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Investigations into the diving response of northern elephant seals (Mirounga angustirostris) using magnetic… Thornton, Sheila J. 2000

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INVESTIGATIONS INTO THE DIVING RESPONSE OF NORTHERN ELEPHANT SEALS (Mirounga angustirostris) USING MAGNETIC RESONANCE IMAGING AND SPECTROSCOPY by SHEILA J. THORNTON B.Sc, The University of Manitoba, 1988 M.N.R.M., The University of Manitoba, 1994 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Zoology) We accept this thesjs-as conforming to ^he-required standard  THE UNIVERSITY OF BRITISH COLUMBIA June 2000 © Sheila Jean Thornton, 2000  In  presenting  degree freely  at  this  the  thesis  in  partial  fulfilment  University  of  British  Columbia, I agree  available for  copying  of  department publication  this or of  reference  thesis by  this  for  his thesis  and study. scholarly  or for  her  financial  of  'C~-C^G»  I_ o-cS M  The University of British C o l u m b i a Vancouver, Canada  DE-6  (2/88)  the  requirements that the  I further agree  purposes  may  representatives.  permission.  Department  of  It  gain shall not  be is  that  Library  permission  granted  by  understood be  for  allowed  an  advanced  shall make for  the that  without  it  extensive  head  of  my  copying  or  my  written  ABSTRACT To examine the physiological and biochemical changes that occur during diving, northern elephant seal pups were subjected to forced dive protocols while undergoing Magnetic Resonance Imaging and Spectroscopic analysis. Splenic volume measurements were obtained in the predive, dive and postdive state. Splenic contraction was initiated immediately upon facial immersion, reducing to 14.0 - 17.8% of its predive volume by minute 3 of the dive. A corresponding increase in hepatic sinus volume occurred, suggesting a direct shift of blood from the spleen to the sinus. In the postdive period, the spleen gradually dilated, achieving its maximum volume at 18-22 minutes after the dive (3.35% of body mass; n = 5). Stroke volume (SV) measurements were obtained during diving using phase contrast M R imaging. Mean resting S V was 104.94 mis ± 4.12 S E M , while SV during the dive increased significantly to a mean value of 126.12 ± 3.93 S E M . These data are in contrast to the findings of previous studies on pinnipeds, which indicate either the maintenance or decline of stroke volume during the dive. Pre-dive, postdive or diving heart rates did not correlate with mass or with stroke volume during any state. However, diving SV correlated with the ratio of diving H R to predive H R (P = 0.01, R = 0.99). These findings indicate that the effect of diving on stroke volume is altered by the degree of bradycardia rather than the absolute HR. M R spectroscopy analysis was used to investigate changes in locomotory muscle phosphocreatine (PCr) levels during diving. End-dive PCr levels ranged from 36.84 - 103.93% of resting (average 80.85% ± 5.89 SEM). Animals who exhibited a significant decrease in PCr during diving showed a correlation between end-dive PCr and intracellular pH values. Plasma lactate levels indicated that all dives were within the aerobic diving limit. This study demonstrates that in northern elephant seals, PCr hydrolysis does occur during aerobic dives, indicating that the depletion of [PCr] is driven by an alteration in muscle [H ] rather than by changes in +  [adenylate]. The decrease in muscle pH is attributed to respiratory acidosis caused by significant C 0 accumulation during hypoperfusion. 2  TABLE OF CONTENTS Abstract  ii  Table of Contents  iii  List of Tables  iv  List of Figures  v  Acknowledgements Chapter 1 General Introduction: Discussion of northern elephant seals and the physiology of diving Chapter 2 2.1 2.2 2.3 2.4 2.5 2.6 2.7 Chapter 3 3.1 3.2 3.3 3.4 Chapter 4 4.1 4.2 4.3 4.4 Chapter 5  vii 1  General Materials and Methods: Magnetic resonance imaging (MRI) and spectroscopy (MRS) of northern elephant seals Experimental equipment and diving protocol Animals - Capture, handling and release Protocol Research and Development Extradural intravertebral vein (EIV): Structure and blood flow Aortic bulb dimensions and descending aortic flow rates Peripheral blood flow quantification: Renal artery Organ imaging  21 22 27 28 31 33 34 36  The spleen and hepatic sinus during forced diving in northern elephant seal pups Introduction Methodology Results Discussion  38 38 46 49 54  The effect of forced diving on cardiac function in northern elephant seal pups Introduction Methodology Results Discussion  62 62 67 70 73  5.1 5.2 5.3 5.4  Phosphocreatine flux in the locomotor muscle of diving northern elephant seal pups Introduction Methodology Results Discussion  82 82 89 93 104  Chapter6  Conclusion  Ill  Literature Cited  116  iii  LIST O F T A B L E S Table 3.1.  Northern elephant seal pup splenic volume during rest and diving obtained from in vivo organ assessment using MRI. Mass of individuals (kg) and mass-specific splenic volumes (ml) are also provided  Table 4.1.  60  Comparison of literature values documenting pinniped cardiac output (1 min" ) and stroke volume (ml) during predive and diving 1  state Table 4.2.  65  Average heart rate (HR), stroke volume (SV) and cardiac output (CO) measurements during the predive, dive and postdive state in northern elephant seal pups (n = 4)  Table 5.1.  71  Heart rate (beats min' ) during the predive, dive and postdive state 1  was obtained during P M R S analysis of northern elephant seal 31  locomotor muscle. Values are the average of all diving trials for each individual (n=6). A l l seals were subjected to 5 dives with the exception of Montague (3 dives) Table 5.2.  94  Northern elephant seal pup respiratory frequency and amplitude during forced diving. Values for predive, dive and postdive state are the average of all diving trials for each individual (n = 6). A l l seals were subjected to 5 dives with the exception of Montague (3 dives)  Table 5.3.  95  Blood lactate (mmol/1) and haematocrit values obtained from northern elephant seal pups during 8 minute forced dives (n = 5)  97  31  Table 5.4.  Locomotor muscle P values obtained from northern elephant seals over the course of a dive (expressed as a % of resting level). Values are the average of all diving trials for each individual (n = 7; Montague - 3 dives, all other seals - 5 dives)  99  LIST O F FIGURES Figure 1.1. A model of the contribution of phosphocreatine to buffering capacity within the cell  18  Figure 2.1. Northern elephant seal in acclimation phase with plexiglas diving helmet in place  22  Figure 2.2. Northern elephant seal pup in the bore of the magnet during a forced dive Figure 2.3. Restraining system for imaging northern elephant seals  23 25  Figure 2.4. E C G leads and respiratory bellows placement for monitoring northern elephant seals during M R imaging Figure 2.5. Extradural intravertebral vein locator sequence and flow image  26 32  Figure 2.6. Thoracic M R images of a northern elephant seal showing the location of the descending aorta (A - sagittal image; B - axial image)  33  Figure 2.7. Descending aortic blood flow (ml) over a cardiac cycle in a human and a northern elephant seal pup  34  Figure 2.8. Blood flow through the renal artery during rest and diving in a northern elephant seal pup demonstrates cessation of flow during the dive  35  Figure 3.1. Sagittal and axial images of the spleen of a northern elephant seal pup  47  Figure 3.2. Haematocrit values obtained from northern elephant seal pups during forced diving (n=3)  50  Figure 3.3. Lumbar (a) and thoracic (b) images of a northern elephant seal during rest and diving  51  Figure 3.4. Northern elephant seal spleen volume during rest (Min 0) and diving (Min 1-7) was obtained using M R imaging techniques  52  Figure 3.5. Graph of an individual northern elephant seal's splenic volume and hepatic sinus volume at each minute of a 7 minute dive  53  Figure 3.6. The hepatic sinus of a northern elephant seal dilates rapidly upon submergence and remains dilated throughout the dive  ,  56  Figure 4.1. M R axial images of a northern elephant seal pup during cardiac phase contrast data acquisition  69  Figure 4.2. Stroke volume data from an individual northern elephant seal during the predive, dive and postdive periods  72  Figure 4.3. Diving SV correlates with the degree of bradycardia expressed during the dive (ratio of mean diving H R to mean dive HR)  76  Figure 5.1. Coronal image of the lumbar region of a northern elephant seal pup  90  Figure 5.2. Muscle PCr values from an individual northern elephant seal during the dive and postdive period  100  Figure 5.3. Representative P MRS data obtained from the locomotory 31  muscle of an individual northern elephant seal during forced diving  101  Figure 5.4. Phosphocreatine signal intensity correlates with intracellular pH in the locomotor muscle of northern elephant seal pups  102  Figure 5.5. End dive PCr and respiratory rate correlations for predive and postdive Minute 2 collected during forced diving  103  ACKNOWLEDGEMENTS Research is seldom a solitary event, and this thesis holds no exception to the rule. Over the course of my degree, I have been continually inspired by the enthusiasm and philosophies of my mentor, Dr. Peter Hochachka. His fascination with science and ability to balance research with the finer things in life exemplify success at all levels. Thank you for your inspiration and support. Through his efforts, I have been privileged to work with a great number of scientists. Collaboration with the University of California, Santa Cruz introduced me to Dr. Burney Le Boeuf, who gave me access to the wonderful world of elephant seals. Dr. Dan Costa provided invaluable logistic and scientific support, supplying equipment, lab space and, when needed, a roof overhead. This study could not have been completed without the significant generosity of graduate students at U C Santa Cruz, who would work endlessly into the wee hours in exchange for pizza. Special thanks to Dan Crocker, for teaching me to stay away from the pointy end of a pinniped, Dorian Houser, for his constant and unwavering willingness to haul, Suzanne Kohin, for showing me how to think like a seal, and Javier Janz, who provided constant assistance and companionship over the years. Thank you to Paul Webb, Sean Hayes, and the many volunteers who all endured significant periods of sleep deprivation in the name of science. My Santa Cruz experience was enhanced considerably by the crew of the SV Talofa, who provided me with friendship, food, a berth to sleep in and the gentle sound of the sea outside my porthole. Fair winds my friends. This study was made possible through the generosity of the Lucas Center for Advanced M R Technology at Stanford University, C A . The knowledge and expertise provided by Drs. Norbert Pelc, Daniel Spielman and Wally Block was exceeded only by their tolerance for marathon magnet sessions, large smelly elephant seals and sleep deprived, equally smelly graduate students. Dr. Thomas Brosnan deserves special mention for his efforts in assisting in flow data analysis. On the U B C physics front, I am deeply indebted to Dr. Frank Linseisen, whose ability to decipher the physics of M R S and translate them into a comprehensible language was crucial to the completion of this thesis, and to Dr. Ken Whittall, for his significant assistance in data translation and transfer. The members of my committee must be commended for their various contributions to the development of this thesis (and for their tolerance for my "work in progress"): thank you to Dr. David Jones, for your considerable foresight and experience; Dr. Bill Milsom, whose impeccable editing skills and clarity of thought kept me on track (and on schedule!); Dr.  vii  Alex MacKay, for your unfailing enthusiasm and willingness to deal with my inability to comprehend K space; and to Dr. Andrew Trites, for introducing me to the world of pinnipeds and keeping me grounded in the world of whole animals. The journey through graduate school was not made alone. M y fellow comrades in education contributed significantly, both personally and intellectually, to the experience. On the, intolerable road to Bamfield, Steve Land regaled me with tales from the Hochachka lab and encouraged me to explore the opportunity. Gary (Fat Boy) Burness and Grant (Rat Boy) McClelland were constant sources of companionship through the range of events, both personal and professional, that encompassed the development of this thesis. Thank you both for being great friends and for not throwing me out the window when times were trying. Special thanks to Gunna Weingartner, my phocid wrestling partner and Withers cipher. Over the course of my degree, I have had the pleasure of interacting with fellow Hochachka-ites Petra Mottishaw, Mark Trump, Jim Rupert, Kevin (Muskrat Boy) Campbell, Charles (Chas) Darveau and Cheryl Beatty, who all have provided me with tremendous intellectual stimulation and significant comic relief over the years. It is a long climb to reach the shoulders of giants, and I could not have made the ascent iwithout the unfailing support of my family and friends. I extend my heartfelt gratitude to Scott Bell, for his tolerance, love, support, and did I mention his tolerance? During the chaos that commonly accompanies forays through the academic jungle, his constant encouragement and faith served as lifelines back to the real world.  viii  'Begin at the beginning' the King said, gravely, 'and go on till you come to the end: then stop.' Lewis Carroll ix  Chapter 1  General Introduction: Discussion of northern elephant seals and the physiology of diving The ability of aquatic organisms to sustain life in the absence of frequent breathing has captured the interest of seagoing humans for millennia. Throughout the logs of the early sailors and explorers we find evidence of mankind's insatiable fascination with diving mammals. As our knowledge in the field of diving physiology progresses, we begin the long road toward understanding the mechanisms by which these animals are able to survive extended submergence.  The field of comparative physiology is based on investigation into the ability of one species to do something another cannot. This system of comparison allows us to elucidate the mechanisms by which organisms react to their environment at a whole animal, tissue, and cellular level. As air breathing mammals, we exhibit an absolute dependence on oxygen for the maintenance of cellular function. Indeed, our definition of death is based on the inability to revive tissues from extended hypoxia and resulting cellular damage. Investigation into the mechanisms employed by marine mammals during breathhold diving may provide clues toward surviving periods of reduced oxygen availability. For this purpose, we have found no better candidate for study than the northern elephant seal, Mirounga angustirostris.  Elephant seals are the largest members of the pinniped family, with males weighing up to 3,700 kg (Ling, 1981). The genus Mirounga is divided into two species, with the southern elephant seal (M. leonina) inhabiting a broad range of latitudes in the southern circumpolar region. The northern species (Af. angustirostris) has a more limited breeding range, with  l  range, with the vast majority of rookeries found along the coastline of California (Radford et al, 1965; Stewart et al, 1994). The proximity of these rookeries to populated areas has resulted in a plethora of research on the behaviour and ecology exhibited in the terrestrial portion of their life history. Over the last three decades, the development of remote telemetry devices has provided scientists with an opportunity to gain significant insight into the natural world of marine mammals. Through the application of time-depth recorders (TDRs) we have been privileged to accompany these animals on their diving bouts. The resulting data have provided a tremendous amount of information on the diving behaviour and ecology of these species. Through the analysis of such data we are able to infer that, even within the world of pinnipeds, the elephant seal's diving ability may be considered exceptional (DeLong and Stewart, 1991; Le Boeuf et al, 1996; Andrews, 1999). When at sea, these animals spend 80-95% of their time submerged (Le Boeuf et al, 1988; Hindell et al, 1992). They follow a pattern of long, deep, continuous dives interspersed with brief surface intervals (1-3 minutes) (Le Boeuf, 1993) and have been recorded to dive for up to 2 hours and to depths of 1.5 km (Stewart and DeLong, 1995). The extraordinary breathhold ability of the northern elephant seal provides us with a unique opportunity to study the physiological mechanisms employed for survival in the undersea world.  The selection of a study species is dependent not only on its physiological characteristics, but also on its availability to the researcher. The life history of the elephant seal includes biannual haulout periods, providing ease of access to the animals. Haulout sites and rookeries consist of sloping sandy beaches and spits, with dominant individuals selecting areas of fine sandy substrate over pebbles or rock. The shores of Afio Nuevo, California offer these characteristics and consequently are home to a small peripheral colony consisting of migrants from larger rookeries in southern California. In addition to the continued flux of immigrants, Afio Nuevo sees the birth of approximately 2,000 pups each year (Le Boeuf and Reiter, 1991). The rookery is in close proximity to research facilities at  2  University of California Santa Cruz (UCSC) Long Marine Laboratory and the Lucas Center for Advanced Magnetic Resonance (MR) Technology at Stanford University, the site of our experiments.  The annual cycle of northern elephant seals at Ano Nuevo is typical of northern hemisphere populations. In December, the arrival of males at the rookery signifies the beginning of the breeding season. These bulls will compete for territory, staking out and defending areas that afford ease of access to incoming females (Le Boeuf and Laws, 1994). Within a month of the males' arrival, the beaches are littered with pregnant females who give birth shortly after landfall. The females remain at the rookery for approximately one month, nursing the pups for 24 to 28 days. Once the females have terminated lactation, they leave their pups behind and return to the sea (Le Boeuf et al, 1994).  The pups remain on the beach for 2-3 months after the departure of their mothers, living off the ample blubber stores afforded by the high fat milk diet of their first month of life (Reiter et al, 1978). They undertake their first foraging trip at 4-5 months of age, where mortality will claim over 50% of the cohort. The animals who survive the initial post-weaning foraging trip will then return to the rookery and haul out for approximately one month before returning to the sea. Juveniles repeat the pattern of five month foraging and one month haul-out until puberty is reached at approximately three years of age (Le Boeuf and Laws, 1994). The life history of the species provides us with an opportunity to access 4-5 month old pups in May, who are preparing for departure to sea, and 9-10 month old pups in October after they have completed their first bout of foraging. In general, the yearlings are less aggressive and more manageable than animals from other cohorts. Although 4 month old pups sampled in the spring have a higher percentage of body fat and lower muscle mass than 9 month old pups sampled in the fall, the overall mass is approximately equal (4 month old 96 kg ± 13 SD; 8 month old 107.2 kg ± 16 SD; Le Boeuf et al, 1996).  3  The equipment selected for the experimental protocols outlined in this thesis will not accept animals over 125 kg, therefore selection of individuals is limited to animals in their first year of life.  At weaning, the pups are effectively cut off from any further nourishment and enter a lengthy post weaning fast. It is believed this fasting period is required in order to develop the skills needed to undertake a long migration to the feeding grounds (Thorson and Le Boeuf, 1994; Thorson 1993). The pups first enter the water at about six weeks of age, learning to swim and dive in the shallows near the rookery. The fraction of time spent in the water rapidly increases from 1.9 to 52.2% over the course of the fast. Blood and muscle oxygen stores rise dramatically (20 and 40% respectively) and mean dive duration increases three fold from 1.9 to 6.1 minutes (Thorson, 1993). The diving pattern of a weanling during its first trip to sea is similar to that of an adult elephant seal (Thorson and Le Boeuf, 1994) and exceeds the depth and duration of most other adult pinniped species (Le Boeuf et al, 1996; Kooyman, 1989).  The rapid development in diving ability exhibited by these pups may be necessary for both initial foraging success and for predator avoidance during the ensuing sojourn to the feeding grounds (Thorson and Le Boeuf, 1994). Predation by white sharks (Carcharodon carcharias) is observed to occur most often within 450 m of shore, with a decrease in attack frequency observed with increasing depth (Le Boeuf and Crocker, 1996; Klimley et al, 1992). Individuals who are less capable divers will spend a greater amount of time in the upper water column and will consequently be exposed to a higher predation risk. The duration of time spent submerged and actively seeking prey is increased as diving ability advances, leading to a higher yield (catch per unit effort) and a greater overall foraging success. Hydrodynamics dictate that it is more efficient for a seal to travel underwater rather than at the air-water interface (Williams et al, 1991), therefore animals who surface  4  more frequently and for longer intervals may have a delayed arrival at the feeding grounds. As these animals have been fasting for 2-3 months and exhibit biochemical changes in blood parameters consistent with entry into phase 3 fasting (Castellini and Costa, 1990), any delay in arrival at the feeding ground may significantly decrease survivorship of an individual.  If we accept the arguments for increased diving ability leading to greater survivorship, we would expect that the principles of natural selection would act against animals who are inferior divers and would select for traits that support extended submergence. As the total age-specific mortality for this cohort is in the order of 60%, with the majority of first year mortality occurring at sea (Le Boeuf et al, 1994; Le Boeuf et al, 1996), it is plausible to assume that inferior diving ability contributes to mortality during the migration. Traits that contribute to survivorship during the first year of life will be actively selected for in a Darwinian fashion, resulting in a strong selective pressure favoring diving ability and hypoxia tolerance. However, the task of separating out the individual physiological traits that contribute to the diving response is quite difficult, as the ability to perform extended dives is based on a myriad of physiological events all working in conceit rather than a single feature. As many of the physiological responses that occur during diving are related to the physiology of oxygen delivery, a closer look at the distribution of oxygen reserves is required.  It is now widely accepted that the diving ability of an organism (in terms of submergence duration) is dependent on i) the volume of accessible oxygen reserves, and ii) the rate at which these reserves are depleted during the dive (Butler and Jones, 1997). The rise in oxygen storage capacity observed in elephant seal pups during their post-weaning fast is characteristic of all pinnipeds and crucial to their development as divers. Diving mammals exhibit higher haemoglobin concentration, elevated blood volume, and greater quantity of  5  muscle myoglobin than terrestrial animals, resulting in a greater mass-specific oxygen storage capacity (Kooyman, 1985). The rise in mass-specific blood volume continues to increase through to adulthood, resulting in a continued elevation of oxygen storage and correlating with increases in diving depth and duration (Le Boeuf et al, 1996). If these young pups were forced to leave the beach prior to the period of time required for these changes to occur, their diving ability would be seriously impaired and survival would likely be compromised.  The total blood volume exhibited by pinnipeds is in the range of 15% of body mass (human blood volume is - 5 - 7 % of body mass) and represents the largest depot of oxygen storage available during the dive (Kooyman, 1985; 1989). In phocids, the proportion of red blood cells ( R B C ) in whole blood gives rise to some of the highest haematocrits recorded in mammals (Lenfant, 1969). In addition to an elevated haematocrit, each corpuscle contains a high concentration of haemoglobin which serves to further increase the oxygen content of the blood (Lenfant et al, 1970). Although increased haematological values augment diving duration and ability by elevating blood oxygen storage capacity, the advantages do not come without a potential cost (Hedrick and Duffield, 1991). A rise in haematocrit is normally accompanied by an elevation in blood viscosity, leading to potential perfusion impairment, R B C damage, vascular stress and increased cost of blood transport (Castle and Jandl, 1966; Wickham et al, 1989). Whether these animals are impervious to the effects of viscosity or have developed mechanisms for coping with the deleterious effects of perfusion impairment has been a recent topic of discussion in the literature (Meiselman, 1983; Hedrick et al, 1986; Hedrick and Duffield, 1991; Meiselman et al, 1992). The variable nature of pinniped haematocrit may provide some insight into the situation.  During diving or apnea, a significant and rapid increase in circulating red blood cells occurs (Kooyman et al, 1980; Castellini et al, 1986; Qvist et al, 1986; Castellini et al, 1988;  6  Castellini and Castellini, 1993). The observed rise in haematocrit suggests that the total red cell mass is not always in circulation, leading researchers to speculate on the means of R B C partitioning (Qvist et al, 1986; Castellini et al, 1988: Zapol et al, 1989; Eisner and Meiselman, 1995). It is widely suspected that the source of the sequestered red blood cells is the spleen (Qvist et al, 1986; Zapol et al, 1989; Bryden and Lim, 1969; Ponganis et al, 1992). The histology and significant size of the phocid spleen implicates it as the likely site for red blood cell (RBC) storage. Anatomically, seals have the largest reported spleen-tobody-weight ratio of any mammal (Qvist et al, 1986). Histological studies indicate that phocid spleens are capable of sequestering significant quantities of erythrocytes and possess contractile properties in both the smooth muscle capsule and the fibroblastic reticulum cells (Schumacher and Welsch, 1987). The anatomical and histological data support the suggestion that the spleen acts as a " S C U B A tank", carrying oxygen bound to red blood cells stored in the spleen and slowly metering them out as the dive progresses (Qvist et al, 1986; Hurford et al, 1996). The design of this organ is such that it would be plausible to envision a large volume of red blood cells stored in the spleen and a subsequent stimulus for contraction causing release of RBCs into the general circulation, resulting in the observed rise in circulating haematocrit.  The importance of the role of the spleen in phocid diving is also supported by phylogenetic studies indicating that relative spleen weight correlates with diving duration (Hochachka and Mottishaw, 1998; Mottishaw et al, 1999). Species with larger spleens are capable of undertaking longer dives, providing strong evidence for the S C U B A tank hypothesis. However, recent proposals have suggested that the primary function of the spleen may not be during the dive per se (Ponganis et al, 1992), but instead its importance may be realized in the postdive period, where sequestration of RBCs would serve to reduce haematocrit and blood viscosity (Castellini and Castellini, 1993; Eisner and Meiselman, 1995). By utilizing the spleen to store RBCs when the seal is not diving, a marked reduction in seal blood  7  viscosity would be realized. Clearly, the function of the spleen in a diving animal requires further investigation. Magnetic resonance imaging (MRI) techniques provide an excellent opportunity for direct observation of the spleen during diving. This thesis set out to quantify splenic volume during the predive, dive and postdive state and correlate these changes with the observed diving-induced rise in haematocrit that is known to occur in phocid seals.  The notable increase in phocid oxygen stores partly explains the exceptional breathhold ability of phocids; however it is the distribution of these stores during diving that is thought to be the cornerstone of the mammalian diving response. In his classical work of 1940, Scholander demonstrated that in many different species of animals, facial immersion is accompanied by an immediate and profound bradycardia and peripheral vasoconstriction (Scholander, 1940). This particular circulatory adjustment allows blood oxygen to be preferentially consumed by the heart and brain, while the hypoxia-tolerant tissues in the periphery are dependent mainly on local oxygen stores, gradually accumulating end products of anaerobic metabolism as local oxygen supplies diminish (Irving, 1934, 1938, 1939). Once the animal surfaces and vasodilation occurs, metabolic end-products formed in ischaemic tissues are "washed out" into the general circulation. In these original studies, Scholander (1940) provided indirect evidence for peripheral vasoconstriction by noting that lactate levels remained low throughout the dive, but rose significantly in the postdive period (as would be expected to occur once circulation to the periphery was resumed). Since the pioneering work of Irving and Scholander, laboratory studies have verified that forced diving stimulates a profound drop in heart rate accompanied by a reduction in perfusion in all tissues except for the brain, heart, lungs and adrenal glands (Bron et al, 1966; Kerem and Eisner, 1973; Zapol et al, 1979; Blix et al, 1976; Hill et al, 1987).  8  Further research on diving mammals reported variations in the degree of bradycardia exhibited during a dive, suggesting considerable plasticity in the response to facial immersion. In early laboratory investigations, forced diving techniques elicited a dramatic and profound reductions in heart rate. When forcibly submerged, many species of seals immediately reduce their heart rate into the 10 - 20 beats per minute (bpm) range. Through advances in microcomputer technology, the ability to acquire heart rate remotely led to a plethora of data from freely diving animals in their natural environment (Guppy et al, 1986; Hill et al, 1987). The most striking difference between field and laboratory data was that although animals in the field occasionally exhibit instantaneous heart rates in the 2-4 bpm range, overall diving heart rates are considerably higher during voluntary diving than those recorded during forced dive experiments (Murdaugh et al, 1961; Eisner, 1965; Fedak, 1986; Jones et al, 1973; Kooyman and Campbell, 1972). With the advent of remote heart rate telemetry equipment, we now have a significant quantity of research from the field showing that phocid species respond to voluntary diving with a more moderate drop in overall heart rate, usually in the range of 30 - 50 bpm (Kooyman and Campbell, 1972; Kooyman et al, 1980; Guppy et al, 1986; Hill et al, 1987; Fedak et al, 1987; Andrews et al, 1997).  It has often been postulated that the stress and restraint associated with forced diving experiments may cause the catecholamine component to be elevated above levels observed during free dives, thus producing the greatest degree of the diving response (Kooyman et al, 1980; Lacombe and Jones, 1991; Robin et al, 1981). Other researchers suggest a conditioning effect, indicating that diving animals may have the ability to modulate their heart rate in response to the length and type of dive they are performing (Jones et al, 1973; McCulloch and Jones, 1990). In a forced dive situation, the animal is unaware of the dive duration and may respond with a maximum physiological response geared toward surviving a potentially lengthy period of submersion. Another possible explanation for the  9  higher heart rates observed in freely diving seals is the increase in oxygen demand resulting N  from the action of locomotory muscles (Williams et al, 1991). There are many studies confirming the difference in bradycardic response to free and forced dives, but the control mechanisms causing the discrepancy remain relatively elusive. However, the importance of bradycardia in facilitating extended diving is undisputed. When bradycardia is abolished or prevented from occurring in a timely fashion, a reduction in survival time during diving is observed (Irving et al, 1942; Murdaugh et al, 1961). Although it appears that bradycardia is a fundamental reflex required for survival during diving, it is not specific to diving animals. The universality of this trait strongly suggests that heart rate reduction is used by many species of air breathing animals during threat of asphyxia. As such, the bradycardic reflex that occurs during diving is considered to be a component of a more general asphyxia response rather than an adaptation specific to the act of diving (Scholander et al, 1942; Eisner and Gooden, 1983; Eisner, 1989). Through the use of phylogenetically independent contrast (PIC) analysis, the bradycardic response to diving was identified as an ancestral condition rather than an adaptation to diving (Mottishaw et al, 1999). To fully appreciate the physiological effect of bradycardia on a diving organism, the mechanistic repercussions of reducing heart rate must be evaluated. When an animal is faced with a decline in heart rate, an increase in stroke volume may occur in order to maintain sufficient cardiac output to meet the oxygen delivery needs of the body. This compensatory mechanism is dictated by the following equation:  Equation 1:  where  Q,, =f  h  x V  h  Q/i  = cardiac output in mis blood/minute  fh  = heart rate in beats/ minute  Vfc  = stroke volume in mis/cardiac cycle  10  In the case of a diving seal, the degree of bradycardia is often too profound to allow for maintenance of cardiac output through the compensatory mechanism of increasing stroke volume. In fact, studies measuring CO in diving pinnipeds indicate that a decrease in S V accompanies diving bradycardia (Sinnett et al, 1978; Zapol et al, 1979; Blix et al, 1983; Ponganis et al, 1990). However, other studies report a reduction in pinniped CO that is of the same magnitude as the decrease in heart rate (Eisner et al, 1964; Murdaugh et al, 1966; Blix et al, 1976), indicating that stroke volume remains stable between the predive and diving state.  A diving animal's cardiac response to facial immersion is driven by stimulation of the trigeminal nerve leading to an almost instantaneous vagal response and reduction in heart rate (Jones, 1992; Daly et al, 1977; Eisner et al, 1977; Daly et al, 1980). Immediate circulatory adjustments are required to prevent a drop in systemic blood pressure normally associated with such a profound reduction in cardiac output (Irving et al, 1942). A significant reduction in blood pressure may severely impair the body's ability to deliver a sufficient quantity of oxygen to the brain, leading to loss of consciousness and eventually, the demise of cellular function. The maintenance of blood pressure in the face of declining cardiac output is achieved through an increase in total peripheral resistance as described in the following equation:  Q = MABP/TPR  Equation 2:  where  h  Qj,  = cardiac output  MABP = mean arterial blood pressure TPR  = total peripheral resistance  11  The resulting adjustment in perfusion and redistribution of cardiac output is thought to be the centerpiece of a mammal's response to diving. Through the combination of increased oxygen stores and prudent regulation of oxygen delivery, these animals are able to sustain periods of apnea and submergence far beyond that of a terrestrial animal.  The mechanistic relationship described in equations 1 and 2 suggest that the physiological events that occur during diving may be elucidated from the determination of heart rate. However, the system is a dynamic one, responding continuously to the changing conditions within the organism. Although much effort has gone toward establishing a correlation between the degree of bradycardia and diving ability, metabolic rate, or blood flow distribution, to date no definitive relationships have emerged (Eisner, 1965; Butler, 1993; Webb, 1994; Andrews, 1999; Mottishaw et al, 1999). In fact, the major conclusion reached by most researchers in the field of diving physiology is that the bradycardic response is considerably more variable than first realized, and extrapolation of heart rate data to other physiological events must be approached with caution.  One of the primary difficulties in interpreting HR data is the lack of detailed information on the relationship between HR and CO. At present, the effect of diving on phocid SV is far from clear. Noninvasive evaluation of SV and C O would provide a stronger physiological basis for the interpretation of diving HRs. As a second major goal, this thesis set out to investigate the inotropic effects of facial immersion using MRI phase contrast analysis techniques. A detailed evaluation of HR and SV in elephant seals during the predive, dive and postdive period is proposed.  The presence of a significant body of knowledge detailing the mechanisms of increased oxygen stores and the physiology of cardiac output redistribution still does not explain how a diving animal can extend its onboard oxygen stores beyond what would normally be  12  consumed during a resting period of equal duration. An estimate of the duration of time an animal would be able to sustain aerobic metabolic function during diving may be achieved by calculating the total amount of accessible body oxygen stores and the rate at which it is consumed (Irving, 1939). Once a tissue has consumed all available oxygen, it must rely on anaerobic metabolism as indicated by the presence of elevated blood lactate levels. This observation resulted in the development of the "aerobic dive limit" (ADL) concept, a term used to indicate the duration of maximum breathhold that an animal could endure without producing a significant elevation in blood lactate concentration during or after the dive (Kooyman, 1980,1983). An estimate of the actual A D L could be achieved by dividing total body oxygen stores by the diving metabolic rate (Kooyman, 1983, 1985). However, by using a resting metabolic rate to calculate the A D L of a Weddell seal (approximately 16 minutes) and then comparing it to actual ADLs, where no significant blood lactate appeared until dives exceeded 20 minutes in duration, a discrepancy was revealed (Kooyman, 1980). These findings indicated that the animals were able to dive beyond their calculated A D L , suggesting that the diving metabolic rate fell below resting values. Differences between calculated and actual ADLs have been documented by other researchers, suggesting that the metabolic rate of a seal is lowered during diving (Castellini et al, 1992; Ponganis et al, 1993).  One of the most persuasive arguments for the existence of hypometabolism comes from Scholander's (1940) seal research involving post-dive excess oxygen consumption and subsequent calculation of a diving metabolic rate. During a quiescent dive, the difference in oxygen consumption between pre-dive and post-dive periods does not account for an elevated metabolic rate during the dive. If the animal struggled during the dive, the amount of energy expended was estimated to be several times greater than that of the pre-dive period, but was not reflected by an equal increase in excess post-dive oxygen consumption. In fact, no significant difference was noted between the postdive oxygen consumption from  13  a tranquil dive and one with considerable struggling (Scholander, 1940). If we accept the arguments for hypometabolism in a diving animal, we are faced with the challenging task of defining the mechanisms by which these animals are able to downregulate their energy needs.  It has been suggested that the reduction in oxygen delivery experienced by the peripheral tissues in a diving animal may be responsible for a drop in overall energy consumption. The most compelling evidence for the existence of hypometabolism by this mechanism comes from studies on terrestrial animals showing a linear decrease in oxygen consumption with decreasing oxygen delivery (Idstrom et al, 1985; Wagner, 1995; Hogan, 1996). When the energy consumption of a cell is increased or decreased, a rapid change in adensosine triphosphate (ATP) production is needed to maintain cellular ATP concentrations. Blood flow varies in relation to the need for aerobic metabolism in the cell, and this relationship holds true over a wide range of activity, suggesting that reduced perfusion results in an overall suppression of metabolism at both the tissue and whole animal level. In the seal, systemic vasoconstriction serves to decrease blood flow to many tissues and restrict access to blood-bound oxygen in the general circulation. By reducing the body's overall need for energy, more oxygen remains in the central circulation for delivery to hypoxia sensitive tissues like the brain and heart. It is likely, albeit difficult to demonstrate, that seals experience a significant reduction in overall metabolic rate related to peripheral vasoconstriction.  Through the measured reduction in heart rate and subsequent drop in cardiac output we are able to infer an increase in total peripheral resistance, but we do not have sufficient information to support definite conclusions as to which tissues are experiencing ischaemia at any given time during the dive. As demonstrated by Zapol et al (1979), Weddell seals are capable of profound peripheral vasoconstriction during forced dives, with a greater than  14  90% reduction in blood flow to the splanchnic and peripheral vascular bed. It is plausible to suggest that peripheral tissues may experience some degree of pulsatile flow, the existence of which would not be clearly illustrated through common perfusion measurement techniques, such as microspheres or flow transducer implantation on large arteries (Zapol et al, 1979; Blix et al, 1983; Cherepanova et al, 1993). Using LaserDoppler flowmetry, Ponganis et al (1999) evaluated muscle blood flow (MBF) during 8-12 minute spontaneous apneas in unrestrained northern elephant seals. Apneic M B F decreased progressively, averaging 52% of eupneic blood flow. Occasional transient increases in flow were noted, suggesting that peripheral vasoconstriction is not constant. However, little is known about the degree of peripheral vasoconstriction experienced by a freely diving animal.  In freely diving animals, higher diving HRs are likely accompanied by a less profound increase in total peripheral vasoconstriction (Equation 2). This concept is supported by Near Infrared Spectrophotometry (NIR) experiments conducted on freely diving Weddell seals, which indicate that end-dive myoglobin saturation was typically 40-60% of resting, even when the dive exceeded the A D L (-17 minutes). Phocid muscle is rich in myoglobin, containing approximately ten times the amount found in human muscle (Kooyman, 1981). As myoglobin's affinity for oxygen is approximately 10 fold higher than that of haemoglobin, the monotonic decline in myoglobin saturation indicates that oxygen delivery to the muscle was not sufficient to maintain 100% saturation of myoglobin, and therefore was not sufficient to meet the metabolic needs of the muscle tissue. However, the rate of desaturation is not consistent with a situation of total ischaemia, where desaturation of locomotory muscle myoglobin should proceed at ~ 20%/minute (Castellini et al, 1992). These findings strongly suggest that some degree of hypoperfusion occurs in freely diving animals and that ischaemia tolerance would be a necessary component of the physiological response to diving.  15  In most vertebrates, the response to ischaemia is an obligatory decrease in aerobic metabolism and a compensatory increase in anaerobic energy production, commonly referred to as the Pasteur effect. This attempt to maintain aerobic rates of A T P turnover results in the production of lactate and requires that the organism have sufficient glucose/glycogen supply to maintain anaerobic glycolysis. However, some animals exhibit a reverse Pasteur effect, whereby tissue ischaemia results in a decrease or maintenance in glycolytic flux (Hochachka and Guppy, 1987). The suppression of anaerobic metabolism combined with overall metabolic downregulation would certainly be a beneficial mechanism for a tissue subjected to frequent ischaemia, but at present we have no firm evidence that this situation occurs in seal muscle during diving. High levels of muscle buffering capacity are usually associated with burst locomotion capabilities or poor capillary circulation, increasing the need for anaerobic metabolic pathways. Although the extensive buffering capacity of phocid muscle tissue has been identified, the degree of anaerobic metabolic activity during ischaemia has not been established (Castellini and Somero, 1981).  When a muscle bed is subjected to hypoperfusion, it has a number of potential biochemical options. It may react with a reduction in ATP consumption resulting in a reduced demand for ATP production and an overall decrease of oxygen consumption. Information as to the rate of perfusion and myoglobin desaturation would provide valuable insight into the aerobic metabolism in seal muscle. A n increase in flux through anaerobic pathways may also occur; however the dynamics of lactate production and metabolism in a diving animal are complex and are not completely understood (Davis, 1983; Castellini et al, 1988; Davis et al, 1991). Another potential anaerobic source of ATP production in muscle is the transfer of a high energy phosphate molecule from phosphocreatine to ADP. Creatine phosphokinase (CPK) in the cytosol catalyses the rapid transfer of a high energy phosphate group from phosphocreatine (PCr) to A D P (Lohman reaction):  16  PCr  2  + MgADP + H  +  o  Cr + MgATP  2  During changes in metabolic demand, cellular A T P concentration may be initially buffered by the creatine kinase system. The breakdown of ATP leads to a rapid reduction in cellular PCr and subsequent resynthesis of ATP. When net high energy phosphate hydrolysis occurs, the PCr pool is depleted to a greater extent than the ATP pool, indicating that phosphocreatine acts as a readily available high energy phosphate store.  To date, two studies examining the role of PCr in diving animals have been conducted. In Pekin ducks (Anas platyrhynchos), forced dives of 6.5 ± 0.3 min resulted in an end-dive PCr value indicating a -22% depletion of total PCr stores (Stephenson and Jones, 1992). These animals also exhibited a significant post dive plasma lactate concentration, peaking at -3-6 minutes postdive at a level of 7.6 + 0.3 mmol/1 above predive levels, indicating simultaneous employment of different anaerobic pathways. The authors conclude that, based on the measured rate of diving ATP consumption and the quantity of PCr remaining, diving could be sustained for another 27 minutes. However, the dynamics of the PCr reaction and the role it plays in maintaining ATP levels indicate that it is not merely a high energy phosphate store. Evaluation of muscle PCr must take the complex role of this metabolite into account when evaluating its role as an energy "stockpile" for use during diving. A second study was undertaken using harbor seals (Phoca vitulina). These animals were conditioned to tolerate periods of facial immersion ranging from 5-12 minutes while lying in a 1.5 Tesla M R magnet. The animals exhibited a PCr decline of -50% during the dive, with a full recovery to resting levels observed within 90 seconds of surfacing (T.M. Williams, unpub data). At present, the contribution of PCr to submergence duration in diving animals has not been fully elucidated.  17  The difficulty in assessing the role of PCr in diving seal energy metabolism is that the adenylate levels are not the only driving force behind PCr hydrolysis. The creatine kinase reaction may be shifted to the right by increases in both [ADP] and proton load. Substantial amounts of H are generated in an ischaemic muscle as the result of lactate +  dehydrogenase reaction, the hydrolysis of adenylates to IMP, and/or C 0 accumulation. 2  One of the principal buffers of muscle proton load is the intracellular phosphate (Pi) derived from both the breakdown of PCr and ATP. The stoichiometry of the reaction is such that cellular PCr could be significantly depleted purely through the effect of proton load, without any appreciable change in A T P flux (Arthur et al, 1997; Fig. 1.1).  ^  60  -j 1.0 - 0.8  40  r—i  " 0.6  O X  g <  0.4  20  | <  S 0.2 0 Figure 1.1.  A model of the contribution of phosphocreatine to buffering capacity within the cell. The concentration of phosphocreatine (PCr, filled circles) and the concentration of protons bound as H2PO4" (filled squares) are shown (left hand axis) as changes in intracellular pH (pHi) cause a shift in the creatine kinase equilibrium. The model indicates that the ratio of change in [H ] to the change in [PCr] (right hand axis) decreases from above 0.8 at pHi 6 to just below 0.2 at pHi 7.5 (open circles) (After Arthur et al, 1997). 1  +  In the ischaemic muscle of a diving seal, substantial myoglobin stores may continue to provide oxygen for oxidative phosphorylation throughout a short dive, negating the need for anaerobic metabolism and thus preventing the formation of lactic acid. However,  18  muscle hypoperfusion may lead to insufficient C 0 clearance and an associated increase in 2  intracellular proton concentration. The interplay between myoglobin, oxidative phosphorylation, lactate production and C 0 accumulation all serve to alter pH and 2  adenylate concentrations within the tissue. By examining the change in muscle [PCr] in a northern elephant seal, the relationship between PCr and muscle metabolism during diving may be elucidated. Through the use of M R Spectroscopy, I propose to measure high energy phosphate flux and pH in the locomotory muscle of a seal during aerobic dives and evaluate changes in muscle metabolism between the predive and forced dive condition.  Through the application of Magnetic Resonance Imaging (MRI) and Spectroscopy (MRS) technology, physiological events are recorded as they occur during the dive, rather than by inference from data collected in the predive and postdive state. In addition, a selected organ or region of tissue is evaluated in situ, providing a considerable increase in accuracy over more traditional methods of surgical implants, biopsy and assay techniques, where stress or disruption of cellular integrity and function occurs. However, as these techniques were developed to evaluate human anatomy and physiology, the difficulties associated with the application of M R technology to a diving seal had to be overcome. In order to achieve the three goals outlined above, significant modification of existing methodologies was required.  This document outlines the development of a methodology for imaging northern elephant seals in a whole body magnet, investigation into techniques and M R sequences that would provide usable images and accurate M R S data from seals, as well as some preliminary results from perfusion studies of diving elephant seal pups (Chapter 2). These preliminary studies revealed a vast potential for research into the mammalian diving response using M R techniques. Although rapid development in the field of medical imaging has resulted in well established protocols for the evaluation of internal organs, these techniques required  19  modification for use with northern elephant seals before acceptable results were obtained. Modified organ imaging sequences were employed to study the effects of forced diving on the spleen of a northern elephant seal (Chapter 3). Advances in phase contrast techniques have provided methods for the quantification of blood flow, allowing for investigation into the inotropic effects of facial immersion and a detailed evaluation of HR and SV in elephant seals during the predive, dive and postdive period (Chapter 4). Through the application of 31  P M R Spectroscopy, alteration in [PCr] and intracellular pH in the locomotory muscle of a diving seal were evaluated (Chapter 5).  20  Chapter 2  General Materials and Methods: Magnetic resonance imaging (MRI) and spectroscopy (MRS) of phocids during forced diving Introduction Clinical applications in the field of medical imaging have provided considerable impetus for accelerated research and development of MRI techniques. Over the last two decades, significant advances have been made in both the hardware and the sequences used to acquire images. Although MRS techniques are not commonly used for medical diagnostic purposes, spectroscopy will likely prove to be a valuable diagnostic tool as the field advances. From a research standpoint, the use of in vivo imaging and spectroscopy offers the advantage of being non-destructive, thereby reducing potential artifacts in the data caused by invasive analysis techniques. As there are no ill effects from experimental manipulation, each animal may act as its own control (Koretsky and Williams, 1992).  To evaluate physiological and biochemical changes that may occur during diving, northern elephant seals were subjected to forced dive protocols while undergoing M R imaging and spectroscopy analysis. The development of the experimental protocol involved designing a system of restraint that would allow the animal to undergo facial immersion in the magnet, yet still provide rapid access to the animal if interventions were required. To ensure the health and well being of the animal and also to provide ongoing physiological data, it was necessary to modify the configuration of E C G leads and respiratory bellows. In addition to the above constraints, the prevention of damage to the equipment resulting from the animal's movements or fluid leakage was of paramount importance. As most imaging sequences were developed for use in humans, some modification of the sequence parameters was required to accommodate the differences in body composition and heart rate exhibited by northern elephant seals.  21  Experimental Equipment and Diving Protocol All imaging and spectroscopy protocols were performed on a high performance 1.5T system (Signal Horizon Echo Speed, G E Medical Systems, Milwaukee, WI) located at the Lucas Center for Advanced M R Technology, Stanford University, California. Respiratory and E C G data were collected on a Macintosh ESI with a 8 channel Powerlab™ (ADInstruments) interface, M L 132 BioAmp and analysed using Chart™ (ADInstruments) software.  The use of forced diving protocols in the magnet required a system that would allow for complete facial immersion yet prevent water from entering the M R unit. To create an air and watertight environment, a Plexiglas helmet (35 cm in diameter) was manufactured to fit over the animal's head (Fig. 2.1).  Figure 2.1. Northern elephant seal in acclimation phase prior to imaging. The foam block situated in the upper margin of the helmet prevents the seal from raising its nostrils into the air pocket formed during exhalation. During acclimation, both valves are in the "open" position and a vacuum hose under the neck seal ensures adequate airflow through the helmet.  22  An inner neoprene seal was glued to the Plexiglas helmet and cut to the animal's neck circumference, and a secondary outer latex neck seal was attached to the helmet to prevent fluid leakage. Distal to the neck seal, two valves were installed in the helmet endplate to allow for rapid draining and flooding during experimental manipulation.  To reduce the risk of water damage from catastrophic failure of the helmet neck seals, a fluid containment unit was manufactured from a 54 cm diameter P V C sewer pipe. The structure was cut in half longitudinally and end caps were plastic welded to ensure a watertight seal (Fig. 2.2).  Figure 2.2.  Northern elephant seal pup in the bore of the magnet during a forced dive. The P V C half pipe prevents water from accessing the magnet and allows for further restraint of the animal during imaging. Positioning of the animal in the "feet first prone" position allowed for continuous visual monitoring of the animal through the plexiglass helmet and facilitated the use of E C G fibreoptic leads and respiratory bellows.  23  Four rope and tubing handles were installed along each side of the 175 cm half pipe and a 2 cm wastewater drain was inserted at the lower edge of the half pipe end wall. A conduit was installed above the wastewater drain to accommodate the helmet outflow valve and allow for placement of an external drain hose.  To assist in restraining the seal, a conical nylon jacket was designed and manufactured such that it would remain in place at the region of the neck caudal to the helmet neck seal and fully envelop the seal's thoracic region, thus restricting use of the foreflippers. A series of nylon webbing straps and buckles sewn on the outside of the jacket allowed for custom adjustment. A fiberglass backboard with a series of 5 cm nylon webbing straps was manufactured to further immobilize the seal and to facilitate transfer of the animal into the magnet (Fig. 2.3). The board was designed to sit inside the half pipe at a level that would allow the helmet to be placed on the animal's head when the half pipe was in position in the bore of the magnet.  Upon arrival at the M R unit, each seal was placed in a preparation room and allowed to leave the transport cage. The animal was manually restrained and rolled over to expose the ventral surface for E C G placement. Using electric clippers, four 3 cm circular areas approximating a 20 cm square surrounding the heart were shaved down to the skin and cleaned with Hibitane and water. To ensure maximum contact of the electrode with the animal's skin, the site was then shaved using a disposable razor. Each site was swabbed with alcohol wipe and allowed to dry before placing electrode conductance gel (Redux Paste, Hewlett Packard, Waltham, Mass.) on the site and then attaching the disposable G E E C G electrode with cyanoacrylate adhesive. Neoprene patches were positioned over the electrodes and were glued to the fur on three sides to prevent displacement of the lead during movement of the animal. The opening of the pocket was positioned on the animal's right side to allow the E C G leads to feed out of the half pipe. The restraint jacket was then  24  applied and the animal was strapped to the restraining board in a prone position. Once placed in the half-pipe, the E C G leads were connected to the electrodes and the strap of the respiratory bellows was threaded under the animal at the level of the diaphragm (Fig. 2.4).  Figure 2.3.  Restraining system for imaging northern elephant seals. A nylon jacket is placed over the animal's body and adjusted to restrict front flipper motion. The seal is then strapped to the restraining board and placed in the half pipe. 25  Figure 2.4.  E C G lead and respiratory bellows positioning for monitoring northern elephant seals during M R imaging. E C G leads are attached to electrodes on the ventral surface. A neoprene patch is glued over the lead to prevent dislodgment during imaging. The respiratory bellows were placed around the animal at the level of the diaphragm. Note that the bellows portion would actually lay across the dorsal surface of the animal.  26  Approximately 30 minutes prior to an experiment, the helmet was placed on the seal's head and secured to both the board and half pipe by means of webbing straps. A vacuum hose was attached to the helmet to ensure sufficient air flow through the open valves, and the animal was left alone for a brief period of acclimation. This also allowed time for the evaluation of E C G signal quality and respiratory bellows function prior to data acquisition.  Once acclimated, the seal was moved into position in the magnet for acquisition of predive scans. Diving protocol would begin with the removal of the vacuum hose from the helmet, closure of the drain valve and placement of a nylon cover sheet over the helmet to prevent spray in the event of a neck seal rupture. The helmet was then slowly flooded by means of gravity feed from a plastic water container, and a small hose was inserted under the neck seal to allow the air within the helmet to escape. By way of a microphone within the magnet, the initiation of the dive would be communicated to the M R physicist at the console and imaging would commence. Complete immersion of the nostrils was considered to be the start of the dive, and the animal's first breath as the helmet drained indicated the completion of the dive.  Animals - Capture, handling and release  Over a two year period, 16 animals were captured from the beaches of Afio Nuevo State Reserve, California (National Marine Fisheries Service Marine Mammal Permit # 7861463) and immediately transported to Long Marine Laboratory, Santa Cruz, CA. At the conclusion of each study, the animals were released at the site of capture. Method of capture depended on ease of accessibility to the selected individual. Preferred methodology was to locate a sleeping animal of appropriate age and condition that was relatively isolated from other individuals. The age class of the animal would be determined by a general assessment of size and body composition, coat quality and characteristics, and dental development. This assessment could often be accomplished while the animal slept,  27  including a brief examination of the canine development by a gentle retraction of the upper lip. If the animal was deemed suitable, the transport cage would be laid down in the sand next to the pup, and the animal would be rolled into the cage, often prior to awakening. If the selected pup was amidst a large group of seals, the warning calls and behaviour of surrounding individuals would prevent deployment of passive capture methods, instead requiring the use of chemical immobilization. Administration of an intramuscular injection of approximately 1 mg kg~l tiletamine HC1 and zolazepam HC1 (Telazol®, Aveco Co., Fort Dodge, IA) provided sufficient immobilization to facilitate transfer to the transport cage.  Upon arrival at Long Marine Laboratory, the animals were transferred to a holding facility with access to water of sufficient depth for diving. As the haulout period of these animals corresponds to their natural fast, they were not provided with food during the study. The animals were transported from the holding facility to the Lucas Center for Advanced M R Technology at Stanford University on each day of an M R experiment.  Protocol Research and Development  In April and October of 1996, one 4 month old male and three 9 month old female elephant seals were collected from Afio Nuevo, California and held at Long Marine Laboratory, UCSC. The animals were held for 27 days and 11 days respectively and were released at the site of capture at the conclusion of the experiment. These animals were used for research and development of M R I sequences to be used in the following studies.  The initial challenge was primarily logistical and involved simple placement of the animal in the magnet to evaluate the effectiveness of the restraining system and diving helmet. The bed limit of the magnet was designated at 150 kg, and clear access to the bed was available only from the front side of the unit. The 54 cm halfpipe was loaded onto the bed in the  28  preparation room, then transferred into the magnet area and inserted into the 55 cm bore of the magnet. Once adjusted, the half pipe was able to fit within the bore and would advance far enough into the magnet such that the region of interest (thoracic to abdominal region) could be placed in the center of the coil yet still allow for access to the animal's head. Animals over 125 kg were unsuitable as the combination of seal and equipment exceeded the weight limit of the bed, and the animal's back would touch the light tracks within the bore of the magnet.  Human imaging is normally conducted in a "head first, supine" position and therefore the magnet is designed for such an orientation. To allow for normal positioning of the seal as well as full access to the helmet, seals were placed in a "feet first, prone" position. This caused some difficulty with E C G lead placement leading to relocation of the leads from a dorsal position, where they were subjected to magnetic inhomogenieties causing signal interference, to a ventral site. The most reliable performance was obtained when the ventral E C G leads were affixed to four sites approximating a 20 cm square surrounding the heart. Neoprene patches were glued over the leads to prevent displacement during movement of the animal.  Imaging technology offered an opportunity to view the events that occur real time, in vivo during a dive. A number of preliminary studies were conducted to establish the suitability of M R analysis for the selected hypotheses. This allowed the physicists to evaluate which studies would yield the most accurate data and provided an opportunity to modify the sequences over the next few months before an actual study was undertaken. The majority of effort was concentrated on manipulating sequences for organ imaging to yield clear and precise delineation of internal structures. This presented a challenge as the sequences were designed for human use and therefore rely on the presence of adipose tissue in the viscera. In humans, organs are encapsulated in a layer of fat which provides a strong M R signal for  29  imaging. The anatomy of a seal is such that the vast majority of adipose tissue is stored in the blubber layer, resulting in limited visceral fat content and poor organ discrimination in the images. Once a suitable sequence for organ image enhancement was developed, the splenic study was undertaken (Chapter 3).  Flow quantification in vessels during the pre-dive and diving period was attempted. Standard flow analysis sequences and programs proved to be suitable for use in the northern elephant seal, allowing for identification of vessels and quantification of blood flow during diving and rest. However, significant alteration to the acquisition protocol for cardiac gating (images of the heart acquired at a given point in the cardiac cycle, usually the R wave) during diving was required. The factory-set parameters restricted gating sequences to heart rates above 30 beats per minute and would not function during bradycardic sequences exhibited by a diving seal. Once the restriction on cardiac gating was overcome, the data outlined in Chapter 4 were acquired.  In addition to the two studies outlined in Chapter 3 and 4, a number of preliminary imaging investigations were undertaken. While time and financial constraints restricted our focus to three studies, all preliminary investigations yielded promising results. Our first preliminary study was to examine the well developed extradural intravertebral venous system which is evident in all species of pinnipeds (Harrison and Tomlinson, 1956). As this vessel is the main conduit for venous return to the heart and is the most accessible vein for research purposes and medical intervention, a greater understanding of flow parameters and anatomy would be beneficial. In addition, it has been previously noted that the flow within the vessel reverses during diving, leading to a unique physiological situation that warrants further investigation (Ronald et al, 1977). The second trial involved imaging the prominent bulbous expansion of the initial segment of the aorta. The aortic bulb provides a mechanism for maintaining arterial diastolic pressure during the period of pronounced  30  bradycardia associated with the diving response (Rhode et al, 1986). The ability to quantify bulb diameter and descending aortic flow using magnetic resonance techniques was investigated. The third study involved quantification of peripheral vasoconstriction by measuring flow through the renal artery during the predive and diving state.  A variety of M R pulse sequences were tested for imaging these animals, including spin echo (SE), fast spin echo (FSE), gradient echo (GRE), Fast (or Turbo) GRE, cardiac gated segmented k-space GRE, and echo-planar imaging (EPI). The use of methods, such as the Diminishing Variance Algorithm, to reduce respiratory motion artifacts was also undertaken. For flow imaging, cine-phase contrast (PC), ungated 2DPC, ungated spiral PC, and cine-spiral PC were evaluated for suitability in the selected studies.  Each animal was studied 3-5 times. This included a total of nineteen dives performed on the two female animals with a maximum dive time of 5 minutes. Although the animal is capable of significantly longer dive times, short dives were conducted in these initial studies. Over the course of six sessions, the animals appeared to acclimate to the process. A notable decrease in struggling was observed and a reduction in the degree of bradycardia was also recorded. The results of these trials are presented below.  Extradural Intravertebral Vein (EIV): Structure and flow parameters An ungated phase contrast spiral sequence was used to image extradural vein flow. Using TR=35 ms and 10 interleaves, images of a single flow direction were obtained with a scan time of 0.7 sec. Axial images were collected with a spatial resolution of 128x128 over a field of view (FOV) of 40 cm, 10 mm slice thickness, and ± 75 cm/s flow encoding. In periods of apnea, EIV flow is reduced throughout the vessel. During the pre- and postdive period, blood flow in the EIV changes in velocity in response to the respiratory cycle. In the lumbar and thoracic regions, flow direction is cranial, however in the cervical region,  31  blood flows caudally. During diving, the E I V flow is maintained in the cervical region, but exhibits a marked decrease in the thoracic and lumbar regions. Analysis of the jugular vein from the same image sequence indicated that a decrease in jugular flow occurs during a dive. Alterations of flow direction and rate in the E I V during diving have been observed in harp seals (Ronald et al, 1977). Our data support these observations and provide further insights into flow dynamics of this vessel. During apnea, a reduction in blood flow in the thoracic and lumbar regions of the E I V was observed. A s this vessel is the major site for intravenous catheter placement and drug therapy, alterations in flow during apnea would have significant repercussions on animal husbandry and research. Initial observations on the anatomy of the E I V indicate the presence of two distinct veins lying dorsal to the spinal cord. In many species of phocids, the vessel is duplicated in the cervical and sacral regions, but is essentially a single vessel in the thoracic and lumbar regions. The E I V was highly variable in each animal examined, with some regions exhibiting a double E I V and others a single vessel (Fig. 2.5).  vsingle EIV double  I V  Figure 2.5.  Extradural vein location in the thoracic region of a northern elephant seal pup. The vessel structure showed considerable variation, fluctuating between a single vessel and two parallel veins running the length of the vertebral column. Location of mage on the right is 12 c m caudal to image on the left.  32  Aortic Bulb Dimensions and Descending Aortic Flow Rates Cardiac output and stroke volume were measured using a conventional 2D cine phase contrast (PC) sequence and also with a cine-spiral PC. The scan time with the conventional sequence limited its use to measurements during apnea. The spiral sequence used 6-10 interleaves, thus measurements could be obtained during the -15 sec inspiratory periods that occur throughout the eupneic breathing episodes of these animals. The spiral sequence was also used to image flow in the descending aorta (Figs. 2.6a & b).  Figure 2.6.  M R images of a northern elephant seal showing the location of the descending aorta (A - sagittal image; B - axial image). Figure 2.6a illustrates the signal loss resulting from ferric sand in the stomach (dark region caudal to the heart). Figure 2.6b is typical of axial slice location used for analysis of descending aortic flow rates.  33  The cardiac gating system is limited to rates > 30 bpm, so cine images could not be obtained during significant bradycardia (diving). Modification of gating parameters was required before accurate diving cardiac assessment could be undertaken. Measurement of descending aortic flow indicated a reduced pulsatility as compared with human flow rates, with significant flow occurring through diastole (Fig. 2.7). Flow rates in the descending aorta were maintained at or above 2000 ml/min during diastole. This is most likely due to the distensability of the aortic bulb. The bulb serves as an important compensating mechanism in the transfer of energy from the left ventricle to the arterial system for the maintenance of systemic arterial pressures and arterial flow during the prolonged diastolic period (Rhode et al, 1986).  12000  Figure 2.7.  Comparison of human vs northern elephant seal pup descending aortic blood flow (ml) over a cardiac cycle (human H R = 77 bpm; seal HR = 48 bpm, seal measurement obtained during apnea). Aortic blood flow in the seal is maintained throughout diastole.  Peripheral Blood Flow Quantification: Renal Artery Flow in the left renal artery, was imaged with an ungated 2DPC sequence using a sagittal plane, 256x128 resolution, 7 mm slice thickness. The scan time per slice was 4.4 sec with  34  TR=17 ms. Six contiguous slices were imaged repeatedly. No measurable flow was observed to occur in the renal arteries during diving. However, flow was maintained in other unidentified peripheral vessels, suggesting that vasoconstriction is selective or pulsatile flow may be occurring (Fig. 2.8). Cessation of renal flow during diving has been observed previously in seals (Bron et al, 1966; Murdaugh et al, 1961). Our data confirmed these findings and suggested possibility of pulsatile flow to peripheral tissues.  Predive Localizer Renal Artery  Figure 2.8.  Cessation of renal flow during forced diving in a northern elephant seal pup. Sagittal localizer images (top) indicate location of renal artery. Simultaneous acquisition of phase contrast flow encoded image provides information on blood flow through vessels. No measurable flow in the renal artery could be detected during the dive. Note the shift in stomach contents during diving. Flow in an unidentified vessel in the caudal (right) region of the image was maintained during diving (as indicated by white spot to the right of phase contrast images).  35  Organ Imaging Eupneic respiration in these animals generally consisted of respiratory pauses of 5-20 seconds during end-inspiration, followed by a rapid exhalation/inhalation sequence. As a result, sequences with imaging times <15 seconds could produce reliable results if suitably timed. The abdominal contents of the animals (believed to be iron-laden sand) produced very large magnetic field inhomogeneities (Fig. 2.6a). The fast GRE methods yielded good motion immunity, but image quality suffered in regions of inhomogeneity. Lavaging the animals prior to imaging removed a large quantity of sand and rocks, resulting in elimination of inhomogeneities in the abdominal region. Mildly T2 weighted FSE sequences were most successful in delineating the abdominal organs, for example for measurement of spleen volume. The D V A method was successful in obtaining high quality images during respiration by retaining data collected during consistent positions. Flow dynamics were most successfully studied with ungated spiral PC and cine-spiral PC. At times, the animals would go into spontaneous periods of apnea which lasted as long as 12 minutes. Excellent image quality can be obtained during these periods using all sequences. Respiratory motion was not a problem during forced dives. However, voluntary motion limited imaging windows to only a few seconds at a time. Repeated scanning using very fast sequences (e.g. ungated spiral PC and fast GRE) provided the clearest images with the least amount of motion artifact.  Modification to existing sequences enhanced the image quality and consequently the accuracy of resulting data. Over the course of this study, physicists at the Lucas Center for Advanced M R Technology at Stanford University were able to develop new sequences for respiratory movement suppression and contrast enhancement that aided significantly in our ability to complete these studies. The sequences used in the splenic and cardiac studies are outlined in the Materials and Methods section in Chapters 3 and 4. General methodology in  36  the spectroscopy study followed the experimental diving protocol discussed above, and MRS sequence parameters are given in the Materials and Methods section in Chapter 5.  I  37  Chapter 3  The role of the spleen and hepatic sinus in northern elephant seal pups during forced diving Introduction Phocid seals exhibit a higher haematocrit during apnea and diving than during periods of eupneic respiration (Kooyman et al, 1980; Qvist et al, 1986; Castellini et al, 1988; Zapol et al, 1989). This variation in red cell mass indicates that seals have some method of sequestering red cells during non-apneic events. Bryden and Lim (1969) found the haematocrit values of elephant seal pups to be highly variable and suggested that the spleen acts as a dynamic reservoir for erythrocytes, releasing RBCs into general circulation when required. Qvist et al (1986) recorded a distinctrisein Weddell seal haematocrit during diving and suggested that the total red cell mass is partitioned between general circulation and the splenic reservoir. The concept of splenic red cell storage is well accepted by many researchers, and a diving-induced sympathetic vasoconstriction is thought to be the stimulus for splenic contraction and subsequent injection of oxygenated RBCs into circulation (Qvist et al, 1986; Zapol, 1989; Hurford et al, 1996; Castellini and Castellini, 1993; Cabanac et al, 1997). Strong evidence supports this line of reasoning. The histological data collected on seal spleens indicate that the organ is capable of considerable RBC storage, reacts to catecholamine stimulation and does indeed contract during diving (Schumacher and Welsch, 1987; Hurford et al, 1996; Cabanac et al, 1997, 1999). The ability of the seal spleen to concentrate RBCs is supported by high haematocrit values obtained from splenic venous blood during catecholamine-induced contraction (88-93% in hooded seals; 82-88% in harp seals; Cabanac et al, 1997). Ultrasound imaging in Weddell seals demonstrates a correlation of the diving-induced rise in haematocrit with a post-dive reduction in spleen size (Hurford et al, 1996). Although these findings support the concept  38  of splenic R B C sequestration and release, the dynamics and repercussions of the resulting haematocrit fluctuation during diving have not been clearly demonstrated.  The structure and function of the mammalian spleen varies from species to species. Comparative histology reveals that the spleen has a role in immunological defense, selection and removal of damaged blood cells, erythropoiesis and concentration and storage of red cells for discharge under sympathetic nervous control (Reilly, 1985; Tischendorf, 1985). In general, mammalian spleens are classified by their dominant function: defense (predominantly white pulp) or storage (predominantly red pulp)(Schumacher and Welsch, 1987). The white pulp tissue of the spleen is rich in lymphatic tissue and has an immunological role while the red pulp is engaged in filtration and lysis of abnormal RBCs from the blood (Hartwig and Hartwig, 1985).  Seal spleens are predominantly red pulp, indicating a significant storage and filtering ability (Schumacher and Welsch, 1987). Histological sections of the red pulp also reveal many areas of erythropoiesis. Although the production of erythrocytes normally occurs within the marrow of long bones, the relatively small skeletal structure of a seal leads to speculation that the spleen may compensate for the reduced marrow (Schumacher and Welsch, 1987). Increased haematocrit results in greater wear and tear of the RBCs, leading to a marked decrease in life span and consequently higher R B C turnover (Castle and Jandl, 1966; Seaman, 1981), which may also contribute to the need for a greater quantity of erythropoietic tissue. Although composed predominantly of red pulp, seal spleens also exhibit a well-developed lymphatic system and areas rich in hemosiderin, suggesting the spleen also plays a role in both immune function and red blood cell lysis. The presence of both red and white tissue suggests that a seal spleen may act in both an immunological and storage capacity.  39  In the seal, the spleen is located in the typical left abdominal region and held in place by loose mesenteric tissue and peritoneal ligaments. The pulp of the spleen is contained within a thin capsule composed of smooth muscle (Cabanac et al, 1999). Numerous trabecular infoldings of the capsule penetrate deep into the pulp (Schumacher and Welsch, 1987). Blood enters the spleen via the splenic artery and leaves by way of the splenic vein, returning to the heart via the mesenteric vein and the inferior vena cava (Cabanac et al, 1999). In addition to the primary splenic nerve that runs parallel to the artery and innervates the capsule, a number of unmyelinated nerves are observed in both the capsule and splenic pulp.  In many species of mammals, whole organ spleen preparations exhibit a-adrenergically mediated capsular contraction (Reilly, 1985). The degree of response varies from species to species and is generally dose dependent. In hooded and harp seals, in vitro plethysmographic measurements demonstrates that a-adrenoceptor activation with epinephrine results in forceful contraction within 1-3 minutes. Stimulation of (3adrenoceptor and cholinergic receptors did not cause capsular contraction. The contractile effect of epinephrine and nor-epinephrine was largely abolished when the a-adrenergic receptors were first blocked with phentolamine (Cabanac et al, 1997). Hurford et al (1996) found that in vivo, Weddell seal spleens contract on stimulation by exogenous epinephrine infusion. The splenic dimensions obtained during the post epinephrine injection period were similar to those obtained from the postdive period. However, the doses required to obtain an equal degree of contraction are 400 times resting and significantly higher than observed physiological levels. This suggests that direct neural stimulation plays a considerable role in diving-induced splenic contraction, but to date this has not been investigated (Hurford et al, 1996). Experiments with feline spleens indicate that the capsule is mainly under neural control, demonstrating significant capsular contraction with neural stimulation alone (Greenway, 1979). In a diving seal, the rapidity of contraction  40  indicates that the initial stimulation is likely neural in origin, but contraction may be sustained throughout the dive by circulating catecholamines released from the adrenal gland.  Ultrasound images from free diving Weddell seals reveal that the haematocrit rise is correlated inversely with splenic volume (Hurford et al, 1996). Dive and postdive catecholamine levels collected from free diving Weddell and harbor seals demonstrate a significant increase over resting levels (Hance et al, 1982; Hochachka et al, 1995; Hurford et al, 1996). Although these studies clearly suggest that a diving-induced catecholamine release is followed by contraction of the spleen and release of the stored erythrocytes, a discrepancy exists in the apparent timing of these two events. Complete splenic contraction occurs within 3 minutes of catecholamine stimulation, yet peak haematocrit is not observed until 15-25 minutes after the spleen has contracted (Qvist et al, 1986; Hurford et al, 1996). The delay between splenic contraction and changes in peripheral venous haematocrit may represent an equilibration between the splenic reservoir and the venous circulatory blood pools (Hurford et al, 1996), but to date no clear evidence exists to support this supposition.  Although the spleen is observed to contract in response to facial immersion, its role as a supplemental oxygen store during dives has been debated (Ponganis et al, 1992; Castellini and Castellini, 1993; Eisner and Meiselman, 1995). Growing evidence suggests that seal haematocrit remains elevated during the postdive period, indicating that splenic refill is delayed. In Weddell seals, haematocrit levels remained elevated for 10-12 minutes after diving (Zapol et al, 1989), while northern elephant seals required 7-15 minutes to return to preapneic values (Castellini and Castellini, 1993). In light of the short surface intervals exhibited by seals during extended diving bouts, these findings suggest that haematocrit may stay elevated for prolonged periods. Castellini and Castellini (1993) propose that the spleen may act as a means of reducing haematocrit when the animal is not diving, thus  41  reducing the cost of transporting blood during periods of rest. Eisner and Meiselman (1995) indicate that rheological consequences of elevated haematocrit would adversely affect circulation, thus supporting the advantage of withdrawing a substantial portion of red cells from circulation during non-diving periods.  The negative effects of elevated haematocrit are difficult to evaluate, as the circulatory system is dynamic and possesses many compensatory mechanisms that aid in blood oxygen transport. However, the basic relationship is well established: blood flow is inversely related to viscosity, and viscosity is exponentially related to haematocrit. Hedrick et al (1986) investigated the effects of high haematocrit on viscosity and blood flow in northern elephant seal blood. They concluded that viscosity was indeed higher in elephant seals due to the elevated haematocrit, and the standard compensatory mechanisms for reducing the effects of blood viscosity on flow (increase blood pressure or increase peripheral vasodilation) were not employed. The prediction for animals lacking in compensatory mechanisms is a decreased metabolic scope, however the athletic ability of this species has not been assessed. Studies on harbor seals indicate variable results ranging from an aerobic scope of 4 fold (Eisner and Ashwell-Erickson, 1982) to 9 fold (Ponganis et al, 1990).  Wickham et al (1989) examined the rheological characteristics of seal blood in order to evaluate the effect of increasing haematocrit on blood flow. Blood viscosity is determined by shear rate and strain effects, where increasing shear rate leads to a decrease in viscosity due to changes in the strain and cell/vessel interactions. The relationship is further complicated by the non-Newtonian behaviour of blood and the formation of weak bonds between erythrocytes. These bonds result in loose, rod-like formations called rouleaux, and may contribute to some degree of order in blood flow through the smaller vessels but may also create flow impedance and disruption. Wickham and colleagues found that seal  42  blood was less viscous at low rates of shear than pig blood, despite high mean corpuscular volume, plasma protein levels and white blood cell counts, all factors known to increase viscosity (Chien et al, 1966). They suggest that the relatively low viscosity of harbor and elephant seal blood that accompanies elevated haematocrit may represent a mechanism for the rapid restart of blood flow after diving-related venous pooling.  As blood viscosity and haematocrit are opposing forces in the drive for oxygen delivery, the concept of an optimal haematocrit has been established (oxygen transport is maximized at some particular haematocrit and at lower and higher haematocrits oxygen transport decreases due to reduced oxygen carrying capacity and increased viscosity, respectively) (Crowell et al, 1959; Birchard, 1997). Hedrick and Duffield (1991) compared blood samples from seven marine mammal species and found that the beluga and elephant seal were the only two species that did not exhibit an optimal haematocrit. The increased need for oxygen storage over delivery may be driving the disparity, as both of these species exhibit significantly greater dive durations than all others. In contrast to Wickham et al (1989), they concluded that none of these species have ameliorating mechanisms for the reduction of viscosity.  Meiselman et al (1992) found differences in the rheology of three phocid species, with significant variation in fibrinogen levels, red cell aggregation and aggregate strength. The degree of aggregation was correlated with viscosity, creating widely varying results between the species. However, in spite of conflicting viscosity results in the studies discussed above, these authors agree that an increase in viscosity does occur when shear rates are low (as would occur when H R drops or vasoconstriction is present); it is the degree of increase that is debated (Wickham et al, 1989; Hedrick et al, 1985; Hedrick and Duffield, 1991).  43  The structure of the phocid circulatory system suggests that the spleen may not be the only candidate for red cell storage. The presence of a large venous reservoir, termed the hepatic sinus, provides another possible source of sequestered red blood cells. Formed by the dilation of the hepatic veins, the thin walled sinus lies caudal to the diaphragm, draining from its midpoint through the diaphragm and into the thoracic portion of the posterior vena cava (Harrison and Tomlinson, 1956). The inferior vena cava and the hepatic sinus may contain up to one fifth of the animal's total blood volume and presents a significant storage depot of oxygenated blood during dives. During forced diving in northern elephant seals, the oxygen content of sinus blood was observed to decrease more slowly than arterial blood. As the dive progressed, blood oxygen content of sinus blood exceeded arterial values (6.6 and 4.6 volumes per 100 ml respectively) (Eisner et al, 1964). The behaviour of the sinus and the elevated blood oxygen content in the venous system led some to suggest that the hepatic sinus is a controlled oxygen reservoir for use during diving (Eisner et al, 1964, 1971; Hoi et al, 1975).  Filling of the sinus is dependent on the closure of a muscular vena caval sphincter located on the cranial aspect of the diaphragm. This caval sphincter is, even in young pups, fully capable of occluding the venous return through the posterior vena cava (Harrison and Tomlinson, 1956). Innervation is supplied by a branch of the right phrenic nerve, and the sphincter contracts forcefully when the nerve is stimulated (Harrison et al, 1954; Harrison and Tomlinson, 1956). Placement of a clamp on the vena cava near the sphincter did not alter heart rate appreciably in an anaesthetized harbor seal, suggesting that sphincterinduced venous occlusion does not affect non-diving cardiac frequency (Harrison, 1960). However, prevention of sphincter function via phrenic nerve transsection caused a delay in the onset of bradycardia (Harrison and Tomlinson, 1960), possibly due to the increase in right ventricular end-diastolic pressure that would result from the combination of peripheral vasoconstriction, splenic contraction and unrestricted inferior vena caval flow. It was  44  stated that animals with denervated sphincters "disliked diving for more than 2 minutes", indicating that the perturbation of flow dynamics caused by sphincter dysfunction was significant enough to disrupt the mammalian diving response (Harrison and Tomlinson, 1960).  Angiographic studies enabled researchers to visualize blood flow and hepatic sinus dynamics during diving. Using harbor seals, Eisner et al (1971) demonstrated a divinginduced accumulation of contrast material within the vena cava below the diaphragm. Small quantities of contrast material were observed to pass through the restricted orifice of the sphincter and enter the heart. In experimental dives using harp seals (Pagophilus groenlandicus), Hoi et al (1975) reported a marked constriction of the sphincter occurred 20 seconds after commencement of the dive, with dilation of the posterior caval vein and hepatic sinuses occurring before as well as during the 40 seconds following constriction. The study also demonstrated a temporary relaxation of the caval sphincter during the dive and subsequent mixing of the blood in the sinus with that returning from the anterior part of the body. These findings provide a potential explanation for the Eisner paradox, where blood oxygen content was observed to be higher in the venous system than in arterial vessels. Although these studies reveal a considerable amount of information regarding the dynamics of the sphincter and sinus during diving, its function in phocid physiology has not been clearly defined.  Under most circumstances, accurate measurements of a flaccid blood-filled structure are difficult to obtain, and this task is further complicated when the structure involves contractile smooth muscle. When invasive surgical procedures or stress-inducing methodologies are employed, catecholamine-mediated changes in the smooth muscle are inevitable. Through MRI technology, it is possible to obtain sequential images of the spleen and related vasculature during rest, diving and postdiving periods with minimal  45  perturbation. Volumetric assessment of the spleen and hepatic sinus in vivo during forced diving protocols was achieved through quantitative analysis of these images.  Methodology In April of 1997, four male and one female four-month-old elephant seals were collected from Afio Nuevo, California and held at the Long Marine Laboratory, UCSC for the duration of their captivity (mean = 94 kg ± 7.65 SD). On the day of the experiment, animals were transported by truck to the Lucas Center for Advanced M R Technology at Stanford University (transport time ~ 2 hours). Diving protocols, animal restraint and animal husbandry procedures were undertaken as outlined in Chapter 2. Prior to imaging, each seal was fitted with a diving helmet and after a brief period of acclimation, subjected to four sequential dives (diving time ranged from 5 min 38 sec to 7 min 23 sec; interdive time 20 min 13 sec to 26 min 21 sec). Imaging the entire spleen required 28-32 axial images. The acquisition time for each series of splenic images was less than one minute. Images were acquired sequentially during the dive (initiated as soon as the animal's nostrils were completely submerged) and continued throughout the dive at one minute intervals until the helmet was drained and the animal took its first breath. Images were also obtained in the pre- and post-dive periods.  A l l images were collected using a high performance 1.5 T system (Signal Horizon Echo Speed, G E Medical Systems, Milwaukee, WI). A single shot fast spin echo (SSFSE) pulse sequence was used in the sagittal plane to localize the splenic anatomy (Fig. 3.1). Images for analysis were obtained using SSFSE in the axial plane. Scan parameters used were a flip angle of 90, T E 185, 0.5 N E X , 48x48 field of view, 26-32 contiguous 15 mm thick slices with no overlap, 256x256 matrix.  46  Figure 3.1.  Sagittal and axial images of the spleen of a northern elephant seal pup. Sagittal localizers were used to define the upper and lower image slice location and calculation of the number of axial slices required to image the total spleen. A series of 29-34 1.5 cm "slices" were used to image the organ completely, requiring less than a minute of scan time. Image A is from 5 cm left of the midline (spine) and image B is 8 cm below the diaphragm.  47  Images were analysed using NIH Image 1.6.1. The border of the spleen or hepatic sinus was drawn on each image, and the area of the selected region was derived (1 pixel = 0.1875 cm ). Volume of the selected region was calculated by multiplying the area of the 2  region by 1.5 (slice thickness was 1.5 cm). Organ or sinus volume was obtained by summing the volume of all slices; images were analyzed out of sequence to avoid bias. Statistical analysis was conducted using JMP 3.2.1.  Collection of blood samples during diving could not be achieved in the M R unit, as the caudal end of the animal was not accessible during MRS data acquisition. Consequently, the restraint procedure and diving protocols were repeated at Long Marine Lab, and blood samples were drawn from the extradural intervertebral vein during the predive, dive and postdive period (predive samples were obtained during initial needle placement; dive samples were drawn at 1, 3, 5 and 6 min; postdive samples at 2, 5 and 10 min). Serial blood samples were collected using a 14 gauge 13 cm spinal needle inserted between the lumbar vertebrae (approximately L4-L5, 5 cm cranial to the pelvis). A vacutainer blood collection tube holder was attached to the needle via a multisampler hub, and samples were collected into a heparinized Vacutainer tube (haematocrit) and a non-additive tube (lactate) for analysis.  The heparinized tubes were placed on a blood tube oscillator to prevent clotting and red cell settling prior to the transfer of a subsample into heparinized microhaematocrit tubes. Once the microhaematocrit tube was sealed (Critoseal), the samples were centrifuged at 4500 rpm for 15 minutes, and the haematocrit reading was obtained via comparison of the packed red cell level with a standard haematocrit card.  48  Results Haematocrits obtained during forced diving in three animals showed a significant increase over resting in Dive Min 5 and 6. Haematocrit values returned to resting by postdive Min 10 (ANOVA, F  (3i8)  = 11.01, P = 0.003; Tukey Kramer HSD, P = 0.05). Values show a  trend toward a continued increase for the duration of the dive (Fig. 3.2).  Resting splenic volume measured 3.35% of body mass ( ± 0.33 SD, n = 5). In all animals, splenic contraction was initiated immediately upon facial immersion (Fig. 3.3). By minute 3, the spleen had dramatically contracted to 14.0 -17.8% of its predive volume and remained contracted until the dive was terminated (Fig. 3.4; A N O V A  F  ( 6 j 2  8)=  33.94, P  < 0.0001; Tukey Kramer HSD, no significant decrease in volume after Min 2, P = 0.05). A corresponding increase in hepatic sinus volume occurred concommitant with splenic contraction, suggesting a direct shift of blood from the spleen to the sinus (Fig. 3.5; sinus volume + spleen volume is not significantly different from resting volume at any point in the dive; A N O V A , -F  (7;32)  = 0.24, P = 0.97; comparison to baseline, Dunnett's Method, P =  0.05). Once dilated, the hepatic sinus volume remained constant throughout the dive (no significant change in volume after Min 1; A N O V A , F  ( 52  4>=  0.46, P = 0.80, Tukey-Kramer  HSD, P = 0.05)(Fig. 3.5).  In the postdive period, the spleen gradually dilated, achieving its maximum volume at 1822 minutes after the dive. Immediately after the dive, hepatic sinus volume quantification was hampered by increased respiratory motion and diaphragm movement. However, by post-dive minute four, tachypnea had subsided and no evidence of hepatic sinus dilation or vena caval distension was observed. These images are the first clear evidence demonstrating a functional relationship between the spleen and hepatic sinus and provide evidence for a model to explain the circulatory adjustments observed during diving in phocids.  49  3  5  6  PD2  PD5 P D 1 0  Dive/Postdive Time (min)  Figure 3.2.  Haematocrit values obtained from northern elephant seal pups during forced diving (n = 3). Diving haematocrit is significantly different from predive in Min 5 and 6. A trend toward a continued increase in haematocrit during the dive is observed and values returned to predive levels by PD Min 10 ( A N O V A , F = 11.01, P = 0.003; Tukey Kramer HSD, P = 0.05). (3j8)  50  Resting  Hepatic sinus  Spleen  Dive minute 3  Dive minute 3 . . ;;;:,s;:^;i^:::;.  Spleen  Figure 3.3. Thoracic images of a northern elephant seal during rest and diving. Images on the left are from the region immediately caudal to the diaphragm; images on the right are 12 cm caudal to the diaphragm. Rapid contraction of the spleen and simultaneous filling of the hepatic sinus are observed.  51  3000  2500  2000  _5  1500  o  >  1000  500  0 6 0  3  4  5  Dive Time (minute)  Figure 3.4. Northern elephant seal spleen volume during rest (Min 0) and diving (Min 17) was obtained using M R imaging techniques (n = 5, each individual's value is the average of four dives). Splenic volume does not decrease significantly after minute 2 ( A N O V A , F , = 33.94, P < 0.0001; Tukey Kramer HSD, P = 0.05). Error bars indicate SD. (6  28)  52  4000 3500 3000  CD  2500  E O >  2000 H  <D C> CL  1500  c  W  1000 500 H  Rest  2  3  4  5  Dive time (minute)  Figure 3.5. Graph of an individual seal's splenic volume and hepatic sinus volume at each minute of a 7 minute dive. Combined volume of spleen and hepatic sinus at each minute does not differ significantly from spleen volume at rest A N O V A , F 32) = 0.24, P = 0.97; comparison to baseline, Dunnett's Method, P = 0.05). ( 7  53  Discussion The M R data confirm that forced diving results in contraction of the spleen and emptying of the concentrated RBCs into the venous system. The rapid reduction in splenic volume presents an apparent contradiction to haematocrit data collected from elephant seal pups in the lab (Fig. 3.2) and field data from Weddell seals, where the diving related rise in haematocrit has been observed to be a slow gradual process (Qvist et al, 1986). The delay in appearance of the RBCs into general circulation may be explained by the presence of a diving-induced vena caval occlusion. It has been previously shown that the phocid caval sphincter contracts during facial immersion, restricting vena caval blood flow at the level of the diaphragm and causing the hepatic sinus to dilate (Harrison and Tomlinson, 1956; Murdaugh et al, 1962; Eisner et al, 1971; Hoi et al, 1975; Ronald et al, 1977). Relaxation of the sphincter allows small quantities of blood to escape, leading to a gradual increase in circulating haematocrit (Harrison and Tomlinson, 1956; Eisner et al, 1971; Hoi et al, 1975; Ronald et al, 1977).  By demonstrating an inverse relationship between hepatic sinus volume and splenic volume, a transfer of erythrocytes from the spleen to the sinus is inferred. At the beginning of a dive, contraction of the spleen would result in an increase in vena caval volume at an approximate rate of 25 ml/second. The unfettered flow of splenic RBCs into the right ventricle at such a rate would have deleterious effects on cardiac function. By relocating the RBCs from the spleen into the sphincter-controlled venous sinus, a gradual metering of blood into the heart would occur (Eisner et al, 1971), protecting it from a drastic increase in right ventricular pressure at a time when diving bradycardia is most profound (Ronald et al, 1977). The action of the sphincter would also serve to slowly infuse oxygenated RBCs into the central circulation, supplementing the oxygen content of circulating blood (Kooyman et al, 1980). At the beginning of a dive, hepatic sinus haematocrit would be higher than that observed in general circulation due to the release of concentrated splenic  54  RBCs (Schumacher and Welsch, 1987; Cabanac et al, 1997). As the dive progressed, pulsatile release of oxygenated venous blood into circulation and subsequent replacement by less concentrated (and less oxygenated) blood would result in an overall dilution of the hepatic sinus contents. This situation supports Eisner et al's (1964) observations of a higher oxygen content in the sinus than that observed in general arterial circulation. The maintenance of hepatic sinus volume over the course of the dive indicates that flow into the sinus must be approximately equal to the flow leaving the sinus through the caval sphincter. Although volume of the sinus remains relatively stable in the latter part of the dive, a distinct loss of signal intensity is observed (Fig. 3.6). This loss of signal intensity may be due to a reduction in oxyhaemoglobin signal.  When water protons in blood are subject to a M R pulse sequence, the protons respond by precessing rapidly at a rate determined in part by the precise value of its local magnetic field. Slight differences in local fields cause the protons to precess at different rates, leading to a rapid loss of signal as they cancel one another. The time until cancellation, called T2*, is shortened by the presence of deoxyhemoglobin due to the wide variations in magnetic field caused by the iron atoms. The oxygen-carrying form of hemoglobin is not magnetic, so oxygen-rich blood should have longer T2* times, and thus an increased signal intensity. The loss of signal intensity within the sinus over the course of the dive may indicate an increase in the amount of deoxyhaemoglobin. Deoxygenation of sinus blood may be the result of a haematocrit reduction, desaturation of the haemoglobin, or a combination of both. Without further experimental analysis, the specific cause of signal loss in this instance cannot be ascertained, but one plausible explanation is a reduction in oxygen content within the sinus over the course of the dive.  55  Figure 3.6. The hepatic sinus dilates rapidly upon submergence and remains dilated throughout the dive. Loss of signal intensity is observed as the dive progresses. A x i a l images were obtained from the thoracic region immediately caudal to the diaphragm. Dark crescent shape in the upper left quadrant is the spleen; the margins of the hepatic sinus are outlined in white.  56  In the postdive period, a reduction in sinus volume and a gradual increase in splenic volume is noted, but the timing of these events is disparate. By postdive minute 4, a delay in splenic expansion and absence of blood in the hepatic sinus was noted, suggesting that the caval sphincter is no longer occluding venous flow and the entire blood volume has entered active circulation. Tachycardic respiratory motion prevented acquisition of clear M R images in the immediate post dive period, but previous research indicates a rapid caval sphincter relaxation and release of sinus blood into general circulation (Hoi et al, 1975; Eisner et al, 1971). To maintain blood pressure, a decrease in total peripheral resistance occurs simultaneously with the tachycardia-induced rise in cardiac output. This global vasodilation benefits the animal during short surface intervals through the hypoperfusion of tissues, facilitating rapid replenishment of haemoglobin and myoglobin oxygen stores and removal of metabolic byproducts from previously ischaemic areas (Castellini, 1994). Rapid dilation of the spleen and removal of red blood cells from active circulation at this time might in fact be detrimental to the animal, delaying reoxygenation of the total red cell mass.  The slow rate of splenic refill combined with a short surface interval also creates a situation whereby the animal would maintain an elevated blood volume throughout a diving bout (Qvist et al, 1986; Castellini and Castellini, 1993). Elevated haematocrit through diving bouts has been observed in phocids both in the field (Weddell seals; Kooyman et al, 1980; Qvist et al, 1986; Castellini et al, 1988) and the laboratory (northern elephant seal pups; Thorson, 1993). As it was previously thought that the spleen supplemented circulating blood with oxygenated RBCs during the dive, the inability to resequester RBCs during the surface interval brings this role into question. It has been suggested that these animals may utilize the spleen to sequester RBCs when the benefit of increased oxygen is offset by the cost of transporting blood of higher viscosity (Castellini and Castellini, 1993).  57  During diving, periods of high haematocrit are accompanied by vasoconstriction and bradycardia. The combined effect of these events would serve to decrease shear rate and result in elevated viscosity. The maintenance of an elevated haematocrit would therefore cause a potential trade-off whereby elephant seals experience a decreased rate of oxygen delivery at the expense of increasing onboard oxygen storage capacity. This would likely be beneficial during a dive, as a reduction in oxygen delivery would lead to metabolic suppression. The question then becomes why maintain a high circulating haematocrit during the haulout, where oxygen is not limiting?  One could argue that the cost of maintaining an elevated red cell mass during the 3-4 month haul-out period would not be an optimal use of the animal's energy, especially during energetically expensive periods such as moult, parturition or nursing. However, as these animals move biannually between terrestrial and aquatic environments, the erythropoietic mechanisms observed in high altitude acclimation may be partly responsible for the maintenance of red cell mass. The presence of hypoxia-induced erythropoiesis in harbor seals was demonstrated by Kodama et al (1977). Exposure of harbor seals to a 3.1 km elevation over an 85 day period resulted in elevation of haematocrit and a decrease in resting P A 2 , suggesting a standard high altitude response to hypoxia. The frequent and C0  lengthy periods of apnea exhibited by a fasting elephant seal may provide continued stimuli for erythropoiesis. At birth, an elephant seal pup's haematocrit is considerably lower than at 4 months of age (nursing pups (n = 10), % Het 53 ; 12 week old pups (n = 18), % Het 61.6 ± 4.2 SD) (Thorson, 1993). Over the course of a 3-4 month fast, extended apneic bouts would lead to reduced blood CA02 and potentially stimulate red cell production. The life history of an elephant seal would continue to provide extended periods of reduced blood C A 2 both when diving and when on land, serving to maintain a high level of 0  haematocrit. It would be interesting to evaluate the relationship between apneic bout length  58  and circulating haematocrit within the phocid species, but at present the existence of a causal relationship is highly speculative.  Conversely, retention of a high red cell mass during the haul-out period would enable the seal to increase circulating oxygen stores during apnea (Castellini et al, 1986). The rise in available on-board oxygen stores resulting from splenic contraction would effectively extend the period of nonventilation and allow the animal to reduce the overall respiratory rate. These periods of apnea are important during the fast, as a significant quantity of water is conserved through apneustic breathing patterns (Blackwell, 1996). The reduction in respiratory water loss may provide a positive selective force for the continued availability of these sequestered RBCs. Significant metabolic savings are also realized through apneustic breathing patterns, as an overall reduction in heart rate and oxygen consumption occurs (Kohin et al, 1999). When an increase in oxygen delivery to the muscles is required, brief periods of high viscosity may be tolerable due to an increase in blood velocity. In a manner similar to that observed in race horses, the seal may increase blood volume and oxygen availability to the tissues through splenic contraction, reducing viscosity by increasing shear rate through vasodilation and tachycardia (Persson, 1983; Snow et al, 1983; Pelletier et al, 1987). The explanation for retention of red cell mass during the haulout then becomes teleological, as it is difficult to ascertain whether apneic bouts cause an increase in RBCs, or the presence of an increased R B C mass facilitates apneic bouts.  The demonstrated relationship between splenic contraction and hepatic sinus volume in northern elephant seal pups reveals the functional significance of the sinus and the dynamics of the spleen during forced diving. Although the animals used in this study had not been to sea for an extended period, they are fully capable divers who, upon leaving the beach, immediately exhibit a diving profile similar to adults (Thorson and Le Boeuf, 1994; Le Boeuf et al, 1996). Dissections of pups ranging from 1 month to 16 months of age  59  indicate that the hepatic sinus is present and well developed in all age classes (Thornton, pers. obs). Splenic volumes obtained in this study indicate that the spleen to body weight ratio is high (3.12% to 3.88% of body weight; Table 3.1), suggesting that splenic blood storage is a well developed feature from a young age. As their dive profile, anatomy and mass specific diving ability rivals that of an adult, their circulatory pattern is likely representative of the species. Species within the phocid family exhibit a high spleen-tobody mass ratio (Qvist et al, 1986) and haematocrit shifts. They are the only animals known to possess a well developed hepatic sinus and a complete caval sphincter. The uniqueness of these features to phocids indicates that the basic pattern of circulatory partitioning described above is likely found in all true seals. Table 3.1.  Summary of northern elephant seal pup splenic volumes obtained from M R images during forced rest and forced diving.  Individual  Mass (kg)  Ave Splenic Volume (ml) rest  Minimum Splenic Volume (ml)  Spleen (resting vol) as % body mass  Basil  89.3  2787.39  444.19  3.12  Bogart  83  2749.10  491.34  3.31  Boris  97.3  3033.68  536.03  3.12  Norman  101.5  3938.94  644.43  3.88  Stella  98.9  3352.24  595.91  3.39  Mean ± SD  94 ± 7.6  3172.27± 491.6  542.38 ± 79.94  3.36 ± 0.28  In summary, the data presented demonstrate a direct relationship between the spleen and hepatic sinus in a northern elephant seal and provide us with a model to explain haematocrit shifts observed in diving phocids. The elegant system of storage, transfer, and metering of RBCs provided by the spleen/sinus interaction allows the seal to maintain a higher  60  circulating haematocrit during periods of hypoxia, yet effectively reduce haematocrit and circulating blood volume when oxygen is not limiting, thus avoiding any deleterious effects of increased blood viscosity.  61  Chapter 4  The effect of forced diving on cardiac function in northern elephant seal pups Introduction In 1870, Paul Bert published the first scientific study on the cardiovascular effects of diving (Bert, 1870). Using forced dive techniques in ducks, he observed a dramatic slowing of the heart rate from 100 to 14 beats per minute during a dive. In 1899, further research indicated that diving bradycardia could be abolished by vagotomy or atropine administration (Richet, 1899). The publication of Irving and Scholander's preeminent work on diving physiology set the field in motion, stimulating a considerable quantity of research investigating the cardiovascular changes that accompany diving (Irving, 1939; Scholander, 1940).  Control over cardiac function, specifically heart rate and stroke volume, involves complex interactions of humoral and neural stimuli. Intrinsic heart rate is mitigated by the action of the vagal nerve, while a myriad of humoral factors influence rhythm and force of contraction. In a diving animal, facial immersion initiates a chain of events through stimulation of trigeminal receptors causing apnea, usually in the expiratory position (Angell-James and Daly, 1972). Trigeminal stimulation of the cardio-inhibitory center results in vagally-mediated bradycardia, while vasomotor stimulation leads to vasoconstriction in the periphery (Eisner and Gooden, 1983; Butler and Jones, 1997). The reduction of stimuli from pulmonary stretch receptors (caused by exhalation early in the dive) serves to augment the bradycardic event (Angell-James et al, 1981; Daly et al, 1984).  In adult harbor seals, bilateral vagotomy increased the surface heart rate from 60 to 90 bpm, demonstrating the attenuating effect of the vagus (Harrison and Tomlinson, 1960).  62  The absence of diving bradycardia in vagotomized seals implicated the vagus as the primary efferent stimuli mediating the profound drop in heart rate. When the vagi were severed in a submerged bradycardic seal, an immediate and sustained rise in heart rate was observed, reaching levels that surpassed predive rates. The afferent pathway has been identified as the trigeminal nerve, with branches in the nasal mucous membrane and orbital region (Dykes, 1973). Pharmacological blockage of these afferent pathways results in a diving bradycardia equivalent to that observed during surface apneas. If the animal is forced to respire prior to immersion, a drop in heart rate was not observed (Angell-James and Daly, 1972).  As the dive progresses, increasing levels of hypoxaemia and hypercapnia further stimulate the cardio-inhibitory and vasomotor centers via arterial chemoreceptors, reinforcing the bradycardic response and maintaining vasoconstriction. However, the gradually increasing chemical drive also provides a powerful ventilatory stimuli which must be abrogated in order to maintain diving apnea (Angell James and Daly, 1972; Eisner et al, 1977). The continued presence of vagal inhibition overrides the carotid body ventilatory drive while simultaneously facilitating the cardioinhibitory response, resulting in the maintenance of bradycardia and diving apnea (Daly et al, 1977).  Phocid seals demonstrate a rapid and progressive rise in circulating norepinephrine and epinephrine levels during diving (Hance et al, 1982; Hurford et al, 1996). Circulating catecholamines have been shown to induce splenic capsule contraction (Hurford et al, 1996; Cabanac et al, 1997) and support the maintenance of peripheral vasoconstriction during diving; however during diving the heart appears to be relatively unresponsive to the chronotropic and inotropic effects normally associated with circulating catecholamines. This may again be due to the dominance of the neural stimuli in initiating and maintaining bradycardia.  63  Prior to deciphering the complex interactions that dictate blood flow and oxygen distribution in a diving seal, we are faced with the challenge of obtaining accurate physiological measurements of heart rate (HR), stroke volume (SV) and cardiac output (CO). Electrocardiograph (ECG) measurements in the lab and remote monitoring of pinniped HRs in the field have provided a plethora,of information on the frequency of cardiac contraction under different conditions. However, without concurrent knowledge of stroke volume, it is difficult to make broad physiological conclusions based on H R alone. Traditionally, SV is calculated from CO and HR measurements rather than measured directly from each cardiac cycle. Methods of determining CO include the Fick principle, dye dilution, thermodilution, and Doppler or transducer flow evaluation, making this a difficult and invasive measurement to obtain both in the field and under controlled laboratory conditions. In order to more accurately infer metabolic rate and energetic costs of diving from heart rate data, a greater understanding of the interactions between cardiac factors is required and variation of SV during diving must be evaluated. At present, there are seven publications reporting CO measurements obtained from marine mammals. In these studies, C O was evaluated by a pulmonary artery Doppler flow meter (Eisner et al, 1964), thermodilution (Swan-Ganz catheter insertion) (Zapol et al, 1979; Sinnett et al, 1978; Ponganis et al, 1990), microsphere injection (Blix et al, 1976; Zapol et al, 1979; Blix et al, 1983) and dye dilution (Murdaugh et al, 1966) techniques. In all cases, SV was not measured, but instead was calculated from simultaneous heart rate measurements (SV = CO/HR). These studies all indicate that diving-induced bradycardia results in a decreased CO. However, reported SVs provide disparate results, with some experiments indicating that SV remained relatively constant, while others indicate a divingrelated decline (Table 4.1).  64  c •3  O Ov Ov  -a s  >  a  o &  60  C3  p  N  c  c  o o o  2 «>  •8, •S 6  3  aO  o  d +l  • 'e  a  CO  OS U  <j  _  O  <D ft  'S c  +1  " f t  > a S  +i  roq in vo cn  rOv vd  +1  oo cn od r—<  rvd <N  OJ  <  G 1)  s  SB y  o  c/3 a  3 O  00  T3  D 3  >  a 3  <N  +1  rt rt  co  O-S U  -  +1 +1 Tf  ov o cn  Ov vd  ^  bt)  cn  C  >/•>  •> -a .3  G c3  +1  > a  Ml  oo cn  Tf Ti en  o Ov Ov  cn  u  I  vd  <N ov d o  CN vo vd H (N rt  I  ft  Tf  +1 +1  rt  vo Ov  U  cn VO  |l o  •1  ac  ca U C  '•ai  u W  In 1964, Eisner et al reported the first CO measurement from a diving marine mammal. Working with a trained California sea lion (Zalophus californianus), pulmonary artery flow and CO were determined during voluntary head immersions of up to 2 minutes. Cardiac output varied in close accordance with heart rate, while SV remained essentially unchanged. Murdaugh et al (1966) reported a dramatic reduction of CO during the dive and a subsequent rise in the postdive period in five young harbor seals (Phoca vitulina). Heart rates obtained from these animals showed a profound bradycardic response to the forced dive protocol. Calculated SV was highly variable, with measurements ranging from 5.3 to 108.4 ml/beat. During the dive, stroke volume did not change in a consistent manner, with some experiments showing an increase over predive SV, while others remained the same or decreased when compared to predive volumes. However, mean SV during the predive and the diving period were essentially the same. Using grey seals (Halichoerus grypus), Blix et al (1976) also reported that S V was essentially unchanged during diving, while C O decreased more than 90% in response to submersion.  In contrast to earlier studies, Sinnett et al (1978) reported that forced dives on harbor seals performed under various experimental conditions resulted in an approximate SV reduction of 66%, while C O during the dive declined roughly 83%. In experiments conducted on Weddell seals (Leptonychotes weddelli), Zapol et al (1979) reported an 86% decrease in CO during the dive. The mean decrease in HR with diving was accompanied by a 56% drop in SV (calculated from reported HR and CO measurements). Results from spotted (Phoca vitulina largha) and grey seal experiments also indicate a significant decline in diving CO when compared to resting (90%), as well as a 57% decline in calculated S V during the dive (Blix et al, 1983). Blix and his colleagues concluded that a decrease in SV would be advantageous due to reduced myocardial fiber shortening and cardiac work required to supply the total systemic capillary blood flow during diving.  66  Ponganis et al (1990) conducted thermodilution C O measurements on harbor seals at various levels of work effort during swimming both at the surface and while submerged. Although not directly comparable, the data collected in this study indicate that SV remained relatively constant throughout the range of low work loads. However, submerged swimming seals exhibited a SV of 50-70% of eupneic SV. These findings suggest that the factors dictating cardiac function during submerged exercise may be overridden by the diving response, preventing significant alteration in SV over the lower portion of their work range. The authors suggest that the mechanism for SV reduction during diving likely involves decreased venous return and cardiac filling due to the hepatic sphincter contraction and possibly a decreased ejection fraction secondary to a decreased cardiac inotropic state (Ponganis et al, 1990).  In light of the variable nature of CO measurements and SV calculations in diving pinnipeds, SV was measured directly to investigate the effect of diving on cardiac dynamics. Through the use of M R Imaging and phase contrast flow analysis, accurate flow measurements were obtained from the base of the aorta, and then cardiac output during the predive, dive and postdive period was calculated.  Methodology In October of 1997, four 10 month old northern elephant seal pups were collected from Afio Nuevo, California and held at Long Marine Lab, UCSC for the duration of their captivity (mean mass = 102.2 kg ± 3.58 SD; 2 males and 2 females). These animals were subjected to a total of 11 dives (mean dive time = 6 min 28 sec ± 1 min 07 sec SD). These animals were subjects in the PCr study outlined in Chapter 5, and therefore had prior exposure to the experimental diving protocol and handling procedures. Measurements were collected during the predive, dive and postdive periods. Values referred to as "resting" were obtained during the quiescent 5 minute period immediately preceding a dive.  67  On the day of the experiment, animals were transported by truck to the Lucas Center for Advanced M R Technology at Stanford University (transport time ~ 2 hours). Diving protocols, animal restraint and animal husbandry procedures were undertaken as outlined in Chapter 2. Prior to placement in the magnet, each animal was outfitted with E C G leads and respiratory bellows as described in Chapter 2. Heart rate data were collected on a Macintosh IISI with a 8 channel Powerlab™ (ADInstruments) interface and a M L 132 BioAmp. Heart rate was analysed by recording interbeat intervals using Chart™ (ADInstruments).  A l l images were collected using a high performance 1.5 T system (Signal Horizon Echo Speed, G E Medical Systems, Milwaukee, WI). Stroke volume was measured using a conventional 2D cine phase contrast (PC) sequence and a cine-spiral PC. Localizer images were acquired in the axial plane at the base of the aortic bulb (Fig. 4.1). Scan parameters used were TR 55, T E 6.2, 1 N E X , 48 x 48 FOV, 7 mm thick slice. The spiral sequence used 12-16 interleaves, allowing for rapid data acquisition.  Flow data were analysed using XCinema V . 6.46 in conjunction with custom flow analysis software designed at Stanford University's Lucas M R Center. Statistical analysis was conducted using JMP 3.2.1.  68  A  Figure 4.1.  M R axial images of a northern elephant seal pup during cardiac phase contrast data acquisition. Localizer image (A) shows the location selected for flow analysis and the position of the aorta. In Phase Contrast images A and B, image intensity is proportional to the velocity component of the blood along the superior/inferior direction. Image B was obtained during systole (Frame 4 of 36), and Image C is near the end of diastole (Frame 27 of 36). Black indicates superior (cranial) flow and white represents flow in the inferior (caudal) direction.  69  Results Heart Rate (HR) Heart rate was recorded from four animals throughout the experimental protocol, with predive heart rates corresponding to the five minute acclimation period immediately proceeding the first dive. Heart rates at all times were quite variable, both within and between individuals. Mean pre-dive heart rate was 61.42 bpm ± 6.07 S E M . End dive heart rate (Min 5) was 29.17 bpm ± 4.90 S E M , with an average diving heart rate (Min 1-5) of 31.83 bpm + 3.59 SEM. Mean diving heart rate was not significantly different from diving heart rate at any given minute during the dive (Min 1-5, F  (5jl8  )= 0.61; P = 0.69). Pre-dive,  postdive or diving heart rates did not correlate with mass or with stroke volume during any state. However, diving SV correlated with the ratio of diving HR to predive HR (P = 0.01, R = 0.99; ratio is indicative of the degree of bradycardia expressed by the individual: mean diving HR/predive HR; Fig. 4.3).  Stroke Volume (SV) Mean resting SV was 104.94 ml ± 4.12 S E M , while S V during the dive increased significantly to a mean value of 126.12 ± 3.93 S E M (Table 4.2). These data are in contrast to the findings of previous studies on pinnipeds, which indicate either the maintenance or decline of stroke volume during the dive (Table 4.1). A significant decrease in SV from resting was observed during the postdive phase, with mean values of 81.81 + 3.34 S E M ( A N O V A , P < 0.0001, F  (2  ,9)=  33.85). Stroke volume data from one animal (Kate) was  acquired rapidly enough to facilitate flow pattern evaluation during individual cardiac cycles (Fig. 4.2). Blood flow (ml) over a cardiac cycle during the dive exhibited a distinct peak in the first quarter of the cardiac cycle, then decreased momentarily before leveling off. In the second half of the cardiac cycle (frames 17-32), flow did not fall to zero at any point (average flow/frame 2.23 ml ± 0.08 SEM). Postdive SV exhibited a diminished peak and did not show a distinct post-peak reduction.  70  CO XI  oo  >  CN  tin  cu  •3  o  C .£  15  s  CN VO Ov VO  o  • C XJ  ft  7 3  3  ov vol  x) 4)  O 3 U  OV  cu  o 0) & u.rt rt rt ,|_» O 00 X)  c rt  rt c  •c  > JSrt<uX)rt3„ w 4) bO c  £  'S  o ^ Jrt3 3  w  Sv CD  (U  CO  OO  •3 IS cn u  I  vo o  vS  VO cn  lis  X <D  rt «s X) rt  rt X l  rt o  x)  rt trt l_L|  3 XJ  > ^ X!  IS  >>  cn £  cu  vS  CN  o al  > ^ rt o 2 •£> 1- CD  ISI  > -3  O 1) 1)  cu  al  vol  >  C ^5 X! X!  ool  ml  CN  Q  rt H  cn l+l  71  TJ  a  P  p  -§ 3 D > o  "2 *— CNJ CO T}" I l I I CM CM CNJ (NJ  2 a  (D CD CO CD CD « > > > > > Q. Q Q b Q b  1 0  § W "CO O° E  3 (DC rt * ** f i > .s ®  2 7  3  J3  ^  p rt n_ p : P O SS  I l l s 5 *  P  c  7  en  £ oa X l <3  CN  CD JZ «" « J r-  O P XJ w> *  •—  () S = C < —J= — 1 cd ^ IS « Jj 6 >  5* Sp \  rt rt "5 £ 3 —» o  1  rt  £ o3 2  Sgf £ i l l !  2 O S § 2 rt  — o TJ  4>  rrt  >  -  O |  O  cu *c o ,rt g C P  C v—«x» rt 3 O  oo  to  o  co  CD  CNJ  r-  I g  CN  (|LU) aujn|0A '—  3 bJQ  E  72  C°  Cardiac Output (CO) Cardiac output in these experiments was derived from SV and HR. In contrast to previous studies, the data presented here do not show a significant difference between diving CO (4010.55 + 387.49 SEM) and resting C O (6530.01 ml ± 1017.85 SEM)(t-test, P = 0.06, t = 2.31). However, lack of statistical significance is likely due to a high variability within the data and small sample size rather than a lack of difference between the two situations. In addition, these animals did not exhibit a dramatic decrease in heart rate, which would in turn result in a less profound change in cardiac output from the predive to diving periods.  Discussion Diving heart rate data obtained from elephant seals during the predive, dive and postdive periods were surprisingly high, with a mean diving HR of 31.83 bpm + 3.59 S E M . The animals were remarkably calm during the experimental procedure, often voluntarily placing their nostrils underwater as the helmet filled, then "surfacing" into the air pocket for one last breath just prior to complete filling of the helmet. This may be due to the experience obtained during the PCr experiment (Chapter 5), where they were exposed to the same handling procedures and forced dive protocol.  The calm demeanor of the seal may partially explain the lack of profound bradycardia that normally accompanies forced diving protocols, however a more physiological explanation may be related to thermoregulation. The restraining jacket, board and halfpipe restricted cutaneous heat loss from the animal and may have resulted in an elevated core temperature. As the animal was only subject to facial immersion and not whole body submergence, heat retention may have become a physiological challenge over the course of the experiment. Temperature regulation in phocids is accomplished primarily through peripheral vasoregulation (McGinnis et al, 1971), therefore a decrease in total peripheral resistance due to heat dissipation may have resulted in a higher diving heart rate (Q = MABP/TPR). ft  73  However, experiments by Eisner et al (1975) showed that diving-induced peripheral vasoconstriction in a harbor seal overrides the increased superficial blood flow normally elicited in the presence of a thermal load. These experiments involved a single animal subjected to environmental temperatures up to 25° C and hypothalamic temperature manipulation, therefore the findings may not be directly applicable to the situation described above.  The most striking feature of this study is the elevation of SV during the dive. To date, all pinniped CO studies have indicated either an overall maintenance or a reduction in diving SV when compared to resting levels. Elevation of SV from a resting mean of 102.94 mis ±4.12 S E M to a diving mean of 126.11 mis ± 3.93 S E M suggests that inotropic stimuli during the dive are more significant (or less inhibited) under these experimental conditions than in previous studies. In all previous experiments reporting pinniped SV calculations, the degree of bradycardia was considerably greater than the data presented here. A n increased strength of myocardial contraction is normally thought to accompany bradycardia and compensate for the reduction in cardiac output caused by a decrease in cardiac frequency. This positive inotropic effect is achieved through the increased left ventricular filling time that is associated with extended diastole, causing increased myocardial preload and ventricular performance (Starling relationship). A n increased ventricular volume also stimulates the ventricle to contract more rapidly, further augmenting the interbeat filling time (Frank-Starling Law). However, this expected characteristic relationship between HR and SV is disputed by Eisner et al (1985), who support the existence of a diminished strength of myocardial contraction with reduced frequency (Koch Weber and Blinks, 1963; Eisner et al, 1985). This relationship is known as the treppe effect, where an increase in HR progressively enhances the force of ventricular contraction (SV) and leads to a higher oxygen demand (Opie, 1997). The resulting changes in preload that accompany elevated HR invoke the Frank-Starling law, which opposes the force-frequency effect and corrects  74  for the treppe effect. The degree of influence that each of these effects has on pinniped S V is not known.  Elephant seal SVs do not correlate with HR during the predive, dive or postdive state. However there exists a correlation between S V and the ratio of diving HR to predive HR (P = 0.01, R = 0.99; ratio is indicative of the degree of bradycardia expressed by the individual: mean diving HR/predive HR). Animals who exhibit a more profound bradycardia have a higher SV than individuals who express a less profound reduction in HR (Fig. 4.3). Although the degree of bradycardia is correlated with SV, the absolute HR during the dive is not, suggesting that it is the degree of cardiac response to diving that is driving the increase in S V rather than the absolute rate of contraction.  If a diving seal is able to maintain cardiac contractility during the systolic phase, then the responsibility for altering stroke volume is shifted to the flow of blood entering the ventricle. The extended diastolic filling time that accompanies bradycardia may partially account for the increase in SV, but the reasons for the relationship are likely to be more complex. Right ventricular dilation is observed during diving, indicating a significant increase in preload (Hoi et al, 1975; Blix and Hoi, 1973). Contraction of the caval sphincter that occurs during diving in phocid seals (Chapter 3) may serve as a mechanism for controlling the level of preload. Hepatic sinus filling accounts for the increased volume of blood resulting from splenic contraction, but may not attenuate the increased preload resulting from peripheral vasoconstriction. Thus during diving, some increase in ventricular pressure would be anticipated, but separating the effects of peripheral vasoconstriction from the abrogating action of the caval sphincter are beyond the scope of these data.  75  115 0.30 L  ' 0.40  1  '  '  0.50  0.60  0.70  1  0.80  Bradycardia (mean diving HR/predive HR)  Figure 4.3.  Diving SV correlates with the degree of bradycardia expressed during the dive (ratio of mean diving H R to mean predive H R (P = 0.01, R = 0.99). Although the degree of bradycardia is correlated with SV, the absolute HR during the dive is not, suggesting that it is the degree of cardiac response to diving that is driving the increase in SV rather than the absolute rate of contraction.  76  Pressure sensing corpuscular bodies are found in the phocid caval sphincter, and may act to regulate flow through the vena cava and into the heart (Blessing et al, 1969). An extended diastole may lead to a longer period of caval sphincter relaxation, increasing ventricular filling and supporting the relationship of increased SV with increased diving bradycardia. However, as there is little evidence to suggest the coordinated efforts of the sphincter and cardiac rhythm during a dive, this suggestion is highly speculative.  Previous studies of pinniped COs all report significantly higher values than those predicted by allometric formulas (Table 4.1; Stahl, 1967). Although it was originally proposed that the metabolic rates of marine mammals scale differently in relation to body mass than terrestrial mammals (Piatt and Silvert, 1981), this proposition was soundly disputed (Lavigne et al, 1985; 1986). Eupneic HRs in phocids are reportedly higher than the predicted relationship (M "° ; Castellini and Zenteno-Savin, 1997), however the increase 25  b  in HR is not enough to account for the elevated CO values. A n increase in SV over predicted values is required in order to produce the resting C O values reported for pinnipeds. As seal hearts scale isometrically with body mass, it is unlikely that resting SVs would be 2-3 times higher than predicted values. One explanation for the discrepancy in measured vs predicted values is that the allometric equation is based on resting measurements from a myriad of species. The definition of the phocid "resting" condition is elusive, as many species spend a considerable amount of time in the submerged, apneic and bradycardic condition. In general, most studies (including this one) loosely define a quiescent eupneic period as "resting", introducing considerable variability into this evaluation. It is plausible that the CO measurements from previous pinniped studies were obtained during a time when the animal was not in a "resting" state, thus creating a disparity between the predicted CO and the measured value.  77  The possibility of error in CO measurements using thermodilution or indocyanine green dye technique is discussed by Sinnett et al (1978). Comparison of CO results from dye vs thermodilution techniques during the dive indicated disparate results, likely caused by an extremely rapid recirculation of dye through coronary circulation. Sinnett et al (1978) stated that interpretation of dye curves was difficult, if not impossible, and may explain the significant variance found in Murdaugh et al's (1966) SV data. Thermodilution techniques are subject to potential error caused by changes in blood temperature, which has been observed to occur in diving seals (Sinnett et al, 1978). In Ponganis et al (1990), the level of work required by the animals may have elevated the temperature of circulating blood, introducing potential error in thermodilution measurements.  This is not to say that M R assessment is without error. Inconsistencies in the gating ability of the experimental setup resulted in some loss of accuracy in the SV and C O measurements. Although the SV data are in close agreement between individuals, resolution of individual stroke volume flow pattern was diminished by the inability of the M R unit to recognise the QRS complex and trigger an acquisition. Almost all cardiac MRI techniques employ a data acquisition protocol that is gated by E C G which serves to eliminate motion artifacts due to the contractile motion of the heart. However, gated cardiac studies are reliant on the ability of the M R system to accurately assess each point in the cardiac cycle and require a significant amount of time to acquire a series of images. In this study, a spiral cine sequence using 16 interleaves was selected, where each of the 32 frames represents a different point in the cardiac cycle. Application of this technique to 2D slice data allows for computation of flow over the entire cardiac cycle, but reconstructs the cycle by interleaving data from different beats. This method provides excellent results when heart rate is consistent, but exhibits ah inherent degree of error when heart rate is erratic. For the most part, the animals used in this study maintained a steady HR during the dive except during periods of struggling, where HR would increase during movement, then  78  drop for a period of 5-10 seconds in the post-movement phase before leveling off. However, the advantage of the interleaved method is the reduction in acquisition time, increasing the likelihood of acquiring a complete cardiac series between momentary bouts of struggling. When the acquisition of a clear E C G signal was occasionally impaired by animal movement or lead displacement due to movement, the gating sequence would "stall" until a suitable E C G signal was obtained. The reconstructed cardiac cycle would then consist of images obtained over a period of one to two minutes.  In one animal (Kate), the combination of a clear E C G signal and lack of struggling during the dive resulted in rapid acquisition of SV measurements (Fig 4.2). Aortic blood flow during the dive peaked following ventricular contraction and aortic valve opening. Peak flow was followed by a deflection of flow approaching zero, which corresponds to closure of the aortic valve and phase lag effects (Nichols and O'Rourke, 1990). Flow was then maintained at a relatively constant rate throughout diastole, as evidenced by the latter half of the flow trace. The maintenance of flow during diastole is largely due to the windkessel effect of the aortic bulb, which continues to deliver blood via the elastic recoil of the stretched arterial walls (Jones, 1992).  Postdive SV values are significantly different from those acquired in the predive and dive periods ( A N O V A , F  (2j6  ) = 33.85, P <0.0001). A blunted peak flow is observed in the first  10 frames of the cardiac cycle, and the post-peak reduction in blood flow was notable in its absence when compared to the diving flow trace. The reduced volume of blood moving through the aorta during a cardiac cycle in the postdive period may be related to higher frequency of contraction, where ventricular filling may be restricted. Eisner et al (1985) found that myocardial segment dimensions were significantly reduced during diving and decreased progressively with increasing dive time. Myocardial segment dimensions are indicative of the force of contraction exerted by the cardiac muscle and are used to represent  79  contractility of the ventricle. Eisner et al's study also showed that the major reduction in segment dimensions occurred in diastole, while systolic dimensions remained relatively unchanged. A possible explanation for elevated diving SV is that the maintenance of myocardial segment dimension (and thus cardiac contractile force) combined with longer ventricular filling time could result in higher diving stroke volumes. However, these findings also indicate a higher myocardial distensability during diastole in the nondiving period, which would suggest a greater ventricular filling and would conceivably result in a higher SV.  One salient detail that has been overlooked in previous studies is the effect of diving on the functional aspect of CO. In most systems, comparison of CO between a control and experimental condition will illustrate differences in systemic oxygen distribution and availability. These comparisons are based on the reasonable assumption that the oxygen content  (CA02)  of arterial blood leaving the heart remains relatively constant. However,  when examining variations in phocid cardiac output, one must take into account the effect of increased haematocrit and continued haemoglobin desaturation that accompanies diving. The blood that is expelled from the heart in the predive state does not carry as much oxygen as that expelled in the first few minutes of diving, therefore with respect to oxygen distribution, SV and CO are not directly comparable between control and experimental conditions. This is further complicated by the fact that, after an initial rise in C A 0 2 caused by the increase in haematocrit, the blood oxygen content in the latter part of the dive continuously decreases as haemoglobin desaturation occurs (Qvist et al, 1986). Although the effect of decreased C A 2 on cardiac contractile force in seals is not known, any 0  chemoreceptor effect elicited by a reduced oxygen content would likely be overridden by the vagal input.  80  The question still remains as to what would be the most beneficial condition for a diving seal: increase SV and incur the cost associated with a greater force of contraction, or maintain a higher heart rate and incur the cost associated with an increased frequency of contraction. Clearly, a reduction in cardiac frequency occurs during diving and is accompanied by an increase in total peripheral resistance. However, in these experiments, the degree of bradycardia is considerably less than that reported in the seven previous studies identifying CO measurements for diving pinnipeds and may be the reason for the observed disparity in SV results. As the diving HR values recorded in this study are close to those observed in freely diving animals, the data presented suggest that SV may increase during free dives when bradycardia is less profound, rather than decrease as suggested by previous studies. In addition, the likelihood of error in SV measurement due to technical culpability is reduced using MRI, and reported CO measurements fall much closer to predicted allometric values, supporting an increased S V during diving. These findings indicate that the effect of diving on stroke volume is correlated to the degree of bradycardia, suggesting that HR alone may not be an accurate indicator of oxygen distribution in a diving animal.  81  Chapter 5  Phosphocreatinefluxin the locomotor muscle of diving northern elephant seal pups Introduction Skeletal muscle comprises approximately 35% of a seal's total mass and exhibits a large dynamic work range (Ponganis et al, 1993). The ability of the muscle to respond to rapid changes in energy demand whilst suffering the metabolic consequences of diving-induced peripheral vasoconstriction provides an interesting opportunity for the evaluation of high energy phosphate flux during ischaemia. To evaluate the myriad of reactions involved in maintaining muscle cellular [ATP] during ischaemia, the relative contribution of each component must be assessed. The extent of aerobic metabolism may be elucidated through the rate of myoglobin desaturation and perfusion, while the anaerobic contribution is related to the depletion of PCr as well as the formation of lactic acid.  Diving mammals exhibit myoglobin concentrations that are considerably higher than terrestrial animals (Castellini and Somero, 1981; Kanatous et al, 1999). As a crucial aspect of the diving response involves adjustments in cardiac output distribution favoring hypoxia sensitive tissues, it follows that muscle tissue is often left with reduced blood oxygen delivery. The presence of relatively large local oxygen stores in the form of myoglobin would extend the aerobic metabolic ability of the tissue and lead to a reduced dependence on anaerobic metabolism. Scholander and his colleagues (1942) observed that muscle oxygen content approached zero after ~8 minutes of forced submersion in harbor seals, with a rise in lactate occurring only after all muscle oxygen stores were consumed. These findings indicate that blood flow to the periphery, and hence resaturation of myoglobin, did not occur during the forced dive situation. Microsphere studies of forcibly dived Weddell seals indicate that blood flow to the periphery drops to less than 10% of resting, supporting  82  the suggestion of profound muscle ischaemia (Zapol et al, 1979). During total ischaemia, the time required to completely desaturate the myoglobin stores may be equated with the tissue's metabolic rate if no anaerobic metabolic byproducts have appeared. However, although it is readily apparent that anaerobic pathways are employed when muscle oxygen is depleted, the point at which the tissue begins to use these pathways may occur prior to total oxygen depletion, thus complicating the evaluation of muscle metabolism in ischaemic muscle.  In addition to increasing absolute oxygen concentration within the muscle, myoglobin also facilitates the diffusion of oxygen through solution and may have a significant role in enhancing oxygen flux between the cytoplasm and mitochondria (Honig et al, 1991). The presence of myoglobin may also assist in buffering intracellular oxygen concentrations during different metabolic states. In a diving seal, peripheral vasoconstriction and progressive hypoxemia leads to a reduction in oxygen delivery and eventually results in a drop in cellular oxygen content. The ability of myoglobin to extract oxygen from the blood and facilitate transport through the cytoplasm may assist in maintaining sufficient cellular P 0 levels and oxygen delivery to the mitochondria when there are changes in oxygen 2  supply and demand (Honig et al, 1991).  During free dives, it is thought that a seal maintains some degree of blood flow to the locomotor muscle. As heart rates recorded from freely diving seals exhibit a less profound degree of bradycardia than those recorded during forced dives, flow studies on forcibly dived pinnipeds may overestimate the degree of vasoconstriction. The higher heart rate experienced by free diving seals results in a comparatively higher cardiac output, supporting the concept of reduced total peripheral resistance. In contrast to the forced immersion of a restrained seal, the locomotor muscles of freely diving animals experience an increased oxygen demand, requiring either some degree of maintained blood flow,  83  pulsatile resaturation of oxygen stores, or an increased reliance on anaerobic pathways. In experiments conducted on freely diving Weddell seals (Kooyman et al, 1980; Qvist et al, 1986), elevated blood lactate levels are not observed when the total dive time falls within the theoretical aerobic diving limit, suggesting.that aerobic metabolism provides sufficient energy to meet the rise in demand caused by locomotor muscle contraction. Myoglobin stores provide -60 ml 0 /kg (Guyton et al, 1995), which would support the metabolic rate 2  of actively contracting locomotive muscle for approximately 7.3 minutes (active muscle metabolic rate = - 14 ml 0 /min/kg; Castellini et al, 1992), yet these animals consistently 2  demonstrate aerobic dives in the 20-25 minute range. The combination of increased heart rate, an inferred decrease in total peripheral resistance, and an assumed rise in ATP consumption due to muscular contraction all point toward the presence of some degree of oxygen delivery to the muscle during a dive.  This supposition is supported by Near Infrared Spectrophotometry (NIR) studies by Guyton et al (1995) on Weddell seals, which indicate that the end-dive myoglobin saturation was typically 40-60% of resting, even when the dive exceeded the aerobic diving limit. The maintenance of a constant partial saturation of myoglobin suggests that some degree of sustained oxygen exposure is occurring or that total desaturation of myoglobin is somehow inhibited. In addition, the measured rates of desaturation that were recorded during diving (dives < 17 min, 5.12 ± 4.37%/min; dives > 17 min, 2.54 + 1.95%/min; Guyton et al, 1995) are not consistent with a situation of total ischaemia, where desaturation of active locomotor muscle should proceed at -20%/minute (Castellini et al, 1992). In some instances, a reversal of desaturation during the dive is observed, most likely the result of a brief alleviation of vasoconstriction. These findings provide some support for the suggestion of pulsatile flow to the muscle tissue during diving.  84  Preliminary ' H (proton) MRS investigations of seals indicate that M R technology may be used to monitor myoglobin levels in locomotor muscle. During spontaneous 8-12 minute apneas, northern elephant seals exhibited nonlinear myoglobin desaturation with transient resaturation events. This pilot study demonstrated that during apnea, reduction in blood flow to the muscle is accompanied by desaturation of myoglobin (Ponganis et al, 1999).  Although the dynamics of myoglobin in oxygen transfer are not completely understood, it has become apparent that under conditions of increased oxygen'demand (exercise), reduced delivery (hypoxemia, anemia), or a combination of both, muscle tissue remains at a myoglobin desaturation level of -50%. In studies conducted on human quadricep muscle, a 10 minute period of cuffed ischaemia resulted in a steady increase in the deoxymyoglobin signal, plateauing at the six minute mark (Richardson et al, 1995). It is assumed that during total ischaemia, complete desaturation of myoglobin would occur with the maximum signal equating to 100% desaturation. During unweighted exercise (-30% V 0  2 m a x  ) , the  deoxymyoglobin signal rose and plateaued rapidly at 38%. When work rate was increased to yield 50% V 0  2 m a  x,  the deoxymyoglobin signal rapidly increased, plateauing within 20 s  to - 50% of the maximum signal and maintaining this pattern through four stepwise progressive increases in W R until W R  m a x  was achieved. In hypoxic exercise (12% 0 ) , the 2  deoxymyoglobin signal also increased rapidly, plateauing at -60% of the maximum. This pattern was seen at all work rates to W R  m a x  . In both hypoxic and normoxic conditions,  cessation of exercise produced a rapid resaturation of myoglobin (within 20 s) (Richardson etal, 1995).  Mole et al (1999) conducted a similar study on humans, but selected calf muscle as the site of evaluation and used a plantar flexion exercise protocol to reduce motion artifact. In contrast to the findings of Richardson et al (1995), this study reported that the deoxymyoglobin signal intensities change in proportion to power output or V 0 . 2  85  However, their V 0  2 m a x  results are similar, yielding a maximum myoglobin desaturation of  48% (Mole et al, 1999) and 51% (Richardson et al, 1995). This level of desaturation equates to a maintained intracellular P 0 of 2.9-3.1 Torr, providing sufficient oxygen for 2  cytochrome oxidase activity in the mitochondria. Although these studies provide contrasting results with respect to the relationship between work and myoglobin desaturation, it is the striking similarity of -50% myoglobin desaturation at V 0  2 m a x  that  provides the most provocative finding : oxygen availability does not appear to limit V 0  2  during exercise.  The results of these experiments echo the findings of phocid locomotor muscle research, with cuffed ischaemia being analogous to a forced dive situation (total myoglobin desaturation) and exercising muscle paralleling the situation of a free diving seal, achieving a maximum myoglobin desaturation of 40-60%. However, in contrast to the rapid onset of desaturation reported for exercising human muscle, Guyton et al (1995) found that seal myoglobin saturation declines monotonically throughout the dive. As the myoglobin levels in a seal are considerably higher than in a human, the progressive decline may be indicative of a steady rate of oxygen consumption from the myoglobin stores, leading to the eventual plateau. The estimated oxygen consumption rate of a contracting locomotory muscle exceeds the measured desaturation rate, suggesting that the muscle bed is likely subject to continuous low level perfusion. The inevitable systemic hypoxemia would also contribute to the decline in myoglobin saturation as the dive progressed, leading to a reduction in oxygen delivery for replenishment of myoglobin stores.  Preservation of oxygen availability to the mitochondria is instrumental in maintaining cellular [ATP] over an extended period. When the process of oxidative phosphorylation is compromised by a final electron acceptor insufficiency, the cell must undertake immediate action to prevent a deleterious reduction in [ATP] and potential loss of cellular function. In  86  spite of a relatively small cellular pool of ATP and dramatic changes in energy demand, [ATP] is maintained over a large range of activity. When ATP production is not sufficient to meet consumption (e.g. in early stages of exercise), cellular homeostasis is achieved through the continuous and efficient replenishment of ATP by the creatine phosphokinase reaction. The transfer of a high energy phosphate from PCr to A D P acts as the main ATP regeneration pathway when cell activity exceeds the rate of ATP synthesis by oxidative phosphorylation (Walliman et al, 1992).  PCr " + MgADP" + H 2  +  <=> Cr + MgATP ' 2  Synthesis of ATP through the C P K reaction (30 umol/s/g) is much higher than the maximal rate produced by oxidative phosphorylation (2.5 umol/s/g), and is therefore a more rapid means of replenishing A T P consumed during high levels of cellular activity, such as rapid and continuous muscle contraction. Once oxygen delivery has increased to meet demand, a new steady state may be achieved.  In addition to buffering [ATP] within the cell, the CK/PCr system acts as a proton buffer, consuming H ions and thus preventing acidification within the cell. The C P K reaction +  also prevents a rapid rise in free cellular [ADP] that would normally accompany high ATP turnover, reducing the net loss of cellular adenine nucleotides through the IMP deamination process and avoiding the inhibition of cellular ATPases (Walliman, 1992). Armed with the kinetics of the C P K reaction, it is a logical assumption that when a cell is faced with rates of ATP consumption that exceed the maximal rate of production by oxidative phosphorylation, cellular [PCr] will decline.  The concept of PCr as a "temporal" buffer of ATP and A D P levels during high ATPase activity is well accepted, but the transport effect or "spatial" buffering is the focus of debate  87  in the literature (Meyer et al, 1984; Sweeney, 1991). Some follow the doctrine of pure kinetic models, while other research supports the facilitated diffusion effect of PCr in "shuttling" high energy phosphates from the mitochondria to the site of A T P hydrolysis (Bessman, 1972; Walliman et al, 1992). The identification of C P K isozymes and variations in subcellular location of the enzyme led to the suggestion that PCr acts as a link between sites of ATP production and consumption, representing an integrated 'energy distribution network' connecting sites of A T P production (mitochondria and glycolysis) with sites of ATP consumption (ATPases)(Walliman et al, 1992).  Both the temporal and spatial buffering models of PCr dynamics involve the effect of cellular adenylate concentration on the creatine kinase reaction. However, PCr depletion will also occur in response to an increased proton load within the cell. Alteration of the intracellular [H ] occurs as a result of A T P hydrolysis, inosine monophosphate (IMP) +  formation, C 0 accumulation, and lactic acid formation. Arthur et al (1997) modeled the 2  effects of intracellular metabolites on pH in fish white muscle and discussed the stoichiometric relationships involving PCr and [H ] within the cell. Their model suggests +  that PCr is profoundly affected by alteration of intracellular pH, indicating that an observed decline in PCr is not necessarily indicative of a change in cellular ATP consumption (Fig. 1.1).  Evaluation of phosphagen kinetics in the locomotor muscle of diving elephant seals revealed information on muscle metabolism during diving-induced hypoperfusion. In the ischaemic muscle of a diving seal, aerobic metabolism is sustained through the substantial oxygen stores located in the myocytes. If the period of submergence is within the animal's aerobic diving limit, cellular acidosis resulting from lactate formation may be avoided. However, oxidative phosphorylation generates C 0 , which would accumulate in the 2  88  hypoperfused muscle. The increase in cellular P 2 results in respiratory acidosis, C 0  increasing the [H ] within the cell and driving the creatine kinase reaction to the right. +  M R spectroscopic analysis during predive, dive and postdive periods was undertaken to evaluate the effect of diving on intracellular [PCr] and pH in locomotor muscle during ischaemia.  Methodology In October of 1997, 2 male and 5 female ten-month-old elephant seals were collected from Afio Nuevo, California and held at Long Marine Lab, U C S C for the duration of their captivity (average = 97.4 kg ± 4.25 SEM). On the day of the experiment, animals were transported by truck to the Lucas Center for Advanced M R Technology at Stanford University (transport time ~ 2 hours). Diving protocols, animal restraint and animal husbandry procedures were undertaken as outlined in Chapter 2. Six seals were subjected to five sequential dives and one seal (Montague) was subjected to three sequential dives (diving time ranged from 7 min 15 sec to 9 min 35 sec; interdive time 19 min 27 sec to 24 min 26 sec). A l l seven seals were used in the analysis of PCr depletion and intracellular pH during diving, but the lack of heart rate and respiratory data from one seal (Ophelia) reduced the sample size to 6 for the respiratory analysis.  Prior to placement in the magnet, each animal was outfitted with E C G leads and respiratory bellows as described in Chapter 2. Respiratory and E C G data were collected on a Macintosh HSI with a 8 channel Powerlab™ (ADInstruments) interface and a M L 132 BioAmp. Heart rate, respiratory frequency and amplitude were analysed using Chart™ (ADInstruments). Heart rate was analysed by recording interbeat intervals. Respiratory frequency was obtained by measuring the peak to peak interval from maximum amplitude data. Maximum-minimum differences were used to calculate respiratory amplitude.  89  P-NMR measurements were performed on a 1.5T system (Signal Horizon Echo Speed, G E Medical Systems, Milwaukee, WI) operating at 25.86 M H z for PCr. Data were acquired using a transmit/receive surface coil (diameters 20 cm and 10 cm respectively) placed on the dorsal surface of the animal. The lumbar region of the longissimus dorsi muscle was selected as the site of interest as it is the largest muscle involved in phocid locomotion (Tarasoff et al, 1972). By selecting a site distal to the diaphragm, respiratory motion artifacts were reduced. Localizing images in the axial plane were obtained to ensure the site provided sufficient muscle mass for spectroscopy. The coil was positioned over the longissimus dorsi muscle with the center of the coil located 10 cm off the midline and 10 cm cranial to the ischial tuberosity (Fig. 5.1). A mark was placed on the fur of the seal corresponding to the center of the coil, and three female ends of plastic clips were threaded onto webbing and were then epoxied over the mark. The male ends of the clips were attached to webbing and epoxied 12 cm out from the female clips. The coil could then be attached firmly to the animal, ensuring repeatability and site fidelity between sampling dives.  Figure 5.1.  Coronal image of the lumbar region of a northern elephant seal pup. P coil was placed cranial to the white line, approximately 10 cm off the midline.  90  Data were acquired continuously during the 5 minutes immediately proceeding the dive, throughout the 8 minute dive and continued for a minimum of 7 minutes during the postdive period (20 minute acquisition). The spectral width was 5 KHz with a TE of 7 seconds and a TR of 1 second at 8 N E X , resulting in a single spectrum generated every 8 seconds. As a result, the 20 minute experimental session produced 150 lines of 1,024 complex points. Analysis of PCr spectra was undertaken using Igor Pro 3.13.  Intracellular pH was determined by calculating the distance between the maximums of the Pi and PCr peaks (3) as follows:  pH = 6.75 + log [(d - 3.27)/(5.69 - d)]  where d is given in ppm.  Collection of blood samples during diving could not be achieved in the M R unit as the caudal end of the animal was not accessible during M R S data acquisition. Consequently, the restraint procedure and diving protocols were repeated at Long Marine Lab and blood samples were drawn from the extradural intervertebral vein during the predive, dive and postdive period (predive samples were obtained during initial needle placement; dive samples were drawn at 1, 3,5 and 7 min; postdive samples at 1,3,5,7,9 and 15 min). Serial blood samples were collected using a 14 gauge 13 cm spinal needle inserted between the lumbar vertebrae (approximately L4-L5, 5 cm cranial to the pelvis). A vacutainer blood collection tube holder was attached to the needle via a multisampler hub and samples were collected into a heparinized Vacutainer tube (haematocrit) and a non-additive tube (lactate) for analysis.  91  The heparinized tubes were placed on a blood tube oscillator to prevent clotting and red cell settling prior to the transfer of a subsample into heparinized microhaematocrit tubes. Once the microhaematocrit tube was sealed (Critoseal), the samples were centrifuged at 4500 rpm for 15 minutes and the haematocrit reading was obtained via comparison of the packed red cell level with a standard haematocrit card. Blood samples in the non-additive tubes were allowed to clot for 30 minutes, then spun at 4000 rpms for 10 minutes and the plasma was pipetted into plastic cryosample vials and placed in a -80°C freezer. The samples were subsequently shipped on dry ice back to the University of British Columbia for lactate analysis using U V spectrophotometric assay. Plasma samples were combined with a hydrazine-containing glycine buffer (pH 9.2; Sigma 826-3), NAD (N-7004,MW 663.4) +  and distilled water. After initial spectrophotometric analysis and the addition of lactate dehydrogenase, the following reaction was run to completion (45 min at room temperature):  lactate + N A D + hydrazine -V +  actat£  tW *™™^ 0  pyruvate hydrazone + N A D H + H 0 2  The lactate concentration was then determined by the change in absorbance caused by the formation of N A D H .  Statistical analysis was conducted using JMP 3.2.1. Relationships among heart rate, respiratory frequency and end-dive PCr were determined by A N O V A and Tukey-Kramer HSD. Significance of differences among heart rate, respiratory frequency, and PCr levels were evaluated using pair-wise analysis of variance and paired t-tests.  92  Results Heart Rate Heart rate was recorded from six animals throughout the experimental protocol, with predive heart rates corresponding to the five minute period immediately preceding the first dive. Mean pre-dive heart rate was 58.41 ± 4.62 S E M and declined from Dive Min 1 through to Dive Min 7 in all individuals (Table 5.1). Near the end of the dive (Min 7), mean heart rate was 23.56 bpm ± 3.71 S E M . Pre-dive, postdive or diving heart rates did not correlate with PCr signal intensity, PCr/Total P ratio, mass, pre-dive respiratory rate or postdive respiratory rate. Pre-dive heart rate correlated with postdive heart rate at Min 1-7 inclusive (P < 0.003 - 0.039; R = 0.74 - 0.90, N = 6). Reduction in diving heart rate expressed as % of pre-dive heart rate averaged 39.43% ± 3.74 S E M for Dive Min 7.  Respiratory Frequency and Amplitude Respiratory frequency (breaths per minute) in the pre-dive period averaged 5.86 + 1.09 S E M (Table 5.2a). In the postdive recovery period, respiratory frequency increased to an average of 15.57 breaths per minute ± 1.04 S E M during postdive Min 1. Breathing frequency decreased significantly during the postdive period and after Min 3 it was not significantly different from predive levels ( A N O V A , F  ( 5 < m  = 7.12, P = 0.0002; Tukey  Kramer HSD, P = 0.05). Respiratory amplitude during the predive period averaged 2.44 + 0.10 S E M . As the bellows were not calibrated to tidal volume, the units are arbitrary and serve only as a comparative index for assessing changes between sampling periods. The initial 4 or 5 breaths following the dive were of the highest amplitude, exhibiting a rapid reduction in amplitude as the postdive period progressed. Over the first seven minutes of the postdive period, respiratory frequency declined and amplitude increased (Table 5.2b). However, respiratory amplitude and respiratory frequency were not correlated during either the predive or postdive period. 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PD  § rt  32  < Oi  COV  CO CO OV o CS  rt rt  oo CS  >n Tt CS o CS o l CS oo oo in —< CS  CS  CS  rt  lH  oi  oi  r~  t Tt  oi  oi  M  U  Z, rt £ CN  O  o  r-  vo  Oi  CS  CN  CS  oi  co  in  CO oi  in  u Oi u ft.  ft.  u e  <  <  S O*  o»  rt H  <  Ariel Hamlet Hecate Juliet Kate lontague VERAGE  (8  mplitude  «  Dive  "p .« x p  h  95  Struggling During the dive, deflections in the amplitude of signal obtained from the respiratory bellows reflected periods of struggling. The amount of struggling (min) that occurred during a dive was not correlated with changes in PCr, respiratory frequency or amplitude, or heart rate. However, the amount of struggling was positively correlated with the mass of the animal and is the only parameter measured that significantly correlates with mass (P < 0.018; R = 0.79). 2  Lactate Formation Mean predive blood lactate values were 2.92 mmol/1 ±0.19 SEM). The elevated level of blood lactate in the predive state likely reflects the muscular contraction of struggling during the restraining procedure. Once restrained, the animal was usually calm but struggled considerably during the insertion of the needle. Predive samples were slightly but not significantly higher than dive Min 1 (2.76 mmol/1 ± 0.17 SEM), likely due to the stress of restraint and needle placement. Blood lactate at postdive Min 3 provided the highest values (mean 3.62 mmol/1 ±0.12 SEM) indicating a rise of 0.7 mmol/1 from predive levels (Table 5.3a). Overall lactate formation (peak washout value - predive value) was 0.70 mmol/1 ± 0.26 SEM. Lactate concentration does not vary significantly at any point throughout the experiment ( A N O V A ,  F  (10  , 3) 4  = 2.30, P = 0.028).  Haematocrit Predive mean haematocrit levels were 67.4 ±1.71 S E M . Haematocrit levels showed a general trend toward elevation with the highest values recorded during postdive Min 1 sampling (69.5 ± 1.09 S E M ; Table 5.3b) but no significant differences in values was observed, possibly due to the elevation of predive haematocrit values resulting from the stress of forcible restraint and needle placement. Blood samples were obtained shortly after restraint, reducing acclimation time between handling and sampling periods.  96  l/l  cfl  >  O-S  Tf  >n  VO cs CO CO  o  CO  o  CO  CO  o  CO  CN  m  oi  ro C H £  O  Ov  o-S  _C  "C  >n  CN CO  >o CO  cuS  3 T3  OV  O  Tf  CO  o  CN  CH  co co co  in  3  73  Ov  T) in I CN  C ed  •J3  CO  OH  13  CM  Tf co  ro  •e  Q-2  o c  o  t-; CO  oo CO CO  *i n  oo  ^3 CD  c  o  00  CD 3  cfl  "S3 §  rH  CM  vo > cn +1 •c o o CO cfl o 73  Tf  m  VO CO CO  CN © CO  m  OV CO  o  d  i—i  ro  Tf co  Ov Ov CO  CO  CN VO  oo  CO  ro  Tf  CN  CO CO CO  S CN  in  co  O  Tf ro  oo vq CO  in  CO CO  CO  bt) CS sCO > 03  l-H  CO Ov CN  >n  OV CN  Tf  oo  ro ro ro ro  S  VO  in >  B  co CO  Tf ro  VO  Ov T Tff ro  CO  CO  oo  q  ro  >  r-  B  ir,  Ti-  CN  ro ro  o  OV CO  in  00  CN  r~-  ro a > fi  Ol  ro  CN  r-  oo CO  CN CN CO  rvo CN  CO  VO Tf Tf t> oi  >- s  oi  Si  VC VO  TJ  Blood Lactate  Ki  H  c-  s i CCN  Os q  co  co  CO CO  >n  OV q  CN OV  oi ro oi  O N  d  e u  cn in u  CN  s i  > fi oi  Pre div  >  §  Si  i—  CO  CN  S'S Dive Min 3  'ii  d  f-  C  oo O c o  >n  ro  5§ > fi  m I  §  ro ro  CM  00 CO  >  S  CM  oo  -3  S  OV VO  ro  CM  S  cfl  O O  CM  © CO CO  r-  S  ro  CH£  s  +->  CN Ov CN  S  e  CD -C  §  CN  <=>. I  CO VO  CM  CD cfl  -u*  -a c a  ro ro oi ro Ov VO  CM  cfl Cu  e  r-  VO  T3 CD  o  >n  0)  ii  'Z  a  CS  a ii s u cu 4 H o  W =. SS  W a a O wo <  Oi  > <  a CO  "H.  T3  *  a s  cu  s  CN  I  High Energy Phosphate dynamics PCr and Pi signal intensity levels at rest were consistent both within and between animals (resting PCr = 52% ± 0.007 S E M of total P pool (PCr + Pi + ATPp); n = 7). Cellular [ATP] remained stable throughout all of the trials. During diving, end dive PCr levels were variable both within and between animals, with values from individual dives ranging from 36.84 - 103.93% of resting. The average end dive PCr for all dives was 80.85% + 5.89 SEM. In one animal, all five dives resulted in a significant decline in PCr (Hamlet: PCr of lowest individual dive 36.84% of resting, average of dive Min 8 over 5 dives: 54.17% + 4.30 S E M of resting value, Fig. 5.2). Three animals exhibited varying degrees of individual end dive PCr levels ranging from 101.97% to 39.54% of resting values (Mean values are presented in Table 5.4; Ariel, Kate, Ophelia). No significant variation in [PCr] was observed in the remaining three animals (Hecate, Juliet, Montague). In all dives, Pi was inversely correlated to PCr at all points (Fig. 5.3). Recovery of PCr to resting values occurred by postdive Min 1 (Postdive Min 0 PCr values are significantly different from resting; A N O V A , F^M) = 4.6, P = 0.001, comparisons with predive values conducted using Dunnett's Method, P = 0.05). End dive phosphocreatine values correlated with predive respiratory frequency (P < 0.01, R = 0.84) and postdive respiratory frequency for 2  min 1-7 (P < 0.004 - 0.05, R = 0.68-0.90; Fig. 5.4). 2  pH The pH values obtained through evaluation of PCr and Pi peaks are not all consistent with expected intracellular levels, however the range between resting pH and diving pH values is within physiological limits. Individuals who exhibited a significant decrease in PCr during diving (Ariel, Hamlet, Kate and Ophelia) demonstrated a significant correlation between PCr decline and intracellular pH values (P < 0.0001, R = 0.73; Fig. 5.5). No 2  significant variation in pH values was observed in animals where muscle [PCr] was maintained during diving (Montague, Hecate and Juliet).  98  T3 ft cn > CU TJ X  i  vS  CU  o  cn vd as  I  Si  cd • ° cu  O in  Tt  Tf  00  OV  p-  o c cj ^ cu "c3  a. 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I co  co  CO Ov  m <N m d o <N  I CN  Tf  Tt  u  s  s  Ii  |ve  e  s  s  I  o  8  CS |ov Ov Ov  vo B  IS Q CU  Q CU  99  c  $  T t  00  vd  .23 > CR  J3 **  CJ >  •c a > .5 3 ts  2 £• «x <- >  « ci  I  M  If o •C £ ^ 53  .g cn  §J  -H  IIS  = £* CJ  TJ  O  c u  ! ^ TJ Q •-H  3  C  8 >•« •K TJ CO  1H  § >° > 3  »a,  rt  C3  h  2 «  3 T^J ^  (letuu! io <&) d±V+!d+-Od/-Od in cu IH  3 - i—t  PH  100  co-  bO  (so c C SS 3  g  XJ  w  «CJ g2 c  eg 3  JZ  bB 3  O  c  Cd cd  §"8 CJ  £  _ CJ OJ X>  £  XJ  t>  c w c  2 e  >  O  OJ  IS eg XJ  OJ  c  '3  cd JB  P1 c  o u.  . 3 x i _cd 3 *S cd C  O Sa OJ cfl CJ cfl O  c E,  o  i »-  o  U  CO. CM  H  •4—)  O  E o  E  "5  8 s CO  si XJ  C  < +  E + u  u  "o o OH  OJ OJ  CM  cd •— — cd  X ) cd O C  H—•  cd . £ ? , cd  o  M  S '  7 3  IJ  CM  m  CM CJ  .  > bO OJ XJ cfl _ _ OJ XJ c  OH  (dlV+!d+-Od) l e w p %  OJ  oj  P  8 ; o  5  CN  cfl  II -c u OH  cn «n cj |M  3  bO  E  101  a £  120  R = 0.89 P < 0.016  no £  100  £  90  S. 8 0 cu >  70  T3  60 50 4  6  8  10  12  14  16  18  20  22  20  22  Predive Respiratory Rate (breaths per minute)  120 110 GO  100 I  *  90 P C L  80  CD > Q  T3  70 60  I  R = 0.94 P < 0.006  50 2  4  6  8  10  12  14  16  18  Postdive Minute 2 Respiratory Rate (breaths per minute)  Figure 5.4.  End dive PCr and respiratory rate correlations for predive and postdive Minute 2 collected during forced diving in northern elephant seal pups. Data points represent mean values from six animals. Each animal was dived a minimum of 3 times. Error bars indicate SD.  102  0.6  0.55  D_  I— <  0.5  + CL  + CJ CL  0.45  C L  0.4 R2 = 0.73 P < 0.0001  0.35 -0.25  -0.20  -0.15  -0.10  -0.05  0.00  0.05  0.10  0.15  A pHi  Figure 5.5.  Phosphocreatine signal intensity correlates with intracellular pH in the locomotor muscle of northern elephant seal pups. Data were collected at predive, dive minute 0 through 8 and postdive minute 0 through 6(17 data points). Values are the mean of the four animals who exhibited a decline in PCr during diving (Ariel, Hamlet, Kate and Ophelia; five dives per animal).  103  Discussion Extended periods of ischaemia may lead to a disparity between the aerobic production of A T P and the level required by cellular ATPases. The loss of ATPase function leads to membrane depolarization and an increased permeability to C a , causing cellular 2+  dysfunction and destruction of cell membrane phospholipids. In the muscle of a diving seal, the frontline defense against reduction in oxygen delivery is the maintenance of substantial intracellular oxygen stores in the form of myoglobin. Although the function and dynamics of myoglobin are not completely understood, it is accepted that its contribution to extending oxidative phosphorylation in seal muscle is significant. The ability of an animal to supplement oxidative phosphorylation with anaerobic pathways also contributes to its survival during episodes of reduced oxygen availability. Evaluation of blood [lactate] and muscle [PCr] during forced diving provide information on the contribution of anaerobic metabolism to muscle function in a seal locomotor muscle.  Blood samples obtained from five animals showed no significant increase in blood [lactate] during the dive or in the postdive period, suggesting that either the oxygen stores are sufficient to support the metabolic needs for the duration of the dive (~ 8 minutes); or that the muscle tissue's metabolic requirements are supplemented through PCr hydrolysis. The utilization of muscle phosphocreatine during forced diving in northern elephant seals is highly variable both within and between individuals. However, a strong correlation exists between the decline in [PCr] and the intracellular pH (Fig. 5.5). The lack of increase in blood [lactate] rules out lactic acid as a significant contributor to the observed decrease in muscle pH during diving in these experiments and focuses attention on C 0 accumulation 2  as the cause of increased proton load during the dive.  During ischaemia or hypoperfusion, an accumulation of C 0 in the muscle bed leads to an 2  increase in proton load due to a shift in the bicarbonate buffering reaction ( C 0 + H 0 2  2  104  yields H C 0 , which exists as H C 0 " and H ). The elevation of cellular [H ] drives the +  2  3  +  3  creatine kinase reaction to the right, consuming PCr and H , leading to the fixing of +  metabolic C 0 as HCGy within the muscle tissue. The hydrolysis of PCr would then result 2  in an increased C 0 storage ability within the muscle (Chuang et al, 1999). 2  These findings may explain the paradoxical P A  C G 2  values observed by Kooyman et al  (1973) in freely diving Weddell seals. End-tidal gas tensions obtained immediately after short dives exhibited the lowest P A long dives (> 20 min; P A  C Q 2  C Q 2  values (dives < 20 min; P A  C Q 2  = 27 mm Hg), while  = 36 mm Hg) and apneas (3.4 - 5.7 min apnea; P A  mm Hg) demonstrated higher P A  C Q 2  = 50  C 0 2  values. In a non-breathing animal, it is expected that  the quantity of accumulated C 0 increases with the duration of breath hold, therefore an 2  end-tidal C 0 gas tension obtained from a diving seal after 20 minutes of submergence is 2  expected to be higher than that obtained after a 5 minute apnea. However, through the buffering action of the creatine kinase reaction, storage of aerobically generated C 0 may 2  occur (Chuang et al, 1999). As a seal may exhibit a greater degree of hypoperfusion and thus a greater muscle C 0 accumulation during diving than during apnea, the increase in 2  C 0 may be "hidden" from the blood via the hydrolysis of PCr and the increased fixing of 2  metabolic C 0 as HC0 ". Thus at the conclusion of the dive, the initial blood PAc 2 is 2  3  0  low. Hyperperfusion of the muscle in the postdive period results in restoration of PCr via oxidative phosphorylation and the release of C 0 from H C 0 , but the total quantity of 2  3  stored C 0 would not be released until muscle pH has been reestablished at resting levels. 2  The creatine kinase reaction may explain the unexpected disparity between apneic and enddive P A c  0 2  values, but does not account for the observation that the end-dive PA o2 values C  are higher in dives > 20 minutes than in dives < 20 minutes. In Weddell seals, dives > 20 minutes are outside the aerobic diving limit and would result in lactate production in the muscle. The proton load within the cell would again increase, but at this point, PCr stores  105  are likely depleted and the intracellular buffering capacity is reduced. The increase in H  +  within the cell resulting from anaerobic glycolysis will shift the carbonic anhydrase reaction to the left, releasing C 0 from HC0 " as HC0 " buffers the newly formed lactic acid 2  3  3  (Chuang et al, 1999). The shift in intracellular pH during the anaerobic phase of the dive would account for the higher P A ^ observed in dives > 20 minutes.  Kooyman et al (1973) also reported respiratory exchange ratio (RER = C 0 produced / 0 2  2  consumed) values for freely diving Weddell seals. The data indicate that postdive RER values continue to rise for approximately 5 minutes. The rise in RER is likely driven by the release in C 0 from bicarbonate stores during postdive hyperperfusion. Over the postdive 2  period, oxidative phosphorylation will produce ATP, shifting the creatine kinase reaction to the left, generating both PCr and H . Release of imadizole-bound hydrogen ions will be +  facilitated by reoxygenation of muscle myoglobin and deoxyhaemoglobin, further increasing the presence of H within the muscle, stimulating the release of stored C 0 +  2  (Kooyman et al, 1973). This pattern of elevated postdive RER has also been observed in grey seals (Reed et al, 1994) and is consistent with C 0 storage during diving. 2  Although the correlation between muscle pH and PCr depletion provides evidence for the cause of PCr decline, it does not explain why a decline in PCr was observed in some but not all animals. In the individuals who maintained constant muscle [PCr] over the course of a dive, muscle pH levels also remained relatively stable, suggesting that intracellular C 0  2  levels (and the resulting H ) are not high enough to shift the creatine kinase reaction. +  Variation in the respiratory pattern of these animals provides some insight as to the cause of pH decline.  When examined together, the respiratory frequency and amplitude data provide information on the potential for oxygen exchange in each animal. Animals who exhibit a higher  106  respiratory frequency are likely exchanging a greater volume of air and consuming a higher quantity of oxygen over a given period of time. Although not measured, an increased oxygen consumption in animals with higher respiratory frequency may be inferred. It is possible that animals who employ a higher frequency of breathing have a smaller lung volume and breathe more shallowly, resulting in the same total ventilation when compared to animals with a lower frequency of breathing. However, the data show that frequency does not correlate with respiratory amplitude, strengthening the inference stated above. It is interesting therefore to consider that the decrease in PCr stores during a dive is correlated with predive respiratory frequency (P < 0.01, R = 0.94; Fig. 5.4). The implication is that animals who exhibit a higher oxygen consumption, and hence a higher rate of oxidative phosphorylation, produce more C 0 over the course of a dive. The resulting increase in 2  muscle C 0 would lead to respiratory acidosis and increased [H ], causing the hydrolysis +  2  of PCr.  A correlation between the degree of PCr depletion during the dive and postdive respiratory frequency is also observed (P < 0.004 - 0.05, R = 0.68-0.90). Again, the obvious 2  inference would be that animals who deplete PCr during a dive require greater oxygen delivery in the postdive period to meet the increased ATP demand resulting from PCr replenishment. The correlation is complicated by the fact that animals who have a high predive respiratory frequency will likely have a higher postdive frequency. Indeed, these two factors are highly correlated (Predive RR to postdive RR Min 1- 6: P < 0.004 - 0.05, R = 0.68-0.90). However, the correlations between end dive PCr levels and both pre and postdive respiratory frequency exist independent of the respiratory frequency correlation. This indicates that two or more factors may be driving these relationships: 1) the "predisposition" to elevated PCr hydrolysis during the dive by a higher predive metabolic rate and thus a higher C 0 production; and 2) the increase in postdive respiratory frequency 2  resulting from an increased PCr depletion during the dive.  107  A number of factors need to be addressed in order to clarify the relationship between PCr depletion and oxygen consumption. The possiblity of a mass effect on metabolic rate, and hence PCr hydrolysis, does exist. The lack of correlation between mass of the individual and PCr consumption, respiratory frequency or heart rate indicates that the variations observed in these factors are not mass-related. However, the variation of mass (77.1 to 105.9 kg) observed in the seal pups was mainly due to variation in blubber layer thickness, suggesting that total body mass may not be the best indicator of the quantity of metabolically active tissue. The lack of lean body mass data provides a certain degree of unreliability, therefore a mass effect cannot be completely ruled out. Interestingly, the one factor that is shown to correlate with mass is the amount of struggling, with larger animals exhibiting a significantly greater amount of movement during diving than smaller animals. This may reflect a behavioural response, as aggression and dominance within the elephant seal world are size-related attributes.  In addition to mass, the degree of activity exhibited by each individual may also play a role in PCr depletion. Logically, one would assume that animals who exhibit considerable muscular contraction during the dive would have a higher tissue A T P demand, and therefore may utilize a greater quantity of PCr, as evidenced by a myriad of ischaemic exercise protocol experiments (Systrom et al, 1990; Blei et al, 1992; Kent-Braun et al, 1993; Kemp, 1997). Struggling during the dive was quantified by summing the periods of signal deflection from the respiratory bellows, where any gross motor movement of the animal would result in oscillation of the respiratory bellows signal. Alteration in PCr desaturation rate was not observed to correlate with the timing of a struggling bout. In addition, PCr depletion did not correlate with the total amount of time spent struggling during the dive. The lack of relationship between these factors may be due to the particular muscle group utilized during the struggling bout. As PCr measurements were recorded from a select region of the longissimus dorsi, they are not indicative of whole body muscle  108  phosphate flux and therefore would not reflect the change in ATP consumption (and subsequent PCr depletion) that occurs in another muscle group. It is possible, albeit unlikely, that the animal recruited other muscles during struggling bouts.  A more plausible explanation is that the increased ATP consumption resulting from muscular contraction during struggling was met through aerobic metabolism, which is not limited by the usual constraints of respiratory insufficiency and oxygen delivery restrictions found in an exercising animal. During exercise, muscle activity causes a substantial and rapid increase in ATP demand within the cell. In a respiring animal, the rapid initial rise in ATP consumption is met through PCr hydrolysis and anaerobic utilization of muscle glycogen. Eventually, the animal's respiratory rate and cardiac output will increase sufficiently to meet the rise in ATP demand, and a new steady state of oxidative phosphorylation is achieved (Idstrom et al, 1985; Sweeney, 1994; Kent-Braun et al, 1993). In a forced diving situation, the quiescent phocid muscle is presented with a similar challenge, i.e. the maintenance of [ATP], but the urgency of the situation is reduced. Using substantial oxygen stores found in myoglobin, the animal may be capable of continuing aerobic function for an extended period and would not be subject to the delay in oxygen delivery experienced by an exercising muscle in a respiring organism.  If the rate of oxidative phosphorylation is sufficient to maintain muscle metabolism over the course of an 8 minute dive, PCr hydrolysis is not likely to be driven by an increase in [ADP]. To establish the likelihood of insufficient oxidative phosphorylation, a rough estimate of the aerobic capacity of quiescent muscle is obtained by dividing the oxygen content of locomotor muscle by the resting muscle metabolic rate (1.5-2.0 ml 0 min" kg" 1  2  !  ; McGilvery, 1979). Assuming an oxygen store of 60 ml kg" ((45 g myoglobin kg" 1  ')x(12.7 ml 0 min'kg^jCastellini et al, 1992), oxygen stores in a resting muscle should 2  meet aerobic demands for approximately 30 minutes. In fact, during total ischaemia,  109  muscle oxygen stores in contracting locomotory muscle would be sufficient for 4.3 minutes of aerobic metabolism (active locomotor muscle metabolic rate: -14 ml 0 min" kg" ). As 1  1  2  the level of muscle oxygen is more than sufficient to support aerobic metabolism over the course of the experimental dive (and blood lactate levels were not significantly elevated), it is unlikely that changes in cellular adenylate concentrations are driving the creatine kinase reaction. Whether PCr is hydrolyzed due to increased [ADP] or increased [FT] does not alter the end result: the contribution to cellular [ATP] will aid in extending the period of time before anaerobic glycolysis is required. However, as muscle myoglobin supports extended periods of oxidative phosphorylation in a diving seal, it is plausible that muscle PCr may be completely hydrolyzed due to the formation of C 0 before any change in 2  adenylate concentration is realized, thus suggesting that the primary function of PCr in seal muscle is that of a H buffer rather than as a high energy phosphate source. +  In conclusion, the data support the role of PCr as a H buffer in seal locomotor muscle +  during forced diving. Northern elephant seal pups are capable of maintaining the metabolic needs of quiescent locomotor muscle through aerobic pathways during the dive. In some animals, use of PCr stores during diving was required to buffer the progressive accumulation of C 0 as evidenced by a reduction in [PCr] within the locomotor muscle and 2  the associated decline in pH. These animals also exhibited higher predive respiratory rate, suggesting that a higher metabolic rate may cause PCr stores to be depleted earlier in the dive. During recovery, PCr stores are rapidly replenished through oxidative phosphorylation. A correlation between the end-dive muscle PCr levels and the respiratory rate in the postdive period represents the increase in postdive oxygen consumption required for replenishment of this high energy phosphate. Greater experimental control is required in order to separate the effects of predive respiratory rate on PCr consumption from the effects of PCr depletion on postdive oxygen consumption.  no  Chapter 6  Conclusion The primary focus of this thesis has been to apply M R technology to the investigation of the mammalian diving response in northern elephant seals and evaluate splenic function, stroke volume and muscle phosphocreatine flux during forced diving. When examining the physiological response to diving, as with many aspects of biology, it is often difficult to separate the effect of the experimental methodology from the results of the experimental manipulation. Through the use of M R techniques, a reduction in potential artifacts normally associated with invasive procedures was achieved by interrogating a selected organ or region of tissue in situ, providing a considerable increase in accuracy over more traditional methods, where stress or disruption of cellular integrity and function occurs. In addition, physiological events were recorded as they occurred during the dive, rather than by inference from data collected in the predive and postdive state, further increasing the potential accuracy. I will conclude this thesis by providing a summary of novel findings from each research chapter. Three distinct studies were undertaken and are discussed below.  1) Changes in splenic volume over the course of a dive were evaluated and discussed in relation to the diving-induced rise in haematocrit that is known to occur in phocid seals (Chapter 3).  Over the last two decades, observations on phocid haematocrit have indicated that a distinct rise in R B C mass occurs during diving, leading many researchers to implicate the spleen as a storage reservoir for RBCs. The rate of haematocrit increase suggested a slow controlled contraction of the spleen occurred during the dive. However, M R images of northern  in  elephant seal spleens acquired during forced diving indicate that contraction occurs immediately upon submersion, and reaches maximal contraction by minute 2 of the dive. The delay in appearance of the RBCs into general circulation may be explained by the presence of a diving-induced vena caval occlusion. It has been previously shown that the phocid caval sphincter contracts during facial immersion, restricting vena caval blood flow at the level of the diaphragm and causing the hepatic sinus to dilate. The images obtained during diving indicate that splenic contraction is accompanied by a dilation of the hepatic sinus, confirming the role of the sphincter in reducing blood flow to the heart during bradycardia and establishing a direct relationship between spleen and sinus. By demonstrating an inverse correlation between hepatic sinus volume and splenic volume, a transfer of erythrocytes from the spleen to the sinus is inferred. These data provide a model for phocid circulation during diving and concur with previous studies that implicate the spleen and hepatic sinus as mechanisms for the alteration of R B C distribution during the dive.  The functional significance of splenic contraction during repetitive dives was also addressed. Although the spleen is observed to contract in response to facial immersion, its role as a supplemental oxygen store during dives has been debated (Ponganis et al, 1992; Castellini and Castellini, 1993; Eisner and Meiselman, 1995). Growing evidence suggests that seal haematocrit remains elevated during the postdive period, indicating that splenic refill is delayed. The slow rate of splenic refill observed in this study concurs with the findings of the above researchers, supporting the concept of the spleen as a means of reducing haematocrit when on land rather than increasing haematocrit when submerged.  2) Investigation of the inotropic effects of facial immersion using MRI phase contrast analysis techniques was undertaken and detailed evaluation of HR and SV in elephant seals during the predive, dive and postdive period was conducted (Chapter 4).  112  In addition to increasing oxygen availability through splenic contraction, mammalian divers alter the distribution of blood oxygen to favour hypoxia sensitive tissues. Although a significant body of literature exists detailing the effect of diving on heart rate in both forced and free diving situations, there is a paucity of data on stroke volume and cardiac output during diving. The functional significance of diving bradycardia is primarily concerned with its effect on oxygen distribution. However, without concurrent knowledge of stroke volume, it is difficult to make broad physiological conclusions based on heart rate alone. At present, there are seven studies detailing the effect of forced or trained diving on pinniped cardiac output. The results of these studies indicate that stroke volume during diving is either reduced below or maintained at predive levels. Contrary to previous research, M R phase contrast analysis of northern elephant seals revealed a diving-induced increase in stroke volume. Heart rate data indicate that the degree of bradycardia exhibited by these animals was considerably less than in previous forced diving studies, suggesting that the reduction in vagal input may contribute to the observed increase in stroke volume. The heart rates observed in this study are comparable to heart rates obtained from freely diving seals, suggesting that stroke volume data presented here may be more representative of the natural physiological response. These findings indicate that the effect of diving on stroke volume is altered by the degree of bradycardia and suggest that caution must be exercised when extrapolating diving heart rates to other physiological characteristics such as metabolic rate or cardiac output.  3) Evaluation of intracellular pH and phosphocreatine flux in the muscle of a diving seal was undertaken using P M R S and the relationship between PCr hydrolysis, H and C 0 3 1  +  2  was established (Chapter 5).  The final question addressed by this thesis is the relationship between PCr and muscle metabolism during diving in northern elephant seals. Many studies have assessed the  113  submergence capacity of diving mammals by calculating the oxygen and energy storage capacity of a seal and speculating on the length of time these stores would support metabolic function under various conditions. Historically, phosphocreatine has been overlooked in these calculations. The development of noninvasive magnetic resonance spectroscopy techniques has resulted in a plethora of phosphocreatine studies in a number of different species, leading to speculation on the role of PCr in a diving animal (Stephenson and Jones, 1992). In animals diving within their aerobic dive limit, the lack of significant increase in blood lactate levels indicates that muscle metabolism is supported through oxidative phosphorylation. The concept of PCr as a temporal ATP buffer suggests that PCr depletion should not occur until an increase in [ADP] was realized. In fact, this study demonstrates that in northern elephant seals, PCr hydrolysis does occur during aerobic dives, suggesting that the depletion of [PCr] is driven by an alteration in muscle [H ] rather than by changes in [adenylate]. The correlation between muscle pH and PCr +  supports the concept of [H ] as a driving force for PCr hydrolysis. In the hypoperfused +  muscle of a diving animal, accumulation of C 0 in the tissue leads to an increase in proton 2  load due to a shift in the bicarbonate buffering reaction ( C 0 + H 0 yields H C 0 , which 2  2  2  3  exists as H C 0 " and H ). The elevation of cellular [H ] drives the creatine kinase reaction +  +  3  to the right, consuming PCr and H , leading to the fixing of metabolic C 0 as HC0 ". +  2  3  Regardless of the cause of PCr hydrolysis in seal muscle, the end result is an anaerobic contribution to ATP production within the muscle. However, as muscle myoglobin supports extended periods of oxidative phosphorylation in a diving seal, it is plausible that muscle PCr may be completely hydrolyzed due to the formation of C 0 before any change 2  in adenylate concentration is realized, thus suggesting that the primary function of PCr in hypoperfused seal muscle is that of a H buffer rather than as an immediate high energy +  phosphate source.  114  In conclusion, the application of M R imaging and spectroscopy techniques to forcibly dived northern elephant seal pups has provided substantial information on the physiological effects of diving. The data presented here serve to advance our understanding of phocid diving physiology and support the concept of a diving seal as an organism capable of avoiding tissue hypoxia through physiological manipulation of oxygen stores rather than exhibiting specific cellular protection against oxygen lack. 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