Thesis for the degree of Doctorate in Biology, Östersund 2008

HEMATOLOGICAL CHANGES ARISING FROM SPLEEN CONTRACTION DURING APNEA AND ALTITUDE IN HUMANS

Matt X. Richardson

Supervisors: Prof. Erika Schagatay Dr. Johan Andersson

Department of Natural Sciences, Engineering and Mathematics Mid Sweden University, Östersund, Sweden

ISSN 1652-893X, Mid Sweden University Doctoral Thesis 57 ISBN 978-91-86073-03-9

Akademisk avhandling som med tillstånd av Mittuniversitetet i Östersund framläggs till offentlig granskning för avläggande av doktorsexamen i Biologi, den 19e september 2008, kl. 10 i sal F234, Ridhuset, Mittuniversitetet, Östersund. Seminariet kommer att hållas på engelska.

HEMATOLOGICAL CHANGES ARISING FROM SPLEEN CONTRACTION DURING APNEA AND ALTITUDE IN HUMANS

Matt X. Richardson

© Matt X. Richardson, 2008

Department of Natural Sciences, Engineering and Mathematics Mid Sweden University, Östersund Sweden

Telephone: +46 (0)771-975 000

Printed by Kopieringen Mid Sweden University, Sundsvall, Sweden, 2008 Cover photographs by Annelie Pompe (World freediving championships, Egypt) and Mike Callaghan (Lobuche Peak, Nepal).

i HEMATOLOGICAL CHANGES ARISING FROM SPLEEN CONTRACTION DURING APNEA AND ALTITUDE IN HUMANS

Matt X. Richardson

Department of Natural Sciences, Engineering and Mathematics Mid Sweden University, Östersund, Sweden ISSN 1652-893X, Mid Sweden University Doctoral Thesis 57; ISBN 978-91-86073-03-9

ABSTRACT

The spleen acts as a storage site for red blood cells, and contraction of the spleen is for many mammals a way of coping with situations where increased blood oxygen storage is beneficial. This ability has also recently been found in humans, where most individuals respond with an increase in circulating erythrocytes in response to environmental or physiological stress, the majority of which can be attributed to the spleen. The initiation of this response, however, is unclear, as is the variability of the response’s development among individuals. This thesis identifies that there is a strong correlation between spleen contraction and circulating hemoglobin (Paper I), that release of these erythrocytes from the spleen is more pronounced in those trained in breath-hold diving than in elite cross-country skiers and untrained persons (Paper II), and that the response develops even in oxygen-poor environments such as at high altitude (Paper III). This is due to the hypoxic stimulus that exists such environments, which acts as an initiator of the response (Paper IV), although even high levels of carbon dioxide are important (Paper V). The size of world championship apnea divers’ spleens is also predictive of their apneic performance, showing that spleen contraction is an important factor in natural diving ability (Paper VI). In conclusion, the contractile response of the spleen, along with the previously described “diving response” helps humans to better endure low-oxygen situations, but despite their common function these responses are not initiated and do not develop in the same manner (Paper VII).

Keywords: hypoxia, hypercapnia, hemoglobin, hematocrit, breath-hold diving

ii SAMMANDRAG

Mjälten innehåller ett extra lager av röda blodkroppar, och mjälteskontraktion är ett sätt för många däggdjur att anpassa sig till situationer där en ökad lagring av syre i blodet är en fördel. Denna förmåga har relativt nyligen upptäckts hos människan där flesta personer svarar på fysiologisk eller omgivningsrelaterad stress med en ökning av mängden erytrocyter i cirkulationen vilket till stor del antas komma från mjälten. Det är dock inte känt hur responsen startas och varför den är olika väl utvecklad hos olika personer. Denna avhandling visar att det finns ett nära samband mellan mjälteskontraktionen och blodets Hb (Paper I), att utsöndring av erytrocyter är större bland tränade apnédykare än bland elitskidåkare och otränade personer (Paper II) och att responsen uppstår även i syrefattig miljö t ex på hög höjd (Paper III). Det beror på att syrebristen i sig är en viktig stimulus för att initiera responsen (Paper IV), men även höjd koldioxidnivån är en viktig faktor (Paper V). En korrelation upptäcktes mellan storleken på apnédykares mjältar och deras resultat på ett världsmästerskap vilket visar att mjälteskontraktionen är viktig för människans naturliga dykförmåga (Paper VI). Slutsatsen är att mjältesresponsen tillsammans med den tidigare beskrivna ”dykresponsen” hjälper oss att bättre klara perioder med syrebrist, och att dessa responser samverkar funktionellt men inte startas på samma sätt (Paper VII).

Nyckelord: hypoxi, hyperkapni, hemoglobin, hematokrit, apnédykning

iii TABLE OF CONTENTS

ABSTRACT ...... II

SAMMANDRAG...... III

TABLE OF CONTENTS...... IV

LIST OF PAPERS ...... VI

INTRODUCTION...... 1

What is apnea?...... 1 What is hypobaria?...... 4 Differences in adaptation to hypoxia in humans ...... 4 Morphology of the spleen...... 5 Functions of the spleen...... 6 Contraction of the spleen in non-human species...... 8 Contraction of the spleen in humans...... 10 Initiation of spleen contraction...... 11 Effects of the spleen on cardiovascular function...... 13

AIM OF THE THESIS...... 14

METHODS...... 14

MEASUREMENT OF BLOOD VARIABLES...... 14 Venous capillarisation for Hb/Hct...... 14 MEASUREMENT OF SPLEEN SIZE...... 15 MEASUREMENT OF OTHER PARAMETERS...... 16 Arterial oxygen saturation ...... 16 Mean arterial pressure...... 17 Skin blood flow...... 17 Respiration and gases...... 18 APNEA AND HYPOXIA EXPERIMENTAL PROTOCOLS ...... 19 Apnea series ...... 19 Inspired gases and hyperventilation...... 20 STATISTICAL APPROACHES...... 20 Publication statistical analysis method...... 20 Additional statistical analysis method...... 21

SUMMARY OF RESULTS ...... 23

OCCURRENCE OF SPLEEN CONTRACTION...... 23

iv INITIATION OF SPLEEN CONTRACTION ...... 25 CONSEQUENCES AND APPLICATIONS OF SPLEEN CONTRACTION ...... 27

DISCUSSION...... 28

OCCURRENCE OF SPLEEN CONTRACTION...... 28 INITIATION OF SPLEEN CONTRACTION ...... 29 Stimulus detection by chemoreception ...... 30 Output following chemoreception...... 31 Other potential output mechanisms...... 34 CONSEQUENCES AND APPLICATIONS OF SPLEEN CONTRACTION ...... 34 In apneic performance...... 35 In acute adaptation to high altitude ...... 36 During exercise...... 37 Problems with Hct limits in competition...... 37 Relation to the diving response...... 38 FUTURE DIRECTIONS...... 39 Other measurement techniques...... 39 Maximizing splenic contraction through training ...... 39 Investigation of other possible sources of stored erythrocytes...... 40

ACKNOWLEDGEMENTS...... 40

REFERENCES...... 41

PAPER I...... 50

PAPER II...... 55

PAPER III...... 63

PAPER IV...... 73

PAPER V...... 85

PAPER VI...... 98

PAPER VII...... 113

v LIST OF PAPERS

This thesis is based on the following seven papers, hereinafter referred to by their Roman numerals:

Paper I Correlation between spleen size and hematocrit during apnea in humans. MX Richardson, R deBruijn, S Pettersson, J Reimers and E Schagatay. 2006. In: Lindholm, P, Pollock NW, Lundgren CEG, eds. Breath-hold diving. Proceedings of the Undersea and Hyperbaric Medical Society/Divers Alert Network 2006 June 20-21 Workshop, p 116-119.

Paper II Increase of hemoglobin concentration after maximal apneas in divers, skiers and untrained humans. 2005. MX Richardson, R deBruijn, HC Holmberg, G Björklund, H Haughey and E Schagatay. Canadian Journal of Applied Physiology, 30(3): 276-281.

Paper III Short-term effects of normobaric hypoxia on the human spleen. 2008. MX Richardson, A Lodin, E Schagatay and J Reimers. European Journal of Applied Physiology, published online Nov 28 2007, ahead of print.

Paper IV Hypoxia augments apnea-induced increase in hemoglobin concentration and hematocrit. 2008. MX Richardson, R deBruijn, and E Schagatay. Submitted for publication in the European Journal of Applied Physiology.

Paper V Hypercapnia moderates hemoglobin increases during apnea. 2008. MX Richardson, A Lodin, H Engan, and E Schagatay. In manuscript.

Paper VI Spleen and lung volumes correlate with performance in elite apnea divers. 2008. E Schagatay, MX Richardson and A Lodin. Submitted for publication in the Journal of Sports Sciences.

Paper VII Cardiovascular and hematological adjustments to apneic diving in humans: Is the spleen response part of the diving response? 2006. E Schagatay, MX Richardson, R deBruijn and JPA Andersson. In: Lindholm, P, Pollock NW, Lundgren CEG, eds. Breath-hold diving. Proceedings of the Undersea and Hyperbaric Medical Society/Divers Alert Network 2006 June 20-21 Workshop, p 108-115.

vi

vii

INTRODUCTION

It is human nature to explore the environments around us, no matter how remote or challenging. In these cases, humans voluntarily subject themselves to hostile or dramatically changing environmental factors such as temperature extremes or alterations in atmospheric composition, despite the fact that such actions threaten their normal functioning, or in some cases, their survival. Luckily, humans are likely the only species capable of adapting to virtually every environment on the planet, by virtue of our intelligent use of tools and therein the ability to produce clothing, shelter, preserve foodstuffs and arrange effective transport methods.

Strip away the largely “technological” advances and humans are considerably less impervious to their surroundings. A naked diver in the cold North Atlantic, for example, will hardly last a tenth of the time of his drysuit-clad equivalent. Other environments that pose particularly acute problems for the human are those of reduced oxygen. Depending on the severity of the reduction, vital bodily functions can become compromised on a very rapid time scale (minutes), although humans show wide variation in their ability to adapt to such reductions. These environments are generally encountered in two forms: 1) during apnea, whether voluntary or involuntary, and 2) in hypobaria at high altitude.

Yet even the best mountaineers in the world, without bottled oxygen, begin to drown their lungs and brains with oedema at our planet’s highest altitudes within hours. Some individuals can adapt to these situations on a purely physiological level and may thus endure these environments for short periods. Strong evidence for this is provided in the current world record for apnea, which at over 9 minutes is far in excess of commonly reported asphyxia survival times. Those that cannot adapt face a considerably less auspicious outcome. These differences in adaptation that determine an individual’s fate are still largely unknown. The intent of this thesis is to shed some light on one piece of this adaptive puzzle.

What is apnea?

Apnea is, simply put, the cessation of breathing. When apnea is voluntary it is also often referred to as breath holding; examples of this behaviour among humans occur in harvest divers, spear fishers, and competitive free divers. Gas exchange with the environment is halted and the body must make do with the oxygen (O2) stored in the body i.e. in the lungs, the blood, and the tissues. This 1

reduces the amount of O2 in the body with time, and at the same time increases the amount of carbon dioxide (CO2). The ongoing metabolic processes in the body will continue to use these O2 stores (and produce CO2) until, in voluntary cases, breath holding ends due to discomfort with the increasing urge to breathe, and gas exchange with the environment resumes.

Depending on the tenacity of the individual, the amount of O2 reaching the various tissues of the body during breath holding may become insufficient in order for them to continue their metabolic processes normally, known as hypoxemia.

The oxygen saturation level of the arterial blood (SaO2) provides a good indication of the severity of hypoxemia attained in the body [1], as the partial pressure of O2 in the alveoli (PAO2) and in the arteries (PaO2) continues to decrease in a constant relationship to each other throughout apnea [2]. Normal

SaO2 values during breathing are typically 97-99% but individuals trained in breath holding (known as apneists or freedivers) can reduce values to less than 50% during extended breath holding.1 The desaturation rate is more rapid when the initial lung volume is smaller [1] and when initial mixed venous O2 saturation is lower [4].

A marked slowing of heart rhythm (bradycardia) mediated via parasympatetic vagus nerve activity and a progressive increase in mean arterial pressure (MAP) occur within 30 s of breath holding [5], along with a sympathetically mediated reduction of blood flow to e.g. the extremities and skin via narrowing of the blood vessels (peripheral vasoconstriction). This series of measures helps in conserving the diminishing O2 for extremely vital processes such as brain and heart function – at the expense of other tissues and organs – and are collectively known as the human diving response.2

The lack of gas exchange during apnea also means that the CO2 produced by the metabolic processes of the body cannot be removed from the venous blood. This causes subsequent elevation of CO2 in the arterial blood, known as hypercapnia.

1 To put this in context, values of less than 90% define clinical hypoxemia, and in a hospital setting are typically considered an acute problem in patients [3].

2 While some experiments described in this dissertation refer to or compare the parameters that make up the diving response in apneic situations, it is not the focus of this work. For a more detailed review, the theses by Schagatay [6] and Andersson [7] and review by Foster and Sheel [8] provide thorough discussion of the human diving response.

2

The hypercapnia is augmented by shrinking lung volume and the Haldane effect from oxygenation of hemoglobin and liberation of hydrogen ions [9]. Venous blood experiences a lesser rise in CO2 partial pressure (PCO2) due to the Haldane effect, which in venous blood works in the opposite direction to arterial blood, and due to buffering of CO2 by the tissues. CO2 output into the lungs reduces progressively and eventually stops; continued breath holding reverses the cycle and sees CO2 move from the lungs into the arterial blood [2, 9].3

As arterial PCO2 is the main determinant of voluntary apneic duration, pre- apneic hyperventilation can prolong apnea by reducing the CO2 content of the lungs, blood, and eventually the tissues (ref e.g. Lin). This allows the “breaking point” of apnea, where PCO2 reaches a critical level that can no longer be voluntarily tolerated, to be reached later. Pre-apnea breathing of O2 can also increase apneic duration, as the arterial blood will remain normally saturated until the lungs start to become depleted of oxygen [9]. Thus, the development of a hypoxic stimulus and the onset of tissue hypoxemia are delayed. Klocke and

Rahn [12] found that combining hyperventilation and O2 breathing could result in breath-holding times up to 14 minutes with the entire lung vital capacity volume absorbed during apnea in some subjects.

Repetition of apnea with short rest intervals (2-3 minutes) also results in an increase in apnea time and the time until the “physiological breaking point”, defined as the onset of involuntary breathing movements [13]. This occurs despite unchanging pre-apneic arterial PCO2 and lung volume, which indicates that other factors are responsible.

As a physiologically stressful experience, sympathetic activity is also increased in various physiological systems during apnea. Renal sympathetic nerve activity and MAP were shown to increase with apneic duration in cats, although these could be reduced by 80% and 100%, respectively, when pre-ventilated with O2 and a matched level of PaCO2 [14]. Resumption of breathing caused an immediate fall in sympathetic nerve activity. Leunenberger and associates found tha t sympathetic nerve activity was greatest during hypoxic apnea and attenuated

3 The abovementioned CO2 dynamics are only relevant for surface breath holding.

In the case of diving to depth, CO2 moves from the alveoli into the blood due to lung compression, which increases the PCO2 in the lung above that of the venous blood; during ascent, the opposite occurs [10]. The extent to which the blood acts as the CO2 store during continued breath-holding at the bottom is dependent upon the relationships between the alveolar PCO2 and the PCO2 in the tissues [11].

3 during hyperoxic apnea [15], and was also increased during intermittent hypoxia without apnea [16].

What is hypobaria?

Hypobaria occurs naturally as we ascend to altitude, where the pressure of the atmosphere (and thus the partial pressures of the gases that it is composed of) is lower than at sea level. Humans exposed to such situations include high altitude natives and residents, mountain climbers, and aviators, among others. It is also possible to artificially impose a reduced amount of O2 while maintaining a normal atmospheric pressure (i.e. 760 mmHg, or sea level) by filtering out O2 and replacing it with another gas, typically nitrogen. This is called normobaric hypoxia.

While most known adverse effects of high altitude are ascribed to the reduction in ambient O2, there may be some differences between hypobaric and normobaric hypoxia. The former leads to a slightly greater hypoxemia, reduction of CO2 in the blood (hypocapnia), blood alkalosis and lowering of SaO2; however, the end- tidal partial pressure of O2 and CO2 are similar between the two conditions [17]. Despite these differences, it is common to study most effects of altitude by simulating it in normobaric hypoxic environments.

When the onset of hypoxia is rapid, the human body follows a time dependent series of adaptations, which on the shortest time scale (minutes) begins with increased ventilation and abrupt changes in cardiovascular dynamics [18]. These include an increase in sympathetic activity in the body, resulting in increased cardiac output, centralization of blood volume, and constriction of the venous capacitance vessels [19-21]. An increase in hematocrit is also noted. Chronic hypoxia further elevates hemoglobin via release of erythropoietin, and production of new red blood cells within days. However, the effect of the responses to both acute and chronic hypoxia is an attempt to increase O2-carrying capacity.

Differences in adaptation to hypoxia in humans

While the physiological adaptation to hypoxia is similar among most individuals, the magnitude or effectiveness of some aspects of this response are not. Both acute and acclimatory adaptive responses to hypoxia appear to have certain characteristics that vary amongst individuals or groups of individuals. According to Hochachka [22], these include vaoscontrictive responses mediated by lung vasculature O2 sensors, production of new blood vessels, and maintained 4

erythropoeisis via kidney O2 sensor output, leading to increased red blood cell

mass and O2-carrying capacity. Andean and Himalayan natives show blunted responses to acute hypoxia compared to lowlanders, suggestive of a high-altitude phenotype in, for example, Quechuas and Sherpa populations. Although difficult to quantify, genetic and environmental contributions to the variation in these response characteristics are generally considered to be equal [23, 24], leaving a fair amount of “flexibility” in the extent to which individuals sense and respond to Figure 1. The spleen, hypoxia [25, 26]. illustrated with vein and artery. From Gray’s Anatomy, An increase in the amount of circulating O2 1918. in the body is, at its most essential level, necessary to improve oxygenation at the tissue level in oxygen-poor environments where breathing is still permitted [27]. While this is possible at altitude, in the case of apneic hypoxia ventilatory responses are not immediately possible during apneic hypoxia. Here, decreases in heart rate are instead noted, although an increase in hematocrit is still present. The increase in hematocrit (Hct) and hemoglobin concentration (Hb) noted in both cases of acute hypoxic exposure is not well understood. On such a short time scale, where erythropoietin-stimulated red blood cell production is not yet realized, the intervention of a source of stored blood cells may be one possible explanation yet to be explored in humans. And the spleen, an organ that throughout history has had a multitude of afflictions and functions attributed to it, may be an ideal candidate for such a storage function. The spleen’s prospective role in this adaptation to these environmental stressors is the main focus of this thesis.

Morphology of the spleen

Found in humans beneath the diaphragm in the upper left abdomen, the spleen is a uniquely complex organ with both immune and circulatory functions. According to Gray’s Anatomy it normally makes up approximately 0.25% to 0.29% of total body weight, and has a volume of about 200-250 cm3 in adults [28]. Two coats surround the organ: a smooth external coat or tunica serosa, and an internal fibroelastic coat or tunica albuginea. The internal coat reflects inwards as sheaths 5 which in turn branch into numerous small fibrous bands, or trabeculae, forming the framework of the spleen. The small spaces created within this trabecular framework constitute the spleen’s pulp.

The pulp itself can be grossly divided into white and red sections, their respective colours obtained by the presence of a large number of lymphocytes and erythrocytes. Not surprisingly, the white pulp performs immunological functions as part of the largest lymphoid organ in the body, while the red pulp is the largest filter of blood in the human [29]4. Arterial blood, brought to the spleen via the splenic artery, is transported through a series of cords and then into venous sinuses of the red pulp. The endothelial lining of the sinuses contains non- striated muscular filaments, and with its cells in parallel formation has the ability to force blood from the cords to the sinuses through fibrous slits [31]. Older, less-flexible erythrocytes may become trapped at this stage and are “pitted” for recycling of their heme components [32]. However, the flexibility and contractility of these fibres also appear to help retain more viable erythrocytes in the spleen, forming a reservoir. Via this meshwork, the spleen concentrates blood in the reservoir to about twice that of the arterial hematocrit circulating in the rest of the body, from normally 40% to approximately 78% [33], though others have estimated this value as high as 96% [34]. Although the quantity of this reservoir can vary greatly dependent on the size of the organ, it is generally believed to contain on average 200-250ml of concentrated blood [35, 36].

Functions of the spleen

The early medical practitioner Galen (circa 200 AD) identified the spleen as the source of “black bile” in the body, up until the Middle Ages considered to the most noxious substance in the body, the release of which both anger and melancholy was often associated to5.

4While the spleen’s immune functions are considerable, it is not the focus of this dissertation and therefore will not be discussed in detail. For more information about this aspect of spleen function, Mebius [30] provides a thorough review.

5 Indeed, the contents of the spleen may appear black (for example, upon splenectomy or autopsy) but this is almost certainly due to the elevated hematocrit level, which differentiates it from normally circulating arterial blood that is more reddish on account of its higher dilution with plasma.

6

As medical science modernized, late 19th and early 20th century experimentation on the circulatory roles of the spleen involved domesticated animals, typically dogs and cats. In these species, it was established that the spleen’s volume was variable, capable of rapid contraction resulting in a discharge of its stored contents, increasing both circulating red blood cells and arterial pressure [37-39]. It was also found to contract and dilate rhythmically under normal physiological conditions, albeit to a lesser extent [40, 41]. Later experimentation seemed to confirm the role of the spleen as a storage site, as blood volume returned to normal only minutes after large infusions of saline, dextrose and whole blood in intact dogs, but not in splenectomised dogs [42]. There was also evidence for the discharge of vasoactive substances from the spleen itself during its contraction, which might further affect arterial pressure above and beyond the effects of the spleen’s expelled volume [43]. More recently, in rats there is evidence that it may be capable of rapidly filtering out plasma from the blood and into the lymphatic system when blood volume is expanded [44]. The storage function of the spleen is also considered important in animals for survivability in cases of severe haemorrhage or shock, and restoring volume homeostasis when such interventions were not required [45, 46].

Despite such research findings, however, the concept of similar functions in the human spleen was disregarded in many medical textbooks as late as the 1980s. This was undoubtedly due to the frequent observation that splenectomy, a common procedure in traumatic injury, could be performed without noted adverse long-term effects for the individual. Later long-term studies showed, however, that while not necessary for human survival per se, removal of the spleen increases the risk for life-threatening bacterial infections [47]. The more recent observation that the human spleen does contract [48-50], on the other hand, is still largely unknown to most practitioners of medicine, and the phenomenon is not well investigated or understood.

In humans, 70-80% of the total circulating blood volume is in the venous system at any given time, of which the largest pools can be found in the cutaneous and splanchnic circulation - the latter composed of the gastric, small intestinal, colonic, pancreatic, hepatic, and splenic circulations, arranged in parallel with one another. As part of the splanchnic circulation, the spleen contributes to homeostatic regulation of both blood volume and pressure and acts as an “autologous blood bank” in times of depleted intravascular volume, and also unloads the circulation by absorbing excess volume [51]. The spleen is also considered important in regulating blood hematocrit, itself a major determinant of blood viscosity and factor responsible for of the O2-carrying capacity of the blood. This may occur both through filtration but also through plasma volume

7 regulation [51]. Possibly as an indication of these functions, the spleen receives a relatively large percentage of the cardiac output (up to 5%) for its size, with a flow per gram of tissue 10 times higher then that of resting skeletal muscle and almost similar to the blood flow to the heart muscle [52]6. Only 75% of this volume is returned to the splenic vein at any given time, likely due to the filtration of plasma into the lymphatic system, which occurs in the red pulp and results in an elevated hematocrit in the spleen’s stored contents [51]. Blood viscosity and hematocrit have been shown to increase in humans after splenectomy [50, 70], likely due to the absence of the spleen’s filtering and storage functions.

Contraction of the spleen in non-human species

Spleen size has been found to correlate with dive capacity i.e. the duration of diving in pinnipeds, and this also holds true across the phylogenetic tree – species which have larger spleens have also longer dive times [53, 54]. The Weddell seal is arguably most well endowed in terms of spleen size, with approximately two-thirds of the seal’s red blood cells sequestered in the spleen during rest [55], and can also be considered one of the most accomplished divers amongst seal species. Qvist and colleagues [55], as well as Thornton and colleagues [56], the latter using magnetic resonance imaging (MRI), demonstrated a fast contraction of the spleen during diving in seals, with concomitant increase in red blood cells ejected into the circulation. This conceivably allows for an increase in O2 storage capacity during diving and more effective O2 replenishment during rest periods between dives [57]. Both the Qvist and Thornton groups also noted that a re-uptake of the ejected blood occurs within 15 minutes post-dive. This recovery of the ejected blood by the spleen during non-diving periods would prevent excessive circulatory viscosity when O2 was not limiting [58].

Many varied stimuli have been shown to cause a reduction in spleen volume in mammals. In dogs, rebreathing, injection of epinephrine, and fright from loud noises caused significant transient decreases in spleen size [59]. Further experimentation in dogs confirmed that the blood volume increases seen after fright, exercise and hypoxia were exclusively associated with spleen contraction, and not the liver [46], although Carneiro and Donald [45] did find tha t haemorrhage caused decreased blood volume in the liver as well, albeit about half of the loss from the spleen. It was also found that an increase in blood

6 The spleen is also therefore particularly troublesome when ruptured or damaged, as it can lead to massive internal blood loss and death.

8 viscosity also followed “emotional excitement” in rats and rabbits but that this increase was far smaller if their spleens were removed [60]. Sheep [61], goats [62], pigs [63] and horses [64] all show significant elevations of hematocrit during exercise, which can be eliminated via splenectomy. Conceivably the purpose underlying increases in circulating red blood cells is the same in virtually all situations named above – a resultant increase in O2 uptake and/or an ability to recover quickly during rest, thus reducing fatigue and improving performance during physiologically stressful or demanding situations.

Kramer and Luft [65] found that hypoxia also caused appreciable reductions in spleen mass in dogs, calculating blood expulsion in the order of 16-20% of circulating blood volume. This effect was reversible with the removal of hypoxia. Tellingly, the hematocrit measured in the splenic vein during expulsion was on average 80% higher than in arterial blood, but during recovery from hypoxia this pattern was reversed, providing evidence for the storage of red cells by the spleen7. Furthermore, the ejected blood in the splenic vein maintained consistently higher O2 saturation than the arterial blood in the hypoxic dogs, suggesting that stored cells in the spleen were well oxygenated. These findings were later confirmed in cats, whereby rapid reduction in ambient O2 levels to 3.5% caused an increase in hematocrit as well as survival time, compared with splenectomised cats [67]. Plasma protein values were not different between the two conditions, suggesting circulating plasma volume was unaffected. Hoka et al [68] confirmed that severe hypoxia was accountable for reductions in spleen volume, of which 60% was accounted for by active spleen contraction due to sympathetic efferent nervous discharge to the spleen. This sympathetic nervous discharge increased six-fold over baseline activity during hypoxia in dogs. Increases in Hb from the spleen were also noted in rats when exposed to hypoxia after undergoing 1 h of hypoxia a day for 4 days, but not in rats exposed for only 1 day [69]. However, after the spleen contraction response was established, the ability to elicit it again with a single hypoxic stimulus remained for 60 days thereafter.

7Radioactive labelling in dogs showed that tagged cells rapidly reach an equilibrium concentration in the spleen [66], making it difficult to distinguish changes in red cell volume calculations attributable to spleen contraction when measured on a total-body scale. Targeted or regional analysis of radioactivity when using this method is required to analyse blood reservoir shifts. 9

Contraction of the spleen in humans

During exercise8

Sandler and associates [73] used radioactive labelling to demonstrate spleen shrinkage during postural changes (from supine to standing) and during supine exercise, resulting in a reduction in splenic radioactivity and increase in blood radioactivity and red blood cell count. Spleen volume was found to decrease also during upright exercise [74, 75] while kidney and liver volume showed mixed results, with some showing no change [75] and others showing decreased volume, though to a lesser extent than the spleen [74]. Plasma norepinephrine and epinephrine levels have also been shown to closely correlate with spleen volume during graded exercise [49].

Wolski [34], in her investigation of spleen responses to exercise, noted an approximately 9% reduction the total red cell volume of untrained subjects’ spleens after exercise, with an overall organ volume reduction from 338 mL to 143 mL. The proportion of red blood cells released by the spleen did not differ between untrained or aerobically trained subjects and even exercise in hypoxia did not increase the amount of red blood cells released.

Stewart and associates [72] found reductions in spleen volume from cycling at 60% maximum O2 uptake after 5 min (28% reduction), 10 min (30%), and 15 minutes (36%). An incremental ride to exhaustion resulted in a 56% reduction in spleen volume. They suggested that the spleen regulates its volume during exercise in response to an intensity-dependent signal, in which plasma catecholamines may play a role.

During apnea

The human spleen has also been found to contract during apnea or breath holding, with a subsequent increase in both Hb and Hct [48, 50, 76, 77] that is not caused by hemoconcentration [50, 76]. Splenectomised subjects do not demonstrate these hematological changes after apnea, and also lacked the typical prolongation of

8General points regarding exercise: plasma volume decreases by 10 to 20% during exercise [71], causing an increase in hematocrit and blood viscosity. This is caused by ultrafiltration of plasma extravascularly and the movement of plasma to muscle, as well as through fluid loss. However, according to Stewart, it appears unlikely that splenic release of erythrocytes during exercise has any effect on indirect measures of plasma volume [72]. 10 repeated apneas, suggesting the effect is mainly attributable to the spleen [50, 76].

Maximization of spleen contraction through apnea appears to require more than one apnea, with maximum contraction and Hb/Hct increase typically occurring after 3-5 apneas [13, 78], although one study has suggested that maximal contraction is perhaps attainable after a single apnea [35].

Even very short breath holds (30s) have been shown to elicit reductions in spleen volume in humans, and the amount of hematological increase is related to the degree of contraction [77]. There is, however, uncertainty regarding the influence of apneic duration on degree of contraction of the spleen. One study has demonstrated increased spleen contraction with increased apneic duration [77] while other work has demonstrated powerful contraction also in time-limited apneas [79].

Some benchmark studies of spleen contraction have reported mean reductions in spleen volume of 18% [79], 20% [48], and 25% [35]. It has been shown that this contraction is likely actively initiated, that is, not due to passive collapse or reduced flow in the splenic artery [35]. The spleen remains contracted even between apneas with short rest periods (2-3 minutes) in between [35, 50] but typically returns to its relaxed size and volume within 10 minutes barring further apneic intervention [35, 50, 77, 79]. This is also the duration known to reverse the increase in apneic duration during repetitive attempts [79]. While the observations by Hurford et al [48] suggested that only trained Ama divers responded with a spleen contraction, other studies reveal that both individuals trained and untrained in breath-hold diving demonstrate splenic contraction and increases in hematological parameters [50, 77], although differences between those trained or not trained in breath holding have also been demonstrated [76].

Initiation of spleen contraction

There is virtually no consensus about the “trigger(s)” of splenic contraction. Much research points to the possibility of an underlying active, sympathoadrenergic mechanism. The earliest work demonstrated splenic contraction after stimulation of the splenic nerve in dogs [39]. This was again confirmed in later work [80, 81] which observed that stimulation of the sympathetic nervous input, and not mechanical restriction of inflow, to the dog spleen caused it to expel its stored blood. Spleen size in Weddell seals has been reported to be correlated to plasma catecholamine concentrations during diving and also contracts in response to exogenous epinephrine infusion [82]. Innervation of the dog, cat, rat, mouse, and

11 human spleen are all adrenergically (sympathetically) controlled while cholinergic (parasympathetic) and sensory/tactile innervation is limited or non- existent [83]. The spleen belongs to the most densely sympathetically innervated organs, with a norepinephrine turnover several times higher than the liver or the lungs [84].

Accordingly, epinephrine, norepinephrine, and neurostimulation all cause alpha-mediated vasoconstriction and contraction of the splenic capsule in both non-humans and humans [82, 85-88]9. Indeed, Shimizu and associates [89] using microdialysis in conscious rats found that norepinephrine recovered in the spleen was derived from the nerve terminals of the splenic sympathetic nerve. Rogausch and associates [52] found that removal of noradrenergic sympathetic transmission to the spleen resulted in increased spleen blood flow and cell retention. The α1- adrenergic receptors appear to be activated during spleen contraction in rats [90]. Numerous and varying stimuli ranging from corticotrophin-releasing factor from the brain [89], cocaine administration [91], to vasoactive amines [92, 93] may have the same catecholamine-releasing effects resulting in splenic contraction. In the case of repetitive exposure to short-term hypoxia, observations in rats suggests that splenic contraction is enabled by an increased response of the α2-adrenergic receptors to their catecholamine agonists [69]. But there is little support for other vasoactive substances such as nucleotides, ions, angiotensin II etc. influencing spleen volume through direct effects in humans [83].

Another possibility is that the mechanisms underlying splenic contraction also have their roots at least partially in chemoreception. Hoka and associates’ study [68] of hypoxia in dogs suggested that both “early” sympathetic nerve activity (1 minute after initiation of hypoxia) and “late” activity (2.5 minutes after hypoxia) could be explained by chemoreceptor stimulation and central nervous system (i.e. medullary) stimulation, respectively. The latter, also known as the central pressor effect [94] may be augmented by decreased cerebral perfusion pressure. Cutler and associates [95] found that periods of intermittent hypoxic apnea could alter chemoreflex control of muscle sympathetic nerve activity in humans, elevating the baseline sympathetic output. Hypoxia appeared to be the primary stimulus for this response. While sympathetic nervous output to the spleen was not directly investigated, in light of Hoka and

9 An additional rhythmic contraction of the spleen, at least in dogs, is known to occur even after denervation, and is likely a result of variations in blood flow to the splenic circulation [41]. These rhythms, however, are not present during contraction of the spleen caused by rebreathing or catecholamine injection.

12 associates [68] previous research in dogs suggests that altered chemoreflex control may also affect spleen volume.

Additional axoaxonal innervation along the length of the splenic nerve may adjust its ability to influence spleen contraction. Quatacker and associates [96] found adrenergic, post-ganglionic inputs on bovine splenic nerves, allowing direct (and most likely inhibitory) modulation of the splenic nerve axons. Direction associations between noradrenergic fibres of the sympathetic nervous system and lymphocytes in the spleen’s white pulp provide a route for direct influence of the autonomic nervous system [97]. While these are postulated to serve as interactions between the nervous system and the spleen’s immune functions, it is not improbable that collateral influence on the spleen’s contractile functions could also occur.

Effects of the spleen on cardiovascular function

Herman and associates [88] found in dogs that splenic afferent nerve activity followed unmyelinated C-fibres, a relatively slow-firing nerve type that lends itself to tonic firing rates and over longer durations. Perhaps more interestingly they were the first to demonstrate low-pressure baroreceptors in the spleen’s venous side that responded to increases in spleen venous blood pressure. These receptors in turn appeared to produce reflex alterations in cardiopulmonary and renal sympathetic nerve activity via spinal sympathetic pathways, resulting in increases in heart rate and blood pressure. The existence of such spleen receptors was later confirmed in cats by Calaresu and associates [98], and when stimulated, resulted in increased splenic efferent nervous activity and venous pressure and furthermore elicited reflex cardiovascular responses. These reflex responses were achieved through stimulation of the receptors by distension, bradykinin and capsaicin, but not through contraction of the spleen by norepinephrine injection. Both the Herman and Calaresu groups suggested that stretching of the spleen, but not contraction of the spleen, resulted in the reflex cardiovascular adjustments. However, there was disagreement amongst these groups about the presence of myelinated fibres in the splenic nerve, something that Calaresu’s group appeared to confirm.

Furthering the issue of baroreception in the spleen, Deng and Kaufman [99] highlighted the effects of splenic control of arterial pressure, where they found that intrasplenic nitric oxide reflexly altered MAP, an effect that could be abolished by splenic denervation. Further research by the same group [100] showed that splenic venous pressure stimulated splenic afferent nerve activity and activated a splenorenal reflex, which might account for this effect. Thus it

13 appears that the spleen, along with the liver and kidneys, is integrally involved in cardiovascular homeostasis via afferent nervous activity.

AIM OF THE THESIS

With such diverse roles indicated for it, and with such integrated physiological mechanisms associated with it, it is surprising that the spleen has not received more recognition as a useful, functional organ. This may indeed be due to the elusiveness of identifiable mechanisms behind its various functions, and its relatively newly found applications for the healthy human.

One of these recently established applications is the spleen’s ability to contract in certain situations to inject a stored amount of red blood cells [48, 49, 101]. In some non-human species, especially mammalian expert runners and divers, the involvement of sympathetic pathways in this contraction is fairly established and similar processes may be at work in humans as well. What is not well understood is the range of situations in which spleen contraction may occur: besides voluntary apnea and hard physical work, there are a number of situations where an increase in blood gas transportation capacity would be beneficial. The “triggers” of the spleen contraction also remain to be clarified. Nor is it clear which groups might draw benefits from the effects of spleen contraction, or to what extent. The ability to alter, adapt or maximize the contraction of the spleen to obtain greater effects for the individual is also unclear.

This thesis addresses these issues through a series of experimental studies aimed at better understanding the triggers, effects, and potential uses of the spleen’s hematological contributions in different environmental conditions and activities.

METHODS

Measurement of blood variables

Venous capillarisation for Hb/Hct

The main hematological measures used in experimentation were Hb (reported in g/L) and Hct (reported in % blood volume, BV). These measures were obtained through closed-system antecubital venous sampling in all studies, although in

14 the study of trained divers, skiers and untrained humans [102] they were also obtained through capillary puncture.

Hb is normally the most important factor in determining blood O2 content, and accordingly the higher the Hb, the greater the O2 content of blood at a given PO2. Normal values are 130-180 g/L for males and 120-160 g/L for females. Hct or packed cell volume (PCV) measures the proportion of blood volume that is occupied by red blood cells, and is also an important determinant of blood viscosity and microcirculatory flow changes. Hct normal values (±SD) are 45±7% BV for males and 42±5% BV for females.

Hb was measured via automated blood analyzer using the cyanomethemoglobin method, where a lysing agent is added to a sample of diluted blood, disrupting the red cells in the sample and releasing the hemoglobin into the fluid. The hemoglobin is converted to cyanomethemoglobin and the concentration is read by a spectrophotometer and calculated from the optical density of the solution. Hct is calculated by multiplying the red cell count and the mean red cell volume, both of which were measured directly by automated blood analyzer. Studies that analyzed blood samples in-house used the Micros 60 automated blood analyzer (ABX Diagnostics, Montpellier, France) while studies that sent blood samples to hospital for analysis used a similar apparatus with the same measurement mechanism.

Hemoglobin concentration and red blood cell mass are independent of blood volume, and neither is associated with spleen size in mammalian hypobaric hypoxic responses [103].

Measurement of spleen size

Spleen size was measured in four studies in the current thesis (Papers I, III, V and VI). During spleen measurements, triaxial ultrasound measurements were obtained via stationary ultrasound apparatus (HDI 3000, ATL A, Philips Company, Bothwell, WA, USA) or via portable ultrasound apparatus (Mindray DP-6600, Shenzhen Mindray Bio-Medical Electronics Co., Ltd., Shenzhen, China). These measurements were then used as a basis for spleen volume calculations, using a formula developed by S. Pilström: Lπ(WT-T2)/3, where (L) is spleen length, (W) width and (T) thickness. The formula is based on the average shape of the spleen observed on the ultrasonic images and describes the difference between two ellipsoids divided by two. This formula was compared to one previously used by Breiman and associates [104] and Sonmez and associates [105], where volume is calculated by spleen length x width x thickness x 0.523, 15 and the two methods on average differed by less than 4 mL, or approximately 1% of the measured volume.

Ultrasound measurement of spleen volume has demonstrated low intra- and interobserver variability as well as excellent correlation with computerized tomography [106, 107]. In vivo ultrasound measurements of spleen volume have been shown to correlate well with the weight [108] and volume [109] of the resected spleen.

Measurement of other parameters

Arterial oxygen saturation

Arterial oxygen saturation (SaO2) measures the percentage of hemoglobin binding sites in the arterial bloodstream occupied by O2. This measurement was, in the works of this thesis, obtained through pulse oximetry, although it can be obtained via other methods as well. Pulse oximetry uses a light sensor containing two sources of light (red and infrared) that are absorbed by hemoglobin and transmitted through tissues to a photodetector. The amount of light transmitted through the tissue is then converted to a digital value representing the percentage of hemoglobin saturated with O2.

Pulse oximetry has been shown to have a slightly positive bias in critically ill patients, whereas in healthy patients the magnitude of differences between pulse oximetry SaO2 readings and SaO2 values obtained from blood samples are small [110] and should be considered reliable. The averaging window for the raw data logged from the pulse oximeters used in the current studies (Biox 3700e, Ohmeda, Madison, WI, USA and WristOx 3100, Nonin Medical Inc., Plymouth, Minnesota, USA) was 2 seconds. Heart rate was also measured simultaneously from the same machines.

Normal SaO2 values are 97% to 99% in the healthy individual, whereas 90% or lower is defined as clinical hypoxemia [3]. Using the oxyhemoglobin dissociation curve, an O2 saturation value of 90% is generally equated with a PaO2 of 60 mm

Hg, depending on e.g. the PaCO2.

The rate of arterial oxyhemoglobin desaturation during apnea is correlated to lung gas volume at apnea onset, resting O2 consumption, and pre-apneic SaO2 [111]. The depth of desaturation is dependent upon the duration of the apnea and the rate of desaturation, however these two items do not always appear to be

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correlated to each other [111]. Central venous blood O2 levels at apnea onset play a major role in determining the rate of desaturation, however [111].

Mean arterial pressure

The mean arterial pressure (MAP), or the average arterial pressure during a single cardiac cycle, depicts the average blood pressure of the individual, according to the following equation at normal or low heart rates: MAP = DP + 1/3 (SP-DP), where SP and DP are systolic and diastolic pressures, respectively. MAP is also considered to be the perfusion pressure experienced by organs in the body, including the spleen.

Cardiac output and systemic vascular resistance affect MAP, which is to say that everything that affects these two parameters also affects MAP i.e. stroke volume is affected by ventricular preload, venous compliance, and blood volume, thus MAP as well.

In those studies where it was measured, MAP was measured continuously from the finger via automated sphygmanometer (Finapres 2300, Ohmeda, Madison, WI, USA). This non-invasive device has shown good correlation with invasive (i.e. intra-radial) measurements of MAP not occurring during anaesthesia [112], and also for measurement of MAP according to the aforementioned equation at rest or during light exercise [113]. Reliability of continuous MAP measurement from the finger can be influenced by changes in limb posture (unpublished observations) and limb cooling [114]. In our studies these two items were held constant and thus should not have affected recording reliability. The Finapres does, however, have a tendency to underestimate MAP during extreme peripheral vasoconstriction [115] and thus it is recommended to monitor peripheral vasoconstriction simultaneously, using skin blood flow measurements, for instance.

Skin blood flow

Skin blood flow (SkBF) measurements in the works included in this thesis were intended to represent peripheral vasoconstriction status. Vascular factors such as nitric oxide, endothelin, and prostacyclin all have influences on vessel diameter, as well as tissue factors such as adenosine, K+, histamines, H+ etc. However, myogenic mechanisms that, intrinsic to vascular smooth muscle, alter vessel diameter are the intended parameter for measurement in skin blood flow recordings. These alterations are primarily regulated by arterial baroreceptors, and to a lesser extent, chemoreceptors via neurohumoral pathways, which further affect venous compliance, systemic vascular resistance, heart rate, and 17 stroke volume. Thus SkBF is almost certainly integrally related to effects on MAP.

SkBF in the works of this thesis was measured via laser Doppler flowmetry (Periflux System 5000, Perimed, Järfälla, Sweden). The laser Doppler technique measures blood flow in the very small blood vessels of the microvasculature, primarily the low-speed flows in capillaries close to the skin surface. The technique depends on the Doppler principle whereby low power light from a laser is scattered by moving red blood cells and as a consequence is frequency broadened, and then sent to the machine via fibre-optic probe. In the current studies, the laser-Doppler probe was medially attached to the first joint of the thumb. Continuous recordings were made throughout the duration of the experimental session.

Of all the recorded parameters in the studies in this thesis, however, it is also most unstable due to its substantial temporal and spatial variability. Consequently, the comparatively small sampling volume (about 1 mm3) of fibre- optic-based laser-Doppler flowmetry instruments constitutes one of the main limitations of this technology: with the spatial variability in tissue blood perfusion, gross differences in perfusion readings may appear at recordings from adjacent sites. Temporal variability, however, is recorded effectively from single sites, and thus gives a good picture of changes in peripheral vasoconstriction.

Respiration and gases

In the studies where it was measured, the mechanical characteristics of breathing before, between and after apneas was measured via lab-developed chest bellows, placed around the circumference of the thorax just below the pectoralis major muscles. The pneumatic chest bellows produce a pressure change with every chest movement that is then amplified and converted to a digita l signal for online recording. The chest bellows were used during apnea to designate the onset of involuntary breathing movements, often referred to as the physiological breaking point of the apnea. They were also used for comparison of respiration during non-apneic periods and could thus provide a gross measure of ventilation rate and depth.

Some of the studies included in this thesis also measured inspired and expired percentages of O2 and CO2 via gas analyzer (Normocap Oxy, Datex-Ohmeda, Helsinki, Finland). One of the main intentions with the measurement of expired

CO2 pre-apnea was to ensure primarily that its level was equivalent for repeated

18 apneas, thus ensuring that hyperventilation did not occur, or in the cases where it was included in the protocol that hyperventilation occurred to a similar level in each apnea. Similarly, inspired and expired O2 levels are measured in Papers IV and V to confirm that any hypoxic influence was absent when it was not wanted.

Expired CO2 post-apnea also provided an indication of CO2 production and transfer to the alveoli during apnea.

Apnea and hypoxia experimental protocols

Apnea series

The experimental protocol for apnea in all of the current studies except Paper III followed essentially the same steps. Subjects began with a rest period, typically 20 minutes, in a prone or supine position (depending on the study) to ensure blood mixing and stabilization of the transcapillary fluid exchange [116]. During this time all required equipment probes were placed, as well as catheterization of the arm when venous blood sampling was required.

All apneas were preceded by a 2-minute countdown. During this time before each apnea, the “control” period was established, typically a 30s or 60s window, which ended a minimum of 30s before the onset of apnea. This was used in later analyses to denote changes occurring due to apnea, and also for comparison between subsequent apneas i.e. recovery characteristics. Three apneas were standard in the current studies, each spaced by 2 minutes. A nose clip was applied in the final minute before apnea onset, and a mouthpiece or mask, dependent on whether the protocol calls for pre-apneic inspiration of special gas mixtures, was placed for measurement of inspired and expired gases. The subject determined the onset of apnea at or near the end of an audible 10s countdown demarcating the end of the 2-minute countdown period. Each apnea was preceded by a full exhalation, followed by a deep but not maximal inspiration, as instructed to fall in the range of 85% range of vital capacity. Self selection of the final inhalation depth has been shown to yield reliable and repeatable results in our numerous studies following the same protocol (see Schagatay et al, 2001, Table 2) and maintains subject comfort. If pre-apneic hyperventilation was required, the subject was given audible feedback on rate and depth of breathing by the experimenter during the hyperventilation period, but the apnea began in the same manner as in non-hyperventilation trials.

The provision of time cues during apnea was dependent upon the protocol, but maximum duration apneas typically occurred without time cues. Upon completion of apneas, subjects expired fully through the mouthpiece or mask and then resumed normal breathing. 19

An online data acquisition system (BioPac MH100A CE multi-channel data acquisition system, BioPac Systems Inc., Goleta, CA, USA) recorded some or all of the following parameters continuously from the start of the first 2-minute countdown until the end of the protocol: SaO2, HR, MAP, SkBF, O2, CO2, breathing movements from the chest bellows, and apnea time.

Inspired gases and hyperventilation

In some of the experiments included in this thesis, inspired gases and breathing rates were altered in order to maintain a specific physiological state or environment. These experimental interventions included:

1) the use of 100% inspired O2. This intervention was employed in Papers IV and V to remove any hypoxic stimulus that would normally result from

apnea. The intervention was achieved by providing medical O2 through a non-rebreathing mask at 5-10L/minute.

2) the use of 12.8% inspired O2. This intervention was employed in Paper III to induce a hypoxic stimulus that was not present when breathing normal air. This intervention was achieved via hypoxicator, which is a

machine with a metabolic filter that replaces O2 with nitrogen without changing the ambient pressure.

3) the use of 5% inspired CO2. This intervention was employed in Paper V to induce a greater hypercapnic effect during apnea. This intervention was

achieved through a mixture of CO2 in medical O2 and via non- rebreathing mask at 5-10L/minute. 4) the use of hyperventilation. This intervention is employed in Paper V to

reduce the body’s CO2 stores prior to apnea, to reduce the effect of hypercapnia during apnea. This intervention is achieved by advising the subject on increased rate and depth of breathing according to online

feedback of breathing movement and expired CO2 percentage, for 1 min prior to each apnea.

Statistical approaches

Publication statistical analysis method

All experiments in the current study were controlled trials i.e. subjects were measured at least once both pre- and post-treatment, and can also be considered crossover trials, i.e. all subjects underwent all control and experimental treatments. The final outcome statistic is thus the mean change between treatments or from control. Statistical approaches vary amongst the studies in the current thesis dependent on the analysis required, however there are some common methods between them that can be described.

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1) Intra-individual comparisons: these comparisons denote any changes that occur within a single trial or between trials of each individual. For example, a comparison of hemoglobin increase might occur for one individual between their control sample taken 2 minutes prior to the first apnea, and immediately following all subsequent apneas. Another example would include the comparison of hemoglobin increase between a

trial with inspired O2 and one without. Paired t-tests are used in all comparisons with Bonferroni corrections for multiple comparisons when applicable. The level accepted for statistically significant change is P≤0.05. Non-significant trends are denoted for P<0.1 but >0.05. 2) Inter-individual comparisons: these comparisons denote any changes between groups of individuals. For example, a comparison of hemoglobin increase during a trial amongst trained divers might be compared with increases amongst untrained individuals completing the same trial. Control values are also similarly compared between groups as well. Unpaired Student t-tests (with corrections for multiple comparisons, when appropriate) are used in these comparisons and the level accepted for statistically significant change is P≤0.05. Non-significant trends are denoted for P<0.1 but >0.05.

Inter-individual comparisons of control values were usually compared as absolute values i.e. using the units and scales in which the values themselves are obtained. Intra-individual comparisons on the other hand used relative values i.e. percentage changes from control values where the established control value represents zero change. In this manner, subjects served as their own controls and the non-uniformity of individual residuals was reduced.

Analyses of correlations between various parameters e.g. spleen size and diving performance were achieved using Pearson’s product-moment correlations. The level accepted in correlation analyses for presence of significant change is P≤0.05.

Additional statistical analysis method

An additional statistical analysis method (a priori or post-hoc, depending on the study) has been used in all experiments in the current thesis in addition to the methods listed above or in the publications, regardless of whether this second method has been included in the published material or not. These analyses are also based on paired t-statistics for crossover experimental design, but with some deviation from the above descriptions.

a) Firstly, the “relative” values are brought forth using log transformation using the natural logs of the values of the variable – thus removing the

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non-uniformity of the residuals entirely. Standard deviations in these cases are a percent variation or coefficient of variation. b) Secondly, the level accepted for presence of significant change is not determined via P-value, but rather on Cohen’s thresholds for clinical significance [117]. Based upon the smallest clinically relevant values of the effect in question, the Cohen’s thresholds attribute size characteristics to changes i.e. small (0.2-0.5), moderate (0.5-0.8), large (0.8-1.2), very large (1.2 and greater), and can then be extrapolated into a percent probability that the change in measured parameter is indeed clinically significant. In the case of Hb and Hct, the smallest significant change was deemed to be 2% and 0.5% respectively, based upon initial measures of normal responses of average subjects [78, 118] and clinical guidelines [119].

A relevant situation might be as follows: A group of subjects demonstrates a 2.3% increase in Hb after 3 breath-holds. The P-value for the paired t-test comparison between control and post-apnea Hb is 0.06, which according to our set criteria can only conclude that there was no statistically significant change. The Cohen’s threshold is 0.24, and indicates a small but clinically significant increase in Hb, with a 90% confidence interval of ±0.8% i.e. there is a 90% chance that the true Hb increase is between 1.5% and 3.1%.

In this case of the P-value, it must be concluded that no change has occurred, as the P-value falls outside the accepted level of statistical significance. However, a 2.3% change in Hb should not be considered as zero change. Indeed, a 2% increase in Hb would by many physiological standards (and the author) be considered a vital improvement. The Cohen’s threshold illustrates this, and the confidence interval gives an estimation of the uncertainty behind the statistical analysis.

This method is, unfortunately, not yet widely accepted by journal reviewers and editors, and therefore does not constitute the majority of published statistical analyses. However, considering that statistical significance focuses on the mathematical null value of the effect, while clinical significance provides an measure of probability that any effect might have in terms of benefiting or hindering the individual e.g. improving or reducing performance, it can be argued that the latter approach is considerably more valuable and interesting to both physiologists and experimental subjects. For this reason, this second method was used for comparison with the “traditional” statistical analyses. Any deviations or differences between the two approaches are reported in the results section of this thesis, otherwise in can be assumed that the two methods are in agreement.

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SUMMARY OF RESULTS

Occurrence of spleen contraction

PAPER I: Correlation between spleen size and hematocrit during apnea in humans.

Main conclusion: Spleen contraction and circulating erythrocyte increase develop in close association, with the latter arising primarily due to spleen contraction.

In this study, which aimed to further investigate the relation between spleen volume, Hb and Hct during serial apneas, 3 male subjects and 1 female subject performed 3 maximal-duration apneas spaced by 2-minute pauses. Ultrasound measurements revealed a mean reduction in spleen volume of 34% (p<0.01), from 338 ± 93 ml before apnea to 223 ± 66 ml after 3 apneas, while simultaneous increases in Hb and Hct from control values of 2.5 ±1.5 % and 3.1 ± 2.8%, respectively (p<0.05 and p=0.07) were observed. The correlation coefficient mean (with 90% likely range values) between spleen volume and Hb was -0.88 (-1.0 to - 0.26), and between spleen volume and Hct the correlation coefficient was -0.85 (- 0.99 to -0.37). Splenic volume, Hb and Hct had returned to pre-apnea levels within ten minutes after the third apnea. Based on 80% splenic hematocrit estimation, and blood volumes of 8% body weight, the relative contribution of the observed spleen shrinkage to change in Hct was calculated to be 69%. It was concluded that spleen contraction and Hb and Hct increases develop in close association, with the erythrocyte increase arising due primarily to the spleen contraction.

Two additional male subjects were added to the study after publication of Paper I, which confirmed the findings with four subjects (Figure 2). Spleen volume decreased 29% (p<0.01) from 353 ± 101 ml before apnea to 251 ± 76 ml after 3 apneas, while simultaneous increases in Hb and Hct from control values of 3.8 ± 2.3% and 4.3 ± 2.2%, respectively (both p<0.05) were observed. Splenic volume, Hb and Hct had returned to pre-apnea levels within ten minutes after the third apnea. The correlation coefficient means between spleen volume and Hb was - 0.81, and -0.82 between spleen volume and Hct (p<0.05).

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Figure 2. Mean spleen volume (mL) and Hb (g/L) before apneas (Control), after Apnea 1 and Apnea 3, and during the 10 minutes following Apnea 3. N=6. Correlation between spleen volume and Hb is -0.81 (p<0.05)

PAPER II: Increase of hemoglobin concentration after maximal apneas in divers, skiers and untrained humans.

Main conclusion: Non-diving athletes, untrained subjects and apneic divers all respond with spleen related Hb increases during apnea. Trained divers have a stronger response.

A comparison of 29 male and 4 female trained apneists, 13 male elite cross- country skiers, and 23 male and 9 female untrained individuals showed that the apneists tended to have a higher baseline Hb (150.1 g/L) than the other groups (145.5 g/L and 146.9 g/L, respectively). All groups showed an increase in Hb after 3 apneas, but the largest relative increase was in divers (2.7%) compared to the other groups (2.1% and 1.4%, respectively). By 10 minutes after apneas, all groups had returned to baseline Hb values.

The apneic duration for the first apnea was also longest in the divers (144s) compared to the skiers and untrained individuals (87s and 94s, respectively), and while all groups increased their apnea duration across the series, the divers showed the largest increase (20%, compared to 12.2% and 15.5%, respectively).

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There was no correlation between individual apneic duration and increase in Hb over the series in any group.

PAPER III. Short-term effects of normobaric hypoxia on the human spleen.

Main conclusion: Eupneic hypoxia initiates spleen contraction and Hb increase, suggesting a role of spleen contraction for Hb elevation in the early phase of altitude acclimatization.

A pilot study at altitude was completed with three male subjects [120] revealing that the Hb increase normally associated with apnea was reduced and eventually diminished at 5000 m.

In a following laboratory study using 20 min exposure to a low-O2 (12.8%) but normocapnic environment, 4 males and 1 female untrained in apnea showed a reduction of SaO2 to 64.4% and a simultaneous decrease of spleen volume by 17.6%. Hb and Hct simultaneously increased by 2.1% and 2.1% respectively, and all parameters reverted to normal within 10 minutes from removal of the hypoxic environment. Negative correlations between spleen volume and both Hb and Hct (r=-0.51 and r=-0.42, respectively) were found.

Initiation of spleen contraction

PAPER IV: Hypoxia augments apnea-induced increase in hemoglobin concentration and hematocrit.

Main conclusion: Hypoxia augments spleen contraction during apnea, but there is some residual contraction even without hypoxia indicating that other factors are involved

After 3 apneas in oxygen and without desaturation, 10 male untrained subjects showed lesser increases in Hb and Hct (2.0% and 2.0% respectively) than when they performed apneas of the same duration in air with desaturation to 84.7%

(3.1 and 3.0%, respectively). Expired CO2 after apneas was identical in both trials. The Hb values returned to normal within 10 minutes after the final apnea. There were no differences in the diving response between the trials.

Secondary statistical analysis using Cohen’s thresholds calculated that in the air group, Hb and Hct were still elevated by a small amount after 10 minutes, with 63% and 55% likelihood, respectively.

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PAPER V: Hypercapnia moderates hemoglobin increases during apnea.

Main conclusion: Hypercapnia during apnea causes increases in hemoglobin and hematocrit in the absence of hypoxia, but not solely through breathing high levels of carbon dioxide.

Four male and four female subjects conducted four separate 3-apnea series, each in hyperoxia but with varying levels of CO2 – a series 1) with pre-apneic hypercapnic inhalation, 2) after normocapnic inhala tion, and 3) at hypocapnia achieved via hyperventilation. In addition three test-periods were performed 4) without apnea but breathing 5% CO2 in O2 for an equivalent duration as the apneas in the other trials. Hb increased most with pre-apneic hypercapnic inhalation (by 4.0%) than in the normocapnic, hypocapnic, and eupneic trials (which had Hb increases of 3.0%, 1.8% and 0.8%, respectively). The physiological breaking point of apnea was shortest in the hypercapnia trial, while at hypocapnia it was the longest. Expired CO2 followed a similar pattern as the Hb increases – greatest in the hypercapnia trial, and lowest in the non- apneic trial. No desaturation occurred in any trial.

Three subjects had spleen measurement completed during all of the trials. The change in spleen size after apnea 3 for the hypercapnic, normocapnic, hypocapnic and eupneic trials was -32.5%, -9.4%, 30.4% and 13.0% from control, respectively (Figure 3 a-d). Ten minutes following the final apnea, spleen size showed a return to control values in all trials.

Secondary statistical analysis using Cohen’s thresholds calculated that the increase in Hb and Hct in the hypercapnia trial was greater than the hyperventilation and eupnic trials by a large amount (94% and 97% likelihood, respectively), but only by a small amount in comparison with to the normocapnia trial (53% likelihood). Hct in the hypercapnia trial was greater by a large amount compared to the hyperventilation and eupnea trials (87% and 89% likelihood, respectively). It was lower than in the normocapnia trial by a small amount (53% likelihood), however.

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Figure 3 a-d. Mean spleen volume (mL) and Hb (g/L) before apneas (Control), immediately following Apnea 1 and Apnea 3, and 10 minutes following Apnea 3, for hypercapnic, normocapnic, hypocapnic, and eupneic series. N=3.

Consequences and applications of spleen contraction

PAPER VI: Spleen and lung volumes correlate with performance in elite apnea divers.

Main conclusion: The size of the spleen correlated with competitive apnea performance in elite divers. This study suggests that the spleen volume as well as the lung volume may predict apnea performance.

In studying 14 male elite apneists competing at a world championship competition, it was found that their spleen volume (both absolute and corrected for anthropometric data) was correlated with their combined three-discipline competition score, but not with their height or weight. Their competition score was not correlated with height, weight, hours of training or BMI, either. Lung volume was also correlated with performance.

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The three highest scoring divers at the competition had a mean spleen volume of 538 ml, the largest volumes measured amongst the competitors. They also appeared to have a very large contraction during a single 2-minute apnea, showing a volume reduction to 258 ml. In contrast, the three lowest scoring divers had a mean spleen volume of 270 ml. The extra apneic duration allowed by the spleen contraction of the 3 best divers was estimated to be 30s.

PAPER VII: Cardiovascular and haematological adjustments to apneic diving in humans: Is the spleen response part of the diving response?

Main conclusion: The spleen response and the cardiovascular diving response to apnea are not parts of the same response, and should be considered as separately occurring entities.

While the apnea-induced cardiovascular diving response and spleen contraction both have dive-prolonging effects, these two phenomena should not be considered as functionally connected parts of a single general response to apnea. The increase in Hb and Hct from spleen contraction is not augmented by facial chilling, and does not develop fully during a single apnea, but rather over a series of apneas, and is not reversed until 10 minutes without apnea. The cardiovascular diving response, in contrast, is fully developed within 30 s during each apnea, recovers rapidly following apnea, and is also stronger with facial chilling. The effects of these two phenomena are also realized through different mechanisms – the diving response acts through increased O2 conservation, while the spleen response exerts its influence through increased O2 storage and CO2 buffering capacity.

DISCUSSION

Occurrence of spleen contraction

The results presented in this thesis clearly show that humans respond with elevations in Hct and Hb during apnea, and these elevations correlate closely with splenic contraction. This response is also active during normobaric hypoxia, which may explain the early elevation of Hb and Hct seen at altitude exposure. The contribution of the spleen to these hematological changes is calculated to be as high as 69%, while the remaining contributions may possibly arise from other pooled blood stores with high hematocrit in the abdomen, such as the liver [75], or from plasma extravasation [44].

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The presence of spleen-related hematological increases in apnea divers is a finding similar to that of Hurford and associates [48]. However, the work in this thesis described significant levels of spleen contraction even in individuals with no experience in apnea, in accordance with the findings of Schagatay et al [50], Espersen et al [77] and Bakovic et al [76]. However, the work in this thesis also demonstrated that the spleen response was more pronounced in apnea divers, not only in comparison to sedentary subjects, but also elite aerobic endurance athletes. This suggests either a specific apnea training effect on the spleen, or predisposition.

The novel finding that simulated altitude by normobaric hypoxia induces spleen contraction is interesting as it suggests a role of the spleen in the Hb elevation often observed in the early phase of altitude exposure. Sonmez et al [105] had previously noted a reduction in spleen size in humans after prolonged residence at high altitude, but such a response would perhaps be even more beneficial when compensating for the acute effects of a transition to a lower O2 environment.

Identification of these situations and groups in which spleen contraction occurs provided a background for further investigation of the mechanisms underlying the response pattern.

Initiation of spleen contraction

That both hypoxia and hypercapnia play a role in triggering spleen contraction are also major novel findings. Hypoxia was found to cause spleen contraction regardless of breathing state – both during apnea and normal ventilation the spleen responded to hypoxia. Hypercapnia, on the contrary, appeared to only play a role during apneic states; normal breathing of CO2, at least under shorter periods, does not appear to cause spleen contraction at all. It is, however, a residual effect of apnea without excessive hyperventilation, and thus underlies essentially all apneic periods even when hypoxia is eliminated. Combined, hypoxia and hypercapnia may even achieve a similar contractile state as either can alone, suggesting that there may be a threshold level of contraction i.e. the spleen becomes fully contracted by a certain amount of either stimulus and no further contraction can be obtained with additional stimulus load.

That hypoxia and hypercapnia play a role in spleen contraction during apnea infers that some form of chemoreception must also be involved in initiating the contraction process. While no direct evidence of the input of these physiological systems can be derived from the current studies, the possible chain of events from stimulus detection to effect is a worthwhile discussion point.

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Stimulus detection by chemoreception

The body has at least two highly sensitive receptor systems that initiate system-wide nervous feedback during changes in CO2 and O2 respectively: the central chemoreceptors located in the pontomedullary region of the brain which are stimulated by pH changes caused by diffusion of CO2 into the cerebrospinal fluid, and the carotid bodies, themselves only a few micrograms in weight, which are stimulated by changes in PO2 in the plasma (though CO2 may also have minor effects here as well, in the order of 10-20% [121]). Comparisons of the speed of response and relative gains of these two systems have been studied for decades and are still embroiled in controversy.

Hypoxia augments the sensory discharge of the carotid bodies before it reaches the central nervous system, and then triggers the CNS to initiate autonomic changes such as increased breathing rate and blood pressure. Although there is debate about the mechanisms behind arterial PO2 sensing, it is currently thought that activation of the afferent nerve ending in the carotid body is responsible for detecting even modest changes (i.e. PO2 of 100 to 80 mmHg) within seconds, via ion transporters, mitochrondrial cytochromes or a combination of the two [122]. Cellular responses in the rest of the body often require far more severe hypoxia (ca 40 mmHg) before initiation, and take minutes to hours to develop. Thus it can be logically suggested that apneic interventions resulting in hypoxia or acute hypoxic environments stimulate the carotid bodies, which in turn elicit subsequent systemic effects. The results from the studies in this thesis examining the effects of hypoxia during apnea and during normobaric hypoxia follow this line of reasoning.

Changes in alveolar CO2, on the other hand, are reflected in brain extracellular fluid pH on a time course consistent with circulation time i.e. a few seconds [123]. Nonetheless, when peripheral receptors are bypassed, ventilatory response is about 58% slower in dogs [124]. The same research in dogs also demonstrates wide individual variation in the relative contribution of central vs. peripheral chemoreceptors to gain effects, although central reception appears dominant i.e. approximately 60%. This holds true in other studies of dogs [125] that experienced hypercapnia in hyperoxia, a method similar those used in Paper V included in this thesis.

Earlier research demonstrated the cross-influence of hypoxia and hypercapnia

[126]. For example, the higher the PCO2, the greater the hypoxic sensitivity of the carotid body [127]. Along the same lines, low O2 potentiates the ventilatory

30 response to hypercapnia. The carotid body chemoreceptors are the site of hypoxic-hypercapnic interaction [128] and also control the increased ventilatory response post-apnea. Indeed, these receptors appear to be essential for the initiation of involuntary apnea, for example, during sleep apnea and periodic breathing due to low CO2 [129]. Thus a major role for carotid body chemoreceptors could be theorized in terms of spleen contraction when its initiation follows chemoreceptive stimuli. CO2 elevation always develops during apnea, even in hyperoxia, and thus stimulation of the chemoreceptor system is inevitable.

This is not the case, however, for normobaric hypoxia without apnea. Even without hypercapnia or cessation of breathing a notable spleen contraction and Hb/Hct elevation is evident. Here it is hypoxia that is the sole stimulus, and it produces a spleen contraction-related elevation in Hb similar to those attainable during the apnea trials without hypoxia (but not with hypoxia). It is logical that peripheral chemoreceptor stimulation is the primary system affected in these cases. However, neither normobaric hypoxia, nor apnea without hypoxia reached the same levels of Hb/Hct elevation that apnea with hypoxia did. This provides further evidence for the synergistic effect of hypoxia and hypercapnia in producing maximal spleen-related Hb/Hct elevation. Indeed, central and peripheral mechanisms may act in a redundant fashion to reconfigure ventilatory responses after intermittent exposure to altered blood gases, a finding which Cummings and Wilson [130] found in their own studies in rats.

The co-release of transmitters at the carotid body may also signal an interaction of O2 and CO2. ATP appears to play a critical role in subsequent nerve activation by hypoxia [122]. However, acetylcholine has also been implicated as a co- release mechanism with ATP, and the release of this neurotransmitter appears to be facilitated by CO2. Blockade of ATP receptors at the carotid body also appears to prevent sensory excitation by CO2 [131].

Output following chemoreception

Triggering of the chemoreceptors then requires an effector signal, in an attempt to adapt to the environmental conditions, a response in which spleen contraction is likely included. Considering that in most mammals the splenic nerve is 98% composed of sympathetic nerve fibres [132], and that the sympathetic nerve terminals in the spleen store, take up, and release norepinephrine in response to stimulation [133], it is likely that neuronally-derived catecholamine release is the end-point in the chain of events resulting in spleen contraction. While the onset of this could potentially occur relatively quickly, as the nervous system works at greater speeds than systemic circulating hormonal or other chemical

31 release acting upon local receptors, the actual contractile effect may also follow a slower time pattern more dependent on the contractile properties of the spleen’s musculature and the progressive development of asphyxia.

Whether the nervous input exerts its effects independent of, or as a result of, changes in chemoreception, is debatable. It is possible that splenic contraction signals come directly from higher neural input. The ventrolateral medulla, which contains a group of neurons projecting to spinal sympathetic preganglionic neurons, has been indicated in splenic excitatory responses [134]. Such input might be expected to be responsible if cessation of breathing were enough to trigger medullary impulses for splenic contraction, in the absence of changes in blood gases.

But the evidence for influence of chemosensitive mechanisms on neural input is far more extensive. The idea that CO2-sensitive neuronal mechanisms might be involved in the generation of sympathetic tone is not new [135]. More recent studies have shown that 30s of intermittent hypoxic apneas for 20 minutes [136] and 20s of intermittent hypoxic apneas for 30 minutes [16] increase muscle sympathetic nervous activity (MSNA), an alteration of chemoreflex control tha t appears to be mediated by hypoxia. In the latter study, MSNA remained elevated for 30 minutes post-apneas, a finding which has been noted in obstructive sleep apnea patients. Though, other recent work [137] found this not to hold true in elite breath-hold divers’ resting MSNA values, suggesting that the effect of apneas on sympathetic outflow may not be chronic.

Work by O’Donnell and associates [14] showed that renal sympathetic nerve activity increased during apnea and was correlated to directly recorded carotid chemoreceptor activity. Potentiation of the chemoreflexes also appears to occur when the inhibitory influence of stretch receptors is eliminated, as during apnea, and this potentiation is greater on the peripheral side [138].

Which of O2 and CO2 has the greater influence on eventual neural output to the spleen is also discussible. Earlier work in sleep apnea patients showed that 100%

O2 results in reductions in central sympathetic outflow [139]. Augmentation of nerve activity during apnea has been shown to be much greater with hypoxia than with hypercapnia [121]. In Paper IV in this thesis, hypoxia caused spleen contraction even in the absence of apnea. However, similarly large increases in Hb and Hct occurred in hypercapnia without hypoxia, suggesting that the chemoreceptive output from either trigger is equally great. Nonetheless, recent studies in this author’s laboratory also showed that a 2 min period of apnea (hypercapnic hypoxia) induced a spleen volume reduction twice as large as in

32 eupneic normobaric hypoxia (normocapnic hypoxia) despite similar levels of hypoxia (in manuscript).

There is also little argument that catecholamines are released systemically during hypoxia, but whether these are responsible for the initiation of spleen contraction and/or the maintenance of spleen contraction remains to be seen. Earlier research [140] showed that in both arterial and primary tissue hypoxia the sympathetic nerves played a more important role in circulatory responses than the adrenal medullary hormones, and that sympathetic nerve control alone produced more peripheral vasoconstriction than increased adrenal catecholamine secretion.

The time scale for systemic catecholamine release is not as fast as neural conduction. This also holds true for local catecholamine output, such as in the carotid bodies, which release catecholamines in response to hypoxic insult. However, it has been shown that this release is not required for hypoxia-induced chemosensory excitation in the carotid body, as it typically occurs 1-3 minutes following the initial chemoexcitation [141]. Sonmez and associates [105] study showed a persistent reduction in spleen volumes after chronic exposure to altitude after individuals moved there from sea level. The increase in catecholaminergic discharge at high altitude [142] may be one explanation, whereby a low-O2 environment may continue to stimulate catecholamine secretion and subsequent spleen contraction even over long periods.

It seems likely that a combination of neural input and catecholaminergic release resulting from chemoreceptive stimuli initiates and prolongs the splenic contraction, respectively. Ojiri and associates [90] showed that α1-adrenergic blockers prevented increases in Hb and Hct in resting dogs induced by splenic nerve stimulation and epinephrine and norepinephrine infusion. In humans, α- adrenoreceptor blockers abolished the vascular responses of the spleen to norepinephrine and direct neural stimulation [85]. Thus it appears that both neural and catecholaminergic innervation are susceptible to blockade when α1- receptors are antagonized. Thus both may affect the spleen, but perhaps on separate time scales. Catecholamines circulating systemically have a half-life of about 2 minutes, whereas nervous impulses can be ceased immediately. The delay in complete recovery of the spleen following apnea (up to 10 minutes) despite removal of chemoreceptive stimuli could be accounted for by residual systemic catecholamine presence.

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Other potential output mechanisms

That other basic autonomic mechanisms could lead to spleen contraction is also a possibility. Apneas held at inspiratory capacity have been shown to cause increases in muscle sympathetic nerve activity [143], which may be related to the unloading of low-pressure baroreceptors located in the atria and pulmonary veins. These baroreceptors respond to small changes in absolute venous pressure – likely to occur during breath holding at larger lung volumes – and may send signals to neural regions responsible for control of sympathetic activity. Spleen contraction may be affected as a result of the increased sympathetic output, through mechanisms already named. Macefield and associates [144] used brain imaging techniques to study exactly this phenomenon during 40s apneas, but used calculation methods designed to eliminate the effects of hypercapnia, making theoretical interpretation of the results difficult in the context of this thesis.

Palada and associates [145] also indicated downloaded baroreflex signals as a possible mechanism of splenic contraction during apnea at large, but not small lung volumes (although spleen contraction still occurred even at small volumes). Their own previous work suggesting an active contractile response during apnea suggests that the neural functions are still involved via sympathetic output, but that baroreception, similar to chemoreception, may also be implicated. Kaufman’s group have also explored similar avenues of spleen function, showing that the spleen may play a role in tonic maintenance of blood pressure in normal physiological conditions [99], and that afferent splenic nerve activity is increased by augmented splenic venous pressure – and not blood flow [100]. Their findings lend weight to the possibility of local baroreception playing not only a role in systemic blood pressure regulation, but even sympathetic nerve activity in local circuits between the spleen and the liver, kidneys, and perhaps itself. Whether such pressure changes may affect spleen contraction via these routes is not certain, but certainly justifies further investigation.

Consequences and applications of spleen contraction

Another novel finding in this thesis is the association between spleen size and apneic performance during competitive apnea, indicating the beneficial effects of a large spleen on overall diving performance. This is perhaps the most applicable finding of the thesis, whereby the maximization of spleen contraction during breath holding is of particular value. This author has previously shown

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[78] that the level of spleen contraction varies greatly between individuals, and Paper II and Paper VI in this thesis suggest that trained apneists are among those who the most powerful elevation of hematocrit related to spleen contraction during apnea. There are thus several ways in which apnea training could lead to performance-boosting hematological effects.

The presence of higher resting Hb levels in trained apneists also points to a possible training effect specific to breath holding, most likely due to long-term effects. The levels of hypoxia obtained during voluntary (and often involuntary) breath holding are typically greater than can be experienced even at maximal levels of exercise [146] and in many cases equal or exceed those attainable at high altitude.10 Thus, regular exposure to hypoxia through breath-hold training may have any number of the following effects: an increased stimulus for erythropoeisis, an increase in the ability to contract the spleen, and/or an increase in spleen size and therein storage capacity. The erythropoietic effect is likely, considering that this author’s research identified increases in endogenous EPO after a single apnea training episode [147] (paper not included in this thesis), which in other circumstances such as altitude training has been shown to lead to increased novel Hb production. The second effect also appears to be confirmed in the first two studies of this thesis. The final possible effect, spleen growth, is not as certain. In the study of the world championship divers, apneic training hours leading up to the competition did not correlate with spleen size or performance. Spleen size can indeed change in individuals, as shown in studies of splenic regeneration after splenectomy and by splenomegolamy, but that its size can be directly affected by training is still uncertain.

Nonetheless, the spleen size in divers likely confers an advantage in terms of breath holding time, and the same might be said for athletes of any sort that consider maximal O2-carrying capacity a limiting factor in performance. A greater ejection of red blood cells in the spleen leads to increased oxygen stores and an improved CO2 buffering capacity.

In apneic performance

The most obvious applications of spleen contraction are probably in apneic situations. Here, enhanced convective O2 transport through release of stored Hb

10 Unpublished observations. However, the author has personally experienced an arterial oxyhemoglobin saturation level of 44% during breath holding, and is not considered a trained apneist. Even at altitudes about 6000m he has never attained this level of desaturation in non-apneic situations. 35

and Hct results in increased O2 carrying capacity, and an improvement in CO2 buffering via increased availability of carboxyl group binding on Hb. The work contained in this thesis has shown that apneists with larger spleens hold their breath for longer, and that the contracted spleen in such cases offers a theoretical advantage of 30s of additional breath holding time, based solely on O2-carrying criteria i.e. with no respect to effects on CO2. In untrained individuals, this time extension was in the order of 18s, itself a considerable advantage when considered in survival terms.

However, the transient increases in Hb and Hct conferred through spleen contraction should not only be considered in terms of O2-carrying capacity. Dane and associates [148] have shown in dogs that the autologous blood infusion from the spleen also enhances diffusive O2 transport in the lung, maintaining alveolar

O2 transport in the presence of ambient hypoxia. Although dogs have considerably larger splenic infusions than humans (in the order of 20% BV increase in Hct), it is conceivable that even the lesser increases in Hct observed in our lab of up to 5% may have positive effects in this regard. Transient Hct increases of up to 10% have been noted in some individuals during this author’s work.

In acute adaptation to high altitude

While spleen contraction may convey advantages at sea level, the new discovery of effects in normobaric hypoxia suggests that it may also play a role in “fast” adaptation to altitude or other ambient hypoxic conditions, serving as a compensatory mechanism by bridging the gap between non-acclimatization and altitude-induced polycythemia. The latter can be achieved within days to weeks depending on the severity of the hypoxia but on its own would not assist the individual in the immediate short-term. An early increase in Hb during altitude exposure has often been reported, but it has been explained to be a side effect of increased ventilation or to altitude-induced hormonal changes [149]. This thesis has added a functional explanation to this phenomenon, which could perhaps also explain individual differences in susceptibility to altitude sickness.

Dogs, with their significant splenic contributions, acclimatize within minutes to altitude allowing them “to chase their prey up and down a mountain with minimal changes in hypoxic acclimatization during the exertion” [148]. Though the human contractile response, comparatively speaking, may or may not occur to the same magnitude as in dogs, it has been shown to be progressive with increasing altitude [120]. This small but likely appreciable advantage may reduce the tissue hypoxia until new erythrocytes can be produced.

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During exercise

That exercise induces spleen contraction is certainly known, but the effects are not. Previous research regarding blood manipulation by venesection or autologous transfusion in human subjects has shown variable increases in O2 uptake in the order of 10-13 % [150, 151], and following splenectomy, maximal O2 uptake is reduced in dogs [148] and horses [152]. A yet unpublished study by this author’s research group has observed spleen contraction during maximal O2 uptake tests in elite cross-country skiers, similar to the findings of Froelich and associates [75] and Laub and associates [49]. In this study, skiers performing apneas prior to the maximal test were compared to a second performed trial without prior apneas.

Both trials achieved the same end-test increase in Hb/Hct as well as VO2 kinetics, although the apnea trial had a “head start” regarding these parameters in the early phase of the trial. Theoretically, a spleen contraction that was “pre-activated” in this regard could serve to improve performance over shorter events i.e. 2-3 minutes, as it likely requires at least this long to become fully initiated. Further investigation of this possibility is certainly warranted.11

Problems with Hct limits in competition

One issue that may arise from the autologous “blood doping” effects of spleen contraction is the presence of periods of naturally-occurring, but higher than normal (i.e. resting) Hct and Hb. The effects of hypoxia, prior exercise, stress (resulting in catecholamine release), apnea and even body position could a ll produce a spleen-related temporary increase in blood parameters that may pose problems in, for example, doping controls in sporting events. In both cycling and cross-country skiing, maximum Hct and/or Hb levels are delineated in order to reduce the risk of cardiovascular complication due to high blood viscosity. While the spleen contributes to such increases in viscosity it also eliminates such increases rapidly when they are not required, allowing a more “risk-free” increase in red blood cell content. Nonetheless, such a transient increase would not be desired when doping controls are being performed. In fact, any athlete could possibly be at higher risk of being detected when tested, if he or she is stressed or nervous during the testing! It may be judicious to inform athletes and officials of the risk of such effects.

11 As an interesting although perhaps coincidental aside, the individual with the clearly largest spleen response and apneic duration during this study, is currently making his mark on the world stage in cross-country skiing – those with lesser responses have yet to achieve the same level of performance. 37

Relation to the diving response

The studies in this thesis suggest that the spleen contraction seen during breath holding is not an integral part of the classical human “diving response” i.e. bradycardia and an increase in peripheral vasoconstriction. This is a topic currently under debate, where Espersen and associates [77] claimed that spleen contraction “should be included in the diving response on equal terms with bradycardia, decreased peripheral blood flow and increased blood pressure”. Palada and associates [145] have also suggested that the onset of spleen contraction is immediate and is facilitated by facial cooling, similar to the diving response.

However, this author’s own research group has shown that facial chilling does likely not enhance spleen contraction-related increases in Hb and Hct, whereas the diving response which can be elicited to a certain extent in this manner even without breath holding. Elsner and associates [153] earlier showed that an equally strong human diving response occurred irrespective of arterial hypoxia or hypocapnia. That the diving response thus appears directly at apnea onset, and independent of blood gas changes and peripheral chemoreception further dissociates it from spleen contraction, shown in this thesis’ studies to be highly sensitive to and most likely initiated by such changes.

But perhaps the strongest evidence for dissociation between apneic spleen contraction and the human diving response is the time scale of development of these responses. Indeed, spleen contraction may begin immediately upon breath holding, but it does not develop fully until after 3-5 apneas, whereas the diving response is fully achieved in the first and essentially every apnea thereafter. This may be due to the fact that the spleen response serves a different function – rather than sparing O2, which is the conceivable function of the diving response, it more likely serves to increase O2 storage and buffer CO2, and functions most effectively during repeated apneas as is seen in diving mammals. From an functional perspective, these effects would be best achieved during surface periods between dives where it facilitates recovery.

Spleen contraction in other situations also varies from the typical diving response. High intensity exercise, for instance, as well as exposure to altitude or eupneic normobaric hypoxia both elicit contractions of the spleen in the absence of any sort of diving response – in fact, tachycardia is more prevalent in these situations.

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Future directions

Other measurement techniques

Splenic contraction in humans has now been measured using radionucleotide imaging [77, 154], CT scan and multi-axial ultrasound. While these measures prove effective in measuring size and volume reduction, they still do not constitute a “gold standard” in terms of three-dimensional quantification with good temporal resolution. For that to be achieved, studies of spleen contraction must turn to magnetic resonance imaging (MRI). MRI magnetizes its target tissue or organ externally, and then passes a radio frequency pulse through the tissue, exciting hydrogen ions in the process to produce images of great detail. Furthermore, extremely quick modern MRI techniques such as single shot fast- spin echo sequences can yield multiple images in fractions of a second, providing an extremely accurate measure of organ volume and in some methods regional blood flow characteristics as well. Thornton and associates [56] used such methods in seals to achieve the most accurate determinations of spleen contraction during diving in mammals to date.

The local chemical and neurotransmitter effects on the spleen are also an area not yet examined in humans in vivo, although this has been achieved in rats using microdialysis [89]. In that study, this technique, which uses tiny needles with membranes semi-permeable to extracellular chemical substances, was used this technique to show the presence and depletion of neuronally derived catecholamines associated with the spleen. Similar work, if ethically possible in humans, would offer much information regarding local mechanisms at work near the spleen.

Maximizing splenic contraction through training

With the correlation between apneic performance and spleen size now established, an obvious direction for future work is investigating the potential ability to increase spleen size through training. While this still remains a purely theoretical pursuit, there may be ties to altitude- or hypoxia-based training in achieving this. This author’s altitude field study [120] showed tha t after altitude-induced polycythemia, descending to lower altitudes resulted in a larger splenic contribution during apnea than during the same altitudes during ascent. Thus, the spleen’s role as a regulator of excess blood viscosity may also render it “fuller” after periods of excess blood building. This could be viewed as an augmentation of its autologous blood doping function – in effect, making the spleen a larger source of stored erythrocytes. In either case, this avenue has yet to be sufficiently explored. 39

Investigation of other possible sources of stored erythrocytes

No study has found that 100% of the increase in Hct and Hb during apnea, exercise or altitude exposure is spleen-derived. And although the liver and kidneys have shown only minor or no reduction in blood volume during such interventions, the door is still open to the possibility of additional reservoirs of blood in the human body that can be remobilized when required. Due to its size, the liver is probably the most likely candidate. More detailed and temporally accurate imaging techniques may assist in this pursuit, and certainly represent a potential area for future exploration.

ACKNOWLEDGEMENTS

The author would like to express his sincere thanks to the following individuals who assisted in the work leading to the production of this document: to Professor Erika Schagatay, for her patient guidance in times of both success and frustration; to colleagues and co-authors Robert de Bruijn, Angelica Lodin, Dr. Sofia Pettersson, Helena Haughey, Dr. H.-C. Holmberg, Dr. Jenny Reimers, Glenn Björklund and Harald Engan for their unselfish assistance and attention to detail; to Torborg Jonsson, Håkan Norberg, and Siw Stenlund for their technical, logistical and administrative expertise; to Dr. Johan Andersson and Dr. Mats Liner for their help in revising the text; to the Jönsson, Norström, Borg, Schagatay, Richardson and Grousset families for their exceptional hospitality and kindness both home and away; and not least to our subject volunteers, who, often under great physical duress, allowed the author to take pictures of their internal organs and drain their vital fluids for no more than a handshake and perhaps a free lunch.

Special personal gratitude is extended to Sara Norström, whose sharing in these endeavours has made them especially meaningful, and to Sarah, Paul and Mary Anne Coderre Richardson for their lifelong moral support and encouragement in maintaining a balance.

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PAPER I.

Correlation between spleen size and hematocrit during apnea in humans. MX Richardson, R deBruijn, S Pettersson, J Reimers and E Schagatay. 2006. In: Lindholm, P, Pollock NW, Lundgren CEG, eds. Breath-hold diving. Proceedings of the Undersea and Hyperbaric Medical Society/Divers Alert Network 2006 June 20-21 Workshop, p 116-119.

Correlation between spleen size and hematocrit during apnea in humans

Richardson M1, de Bruijn R1, Petterson S1, Reimers J2, Schagatay E1 1) Environmental Physiology Group, Mid Sweden University, Sundsvall, Sweden. 2) X-ray Clinic, Härnösand Hospital, Härnösand, Sweden.

Introduction

Increases in hemoglobin concentration (Hb) and hematocrit (Hct) have been observed after both solitary (3) and repeated apneas spaced by short intervals (7; 2). In the latter case, the increase in Hb and Hct reaches a maximum after 3 – 4 maximal effort apneas (7). This increase is reversed within 8-9 minutes after cessation of apneas (8). The return to baseline of Hb shows that the response is not due to increased diuresis or sweating, and constancy of protein levels shows that it is not due to extravasation of plasma by increased filtration (7). In diving mammals, such as seals, increases in Hb and Hct have also been observed during dives and this has been attributed to a simultaneous reduction in spleen size (4). It is thus conceivable that the increase in Hb and Hct seen during apneas in humans is, at least partly, caused by a similar contraction of the spleen. In humans, as in other mammals, the spleen apparently retains erythrocytes and forms a reservoir of red blood cells while reducing blood viscosity during rest (10), but the size of this reservoir still needs to be determined. Contraction of the spleen during apnea has been shown to be an active process, largely independent of arterial inflow, suggesting the presence of a centrally-mediated feed-forward mechanism (1). No direct assessment has been made of splenic volume changes in relation to increases in hematocrit during apnea, and the indirect estimations have varied. In this study we aimed to further investigate the relation between spleen volume, Hb and Hct during serial apneas.

Methods

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Four healthy subjects volunteered to perform a series of 3 maximal apneas, spaced by 2 min pauses. Ultrasonic measurements of spleen diameter and venous blood samples, for Hb and Hct analysis, were taken pre-series, after the 1st and 3rd apnea, and at 2, 4, 6, 8, and 10 minutes following apnea 3. Ultrasonic measurements of spleen maximal length (L), thickness (T) and dorsoventra l width (W) were used to calculate spleen volume using the following equation: Lπ(WT-T2)/3. Comparisons between pre- and post-series measurements were made for splenic volume, Hb and Hct using paired Students t-test. Correlations between spleen volume and Hb and Hct across the series were calculated using Pearsons test. An estimation of the spleen contribution to observed Hct changes was made, based on an estimated blood volume of 8% of body weight, a spleen hematocrit of 80% (5) and the spleen volume and Hct observed before and after apneas.

Results

A reduction in spleen volume during apneas was seen in all subjects, on average from 338 ml pre-series to 223 ml directly after apnea 3 (by 34%; P=0.01). Simultaneously increases in Hb, from 133 g/L to 136 g/L (P=0.02), and in Hct, from 38.6% to 39.9% (P=0.07), were observed (Figure 1). The Pearson´s correlation coefficients for spleen volume and Hb and Hct were -0.88 and -0.85, respectively (both P<0.01). The reduction in spleen volume was estimated to account for 70% of the observed increase in Hct. The apnea-induced changes of Hb, Hct and spleen volume were completely reversed within 10 minutes after the last apnea.

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Figure 1. Mean spleen volume (ml) and hematocrit (Hct: %) from 4 subjects before apneas, after apnea 1, apnea 3, and during 10 min recovery after apneas. ** denotes significance at P=0.01 for spleen volume after apneas, compared to control. The P value for Hct was 0.07.

Discussion

The inverse correlation between spleen volume and both Hb and Hct during the apnea series suggests a causative relation between the parameters. The estimation that the observed reduction in spleen volume could account for 70% of the observed Hct increase, further supports this conclusion. This is a higher value compared to previously reported estimations of 60 % (7) and 37 % (8). One difference may be the timing of post apnea spleen volume measurement, which in the present study is directly after apnea termination. We speculate that the remaining increase in Hb and Hct could arise from either extravasation of plasma, or possibly from venous blood pools with high hematocrit in the abdomen such as the liver. The transient increases in circulating erythrocyte volume will increase oxygen storage capacity as well as carbon dioxide buffering during subsequent apneas, both with a dive prolonging effect. The spleen related increases in Hb and Hct may be an explanation for the increases in apneic duration in successive apneas (7), previously thought to be due to 52 hyperventilation. Some observations indicate that this spleen and Hb response may be trainable: The increase in apneic duration across serial apneas tended to be stronger after apnea training (9), and trained apneists were found to have a larger increase in Hb during apneas, compared to untrained subjects and elite cross-country skiers (6), which is linked to spleen contraction by the present study. Further studies will reveal whether the spleen response can be trained, and what benefits it may offer in different sports.

Conclusion: We conclude that spleen contraction and Hb and Hct changes develop in close association and that the main cause of the rise in Hb and Hct observed after repeated apneas is spleen contraction.

References

1. Bakovic D, Eterovic D, Saratlija-Novakovic Z, Palada I, Valic Z, Bilopavlovic N and Dujic Z. Effect of human splenic contraction on variation in circulating blood cell counts. Clin Exp Pharmacol Physiol 2005; 32: 944-951. 2. Bakovic D, Valic Z, Eterovic D, Vukovic I, Obad A, Marinovic-Terzic I and Dujic Z. Spleen volume and blood flow response to repeated breath-hold apneas. J Appl Physiol 2003; 95: 1460-1466. 3. Espersen K, Frandsen H, Lorentzen T, Kanstrup IL and Christensen NJ. The human spleen as an erythrocyte reservoir in diving-related interventions. J Appl Physiol 2002; 92: 2071-2079. 4. Hurford WE, Hochachka PW, Schneider RC, Guyton GP, Stanek KS, Zapol DG, Liggins GC and Zapol WM. Splenic contraction, catecholamine release, and blood volume redistribution during diving in the Weddell seal. J Appl Physiol 1996; 80: 298-306. 5. Laub M, Hvid-Jacobsen K, Hovind P, Kanstrup IL, Christensen NJ and Nielsen SL. Spleen emptying and venous hematocrit in humans during exercise. J Appl Physiol 1993; 74: 1024-1026. 6. Richardson M, de Bruijn R, Holmberg H-C, Björklund G, Haughey H and Schagatay E. Increase of hemoglobin concentration after maximal apneas in divers, skiers, and untrained humans. Can J Appl Physiol 2005; 30: 276-281. 7. Schagatay E, Andersson JP, Hallen M and Palsson B. Selected contribution: role of spleen emptying in prolonging apneas in humans. J Appl Physiol 2001; 90: 1623- 1629; discussion 1606. 8. Schagatay E, Haughey H and Reimers J. Speed of spleen volume changes evoked by serial apneas. Eur J Appl Physiol 2005; 93: 447-452.

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9. Schagatay E, van Kampen M, Emanuelsson S and Holm B. Effects of physical and apnea training on apneic time and the diving response in humans. Eur J Appl Physiol 2000; 82: 161-169. 10. Stewart IB and McKenzie DC. The human spleen during physiological stress. Sports Med 2002; 32: 361-369.

54

PAPER II.

Increase of hemoglobin concentration after maximal apneas in divers, skiers and untrained humans. 2005. MX Richardson, R deBruijn, HC Holmberg, G Björklund, H Haughey and E Schagatay. Canadian Journal of Applied Physiology, 30(3): 276-281. This article is reproduced with the kind permission of NRC Research Press.

Increase of Hemoglobin Concentration After Maximal Apneas in Divers, Skiers, and Untrained Humans

Matt Richardson1, Robert de Bruijn1, H.-C. Holmberg2,3, Glenn Björklund1,4, Helena Haughey1, and Erika Schagatay1

1Dept. of Natural and Environmental Physiology, Mid Sweden University, 85170 Sundsvall, Sweden; 2Dept. of Physiology & Pharmacology, Karolinska Institut, Stockholm, Sweden; 3Swedish Olympic Committee, Sofiatornet, Olympiastadion, Stockholm, Sweden; 4National Winter Sport Center, Östersund, Sweden.

Abstract/Résumé

Human splenic contraction occurs both during apnea and maximal exercise, increasing the circulating erythrocyte volume. We investigated the hematological responses to 3 maximal apneas performed by elite apneic divers, elite cross-country skiers, and untrained subjects. Post-apnea hemoglobin concentration had increased in all groups, but especially in divers. The increases disappeared within 10 min of recovery. Apneic duration across apneas also increased the most in divers. Responses in divers could be more pronounced as a result of apnea training.

La contraction de la rate chez l’être humain qui se produit pendant l’apnée et au cours d’un exercice maximal augmente le nombre d’érythrocytes en circulation. Nous avons analysé les ajustements hématologiques de plongeurs en apnée de haut niveau, de skieurs de fond de haut niveau, et de sujets non entraînés au cours de trois apnées maximales. On remarque après une période d’apnée une augmentation de la concentration d’hémoglobine dans les trois groupes, mais de façon plus marquée chez les plongeurs. L’augmentation s’estompa pendant les 10min de 55 récupération. La période apnéique fut plus marquée chez les plongeurs. Les adaptations plus importantes chez les plongeurs sont peut-être dues à l’entraînement à l’apnée.

Key words: breath hold, serial apneas, spleen contraction, cross-country skiing Mots-clés: apnée, apnée en série, contraction de la rate, ski de fond

Introduction

Oxygen storage capacity is a vital component both for endurance performance in athletes and for apneic duration in apneic divers. One way to increase oxygen storage capacity is to increase the circulating hemoglobin volume by various means such as altitude training or even blood doping (Ekblom, 2000). Several studies have shown that spleen contraction is associated with near-maximal or maximal exercise (Froelich et al., 1988; Laub et al., 1993; Wolski, 1998). This spleen contraction during exercise elicits a transient increase in hemoglobin concentration (Hb) and hematocrit (Laub et al., 1993; Wolski, 1998), which may, without changes in other blood components, increase oxygen carrying capacity. An increase in circulating Hb can also be achieved by apnea-induced splenic contraction (Schagatay et al., 2001), leading to an increase in oxygen carrying capacity. Repeated apneas in series with short rest intervals increase in duration (Schagatay et al., 1999), which has been attributed to the increase in Hb due to spleen contraction (Schagatay et al., 2001). This prolongation may reflect both increased oxygen storage and improvements in CO2 buffering capacity on repeated apneic attempts. Hb returns to normal values within 10 minutes of normal breathing, which is also the time known to reverse the increase in apneic duration at repetition. The return of Hb to baseline shows that the response is not due to increased diuresis or sweating, and the constancy of protein levels shows that it is not due to extravasation of plasma by increased filtration (Schagatay et al., 2001). Apnea-induced splenic contraction has been shown to be present in trained apneists (Bakovic et al., 2003; Hurford et al., 1990), and a spleen- attributable increase in Hb after apneas has been documented in untrained subjects (Schagatay et al., 2001), but it is unknown whether this response can be augmented through apneic training. It is also unknown whether spleen contraction resulting in increased Hb can be triggered in endurance athletes by apnea alone, and how this compares to the response in apneic divers and untrained subjects. The present study was aimed at describing the hematological response pattern associated with repeated maximal apneas in healthy non- divers (untrained), nondiving elite cross-country skiers (skiers), and elite apneic divers (divers).

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Methods

Subjects A total of 78 volunteers from specific populations, ages 18 years and older, were recruited into three groups: elite divers, elite skiers, and untrained subjects. Of the 33 elite divers, 29 men and 4 women (30.3 ± 10.6 yrs, 75.9 ± 12.1 kg, 181.5 ± 8.5 cm), 17 were national team members in apnea sports from six countries, 9 were Swedish underwater rugby players, and 7 were apnea or free diving instructors from Sweden. They trained an average of 5.5 ± 4.0 hrs a week in apneic diving and apnea related activities, and also an average of 6.5 ± 6.1 hrs a week of physical training. No divers had been residing or training at altitude in the 6 months prior to the study, but some of the national team members had been competing in breath-holding in the month leading up to the testing period.

The 13 skiers, all male (20.2 ± 3 yrs, 72.1 ± 5.5 kg, 180.6 ± 6.2 cm), were all members of the Swedish national senior (n = 4), under-23 (n = 4), and junior (n = 5) cross-country ski teams. They were tested in their off-season during which they trained 13.5 ± 3.2 hrs per week of ski training or skiing related physical activities. They had little or no experience in breath-holding. There had been no competi- tions in the previous month before testing, and none of the skiers had been living or training at altitude in the 6 months prior to the study.

The 32 untrained subjects (30.0 ± 6.3 yrs, 71.6 ± 8.4 kg, 177.6 ± 8.0 cm) were 9 women and 23 men who had little or no experience in breath holding. On average this group completed 2.5 ± 3.1 hrs per week of physical training or athletic activities, but none were competitive athletes in any sport, and none were living at altitude.

Experimental Procedure The subjects reported to the lab after 2 hrs without heavy meals and 1 hr without any meals or liquid intake. They signed informed consent documents for the test protocol, which was approved by the regional human ethics board. Following 20 min of horizontal rest, each subject performed 3 apneas of maximal, voluntary duration without time cues, spaced by 2 min of rest and normal breathing. Subjects were instructed not to hyperventilate and to immediately precede apneas by a total expiration and a deep but not maximal inspiration.

Measurements Blood samples for hemoglobin values were taken by trained personnel via intravenous catheter (Venflon Pro, Beckton-Dickson AB, Helsingborg, Sweden) and analyzed via automated blood analysis unit (Micros 60 Analyzer, ABX Diagnostics, Montpellier, France), or via capillary blood samples and analyzed by hemoglobin analyzer (B-Hemoglobin Photometer, Hemocue AB, Ängelholm,

57

Sweden), all analyzed in triplicate. These two methods have been shown to produce similar values in previous studies (Neufeld et al., 2002; Paiva Ad Ade et al., 2004) and in our own pilot study data (unpublished). Methods were consistent within individuals and each person served as his or her own control. Blood samples were drawn immediately before the onset of the 2-min countdown for the first apnea, immediately following the final apnea, and 10 min after the final apnea. Maximum volume drawn for catheterized subjects was below 15 ml, and fingertip samples consisted of several drops from a new fingertip each time collected in the Hemocue cuvettes.

Analysis The values of Hb and apneic duration were compared within each group via a Student paired t-test, and the effects of apneas between groups were compared via unpaired t-test. T-tests were corrected for multiple comparisons using Bonferroni correction. Statistical significance was accepted at p < 0.05. Values are expressed as absolute values and, when appropriate, also as percent changes from reference. A Pearson correlation was used to analyse the relationship between apneic duration and Hb increase across the apnea series.

Results

Baseline Hb was similar and normally distributed in all groups, with divers tending to have a higher value (divers 150,1 g/L; skiers 145,5 g/L; untrained 146.9 g/L) (Figure 1). After the apneas, all groups responded with an increase of Hb (p < 0.001; Figure 1). The largest increase was seen in divers (4.0 g/L–1; 2.7%) compared to skiers (3.0 g/L–1; 2.1%), and untrained (2.1 g/L–1; 1.4%, p < 0.05), while skiers did not differ from either divers or untrained (Figure 1). All groups returned to baseline Hb values within 10 minutes. When the data from skiers and untrained subjects was pooled into a single non-diver group, Hb was higher in divers than in the non-diver group after 3 apneas (p < 0.05). Duration of the initial apnea was higher for divers (144 s) than for both skiers (87 s; p < 0.001) and untrained (94 s; p < 0.01; Figure 2), but the durations were the same for skiers and untrained.

The apneic duration increased in all groups across the 3-apnea series (p < 0.01 in skiers, and p < 0.001 in divers and untrained; Figure 2). The increase was more pronounced in divers (43 sec, 20%) compared to skiers (26 sec, 12.2%, p < 0.05) and untrained (24 s, 15.5%, p < 0.01; Figure 2). The apneic duration of the last apnea of the series was 187 s in divers, 111 s in skiers, and 121 s in untrained. Four subjects in the divers’ group performed near-maximal apneas instead of maximal apneas and were excluded from duration analysis. However, no correlation was found

58 between apneic duration and Hb increase across the apnea series in any group.

Figure 1. Hb values (mean ± SE) before the first apnea (A1), after Apnea 3 (A3), and 10 min after apneas in divers (n = 33), skiers (n = 13), and untrained subjects (n = 32). ***Significant changes of Hb from pre-apnea value within groups, p < 0.001; a Significant difference between divers and pooled non-divers, p < 0.05.

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Figure 2. Duration of apneas performed by divers (n = 29), skiers (n = 13), and untrained subjects (n = 32). Significant changes of duration across 3 apneas within groups: ***p < 0.001; **p < 0.01. Difference between: a divers and untrained; b divers and skiers.

Discussion

This study shows that elite divers, elite skiers, and untrained subjects all respond with elevated Hb to 3 maximal effort apneas, but with divers showing a more powerful response. Since Hb increase after apneas has previously been shown to be absent in splenectomized subjects (Schagatay et al., 2001), we suggest that splenic contraction is likely to be the major factor behind the increase in Hb seen in the current study. This is the first observation of an apnea-induced elevation of Hb in non-diving endurance athletes, although the response is no greater than that of untrained subjects. The higher increase in Hb after apneas in the divers suggests that regular apnea practice could impart a specific training effect, affecting haematological responses to apnea in a manner that differs from that of exercise training. It may be that repeated attempts to a level of transient hypoxia, which is not achieved during sub-maximal or even maximal exercise, are needed to evoke a greater splenic response.

The apneic duration was higher in the trained divers than in other groups, and it could be argued that longer apneas caused the further increase in Hb. However, it has previously been observed in untrained subjects that a longer duration of

60 individual maximal apneas did not affect the magnitude of Hb and Hct increases (pilot study reported in Schatagay et al., 2001). The lack of correlation between duration of apneas and magnitude of Hb increase within groups is in accordance with these earlier findings. The pronounced hematological response in divers could conceivably be a function of increased splenic contractility or a greater amount of stored red blood cells prior to splenic contraction after apnea training, or both. However, differences could also be due to specific genetic traits of persons who choose diving as a sport. Known effects of apnea training include increased cardiovascular diving response and apneic duration (Schagatay and Andersson 1998), but it is not known whether the spleen response can similarly be trained. Longitudinal studies of apnea training in previously untrained subjects are needed in order to explore this possibility further.

In conclusion, all groups responded to apnea with an increased Hb, likely due to spleen contraction, but this was most prevalent in divers, suggesting that these responses may be enhanced by apnea training but not by endurance ski training.

Acknowledgments

This study was supported by The Swedish National Center for Research in Sports, Länsstyrelsen Västernorrlands län and Sundsvalls Sjukhus. All subjects participated voluntarily and the study was conducted in compliance with Swedish laws and ethical standards. We would like to thank all participating subjects, Dr. Johan Andersson, the National Winter Sports Center in Östersund, and all our laboratory personnel.

References

Bakovic, D., Valic, Z., Eterovic, D., Vukovic, I., Obad, A., Marinovic-Terzic, I., and Dujic, Z. (2003). Spleen volume and blood flow response to repeated breath-hold apneas. J. Appl. Physiol. 95: 1460-1466. Ekblom, B.T. (2000). Blood boosting and sport. Baillieres Best Pract. Res. Clin. Endocrinol. Metab. 14: 89-98. Froelich, J.W., Strauss, H.W., Moore, R.H., and McKusick, K.A. (1988). Redistribution of visceral blood volume in upright exercise in healthy volunteers. J. Nucl. Med. 29: 1714-1718. Hurford, W.E., Hong, S.K., Park, Y.S., Ahn, D.W., Shiraki, K., Mohri, M., and Zapol, W.M. (1990). Splenic contraction during breath-hold diving in the Korean ama. J. Appl. Physiol. 69: 932-936. Laub, M., Hvid-Jacobsen, K., Hovind, P., Kanstrup, I.L., Christensen, N.J., and Nielsen, S.L. (1993). Spleen emptying and venous hematocrit in humans during 61 exercise. J. Appl. Physiol. 74: 1024-1026. Neufeld, L., Garcia-Guerra, A., Sanchez-Francia, D., Newton-Sanchez, O., Ramirez- Villalobos, M.D., and Rivera-Dommarco, J. (2002). Hemoglobin measured by hemocue and a reference method in venous and capillary blood: A validation study. Salud. Publica Mex. 44: 219-227. Paiva Ad Ade, A., Rondo, P.H., Silva, S.S., and Latorre Md Mdo, R. (2004). Comparison between the HemoCue(R) and an automated counter for measuring hemoglobin. Rev. Saude Publica 38: 585-587. Schagatay, E., and Andersson J. (1998). Diving response and apneic time in humans. Undersea Hyperb. Med. 25: 13-19. Schagatay, E., Andersson, J.P., Hallen, M., and Palsson, B. (2001). Selected contribution: Role of spleen emptying in prolonging apneas in humans. J. Appl. Physiol. 90: 1623-1629. Schagatay, E., van Kampen, M., and Andersson, J. (1999). Effects of repeated apneas on apneic time and diving response in non-divers. Undersea Hyperb. Med. 26: 143-149. Wolski, L. (1998). The impact of splenic release of red cells on hematocrit changes during exercise. PhD thesis. Vancouver: University of British Columbia.

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PAPER III.

Short-term effects of normobaric hypoxia on the human spleen. 2008. MX Richardson, A Lodin, E Schagatay and J Reimers. European Journal of Applied Physiology, published online Nov 28 2007, ahead of print. This article is reproduced with kind permission of Springer Science and Business Media. The European Journal of Applied Physiology can be found online at: http://www.springer.com.

Short-term effects of normobaric hypoxia on the human spleen

Matt X. Richardson1, Angelica Lodin1, Jenny Reimers2 and Erika Schagatay1

1) Department of Natural Sciences, Mid-Sweden University, Sundsvall, Sweden 2) X-ray clinic, Härnösand Hospital, Härnösand, Sweden

Abstract

Spleen contraction resulting in an increase in circulating erythrocytes has been shown to occur during apnea. This effect, however, has not previously been studied during normobaric hypoxia whilst breathing. After 20 min of horizontal rest and normoxic breathing, 5 subjects underwent 20-minutes of normobaric hypoxic breathing (12.8% oxygen) followed by 10 minutes of normoxic breathing. Ultrasound measurements of spleen volume and samples for venous hemoglobin concentration (Hb) and hematocrit (Hct) were taken simultaneously at short intervals from 20 min before until 10 min after the hypoxic period. Heart rate, arterial oxygen saturation (SaO2) and respiration rate were recorded continuously. During hypoxia, a reduction in SaO2 by 34% (P<0.01) was accompanied by an 18% reduction in spleen volume and a 2.1% increase in both Hb and Hct (P<0.05). Heart rate increased 28% above baseline (P<0.05). Within 3 min after hypoxia SaO2 had returned to pre-hypoxic levels, and spleen volume, Hb and Hct had all returned to pre-hypoxic levels within 10 min. Respiratory rate remained stable throughout the protocol. This study of short-term exposure to eupneic normobaric hypoxia suggests that hypoxia plays a key role in triggering spleen contraction and subsequent release of stored erythrocytes in humans. This response could be beneficial during early altitude acclimatization.

Keywords: hypoxia, spleen, hemoglobin, hematocrit, ultrasound

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Introduction

The spleen is known to serve as a dynamic reservoir for red blood cells in many species (Barcroft and Stephens 1927; Kramer and Luft 1951; Hurford et al 1996). In humans, the splenic reservoir is relatively small compared to body size but still augments the body’s circulating hematocrit (Hct) and hemoglobin (Hb) concentration when the spleen contracts. These spleen-associated increases have been shown to occur during apnea (Schagatay et al 2001; Bakovic et al 2005) and are reversible within minutes (Schagatay et al 2005). Similar effects occur during high-intensity exercise (Laub et al 1993). The benefit of such a transient increase is an improved oxygen carrying capacity during situations where it would be particularly helpful, while avoiding polycythemia when it is not necessary through expedient storage of extra red blood cells by the spleen.

One such situation where fast Hb elevation would be helpful is acute hypoxia. Rats have shown an increase in Hb originating from the spleen in response to acute intermittent hypoxia, which is also reversible and mediated by alpha2- adrenoreceptors (Kuwahira et al 1999). Kramer and Luft (1951) also showed in a study on dogs that Hb increased directly correlated to the size of the spleen following severe hypoxia. In humans, Richardson et al (2005a) have suggested hypoxia to be involved in initiating the spleen-associated Hb and Hct increase during breath holding, together with one or more other stimuli.

Human splenic contraction is not well-investigated in general hypoxic situations, and previous work has focused on chronic exposures to altitude. Using ultrasonography, Sonmez et al (2007) confirmed that 3-6 months exposure to altitude through relocation of lowlanders resulted in reduced splenic volume and concomitant increases in Hb and Hct, but no specific focus was directed toward the ascent and descent phases. Richardson and associates (2007) discovered that the transient elevation in Hb during apnea was attenuated and eventually disappeared as individuals travelled to progressively higher altitudes, during which time an overall polycythemia occurred. During descent, the Hb increase during apnea was greater than at similar altitudes during ascent. This suggests that the spleen may contract tonically with increasing altitude, eventually reaching a “fully contracted” state which would not produce any further Hb elevation even during additional hypoxic insult i.e. apnea. During descent, after three weeks of increasing altitude, splenic reuptake of unnecessary red blood cells may have resulted in a greater Hb increase through expulsion of its contents during apnea.

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There has been no previous study of the short-term effects of a hypoxic stimulus during regular breathing on the spleen. The aim of this study was therefore to reveal whether spleen contraction and related increases in Hb and Hct occur during eupneic exposure to short-term normobaric hypoxia.

Methods

Subjects

Five volunteer subjects (4 men, 1 woman), of mean±SD age 27±3 years, height 1.85±0.1 m, and mass 89±21 kg participated in the study. All were healthy non- smokers who trained 5±3 hours per week for physical fitness, and had a vita l capacity of 6.1±0.7 L. None had been at high altitude for at least three months prior to the study. The study protocol was approved by the ethics committee at Umeå University, Sweden and complied with the 2004 Declaration of Helsinki for research involving human subjects. Experimental protocol

Testing took place at Härnösand regional hospital. After 20 min of horizontal rest and normoxic breathing at sea level, subjects underwent 20-minutes of hypoxic breathing (hypoxia) followed by 10 minutes of normoxic breathing. The hypoxia was induced via mask (Hypoxico, Hypoxico Inc., New York USA) to an oxygen content of 12.8%, which simulated approximately 4100m above sea level. All subjects remained prone and resting during the entire protocol. For analysis of Hb and Hct 1.5 mL blood samples were taken via intravenous catheter (Venflon Pro, Beckton-Dickson AB, Helsingborg, Sweden) placed at the antecubital region, at the time periods illustrated in Figure 1. Ultrasound measurements of the spleen (via HDI 3000, ATL A, Philips Company, Bothwell, WA, USA) were taken in three axes by a trained professional, simultaneously with the blood samples.

Arterial oxygen saturation (SaO2) and heart rate (HR) were measured continuously throughout the protocol via pulse-oxymeter (WristOx 3100, Nonin Medical Inc., Plymouth, Minnesota, USA). Breathing movements were also recorded through the protocol using a lab-developed pneumatic chest bellows.

The total volume of blood (including waste volume) obtained from each subject was approximately 40 mL, and the injected saline was approximately 20 mL. The analysis of Hb and Hct was conducted via automated blood analysis unit (Micros 60 Analyzer, ABX Diagnostics, Montpellier, France).

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Figure 1. The time course of the test protocol, illustrating the hypoxic and normoxic periods and sampling points for blood and ultrasound measurements.

Data analysis

Spleen volume was calculated using a formula developed by S. Pilström (personal communication): Lπ(WT-T2)/3, where (L) is spleen length, (W) width and (T) thickness obtained from the ultrasound measurements. The formula is based on the average shape of the spleen observed on the ultrasonic images and describes the difference between two ellipsoids divided by two. Control values for spleen volume, Hb and Hct were established immediately prior to the onset of hypoxia, and then compared with values from hypoxia and post-hypoxia. For SaO2 and HR, the control value was established as a 60 s average recorded 90-30 s before the onset of hypoxia, and compared with 30 s average values taken at the same time points as the ultrasound and blood measurements. All values were log- transformed before comparison via t-test. 90% confidence intervals and p-values were calculated for all comparisons. Pearson r correlations were performed between spleen volume and blood parameters, and both p-values and the likelihoods for true values based on Cohen’s threshold (Cohen 1988) were calculated via purpose-made spreadsheet (Hopkins 2002).

Individual observed reductions in spleen volume during the hypoxic period were used to represent expelled blood volumes, with a Hct of 80% (Laub et al 1993). The effects of adding this to the circulating blood was calculated based on the measured individual Hct before hypoxia and an estimated circulating blood volume based on height and weight (Nadler 1962). The mean estimated Hct

66 increase for all measurements during the hypoxic period was then compared to the measured increase in Hct to calculate the contribution from the spleen.

Results

Results are reported as mean±SD for anthropometric data, and as mean (90% confidence limits) for changes and comparisons, with p-values.

Pre-hypoxia

Control values were established for SaO2 at 96.6±2.0%, HR at 62±17 bpm, and respiration rate at 9.5 breaths per minute. During the pre-hypoxic 20 min period of horizontal rest, spleen volume increased and Hb and Hct decreased as the subject’s blood volume and capillary exchange equilibrated as a result of the change in position. Prior to the onset of hypoxia, spleen volume was 405±133 mL, Hb was 134.4±9.0 g/L and Hct was 39.8±3.2 %BV.

Hypoxia

SaO2 fell and HR increased promptly during the initial 5 min of hypoxic exposure, to reach 33.9% (17.2) below baseline for SaO2 (64.4%; P<0.01) and 28.1 (19.5)% increase above baseline for heart rate by 20 min (P<0.05). Respiration rate was 10.3 breaths per minute by the end of the hypoxic exposure (NS).

Mean spleen volume was reduced by 13.3% (6.4) after 3 min of hypoxic breathing (P<0.01; Figure 2). At 15 min it had been reduced by 17.9% (8.7) and after 20 min the volume was reduced by 17.6% (8.7) below the control value (both P<0.01).

Hb had increased by 2.1% (1.0) to 137.2±9.0 g/L at 3 min (P<0.05), and was 136.2±9.9 g/L after 20 min of hypoxic breathing (Figure 2). Hematocrit followed the same response pattern as Hb, increasing by 2.1% (0.9; P<0.01) to 40.6±3.4 %BV at 3 min hypoxia and was 40.2±3.3 %BV after 20 min of hypoxic breathing.

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Figure 2. Spleen volume and Hb during pre-hypoxia, hypoxia and post-hypoxia for N=5. For comparison to control values, P<0.05 is indicated by * and P<0.01 by **.

Post-hypoxia

There was a prompt return of SaO2 and HR to the control values following the onset of the post-hypoxic period, and HR values were -3.8% (10.6; NS) and - 12.7% (11.5; NS) compared to control at 5 and 10 minutes post-hypoxia, respectively. Respiration rate was 9.2 breaths per minute ten minutes after exposure.

Spleen volume was still reduced by 22.2% (11.3; P<0.01) and 13.6% (11.0, P<0.05) at 1 and 5 min post-hypoxia, respectively (Figure 2). Corresponding SaO2 values were -6.1% (3.6; p=0.05) and -2.9% (4.5; NS) compared to the control value. At 10 min post-hypoxia spleen volume was -5.8% (9.7; NS) and SaO2 was -1.4% (1.8; NS), compared to control values.

Compared to the control value, Hb was still elevated by 3.4% (2.0; P<0.05) and 3.3% (2.0; P<0.05) at 1 and 5 min post-hypoxia, respectively, but gradually decreased throughout the post-hypoxic period and by 10 min post-hypoxia was 1.8% (2.2; NS) above control value (Figure 2). Hct followed a similar pattern,

68 with an increase of 3.2% (2.1; P<0.05) and 2.6% (±2.1; NS) at 1 and 5 min post- hypoxia, respectively, gradually decreasing to 0.9% (1.7; NS) from the control value at 10 minutes post-hypoxia.

Correlation between spleen volume and blood parameters

The Pearson r correlation between spleen volume and Hb was -0.51 (P<0.05), representing 74% likelihood that the true value of the correlation is clinically negative according to Cohen’s threshold of 0.1. For the correlation between spleen volume and Hct of -0.42 (P<0.1), this represents 69% likelihood that the true value of the correlation is clinically negative according to Cohen’s threshold of 0.1. The spleen was estimated to contribute to 60% of the observed overall increase in Hct during hypoxic exposure.

Discussion

This study shows that spleen contraction develops rapidly upon onset of normobaric hypoxia in humans. An ejection of stored erythrocytes results, as demonstrated by the corresponding elevations in Hct and Hb. These responses are reversed within approximately 10 minutes upon return to normoxia. This result suggests, together with other recent observations (Richardson et al 2007) that spleen contraction may be part of the early acclimation to altitude in humans, whereby increases in circulating hematocrit could help overcome the initial phase of hypoxia before erythropoietic effects may develop. These early elevations in Hct and Hb were previously suggested to be associated with changes in plasma volume caused by the hypoxia-induced responses of sodium- and water-regulating hormones (Schmidt 2002) or dehydration due to increased ventilation (Heinicke et al 2003). However, in the current study it appears that the spleen is an important source of extra circulating erythrocytes during hypoxia.

The spleen contraction generally follows the development of arterial hypoxia suggesting, in accordance with earlier findings, that hypoxia may be an important input for its development (Richardson et al 2005a). In that study, removal of the apneic hypoxia by inspiration of oxygen attenuated the rise in Hb and Hct, but some response remained showing that other factors are also involved. A possible contributing factor in the apneic situation could be hypercapnia, but in the current context hypercapnia is highly unlikely, as the respiration rate was unchanged during the hypoxic exposure. Catecholamines or sympathetic nervous system activity related to stress might also contribute to splenic contraction, although the conduct of the current experiment was not

69 considered by subjects to be stressful (personal communication). Apneic situations are likely perceived as more stressful, but changes in Hb and Hct in some previous apneic studies (Schagatay et al 2005; Richardson et al 2005b) were of a similar magnitude as in the present study.

The decrease in SaO2 was most intense during the initial 5 min, which may explain the powerful spleen responses during this period. Interestingly, it appears that both the initiation and interruption of the hypoxic exposure initially evoke a spleen contraction, as indicated both by the spleen volume and blood boosting response. We speculate that this could either be a response to the change in inspired oxygen, irrespective of direction, or a general arousal response associated with the placement and removal of the breathing mask. However, the direction of the response after the initial phase in these two situations is clearly different, showing that during hypoxia spleen contraction prevails.

The question if there are any physiological differences between hypobaric hypoxia compared to normobaric hypoxia for the same ambient PO2 is still under debate. In addition to lowered SaO2, Savourey and associates (2003) reported changes in mainly respiratory parameters at 4500 m altitude, such as increased breathing frequency, tidal volume and minute ventilation, resulting in hypocapnia and a higher blood alkalosis. While blood chemistry and tidal volume were not measured in the current protocol, respiration rate remained unchanged during the hypoxia, suggesting that no hypercapnia occurred.

This laboratory study supports the observations of a previous study in 3 climbers (Richardson et al 2007) where apnea-induced elevations in Hb were found to diminish with increased altitude, conceivably due to emptying of the spleen with increasing hypoxia. The present study suggests that even during normobaric eupnea, hypoxia is a trigger for spleen contraction and increased volume of circulating erythrocytes. This may provide a rapid means of increasing oxygen carrying capacity during early phases of altitude adaptation as well as during acute hypoxic situations.

Acknowledgements

The authors would like to thank our volunteer subjects for participating, Mr. Ka- Yu Law, Ms. Torborg Jonsson and Mr. Håkan Norberg for technical assistance, and the Swedish National Center for Research in Sports (CIF) for financial support. This study complies with Swedish laws and ethical standards.

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References

Bakovic D, Eterovic D, Saratlija-Novakovic Z, Palada I, Valic Z, Bilopavlovic N, Dujic, Z (2005) Effect of human splenic contraction on variation in circulating blood cell counts. Clin Exp Pharmacol Physiol 32: 944-951 Barcroft J, Stephens J (1927) Observations upon the size of the spleen. J Physiol 64: 1-22 Cohen J (1988) Statistical power analysis for the behavioral sciences. 2nd edition. Lawrence Erlbaum, New Jersey Cook S, Alafi M (1956) Role of the spleen in acclimatization to hypoxia. Am J Physiol 186(2): 369-372 Heinicke K, Prommer N, Cajigal J, Viola T, Behn C, Schmidt W (2003) Long-term exposure to intermittent hypoxia results in increased haemoglobin mass, reduced plasma volume, and elevated erythropoietin plasma levels in man. Eur J Appl Physiol 88: 535-543 Hopkins W (2002) Calculating likely (confidence) limits and likelihoods for true values (Excel spreadsheet). In: A new view of statistics, sportsci.org: Internet society for sport science, sportsci.org/resource/stats/xcl.xls Hurford W, Hochachka P, Schneider R, Guyton G, Stanek K, Zapol D, Liggins G, Zapol W. Splenic contraction, catecholamine release, and blood volume redistribution during diving in the Weddell seal. J Appl Physiol 80(1): 298- 306 Kramer K, Luft U (1951) Mobilization of red cells and oxygen from the spleen in severe hypoxia. Am J Physiol 165: 215-228 Kuwahira I, Kamiya U, Iwamoto T, Moue Y, Urano T, Ohta Y, Gonzalez N (1999) Splenic contraction-induced reversible increase in hemoglobin concentration in intermittent hypoxia. J Appl Physiol 86(1): 181-187 Laub M, Hvid-Jacobsen K, Hovind P, Kanstrup I, Christensen N, Nielsen S (1993) Spleen emptying and venous hematocrit in humans during exercise. J Appl Physiol 74: 1024-1026 Nadler S, Hidalgo J, Bloch T (1962) Prediction of blood volume in normal human adults. Surgery 51: 224-232 Richardson M, de Bruijn R, Schagatay E (2005a) Hypoxia – a trigger for spleen contraction? In: Desola J (ed) ICHM-EUBS Proceedings, Barcelona, Spain. Richardson M, de Bruijn R, Holmberg HC, Björklund G, Haughey H, Schagatay E (2005b) Increase of hemoglobin concentration after maximal apneas in divers, skiers and untrained humans. Can J Appl Physiol, 30(3): 276-281 Richardson M, Schagatay E (2007, in publication) Altitude attenuates apnea- induced increase in hemoglobin concentration. In: Mejkavic E (ed) ICEE conference proceedings, Portoroz, Slovenia.

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Savourey G, Launay J, Besnard Y, Guinet A, Travers S (2003) Normo- and hypobaric hypoxia: are there any physiological differences? Eur J Appl Physiol 89: 122-126 Schagatay E, Andersson J, Hallén M, Pålsson B (2001) Selected contribution; Role of spleen emptying in prolonging apneas in humans. J Appl Physiol 90: 1623-1629 Schagatay E, Haughey H, Reimers J (2005) Speed of spleen volume changes evoked by serial apneas. Eur J Appl Physiol 93: 447-452 Schmidt W (2002) Effects of intermittent exposure to high altitude on blood volume and erythropoietic activity. High Alt Med Biol 3: 167-176 Sonmez G, Ozturk E, Basekim C, Mutlu H, Kilic S, Onem Y, Kizilkaya E (2007) Effects of altitude on spleen volume: sonographic assessment. J Clin Ultrasound 35:182-185

72

PAPER IV.

Hypoxia augments apnea-induced increase in hemoglobin concentration and hematocrit. 2008. MX Richardson, R deBruijn, and E Schagatay. Submitted for publication in the European Journal of Applied Physiology.

Hypoxia augments apnea-induced increase in hemoglobin concentration and hematocrit

Matt X. Richardson, Robert de Bruijn, and Erika Schagatay Environmental Physiology Group, Mid Sweden University, Östersund, Sweden.

Abstract

Increased hemoglobin concentration (Hb) and hematocrit (Hct), attributable to spleen contraction, raise blood gas storage capacity during apnea, but the mechanisms that trigger this response have not been clarified. We focused on the role of hypoxia in triggering these Hb and Hct elevations. After horizontal rest for 20 min, 10 volunteers performed 3 maximal apneas spaced by 2 min, each preceded by a deep inspiration of air. The series was repeated using the same apneic durations but after 1 min of 100% oxygen (O2) breathing and O2 inspiration prior to each apnea. Mean apneic durations were 150s, 171s, and 214s for apneas 1, 2, and 3, respectively. Relative to pre-apnea values, the mean post-apneic arterial O2 saturation nadir was 84.7% after the air trial and 98% after the O2 trial. A more pronounced elevation of both Hb and Hct occurred during the air trial: after apnea 1 with air, mean Hb had increased by 1.5% (P<0.01), but no clear increase was found after the first apnea with O2. After the third apnea with air Hb had increased by 3.0% (P<0.01), and with O2 by 2.0% (P<0.01). After the first apnea with air Hct had increased by 1.9% (P<0.01) and after 3 apneas by 3.0% (P<0.01), but Hct did not change significantly in the O2 trial. In both trials, Hb and Hct were at pre-apneic levels 10 min after apneas. Diving bradycardia during apnea was the same in both trials. We conclude that hypoxia contributes to spleen contraction during apnea, likely through chemosensor- related sympathetic output. There are, however other factors involved that trigger spleen contraction even in the absence of hypoxia.

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Key Words: Breath-hold, oxygen saturation, spleen contraction, diving response, diving reflex

Introduction

Diving mammals, e.g. the Weddell seal, are known to expel red blood cells stored in the spleen during dives, which raises the circulating hemoglobin concentration (Hb; [82]. In humans, an increase in circulating Hb and hematocrit (Hct) can also be achieved by apnea-induced splenic contraction [155-157]. This increase results during both solitary [77] and repeated apneas spaced by short intervals [50, 76]. In the latter case, the increase in Hb and Hct reaches a maximum after 3 – 4 maximal effort apneas [50]. Previous work [78] described a normal distribution of Hb and Hct responses to a three-apnea series in untrained breath-holders, where the 25th to 75th percentile showed a 0.7% to 3.7% increase in Hb, and 0.4 to 4.0% in Hct. Spleen contraction has been shown to account for approximately 60% of these increases (Richardson et al. 2008; Schagatay et al. 2001). A reversal of the spleen contraction is seen 8-9 min after cessation of apneas [158], with a concomitant return to baseline of Hb and Hct. This shows that the Hb and Hct increase is not due to increased diuresis or sweating, and constancy of protein levels shows that it is not due to extravasation of plasma by increased filtration [50]. The increased circulating Hb and Hct may also explain the prolonged apneic duration in subsequent attempts, possibly through an increased capability to take up and store oxygen (O2) and also to buffer carbon dioxide (CO2).

Bakovic and associates [35] have shown that the mobilization of blood from the spleen during apnea is an active process in humans largely independent of arterial inflow, suggesting the presence of a centrally-mediated feed-forward mechanism. Their later work suggested that the initial contraction was, at least during apneas at large lung volumes, caused by downloaded baroreflex during decreases in blood pressure and cardiac output (Palada et al. 2007). Eupneic hypoxia in cats and dogs appears to have similar effects as electric stimulation of the splenic nerve which results in an emptying of the spleen of its stored blood contents [81, 159]. Hoka and associates [68] suggested that the sympathetic efferent nerve activity, augmented by the stimulation of chemoreceptors, contributes greatly to those hypoxic changes. Human spleens possess adrenoreceptors in the vasculature and capsule and injection of epinephrine has shown to cause contraction (Ayers et al. 1972). Still, there is little consensus about the actual trigger of human splenic contraction during apnea.

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The decrease in SaO2 during prolonged apnea could be a possible mechanism tha t triggers spleen-related increases in Hb and Hct. Qvist and associates [160] completed studies of Korean ama divers’ hematological characteristics both during diving to depth and breath holding in air. Although it was not the main focus of their study, during the repeated working dives to depth that produced a reduction in SaO2, they noted increases in Hb and Hct in the divers. However, these increases were not present in resting breath-holds of similar duration in air, which showed no decrease in SaO2.

Pre-breathing of oxygen has been shown to extend breath-hold duration [12, 161] and thus delays the development of a hypoxic stimulus. The total body storage of

O2, including tissue, blood, and lung compartments, of a 70 kg male, is approximately 1500 mL, with ~370 mL in the lungs and ~280 mL in the blood

(Rahn 1964). Breathing 100% O2 increases these total body stores through replacement of nitrogen in the lungs with O2, which would be reflected as a slower depletion of SaO2. O2-enhanced breath holding does not, however, appear to affect CO2 production, which appears to develop similarly as during breath holding without O2, as demonstrated by Corkey and associates [162]. They observed that the amount of CO2 added to the lungs during resting apneas was -1 relatively constant at a rate of 0.32 L min , independent of inhaled O2 concentration (21%, 50% or 100%).

In this study, we examined the possibility that desaturation during prolonged apnea is involved in triggering the spleen related Hct increase. In order to test this, we eliminated the desaturation during apnea by pre-apnea O2 breathing in one trial, and compared the hemoglobin response to a trial with subjects breathing air before apneas. The cardiovascular response to apnea was also compared between these two conditions.

Methods

Subjects Ten healthy male subjects of mean ± SD age 24 ± 5 years, mass 80.6 ± 14.8 kg, and height 1.83 ± 0.72 m volunteered for this study. Subjects signed a consent form after being informed of the experimental protocol, which had been approved by the regional human research ethics board at Umeå University, Sweden and in accordance with the Declaration of Helsinki. All were non-smokers and trained 7.3 ± 7.0 h per week for general fitness. Subjects had experience in breath holding at a level averaging 3 on a scale of 1 to 5 (1=no breath-holding experience, 3=regular breath-hold diving but less than 2h per week, 5=world-class apneist; Schagatay 1996). Vital capacity was 6.2 ± 1.0 L in the standing position and 5.8 ± 1.1 L in the experimental, prone position. 75

Experimental procedure

The subjects completed 2 experimental trials spaced by at least 4 hours. The first trial consisted of apneas of maximal voluntary duration after breathing air prior to each apnea (air). In the second trial, the apneic times from the first session were reproduced, with the subjects breathing 100% O2 at a normal ventilatory rate for 1 min prior to each apnea (oxygen). This pre-breathing of oxygen was intended to maintain a higher SaO2 during apnea than in the air trial. Due to the likelihood of greatly prolonged breath-holding durations in oxygen [12, 161], the air trial was completed first so that the apneic duration could be reproduced.

Subjects reported to the laboratory without having eaten or consumed caffeine at least 2 h prior to testing. The vital capacity of the subject was measured via spirometer (Compact II, Vitalograph, Buckingham, England) and an intravenous catheter (Venflon Pro, Beckton-Dickson, Helsingborg, Sweden) was placed in the antecubital region.

Subjects were prone for the entire duration of the trials, beginning with a 20 min period of horizontal rest, followed by a 2 min countdown to the first of three apneas. In the air trial, a noseclip was placed at 30 s remaining in the countdown, and with 15 s remaining in the countdown the subject was administered a mouthpiece for measurement of inspired and expired gases. In the O2 trial, a nose clip was administered just prior to 60 s remaining in the countdown, after which subjects were administered a mask for breathing oxygen with flow rate at ~10 L min-1. For both trials, at the end of the 2 min countdown, the subject exhaled fully followed by a deep but not maximal inspiration and then began the apnea. Hyperventilation was not permitted in either of the series. Upon completion of the apnea, subjects expired fully through the mouthpiece and then resumed normal breathing. All apneas were spaced by 2 min of rest. In the air trial, apneas were conducted without time cues and to maximum duration, while the O2 trial reproduced the times achieved during the air trial and the subject was informed when to stop the apneas.

Blood samples (2 ml) were taken via the intravenous catheter 2 min before the first apnea, immediately after the first apnea, and immediately after as well 10 min following the third apnea. The catheter was rinsed with 2 ml saline following each sample. The total volume of blood (including waste volume) removed for each subject was approximately 25 ml, and the injected saline was approximately 12 ml. Blood samples were analyzed for Hct and Hb via

76 automated blood analysis unit (Micros 60 Analyzer, ABX Diagnostics, Montpellier, France).

From 2 min prior to apneas until 2 min post apneas SaO2 and heart rate (HR) were measured via pulse oximeter (Biox 3700e, Ohmeda, Madison, WI, USA) and using a BioPac MH100A CE multi-channel data acquisition system (BioPac Systems,

Goleta, CA, USA. Inhaled and exhaled O2 and CO2 were measured before and after each apnea via gas analyzer (Normocap Oxy, Datex-Ohmeda, Helsinki, Finland). A lab-developed chest bellows was used to monitor the depth and frequency of breathing movements.

Analyses

The values used for nadir SaO2 were the lowest obtained 2-second averages after apneas. For respiratory parameters O2 and CO2 percentages in the last inspired breath before and the first expired breath after the apneas were used. Hb and Hct obtained 2 min before the first apnea were used as control. Mean changes from control were used to compare changes within each trial, and pooled mean changes from apneas were used to compare between trials.

Control values for cardiovascular and respiratory parameters were calculated as averages from the period 60-30 s before each apnea. Apneic values for HR were obtained from the period from 30 s after breath-hold onset until the cessation of the breath-hold, in order to obtain a fully developed diving response (Jung and Stolle 1981). The mean changes from all apneas were used to calculate individual means.

Subjects served as their own controls and effects were expressed as percent changes from control. All variables were log transformed before analysis to reduce non- uniformity of error. Excel templates were used for all calculations, purpose- designed for analyses using physiological data [163]. Within-trial comparisons for changes resulting from apneas were completed with control values using ANOVA-based pair-wise comparisons with Bonferroni corrections for both a ir and oxygen trials. Separate between-trial comparisons were conducted using ANOVA-based pair-wise comparisons with Bonferroni corrections for Hb and Hct after the first and third apneas. Cardiovascular changes were averaged over the three apneas and then compared between trials using the same method. Acceptable differences were set at p<0.05 for in the comparisons.

Results

Apneic duration 77

All subjects increased their apneic duration (P<0.01) across the three air trials with mean ± SD apnea times of 150 ± 68 s, 171 ± 63 s, and 214 ± 106 s for apneas 1, 2, and 3, respectively. All subjects successfully repeated these durations during the O2 trial.

Arterial desaturation

Mean ± SD control values for SaO2 in the air and oxygen trials were similar prior to the start of apneas, at 96.8 ± 1.1 % and 98.5 ± 0.6 %, respectively (NS). The mean saturation nadir for apneas with air was 84.7 ± 9.1 % (P<0.01), but SaO2 did not change from control in the O2 trial, at 98.4% ± 0.8. During apneas, SaO2 values were lower in the air trial compared to the O2 trial (P<0.01).

Hematological response

One subject’s blood samples are not included in the hematological analysis due to catheter coagulation during the air trial, and N=9 for this section. Mean ± SD control values for Hb before apneas for air and oxygen trials were not different, at 134.1 ± 0.8 g/L and 131.7 ± 0.8 g/L respectively. After apnea 1 with air, mean Hb had increased (P<0.01), but no clear increase was found after the first apnea in the O2 trial (Figure 1). After the third apnea Hb had increased from control in both the air trial and the O2 trial (P<0.01). The increase in Hb resulting from apneas was greater in the air trial (p<0.05). 10 min after apneas, Hb was not different from control values for either air or oxygen trials, nor were they different from each other.

Control values for Hct were 40.9 ± 2.0 %BV and 40.6 ± 2.4 %BV for air and oxygen respectively, and not different from each other. After apnea 1 with air Hct had increased (P<0.01) and continued this increase after 3 apneas (P<0.01), but Hct did not change significantly in the O2 trial (Figure 2). In both air and oxygen trials Hct values were at control levels 10 min after the final apnea, and values were the same for the air and oxygen trials.

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Figure 1. Mean ± SD percent changes in Hb from control pre-apneic values for 9 subjects. Blood sampling time points are labelled on the horizontal axis. Significant differences from control are indicated by ** for p<0.01.

Figure 2. Mean ± SD percent changes in Hct from control pre-apneic values for 9 subjects. Blood sampling time points are labelled on the horizontal axis. Significant differences from control are indicated by ** = p<0.01.

Cardiovascular Responses

Mean ± SD control values of HR for the air and oxygen trials were not different at 77.5 ± 16.9 bpm and 76.0 ± 8.5 bpm , respectively. The HR showed similar reductions from control values during apnea in the air trial, at 14.8 ± 8.8 %, and in

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the O2 trial, at 14.6 ± 9.8 %. Between apneas HR was restored to the resting eupneic level in both trials, and there were no changes of apneic or recovery levels throughout the series. During apneas, there was no difference in HR between the air and oxygen trials.

Respiratory Parameters

Inspired and expired O2 and CO2 percentages before and after apneas are listed in Table 1. Both inspired and expired O2 were higher in the O2 trial (p<0.01). Inhaled and exhaled CO2 were similar between the two trials.

Table 1. Inspired and expired O2 and CO2 percentages before and after apneas for 10 subjects.

AIR OXYGEN

Inspired O2 20.7% (0.04) 87.0% (1.4)**

Expired O2 10.0% (1.0) 42.4% (11.9)**

Inspired CO2 0.1% (0.02) 0.1% (0.03)

Expired CO2 7.2% (0.2) 7.5% (0.2) **P<0.01

Discussion

The greater increase in Hb and Hct after apnea with air suggests that the associated decrease in SaO2 imposes an essential stimulus for the increase in circulating erythrocytes. Previous studies have shown that these Hb and Hct elevations during apnea are mainly due to spleen contraction (Bakovic et al. 2005; Espersen et al. 2002; Schagatay et al. 2001). The recovery after 10 min shows that these elevations are not an effect of increased diuresis, which is in accordance with earlier findings [50]. A constant total protein level across serial apneas indicates that changes in Hb and Hct are not a result of hemoconcentration caused by extravasation of plasma due to increased filtration (Schagatay et al. 2001).

Thus, a decrease in SaO2 appears to be a major stimulus evoking spleen contraction in humans.

In mammals, it has been shown that the spleen contracts in response to sympathetic nervous stimulation (Donald and Aarhus 1978; Greenway and Stark 1968; Hurford et al. 1996), something that can also be induced by hypoxia (Hoka et al. 1989). As an example, renal sympathetic nerve activity has also been shown to increase during apnea, and was directly correlated to carotid chemoreceptor activity (O’Donnell et al. 1996). The splenic nerve, itself composed almost entirely of sympathetic nerve fibers, may thus be affected by increased hypoxia-induced sympathetic output, resulting in contraction. The

80 carotid bodies are thought to react within seconds to even small changes in PO2, whereas other hypoxic related cellular responses in the rest of the body work on a considerably longer time scale i.e. hours (Prabhakar 2006), making any hypoxia-related spleen contraction more likely to be associated with chemoreceptor-related output.

This would not explain the increased in Hb in the O2 trial, however, as any prospective carotid chemoreceptor stimulation would have been removed by virtue of the hyperoxic intervention and lack of SaO2 change. An additional factor such as arterial pCO2 may play a role however, since a residual carbon dioxide level is always present during apnea, even in hyperoxia (Lin et al. 1983,

Corkey et al. 1998), and thus stimulation of CO2-sensitive central chemoreceptors may also occur, even in the absence of peripheral chemoreceptor responses. CO2 sensitive neuronal mechanisms have been suggested to generate sympathetic tone (Trzebski and Kubin 1981). Lin and associates [164] hypothesized that hypercapnia may enhance sympathoadrenal catecholamine release. Thus it is also conceivable that sympathetic output from central chemosensation of increasing CO2, which also occurs on an rapid time-scale. can result in splenic stimulation resulting in contraction.

The function of the spleen related Hct and Hb release in diving mammals is likely to prolong aerobic dive duration (Cabanac et al. 1997) and there is also evidence of an apnea prolonging effect in humans across repeated apneas (Schagatay et al. 2001; Bakovic et al. 2003). The greater increase in Hb and Hct found in the air trial could thus be assistive in subsequent apneic attempts. The release of additional red blood cells from the spleen during sensed reductions in hemoglobin saturation would provide additional O2 storage and buffering capabilities to prolong subsequent apneas, which is typical in repeated apnea attempts (Schagatay et al. 1999; 2001; 2005). The circulating red blood cells would also accelerate recovery between apneas and allow apneas to be spaced by short pauses. Without hypoxia, as during apnea in the O2 trial, an injection of red blood cells of similar magnitude would not be required, as O2 is no longer a limiting factor. In this way an unnecessary increase in blood viscosity and cardiac strain would be avoided.

In this study the total apneic duration increased by 64 s between the first and third apnea, but it has previously been shown that a prolonged “struggle phase” of the apnea accounts for a large portion of the apnea time increase, due mainly to psychological factors (Schagatay et al. 1999). Nonetheless, a typical ejection of stored blood in the spleen in a human with 5L blood volume could increase arterial O2 content by 2.8 to 9.6% (Stewart and McKenzie 2002), an appreciable

81 effect for those undertaking acute hypoxic activity. The 3% increase in Hct observed in the present study falls within this range, and assuming a 400 ml spleen volume (Richardson et al. 2008) with 50% reduction after apnea 3, and a

VO2 of 215 ml/min (Byrne et al. 2005), the increase in apneic duration attributable to the increase in arterial O2 supply could be approximately 18 s.

Added to this would be the effect of the increased CO2 buffering capacity, thus a significant part of the increase in apneic duration could be attributable to the observed spleen contraction induced Hct increase.

Conclusions

We conclude that hypoxia is an important factor for development of the spleen- related Hb and Hct increase during apnea, beyond additional apneic co- stimulus/stimuli. Hypercapnia is one such possible co-stimulus, which may also provide an initiating or moderating influence on these increases.

Acknowledgements

The authors would like to thank our volunteer subjects for participating, Dr. Jenny Reimers, Ms. Torborg Jonsson and Mr. Håkan Norberg for technical assistance, and the Swedish National Center for Research in Sports (CIF) and the County Administrative Board of Västernorrland, Sweden for financial support. This study complies with Swedish laws and ethical standards.

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Prabhakar, NR (2006) O2 sensing at the mammalian carotid body: why multiple

O2 sensors and multiple transmitters? Exp Physiol 91:17-23. Qvist J et al (1993) Arterial blood gas tensions during breath-hold diving in the Korean ama. J Appl Physiol 75:285-293 Rahn H (1964) Oxygen stores of man. In: Oxygen in the Animal Organism, Dickens E (ed), Pergamon/MacMillan, Oxford, pp 609-618 Richardson MX et al (2003) Hematological response pattern associated with maximal-duration apnea series in untrained subjects. In: Proceedings of the 24th Annual Scientific Meeting of the European Underwater and Baromedical Society, Jansen EC, Mortensen CR, Hyldegaard O (eds), EUBS, Copenhagen, pp 39 Richardson MX et al (2008) Short-term effects of normobaric hypoxia on the human spleen. Eur J Appl Physiol, DOI 10.1007/s00421-007-0623-4

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Schagatay E (1996) The human diving response: effects of temperature and training [Doctoral thesis]. Lund: Department of Animal Physiology, Lund University Schagatay E, van Kampen M, Andersson JPA (1999) Effects of repeated apneas on apneic time and diving response in non-divers. Undersea Hyperb Med 26:143-149 Schagatay E et al (2001) Role of spleen emptying in prolonging apneas in humans. J Appl Physiol 90:1623-1629 Schagatay E, Haughey H, Reimers J (2005) Speed of spleen volume changes evoked by serial apneas. Eur J Appl Physiol 93:447-452 Stewart IB, McKenzie DC (2002) The human spleen during physiological stress. Sports Med 32:361-369 Trzebski A, Kubin L (1981) Is the central inspiratory activity responsible for pCO2-dependent drive of the sympathetic discharge? J Auton Nerv Syst 3:401- 420.

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PAPER V.

Hypercapnia moderates hemoglobin increases during apnea. 2008. MX Richardson, A Lodin, H Engan, and E Schagatay. In manuscript.

Hypercapnia moderates hemoglobin increases during apnea

Matt X. Richardson, Angelica Lodin, Harald Engan, Erika Schagatay Environmental Physiology Group, Mid Sweden University, Östersund, Sweden.

Abstract

Spleen contraction associated with apnea causes increased Hb and Hct, an effect that may promote prolonged breath holding. Hypoxia has been shown to augment this effect, but hypercapnic influences have not been investigated previously. Eight non divers performed three different series of three apneas spaced by two minutes on separate days: one with pre-breathing of 5% CO2 in oxygen (Hypercapnic), one with pre-breathing of 100% oxygen (Normocapnic), and one with hyperventilation of 100% oxygen (Hypocapnic). The apneic durations were repeated identically in all trials, determined from the maximum duration attained in the CO2 trial. A fourth Eupneic hypercapnic trial breathing

5% CO2 in oxygen for the same duration as in the apneic trials was also performed. Hb increased by 4% after apneas in the Hypercapnic trial (p<0.01), followed by a 3% increase in the Normocapnic trial (p<0.01) and non-significant changes in the Hypocapnic and Eupneic hypercapnic trials. The “easy phase” of apnea i.e. without involuntary breathing movements was longest in the Hypocapnic trial and shortest in the Hypercapnic trial. Changes in cardiovascular parameters during apnea did not differ between the apneic trials.

There appears to be a dose-response effect of CO2 on triggering spleen contraction during apnea, even in the absence of hypoxia.

Key Words: Breath-hold, oxygen saturation, spleen contraction, hypercapnia, hypoxia

Introduction

Apneic diving is associated with several adjustments in order to secure brain and heart function during interrupted gas exchange with the environment, the most well-described of which is the “diving response”, consisting of bradycardia and 85 an increase in peripheral circulatory resistance, and which is oxygen-conserving [165]. A more recently discovered response is spleen contraction [48], which serves to elevate body gas storage capacity by elevating circulating hematocrit [50]. An increase in haemoglobin concentration (Hb) and hematocrit (Hct) has been demonstrated during both solitary [77] and repeated apneas performed with short intervals between [50, 76]. The increase in Hb and Hct is related to contraction of the spleen [48, 50], an effect that is maximized after 3-5 apneas and reversed within 8-9 minutes after cessation of apneas [50, 79]. The changes in these blood parameters may increase oxygen carrying capacity and carbon dioxide buffering during apnea.

The correlation between changes in Hb and Hct and spleen contraction is strong [166], and it is estimated that approximately 60% of the change in these parameters during apnea can be directly attributed to the emptying of the spleen’s stored contents [50, 167]. This response also does not appear to be affected by facial immersion, as apnea with or without facial immersion results in similar Hb increases [168]. This makes the spleen response differ from the cardiovascular diving response, which is fortified by facial immersion [165].

It was recently shown that the magnitude of the spleen-related Hb increase is augmented by hypoxia [169], but there appeared to be other residual apnea- related components that still cause a certain degree of contraction even in the absence of any hypoxia. Of these, hypercapnia is a strong candidate, as it is a largely unavoidable consequence during cessation of breathing.

Previous research shows that reaching a threshold level of CO2 initiates both the “struggle phase”, defined as the onset of involuntary breathing movements, and the eventual breaking point of any apnea [170]. Hyperventilation can therefore prolong apneic duration, by reducing the CO2 content of the tissues and blood so that the breaking point of apnea is reached later. Repetition of apnea with short rest intervals (2-3 minutes) also results in an increase in apnea time and the time until the struggle phase is reached [13], which may be at least partially attributed to concomitant increases in circulating erythrocytes ejected from the spleen. It is still unclear, however, if hypercapnia can stimulate this splenic contraction on its own, or in tandem with hypoxia.

Both hypercapnic chemosensory responses, which the central medullary chemoreceptors are primarily concerned with, and hypoxic chemosensory responses, mediated mainly by the peripheral carotid body receptors, and are triggered by extended apnea. There is also, however, some “crossover” effects whereby peripheral receptors respond to CO2, and hypoxia affects central

86 chemoreception via alterations in cerebral blood flow [171-173]. In most individuals, hyperoxia (PO2 = 150 mmHg) effectively silences the peripheral response to CO2 [174, 175], and would also avoid significant changes in cerebral blood flow due to hypoxia. In this manner, the effect of capnic stimuli on central chemoreception can be generally isolated for study.

In order to reveal any effect of the capnic situation on spleen contraction, we examined changes in hematological parameters and spleen volume during apneas conducted at varying hypercapnic levels. Hypoxia was eliminated by oxygen breathing and apnea times held constant in all tests allowing the capnic influence to vary independently. In order to reveal any effect of hypercapnia without apnea, a test using eupneic hypercapnia was included.

Methods

Subjects

Four male and four female subjects of mean(SD) age 28(7) years, mass 78,3(19,1) kg and height 175,9(10,7) cm volunteered for the study. Subjects signed a consent form after being informed of the experimental protocol, which had been approved by the regional human research ethics board at Umeå University, Sweden and in accordance with the Declaration of Helsinki. All were non-smokers although one subject used snuff, and trained 2,9(2.7) h per week for general fitness. Subjects had experience in breath holding at a level averaging 2 on a scale of 1 to 5 (1=no breath-holding experience, 3=regular breath-hold diving but less than 2h per week, 5=world-class apneist; [6]). Lung vital capacity for the subjects was 5.0(1.0) L.

Experimental procedure

The subjects completed four experimental trials spaced by at least 24 hours. Each trial consisted of three apneas preceded by a 2-minute countdown. The first trial consisted of apneas of maximal voluntary duration after breathing 100% oxygen for 1 min 30s, followed by a 5% CO2 95% O2 gas mixture for 30 s prior to each apnea (Hypercapnic). In the subsequent trials, which were randomized in order, the apneic times from the first session were reproduced with the following interventions: breathing of 100% oxygen at a normal ventilatory rate for two minutes prior to each apnea (Normocapnic), breathing of 100% oxygen at a normal ventilatory rate for 1 min 30 s followed by 30s of hyperventilation prior to each apnea (Hypocapnic), and breathing 100% oxygen for 1 min 30s followed by a

30s of 5% CO2, 95% O2 gas mixture, and continued breathing of this mixture thereafter for a similar period of time as the apneas in the Hypercapnic trial

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(Eupneic hypercapnic). Subjects were unaware of which gas was being inspired at which time and during which trial.

Subjects reported to the laboratory without having eaten or consumed caffeine at least 2 hours prior to testing. The vital capacity of the subject was measured via spirometer (Compact II, Vitalograph, Buckingham, England) and an intravenous catheter (Venflon Pro, Beckton-Dickson AB, Helsingborg, Sweden) was placed in the antecubital region.

Subjects were prone for the entire duration of the trials, beginning with a 20- minute period of horizontal rest, followed by a 2-minute countdown to the first of three apneas. In all trials, a noseclip was placed prior to the first 2-minute countdown and remained in place until 2 minutes after the final apnea. Subjects were administered a mask for breathing the various gas mixtures with flow rate at ~10L/min during the two minute countdown periods. For all trials, at the end of the 2-minute countdown, the subject exhaled fully followed by a deep but not maximal inspiration and then began the apnea. Hyperventilation was not permitted with the exception of the final 30s of the countdowns in the Hypocapnic trial only. Upon completion of the apnea, subjects expired fully into the mask and then resumed normal breathing. In the Hypercapnic trial, apneas were conducted without time cues and to maximum duration, while in all subsequent trials the subjects reproduced the times achieved during the air trial and were informed when to stop the apneas.

Blood samples (2 ml) were taken via the intravenous catheter 2 minutes before the first apnea, immediately after the first and third apneas, as well as 10 minutes following the third apnea. The catheter was rinsed with 2mL saline following each sample. The total volume of blood (including waste volume) removed for each subject was approximately 15 ml, and the injected saline was approximately 12 ml. Blood samples were analyzed for Hct and Hb via automated blood analysis unit (Micros 60 Analyzer, ABX Diagnostics, Montpellier, France).

From 2 min prior to apneas until 2 min post apneas the following parameters were also measured online via a BioPac MH100A CE multi-channel data acquisition system (BioPac Systems Inc., Goleta, CA, USA): arterial oxyhemoglobin saturation (SaO2) and heart rate (HR) via pulse oximeter (Biox 3700e, Ohmeda, Madison, WI, USA), mean arterial pressure (MAP) via continuous plethysmanometer (Finapres 2300, Ohmeda, Madison, WI, USA), skin blood flow (SkBF) via laser Doppler (Periflux System 5000, Perimed, Järfälla, Sweden), and breathing movements recorded via lab-developed chest bellows. Inhaled and

88 exhaled O2 and CO2 were measured before and after each apnea via gas analyzer (Normocap Oxy, Datex-Ohmeda, Helsinki, Finland).

Spleen measurement

Three subjects also had triaxial measurement of the spleen simultaneous with all blood-sampling occasions, for all four trials. Ultrasonic measurements of spleen length (L), width (W) and thickness (T) were used to calculate spleen volume according to the Pilström equation: Lπ (WT-T2)/3. The pre-apnea control measurement was compared with the measurement taken after the third apnea and with the 10-minute post-apneic measurement. Due to the small N value, no statistical analyses were conducted on spleen volume changes.

Analyses

The Hb and Hct values obtained 2 min before the first apnea were used as control values. Mean changes from control were used to compare changes within each trial, and pooled mean changes from apneas were used to compare between trials.

Control values for cardiovascular and respiratory parameters were calculated as averages from the period 60-30 s before each apnea. Apneic values for HR, MAP and SkBF were obtained from the period from 30 seconds after breath-hold onset until the cessation of the breath-hold, in order to obtain a fully developed diving response [176]. The mean changes from all apneas were used to calculate individual means. SaO2 values from the 30s following each apnea were analyzed to determine if any desaturation occurred as a result of apnea, and compared to both control and apneic SaO2 values. Expired O2 and CO2 percentages from the first breath following each apnea (and prior to gas mixture inhalation) were compared between trials. Breathing movements during apnea were divided into easy phase (prior to the onset of involuntary breathing movements) and struggle phase (after the onset of involuntary breathing movements) durations and compared between trials.

Subjects served as their own controls and effects were expressed as percent changes from control. All variables were log transformed before analysis to reduce non- uniformity of error. Excel templates were used for the calculations, purpose- designed for analyses using physiological data [163]. All values were compared between the four trials, as well as within each trial via Student´s t-test, with p<0.05 set as acceptance for differences in the comparison.

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Results

All results are reported as mean (SD) for point values, and as mean (±90% confidence interval) for comparisons. One subject’s blood values were lost for the eupnea trial due to catheter failure and are not included in the blood analysis.

Apneic duration

All subjects successfully repeated the following apnea times (SD) in all trials: 216 (68) s for apnea 1, 222 (80) s for apnea 2, and 245 (55) s for apnea 3. There was a trend (p=0.07) towards prolonged apneic duration from the first to the third apnea.

The “easy phase” of the apneas, where involuntary breathing movements were not present, was shortest in the Hypercapnic trial at 90 (27) s, followed by the

Normocapnic trial at 103 (47) s and longest in the Hypocapnic trial at 132 (43) s. The easy phase was significantly longer in Hypocapnic trial than in Hypercapnic trial (p<0.01). There were no significant differences in struggle phase duration the Hypercapnic trial (134 (38) s), the Hypocapnic trial (118 (44) s) and the Normocapnic trial (112 (48) s).

Hematological Parameters

Baseline values of Hb and Hct before the apneas were the same for all conditions. After the first apnea, Hb had increased in the Hypercapnic and Normocapnic trials (both p<0.05), while the Hypocapnic and Eupneic hypercapnic trials were still similar to control values (Figure 1). After the final apnea Hb had further increased in the Hypercapnic trial (p<0.01), followed by the Normocapnic trial (p<0.01), while the Hypocapnic and Eupneic hypercapnic trials showed no significant changes.

A comparison between trials showed that after 3 apneas the increase in Hb in the Hypercapnic trial was 9.1% greater than in the Normocapnic trial, 71.1% greater than in the Hypocapnic trial, and 87.5% greater than in the Eupneic hypercapnic trial (p<0.05).

Hct values showed a similar pattern and increased after the first apnea in

Hypercapnic and Normocapnic trials by 1.8 (1.0)% and 3.0 (1.6)% (p<0.05 and p<0.01, respectively) while the Hypocapnic and Eupneic hypercapnic values were still similar to control. After the final apnea Hct further increased in the Hypercapnic and Normocapnic trials to 3.3 (2.0)% and 3.5 (1.8)% above control

90 values, respectively (both p<0.05), while the Hypocapnic and Eupneic hypercapnic values were unchanged from control. Ten minutes after apneas, Hb was not different from control values for any of the trials, nor were they different from each other.

A comparison between trials showed that after 3 apneas the increase in Hb in the Hypercapnic trial was 27.9% and 30.1% greater than in the Hypocapnic and Eupneic hypercapnic trials, respectively. The increase in Hb in the Normocapnic trial was 6.9% greater than in the Hypercapnic trial.

Figure 1. Changes in Hb from control after apnea 1 (A1), after apnea 3 (A3), and 10 minutes following A3. * indicates p<0.05 and ** p<0.01 for comparisons with control. N=7 for Eupneic hypercapnic trial, N=8 for all other trials.

Spleen size

The largest reduction in spleen size after apnea 3 was seen in the CO2 trial, at -

32.5% from control, followed by the O2 trial at -9.4% from control (Figure 2). The HV and eupnea trials saw increases in spleen size of 30.4% and 13.0% from control respectively. Ten minutes following the final apnea the spleen contraction had reduced to -6.7% from control in the CO2 trial, and in the O2 trial the spleen had reverted to 9.3% above control. The HV and eupnea trials also showed a return to

91 control values at 21.5% above control value and 5.7% below control values, respectively.

Figure 2. Changes in spleen volume from control after apnea 1, after apnea 3, and 10 minutes following apnea 3. N=3.

Arterial oxyhemoglobin saturation

Control values for SaO2 were 98.7 (1.0)% for the Hypercapnic trial, 98.7 (1.1)% for the Normocapnic trial, 99.3 (0.6)% for the Hypocapnic trial, and 99.2 (0.6)% for the Eupneic hypercapnic trial. SaO2 did not change from control values during apneas in any of the trials, or during any post-apneic period. It was also not different between trials for the control, apneic or post-apneic periods.

Respiratory Parameters

Post-apneic expired CO2 was greatest in the Hypercapnic trial at 7.6 (1.3)%, followed by the Normocapnic trial at 7.4 (1.8)%, and the Hypocapnic trial at 7.0

(1.5)%. In the Eupneic hypercapnic trial, the expired CO2 level following the breathing period equivalent to the apneic duration was 4.6 (1.0)%. Expired CO2 in the Hypercapnic trial was higher than the Hypocapnic trial (p<0.05), and the Eupneic hypercapnic trial was lower than all other trials (p<0.01).

Post-apneic expired O2 was highest in the Hypocapnic trial at 58.4 (6.0)%, followed by the Normocapnic trial at 45.8 (6.1) % and the Hypercapnic trial at

45.9 (11.5)%. The expired O2 percentage in the Eupneic hypercapnic trial following the breathing period equivalent to the apneic duration was 72.2

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(2.0)%. Eupneic hypercapnia was higher than all other trials (p<0.01) and the Hypocapnic trial tended to be higher than the Hypercapnic trial (p=0.05).

Cardiovascular diving response

No changes in HR were noted from control values during apnea. When comparing trials, HR during apnea was lower in the Eupneic hypercapnic trial (p<0.05) at 57.3 (3.7) beats per minute (bpm) than in the Hypercapnic trial at 73.9 (8.8) bpm,

Normocapnic trial at 70.9 (8.3) bpm and Hypocapnic trial at 70.1 (8.0) bpm.

MAP increased from control in the Hypercapnic trial by 35.3% (p<0.01), in the Hypocapnic trial by 23.1% (p<0.05), and in the Eupneic hypercapnic trial by 4.2% (p<0.05), while the Normocapnic trial showed a trend towards increase from control by 14.8% (p=0.06). There were no differences in MAP between trials, although the Eupnic hypercapnic trial tended to be lower than the Hypocapnic trial (p=0.06).

SkBF was not different from control during apnea for any of the trials, nor was it different between trials.

Discussion

Increases in Hb in the current study were greater in trials with increased hypercapnic stimulus. The increase in Hb was stepwise according to increasing hypercapnic levels: the 5% CO2 pre-apneic inhalation trial elicited the greatest response, which added an extra CO2 “load” prior to the breath-hold. This was followed by the O2 trial, which allowed CO2 to be produced during the apnea but without a pre-apneic load. The increase in Hb during apnea was lowest in the

HV trial, which inferred a reduced pre-apneic CO2 load through hyperventilation. Hct followed a similar pattern as Hb, with the exception that the greatest increase was seen in the O2 trial, followed closely by the CO2 trial. All apneic trials occurred without any hypoxic stimulus, as indicated by the lack of change in oxyhemoglobin saturation level throughout the trials.

The greater stepwise influence of CO2 was also apparent in the relative division of the struggle phase and easy phase during apnea. The CO2 trial had the shortest easy phase while HV had the longest. This further confirms the “pre- loading” effect of CO2, along with the expired CO2 percentages that also follow a similar pattern.

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The apparent role that hypercapnia plays in spleen contraction and subsequent increases in Hb and Hct infers that some form of capnic chemoreception must also be involved in the contraction process. While no direct evidence of the input of these physiological systems can be derived from the current studies, the possible mechanisms can be discussed.

Changes in alveolar CO2 are reflected in brain extracellular fluid pH on a time course consistent with circulation time i.e. a few seconds [177]. This central stimulation differs from that of hypoxia, which affects mainly peripheral receptors in the carotid bodies. The likely prevention of significant peripheral chemoreceptor input, due the hyperoxia imposed by our protocol, makes it conceivable that the chemoreceptive stimulus created by CO2 alone is sufficient to elicit a stimulus leading to spleen contraction.

Although the mechanisms leading to spleen contraction are not well defined, they almost certainly involve some kind of sympathetic innervation. The splenic nerve is almost entirely adrenergic in composition, and has been shown to respond powerfully to sympathetic discharge and related catecholaminergic output [85,

88, 178]. That CO2-induced central chemoreceptor activation could cause such sympathetic output has been stated previously (see review article by [179]), through direct action on the adrenal medulla [180] and resulting in, among others, increased blood pressure and renal nerve sympathetic activity [181]. Hoka and associates [182] also noted marked changes in blood volume following hypercapnia in spleen-intact dogs, whereas this response was considerably decreased in splenectomised dogs. Similar sympathetic activity on the part of the splenic nerve in humans is thus a real possibility.

That said, circulatory responses are also greatly modified by local peripheral effects of hypoxia, hyperoxia, hypo- and hypercapnic stimuli on the cardiovascular system [181]. That no differences in the present study were seen in heart rate, blood pressure or skin blood flow between the apneic series suggests that the prospective “removal” of peripheral chemoreceptor input via hyperoxia may have also nullified expected variations in these parameters by different CO2 levels. An indication of hyperoxic effects on cardiovascular output may also be the lack of bradycardia seen during apnea, which is typically present in non-hyperoxic breath holding.

Eupneic breathing of 5% CO2 does not appear to cause an increase in Hb or Hct, or a concomitant spleen contraction, at least on the same time scale as apneic intervention does. This may be due to the fact that CO2 does not reach similar levels (closer to 7%) that are observed at end-apnea expiration. However,

94 splenic contraction has been noted in this research group’s previous work

(unpublished observations) in breath-holding trials with expiratory CO2 values of less than 5%. It may be that the constant presence of CO2 in the lung and bloodstream not seen in eupnea, and potentially greater uptake by tissues creates the greater chemoreceptive stimulus for spleen contraction and subsequent hematological changes.

Divers engaging in breath holding activities or competitions likely benefit from a strong splenic contraction, as this would result in increased oxygen storage capabilities, increased CO2 buffering capacity, and a speedier recovery from hypoxia between apneas. Many divers employ specific “warm-up” activities, following various breath-holding routines often involving different lung-volumes or hyperventilation regimes. Based on the observations in this study, an increased capnic stimulus during apnea may elicit a stronger spleen response and subsequent Hb increase than apnea preceded by hyperventilation.

In conclusion, the spleen contraction associated with apnea appears to be triggered by hypercapnia, even exclusively without hypoxic input.

References

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7. Espersen K, Frandsen H, Lorentzen T, Kanstrup I-L, and Christensen NJ. The human spleen as an erythrocyte reservoir in diving-related interventions. J Appl Physiol 92: 2071-2079, 2002. 8. Fukuda Y, Sato A, Suzuki A, and Trzebski A. Autonomic nerve and cardiovascular responses to changing blood oxygen and carbon dioxide levels in the rat. J Auton Nerv Syst 28: 61-74, 1989. 9. Herman NL, Kostreva DR, and Kampine JP. Splenic afferents and some of their reflex responses. Am J Physiol Regul Integr Comp Physiol 242: R247- 254, 1982. 10. Hoka S, Arimura H, Bosnjak ZJ, and Kampine JP. Regional venous outflow, blood volume, and sympathetic nerve activity during hypercapnia and hypoxic hypercapnia. Can J Physiol Pharmacol 70: 1032-1039, 1992. 11. Hopkins W. A new view of statistics. Internet Society for Sport Science http://www.sportsci.org/resource/stats/, 2000. 12. Hurford WE, Hong SK, Park YS, Ahn DW, Shiraki K, Mohri M, and Zapol WM. Splenic contraction during breath-hold diving in the Korean ama. J Appl Physiol 69: 932-936, 1990. 13. Jung K and Stolle W. Behavior of heart rate and incidence of arrhythmia in swimming and diving. Biotelem Patient Monit 8: 228-239, 1981. 14. Kara T, Narkiewicz K, and Somers VK. Chemoreflexes-- physiology and clinical implications. Acta Physiol Scand 177: 377-384, 2003. 15. Lin YC, Lally DA, Moore TO, and Hong SK. Physiological and conventional breath-hold breaking points. J Appl Physiol 37: 291-296, 1974. 16. Lloyd BB, Jukes MG, and Cunningham DJ. The relation between alveolar oxygen pressure and the respiratory response to carbon dioxide in man. Q J Exp Physiol Cogn Med Sci 43: 214-227, 1958. 17. Meadows GE, O'Driscoll DM, Simonds AK, Morrell MJ, and Corfield DR. Cerebral blood flow response to isocapnic hypoxia during slow- wave sleep and wakefulness. J Appl Physiol 97: 1343-1348, 2004. 18. Mohan R and Duffin J. The effect of hypoxia on the ventilatory response to carbon dioxide in man. Respir Physiol 108: 101-115, 1997. 19. Poulin MJ, Liang PJ, and Robbins PA. Dynamics of the cerebral blood flow response to step changes in end-tidal PCO2 and PO2 in humans. J Appl Physiol 81: 1084-1095, 1996. 20. Richardson M, de Bruijn, R., Schagatay, E. Hypoxia - a trigger for spleen contraction? International Conference on Diving and Hyperbaric Medicine, Barcelona, Spain, 2005. 21. Richardson M, deBruijn R, Pettersson S, Reimers J, and Schagatay E. Correlation between spleen size and hematocrit during apnea in humans.

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Proceedings of the Undersea and Hyperbaric Medicine Society Annual Conference, Orlando, FL, 2006, p. 116-119. 22. Richardson MX, Lodin A, Reimers J, and Schagatay E. Short-term effects of normobaric hypoxia on the human spleen. Eur J Appl Physiol, 2007. 23. Schagatay E. The human diving response: effects of temperature and training (Doctoral thesis). Lund, Sweden: Department of Animal Physiology, Lund University, 1996. 24. Schagatay E and Andersson J. The respiratory, circulatory and hematological effects of repeated apneas in humans. Undersea Hyperb Med 26 (Suppl): 29-30, 1999. 25. Schagatay E, Andersson J, and Nielsen B. Hematological response and diving response during apnea and apnea with face immersion. European Journal of Applied Physiology 101: 125, 2007. 26. Schagatay E, Andersson JPA, Hallén M, and Pålsson B. Selected Contribution: Role of spleen emptying in prolonging apneas in humans. J Appl Physiol 90: 1623-1629, 2001. 27. Schagatay E, Haughey H, and Reimers J. Speed of spleen volume changes evoked by serial apneas. In: European Journal of Applied Physiology, 2005, p. 447-452. 28. Vovk A, Cunningham DA, Kowalchuk JM, Paterson DH, and Duffin J. Cerebral blood flow responses to changes in oxygen and carbon dioxide in humans. Can J Physiol Pharmacol 80: 819-827, 2002.

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PAPER VI.

Spleen and lung volumes correlate with performance in elite apnea divers. 2008. E Schagatay, MX Richardson and A Lodin. Submitted for publication in the Journal of Sports Sciences. This is a preprint of an article submitted for consideration in the Journal of Sports Sciences© 2008, copyright Taylor and Francis; The Journal of Sports Science is available online at: http://www.informaworld.com.

Spleen and lung volumes correlate with performance in elite apnea divers

Erika Schagatay1,2, Matt X Richardson1, Angelica M Lodin1

1Department of Engineering and Sustainable Development Mid Sweden University, Östersund, Sweden 2 National Winter Sport Center, Östersund, Sweden

The study was supported by the Swedish National Center for Research in Sports (CIF) and Mid Sweden University research funds.

Abstract

Humans and diving mammals share the ability to contract the spleen and increase circulating hematocrit. Human spleen volume is highly variable, and our aim was to determine whether spleen and lung size correlates with performance in elite apnea divers. Anthropometric data were measured in 14 male apnea world championship participants. Spleen volume was calculated from spleen length, width and thickness measured via ultrasound. Tota l competition scores from dives of maximal depth, time and distance were compared to anthropometric measurements and training data. Subjects’ mean height was 184(2) cm, weight 82(3) kg, vital capacity (VC) 7.3(0.3) L and spleen volume 336(32) ml. Mean dive depth was 75(4) m for constant weight, duration 5min 53(39)s for static apnea and distance 139(13) m for dynamic apnea. Spleen volume did not correlate with subject height or weight, but was positively correlated with competition score (r=0.57; P<0.05). Score was also positively correlated with VC (r=0.54; P<0.05). In comparison to the 3 lowest scoring divers, the estimated time advantage during static apnea due to a greater spleen volume (by 268 ml) and VC (by 1.2L) in the 3 highest scoring divers was 15 s and 60s, respectively. We conclude that both spleen- and lung volume may predict apnea performance in elite divers.

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Key Words: Spleen contraction, hematocrit, diving response, breath-hold, diving

Introduction

Humans share with diving mammals the ability to contract the spleen and increase circulating hematocrit [48] with a possible apnea-prolonging effect [50]. Spleen size varies greatly between mammalian species and in some deep diving mammals like the Weddell seal it is exceptionally large with the ability to store 2/3 of the body´s erythrocytes during rest [55]. In highly aerobic species like the horse and the dog, the spleen contracts and elevates hematocrit during exercise [183]. The human spleen also contract, increasing circulating Hb and Hct during both apnea [50] and exercise [36, 49]. This contraction is probably active i.e. not due to reduced blood flow to the organ [35] and the elevations of Hct and Hb are not due to hemoconcentration by extravasation of plasma [50, 184]. Spleen size shows great individual variation in humans [185], and also its ability to contract varies between individuals [79] but it has never been determined whether these factors correlate with individual apneic diving ability.

The ability to perform extended apnea depends mainly on three factors: 1) gas storage capacity, or the total body storage in lungs, blood and tissues; 2) asphyxia tolerance, or the tolerable levels of hypoxia and hypercapnia as determined by e.g. brain hypoxia tolerance and the CO2 buffering capacity; and 3) metabolic rate, which is determined mainly by work economy and the ability to restrict metabolism via the diving response. A large spleen with the ability to eject a great quantity of stored erythrocytes into circulation would be beneficial for apneic duration both by increasing blood oxygen storage and enhancing buffering capacity.

Another way to increase gas storage capacity would be to maximize lung volume, thereby increasing lung oxygen stores and allowing more CO2 to be transferred to the lungs during apnea via dilution effect. In addition, large lungs with small residual volume also help the diver to attain greater depth without risking “squeeze”, as the depth at which compression to residual volume is reached would be greater. Increased lung volume furthermore facilitates equalization of middle ears and sinuses. Breath hold divers are known to overfill their lungs before diving via buccal pumping or “lung packing” [186] i.e. by pumping air into the lungs via a swallowing motion. However, there are also negative effects of large lung volume including increased buoyancy as well as a high intra-thoracic

99 pressure, with a negative effect on venous return and increased risk of syncope [187]. The diving response may also be decreased by a large inspired lung volume [188], and since the diving response has been shown to conserve oxygen [189], over filling of the lungs could elevate metabolism during diving, at least while the diver is not at depth. Trained apnea divers have previously been reported to have large lungs [190] suggesting that the positive effects may outweigh the disadvantages. However, the lungs of typical marine mammals are no larger than those of terrestrial, and e.g. the deep diving Weddell seal exhales before diving and relies mainly on blood- and tissue (mainly muscle myoglobin) oxygen storage during diving [191]. Conversely, competitive apneists are known to train to increase lung volume, by stretching and lung packing maneuvers, but to our knowledge no systematic study of the effect of lung volume on competitive apnea diving performance in elite apneists has been performed.

We therefore aimed to compare spleen and lung volume, two possible dive- prolonging characteristics, to the individual scores in an apnea world championship competition. We hypothesized that scores in the competition would be positively correlated with both spleen and lung volume.

Methods

Participants and setting This study complied with the Declaration of Helsinki and was approved by the human ethics board of Umeå University, Sweden. Information about the study was posted in the competition area of the World Championship in Hurghada 2006. Fourteen male subjects from 10 different nations volunteered for the study and, after receiving more detailed information about the study, signed an informed consent form. Their mean(SE) age was 29(2) years, and several were individual world record holders. The participants had an average of 5.8(1.2) years of experience in apnea training. Their apnea training load averaged 6.2(0.6) h per week and other physical training 6.3(0.7) h per week in the month leading up to the competition. Anthropometric and training data is presented in table 1.

The apnea competition consisted of accumulating points from dives of maximal depth, time and distance for a total score. In competitive apnea, points are gained in direct relation to depth, time and distance in the respective disciplines, producing an overall score that renders the winner. The three disciplines were Constant weight deep diving with fins, where 1 m generates 1 point, Static apnea, where 5 s generates 1 point, and Dynamic apnea with fins,

100 where 2 m yields 1 point. The total score was used as a representation of individual overall diving performance.

Measurements Subjects arrived at the laboratory after at least 45 min without apnea training, physical activity or eating. Ambient temperature was 23-25 ˚C. Vital capacity (VC) in the standing position, without lung packing, was measured three times (Microlab spirometer, Micro Medical Ltd., Kent, UK). Subjects’ height and weight were also measured and training data collected. Spleen measurements were obtained via ultrasonic imaging (Mindray DP-6600, Shenzhen Mindray Bio- Medical Electronics Co., Ltd., Shenzhen, China) using the following protocol: Divers were semi-seated vertically on an elevated stool for approximately 3 min while the site for spleen measurements was identified, followed by measurement of spleen maximal length (L), width (W) and height (H) every minute for 7 minutes. From these triaxial measurements, spleen volume was calculated using the Pilström formula: Lπ (WT-T2)/3 [79].

To determine their magnitude of spleen contraction, the 3 highest-scoring divers performed a 2 min apnea out-of-competition, in order to estimate the contribution of the expelled blood volume to apneic duration. The apnea was performed in the sitting position without prior hyperventilation. Triaxial spleen measurements were obtained before and within 30 s after the apnea, and every minute thereafter for an additional 6 min to obtain a recovery profile. For logistical reasons not all competing divers, aside from the six tested, could participate in the out-of-competition apneas.

Analysis To obtain an individual baseline measurement of spleen volume, the mean of the two largest subsequent volume measurements recorded during the 7 min resting period was used, and for lung volume the largest of the three VC measurements was used in the analysis. Spleen and lung volume, height, weight and training data from the divers were compared with the individual total scores in the competition via Pearson product-moment correlation. For an estimation of the relative contribution to apneic duration of spleen- and lung volumes, the mean values from the three subjects with the highest competition scores were compared to those of the three lowest scoring divers. Between-group comparisons were completed using paired Student´s t-tests and within-group tests using unpaired t- tests. The accepted level for differences was p<0.05.

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Results

Diving performance

Mean(SD) competition dive performances of participating subjects were 75(15) m for constant weight deep diving, 332(147) s for static apnea and 139(48) m for dynamic apnea, which are to our knowledge the highest mean performances reported in any subject group studied in apnea physiology. The mean total competition score for the tested divers was 211(8) points.

Correlations between antropometric data and performance.

Spleen volume Mean(SE) spleen volume was 336(32) ml with range 215 – 598 ml. Spleen volume was not correlated with individual height or weight, but was positively correlated with total competition score (r = 0.57; P<0.05).

Lung volume Mean(SE) VC was 7.3(0.25) L with a range from 5.5 to 8.9 ml. No correlation between VC and subjects height or weight was observed. There was a positive correlation between VC and total competition score (r=0.54; P<0.05).

Other factors Subject height, weight and BMI were not correlated with performance (Table 1). There was no correlation between performance and hours of apnea training during the month prior to the competition, but there was a trend towards negative correlation between physical training and performance (r = -0.48; P<0.1; Table 1).

Table 1. Correlation of anthropometric- and training data to scored total points.

n=14 Mean (SE) Min - Max Total points r Height (cm) 184 (1.6) 177 - 196 0,31 Weight (kg) 82 (3.2) 72 - 118 -0,11 BMI (units) 24.2 (0.8) 21.6 - 33.7 -0,29 Spleen volume (ml) 336 (32) 215 - 598 0.57 (P<0.05) VC (L) 7.3 (0.25) 5.5 - 8.9 0.54 (P<0.05) Apnea training h/w 6.2 (0.6) 2.0 - 10.5 0.43 Physical training h/w 6.3 (0.7) 0 - 12 -0.48 (P<0.1)

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Estimated contributions from spleen and lung volumes to apneic performance

Effect of spleen volume The three highest scoring divers (mean 242 pts) had a mean(SD) spleen volume of 538(53) ml and the three lowest scoring divers (mean 172 pts) a volume of 270(71) ml; (Fig 1; P<0.01) while height and weight of the two groups were similar (NS). When the high-scoring subjects performed a 2 min apnea, their mean spleen volume decreased by 260 ml (P<0.05; Fig 2). Based on the spleen volume reduction observed, the splenic blood contribution to apneic duration in high performing divers was estimated. Spleen blood contains at least double the normal hematocrit [36, 49] and with 260ml of spleen blood being added to the circulation, equivalent in Hb content to at least 500 ml of whole blood, it could bind another

100 ml of O2. Estimating VO2 during resting apnea from RMR measurements via 2 indirect calorimetry [192], 2.67 mlO * kg -1*min -1, as approximately 200 ml O2 per minute, the extra O2 stored in the three top-scoring divers would last for 30 s. The smaller spleens in the three lowest-scoring divers would, assuming a similar

50% spleen volume reduction, would contribute extra O2 lasting approximately 15 s. Thus, the three best divers could extend apneic duration by 15 s of apnea in comparison to the three lowest scoring divers, due to the extra O2 storage capacity resulting from the Hb expelled by their larger spleens.

Figure 1. Mean (SD) spleen- and lung volumes in highest (n=3) and lowest (n=3) scoring divers. Significance at P<0.01 is indicated by **.

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Figure 2. Minute-to-minute spleen volume before and after 2 min apnea in the three highest scoring divers. Significance at P<0.05 is indicated by *.

Effect of lung volume Mean VC was 7.9 (0.36) L in the three top-scorers and 6.7 (0.19; P<0.01) in the three lowest-scoring divers (Fig 1). The mean VC in the three best divers was thus more than 1 L greater than in the three lowest-scoring divers, equivalent to approximately 200 ml of extra O2 storage, allowing for an extension of apneic duration by one minute.

Discussion

A significant correlation was found between accumulated competition score and both spleen volume and lung volume, despite the relatively small number of subjects investigated in this study, suggesting that both these factors may predict competitive apneic performance. There was a large span of individual variation in spleen- and lung volume among divers, showing that such traits are not necessarily prerequisite for participation at the elite level. However, from the group correlation analysis it is clear that both these factors were associated with the production of top results. This is a novel finding, which may be useful in predicting performance in apneic divers. An interesting question arising from these findings is whether apnea-specific training can increase spleen and lung size, or if successful divers self-recruit to the sport by virtue of this physiological predisposition.

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Spleen volume Previously reported spleen volumes in the general population vary greatly, and the average in 140 healthy Caucasians of both genders was found to be 215 ml, spanning from 107 to 342 ml [185]. These values appear to be lower than the mean of 336 ml observed in our study’s elite apneists. The span 215 – 598 ml in our study’s divers starts with their normal value. In our study there was no correlation between subject height or weight and spleen volume, in accordance with the study by Prassopoulos and associates [185], which also did not find any difference between genders or age groups. Geraghty and associates reported a mean value of 238 mL in 47 mid-aged males, spanning 76 - 400 ml, but a volume of only 180 ml in females [193]. They had corrected values for height and weight using a standardized body size. When standardized for height and weight, our divers’ mean spleen volume was in the 90th percentile of Geraghty and associates’ observed range and the values of our 3 best divers more than 100 ml above their recorded maximum. Our lowest scoring divers were in the 60th percentile, thus near the median presented by Geraghty and associates. It cannot be ruled out tha t differences in methods between the studies cited above and the present study could yield different results. However, in a study on 10 non-divers of both genders, using the same method for spleen measurements as in the present study but with subjects in a prone position, we found a mean spleen volume of 275 ml, with a range of 185 - 391ml [79]. This is similar to the other values reported for non-divers, but clearly less than the 336 ml found in the elite divers in the present study. Bakovic and associates [35] reported a mean spleen volume in 10 elite divers of 344 ml, which corresponds well to the divers in the present study, but spleen volume in their control group was only slightly smaller.

This is the first report of competitive apneists’ spleen volumes in relation to their performance. An indication that trained apneists may have larger spleens or more pronounced spleen contraction, or possibly a higher spleen hematocrit than non divers, was the greater elevation of Hb observed after 3 maximal apneas in trained apneic divers compared to both non-diving athletes and sedentary non divers [102]. Studies by Hurford et al. [48] Bakovic et al. [35] and Prommer et al. [184] also suggest that spleen contraction is at least somewhat augmented in apneic divers. However, in all these studies the apneas performed by the apneic divers have been longer than in the non-divers, which could account for their more powerful spleen contraction. In order to make a fair comparison of spleen contraction between groups or individuals, they should probably instead be exposed to the same duration of apnea.

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It is not known if training can increase mammalian spleen volume, but the spleen appears to have a high regenerative ability. After the experimental removal of 2/3 of the spleen, the remaining tissue showed compensatory growth in baboons [194]. In humans, growth of a small, accessory spleen after removal of the main spleen has been demonstrated [195] and the intact human spleen increased in volume to 179% its original size within a month in parallel with liver regeneration after part of the liver had been surgically removed [196], suggesting that the same growth factors are involved. Whether our findings indicate training-stimulated growth or reflects individual predisposition can not be determined in this study, but the spleen volume of the three most successful divers is clearly in the upper range of that observed in healthy humans, which may indicate this possibility.

Lung volume Previously reported mean VC in the general adult male population was 4.1L for 225 Caucasian subjects with a mean age of 19 years and height of 162 cm [49, 197] and after correction for differences in height, our subjects’ mean VC was 158% of this reference group. Thus, the 7.3 L mean lung volume, with a span from 5.5 to 8.9 L in our study’s elite divers, was well above normal. Previously reported mean VC in eight male divers was 6.1 L (span 5.6 – 6.7 L; [198]) and 6.3 L in another group of 15 male divers (span 5.0-7.1 L; [199]), where both groups were of similar age, height and weight as in the present study but also included recreational apnea divers. No comparison between competition results and VC among divers has been published previously, but the results are in line with studies comparing groups of non-divers with trained divers and reporting greater lung volumes in the divers (see review by [200]). There is also a report of similar lung volumes between apnea divers and non-divers [35].

Large lungs in skilled divers could be due to individual predisposition, increased respiratory muscle strength or chest flexibility, or it could be due to training- stimulated lung growth. A direct increase in lung volume as a response to training is suggested by the longitudinal changes in divers observed by Carey et al [190]. Other research suggests an enlarging effect on the lungs by swimming and by high altitude exposure, but most likely not by other sports [201]. The presence of stem cells in lung tissue [196], as well as the observation that lobular removal of lung lobes in children may lead to regeneration to normal size within 2 years [202] suggests that lung growth may be induced in man, at least at a young age. It is also suggested by statements from elite divers that lung volume has been increased substantially since they started training in adult age by specific lung training protocols (personal communication) but it can not be determined whether this is due to stretching, respiratory muscle training, or to actual lung growth.

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Longitudinal studies are required to verify this possibility and clarify responsible mechanisms.

Physical- and apnea training The tendency for a negative correlation between time spent with general physical training and apneic performance could possibly reflect a time limitation when divers spent more time with apnea related training during the weeks before the competition, but it is also in line with the previous findings that specific aerobic physical training does not increase the diving response or the “easy phase” of apnea, and only marginally prolongs apneic duration by increasing the “struggle phase” where the diver resists the urge to breathe [203]. Several other studies concluded that there is no increase of the diving response with increasing physical fitness (e.g. [204]). The lack of correlation between time invested in apnea training and competition result was somewhat surprising, as previous findings have found that apnea training enhanced diving bradycardia and prolonged the “easy phase” of the apnea [203]. It is clear, however, that specific apnea training is necessary at this level of performance, as the average time spent on apnea training was >6 h per week.

Contributions from spleen and lung to apneic duration in top athletes Extended apneic duration is required for good performance in all disciplines of competitive apnea. Previous research has shown an apnea-prolonging effect by repeated apneas in intact but not in splenectomised subjects, ascribed to splenic contraction [35, 50]. A larger spleen with the ability to eject more stored red blood cells would be beneficial for the apnea diver, allowing enhancement of both body oxygen stores and asphyxia tolerance via increased buffering of CO2, and subsequent prolonged apneic duration. The estimated contribution of the spleen blood to apneic duration in the most successful divers, compared to the least successful divers was 15 s, based on the difference in measured spleen volume reduction between these groups. Reported spleen volume reductions of between 18 and 35% in other studies [35, 48, 79, 205] shows that the spleen contractions seen in our top 3 divers was higher, and may suggest that the low scorers would probably not display a more powerful contraction. With less relative spleen contraction in low scorers the time gain would be greater. It should further be noted that the single, non-maximal apnea used in this study would, according to other studies, perhaps not have elicited the full spleen response [50, 79]. Metabolic rate during apnea performance is most likely lower than in any other sport, and apnea performance is often preceded by meditation-like stages. We estimate the VO2 during resting apnea based on RMR measurements obtained via indirect calorimetry in 78 healthy males [192], found to be 2.67 mlO2 * kg -1*min -1, which is lower than most RMR values reported. According to this, the divers would use

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about 200 ml O2 per minute. It is even possible that apneic VO2 is lower, as > 20% reduction of the resting aerobic metabolism during apnea on account of the diving response has been demonstrated [206]. In addition to the increased oxygen storage, the increased Hb during apnea would enhance the CO2 buffering capacity and delay the urge to breathe and the individual breaking point. In competitive apnea, the differences in splenic contribution may thus play a decisive role for competition results.

The mean VC in the three most successful divers studied was more than 1 L greater than in the three lowest-scoring divers, likely affecting apneic duration by adding about 200 ml of O2 to the total body stores corresponding an additional 60 s of apnea, which shows that lung volume is a major factor for determining human apneic duration. Aside from enhanced O2 storage, the CO2 storage capacity would be increased by the larger VC by a dilution effect which could prolong apneas further. An enlarged VC could furthermore contribute to faster recovery after apnea by reducing relative dead space and increasing alveolar gas exchange by enlarging minute ventilation at a given respiratory frequency. This could provide additional benefits to competitive apneists, and also be important in repeated diving in for example fishing divers. However, lung volume is not known to be large among diving mammals, and some seals dive after exhalation, likely reducing buoyancy and avoiding decompression illness by limiting alveolar gas exchange when the lungs are allowed to collapse during diving [191]. This may be compared to professional Ama divers, diving with only 85% of VC for repeated sea harvesting at moderate depths [195]. Thus not only positive effects result from using large lung volumes and for sub-maximal dives the optimal volume may be below total lung capacity (TLC). Competitive apneists instead fill their lungs beyond TLC by buccal pumping [186] which has been shown to prolong apneas compared to diving at TLC, which in turn yielded longer apneas than using 85% of TLC [207]. This demonstrates the great importance of lung gas storage capacity for extended human diving. The TLC to residual volume (RV) ratio is also important for reaching great depths and increasing TLC without increasing RV would thus be beneficial for deep diving [208].

Other factors in a young, developing sport Many other physiological factors aside from spleen- and lung volume are likely involved in setting the limits in competitive apnea diving. In the swimming disciplines, technique and work economy are important. Also the magnitude of diving response [189, 209] anaerobic capacity and tolerance of lactic acid accumulation are likely factors involved in determining individual performance. Hematocrit is known to be high in divers [102], a condition which may be induced by apnea training [147]. Depth disciplines are dependent on advanced

108 equalization techniques to avoid squeeze when the lung is compressed below RV, and factors such as capacity for “blood shift” [210] and neuropsychological tolerance to nitrogen narcosis and to hypoxia [211] will be increasingly important with depths increasing beyond 100 m.

Not only physiological features and individual results determine who participates in the World Championship in a young sport like apnea. There are national and regional annual competitions in several countries with the possibility to qualify for participation in the world championships, but the possibility to join clubs and practice varies widely. A team WC and an individual WC are held in alternating years. There is little financial incentive in apneic diving, and the sport is generally not supported by national talent development programs. Therefore, socioeconomic factors also determine the circumstances for training and participation in competitions. Despite limited recruitment, there have been dramatic and continual improvements in elite apneic performance in recent years. Individual spleen- and lung volume measurement may be an effective method of further identifying “hidden” talent on performance. Longitudinal study is necessary to determine whether these features are inherent or acquired by apnea specific training.

Conclusions

This study suggests that spleen volume as well as lung volume may contribute to successful apnea performance in humans. A correlation of spleen volume with apneic performance has never been described before but is functionally logical, as ejection of additional red blood cells to circulation will both enhance oxygen storage and CO2 buffering capacity. Lung volume has previously been reported to be greater in divers than in non-divers, but this is the first report connecting VC to apnea competition results. Studies of performance-predicting factors in competitive apnea will provide a functional evaluation of human apneic diving ability and may aid divers in improving training techniques and safety.

Acknowledgements

We thank the participating divers for their time and effort and the local organizers of the 5th team world championship of apnea 2006 in Hurghada, the national team coaches, and Bill Strömberg, president of AIDA international, for the opportunity to undertake this research. We also thank Staffan Pilström and

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Seved Granberg for statistical advice. The study was supported by the Swedish National Center for Research in Sports (CIF) and Mid Sweden University research funds.

References

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PAPER VII.

Cardiovascular and hematological adjustments to apneic diving in humans: Is the spleen response part of the diving response? 2006. E Schagatay, MX Richardson, R deBruijn and JPA Andersson. In: Lindholm, P, Pollock NW, Lundgren CEG, eds. Breath-hold diving. Proceedings of the Undersea and Hyperbaric Medical Society/Divers Alert Network 2006 June 20-21 Workshop, p 108-115.

Cardiovascular and hematological adjustments to apneic diving in humans – is the ‘spleen response’ part of the diving response?

Schagatay E1, Richardson M1, de Bruijn R1, Andersson J2 1) Department of Natural Sciences, Mid Sweden University, Sundsvall, Sweden. 2) Department of Cell and Organism Biology, Lund University, Lund, Sweden.

Abstract

Adaptive mechanisms, involving both the cardiovascular diving response and the red blood cell-boosting spleen contraction may serve to facilitate apneic diving in humans. Both mechanisms have been shown to have apnea-prolonging effects. The more recently discovered spleen response may or may not be part of the general cardiovascular diving response, discussed based on published and new data. Methods used were mainly simulated apneic diving by serial apneas or apneas with facial immersion, spaced by short pauses, with the registration of cardiorespiratory and haematological parameters. The volunteer participants were healthy untrained subjects and divers of various levels of training. We found that the hematological response was not fortified by facial chilling by immersion like the cardiovascular diving response, but appears to be triggered by the apnea stimulus alone, possibly in part by the hypoxic stimulus. While the diving response reaches its full magnitude during each subsequent apnea with full recovery between apneas, the hematological response developed progressively across several apneas, and is not fully reversed until at least 10 min after apneas. Taken together the results suggest that the hematological response may be closely associated with, but not neurally linked to the human diving response, indicating that there are two different responses associated with human apneic diving. The main functions of the two adjustments may also be different: While the cardiovascular diving response has an oxygen-conserving effect during apnea, the blood boosting spleen contraction will increase blood gas storage capacity, increasing dive oxygen storage as well by facilitating recovery between apneas. 113

Introduction

Cessation of breathing (apnea), whether voluntary or involuntary, is one of the most acutely stressful situations for the organism, which can within short become a threat to survival. This raises great demands on an effective regulation of limited supplies of oxygen in lungs, blood and other tissues, and any increase of the gas-storage potential would also be beneficial. Functions known to serve these regulatory purposes in diving mammals are the cardiovascular diving response and the red blood cell-boosting spleen contraction, which may facilitate apnea and apneic diving also in humans. Previous studies revealed the presence of an oxygen-conserving diving response also in humans, and more recently a hematological response from spleen contraction was described.

Diving response

The diving response, first observed in human divers by Irving (12) can be viewed as a priority system triggered during the threat of apneic asphyxia (hypoxia, hypercapnia and acidosis). The most pronounced physiological adjustments are a reduction of the heart rate (bradycardia) and selective vasoconstriction. The function is to guarantee the oxygen supply to the heart and brain (7), which cannot survive without a constant access to oxygen, while less sensitive organs rely on stored oxygen (e.g., myoglobin in muscles), anaerobic metabolism, or, possibly, hypometabolism. Physiological mechanisms for reducing metabolism during apnea are selective vasoconstriction, causing local reduction in oxygen consumption and the bradycardia in itself, leading to a reduction of the demand of oxygen of the cardiac muscle (15). Both mechanisms have been considered to contribute to prolonged apneic durations in diving mammals (7). It has previously been shown that these factors are at work with a dive-prolonging (19) and oxygen-conserving effect also in humans during apnea (1) and during apnea with exercise (2,3,16).

Spleen response

In diving seals, spleen contraction has been shown to contribute to prolonged apneas by release of erythrocytes (11,17). Spleen contraction has been observed in humans both during physical exercise (14) and diving (10). Hurford observed a decrease in spleen size after long series of breath-hold dives in the Korean Ama by measurements with ultrasonic imaging (10). Little is known about its initiation and function, but the response appears to develop across a number of apneas. In both diving mammals and naturally diving humans, dives are performed in series

114 with the duration of apneas and surface intervals adapted to the working depth. In elite apneists, 'work-up' apneas are used to increase performance, and may yield apneic durations exceeding eight minutes. Repeated apneas with short (<10 min) intervals have been shown to prolong human apneic time (9). The underlying mechanisms were poorly understood until it was discovered that the hemoglobin concentration (Hb) and hematocrit (Hct) were reversibly increased over series of apneas and not accompanied by an increase in plasma proteins, which suggests that spleen contraction had occurred (22). The conclusion was supported by a lack of this response in splenectomized subjects (subjects with spleen removed), which also lacked the increase in apneic time observed in the subjects with spleen (22). A close association was observed between changes in spleen size and Hct and Hb across a series of apneas, which supports the conclusion that spleen contraction is the origin of the hematological effects (18). This indicates that the spleen response can be held responsible for an increase in apneic time at repeated apneas in humans (22).

It seems clear that both the cardiovascular diving response and the spleen response have potentially dive-prolonging effects in diving mammals as well as in humans. Repeated diving may elicit the diving response and spleen contraction at similar or different time frames, by the same or by different mechanisms. If mechanisms for initiation and time for establishment of the adjustments are similar or identical, they could be considered parts of the same general response, and we have to accept a wider definition than the classical of the diving response. But if initiation and development are different, they may be two different responses occurring in parallel but with different physiological origins. The aim is to further study the mechanisms responsible for initiation of the responses and their development, to reveal whether the spleen-response is part of the classical diving response.

Methods

A model for 'simulated diving' by apnea or apnea with facial immersion in cold water has been developed in our laboratory, allowing a high level of control of the behavioral and environmental factors which can affect the associated responses (20). Healthy adult volunteers of both sexes, with categorized diving experience and physical-training background, participated. They performed series of apneas, simulated dives or immersed dives, at rest or during exercise, usually with short (2 min) recovery pauses. Apneas were typically performed after a deep but not maximal inspiration, and preceded by normal respiration. Durations and details in performance depended on the factor being studied. By manipulating the factor of interest we could elucidate the physiological

115 mechanisms responsible for the cardiorespiratory or haematological adjustments. The method developed is based on continuous registration of cardiovascular and respiratory parameters using mainly non-invasive techniques. These methods are combined with the use of invasive techniques, mainly blood sampling and ultrasonic imaging for measurements of spleen size. As a complement to the simulated diving model we have in some studies used field registrations from divers, or experiments in an indoor tank. For details in methods see previous studies and below.

Results and Discussion

Initiation and development of the cardiovascular diving response

The main neural inputs eliciting the cardiovascular diving response are 1) initiation of apnea, most likely mediated via lung stretch receptors, or possibly by direct radiation from the respiratory center to the cardiovascular control centers (7), and 2) chilling of the face (13), mediated by cold receptors in the upper part of the face (24). About half of the response is present at apnea alone, compared to apnea with face immersion (Figure 1). The influence of hypoxia and hypercapnia appear to be of minor importance. The diving response is thereby a preventive system, as the response is triggered prior to the occurrence of asphyxia. The diving response develops rapidly upon apnea onset, and is fully developed within 30 s (Figure 3). The recovery after apnea is also nearly immediate, and the diving response does not change in magnitude across seria l apneas or dives (21; Figure 3). The response is, however, affected by the intensity of the thermal stimulus, i.e., the difference between ambient air and water temperatures (20). It was concluded from that study that the tropical diver will also have a well developed response, as long as there is a temperature difference between air and water. A more recent study revealed that an oxygen conserving diving response resulted with apneic face immersion also in the diver with the body being constantly immersed in 23°C water, despite cold-induced vasoconstriction and other circulatory effects of the immersion (6). The diving response was also found to dominate over the tachycardia response normally associated with immersion of the arm, when both arm and face were immersed in cold water during apnea (4). It is present also during exercise and apnea (2,3). This powerful response thus seems to have the highest priority in cardiovascular regulation, overruling the influence of other strong cardioregulatory stimuli.

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Figure 1. Mean (±SE) heart rate and skin blood flow reduction during apneas (A) and apneas with face immersion (FIA) of the same duration in eight subjects. Significance is indicated by *, for p<0.05, and *** for p<0.001.

Figure 2. Mean (±SE) change in hematocrit (Hct) and hemoglobin concentration (Hb) from eight subjects during apneas (A) and apneas with face immersion (FIA) of the same duration. No differences were observed between A and FIA.

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Figure 3. Spleen volume (mL) and heart rate (beats*min -1) values from one subject across three maximal effort apneas (A1-A3) performed in series interspaced by 2 min pauses. Spleen volume is calculated from three diameters, measured using ultrasonic imaging.

Initiation and development of spleen contraction and hematological response

Spleen contraction and the associated increases in Hct and Hb appear to develop slowly compared to the cardiovascular diving response, and several apneas are required for the full response development (22; Figure 3). The hematological response has been suggested to be initiated by apnea and facial immersion, i.e., by the same factors as the cardiovascular diving response (8). However, we found that the response was the same irrespective of face immersion in cold water or not (Figure 2). It appears that apneic hypoxia augments the response, but some of the Hb response occurs also during normoxic apnea (18), indicating that hypercapnia or the apnea in itself may also be involved. There are indications that catecholamines are involved in inducing the exercise-related spleen contraction, but also that these hormones do not account for all of the changes seen (25). The conclusions from different studies concerning recovery have been very different, likely depending on both the method used for triggering the response and for detecting the spleen volume changes. The recovery process is described as completed within 2 min after apneas when apneas were spaced by at least 10 min of rest (8). This contrasts to the findings by Bakovic and associates (5) where the recovery lasted at least 60 min after serial apneas. Our studies using five apneas spaced by 2 min, suggest an intermediate recovery time, of approximately 10 min 118 after the last apnea (23). However, when we used several such apnea series spaced by 10 min, the recovery was still incomplete after 10 min between series but complete 10 min after the last series (unpublished observations). Further studies will reveal the physiological origin of spleen contraction, but it seems clear that the mechanisms are different from those initiating the diving response.

Conclusions

These results suggest that the hematological spleen response is not neurally linked with the human diving response, nor is it developing in the same time span. The different initiation and development indicate that the diving response and the spleen response should be viewed as two different responses associated with human apneic diving, each with specific effects on apneic performance.

References

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