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The mechanics of breathing in the turtle, Pseudemys scripta Vitalis, Timothy Zoltan 1984

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THE MECHANICS OF BREATHING IN THE TURTLE, PSEUDEMYS SCRIPTA by TIMOTHY ZOLTAN VITALIS B.Sc. (Hons.), University of B r i t i s h Columbia, 1981 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (DEPARTMENT OF ZOOLOGY) We accept t h i s thesis as conforming to the required^tandard THE UNIVERSITY OF BRITISH COLUMBIA October 1984 © T.Z. V i t a l i s , 1984 In presenting this thesis i n p a r t i a l fulfilment of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the l i b r a r y s h a l l make i t freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representa-tives. It i s understood that copying or publication of this thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of Zoology The University of B r i t i s h Columbia Vancouver, B.C. V6T 2A9, Canada i i Abstract Measurements of pulmonary mechanics on anaesthetized specimens of the aquatic t u r t l e Pseudemys s c r i p t a , indicate that the s t a t i c pulmonary mechanics are determined primarily by the mechanics of the body wall of the animal rather than those of the lungs. The mechanics of the body wall are also predominant under the dynamic conditions of pump v e n t i l a t i o n , but only at low frequencies. As pump frequency increases, the multicameral lungs of the t u r t l e begin to contribute an ever increasing portion to the t o t a l mechanical work required to produce each breath as measured from pressure volume loops. The r i s e i n the work performed on the lungs results from an increase i n the nonelastic, flow r e s i s t i v e forces which must be overcome during v e n t i l a t i o n . The respiratory system i s the major s i t e of nonelastic work. The primary bronchus to each lung branches to seven intrapulmonary chambers, and i s the most l i k e l y s i t e of flow-resistance. There i s also a small e l a s t i c component of the work required to ventilate the lung a r i s i n g from forces i n the intrapulmonary septa and the s t r i a t e d muscle surrounding the lungs. The contribution of the body to the t o t a l mechanical work required to generate each breath remains r e l a t i v e l y unchanged with increa-sing v e n t i l a t i o n frequency indicating that the majority of the forces to be overcome i n the body wall are e l a s t i c i n nature. Since the s h e l l of the animal i s r i g i d these e l a s t i c forces must reside i n the flanks and pectoral regions. For a constant alveolar v e n t i l a t i o n rate (V^) as frequency increases, the e l a s t i c work done per minute decreases while the nonelastic work done per minute begins to r i s e . This results i n a 'U'-shaped curve for the t o t a l mechanical work done per minute i n venti l a t i n g the lungs at a i i i * constant V A as breathing frequency increases. There i s then, for any given l e v e l of v e n t i l a t i o n , a combination of t i d a l volume and frequency at which the rate of mechanical work i s minimized. This occurs at a frequency of 35 breaths per minute. The normal breathing pattern of P. s c r i p t a consists-of bursts of continuous breathing separated by variable periods of breath holding. Increases i n pulmonary v e n t i l a t i o n , upon stimulation by hypercapnia (3% CC>2 i n a i r ) or hypoxia (4% Q>2 I 1 1 ^2^* a r e c a u s e c - D v increases i n the number of breaths per minute due to the shortening of the breath hold period. Tidal volume and breath duration remain unchanged. The instantaneous breathing frequency ( f , = 6 0 / T t o t ) corresponds to the pump frequency which minimizes the rate of mechanical work as measured i n anaesthetized t u r t l e s . This indicates that t u r t l e s breathe at a combination of t i d a l volume and f' which minimizes the rate of mechanical work required to ventilate the lungs. To increase v e n t i l a t i o n , the breath hold i s shortened and more breaths are taken at this optimal combination. B i l a t e r a l vagotomy d r a s t i c a l l y a l t e r s the breathing pattern producing an elevation i n t i d a l volume, a slowing of breathing frequency and a prolongation of the breath duration, resulting i n an increase i n the mechanical cost and thus i n the oxidative cost of breathing even though t o t a l minute v e n t i l a t i o n (V £) changes very l i t t l e . These data suggest that periodic breathing i n this species may represent an adaptive strategy which i s under vagal afferent control and which serves to minimize the cost of breathing. Iv TABLE OF CONTENTS Abstract i i L i s t of Tables v i L i s t of Figures v i i Acknowledgement i x Chapter 1 General Introduction 1 Chapter 2 The St a t i c Mechanics of the Respiratory System i n Pseudemys sc r i p t a Introduction 14 Materials and Methods 19 Results 22 Discussion • 30 Chapter 3 The Dynamic Pulmonary Mechanics of the Turtle, Pseudemys s c r i p t a Introduction 35 Materials and Methods 38 Results 45 Discussion 62 Page Chapter 4 The Interrelationship Between Pulmonary Mechanics and the Spontaneous Breathing Pattern in Pseudemys scripta Introduction 71 Materials and Methods 73 Results 76 Discussion 80 Chapter 5 General Discussion and Conclusions 91 Literature Cited 95 v i List of Tables Page Table 2.1 Compliance of the total system (C^), body wall (Cg) and lung (C L) 24 Table 2.2 Compliance of the total system (Crj,), body wall (Cg) for various species 25 Table 2.3 Percentage of the total work required to overcome the elastic forces resisting inflation which arise from the body wall and lung 27 Table 3.1 Values of anatomical dead space determined for specimens of P_. scripta by two different methods 61 Table 4.1 Respiratory variables for spontaneously ventilating turtles breathing air, 3 - 5 % or 4% O 2 in air 78 Table 4.2 Comparison of respiratory variables reported in the literature for turtles 81 v i i L i s t of Figures P a S e Figure 2.1 Schematic representation of a pressure-volume curve 15 Figure 2.2 Pressure-volume curves f o r the t o t a l system, the body wall and lungs 23 Figure 2.3 S i m p l i f i c a t i o n of fig u r e 2.1 to i l l u s t r a t e technique used f o r c a l c u l a t i o n of work required to overcome the e l a s t i c forces r e s i s t i n g a 1 ml i n f l a t i o n 28 Figure 3.1 Schematic diagram of the pressure-volume r e l a t i o n s of the i n t a c t respiratory system during a sin g l e v e n t i l a t i o n cycle 42 Figure 3.2 E f f e c t s of changes i n v e n t i l a t i o n frequency and volume on the pressure-volume r e l a t i o n s h i p associated with a single v e n t i l a t i o n cycle of the t o t a l respiratory system 46 Figure 3.3 E f f e c t s of changes i n v e n t i l a t i o n frequency and volume on pressure-volume loops associated with a s i n g l e v e n t i l a t i o n cycle of the body compartment alone 47 Figure 3.4 The r e l a t i o n s h i p between v e n t i l a t i o n frequency and dynamic compliance at constant volumes 49 Figure 3.5 The r e l a t i o n s h i p between the t o t a l work/breath and minute v e n t i l a t i o n f o r d i f f e r e n t combinations of t i d a l volume and frequency 50 Figure 3.6 The contribution of e l a s t i c and nonelastic work to the t o t a l work/breath 52 v i i i Page Figure 3.7 The contribution of the lung and body to the t o t a l work/breath 54 Figure 3.8 The effect of increasing v e n t i l a t i o n at a constant minute v e n t i l a t i o n on PaC>2> Pa^2» a n c" a l v e ° l a r minute v e n t i l a t i o n 55 Figure 3.9 The relationship between the rate of work and v e n t i l a t i o n frequency for constant levels of minute v e n t i l a t i o n and alveolar v e n t i l a t i o n 56 Figure 3.10 The contribution of e l a s t i c and nonelastic work to the t o t a l rate of work for a constant alveolar v e n t i l a t i o n . . . . 58 Figure 3.11 The contribution of the lung and body compartments to the t o t a l rate of work for a constant alveolar v e n t i l a t i o n . . . . 59 Figure 4.1 Representative flow traces from an (a) intact and (b) vagotomized t u r t l e spontaneously breathing a i r 77 Figure 4.2 The rate of work for constant levels of alveolar v e n t i -l a t i o n and t o t a l v e n t i l a t i o n as a function of pump ve n t i l a t i o n frequency 85 Figure 4.3 The relationship between t o t a l work/breath and pump ve n t i l a t i o n frequency 87 ix ACKNOWLEDGEMENTS I wish to express my sincere appreciation to Dr. W.K. Milsom for his encouragement, support, and advice throughout this study. I also wish to thank Dr. J. Gosline for his help, insights, criticisms, and suggestions. Financial support was provided by Teaching Assistantships from the Department of Zoology, and by the McLean Fraser Memorial Fellowship. Research costs were covered by an N.S.E.R.C. operating grant to Dr. W.K. Milsom. - 1 -CHAPTER 1 GENERAL INTRODUCTION The t r a n s i t i o n from l i f e i n water to l i f e on land required the development of two important structures, limbs and lungs (Romer, 1967). Romer (1972) contends that lungs developed i n f i s h , most probably, as a response to shallow, fresh water environments characterized by hypoxia and periodic drought. The appearance of such lungs gave r i s e to the further p o s s i b i l i t y of l i f e on land. Both aquatic a i r breathing organisms and t e r r e s t r i a l organisms can exploit the use of a i r as a respiratory medium. A i r has the advantage of providing an abundant and stable source of oxygen, but the disadvantage of desication posing a constant and serious problem. To combat the problem of desication, the integument of the t e r r e s t r i a l tetrapods became increasingly keratinized resulting i n a progressive loss of cutaneous gas exchange and an increasing dependence on pulmonary gas exchange (Tenney and Tenney, 1970). Coupled with t h i s increased dependence on pulmonary gas exchange was an increase i n the complexity and surface area of the lungs. Several trends also developed i n the mechanisms employed for v e n t i l a t i n g these lungs. The f i r s t land dwellers were the amphibians. Modern amphibians such as the grass frog, Rana pipiens, rely on cutaneous gas exchange for 34% of t h e i r O2 requirements and 83% of th e i r CO2 loss (25°C) (Hutchinson et a l . , 1968). It i s unclear, however, whether cutaneous gas exchange i n amphibians i s a primitive c h a r a c t e r i s t i c or has evolved l a t e r as a secondary sp e c i a l i z a t i o n to aid i n CO2 elimination (Romer, 1972). The lungs of modern amphibians are simple sacs with l i t t l e infolding to increase surface area (Hughes, 1963). The amphibians ventilate t h e i r lungs using positive - 2 -pressure generated by their buccal cavity (West and Jones, 1975), a process which i s dynamically analogous to g i l l v e n t i l a t i o n (Tenney and Tenney, 1970). "This primitive breathing mechanism has a limited ventilatory capacity and never frees the animal from some dependence on extra-pulmonary gas exchange" (Tenney and Tenney, 1970). Ventilatory capacity i s l i m i t e d , not by respiratory frequency but by t i d a l volume ( V t ) , which i s limited by the size of the buccal cavity. With some modification, t h i s mechanism probably represents a retention of that used by lunged f i s h where g i l l and branchial musculature are used for both g i l l and lung v e n t i l a t i o n . By retaining this mechanism a s h i f t only i n control mechanisms regulating the timing of various portions of the pumping sequence were required (Shelton et a l . , 1984). In r e p t i l e s the skin has been almost completely eliminated as a gas exchange organ. The lungs are now the predominant s i t e of gas exchange though aquatic t u r t l e s can eliminate considerable amounts of CO^ through thei r skin (Jackson et a l . , 1974). In general, the lungs of re p t i l e s have an increased surface area compared to amphibians but this i s not always the case. For instance, some of the less active r e p t i l e s such as Sphenodon have only simple bag-shaped lungs (Hughes, 1963). The lungs i n a l l r e p t i l e s communicate with the pharyngeal cavity by a cartilage-ringed tracheal tube. In most r e p t i l i a n species t h i s tracheal tube divides into primary bronchi which connect the several intrapulmonary chambers of each lung (see chapter 2). The positive pressure buccal pump i s replaced by aspiration breathing which i s characterized by the generation of negative pressure within the lung. A i r i s now drawn into the lung rather than forced i n by positive pressure b u i l t up i n the anterior buccal cavity. The complex co-ordination - 3 -of nasal and g l o t t a l opening and closing i s no longer essential but both sets of openings s t i l l play major roles. During aspiration breathing both sets of openings are open simultaneously allowing for continuous t i d a l v e n t i l a t i o n of the pulmonary gas exchange surface. Once breathing ceases the g l o t t i s closes at end-inspiration. The three classes of modern r e p t i l e s a l l possess different mechanisms of v e n t i l a t i o n which, on a morphological basis, are not homologous to one other (Gans, 1976). The variety of ventilatory mechanisms seen i n the r e p t i l e s suggests that aspiration breathing has evolved many times using different muscles, different mechanics, and different mechanisms to suit the i n d i v i d u a l problems of each group. Lizards and snakes have long f l e x i b l e bodies with ribs for support and costal breathing was an easy adaptation. Crocodiles are much too heavy to l i f t themselves off the ground to breath using r i b movement and have developed a l i v e r - p i s t o n pump. Turtles l i v e i n a box formed by t h e i r s h e l l and use thei r flank cavities and also, p a r t i a l l y , t h e i r fore-limbs to ventilate t h e i r lungs. Ven t i l a t i o n i n l i z a r d s (Hughes, 1963) and snakes (Rosenberg, 1973) i s powered by a costal pump. Inspiration i s an active process brought about by a suction pump powered by the muscles of the r i b s . The in t e r c o s t a l muscles contract, moving the ribs and expanding the r i b cage and the thorax. This produces a negative pressure i n both the thorax and the lungs. Expiration i s passive but may be assisted by the contraction of transverse abdominal muscles (Hughes, 1963). The respiratory movements are tr i p h a s i c while a i r flow i s diphasic. Flow appears to only be tr i p h a s i c following mistiming of g l o t t i s closure i n disturbed animals, or due to the effects of anesthesia (Cragg, 1978; Gans and Clark, 1978). A v e n t i l a t i o n cycle begins - 4 -with expiration. During the period of breath holding which follows i n s p i r a t i o n , pulmonary pressure i s above atmospheric. Inward movements of the thoracic cage preceding expiration causes the pressure within the lung to r i s e further u n t i l the g l o t t i s opens and a i r flows out of the lungs. The inspiratory phase follows immediately whereupon a subatmospheric pressure i s produced i n the lungs due to the outward movement of the thoracic cage. At end-inspiration the g l o t t i s closes and the inspiratory muscles relax causing a s l i g h t compression of the lungs which results i n intrapulmonary pressure being above atmospheric pressure during breath holding. The crocodile, Caiman crocodilus, ventilates i t s lungs by a hepatic piston pump (Gans and Clark, 1976). The ribs are held i n position by the i n t e r c o s t a l muscles while the transverse abdominal muscles s h i f t the l i v e r forward thereby reducing the volume of the pulmonary chamber and causing exhalation. If the muscles contract more strongly, or i f contraction precedes the opening of the g l o t t i s , the lungs may be pressurized leading to a more rapid exhalation. Inspiration occurs after expiration and i s brought about by the contraction of the diaphragmatic muscle. This diaphragmatic muscle i s not homologous to the mammalian diaphragm but inserts on to the l i v e r from i t s o r i g i n on the i l i a and epipubic elements of the pelvic g i r d l e . Contracti on of this muscle s h i f t s the l i v e r posteriorly thus increasing the volume of the thoracic cavity and hence the lungs. The negative pressure generated by the increase i n lung volume causes a i r to flow i n through the open g l o t t i s . The ventral, deep intercostals also contract to maintain the patency of the pulmonary cavity. The pattern of pressure changes generated i n the lung during breathing are similar to those seen i n l i z a r d s and snakes. - 5 -The chelonians have a unique mechanism of aspiration breathing which results from the presence of a rigid shell in these animals. The ribs of the chelonians are fused to the dome of the carapace and therefore there are no intercostal muscles available to power or assist in ventilation. A detailed investigation of the mechanism of ventilation has been carried out on three species of chelonia Malaclemmys centata (McCutcheon, 1943), Testudo  graeca (Gans and Hughes, 1967), and Chelydra serpentina (Gaunt and Gans, 1969). Gans and Hughes (1967) and Gaunt and Gans (1969) investigated the breathing mechanism of turtles by correlating electromyographical data from respiratory muscles with pulmonary pressure recordings in the terrestrial tortoise, Testudo graeca, and the semi-aquatic snapping turtle, Chelydra  serpentina. Both species show triphasic respiratory movements and diphasic air flows with pauses at end-inspiration, between ventilation cycles. There are differences, however, in the rhythm of ventilation and the muscles powering ventilation. The aquatic turtle ventilates i t s lungs intermit-tently with a series of continuous breaths during each breathing cycle. Breathing cycles are separated by long variable periods of breath holding. The terrestrial tortoise, on the other hand, exhibits single ventilation cycles separated by a pause at end-inspiration. The differences are thought by Gaunt and Gans (1969) to be due to the aquatic habitat and predatory l i f e style of Chelydra. A great deal of activity is carried out while the animal i s submerged and when i t breathes, air has to be recycled very rapidly in preparation for the next dive and presumably this requires multiple breaths. Ventilation in the tortoise is powered by the muscles of the - 6 -pectoral g i r d l e and by the abdominal muscles. The muscles of the pectoral g i r d l e involved i n v e n t i l a t i o n are the Muscularis (M) pectoralis which induces inward rotation of the pectoral g i r d l e and the M. serratus magnus (= M.testocoracoideus) which produces outward rotation of the g i r d l e . The position of the pectoral g i r d l e determines the anterior l i m i t of the vi s c e r a l cavity. The pelvic g i r d l e i s immobile and located centrally within the posterior aperture of the s h e l l . The flanks l a t e r a l to the hind limbs are formed by skin, connective tissue, and a thin sheet of muscle. The flanks determine the posterior l i m i t s of the v i s c e r a l cavity. Here l i e the M. obliquus abdominis and the M. transversus abdominus, referred to by Gans and Hughes (1967) as the respiratory muscles. The connective tissue and muscles of the posterior flanks form an anteriorly concave cup that encloses the visera. Contraction of the M. transversus abdominus flattens the curvature thus exerting pressure on the viscera while contraction of the M. obliquus  abdominis increases the curvature and decreases the v i s c e r a l pressure. A respiratory cycle begins with a rapid increase i n pulmonary pressure which i s associated with a high l e v e l of e l e c t r i c a l a c t i v i t y i n the M. transversus abdominus, and a low l e v e l of a c t i v i t y i n the M. pectoralis, a s l i g h t inward movement of the limbs, and e l e c t r i c a l a c t i v i t y i n the d i l a t o r muscle of the g l o t t i s . The second phase begins with a reversal of the pulmonary pressure, a sharp increase i n the EMG of the M. pectoralis and an acceleration of the inward movement of the forelimbs. The pulmonary pressure drops to atmospheric, the M. transversus abdominus and M. pectoralis cease f i r i n g and expiration i s complete. The M. obliquus  abdominus and M. serratus magnus begin f i r i n g at or near the time when - 7 -pulmonary pressure approaches atmospheric pressure. The forelimbs begin a phase of outward movement causing the volume of the body cavity to increase which results i n a subatmospheric pressure being generated i n the lungs. Pressure remains subatmospheric for a variable period and the g l o t t i s opener muscle, the M. obliquus abdominus, and the M. serratus magnus cease f i r i n g as the pressure equilibrates with atmospheric pressure upon the inflow of a i r . Inspiration i s complete when the pressure reaches atmospheric and the g l o t t i s closes. Pulmonary pressure then rises above atmospheric pressure due to a passive compression of the system. Both i n s p i r a t i o n and expiration appear to be active processes. The M. transverse abdominus i s the most important muscle of respiration with the M. pectoralis playing an accessory rol e . If v e n t i l a t i o n i s stimulated by CO2 inhalation or f r i g h t , the pectoral g i r d l e takes on a more active role i n v e n t i l a t i o n . The aquatic t u r t l e , Chelydra serpentina shows the same pattern of triphasic pulmonary pressure changes and diphasic a i r flow as T_. graeca. The differences i n the mechanism of v e n t i l a t i o n seen i n the aquatic t u r t l e involve the muscles powering exhalation and inhalation. The muscles that power exhalation are the M. transversus abdominus which i n t e r n a l l y forms the membrane of the exposed flank, and the M. diaphragmaticus, a muscle not seen i n T_. graeca. The M. diaphragmaticus originates inside the carapace from the margin of the second costal plate and extends to a position anterior to the lung along the f i r s t fused r i b . Contraction of these two muscles compresses the v i s c e r a l mass, increasing the pressure i n the lung. The opening of the g l o t t i s permits a i r flow out of the animal. The pectoral girdle does not play an active role i n exhalation. The muscles powering inhalation include the M. testocoracoideus - 8 -(M. serratus magnus i n T_. graeca) and the M. obliquus abdominus. These muscles begin f i r i n g as the intrapulmonary pressure f a l l s to atmospheric pressure following expiration. Contraction of these muscles serves to increase the volume of the v i s c e r a l mass by causing an outward rotation of the pectoral g i r d l e (M. testocoracoideus) and flattening of the concavity of the flanks (M. obliquus abdominis). This causes the pulmonary pressure to drop below atmospheric pressure causing an inward flow of a i r . Upon closure of the g l o t t i s the pulmonary pressure rises above atmospheric pressure due to passive r e c o i l of the system. There i s also some low l e v e l a c t i v i t y i n the M. diaphragmaticus and M. obliquus abdominis. The actions of the respiratory muscles cause some s h e l l deformation during v e n t i l a t i o n . The M. testoscapularis shows low l e v e l a c t i v i t y at end-expiration and throughout inhalation which helps restore the curvature of the carapace. The M. testoscapularis originates on the carapace near the l a t e r a l t i p of the f i r s t fused r i b and inserts dorsally on the scapula. Contraction of the M. testoscapularis, during the exhalation period when pulmonary pressure drops and the shell's transverse curvature f a l l s , restores the curvature of the carapace. Chelydra appears to minimize the muscular e f f o r t of exhalation by u t i l i z i n g hydrostatic pressure when submerged. In this s i t u a t i o n when the animal i s i n water expiration tends to be passive. More ef f o r t i s , however, required of the muscles generating i n s p i r a t i o n to overcome the hydrostatic pressure head which tends to keep the pressure i n the system above atmospheric. Chelydra also spends time on land and at such times, due to the reduced plastron i n this species, the viscera tend to sag resulting i n subatmospheric pressures i n the lung during the breath-hold period. - 9 -Expiration now takes more effort but inhalation tends to be passive. The presence of the M. diaphragmaticus as a respiratory muscle i n Chelydra has freed the pectoral girdle from i t s active role in expiration which Gaunt and Gans (1969) believe may provide several advantages for this aquatic turtle. First, the limbs are now free for swimming while the animal is ventilating at the surface. While the animal is afloat and stationary, extended limbs would stabilize the animal as i t bobs due to buoyancy changes associated with ventilation. Secondly, the freeing of the limbs from powering ventilation also reduces large amplitude movements of the limbs which might cause prey to react suddenly. This i s of great importance to a predatory species. The significance of these advantages is questionable, however, as even Testudo has no trouble breathing with i t s front limbs retracted and most predatory species feed submerged rather than while breathing at the surface. A l l these mechanisms of breathing involve muscular work. One can measure the total energy required by the respiratory muscles through oxygen consumption measurements, or the mechanical work done by the respiratory muscles which can be estimated through measurements of pressure changes and volume changes within the respiratory system. The mechanical work divided by the total energy requirements of the respiratory system gives an indication of the efficiency of the mechanism of ventilation. The measurement of pulmonary mechanics allows one to analyze in detail the various forces which contribute to the total mechanical work of breathing. Measurement of the oxidative cost of breathing allows one to examine how much of the energy budget of the animal is required for breathing. In general, mechanical work is performed when a force moves - 10 -through a distance, or i n the case of a f l u i d system such as the respiratory system, when a change i n pressure causes a change i n volume. During breathing, i n s p i r a t i o n requires the respiratory muscles to contract thus increasing the volume of the cavity containing the lungs. To increase the volume of the cavity the inspiratory muscles have to overcome the e l a s t i c forces i n the tissues of the lungs and body cavity, and the nonelastic forces which include flow r e s i s t i v e forces i n the airways, viscous forces due to the movement of tissues and the i n e r t i a of the respiratory system. The t o t a l mechanical work required to produce a breath consists of the sum of a l l the e l a s t i c and nonelastic forces during i n s p i r a t i o n and expiration. Estimates of the t o t a l work of breathing and i t s components can be derived from pressure-volume relationships which are described i n chapters 2 and 3. The work of breathing has been extensively studied i n humans (see Otis, 1954; Otis, 1964 for reviews) and other mammalian species ( C r o s f i l l and Widdicombe, 1961). Otis et a l . (1950) developed a model to estimate the mechanical work of breathing i n humans. The model predicts that for a constant l e v e l of alveolar v e n t i l a t i o n there i s a combination of t i d a l volume and breathing frequency which requires minimum work and thus minimizes the oxidative cost of breathing, since the work per unit v e n t i l a t i o n and oxidative cost are proportional (McKerrow and Otis, 1956; M i l i c - E m i l i and P e t i t , 1960). It was found by Otis et a l . (1950) that the spontaneous breathing pattern of humans corresponded very closely with the predicted pattern. C r o s f i l l and Widdicombe (1961) showed for several species of mammal that the combination of t i d a l volume and frequency normally employed at various levels of ve n t i l a t i o n also coincided with the model predictions. These correlations gave r i s e to the suggestion that regulatory mechanisms must exist to regulate breathing frequency so as to minimize the mechanical work (Otis, 1954). Mead (1960) proposed that regulation occurs at the l e v e l of the respiratory muscles to keep the length-tension relationship of the muscles optimal i n order to maximize power output rather than to minimize mechanical work. His argument was that force sensed by "appropriately located stretch receptors, represents a more simple case than work rate, which would require sensing, and appropriate manipulation of, three variables; force, distance, and time." (Mead, 1960) Mead (1960) concedes, however, that upon imposed mechanical loads the p r i n c i p a l receptors for a r e f l e x controlling breathing frequency cannot l i e i n the respiratory muscles, but must l i e i n the lung and this role i s probably performed by the slowly adapting vagal stretch receptors. I t seems unlike l y , however, that the system would regulate breathing frequency by one method at rest ( i . e . receptors i n the respiratory muscles), and by vagally innervated stretch receptors upon changes i n mechanical load. Rabbits subjected to e l a s t i c loading have been shown to increase breathing frequency (McClelland et a l . , 1972) as i s predicted. If the animals are vagotomized there i s no s i g n i f i c a n t change i n breathing frequency upon addition of an e l a s t i c load. This seems to indicate that the Breuer-Hering reflex might i n some way be responsible for regulating and s t a b i l i z i n g v e n t i l a t i o n i n order to minimize the work of breathing. Thus mechanical factors appear to play a major role i n determining the nature of the breathing pattern employed by mammals. Given the di v e r s i t y seen i n the mechanisms powering v e n t i l a t i o n i n the three r e p t i l i a n groups, a s i m i l a r d i v e r s i t y might be expected i n the mechanics of the various systems described above. This possible d i v e r s i t y i n pulmonary - 12 -mechanics may play a role i n establishing the d i v e r s i t y i n breathing patterns seen i n r e p t i l e s which range from single breaths separated by periods of breath holding, as seen for instance i n resting l i z a r d s or tortoises, to bursts of continuous breathing separated by periods of breath holding as seen i n semi-aquatic t u r t l e s and crocodiles, through to continuous or near continuous breathing patterns seen i n predatory species of l i z a r d s and almost a l l r e p t i l e s when excited. L i t t l e work, however, has been done on the pulmonary mechanics of r e p t i l i a n systems. Perry and Dunker (1978, 1980) suggested that resting breathing patterns may be related to s t a t i c mechanics though t h e i r analysis was incomplete. Milsom and V i t a l i s (1984) have investigated the s t a t i c and dynamic mechanics of the l i z a r d Gekko gecko which has very simple sac-like lungs and breathes with single breaths. It was shown, i n this species, that there i s an optimal combination of frequency and t i d a l volume corresponding to minimum mechanical work. In a subsequent study Milsom (1984) showed that the components of time and t i d a l volume of a single breath from a spontan-eously breathing Gekko corresponded to the predicted optimal combination giving minimum work per breath. Upon vagotomy i t was shown that the spontaneous pattern deviated from the optimum pattern. Pseudemys s c r i p t a i s a semi-aquatic t u r t l e which uses a different mechanism of v e n t i l a t i o n from that of G_. gecko, due to the presence of a r i g i d s h e l l enclosing the body cavity as described e a r l i e r . This species of t u r t l e also possesses a more complicated multichambered lung compared to the simple sac-like lung of G_. gecko. One aim of this present study, therefore, was to investigate how these differences i n lung structure and body wall architecture i n P_. s c r i p t a and G_. gecko influence the mechanics of the - 13 -r e s p i r a t o r y system. Another goal was to determine i f d i f f e r e n c e s i n pulmonary mechanics could account f o r the d i f f e r e n c e s seen between the s i n g l e breath p a t t e r n of G. gecko and the p a t t e r n of P. s c r i p t a which c o n s i s t s of bursts of continuous breaths separated by breath-holds of v a r i a b l e d u r a t i o n . - 14 -CHAPTER 2 THE STATIC MECHANICS OF THE RESPIRATORY SYSTEM IN PSEUDEMYS SCRIPTA Introduction The pressure of a volume of gas contained i n the lungs, under s t a t i c conditions with the respiratory muscles relaxed and the g l o t t i s closed, depends upon the surface tension of the lungs and on the e l a s t i c and gravi t a t i o n a l forces operating on the lung and chest wall (Agostoni, 1970). These are the forces which must be overcome to i n f l a t e the respiratory system to this volume. The p r i n c i p a l force generating this pressure, which i s equal to the difference between intrapulmonary and atmospheric pressure, i s that due to e l a s t i c r e c o i l . The mechanical work, which was required to overcome these e l a s t i c forces can be determined from the s t a t i c pressure-volume relationship of the t o t a l respiratory system (the lungs and chest w a l l ) . Figure 2.1 shows a schematic pressure-volume relationship for the t o t a l respiratory system of a t y p i c a l vertebrate (Otis, 1964). Such a curve i s generated by measuring the pulmonary pressure following stepwise i n f l a t i o n and deflation of the lungs starting from resting lung volume (Vy^). Resting lung volume i s the volume of gas i n the lung after a normal expiration when intrapulmonary pressure and atmospheric pressure are equal (A i n figure 2.1). The mechanical work required to overcome e l a s t i c forces r e s i s t i n g an increase i n volume from A to B i s given by the area ABCA i n ml'cmR^O. The slope of the li n e a r portion of the curve (AC which = V/ P) represents the s t a t i c compliance of the t o t a l system ( C T ) . This - 15 -Fig. 2.1 Schematic representation of a pressure-volume curve. For a volume change of AB, the pressure change i n the system would be AD. The work required to overcome the e l a s t i c forces r e s i s t i n g i n f l a t i o n would be represented by area ABCA. The compliance of the system (Cp) i s the slope of the pressure-volume curve which i s linear over the range shown. The o r i g i n of the graph i s V L R at atmospheric pressure. - 15a -- 16 -value i s a measure of the s t i f f n e s s of the system. A low compliance indicates a s t i f f system where the e l a s t i c forces are large, and, correspon-dingly, area ABCA would be large indicating that much mechanical work would be needed to i n f l a t e the system. A high compliance on the other hand, indicates a system where the e l a s t i c forces are small and thus area ABCA would be small and l i t t l e mechanical work would be needed to i n f l a t e the system. The mechanics of the t o t a l respiratory system can also be analyzed i n terms of the mechanics of i t s component parts, the lung and body w a l l . Since the forces exerted by the lung and body on the lung contents act i n series, the compliance of the t o t a l respiratory system i s related to the compliance of i t s component parts by the relationship 1/C^ = 1/C^ + 1/Cg where C^ i s the compliance of the lung, and Cg i s the compliance of the body w a l l . If the compliance of the lung and body wall are each determined, one can derive the r e l a t i v e contributions of each component to the t o t a l e l a s t i c forces within the system. Measurements obtained from such pressure-volume curves also allow comparisons of pulmonary mechanics between animals which exhibit wide variations i n lung structure. R e p t i l i a n lungs, for instance, have been c l a s s i f i e d into three major groups according to their gross anatomy (Perry and Dunker, 1978). One group contains the unicameral lungs which each consist of a single chamber lacking any intrapulmonary septation or intrapulmonary bronchi. In these lungs a r e l a t i v e l y uniform network of trabeculae a r i s i n g from the outer lung wall support the faveolar parenchyma. This honey-comb-like parenchyma bears a matrix of c a p i l l a r i e s on both surfaces and i s the s i t e of gas exchange. Paucicameral lungs are p a r t i a l l y - 17 -divided into chambers but lack an intrapulmonary bronchus. The faveolar parenchyma now arises from the intrapulmonary septa as well as the lung wall and i s deepest immediately caudal to the bronchial entrance. The degree of faveolar partitioning decreases more caudally and disappears i n the extreme caudal region which consists of membraneous f i n g e r - l i k e projections. M u l t i -cameral lungs are completely divided into many chambers connected indepen-dently by a single cartilage ringed intrapulmonary bronchus. The ventral chambers i n the caudal portion of the lung have thin membraneous walls whereas the parenchyma i n the dorsal portion and near the intrapulmonary bronchus i s densely partitioned. A l l three of these types of lungs found among the r e p t i l e s d i f f e r markedly from the alveolar lungs of mammals. The lungs of mammals are enclosed i n a pleural cavity formed by pleural membranes with a muscular diaphragm separating the viscera from the thoracic cavity. This i s unlike the r e p t i l i a n system where the lungs l i e i n the body cavity with only a delicate mesopneumonium preventing the lung from moving too far c r a n i a l l y or ventrally at low v e n t i l a t i o n volumes (Perry and Duncker, 1978). Mammals have a compact lung with a highly branched bronchial tree communicating with a large number of a l v e o l i . Surrounding the a l v e o l i and alveolar ducts are h e l i c a l networks of collagen and e l a s t i c f i b e r s which p a r t i a l l y generate the e l a s t i c r e c o i l of the lungs (Bouhuys, 1977). Besides the differences found i n lung architecture among various groups of animals there i s also tremendous d i v e r s i t y i n the structure of the body wall and i n the mechanisms employed for v e n t i l a t i o n (see chapter 1). One would therefore expect that the s t a t i c mechanics of the various respira-tory systems would also be diverse and r e f l e c t these differences. In this - 18 -chapter, experiments designed to measure the s t a t i c pulmonary mechanics of the t u r t l e , Pseudemys s c r i p t a are o u t l i n e d and the values obtained from these experiments are compared to those obtained from other r e p t i l e s and mammals. - 19 -Materials and Methods Experiments were performed on eight t u r t l e s (726.3 +^  107 grams body weight) which were maintained at room temperature (20-22°C) for several days before the experiments. On the day of an experiment an animal was given on overdose of sodium pentobarbital and when a l l signs of ref l e x a c t i v i t y were absent, the cloaca was sealed to prevent water loss from the bladder which would have altered body volume, intra-abdominal pressure and thus the mechanics of the body w a l l . The trachea was cannulated using P.E. 220-240 tubing with a side-arm close to the trachea leading to a Statham P23 Db physiological pressure transducer to measure intrapulmonary pressure. This signal was amplified and recorded on a Gould series 2600 pen recorder. Compliance of the t o t a l respiratory system (C^) With the tracheal cannula open to atmosphere the lungs were d e f l a -ted, then r e - i n f l a t e d to a volume of 11 ml/100 g. body weight with an a i r f i l l e d syringe. This volume corresponded to the resting lung volume ( V ^ ) as determined for this species by Jackson (1971). This volume was used as a common reference point for the t o t a l system, body w a l l , and lungs. The volume i n each compartment when the lungs were f i l l e d to V L R was referred to as zero volume for each compartment, and the pressure associated with i n each compartment was referred to as zero pressure. Starting from V L R, the lungs were then i n f l a t e d i n 5 or 10 ml steps. Following each step i n f l a t i o n the intrapulmonary pressure was allowed to decay to a new steady l e v e l . Step i n f l a t i o n continued u n t i l the i n f l a t i o n volume approached twice V , A t this volume the pressure increase for each further step inflation became very large and the slope of the pressure-volume curve decreased dramatically. The system was then deflated in a similar stepwise fashion until the lungs were completely deflated. The system was then reinflated to V^. Compliance of the body wall (Cfi) The preparation of the animals for these measurements required the cannulation of the abdominal cavity i n order to measure intra-abdominal pressure rather than intratracheal pressure. The flank of the turtle just anterior to the pelvic girdle was pierced with a 13 gauge hypodermic needle. A saline f i l l e d cannula (PE 190) was passed down the barrel of the needle which was then removed and the cannula was secured to the flank with a purse string suture. This cannula was connected to the Statham P23 Db physiological pressure transducer. The trachea was cannulated as in the previous section except there was no side-arm present in the cannula. The lungs were deflated then inflated to V^. Stepwise inflation and deflation of the body cavity was achieved by inflation and deflation of the lungs in the same manner as described in the previous section. Pressure inside the body cavity was now monitored instead of intratracheal pressure. Compliance of the lungs (C L) Following the measurements outlined above, the lungs of the turtles were exposed to determine the compliance of the isolated lungs. The plastron of a turtle was removed using a necropsy saw, the pelvic girdle was carefully removed from the shell and a l l the viscera including the - 21 -d i g e s t i v e t r a c t , l i v e r , and heart were c a r e f u l l y d i s s e c t e d away from the lungs. Once the v i s c e r a were removed the lungs were i n f l a t e d to V ^ . This volume and i t s associated pressure are again r e f e r r e d to as zero volume and pressure. Stepwise i n f l a t i o n and d e f l a t i o n of the i s o l a t e d lungs were then c a r r i e d out as was done f o r the t o t a l r e s p i r a t o r y system. - 22 -Results The pressure-volume curves for i n f l a t i o n and deflation of the t o t a l system ( t o t a l ) , body cavity (body), and lungs (lung) are shown for a 639 g. t u r t l e i n figure 2.2. A l l curves are generally sigmoid i n shape indicating that i n a l l cases increasing or decreasing volume ultimately leads to disproportionate increases i n pressure. This s i g n i f i e s that at some volume a l l a i r spaces are opened and f i l l e d with a i r and that the e l a s t i c components are approaching t h e i r l i m i t s of e x t e n s i b i l i t y . A l l three curves showed a pronounced hystersis upon deflation. The s t a t i c compliances derived from the slopes of the straight portions of the various curves originating from and zero pressure are given i n Table 2.1 for a l l animals examined. The mean compliance of the t o t a l system ( C T ) was 8.4 ml/cmh^O/kg +_ .65 S.E.M., that of the lungs alone ( C L ) was 35.1 ml/cmH^O/kg + .78 S.E.M., and that of the body cavity alone (Cg) was 11.6 ml/cmR^O/kg +1.35 S.E.M. These values are compared to published values for other r e p t i l e s i n Table 2.2. The values i n Table 2.2 were normalized to body weight and to and grouped according to lung type as c l a s s i f i e d by Perry and Duncker (1978). The percentages of the t o t a l work required to overcome e l a s t i c forces a r i s i n g from the body and the lungs on i n f l a t i o n of the intact respiratory system are shown i n Table 2.3. These values are derived from compliance values given i n Table 2.2 i n the following fashion. Since the compliance values l i s t e d i n Table 2.2 are for the line a r portion of the pressure-volume curves for each respective animal originating from and zero pressure, Figure 2.1 can be simpl i f i e d as shown i n Figure 2.3. The - 23 -Fig. 2.2 A pressure-volume curve for a 639 gram turtle. Curves for the total system (total), the body wall (body) and lungs alone (lung) are shown. Arrows pointing in a positive direction indicate inflation, those pointing in a negative direction indicate deflation. Values for C^, Cg, and C^, the compliances of the total system, the body wall, and lungs respectively were measured from the slope of the linear portion of the pressure volume curve upon inflati o n . - 23a:i-- 24 -Table 2.1 Compliance of the total system (Cj,), body wall (Cg) and lung (C L) in ml/cmh^O/kg body weight for 8 animals. Weight (kg) C T C L Cfi 0.733 9.2 35.8 8.4 0.437 6.8 37.0 12.1 0.639 9.1 34.2 12.4 0.750 9.8 0.485 17.1 0.560 17.3 1.467 10.3 0.739 5.4 x 0.726 8.4 35.1 11.6 +S.E.M. .107 .65 .78 1.35 - 25 -Table 2.2 Compliance of the t o t a l system ( C T ) , body wall (Cg), and lung (C^) for various species. (A) Values standardized to body weight. (B) Values standardized to resting lung volume <VLR>' A. ml/cmh^O/kg Animal Lung Type JB Source Green Lizard unicameral (Lacerta v i r i d i s ) Tokay Gekko unicameral (Gekko gecko) 18.20 62.10 26.70* Perry & Duncker (1978) 47.10 273.40 56.9* unicameral 16.0 201.6 14.5 Milsom & V i t a l i s (1984) Chameleon paucicameral 305.70 759.00 511.8* Perry & Duncker (Chameleo chameleon) (1978) Monitor Lizard multicameral 66.80 365.50 82.20* (Varanus exanthemat i cus) Red Eared Turtle multicameral 10.00 170.00 10.60* Jackson (1971) (Pseudemys scripta ) multicameral 8.40 35.10 11.60 Present study Rat broncho-alveolar 0.91 1.56 2.20 C r o s f i l l & (Ratus sp.) Widdicombe (1961) B. ml/cmH.20/ml.VLR Animal Lung Type Cp C^ Cg Source Green Lizard (Lacerta v i r i d i s ) unicameral 0.53 1.78 0.76* Perry & Duncker (1978) - 26 -Table 2.2 (continued) ml/cmH^O/ml.V]^ Animal Lung Type 0.73 4.07 0.89* 0.25 3.17 0.23 Tokay Gekko unicameral (Gekko gecko) Tokay Gekko unicameral (Gekko gecko) Chameleon paucicameral 2.50 5.73 3.84* (Chameleo chamelean) Monitor Lizard multicameral 0.66 3.27 0.83* (Varanus exanthematicus) Red Eared Turtle multicameral 0.10 1.70 0.11* (Pseudemys scripta) multicameral 0.08 0.35 0.12 Rat broncho-alveolar 0.09 0.12 0.55 (Ratus sp.) Source Perry & Duneker (1978) Milsom & V i t a l i s (1984) Perry & Duncker (1978) Jackson (1971) Present study C r o s f i l l & Widdicombe (1961) * Values for Cg calculated from the formula 1/Cg = 1/Cp - 1/C Other values for C R were derived experimentally. - 27 -Table 2.3 Percentage of the total work required to overcome the elastic forces resisting static lung inflation which arise from the body wall and lung. Calculations were based on compliance values standardized to V T D. Animal Lung Body Source Green Lizard 30% 70% Perry & Duncker (1978) Tokay Gekko 18% 82% 8% 92% Milsom & Vitalis (1984) Chameleon 44% 56% Perry & Duncker (1978) Monitor Lizard 20% 80% •• Red Eared Turtle 8% 92% Jackson (1971) 23% 77% Present study Mammal (Rat) 75% 25% C r o s f i l l & Widdicombe (1961) - 28 -Fig. 2.3 Simplification of figure 2.1 to ill u s t r a t e technique used for calculation of the work required to overcome the elastic forces resisting a 1 ml inf l a t i o n . For a 1 ml inflation the pressure generated i s equal to 1/C.j, where Cj, is the compliance of the system. The work required to overcome elastic forces for a 1 ml inflation i s represented by the area ABCA which equals 1/2CT. - 28a -work required to inflate this system by J\V is given by the area ABCA as stated earlier. This area is equal to A V x ^ P x |« Since compli-ance equals A^/A P» A p i - s equal to A V/compliance. As a consequence, for a 1 ml inflation, the area ABCA becomes 1 ml x 1 ml x 1 or simply 1 ml'cmR^O. C ml 2 2C cmH20 In this way the work required to inflate the lungs alone, the body cavity alone, and the intact respiratory system can be calculated. The values derived this way for the lungs and body cavity are shown in Table 2.3 as percentages of the values derived for the intact respiratory system. Among the reptiles the majority of the elastic recoil resisting inflation arises from the body wall while the highly compliant lungs offer l i t t l e resistance to inflation. In mammals, on the other hand, the majority of the work performed to overcome elastic forces during inflation i s required to expand the relatively s t i f f lungs. Only 25% of the total elastic work is required to expand the body cavity. - 30 -Discussion The compliance of the total respiratory system of the turtle, Pseudemys scripta, measured in the present study, is i n general agreement with results obtained for the same species by Jackson (1971). Jackson (1971) reported values for lung compliance (170 ml/cm H^O/kg), however, which were much higher than those measured in the present study (35.1 ml/cmlbjO/kg). It i s probable that this discrepancy results from differences in methodology. In the study of Jackson (1971), the compliance of the exposed lungs was determined beginning at atmospheric pressure with the lungs completely collapsed. In the present study the static inflation curve was determined beginning with the exposed lungs inflated to V L R. As a consequence in the present study was determined at a greater lung volume which may have corresponded to a s t i f f e r portion of the pressure-volume curve. Since the lungs and the body are placed in series, the algebraic sum of the pressure exerted by these two component parts must equal the pressure exerted by the total respiratory system (Agostoni, 1970). Since compliance i s equal to V/ P i t follows from this that for any V, the sum of the reciprocals of the lung compliance and body compliance must equal the reciprocal of the compliance of the total system (i.e. 1 + 1 = 1 ). C L C B CT If the values of Cj, and Cg measured in the present study are substituted into this equation a calculated value for C^ of 30.5 ml/cmR^O/kg is obtained which is close to the experimentally derived value of 35.1 ml/cmH^O/kg _+ .78 S.E.M. for lung compliance. Thus the compliance of the lung as measured in this study is consistent with the experimentally derived values for body - 31 -compliance and t o t a l respiratory system compliance. The results of the present study further indicate that the compliance of the t o t a l respiratory system of t h i s species of t u r t l e i s determined mainly by the compliance of the body wa l l . Only 23% of the e l a s t i c forces r e s i s t i n g i n f l a t i o n within this system arise from the lungs. Using the values for C^ and Cj, reported by Jackson (1971) one obtains an e l a s t i c contribution by the lungs of only 8% of the t o t a l e l a s t i c forces re s i s t i n g i n f l a t i o n . The percentage of e l a s t i c resistance to i n f l a t i o n offered by the lungs, as calculated i n this study, l i e s well within the range of values calculated for other r e p t i l e s (8%-44%) while the value calculated from Jackson (1971) l i e s at the lower extreme. A comparison of compliance values for the t o t a l respiratory system, body w a l l , and lungs of various species of r e p t i l e s indicates that the respiratory system of t u r t l e s i s r e l a t i v e l y s t i f f (Table 2.2). The li z a r d s for instance, have a highly compliant system two to t h i r t y times more compliant than the chelonian respiratory system. The r e l a t i v e l y s t i f f respiratory system of the t u r t l e can probably be attributed to the animal's s h e l l . The ribs of the t u r t l e are fused to the dome of the carapace and are immobile. The only portions of the animal's body which are capable of distension upon i n f l a t i o n are the flank cavities and the pectoral g i r d l e . The l i z a r d s , on the other hand, have articulated ribs capable of moving as the lungs i n f l a t e and the volume of the body cavity increases. Table 2.2 also indicates that the lungs of the t u r t l e are somewhat s t i f f e r than those of l i z a r d s . This i s probably due to the multicameral nature of the lungs of the t u r t l e , which possess intrapulmonary septation. The lungs of most liz a r d s l i s t e d i n Table 2.2 are unicameral or paucicameral. - 32 -Interspecific comparisons of compliance values normalized to body weight show the compliance of the respiratory system of lizards to be much greater than that of chelonians and the chelonians possessing a more compliant system than mammals (C 2 to 30 x > C 9-10 x > i l i z a r d s xturtles C T ). Comparisons made on a volume basis (normalized to resting xrat lung volume) again show lizards having the highest compliance values but now the chelonian system has a similar value to that of the mammalian system (C 5 to 23 x > C ^ C ). In mammals, however, C T is i l i z a r d H u r t l e s i r a t determined primarily by the lungs which contribute 75% of the elastic forces resisting inflation of the respiratory system. Turtles, on the other hand, have relatively compliant lungs compared to mammals and is predominantly determined by the body wall which contributes 77% of the elastic forces within the respiratory system resisting inflation. Perry and Duncker (1978) claim that high lung compliance is asso-ciated with a high degree of development of caudal and ventral dilations of the lung with the development of these membraneous regions being most exten-sive in the chameleon followed by the tokay gekko and the savanna monitor (Table 2.2). The data of the present study, however, do not support this trend in the relationship between lung volume, general internal architecture of the lung, and lung compliance. Whether the comparisons are made using values standardized to body weight or to resting lung volume the C T of the turtle i s much lower than would be predicted given the lung volume and lung architecture of this species. It may be the case that this trend i s only found in the lizards, however, i t may also be the case that this trend i s - 33 -merely a consequence of the small sample size used by Perry and Duncker (1978). The hysteresis seen i n the s t a t i c i n f l a t i o n - d e f l a t i o n curves has previously been reported for t u r t l e s (Jackson, 1971), l i z a r d s (Perry and Duncker, 1978, 1980; Milsom and V i t a l i s , 1984), amphibians (Hughes and Vergara, 1978) and mammals (Butler, 1957; Mead et a l . , 1957) and has been attributed to v i s c o e l a s t i c effects a r i s i n g from the lungs and body wall (Agostoni, 1970). In the lungs i t has been primarily attributed to surface forces present at the a i r - a l v e o l a r interface. Hughes and Vergara (1978) showed that the hysteresis present during an a i r i n f l a t i o n - d e f l a t i o n cycle of the frog lung almost completely disappeared when the lungs were f i l l e d with saline as was also shown for l i z a r d lungs by Perry and Duncker (1978). This suggests that surface active elements may play a role i n reducing the surface tension of the a i r - a l v e o l a r interface, upon i n f l a t i o n of the lungs, i n lower vertebrates (Hughes and Vergara, 1978) as i n mammals. The hysteresis of the body wall of the t u r t l e i s due to extra-pulmonary hysteresis. This extra-pulmonary hysteresis i s not due to surface tension forces but to the v i s c o e l a s t i c behavior of the distensible elements of the flanks and body wall of the animal, namely s k e l e t a l muscle, connective tissue, and ligaments (Agostoni, 1970). In conclusion, the compliance of the respiratory system of the t u r t l e i s largely determined by a s t i f f body compartment with l i t t l e contribution from the lungs. The reverse i s true i n mammals where the lungs play the predominant role i n determining t o t a l system compliance. On a weight basis the t o t a l system compliance (C-jO of the t u r t l e i s ninefold greater than that of the mammalian (rat) system, whereas values standardized - 34 -to show the chelonian and mammalian systems to be approximately equal. In a l l cases, l i z a r d s had higher values f o r Cj, than e i t h e r t u r t l e s or mammals due to the presence of a very compliant body wall and lungs. Thus the resp i r a t o r y system of t u r t l e s i s s t i f f e r than that of l i z a r d s and behaves s t a t i c a l l y i n a s i m i l a r fashion to the mammalian system. It i s of in t e r e s t then to compare how the t u r t l e system behaves under dynamic conditions with the systems of l i z a r d s and mammals and t h i s w i l l be considered i n the next chapter. CHAPTER 3 THE DYNAMIC PULMONARY MECHANICS OF THE TURTHE, CHRYSEMYS SCRIPTA Introduction Alveolar v e n t i l a t i o n (V^) i s the product of alveolar volume (V^ which equals t i d a l volume (V^ ,) minus dead space volume (V^)) and breathing frequency ( f ) . Any part i c u l a r l e v e l of can be produced by a variety of combinations of and f. In mammals, i t has been shown that for each l e v e l of there i s a combination of and f which requires a minimum of mechanical work to power breathing (Otis, Fenn, and Rahn, 1950), or, a minimum average force from the respiratory muscles to move the lungs (Mead, 1960). I t i s more expensive, i n terms of mechanical work, to ventilate the lungs at frequencies above or below the values associated with minimum work when V^ , i s adjusted to maintain a constant V^. This can be understood i n view of the fact that at a low f, and therefore high V^ ,, the work required to overcome the e l a s t i c forces within the system i s high. At a high f, on the other hand, when V^ , i s small, the e l a s t i c work i s smaller but high a i r flow increases the work required to overcome viscous and turbulent resistance. The result i s a U-shaped curve for the work required to overcome the forces within the respiratory system. The oxidative cost of v e n t i l a t i o n i s proportional to the mechanical work of breathing ( M i l i c - E m i l i and P e t i t , 1960) therefore the combination of V-j, and f which requires minimum mechanical work can be considered an optimal pattern i n terms of energy expenditure and ef f i c i e n c y . - 36 -It has been demonstrated that the spontaneous breathing pattern of many mammals corresponds very closely with the i r predicted optimal patterns ( C h r i s t i e , 1953; Agostoni et_ a l . , 1959; C r o s f i l l and Widdicombe, 1961; Yamashiro et a l . , 1975). Despite t h i s , i t has proven d i f f i c u l t to postulate a control system for the regulation of these optimal patterns i n terms of minimal mechanical work. This stems from the fact work i s an int e g r a l function and therefore would not be sensed by any of the known receptor groups associated with the respiratory system. Pulmonary afferent information does, however, appear to play an important role i n pattern regulation since absence of this information results i n deviation from the optimal pattern (Mcllroy, Marshall and C h r i s t i e , 1954; McClelland et a l . , 1972). In r e p t i l e s the normal resting breathing pattern i s intermittent unlike the continuous breathing pattern of mammals. The r e p t i l i a n pattern i s characterized by single breaths or bursts of breaths separated by breath holds of varying length (McCutcheon, 1943; Gans and Hughes, 1967; Naifeh et a l . , 1970; Gans and Clark, 1976; Glass and Johansen, 1976). The primary variable which i s regulated i n response to respiratory stimulants such as hypoxia, hypercapnia, or increased temperature i s the breath hold period (Glass and Johansen, 1976; Milsom and Jones, 1980; Benchetrit and Dejours, 1980) while the response to exercise i s characterized by increases i n t i d a l volume (Wood and Lenfant, 1976; Cragg, 1978). Very l i t t l e i s known about the influences of mechanical factors on the breathing patterns or respiratory responses of intermittent breathers. The s t a t i c compliances of the lung and t o t a l respiratory system have been measured i n a few species (see chapter 2) and Perry and Duncker (1978, 1980) have attempted to - 37 -correlate resting breathing patterns with these static compliances. Such analysis, however, i s extremely limited. Since respiration i s an active rather than a static process, an understanding of dynamic pulmonary mechanics should provide more useful information for assessing the mechanical factors influencing breathing patterns. To date only one such study has been performed on a non-homeothermic species. Milsom and Vi t a l i s (1984) have shown that for the Tokay lizard Gekko gecko, dynamic pulmonary mechanics were dominated by the mechanics of the body cavity and chest wall. This is unlike the situation i n mammals where the pulmonary mechanics are most strongly influenced by the mechanics of the lungs. Despite this there was s t i l l a combination of t i d a l volume and frequency in the gekko which gave minimum values for mechanical work for each level of V"A. The aim of experiments outlined i n this chapter was to extend such studies on dynamic respiratory mechanics to the turtle, Pseudemys scripta. The results of the previous chapter have shown that although the static mechanics of the total respiatory system of the turtle are very similar to those of mammals, the lungs of turtles are less complex than mammals but more complex than those of lizards. Mechanics of the body wall show that on a volume basis the body wall of the turtle i s s t i f f e r than that of either reptiles or mammals due to the presence of the shell. Given this unique combination of features i t i s of interest to see how the differences in the architecture of the lungs and body wall of turtles is reflected in the dynamic mechanics of the respiratory system. - 38 -Materials and Methods Dynamic Pressure-Volume Relationships a) Total Respiratory System Preparation of animals for measurement of the dynamic pulmonary mechanics of the t o t a l respiratory system was i d e n t i c a l to that used for the determination of the s t a t i c pressure-volume curve of the t o t a l system i n chapter 2. These experiments were performed on three animals with a mean weight of 553 g. + 81 S.E.M. The tracheal cannula was connected to a Harvard Small Animal Respirator (Model 665 Harvard Inc., M i l l i s , Mass., U.S.A.) with a pneumotachograph ( F l e i s h #00) placed i n the airflow l i n e leading from the pump to the tracheal cannula. The pressure drop across the pneumotach screen during tracheal flow was measured with a Validyne d i f f e r e n t i a l pressure transducer (DP103-18) and t h i s flow signal was el e c t r o n i c a l l y integrated (Gould Integrating Amplifier) to give the stroke volume of the pump. Intratracheal pressure was measured from a side-arm i n the tracheal cannula by the Statham P23-Db physiological pressure transducer. Flow, pressure, and volume were recorded on a Gould series 2600 pen recorder. The volume and pressure signal were also recorded simultaneously on an Esterline Angus 575 X-Y pl o t t e r . When volume was plotted against pressure on XY coordinates, through a complete pump cycle, a counter clockwise rotating hysteresis loop was produced (Fig. 3.1). The area within the loop was used to calculate the mechanical work required for a pump cycle. - 39 -(b) Body Cavity The dynamic mechanics of the body cavity alone which were associated with lung ventilation were determined for six animals of a mean weight of 553 g. + 28 S.E.M. The preparation of animals in this experiment involved the cannulation of the abdominal cavity in order to measure intra-abdominal pressure. The cannulation was identical to the proceedure used i n determining the static compliance of the body wall (chapter 2). Intra-abdominal pressure was measured by a Statham P23 DB physiological pressure transducer which was connected to the intra-abdominal pressure cannula. The best position for placement of the intra-abdominal pressure cannula was found to be within the urinary bladder of the animal. This large f l u i d f i l l e d sac prevented the viscera from blocking the tip of the cannula yet transmitted changes i n intra-abdominal cavity pressure. Placement of the cannula within the urinary bladder always gave consistent pressure recordings. The trachea was cannulated as described previously. In these experiments, no side-arm was needed for intratracheal pressure recording. The tracheal cannula was connected to the pump and pneumotachograph as described above and tracheal air flow, pump stroke volume, and intra-abdominal pressure were measured and recorded as described above. The intra-abdominal pressure and the stroke volume were also recorded simultaneously on an Esterline Angus 575 X-Y plotter producing hysteresis loops for the changes in intra-abdominal pressure which accompany changes i n the volume of the body cavity during inflation and deflation of the lungs. - 40 -(c) Protocol The experimental procedure was i d e n t i c a l for measurements carried out on both the t o t a l respiratory system and on the body cavity alone. Starting with the lungs open to atmosphere and, therefore, with i n t r a -pulmonary and intra-abdominal pressure equal to atmospheric pressure, the animals were ventilated with volumes ranging from 1 ml to 6 ml at frequencies ranging from 15 to 60 cycles per minute. Pump volume always started at 6 ml and was sequentially reduced to 1 ml i n 1 ml steps at each pump frequency. Frequencies were sequentially increased from 15 cycles/min. to 20 cycles/min. and then to 60 cycles/min. i n ten cycles/min. steps. (d) Data Analysis During lung i n f l a t i o n i n an intact respiratory system, work i s done to overcome fl o w - r e s i s t i v e , viscous, and e l a s t i c forces (Otis, 1964). The magnitude of the nonelastic forces of flow-resistance and vi s c o s i t y depend on the rates of movement of gas and tissue. Figure 3.1 shows a schematic pressure-volume loop with the arrows indicating the dir e c t i o n of change i n pressure and volume for i n s p i r a t i o n (I) and expiration (E). The horizontal distance between the l i n e , AC whose slope represents the dynamic compliance, and the curved l i n e AIC, represents the pressure gradient required to overcome the nonelastic forces during i n s p i r a t i o n . The area AICA represents the work required to overcome these forces during i n s p i r a t i o n . The area bounded by ACEA represents the work required to overcome the nonelastic forces during expiration. The area ABCA represents the work required to overcome the e l a s t i c forces for one v e n t i l a t i o n cycle. If the area representing the work required to overcome nonelastic forces - 41 -during expiration (ACEA) is within the area ABCA then expiration is passive and powered by the stored elastic energy represented by ABCA. The total work for one cycle, the sum of elastic and nonelastic work, is then represented by the area A1CBA (Otis, 1964). For each pressure-volume loop recorded in the present experiments, a perpendicular line was drawn from the volume axis to the point of peak pressure and volume (line BC in Fig. 3.1). The area contained within the pressure-volume loop (AICEA = nonelastic work of inflation and deflation) and the area within AICBA (total work of one ventilation cycle) were measured using an Apple II microcomputer and graphics tablet. Since i n these experiments, during pump ventilation of the anaesthetized animals, expiration was always passive, nonelastic work of each cycle was calculated as one half of the area of the pressure-volume loop. The work required to overcome the elastic forces in the total respiratory system were calculated by subtracting the nonelastic work/cycle from the total work/cycle. These calculations of total, elastic, and nonelastic work/cycle were made on loops obtained for pump ventilation of the intact system and ventilation of the body cavity alone. The work required to ventilate the lungs alone was then calculated by subtracting the work/cycle required to ventilate the body cavity from the work/cycle required to ventilate the total respiratory system. Dynamic compliance, the slope of the line connecting the two points of zero flow on the pressure-volume curve was approximated by calculating the ratio between ti d a l volume (A v) and the corresponding change in pressure (A p)« - 42 -F i g . 3 .1 Schematic diagram of the pressure-volume r e l a t i o n s of the i n t a c t r e s p i r a t o r y system during a si n g l e v e n t i l a t i o n c y c l e . The loop begins at A and i s generated by a point, moving with time, i n a counter clockwise d i r e c t i o n . Shaded area represents the work required to overcome nonelastic forces during i n s p i r a t i o n . See text f or d e t a i l s . V = i n f l a t i o n volume, P = intrapulmonary or intra-abdominal pressure. - 42a -1 3 5 P (cm H2O) - 43 -Effect of changing v e n t i l a t i o n frequency at constant minute v e n t i l a t i o n (V E) on blood gas p a r t i a l pressures These experiments were performed on s i x t u r t l e s , mean weight 753 + 81 g., to determine the effect of increasing v e n t i l a t i o n frequency, with concomitant deceases i n t i d a l volume to maintain Vg constant, on and on a r t e r i a l and PCG^ The animals were anaesthetized with an i n j e c t i o n of sodium pentabarbital (20 rag/kg i.p.) and a midline i n c i s i o n was made along the neck of the animal and the carotid artery exposed. The artery was cannulated with P.E. 50 tubing f i l l e d with heparinized s a l i n e . The cannula was fed several centimeters down the artery towards the heart to ensure that the blood sampled v i a this catheter was representative of a r t e r i a l blood leaving the heart. The PaC>2 and P aC0 2 of th i s blood was measured using Radiometer electrodes and a Radiometer PHM 71 blood gas analyzer. The trachea was cannulated as described previously. The cannula was attached to a Harvard Small Animal Respirator (Model 665) with a pneumo-tachograph ( F l e i s h #00) i n the a i r flow l i n e to monitor a i r flow. A i r flow and t i d a l volume were measured and recorded as described e a r l i e r . The animal was ventilated at a constant rate of 50 ml/min/kg. Ventilation began at 10 cycles/min. and was increased i n steps of 10 cycles/min. to 60 cycles/min. The animal was ventilated with saturated a i r at room temper-ature (20-22°C). Small quantities of a r t e r i a l blood were removed after 15 minutes of v e n t i l a t i o n at each frequency and analyzed for P0 2 and PC02« The sample was returned to the animal after each measurement. The pump was returned to 10 cycles/min. after the f i n a l measurement and a r t e r i a l blood was again analyzed to ensure that the blood gases returned to o r i g i n a l l e v e l s . If th i s did not occur the data was discarded. - 44 -Measurement of dead space volume (VT)) The anatomical dead space of the respiratory system was determined for s i x animals with a mean weight of 820 +_ 83 g. Anatomical dead space was considered to be the volume of the trachea and primary bronchi from the g l o t t i s to the lung hilus and did not include the volume of the intrapulmonary primary bronchi. Two methods were used to determine V^. One method involved measuring the volume of the trachea by f i l l i n g i t with water after i t had been dissected from the animal. The other method involved measuring the length of the trachea and i t s diameter from which i t s volume was calculated. These measurements were considered to be minimum values for V Q since they did not include any adjustment for physiological dead space. A l l values given i n this chapter are mean values +^  S.E.M. - 45 -Results The dynamic pressure-volume relationships for the t o t a l respiratory system of a 750 g. animal are shown i n Fig. 3.2. These curves are representative of a l l curves measured. The curves i n Fig. 3.2a show that at a frequency of 30 cycles/min., as stroke volume increases, the peak pressure generated within the system r i s e s . The curves i n Fig. 3.2b show that at a frequency of 30 cycles/min., as stroke volume increases, the peak pressure generated within the system also r i s e s . Thus the pressure-volume relationships of the t o t a l respiratory system are both volume and frequency dependent. Figure 3.3 shows representative pressure-volume loops obtained for i n f l a t i o n of the body compartment alone i n a 468 g. animal. At a constant frequency of 30 cycles/min. there i s a strong dependence of the pressure generated on the i n f l a t i o n volume (Fig. 3.3a). For a constant volume of 6 ml, on the other hand, changes vary inconsistantly with changes i n frequency suggesting that the variation seen i s not frequency dependent (Fig. 3.3a). The relationships shown i n Figs. 3.2 and 3.3 for the t o t a l system and the body compartment alone hold true for a l l pump frequencies at a l l i n f l a t i o n volumes. In F ig. 3.4, the changes In dynamic compliance of the t o t a l respiratory system, as a function of pump frequency are shown for volumes of 2, 4, and 6 ms. Each point i s the mean value for 3 animals with standard - 46 -Fig. 3.2 Effects of changes in ventilation frequency (f) and volume (V T) on the pressure-volume (P-V) relationship associated with a single ventilation cycle of the total respiratory system in a 750g turtle, (a) The effect of increasing ti d a l volume from 3 to 6 ml on pressure-volume loops at a constant venti-lation frequency of 30 cycles/min. (b) The effect of increasing ventilation frequency on pressure-volume loops for a constant t i d a l volume of 6 ml. - 46a -QJ O 15 10 J 5 f = 30 (min ) Q VT = 6 m l 15 20 30 40 50 60 A P fcmH20) - 47 -Fig. 3.3 Effects of changes in ventilation frequency (f) and volume (V,jO on the pressure-volume (P-V) loops associated with a single ventilation cycle of the body compartment alone on a 430 g. animal, (a) The effect of increasing ti d a l volume from 1 ml to 6 ml on pressure-volume loops for a constant ventila-tion frequency of 30 cycles/min. (b) The effect of increasing ventilation frequency from 15 cycles/min. to 60 cycles/min. for a constant t i d a l volume of 6 ml. VOLUME (mi) - 48 -error bars included. S t a t i s t i c a l l y the dynamic compliance i s volume independent over the range of volumes recorded although there i s a s l i g h t trend for the system to behave i n a s t i f f e r fashion at higher i n f l a t i o n volumes. The dynamic compliance i s , however, strongly frequency dependent, so that for any given volume, as frequency increases, the dynamic compliance f a l l s . The decrease i n dynamic compliance indicates that the system becomes s t i f f e r with increasing frequency thus for any given volume, as frequency increases, the pressure difference generated by i n f l a t i o n also increases. This apparent s t i f f e n i n g of the respiratory system at increasing v e n t i l a t i o n frequency i s reflected i n the work required to produce each breath. Calculated values of the mechanical work required to produce each breath i n the t o t a l respiratory system, measured from the pressure-volume loops as described i n the methods, are shown i n Fig. 3.5. The work/ventilation cycle (W) i s plotted against Vg i n t h i s figure with isopleths i l l u s t r a t i n g various pump frequencies and pump volumes. Each point i s the mean value for three animals standardized for body weight with standard error bars included. For a constant frequency, increases i n Vg due to increases i n Vj. result i n a li n e a r r i s e i n W on these curves. If Vg i s increased by holding volume constant and increasing frequency, W also rises but i n a nonlinear fashion. Thus the r i s e i n W, with increasing levels of f at constant V T, i s small at f<30 cycles/min. and rises sharply with f>30. The t o t a l work required to produce each breath can be further analyzed i n terms of the work required to overcome e l a s t i c and nonelastic - 49 -F i g . 3.4 The r e l a t i o n s h i p between v e n t i l a t i o n frequency ( f ) and dynamic compliance ( C ^ n ) f o r constant t i d a l volumes of 2 Q ) , 4 ( £ ) ) and 6 (A ) m l . Each point represents the mean value for 3 animals . V e r t i c a l bars i n d i c a t e the S . E . M . 8.00 f ( m i n H ) - 50 -Fig. 3.5 The relationship between the total work/breath (W) and minute ventilation (Vg) for different combinations of tid a l volume (V,j, ml) and respiratory frequency (f in breaths/min.). Data points represent average values from 3 animals with standard error bars. - 50a -V E (ml min"1) - 51 -forces. Figure 3.6 shows the elastic and nonelastic components of the total work required to produce a single breath for tida l volumes of 2 and 6 ml at increasing pump frequencies. For a V T of 2 ml, at low levels of Vg, the work required to overcome elastic and nonelastic forces i s about equal. At • high levels of Vg (f>30) with a V T of 6 ml, the work required to overcome nonelastic forces i s the major portion of the total work required to produce each breath. The work required to produce a single breath can also be analyzed in terms of the work required to inflate the lung and the work required to inflate the body compartment since the pressure within the respiratory system i s the sum of the pressure contributed by the lung and the body wall (Agostoni, 1970). Figure 3.7 shows the contribution of the lung (w"L) and body wall (Wg) to the total work (Wj) required to produce single beaths at ti d a l volumes of 2 and 6 ml with increasing pump frequencies. The work required to overcome the forces within the body wall are volume dependent. The higher the ti d a l volume, the larger Wg becomes. Wg i s , however, relatively frequency independent. As a consequence, rises in Wrj, as Vg increases are a result of frequency dependent increases in W^. Thus although Wg is the major component of Wrj,, for each V^ , shown in Fig. 3.7, i t is the sharp rise in W^ which produces the rise in W-j. during increases in pump frequency. For purely mechanical considerations the effects of changes in f or Vj on the work of breathing have been referenced in terms of their effect on V p. In terms of gas exchange, however, changes in alveolar - 52 -Fig. 3.6 The contribution of elastic (E) and nonelastic (NE) work/breath to the total work/breath (T) for V^ , of 2 ml and 6 ml with changing Vg. Each point represents the mean value of 3 animals with standard error bars included. - 52a -minute v e n t i l a t i o n (V^) are of more concern than changes i n Vg. V A, the product of the alveolar v e n t i l a t i o n volume (V^ = V^ , - dead space (Vp)) and v e n t i l a t i o n frequency ( f ) , decreases as f increases even though Vg i s held constant by adjustments i n V^. Since the dead space volume of the respiratory system i s f i x e d , anatomically, as Vj decreases, • becomes disproportionately small and when V\j = V^, w i l l equal zero. At this point, although much gas i s s t i l l being moved i n and out of the trachea, there i s no gas being turned over i n the lungs. As a consequence, there are l i m i t s to the extent to which V^ , may be reduced before gas exchange i s compromised by the f a l l i n V^. This i s i l l u s t r a t e d i n Fig. 3.8 which shows the importance of holding constant rather than Vg to maintain gas exchange. For a constant Vg of 50 ml/min/kg, V^ f a l l s as f increases. V A values were calculated using a V Q of 0.74 ml/kg from Table 3.1. As V^ f a l l s with increasing f, a r t e r i a l P O 2 levels also steadily decrease from 90 mmHg at an f of 30 cycles/min to 30 mmHg at f of 60 cycles/min. A r t e r i a l P C O 2 steadily rises from 15 mmHg to 29 mmHg over the range of frequencies used. To maintain blood gas levels V^ rather than Vg must be kept constant. For V^ to be kept constant Vg must r i s e along with increases i n v e n t i l a t i o n frequency. This has consequences on the minute work of breathing. The values of work/breath from Fig. 3.5 are replotted i n F i g . 3.9 as minute work (W), the product of the work required to produce a breath (WT) and the number of breaths taken each minute ( f ) . For any given l e v e l of minute v e n t i l a t i o n (Vg) W i n i t i a l l y f a l l s as f increases and V^ decreases u n t i l W reaches a minimum. Further increases i n f and decrease i n - 54 -Fig. 3.7 The contribution of the lung (L) and body (B) to the t o t a l (T) work/breath (W) for t i d a l volumes (V T) of 2 ml and 6 ml with changing minute v e n t i l a t i o n (Vg). Each point for T and L i s the mean for 3 animals +_ S.E.M. Each point for B i s the mean of 6 animals + S.E.M. - 54a -VE (ml • min"') - 55 -Fig. 3.8 The effect of increasing ventilation frequency (f) at a constant minute ventilation of 50 ml/min/kg. on (® •*» P a C 0 2 ( A ) and alveolar minute ventilation ( B I ) . The values for Pa(>2 and ^ a^2 a r e t* i e m e a n f° r ^  animals + S.E.M. V"A was calculated using a value of 0.74 ml/kg + 0.06 S.E.M. for dead space volume (V n). - 56 -F i g . 3.9 The r e l a t i o n s h i p between the rate of work (W) and v e n t i l a t i o n frequency (f i n breaths/min.) f o r constant l e v e l s of minute v e n t i l a t i o n (V £ i n ml/min.) and alveolar minute v e n t i l a t i o n • (V. i n ml/min.). - 56a -f (min-1) V T produce a r i s e i n W. The shape of this curve becomes accentuated and » « • the l e v e l of W increases as V E r i s e s . For V E ranging from 100 to 300 ml/min., W i s minimum at frequencies between 35 and 45 breaths/min. Figure 4 3.9 also shows the effect of changes i n f on W for various levels of constant V A. Note that W i s always greater at any given l e v e l of V A than i t i s at a si m i l a r l e v e l of Vg and that this difference increases as f increases. This stems from the fact that at any given l e v e l of f, V T must be greater to produce a l e v e l of which i s similar to any given l e v e l of V £ since values of V A do not account for dead space v e n t i l a t i o n . This also means that as f increases the difference i n values of V,p required to produce sim i l a r levels of V^ and Vg also increases. The v e n t i l a t i o n frequencies at which W i s minimum for levels of V^ ranging from 100 to 300 ml/min. vary between 30 to 40 breaths/min., s l i g h t l y lower than the minimum frequencies obtained from the plots of W vs. f for constant levels of Vg. Minute work i s analyzed i n terms of the work required to overcome e l a s t i c and nonelastic forces for a V^ of 150 ml/min. i n Fig. 3.10. As pump frequency increases, the minute work done to overcome e l a s t i c forces drops sharply and then ri s e s very s l i g h t l y as f continues to increase. The minute work required to overcome nonelastic forces steadily rises as f increases. The sum of the work rate required to overcome both e l a s t i c and nonelastic forces results i n the 'U' shape of the t o t a l minute work curve. In Fig. 3.11 minute work i s broken down i n terms of the cost of ventil a t i n g the lungs (L) and body cavity (B). The minute work required to overcome the forces within the body cavity for a V^ of 150 ml/min., steadily declines as f increases. The minute work required to overcome the - 58 -Fig. 3.10 The amount of total worK (T) required to overcome elastic (E) and nonelastic (NE) forces at a constant alveolar minute ventilation (V~A) of 150 ml/min 1 with increasing ventilation frequency (f in breaths/min.). - 58a -f (min-') - 59 -Fig. 3.11 The cost of ventilating the lungs (L) and the body (B) compartment compared to the total (T) rate of work for a constant alveolar minute ventilation (V^) of 150 ml/min. with increasing ventilation frequency (f in breaths/min.). - 59a -- 6 0 -forces within the lung drops i n i t i a l l y but begins to rise sharply after the ventilation frequency increases beyond 2 7 cycles/min. The steady decline of minute work required to overcome forces within the body cavity and the i n i t i a l decline in the minute work required to overcome forces within the lung result i n the i n i t i a l decline seen in total minute work. The sharp rise in the minute work required to overcome forces in the lung as ventilation frequency increases, however, causes the total minute work curve to be U-shaped. The values for the anatomical dead space of the respiratory system ( V " D ) are shown for a l l animals in Table 3 . 1 . When V " D was calculated from measurements of tracheal and bronchial length and diameter, a mean value for V " D of 0 . 6 5 +_ 0 . 0 5 8 ml/kg was obtained. Measurements of V " D based on water displacement yielded a mean value of 0 . 7 4 +_ 0 . 0 6 3 ml/kg. With 9 5 % confidence, the difference between the two values was found to be insignificant ( & = 0 . 0 9 ± 0 . 1 5 3 7 ) . Since the volume of the trachea is a minimum value for V " D which does not take into account the volume of the intrapulmonary bronchi or physiological dead space i t was f e l t that the larger value of 0 . 7 4 ml/kg more closely approximated V Q and was therefore used to calculate V . . - 61 -Table 3.1 Values of anatomical dead space determined for specimens of I?. scripta by two different methods. Dead space  Mass (g) A* (ml/kg) B+ (ml/kg) 729 0.74 680 0.66 0.63 755 0.42 1269 0.63 0.95 725 0.55 0.65 759 0.88 0.72 x 820 0.65 ml/kg 0.74 ml/kg S.E.M. 82.7 0.058 0.063 * Calculated from measurements of the length and diameter of the trachea. + Determined by f i l l i n g the trachea with water to determine i t s volume. The difference between the two methods of determining the volume of the trachea is insignificant ( A = «09 + .1537) at the 95% confidence l i m i t . Discussion Under dynamic conditions the compliance of the respiratory system shows frequency dependence, but l i t t l e or no dependence on V^ , over the range studied. The frequency dependence of the dynamic compliance (C^y n) arises from a change in the stiffness of the lungs as there is no frequency dependence seen i n C ( j v n measured for the body cavity alone (Fig. 3.3). The decrease in C ( j v n observed with increasing frequency is not affected by changes in ventilation volume over the range of 2 to 6 ml. These results differ from those obtained for the Tokay gekko where the respiratory system showed a frequency dependent decrease in which was enhanced by increasing ventilation volume at high ventilation frequencies (Milsom and V i t a l i s , 1984). This dependence of ^^yn on tidal volume at high ventilation frequencies in the Tokay gekko was thought to be a result of the visco-elastic properties of the body wall. The absence of such an effect in the present study may be attributable to the presence of the shell in the turtle which does not experience geometric changes with increasing volume and which restricts a l l visco-elastic properties to the membranes of the flank cavities. The ligaments, cartilaginous elements, and muscle fiber arrangements seen in the articulated ribs of the Tokay are absent in the turtle and this could account for the differences seen in the volume effects on the frequency dependence of C^yn in the two animals. It is also possible that the volume effects on the frequency dependence of C (jy n only appear with very high t i d a l volumes. The t i d a l volumes in this study were only 2% to 6% of resting lung volume whereas i n the study on the Tokay gekko the t i d a l volumes used were 10-50% of resting lung volume. The dependence of ^dyn o n r r e c l u e r i c y observed i n the t u r t l e means that a greater change i n pressure w i l l r esult for any given change i n volume as frequency increases. This i s reflected i n the measurements of the t o t a l work/breath obtained for the t o t a l respiratory system. For any given t i d a l volume the work required to produce a single breath rises as frequency increases, with the slope of the r i s e i n being small at low frequencies but increasing sharply above an f of 30 breaths/min. This i s due to an increase i n the work required to overcome the forces within the lung which appear to be predominantly nonelastic i n nature. Despite t h i s , the work required to overcome the forces within the body wall and body cavity account for the majority of the work required to produce a single breath at a l l but the highest frequencies studied. The work required to overcome the forces within the body wall and body cavity are r e l a t i v e l y independent of frequency indicating that these forces are predominantly e l a s t i c i n nature. For any given v e n t i l a t i o n frequency, the work required to produce each breath increases as an exponential function as t i d a l volume increases. This appears to be due to the material properties of the body w a l l , body cavity, and lungs. E l a s t i c work accounts for approximately 50% of the t o t a l work at low t i d a l volumes, but nonelastic work contributes the major portion of the work at high t i d a l volumes due to the necessary increase i n flow rates and thus flow resistance which are a consequence of the need to move more a i r i n the same time i n t e r v a l . The results of the work/breath measurements indicate that i t - 64 -requires less work to increase v e n t i l a t i o n by increasing frequency rather than t i d a l volume at a l l levels of v e n t i l a t i o n studied. This i s because the flow r e s i s t i v e forces r i s e more slowly with increasing frequency over the range studied than the exponential r i s e which occurs i n the work required to overcome e l a s t i c forces when t i d a l volume i s increased and frequency held constant. When levels of minute v e n t i l a t i o n are high, however, although i t s t i l l requires less work/breath to increase v e n t i l a t i o n by holding f constant, the differences are smaller. This i s due to the increase i n flow r e s i s t i v e forces associated with high v e n t i l a t i o n volumes and frequencies which causes the sharp upturn i n the work/breath curves seen i n the larger volume isopleths. The minute work (W) required to maintain a constant l e v e l of » minute v e n t i l a t i o n (Vg) i s a function of the change i n the work/breath associated with different combinations of t i d a l volume and frequency multiplied by the breathing frequency. The slope of the W vs. f curve for a constant Vg decreases at low frequencies due to an exponential drop i n e l a s t i c work as V^ f a l l s and f increases. As f continues to r i s e W begins to increase again due to the steady increase i n nonelastic work caused by the higher vel o c i t y of the a i r flow. This i s also true of the W vs. f curve for a constant l e v e l of V^ but i n th i s curve, Vg also has to increase as f increases since the f a l l i n V^ , cannot completely offset the increase i n f due to the ever increasing r a t i o of dead space v e n t i l a t i o n to t o t a l v e n t i l a t i o n . This causes a further r i s e i n W since W i s proportional to Vg ( M i l i c - E m i l i and P e t i t , 1960). Another consequence of having to increase Vg when f increases i n order to maintain V^ constant i s a sl i g h t l e f t s h i f t i n the frequency corresponding to minimum W. This s h i f t - 65 -occurs as a result of flow r e s i s t i v e forces becoming more important at a lower frequency of v e n t i l a t i o n . « This t o t a l W i s the sum of the rate of work required to overcome the forces within the body wall and within the lung. The majority of W i s required to overcome forces a r i s i n g from the body wall and cavity at v e n t i l a t i o n frequencies below 55 breaths/min. The size of this contribution drops steadily as f increases suggesting that the predominant forces to be overcome within the body wall are e l a s t i c i n nature. The decline, however, i s not exponential as would be the case i n a purely e l a s t i c system suggesting that there are some viscous forces associated with the movement of tissues within the body cavity also. Pa r t i t i o n i n g the amount of the minute work which i s required to overcome the various forces within the lung i s more complex. The curve describing the relationship between W and f for the lungs alone i s also U-shaped with a minimum W occurring at approximately 27 breaths/min. for a V A of 150 ml/min. This curve then ri s e s sharply r e f l e c t i n g the fact that work required to ventilate the lungs becomes the major contributor to the t o t a l W required to ventilate the entire respiratory system at frequencies greater than 55 breath/min. The decline seen i n minute work with increasing frequency, when frequencies are low, indicates that there are e l a s t i c forces which have to be overcome when V T i s high. The sharp r i s e i n W for the lungs above frequencies of 27 breaths/min. i s due to nonelastic forces which are flow r e s i s t i v e i n nature. The shape of the W curve for the lungs i n part r e f l e c t s i t s anatomy. The lungs of the t u r t l e are attached to the dome of the carapace dorsally and to the post-pulmonary septum ve n t r a l l y . The post-pulmonary - 66 -septum i s i n turn attached to the l i v e r and stomach and envelopes the entire lung. The anterior portion of the lung i s covered by a sheath of s t r i a t e d muscle, the striatum pulmonale (Perry, 1978). These connective tissues and str i a t e d muscle along with the intrapulmonary septa are the probable s i t e of the e l a s t i c components of the forces arising from the lungs. The intrapulmonary septa divide the lung into seven chambers which communicate with each other v i a the intrapulmonary bronchi. These intrapulmonary bronchi along with the extrapulmonary primary bronchi and the trachea are the most probable site s for flow resistance i n the lungs of t u r t l e s . Even though t u r t l e s do not possess a highly branched bronchial tree as i n mammals i t i s not surprising that flow resistance i s found i n their lungs. I t has been shown that due to the large cross sectional area of the lower portion of the bronchial tree i n mammals that l i t t l e flow resistance occurs below the medium sized bronchi with the majority of flow resistance being i n the upper airways (Pedley et a l . , 1970). In summary, the mechanical work/breath required to i n f l a t e the respiratory system i n the t u r t l e i s divided equally between overcoming e l a s t i c and nonelastic forces at low frequencies and t i d a l volumes with almost a l l of these forces residing i n the body wall and cavity. Work required to overcome nonelastic forces, however, predominates at high frequencies and t i d a l volumes with the lungs contributing an ever increasing proportion to this work. This difference i n the effect of increasing frequency on the work required to overcome forces a r i s i n g from the lung and body cavity i s responsible for the ove r a l l U-shape of the minute work curve which produces an optimum combination of t i d a l volume and frequency requiring the minimum - 67 -work to maintain a constant level of total ventilation (Vg or V A). The nature of the mechanical work of breathing is heavily influenced by the anatomy of the respiratory system. In lizards, turtles and mammals, differences in the architecture of the lungs and body result i n differences in pulmonary mechanics which are reflected in the work of breathing. For instance, the li z a r d , Gekko gecko possesses single chambered lungs which are fused anteriorly, and share a common bronchial opening arising from a wide bore, undivided trachea. They, consequently, lack an intrapulmonary bronchus and internal lung partitioning consists of a series of dorsolateral ridges with a net-like system of tabeculae arising from the lung wall. These lungs are not enclosed in a pleural cavity but l i e along the dorsal surface of the pleuroperitoneal cavity, held in place dorsally and ventrally, by mesopneumonia. These connections allow free movement of the lungs and establish resting lung volume when the system is open to atmosphere (Milsom and V i t a l i s , 1984). The body wall of Gekko gecko also differs from that of the turtle i n that i t is long, slender and flexible and possesses moveable articulated ribs enclosing the viscera and lungs. As a result of i t s anatomy the majority of the work required to produce each breath (70-90%) in Gekko gecko is required to overcome elastic forces at a l l frequencies and t i d a l volumes (Milsom and V i t a l i s , 1984). Furthermore, almost a l l of these elastic forces arise from the body wall of the animal. The lungs, due to their simple structure, contribute very l i t t l e to the forces resisting inflation and therefore the total work of breathing in Gekko gecko is almost purely required to overcome elastic forces arising from the body wall. Given this, the minute work required to maintain a constant level of minute ventilation (V F) diminishes - 68 -exponentially as a function of increasing breathing frequency. For a constant alveolar ventilation, however, minute work begins to rise after an i n i t i a l decline due to the increase in Vg required to maintain V A constant when ventilation frequency increases. The result i s a U-shaped curve for minute work vs. breathing frequency where a specific combination of t i d a l volume and frequency correspond to the level of minimum work. This 'U'-shaped curve for a constant V^ is here due solely to the increase in Vg required to account for dead space ventilation when frequency increases and not to rising nonelastic forces as is the case in turtles. In the case of mammals, the respiratory system differs in two major aspects from the systems found i n both the turtle and lizard. These differences also influence the mechanics of the system and are i n turn reflected i n the work of breathing. The major anatomical difference influencing the mechanics of the respiratory system in mammals is the structure of the lungs. Mammalian lungs are characterized by a highly branched bronchial tree which communicates with approximately 300 million densely packed alveoli which are each approximately .3 mm in diameter (West, 1979). The alveoli and alveolar ducts are surrounded by an arrangement of collagen and elastic fibers in a helic a l fiber network which provides a large portion of the elastic recoil of the lungs (Bouhuys, 1977). Another important difference i s that the lungs are now isolated in a pleural cavity within the thoracic cage which is separated from the viscera by a muscular diaphragm. Within this cavity the lungs are surrounded by pleural membranes and are unattached to the surface of the thoracic cage. Due to the arrangements of the elastic components, the lungs have a tendency to recoil inwards while the chest wall has a tendency to spring outward. These two forces tend to balance each other under equilibrium conditions and thus the chest i s pulled inward and the lungs outward. The result i s that during i n s p i r a t i o n , because of the natural tendency of the chest wall to spring outwards, work, only has to be done to overcome the forces within the lung. Work i s stored i n the e l a s t i c elements of the lung and expiration i s passive under resting conditions. In humans 60% to 70% of the work of breathing i s required to overcome the e l a s t i c forces within the system (Otis et a l . , 1950) with the majority of these forces residing i n the lungs. The lung i s also a s i t e of flow-resistance which accounts for approximately 25% of the work of breathing with the remainder of the forces to be overcome (££. 5-10%) being viscous i n nature. Here also, as i s the case for l i z a r d s and t u r t l e s , when W i s plotted as a function of f for a constant l e v e l of V^, the curve i s U-shaped. In comparing the mechanics of these three respiratory systems i t can be seen that anatomical factors play an important role i n determining the nature of the work required to i n f l a t e the respiratory system. The work required to i n f l a t e the lungs as a percentage of the t o t a l work of breathing increases i n the sequence Gekko < t u r t l e < mammal. This hierarchy also r e f l e c t s the increasing complexity of the lungs. It i s of interest that an optimal combination of frequency and t i d a l volume exists i n a l l three groups despite the large d i v e r s i t y seen i n the structure and pulmonary mechanics of thei r respiratory systems. This seems to suggest that optimal combinations exist not only because of anatomical factors but also because of the need to maintain alveolar v e n t i l a t i o n (V A) constant rather than t o t a l v e n t i l a t i o n (Vg). I t i s plausible that anatomical dead space and i t s magnitude play as important a - 70 -r o l e i n determining optimal combinations of f and V\j, as the mater ia l and s t r u c t u r a l components of the r e s p i r a t o r y system. CHAPTER 4 THE INTERRELATIONSHIP BETWEEN PULMONARY MECHANICS AND THE SPONTANEOUS BREATHING PATTERN IN PSEUDEMYS SCRIPTA Introduction In the previous chapter i t was shown that a s p e c i f i c combination of t i d a l volume (Vj.) and breathing frequency (f) exists for which a minimum amount of mechanical work per minute (W) i s required to maintain any given constant l e v e l of alveolar v e n t i l a t i o n (V^). This holds true for mammals, t u r t l e s , and gekkos despite great differences i n dynamic pulmonary mechanics. One assumption inherent i n such considerations i s that breathing i s continuous. Due to their low metabolic rates, however, poikilotherms do not need to breathe continuously but instead exhibit arrhythmic breathing patterns. The arrhythmic patterns of poikilotherms are not a l l the same but show some va r i a t i o n . These patterns range from single breaths separated by a variable period of breath holding at end-inspiration as seen i n the gekko to bursts of continuous breaths separated by variable periods of breath holding as seen i n semi-aquatic t u r t l e s . In single breath breathers, i t has been suggested that the work required to take each breath i s a more meaningful criterium for assessing the mechanical e f f i c i e n c y of breathing rather than minute work as i s the case i n continuous breathers (Milsom, 1984). Semi-aquatic t u r t l e s , being half away between single breath breathers and continuous breathers are - 72 -capable of u t i l i z i n g both p a t t e r n s . I t i s of i n t e r e s t , t h e r e f o r e , to analyze the e f f i c i e n c y of the spontaneous breathing patterns seen i n the t u r t l e i n terms of the mechanics d e r i v e d i n chapters 2 and 3. - 73 -Materials and Methods In total, twelve turtles (Pseudemys scripta) of mean weight 536 +_ 40.9 g., were used in this study. The animals were housed in a large circular tank (1.5 m diameter) supplied with flowing tap water (10-15°C) to a depth of 30 cm. Dry areas heated with lamps were provided to allow the animals to bask. Several days before experiments the animals were moved into the laboratory and housed in smaller tanks supplied with water and dry basking areas where they became acclimated to room temperature (20-22°C). A l l animals were in a post-absorptive state when experiments were conducted. Measurement of Ventilation Each turtle was placed in a darkened aquarium with a plexiglass grid covering the surface. A 10 cm. hole was placed in this cover opening into a fit t e d air f i l l e d chamber ( 150 ml) from which the turtle could breath (Glass et a l . , 1983). The chamber was flushed with gas at a rate of 500 ml/min. via inflow and outflow ports on the top. The aquarium was f i l l e d with water to the level of the chamber opening and the animal was allowed free movement within the aquarium and quickly learned to breath from the chamber. A light was placed near the hole to assist the animal i n locating i t . The animal was allowed at least 24 hrs. to become accustomed to the experimental set up. The temperature of the aquarium was room temperature (20-22°C). Inspiratory and expiratory airflows were measured using a Fleish - 74 -pneumotachograph (#00) placed over the outflow post in the chamber arrange-ment. The differential pressure across the pneumotachograph generated by the air flow was measured by a Validyne differential pressure transducer (DP 103-18) and Gould transducer amplifier. The flow of gas flushing the system was offset electronically to read as zero flow. When the turtle breathed in the chamber, inspiratory and expiratory flows were superimposed on this flow of flushing gas and only the inspiratory and expiratory flows were recorded. Inspiratory and expiratory flow were integrated with a Gould integrating amplifier to give t i d a l volume and recorded on a Tanberg Instrumentation Tape Recorder (Series 115) and a Gould 2600 pen recorder. The system was calibrated by injecting known volumes from a syringe. The rate of injection had no effect on the volume recorded. Ventilation was measured while room air, hypoxic (4% O2 in N 2 ) , and hypercapnic (3-5% CO2 in air) gas mixture were used to flush the chamber. These gases were mixed using calibrated flow meters or bottled gases and delivered through the chamber at 500 ml/min. Each animal was allowed to breath each gas for 1 hour before measurements were taken to ensure that steady state conditions were being monitored (Glass et a l . , 1983). After this i n i t i a l one hour exposure to the experimental gas, ventilation was measured for at least 1 hour. Bilateral Vagotomy Turtles were mildly anaesthetized with sodium pentobarbital (20 mg/kg i.p.) and restrained in an upright position. A local anesthetic (lidocaine) was also administered to the neck. An incision was made along one side of the neck and the carotid artery was exposed. The vagus nerve was carefully freed from the carotid artery and approximately 5 mm of nerve - 75 -was removed. The incision was closed and the procedure was repeated on the other side. The animals were allowed to recover for several days and only those animals that were active, showed good reflexes, and were free of infection were used for experiments. These animals were then subjected to the same protocol described above. Calculation of Ventilation A l l calculations of minute ventilation were based on inspired volume. Breathing frequency (f (breaths/min)) was determined by dividing the total number of breaths by the total length of the recording which was usually one hour or more. The instantaneous breathing frequency ( f ) was calculated as the total time required to take a single breath ( T t Q t (sec.)) divided into 60 sec. Total ventilation (Vg (ml/min)) was calcu-lated as the product of ti d a l volume (V T(ml)) and breathing frequency. Average values for V^ , and T t o t were determined for each animal on each gas by analyzing 15 to 20 randomly chosen breaths and mean values for a l l the animals involved in the experiments were then determined. - 76 -Results The respiratory pattern of Pseudemys sc r i p t a breathing a i r , under normal resting conditions consists of series of continuous breaths separated by breath holds of variable duration which are usually associated with diving. This i s i l l u s t r a t e d by Fig. 4.1a which shows a sample trace of respiratory a i r flow from an intact animal. From such traces the respiratory variables were calculated and are shown i n Table 4.1. When respiration was stimulated by hopoxic (4% i n N 2) or hypercapnic (3-5% CC>2 i n a i r ) gas mixtures, t o t a l v e n t i l a t i o n increased by 1.3X and 2.IX respectively. The increase i n t o t a l v e n t i l a t i o n was due solely to an increase i n the number of breaths per minute ( f ) with t i d a l volume (V\j,) and the instantaneous breathing frequency ( f ) remaining unchanged. To increase v e n t i l a t i o n then, the t u r t l e shortened the nonventilatory period (T Nyp) and increased f with V T and T t Q t and, therefore, f' remaining unchanged. The work/breath (W/b) values shown i n this table were taken from Fig. 3.4 i n chapter 3 using the spontaneous values for f 1 and V^ , while the work per minute (W) i s simply the product of W/b and f. Since V^ , and f' remained r e l a t i v e l y unchanged during respiratory stimulation, W/b also remained unchanged, however, W increased when respiration was stimulated by hypoxia or hypercapnia due to increases i n f. The effects of b i l a t e r a l vagotomy on respiratory a i r flow are shown i n F i g . 4.1b while the mean values of the respiratory variables measured under these conditions are l i s t e d i n Table 4.1. During a i r breathing, vagotomy resulted i n large increases i n V T and a reduction i n f - 77 -F i g . 4.1 Representative flow traces from a t u r t l e spontaneously breathing a i r ; (a) i n t a c t animal (b) a f ter b i l a t e r a l vagotomy. i n t a c t i i 10 sec Table 4.1 Respiratory variables for spontaneously ventilating, intact and vagotomized turtles breathing air, 3-5% in air, and 4% O2 in Tidal volume (V^.), breathing frequency (f) and instantaneous frequency ( f 1 = ^ 0/T ^) are expressed as means + S.E.M. for 6 animals. Total minute ventilation (Vg) is the product of V,j, and f. The work/breath (W/b) values are taken from figure 3.5 using spontaneous values for V T and f'. Minute work (W) is the product of W/b and f. air 3-5% C0 2 4% 0 2 Intact v T ml/kg 6.9+1.2 6.8+0.4 6.2+0.8 f breaths/min 2.0+0.7 4.2+1.3 3.0+0.4 VE ml/kg/min 13.8 28.3 18.6 f ' l/min 35+2 34+2 35+2 W/b ml.cmH^O/kg 23 22 19 W ml.cmR^O/min/kg 46 92 57 Vagotomized v T ml/kg 18.0+4.0 28.0+6.0 34.2+6.0 f breaths/min 0.6+0.2 1.6+0.9 0.8+0.2 VE ml/kg/min 10.8 45.4 27.7 f ' l/min 25+3 19+3 21+2 W/b ml.cmH.2O/kg 110 220 340 W ml.cmH20/min/kg 66 352 272 - 79 -and f 1 with t o t a l v e n t i l a t i o n being s l i g h t l y reduced when compared to values recorded i n intact animals. The breathing pattern now generally consisted of single breaths rather than bursts of continuous breathing although t h i s pattern was not seen i n a l l animals. Under conditions of hypoxia and hypercapnia t o t a l v e n t i l a t i o n increased 2.6X and 4.2X respectively. The increase i n v e n t i l a t i o n stimulated by hypoxia was primarily due to a twofold increase i n V T > and a very small increase i n f. There was also a lengthening of each breath so that f was further reduced to 21/min. The Increase i n Vg due to hypercapnia was also due to a large increase i n V T (1.6X) and a 2.7X increase i n f. The instantaneous frequency was also reduced to 19/min. As a consequence of the large increases i n V^ and reductions i n f W / b increased dramatically over the values calculated for intact animals under a l l conditions. The W/b and W values were determined i n the same manner as i n the intact condition. During a i r breathing W/b i n the vagotomized condition increased 5X compared to values calculated for the intact animals but due to the decrease i n f, W rose only 1.8X per unit v e n t i l a t i o n . Upon exposure to hypoxia and hypercapnia W/b increased 4X and 3X respectively over values obtained for the a i r breathing condition. Since f also increased upon exposure to hypoxia and hypercapnia i n vagotomized animals W increased 4X and 5X respectively over the a i r breathing condition. The increases i n W under hypoxic and hypercapnic conditions i n the vagotomized animals when compared to the intact animals were 3.2X and 2.4X greater per unit v e n t i l a t i o n respectively. - 80 -Discussion The breathing pattern recorded in resting, spontaneously breathing Pseudemys scripta in the present study i s similar to that recorded by other researchers in semi-aquatic turtles (see Glass and Wood, 1983; Shelton et a l . , 1984 for reviews). The mean respiratory variables of tida l volume (V^), breathing frequency (f) and total ventilation (Vg) measured in this study are compared to literature values in Table 4.2. The values recorded for f (breaths/min.) f a l l in the upper range of recorded values for semi-aquatic turtles while values of V^ , (ml/kg/min) recorded for P. scripta in this study compare well and are at the low end of previously reported values. The method of measuring ventilation appears to be an important factor determining the variability of values of Vg found in the literature primarily due to variablity in V^. Almost a l l previous measurements of ventilation have involved restraining the animal in some fashion, and/or having the animal out of water and, in some cases, catheterized, a l l of which are unnatural forms of stress. This stress appears to manifest i t s e l f in a variable V,j, and thus Vg. It i s f e l t that the noninvasive method employed i n this study provides a close reflection of the true breathing pattern while other studies have tended to overestimate V T and underesti-mate f. Glass e_t j i l . (1983) using the same method reported identical breathing frequencies, however, V T was 1.6X larger resulting i n Vg being 1.5X larger compared to the present study. The differences may have been due to the presence of arterial catheters in the study of Glass et a l . Table 4.2 Comparison of respiratory variables for turtles taken from literature. Standard error shown when reported. Species Temp. (°C) Respiratory gas (ml/kg) f (min *) VE (ml/min/kg) Source P. floridana 20 Air 12.6 1.4 18.6 Kinney et_ a l . (1977 p. scripta 20 6.9+1.2 2.0+0.7 13.8 Present study p. scripta 20 15.7+2.1 1.6+0.2 23.8+3.4 Jackson et a l . (1974) p. scripta 20 8.8* 1.4+0.15 12.3+1.3 Jackson (1973) C. picta 20 10.7+1.2 1.9+0.27 20.3 Glass et a l . (1983) C. picta 22-25 13.5+0.9 1.8+0.2 24.8+3.4 Milsom et_ a l . (1980) T. horsfieldi 23-25 8.1 1.4 11.5 Benchetrit et a l . (1980) Hypoxia P. scripta 20 3% 0 2 6.8* 2.58+0.25 17.7 Jackson (1973) P. scripta 20 4% 0 2 6.2+0.8 3.0+0.4 18.6 Present study C. picta 20 5% 0 2 18.7 2.16 30.0 Glass et_al. (1983) * calculated from reported values of f and V, Table 4.2 (continued) Species Temp. (°C) Respiratory gas v T (ml/kg) f (min *) (ml/min/kg) Source Hypercapnia P. scripta 20 4% C0 2 29.4+3.6 3.0+0.4 84.4+13.3 Jackson et a l . (1974) c. picta 22-25 5% C0 2 17.5+2.0 3.9+0.6 65.5+11.4 Milsom et a l . (1980) p. scripta 20 3-5% C0 2 6.8+0.4 4.2+1.3 28.3 Present study T. horsfieldi 23-25 4% C0 2 15.7 5.3 82.6 Benchetrit et a l . (1980) - 83 -(1983) or due to species differences. The ventilatory responses to hypercapnia and hypoxia also d i f f e r between various studies. The response to hypercapnia and hypoxia i n the present study i s a stimulation of v e n t i l a t i o n due solely to an increase i n f with V\j, and breath duration, and therefore f' remaining unchanged. Other studies are i n general agreement with hypercapnia (Jackson et a l . , 1974; Benchetrit and Dejours, 1980; Milsom et a l . , 1980) and hypoxia (Jackson, 1973; Glass et a l . , 1983) being ventilatory stimulants and with the former appearing to be the more powerful stimulant of the two. The response to hypercapnia reported i n the l i t e r a t u r e , however, indicates that this response i s due to an elevation of as well as an increase i n f. A comparison of the results of the present study with those of Jackson et_ a l . (1974), Benchetrit and Dejours (1980), and Milsom and Jones (1980) reveals an absence of the large elevation i n V^ , i n the present study though ven t i l a t i o n appears to increase, more or l e s s , to the same extent. This discrepancy may be due to differences i n methodology or i t may be due to a lack of equilibrium between blood gases and inspired gas i n the present study since blood gas values were not measured to confirm equilibrium had been reached. The response to hypoxia (3-5% 0 2) reported i n the l i t e r a t u r e shows increases i n v e n t i l a t i o n of 1.5X (Glass et a l . , 1983) and 1.4X (Jackson, 1973) which are i n agreement with the present study (1.3X). The response reported by Glass et a l . (1983) was due almost solely to an increase i n V T while Jackson (1973) reported that the increase i n Vg upon exposure to hypoxia was due to an increase i n f and a drop i n V j . The present study also showed a s l i g h t drop i n V T with the response being - 84 -due to an elevation in f as was the case in the study of Jackson (1973). Thus although there appears to be some variation in the nature of the response to respiratory stimuli reported in the literature i t i s generally agreed that aquatic turtles increase ventilation by shortening the breath hold period and increasing the number of breaths per minute. This present study confirms this and in addition shows that the characteristics of each breath ( i . e . V\j and T t o t ) remain the same. Semi-aquatic turtles such as P. scripta spend long periods of time underwater and the nonventilatory period i s usually associated with dives. Whatever combinations of respiratory variables i n i t i a t e and terminate ventilatory periods, the longer a turtle spends underwater, the more breaths i t w i l l require to replenish 0 2 stores in the lungs and blood and to eliminate C0 2 when the animal surfaces. During such a long bout of continuous breathing, the work of breathing can be analyzed, as in mammals, in terms of W and f, which during the bout of continuous breathing is equal to f . Measurements made on continously pump ventilated animals show that * the power required to maintain a constant alveolar ventilation rate (V^) is least at pump frequencies betwen 30 and 35 cycles/min. (chapter 3). In Fig. 4.2, W is plotted against f* for various levels of Vg and V A and the shaded area indicates the range of f' calculated for spontaneously breathing animals. Clearly the f' of spontaneously breathing animals corresponds very closely with pump frequencies which generate the minimum rate of work in maintaining a constant level of V^. They correspond less well to those frequencies which correspond to the minimum work required to maintain a constant level of total ventilation (V F). Such considerations - 85 -Fig. 4.2 The rate of total work for constant levels of alveolar ventilation (V^ in ml/min.) and total ventilation (Vg in ml/min). These curves are taken from chapter 3, figure 3.9. The shaded area represents the range of instantaneous breathing frequencies ( f 1 = 60/ T ) measured in spontaneously A t o t breathing animals. - 85a -- 86 -suggest that during the periods of continuous breathing seen i n these t u r t l e s , as i n mammals, spontaneous breathing patterns correspond closely to predicted optimum patterns based on mechanical considerations. Since marine tu r t l e s and tortoises do not breathe i n bursts, as seen i n P. s c r i p t a , but rather take single breaths with periods of breath holding between each breath, i t i s of interest to analyze the mechanical cost of breathing i n terms of the work per breath. Figure 4.3 shows the mechanical cost required to produce a single breath for various levels of as a function of pump frequency. The values of Vrj, and f' from intact animals spontaneously breathing various gas mixtures are placed on the graph for comparison. The work/breath curves shown i n Fig. 4.3 i l l u s t r a t e that for any given frequency, the smaller the V, j , , the lower the mechanical work of each breath. A low V ^ , , however, compromises alveolar v e n t i l a t i o n and gas exchange as was discussed i n chapter 3. These two factors, the need to keep V^, s u f f i c i e n t l y large i n order to maintain alveolar v e n t i l a t i o n on the one hand and the increased mechanical work associated with increases i n V\j, on the other may play important roles i n setting V T > At any given V ^ , the work/breath i s then a function of breathing frequency. For a constant V T the mechanical cost of a breath would be lower the lower the breathing frequency. There are physiological and behavioral factors, however, which would probably r e s t r i c t breath duration from becoming too long. A very long breath duration i n a spontaneously breathing animal would require a slow controlled i n s p i r a t i o n while expiration would have to be actively slowed by the breaking action of the inspiratory muscles. Since the inspiratory muscles would be working against - 87 -Fig. 4.3 The relationship between total work/breath (W) and pump ventilation frequency (f = cycles/min.). Data points for curves represent the mean values for 3 animals taken from figure 3.4, chapter 3. The open symbols represent the mean value + S.E.M. of V T and f' measured from 6 animals spontaneously breathing air (0); or 5% C0? (A ) in air; or 4% 0? (^) in No. - 87a -f (min" ' ) - 8 8 -the passive re c o i l of the system, the stored elastic energy which partially powers both inspiration and expiration would be lost. Although the actual mechanical work of such a spontaneous breath w i l l not be much different from the work recorded when expiration is passive, the oxidative cost would, however, be much greater due to the active muscular work required for the breaking of expiration. This would result i n a drop in the efficiency of ventilation. Another important consequence would be the loss of the nonventilatory period. This would result in the semi-aquatic turtle having to shorten i t s dives and spend more time ventilating at the surface. A further consequence of a long breath duration would be the increased time contribution of the locomotor muscles to their accessory role of ventilation. These factors probably play an important role in restricting the length of breath duration and favouring the retention of a nonventilatory period at the expense of minimizing the work/breath. Inspection of Fig. 4 . 3 shows that the instantaneous frequency (f') of spontaneous breaths in turtles f a l l at or near the inflexion point where further increases i n pump frequency result in a very sharp rise in the work/breath. The difference in mechanical work between a very low frequency and the f of a spontaneous breath i s however very small compared with the large increase in work/breath which occurs when frequency i s elevated above the spontaneous f' • The position of the instantaneous f 1 on the work/breath curve may represent a compromise which allows for a nonventilatory period and maintains gas exchange and ventilatory efficiency with only a small increase in the work/breath over the breath duration which would minimize the mechanical cost of a single breath. From the data presented above, i t appears that in semi-aquatic - 89 -t u r t l e s where continuous breathing i s not required to meet metabolic demands, but where the oxidative cost of breathing i s high (10-30% of t o t a l 0 2 requirements (Kinney and White, 1977)), the most e f f i c i e n t breathing pattern i s an arrythmic breathing pattern. If oxygen requirements r i s e or v e n t i l a t i o n i s stimulated by i t i s better to r e s t r i c t changes i n V T and f' and take more breaths/burst or shorten the T Nyp and take more bursts per unit time. By shortening the breath hold period, or increasing the number of breaths/burst rather than a l t e r i n g V"T or f *, the increase i n the rate of mechanical work i s minimized. Pulmonary vagal afferent information appears to play a role i n regulating the and f' of each breath (Milsom and Jones, 1980). I t could be said then that vagal information plays some role i n determining the mechanical work of breathing even though i t might be an i n d i r e c t one. In spontaneously breathing t u r t l e s , i f vagal information i s removed, the work/breath per unit v e n t i l a t i o n (W/b/Vg) increases s i x f o l d due to an increase i n V\j, and a drop i n f *. This must result i n an increased oxidative cost for v e n t i l a t i n g the lungs. If v e n t i l a t i o n i s stimulated i n a vagotomized animal through hypoxia or hypercapnia, the work/breath increases 2 and 3 f o l d respectively due to further increases i n V^ , and decreases i n f'. This i s unlike the s i t u a t i o n i n intact animals where there are no appreciable changes i n V^ , and f * upon ventilatory stimulation. Although i t i s not t o t a l l y clear what combination of sensory inputs i n i t i a t e and terminate breathing (Lenfant and Johansen, 1968; Lenfant et a l . , 1970; Toews et a l . , 1971; Burggren and Shelton, 1979; Ackerman and White, 1979; B o u t i l i e r , 1981) once breathing i s i n i t i a t e d , the breath length and t i d a l volume appear to be regulated at levels which minimize the work of - 90 -breathing . Whether pulmonary a f ferent information i s d i r e c t l y responsible for the sensing of mechanical work or i f minimal mechanical work i s simply a fo r tu i tous consequence of t i d a l volume regula t ion remains unc lea r . - 91 -CHAPTER 5 GENERAL DISCUSSION AND CONCLUSIONS Analyses of pulmonary mechanics in anaesthetized turtles have shown that the architecture of the body wall and lung determine the overall mechanics of the respiratory system which in turn appears to influence the spontaneous breathing pattern in the animal. The lungs of the turtle are highly compliant as a result of the presence of large membraneous caudal regions (Perry and Duncker, 1978). The majority of elastic forces which must be overcome in the system during ventilation are located within the body wall of the animal. As a consequence the compliance of the body wall is relatively low and is the key factor determining the compliance of the whole system. Under dynamic conditions of continuous pump ventilation the dynamic compliance of the system is independent of stroke volume over a range of volumes around the normal V^ , of these animals but is dependent on ventilation frequency. For any given volume, as frequency increases ^dyn decreases which is reflected in a rise i n the work/breath with increasing frequency. Analysis of the components which contribute to the total work of each breath shows that the majority of the work required to produce each breath at low ventilation frequencies is required to overcome forces within the body wall. If t i d a l volume is held constant and ventilation frequency increased, the proportion of the total work required to overcome forces arising from the lungs steadily rises with these forces being nonelastic, flow-resistive forces. The work required to overcome the the forces within the body does not change as frequency increases suggesting that these forces are predominantly elastic in nature. If frequency is held constant and volume increased then the work required to overcome the forces resisting lung inflation rises exponentially and is predominantly required to overcome increased elastic forces. These effects of changes in respiratory frequency and t i d a l volume on the work required to generate each breath influence the relationship between the power required to ventilate the lungs and the breathing frequency for a constant level of alveolar ventilation. The relationship between the power required to maintain a constant level of alveolar ventilation and breathing frequency takes the form of a U-shaped curve. As frequency increases elastic work decreases due to the necessary drop in V\j, required to maintain constant V"A. With further increases in frequency, however, minute work begins to rise again due to increasing nonelastic forces and the required increase in total ventilation needed to offset dead space ventilation required to keep V~A constant. As a consequence there is a combination of frequency and t i d a l volume at which the work required to overcome the forces associated with ventilation are at * a minimum for each level of V^. The frequency that corresponds to minimum mechanical work is approximately 35 breaths/min. In spontaneously breathing turtles, the respiratory pattern consists of bursts of continuous breathing separated by a variable breath hold period beginning at end-inspiration. The consequence of this breath hold period i s a very low overall breathing frequency of approximately 2 breaths/min. under normoxic resting conditions. The frequency of breathing within the burst, however is 30-35 breaths/min. and is referred to as the instantaneous breathing frequency ( f ' ) . This value corresponds very closely to the ventilation frequency calculated to require the minimum mechanical work, to produce similar levels of V^. If respiration i s stimulated by -hypoxia or hypercapnia V T and f change very l i t t l e and the animal increases ventilation by taking a larger number of breaths at this optimal combination of V\j, and f I f the work of spontaneous breathing is analyzed in terms of a single breath rather than a burst of continuous breaths, f is s t i l l held at a level which tends to minimize the mechanical cost of a single breath. This mechanical analysis of the respiratory system in the turtle has provided insights which may help explain the adaptive significance of arrythmic breathing. Since turtles have a very low metabolic rate continuous breathing is unnecessary. Instead these animals appear to have adopted a strategy of periodic breathing which s t i l l minimizes the mechanical cost of breathing and thus the oxidative cost of breathing. This is important since the metabolic cost of breathing is already very high (10-30% of resting metabolic rate (Kinney and White, 1977)) and because breath hold periods allow these aquatic animals to spend time underwater and s t i l l maintain oxidative metabolism. This type of arrhythmic breathing which minimizes the mechanical cost of breathing appears to be under neural control. If the animals are bilaterally vagotomized, t i d a l volume increases, f' decreases along with f so that total ventilation doesn't change appreciably but the mechanical cost greatly increases. While vagal afferent information may not sense mechanical work directly i t does regulate and f* i n such a way as to minimize the mechanical cost. Under the influence of afferent vagal control - 94 -the response to r e sp i r a to ry s t i m u l i i s to increase the number of breaths but to maintain V"T and f 1 . This serves to minimize the mechanical cost and thus the ox ida t ive cost of the increase i n v e n t i l a t i o n . While the var i ab le s which i n i t i a t e the onset and terminat ion of the v e n t i l a t o r y per iod i n a r rhythmica l ly breathing r e p t i l e s remain unclear mechanical analys i s has provided a framework fo r better understanding the regu la t ion which does occur w i t h i n the breathing episode. - 95 -LITERATURE CITED 1. Ackerman, R.A. and White, R.N. 1979. Cyclic carbon dioxide exchange in the turtle Pseudemys scripta. Physiol. Zool. 52, 378-389. 2. Agostoni, E. 1970. Statics. In The Respiratory Muscles, (eds. G.M.J. Campbell, E. Agostoni and J. Newsome Davis), London: Lloyd-Luke Ltd. 3. Agostoni, E., Thimm, F.F. and Fenn, W.O. 1959. Comparative features of the mechanics of breathing. J_. appl. Physiol. 14, 679-683. 4. Benchetrit, B. and Dejours, P. 1980. Ventilatory CO2 drive in the tortoise Testudo horsfieldi. J_. exp. Biol. 87, 229-236. 5. 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Control of breathing in Acrochordus  javanicus, an aquatic snake. Physiol. Zool. 49, 328-340. 19. Glass, M.L. and Wood, S.C. 1983. Gas exchange and control of breathing in reptiles. Physiological Reviews 63, 232-260. 20. Hughes, G.M. 1963. Comparative Physiology of Vertebrate Respiration, London: Heinemann. 21. Hughes, G.M. and Vergara, G.A. 1978. Static pressure-volume curves for the lung of the frog (Rana pipiens). J_. exp. Biol. 76, 149-165. - 97 -22. Hutchinson, V.H., Whitford, W.G. and Kohl, M. 1968. Relation of body size and surface area to gas exchange i n anurans. Physiol. Zool. 41, 65-85. 23. Jackson, D.C. 1971. Mechanical basis for lung volume v a r i a b i l i t y i n the t u r t l e Pseudemys s c r i p t a elegans. Am. J_. Physiol. 220, 754-758. 24. Jackson, D.C. 1973. Ventilatory response to hypoxia i n t u r t l e s at various temperatures. Respir. Physiol. 18, 178-187. 25. Jackson, D.C, Palmer, S.E. and Meadow, W.L. 1974. 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Quantitative anatomy of the lungs of the red-eared t u r t l e , Pseudemys sc r i p t a elegans. Respir. Physiol. 35, 245-262. 46. Perry, S.F. and Duncker, H.R. 1978. Lung architecture, volume and s t a t i c mechanics i n f i v e species of l i z a r d s . Respir. Physiol. 34, 61-81. 47. Perry, S.F. and Duncker, H.R. 1980. Interrelationship of s t a t i c mechanical factors and anatomical structure i n lung evolution. J_. Comp. Physiol. 138, 321-334. 48. Romer, A.S. 1967. Major steps i n vertebrate evolution. Science 158, 1629-1637. 49. Romer, A.S. 1972. Skin breathing - Primary or secondary? Respir.  Physiol. 14, 183-192. 50. Rosenberg, H.I. 1973. Functional anatomy of pulmonary v e n t i l a t i o n i n the garter snake, Thamnophis elegans. J_. Morph. 140, 170-184. 51. Shelton, G., Jones, D.R. and Milsom, W.K. 1984. Control of breathing i n ectothermic vertebrates i n The Handbook of Physiology ( i n press). 52. Tenney, S.M. and Tenney, J.B. 1970. Quantitative morphology of cold-blooded lungs: Amphibia and R e p t i l i a . Respir. Physiol. 9, 197-215. 53. Toews, D.P., Shelton, G. and Randall, D.J. 1971. Gas tensions i n the lungs and major blood vessels of the Urodele amphibian Amphiuma  tridactylum. J . exp. B i o l . 55, 47-61. - 100 -54. West, J.B. 1979. Respiratory Physiology. Baltimore: The Williams and Wilkins Company. 55. West, N.H. and Jones, D.R. 1975. Breathing movements in the frog, Rana pipiens. I. The mechanical events associated with lung and buccal ventilation. Can. J_. Zool. 53, 332-344. 56. Wood, CM. and Lenfant, CJ.M. 1976. Respiration: Mechanics, control and gas exchange. In Biology of the Reptilia (eds. C. Gans and W.R. Dawson), New York: Academic Press. 57. Yamashiro, S.M., Daubenspeck, J.A., Lauritsen, T.N. and Grodins, F.S. 1975. Total work rate of breathing optimization in CO2 inhalation and exercise. J_. appl. Physiol. 38, 702-709. 

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