure of their activation may contribute to task failureduring inspiratory resistive loading. Therefore, we measured electromyographicactivity in one of the most active expiratory muscles, the transversus abdominis(EMGta) along with the inspiratory activity of the diaphragm (EMGdi) and theparasternal intercostal (EMGps) under extreme inspiratory loading untilrespiratory arrest.MethodsAnimals. Six New Zealand white rabbits were obtained from Geo-Bat Rabbitries(Abbotsford, B.C.) and cared for according to the principles outlined by theCanadian Council for Animal Care at the Animal Resource Unit facility at theUniversity Hospital (U.B.C). Experimental protocols received ethics approvalfrom the University of British Columbia Animal Care Committee.Preparation. The rabbits (mean body weight 3.4 kg, range = 3.2-4 kg) wereanesthetized with i.m. injection of ketamine (Ketavet, Parke-Davis, 30 mg/kg)and a sedative, xylazine (Rompun, Bayer, 7 mg/kg). Anesthesia was maintainedthroughout the studies by supplementing half the initial dose every 30-40minutes. Rectal temperature was continuously monitored and maintainedbetween 38-39°C with a heating pad. Saline was infused via a marginal ear veinto maintain blood pressure. Inspired air was supplemented with oxygen119throughout the study.Rabbits were placed in the supine position and the trachea was cannulated andconnected to a heated pneumotachograph (Fleisch # 00) in series with aminiature two way non-rebreathing valve (Hans Rudolph no. 2814; 2.5 ml deadspace). Pressure across the pneumotachograph was measured with a differentialpressure transducer (± 2 cm H20, Validyne MP-45) and a carrier preamplifier(Gould model 13-44615-35). The carrier output was electronically integrated(Gould integrator amplifier 13-4615-70) to record tidal volume (calibrated fora range of 5-30 ml). Airway pressure (Pao) was measured at the tracheal tubeusing a differential pressure transducer (± 80 cm H20 Validyne, Northridge,CA). An adjustable needle valve was placed at the inspiratory port of the nonrebreathing valve to apply the flow resistive load. During resistive loading, 100%oxygen was provided at the inspired port from a meterological balloon. The leftcarotid artery was cannulated to measure blood pressure and to sample bloodfor blood gas and pH analysis (Model 168 pH/blood gas analyzer, CorningMedical, Medfield, MA).To record the diaphragm electromyogram (EMGdi), a midline upper abdominalincision was made and the uninsulated tips of two multi-stranded stainless steelfine wires (Cooner wire #AS 631) were sutured 1 cm apart into the left costa!hemi-diaphragm midway between the costal margin and central tendon. Through120the same incision, a second pair of wires were sutured 1 cm apart in thetransversus abdominis approximately 2 cm lateral to the linea alba and parallelto the transverse orientation of the muscle fibres to record activity of theabdominal muscles (EMGta). An air filled balloon-catheter assembly wassecured underneath the dome of the right diaphragm above the liver andattached to a differential pressure transducer (± 56 cm H20 Validyne,Northridge, CA) to record abdominal pressure (Pab). The abdominal incision wasthen closed with sutures and surgical staples to restore the fascia, muscle andskin layers respectively. The sternum was exposed by a midline incision alongthe length of the 2nd to 5th rib and the insertion of the left pectoralis musclesdissected away to expose the parasternal muscles between the 3rd and 4th ribs.To record parasternal intercostal muscle activity, a pair of fine wires weresutured 1 cm apart in the parasternal muscles parallel to the muscle fibreorientation and the incision closed as described above.The bipolar EMG signals were amplified (Grass P5 series AC preampliers; GrassInstruments Co., Quincy, MA; band pass 100 Hz-10 kHz), whole wave rectifiedand the moving averages (time constants = 100, 100, 200 msec; EMGps, EMGdi& EMGta respectively) computed using a four pole active filter with a Paynterresponse (EMG Signal Processor, Raytech Instruments, Vancouver, Canada).The moving averages of the EMG signals were further amplified (Gould mediumgain DC preamplifier model 13-4615-10) and recorded on an 8 channel chart121recorder (Gould model 8188-812, Cleveland, OH).ProtocolAfter a 20 minute period of stabilization, baseline measurements were made.Since there is no abdominal EMG activity during eupnea, a qualitativecomparison of the level of activation in the different respiratory muscles fromthe baseline EMG values obtained during eupnea is not possible. Therefore, wedefined maximum EMG activity during exposure to high inspired CO2. Todetermine the maximum peak moving average EMG activities, rabbits wereexposed to an inspired gas mixture of 9% C02, 50% 02 balanced N2 for 10minutes from a meterological balloon in series with the inspired port. The peakmoving average of the EMGs calculated after 10 minutes of breathing this gasmixture was designated in relative units as 100% activity. Rabbits were returnedto breathing oxygen supplemented room air for 20-30 minutes until blood gasesand minute ventilation returned to baseline. Inspiratory resistive loading wasthen applied at end expiration. The inspiratory needle valve was adjustedgradually within 5 minutes to a target peak inspiratory airway pressure (Pao) ofapproximately -55 cm H20. This load was maintained until respiratory arrest(cessation of breathing for > 1 minute). Arterial blood (0.3 ml) was sampledevery 10 minutes in heparinized syringes and arterial blood gases and pHmeasured within one minute of sampling. Expiratory abdominal pressure swings122(Pab), peak inspiratory airway pressure (Pao), the peak moving average activityof respiratory muscles (EMGdi, EMGps, EMGta), airflow, tidal volume andarterial blood pressure were recorded continuously and their mean valuecalculated every 10 minutes during a representative 60 second interval. Theduration of inspiration (TI) and expiration (TE) were determined from theEMGdi recordings. Ti was defined form the EMGd1 as the time from initial riseto the point where a rapid decline was first observed. TE spanned the time fromthe rapid decline in the EMGdi to its next initial rise.Data Analysis.All variables were statistically compared by Friedman’s ANOVA, repeatedmeasures. For variables identified as significantly different, a Wilcoxon signedrank test was used to determine which values differed with time during loadedbreathing (Systat 5.02). Statistical significance was defined as P < 0.05. All valuesin the text represent mean ± S.E.M.ResultsResponses to hyperoxic hypercarbic gas mixture.There was an expected increase in both tidal volume (18± 1 ml at baseline to12328± 1 ml during C02 breathing, p <0.001) and breathing frequency (41± 4breaths/mm at baseline to 58± 5 breaths/minute during C02 breathing p < 0.05)in response to breathing the hyperoxic hypercarbic gas mixture (PaCO2 = 69± 3mm Hg, PaO2 = 239± 13 mm Hg) demonstrating that anesthesia had not abolishedthese responses to chemical stimulation. Increases in tidal volume, breathingfrequency and inspiratory muscle activity (EMGd1, EMGps) had a gradual andearly onset that reached steady state within approximately 5 minutes. The onsetof abdominal muscle activity (EMGta) however had a latency of 2-3 minutes.The relative increase in the EMGdi and EMGps in response to hypercarbia was2 fold reflecting the relative increase in minute ventilation (217± 21 mI/mm/kgto 488± 45 mi/mm/kg). These results are comparable to those obtained inChapter III during steady state after 10 minutes of exposure to hyperoxichypercarbia (FICO2 = 0.10) in a separate group of rabbits.Response to extreme inspirato,y resistive loading.Inspiratory resistive loading led to hypoventilation and severe alterations inblood gases in the anesthetized rabbit (Table VII) as described in Chapter II(Load 4) . The average target peak inspiratory pressure (Pao) of -58 ± 4 cmH20 in this study was maintained for 20 minutes despite severe and progressivehypercapnia and moderate hypoxia.124Respiratory muscle activity increased substantially in all three muscles duringloaded breathing. The increase in the activity of EMGdi, EMGps, EMGta duringloaded breathing was three to four fold that observed during C02 stimulatedbreathing (Figure 19). The inspiratory muscle activity (EMGdi and EMGps)increased six fold relative to activity at baseline during unloaded breathing.There was no expiratory muscle activity (EMGta) observed at baseline during unloaded breathing . With the onset of phasic expiratory muscle activity duringloaded breathing, there was a parallel increase in abdominal pressure swings(Pab) during expiration. Phasic EMGta and expiratory pressure swings weremaintained during the first 20 minutes of loaded breathing (Figure 19).125Table VII: Respiratory variables, arterial blood gases and pH at baseline andduring extreme inspiratory resistive loading.Duration of Loading (minutes)Baseline 20VT (ml) 18(1) 12(2)* 11(1)* 8(1)*tBf (1/mm) 47(6) 28(4)* 25(3)* 21(2)*tVE (mi/mm) 835(107) 322(40)* 276(41)* 155(17)*tVT/TI (ml/sec) 36(4) 14(2)* 15(2)* 12(1)*TI/TrOT 0.37 0.37 0.26j 0.20*1(0.03) (0.04) (0.04) (0.03)Ti (sec) 0.5(0.1) 0.9(0.1)* 0.8(0.1)* 0.7(0.1)*TE (sec) 0.9(0.1) 1.7(0.3)* 2.2(0.3)* 2.8(0.5)*tpH 7.39 7.09* 7.01* 6.86*(0.02) (0.04) (0.03) (0.06)PaCO2 (mm Hg) 47(2) 119(16)*j 151(20)* 187(21)*Pa02 (mm Hg) 157(30) 114(13)* 61(10)* 25(1)*[HC03](mmol/L) 28(1) 33(2)* 34(2)* 34(1)*Values represent mean (± S.E.M.), n=6.<0.05 compared to baseline.<0.05 compared to value at 20 minutes load.p < 0.05 compared to other loaded periods.126Figure 19. Top panel: Peak moving average of the electromyogram from thecosta! diaphragm (EMGdi), parasternal intercostal (EMGps) andtransversus abdominis (EMGta) muscles at baseline and duringloaded breathing. Failure to maintain inspiratory pressure,designated “F” on the abscissa occurs 30-40 minutes after loading.Bars represent mean ± S.E.M.*= significantly different from baseline, p <0.05.I significantly different from other loaded periods, p <0.05.Bottom panel: Peak inspiratory airway pressure, Pao (v)and expiratory swings in abdominal pressure, Pab (.) duringbaseline and loaded breathing. Symbols represent mean ± S.E.M.*= significantly different from baseline, p < 0.05.t significantly different from other loaded periods, p < 0.05.U,-ICDC)>aC)LC:,lii6005000c’JEC)a)DU)C,,a)L.0diI (psta*1Z7*****4003002001 000tBaseline 10 20 FLoaded period (mm utes)**65605550454035302520151050* *•1•Baseline 10 20 FLoaded period (minutes)128After 20-40 minutes of loaded breathing there was progressive drop in EMGtaand a parallel fall in expiratory abdominal pressure swings within a 10 minuteinterval that was associated with a significant drop in Pao from -60 ± 4 cm H20to -49 ± 5 cm H20 and in Pab from 9± 2 cm H20 to 3± 1 cm H20 (Figures 19,period F; Figure 20, panel C). In 4/6 rabbits EMG1a decayed to zero at thistime. Inspiratory muscle activities (EMGdi and EMGps) were maintained duringthis period despite severe hypercapnia and hypoxia (Pa02=25± 1 mm Hg, TableVII). Following decreased inspiratory pressure output, ventilation continued foran average of 5 minutes until respiratory arrest. During this interval, breathingwas characterized initially by periodic clusters during which peak inspiratorypressure (Pao) and activity of the inspiratory muscles (EMGdi, EMGps) weremaintained followed by a parallel decay in peak EMGdI, EMGps, airflow andairway and blood pressures to zero (Figure 21).129Figure 20. Sample tracing of arterial blood pressure (BP), airway pressure (Pao),abdominal pressure (Pab), airflow (7), tidal volume (VT) and moving average ofthe costal diaphragm (EMGdi), the parasternal intercostal (EMGps), and thetransversus abdominis (EMGta) muscles during A) baseline, unloaded breathingB) loaded breathing C) loaded breathing at time of inspiratory pressure failure(task failure). Arrows represent zero.130A B CB P t4J4’50 mm Hg IPao_10 cm H20Pab /5 cm H20/EMGdi/EMGpsEMGta 4•— 5 seconds —+VT 4MEMF’V\ 44’\10 ml I131Figure 21. Sample tracing of arterial blood pressure (BP), airway pressure (Pao),airflow (r), moving average of costal diaphragm (EMGd1) and parasternalintercostal (EMGps) activity in the final minutes leading to respiratory arrest.Activity of transverus abdominis [not shown] decayed to zero in this preparation.Arrows represent zero.BP 5OmmHg.HH,IOcmH20IIIflIjjfljj fl111__________HiiIII<mrnute>-.-—.:111111i’,11_iIIiJjI’I/[EMG_____1[1())133DiscussionResponse to inspirato,y resistive loadingInspiratory flow resistive loading is used to examine the contribution ofdiaphragm fatigue to inspiratory muscle overload. In Chapter II, we demonstratethat neural activation (ENGdi), neuromuscular transmission (M-wave) andactivity (EMGdi) of the diaphragm under inspiratory resistive loads of varyingintensity and duration is maintained in the anesthetized rabbit. However, underextreme inspiratory resistive loading conditions in this animal model, there is asignificant decrease in total respiratory muscle force output (Pao) prior torespiratory arrest and at a time when inspiratory duration Ti is unchanged. Themajor finding in this study is that this decrease in inspiratory pressure generationis associated with a loss of abdominal muscle activity (EMGta) and that theactivity of both inspiratory muscles (EMGdi and EMGps) studied remainscoupled and maintained until respiratory arrest.The parasternal intercostals and the diaphragm are mechanically coupled (DeTroyer and Sampson, 1982) and without their coordinated activity the upperribcage would retract inwards during inspiration. Despite the maintenance ofelectromyographic activity in the two inspiratory muscles, we can not exclude thepossibility that the two inspiratory muscles failed as pressure generators because134we were unable to measure Pdi orPes during this severe load. Pressure failedat a time when TI was unchanged.Therefore, the drop in pressure cannot beattributed to a prolongation or premature termination of Ti, and shouldbe dueto reduced respiratory muscle contraction. The activity of the parasternal andthe diaphragm muscles was not reduced at a time when there was amarkeddecline in abdominal muscle activity.Additionally, airway pressure Pao droppedwhen Pab began to decline. Therefore, we ascribe task failure of the respiratorysystem to generate inspiratory pressures, against severe inspiratory resistive loads,to a failure in diaphragm assist by the abdominal muscles rather than inspiratorymuscle fatigue.It has been established that abdominal muscles are stimulated to contract withphasic expiratory activity during hypercapnic induced hyperpnea (Ainsworth etaL, 1989; Oliven and Kelsen, 1989;Takasaki et aL, 1989; Van Lunteren et at,1988; Wakai et aL, 1992) and under expiratory resistive, elastic and thresholdloaded breathing (Bishop, 1963; Leevers and Road, 1989; Oliven Ct aL 1989;Oliven and Kelsen, 1989). However, the importance of expiratory abdominalmuscle activity in response to inspiratory resistive loading has been underscoredonly recently (Scardella et aL, 1989; 1990).In our model, inspiratory resistivcloading is associated with severe progressive hypercapnia. The recruitment ofabdominal muscles is most likely byvirtue of the excitatory effects of C02 c1’bulbospinal expiratory neurons (Bainton andKirkwood, 1979) and abdomir&l135motoneurons (Ledlie, Pack and Fishman, 1983).Recruitment of abdominal muscles can increase the mechanical efficiency of thediaphragm during stimulated breathing by two mechanisms. The first mechanisminvolves a decrease in end expiratory lung volume (EELV) due to increasedabdominal activity. Decreased EELV associated with the onset of abdominalactivity has been demonstrated during inspiratory resistive and inspiratorythreshold loading in both anesthetized and awake preparations (Mayock et aL,1991; Oliven et aL, 1988; Martin, Aubier and Engel, 1982). A reduction inEELV in turn increases diaphragm length which allows for greater diaphragmshortening and hence increases the pressure generating capability of thediaphragm (Road et aL, 1986; Road and Leevers, 1988). Additionally,abdominal muscle recruitment during stimulated breathing may assist thediaphragm through its relaxation at end expiration resulting in outward recoil ofthe chest wall that can contribute passively to inspiratory mechanical flow(Agostoni and Torn, 1967). Recruitment of abdominal muscles duringinspiratory loading is not limited to studies examining anesthetized animalmodels. In fact, abdominal muscle activity during inspiratory loading is describedin both awake goats and humans (Scardella et aL, 1990; Martin, Aubier andEngel, 1982). In our study, expiratory muscle activity in all the abdominalmuscles is assumed to have decreased as expiratory Pab dropped in parallel withthe drop in EMGta. We did not measure expiratory activity of the rib cage136muscles but expect that their activities were reduced as well since there isevidence that common bulbospinal neurons project to both rib cage expiratorymuscles and the abdominal muscles alike (Road and Kirkwood, 1993).To our knowledge the failure of abdominal muscle assist to diaphragm duringinspiratory resistive loading has not been demonstrated previously. Themechanism(s) underlying a selective decrease in abdominal activity with severeloading remains speculative. Several studies indicate that whereas hypoxicstimulation of ventilation results in an increase in inspiratory muscle activity,expiratory muscle activation is either less, relative to hypercapnia (Brice et aL,1990, Sears Berger and Philipson, 1982; Smith et aL, 1989), or inhibited (Fregosi,1987). In other words, breathing becomes more of an inspiratory act duringhypoxia. Such an inspiratory shift or selective inhibition of expiratory activityduring hypoxia has been attributed to either 1) medullary hypocapnia secondaryto hypoxia (Saupe et aL, 1992); 2) preferential distribution of peripheralchemoreceptor input to inspiratory pre motor neurons (Oyer Chae et aL, 1992)or 3) hypoxic depression of the brain stem (Fregosi et aL, 1987). In ourpreparation, selective inhibition of abdominal muscles during severe inspiratoryresistive loading can not be due to central hypocapnia as progressive severehypercapnia accompanies hypoxemia throughout inspiratory resistive loadedbreathing. The current literature suggests that depression of central neuraloutput during hypoxia is not uniform and there may well exist differences in the137vulnerability of respiratory motor outputs to central hypoxia (Neubauer, Meltonand Edelman, 1990).Given that hypoxemia is progressive and notably severe (PaO2 = 25± 1 mmHg)by the time phasic expiratory abdominal activity (EMGta) and expiratorypressure swings (Pab) are reduced in our preparation, it seems likely thatselective inhibition of the abdominal expiratory muscles reflects the non uniformvulnerability of respiratory nuclei to central hypoxia. In fact, the periodic clusterbreathing (Biot breathing) observed shortly after the decrease in abdominalactivity prior to respiratory arrest is characteristic of severe neural depressionattributed to anoxia or brain stem damage (Plum and Brown, 1963; Webber andSpeck, 1981). Furthermore, the transition from cluster breathing to respiratoryarrest is marked by a parallel decrease in blood pressure and inspiratory muscleactivity. If the vulnerability of respiratory nuclei to central hypoxia is notuniform then it provides the simplest explanation for the sequelae of decreasedabdominal muscle activity, blood pressure and inspiratory muscle activity.According to Neubauer Ct aL (1990), if we consider central outputs other thanthose involved in control of respiration, it seems quite likely that thevulnerability of brainstem nuclei to hypoxia are non uniform. For example,single fibre recordings from the preganglionic cervical sympathetic nerve showseparate populations of fibres that either increase or decrease their activity inresponse to brain hypoxia. Whole nerve recordings from the same nerve suggest138that brain hypoxia selectively depresses the phasic component and increases thetonic component of sympathetic discharge (Wasicko et aL, 1990). Therefore,although depression is a generalized response of the brain stem to CNS hypoxia,there may exist selective vulnerability within this network to hypoxia.In a current conceptual model of respiratory muscle activity during inspiratoryresistive loading, Petrozinno and co workers (1992) suggest that the response ofeach respiratory muscle will represent a balance between load-compensatingreflexes and differential effects of endogenous opioids released during severeresistive breathing on individual respiratory muscles. To what degreeendogenous opioids play a role in the differential response of inspiratory versusexpiratory muscles to severe loads is difficult to ascertain because their levels inthe anesthetized preparation have not been measured directly.Responses to hyperoxic hypercarbic gas mixture. The magnitude of the ventilatoryresponse to the average change in PaCO2 was within the reported range foranesthetized rabbits (Widdicombe, 1969). Although we did not examine the C02threshold for expiratory muscle activity, we found expiratory muscle recruitmentin response to 9% inspired C02 within a 2-3 minute delay in all rabbits. Asimilar delay in expiratory muscle activity to hypercapnia has been documentedin the anesthetized cats (Bishop and Bachofen, 1972). It is important to notethat the magnitude of EMGta in response to conventional C02 stimulated139breathing was significantly less than that observed with severe inspiratoryresistive loading possibly owing to a lower C02 stimulus (PaCO2 = 69± 3 duringC02 breathing versus PaCO2= 119± 16 after 10 minutes of loaded breathing).Additionally, inspiratory resistive loading results in greater electromyographicactivity of the two inspiratory muscles due to an increase in inspiratory time(vagally mediated) and possibly from facilitatory afferent input(s) from chest wallmuscle mechanoreceptors (Sant’Ambrogio and Widdicombe, 1965).The discrepancy between the severity of the CO2 stimulus during CO2 stimulatedbreathing and inspiratory loaded breathing is considerable. In particular, PaCO2reaches narcotic levels (187 mm Hg = 26%) at the time of task failure.Therefore, the central depressant effects of extreme CO2 could potentiallycontribute to task failure as well.In conclusion, anesthetized rabbits increase the phasic activity of both inspiratoryand expiratory muscles in response to severe inspiratory resistive loads. Despitesevere respiratory acidosis and hypoxemia, the phasic inspiratory activity of thecostal diaphragm and parasternal muscles remains elevated throughout loading.The failure to generate inspiratory pressure (inspiratory task failure) results froma decay in abdominal muscle activity during expiration.140References1. Agostoni, E. and G. Torn (1967). An analysis of the chest wall motions athigh values of ventilation. Respii PhysioL 3: 3 18-332.2. Ainsworth, D. M., C.A. Smith, S.W. Eicker, K. S. Henderson and J.A. Dempsey (1989). The effects of chemical versus locomotory stimuli onrespiratory muscle activity in the awake dog. 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The primary aim of this study was to assess central motor output andneuromuscular transmission to the diaphragm during prolonged sustainable andexhaustive inspiratory resistive loads in the anesthetized rabbit. The experimentspresented in Chapter II demonstrated that under all loads examined, includingthe exhaustive load that resulted in task failure, the central motor output to thediaphragm, as assessed by the phrenic electroneurogram, remained elevatedthroughout loaded breathing. There was a linear relationship between theseverity of the target inspiratory pressure achieved with resistive loading and theindices of central motor output to the diaphragm and activity of this muscle.The electromyographic activity of the diaphragm was elevated throughout loadedbreathing. There was no significant change in the relation between centralmotor output to the diaphragm and electromyographic activity of this muscle[ENG:EMG ratio]. Additionally, neuromuscular transmission to the diaphragm,as assessed by evoked compound potentials of the diaphragm, remained intactduring all inspiratory resistive loads. Therefore, we have concluded that centralmotor output and neuromuscular transmission to the diaphragm do notcontribute to task failure during inspiratory resistive loading in the anesthetizedrabbit.Methodological differences may explain the conflicting results between our147findings and those of others (Aldrich, 1987; Bazzy and Donnelly, 1993) inestimating the role of neuromuscular transmission in task failure duringinspiratory resistive loading. These differences are described in detail in ChapterII. By examining inspiratory resistive loads of both greater duration and intensitythan those previously reported we have constructed a clearer picture of theventilator>’ response to inspiratory resistive loading.2. The second aim of our study was to determine the changes in breathingpattern in response to inspiratory resistive loading of different intensities anddurations, ranging from sustainable to exhaustive loads. In Chapter II, wereported that despite significant increases in inspiratory pressure swings duringloaded breathing, ventilation was reduced compared to the baseline valuesobtained prior to loaded breathing. Consequently, arterial carbon dioxidetension rose during all loads examined in our study. Depending on the severityof the load, hypoventilation was due to changes in breathing pattern whichsuggest that an optimization of respiratory work develops early in response toloaded breathing and is achieved through a decrease in central rhythm.Furthermore, we demonstrated that a similar strategy arises upon prolongedexposure to elevated levels of arterial CO2 which develop during prolongedinspiratory resistive loading (Chapter III). Hence, we suggest that although loadcompensating mechanisms are functional at the onset of inspiratory resistiveloading, they compete with load decompensating mechanisms resulting from the148hypercapnia associated with prolonged loading.The changes in breathing pattern under inspiratory resistive loads seemedmechanically appropriate. For example, it has been established that the mostobvious effect of external resistance to air flow in humans is a reduction rate offlow and an increase in the time required for completion of the impeded phase(Zechman, Hall and Hull, 1957). In our rabbit model, during relatively minorloads (Load 1) ventilation was stabilized by a slight drop in breathing frequencydue to an increase in inspiratory time. Increased inspiratory time withinspiratory resistive loading is consistent with a decrease in Hering-Breuerinspiratory inhibitory lung inflation input due to the decreased inspiratory airflow. Moderate inspiratory resistive loads (Load 2) led to a slight decrease intidal volume and a significant drop in breathing frequency. The drop inbreathing frequency resulted from increases in both the inspiratory andexpiratory phase of breathing with moderate loading. Prolonging expiratoryduration increases the time available to the inspiratory muscles to recover beforethe onset of the next inspiration and seems an effective mechanism to optimizethe function of inspiratory muscles. During severe inspiratory resistive loads(Loads 3 and 4) this slow pattern of breathing was maintained until severealterations in arterial blood gases and pH led to periodic cluster breathing andculminated shortly thereafter in respiratory arrest (Chapter IV).149Central modulation of respiratory activity achieved through a reduction inbreathing frequency during inspiratory resistive breathing has been describedpreviously in the awake infant monkey (Watchko et aL, 1988). As thisobservation was limited to an awake preparation, it had been suggested that suchreductions in breathing frequency during inspiratory resistive loading reflectconscious motivational fatigue (Aldrich, 1991). In our study, we have extendedthese findings to the anesthetized preparation to show that this type of centralresponse is not necessarily a result of conscious or behavioral factors.Furthermore, a decrease in central rhythmogenesis is not limited to breathingagainst exhaustive inspiratory resistive loads. Reduced frequency of breathingwas typically seen with all inspiratory resistive loads and was sustained forprolonged periods as well. Additionally, we suggested that this form of centralfatigue represents a means for maintaining respiratory muscle function duringinspiratory resistive loading.3. The third aim of our studies was to determine the target inspiratory resistiveloads which lead to task failure. We were able to document task failure onlyduring the most severe load (Load 4) at target pressures close to the maximumstrength (Pdi max = 55± 9cm H20) previously documented for the rabbitdiaphragm during supramaximal electrical stimulation with a bound abdomen(Aldrich and Appel, 1985; Aldrich, 1987, 1988, Ferguson et aL, 1990). It isimportant to clarify that previous studies had demonstrated a drop in the150frequency-pressure curve of the diaphragm within one hour at target pressuresapproximately one half the intensity of the target pressure that is associated withtask failure in our studies (Aldrich and Appel, 1985; Aldrich 1987, 1988). Wewere able to demonstrate that the rabbit diaphragm is able to maintaintransdiaphragmatic pressure for at least 4 hours undergoing inspiratory resistiveloading of equivalent intensity (Load 2) without task failure. We were unableto determine esophageal pressure during loads of greater intensity due torepetitive swallowing associated with breathing against target inspiratorypressures of Pao = -30 cm H20. Therefore, initially, we could not determine towhat extent the inability of the diaphragm or other respiratory muscles togenerate inspiratory pressure contributed to task failure with target inspiratoryresistive loads in excess of this value (Loads 3 and 4).To assess the contribution of respiratory muscles to task failure duringinspiratory resistive loading, we monitored the electromyographic activity of theparasternal intercostal and the transversus abdominis muscles and the diaphragmduring extreme target inspiratory resistive loads (Pao = -58± 4 cm H20) untilrespiratory arrest (Chapter IV). We described the sequence of changes thatoccur from respiratory muscle task failure to respiratory arrest under severeinspiratory resistive loading as follows. With extreme loading there was agradual decrease in Pa02 followed by a progressive decay in phasic expiratoryabdominal muscle activity and expiratory abdominal pressure swings that led to151a significant decrease in Pao (i.e. task failure). The inspiratory activity of theparasternal intercostals and diaphragm were maintained elevated and coupledduring this phase. Therefore, we attributed task failure during this phase to adecrease in abdominal muscle activation. In the next phase, there was atransition in breathing pattern to cluster breathing which corresponded withsevere arterial hypoxemia (PaCO2 <25 mm Hg) followed by coinciding decreasesin arterial blood pressure and inspiratory muscle activity of the diaphgram andparasternal intercostal muscles, airflow and inspiratory pressure leading torespiratory arrest. We proposed that the selective decrease in abdominal activityduring exhaustive inspiratory resistive loading is most likely due to thedifferential inhibitory effect of progressive hypoxemia (Fregosi, 1987) whichprecedes the reduction in expiratory abdominal activity and expiratory pressureswings.To our knowledge, this study is the first to show that the failure of abdominalmuscle assist to the diaphragm during inspiratory resistive loading precedes taskfailure. As inspiratory resistive loading was associated with progressivehypercapnia in our model, we attributed the recruitment of abdominal musclesduring this type of loading to the excitatory effect of CO2 on expiratorybulbospinal neurons (Bainton and Kirkwood, 1979) and abdominal motoneurons(Ledlie, Pack and Fishman, 1983). We have underscored the importance ofexpiratory abdominal muscle activation in response to inspiratory resistive152loading and characterized the mechanisms by which their recruitment canincrease the mechanical efficiency of the diaphragm (see Chapter IV). A recentstudy on conscious humans subjected to high inspiratory resistive loadingdemonstrates that the ventilatory response to this type of load is achievedpartially through the mechanical assist provided to the diaphragm by theexpiratory muscles (Yan, 1993). Our findings show that failure of expiratorymuscle activity plays a more critical role in task failure than previously believed.Our observation that reduction in activity of the transversus abdominisaccompanies inspiratory pressure failure indicates task failure is central in origin.Although electromyographic evidence showed that activation of the inspiratorymuscles was maintained during task failure under severe inspiratory resistiveloading, myogenic fatigue of the inspiratory muscles during this period can notbe conclusively ruled out. We suspect that pressure loss would develop in ourmodel during supramaximal activation of the diaphragm (Aldrich, 1987) but donot believe that the response of the diaphragm to spontaneous activation againstinspiratory resistive loading contributes to task failure. There is limited data onthe indices of myogenic fatigue in the inspiratory muscles during inspiratoryresistive loading. In contrast to earlier reports of a reduction in glycogen contentof diaphragm during inspiratory resistive loading (Bazzy et aL, 1988), recentreports show no change in the levels of either glycogen, ATP, PCr or lactate inthis muscle after severe and prolonged inspiratory resistive loading (Ferguson et153aL, 1990; Mayock et aL, 1991). Our findings suggest that task failure developsonly when inspiratory muscle function is compromised by severe alterations inblood gases that undermine activation of respiratory muscles (central fatigue).4. The fourth aim of our study was to determine the relationship between taskfailure and hypercapnic ventilatory failure during inspiratory resistive loading.We found that progressive hypercapnia was a salient feature of inspiratoryresistive loading independent of the intensity of the loads examined.Surprisingly, central motor output to the diaphragm (ENGdi) remained constantduring prolonged sustainable loads (Load 1 and 2) despite rising levels of arterialPaCO2. We examined the possibility that prolonged exposure to severehypercapnia alone could suppress ventilatory drive to the diaphragm in ourmodel by considering the time-dependent changes in ventilation duringhypercapnia equivalent to the levels accompanying loaded breathing (ChapterIII). We found a significant reduction in breathing frequency by two hours ofexposure to severe hypercapnia. We discussed several mechanisms that couldpotentially affect the regulation of CO2 chemoreception during prolongedhypercapnia to produce a decrease in central rhythm. We concluded that theprolonged hypercapnia associated with inspiratory resistive loading suppressesdrive to the diaphragm and results in load decompensation.154References1. Aldrich T.K. and D. Appel (1985). Diaphragm fatigue induced by inspiratoryresistive loading in spontaneously breathing rabbits. J AppL PhysioL 59:1527-1532.2. Aldrich, T.K. (1987). Transmission fatigue of the rabbit diaphragm. RespirPhysioL 69: 307-3 19.3. Aldrich, T.K. (1988). Central fatigue of the rabbit diaphragm. Lung 166:233-241.4. Aldrich, T.K. (1991). Central and transmission fatigue. Seminars inRespiratoiy Medicine 12: 322-330.5. Bainton, C.R. and P.A. Kirkwood (1979). The effect of carbon dioxide on thetonic and the rhythmic discharges of expiratory bulbospinal neurones.PhysioL 196: 291-314.6. Bazzy, A.R., S.R. Akabas, A.P. Hays and G.G. Haddad (1988). Respiratorymuscle response to load and glycogen content in type I and II fibres. Exp.NeuroL, 101: 17-28.7. Bazzy A.R. and D.F. Donnelly (1993). Diaphragmatic failure during loadedbreathing: role of neuromuscular transmission. J AppL PhysioL 74: 1679-1683.8. Ferguson G.T., C.G. Irvin, and R.M. Cherniack (1990). Relationship ofdiaphragm glycogen, lactate, and function to respiratory failure. Am. Rev.155Respir Dis. 141: 926-932.9. Fregosi, R.F., S.L. Knuth, D.K. Ward and D. Bartlett, Jr. (1987). Hypoxiainhibits abdominal expiratory nerve activity. J. AppL Physiol. 63: 221-220.10. Ledlie, J.F., A.!. Pack and A.P. Fishman (1983). Effects of hypercapniaand hypoxia on abdominal expiratory nerve activity. J AppL PhysioL 55:1614-1622.11. Mayock, D.E., T.A. Standaert, T.D. Murphy and D.E. Woodrum (1991).Diaphragmatic force and substrate response to resistive loaded breathing iithe piglet. .L AppL PhysioL 70: 70-76.12. Watchko, J.F., T.A. Standaert, D.E. Mayock, G. Twiggs and D.E. Woodrum(1988). Ventilatory failure during loaded breathing: the role of centralneural drive. .L AppL PhysioL 65: 249-255.13. Zechman, F., F.G. Hall and W.E. Hull (1957). Effects of graded resistanceto tracheal airflow in man. J AppL PhysioL 10: 356-362.14. Yan, S., P. Sliwinski, A.P. Gauthier, I. Lichros, S. Zakynthinos and P.T.Macklem (1993). Effect of global inspiratory muscle fatigue in ventilatoryand respiratory muscle responses to CO2. J AppL PhysioL 75: 137 1-1377.

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