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Thesis for Doctoral degree, Östersund 2015

INITIATION OF SPLEEN CONTRACTION RESULTING IN NATURAL BLOOD BOOSTING IN HUMANS

Angelica Lodin-Sundström

Supervisors: Erika Schagatay, Professor. Mats H. Linér, MD., Dr. Med. Sc.

Department of Health Sciences Mid Sweden University, SE-831 25 Östersund, Sweden

ISSN 1652-893X, Mid Sweden University Doctoral Thesis 217 ISBN 978-91-88025-10-4

Akademisk avhandling som med tillstånd av Mittuniversitetet i Sundsvall framläggs till offentlig granskning för avläggande av doktorsexamen tisdag 2 juni 2015, klockan 10:15 i sal F234, Mittuniversitetet Östersund. Seminariet kommer att hållas på engelska.

Academic thesis which by permission of Mid Sweden University in Östersund is presented for public examination on account of doctoral examination Tuesday 2nd of June 2015, at 10:15 in room F234, Mid Sweden University Östersund, Sweden. The seminar will be held in English.

INITIATION OF SPLEEN CONTRACTION RESULTING IN NATURAL BLOOD BOOSTING IN HUMANS

Angelica Lodin-Sundström

© Angelica Lodin-Sundström, 2015

Department of Health Sciences, Faculty of Human Sciences Mid Sweden University, SE-832 25 Östersund, Sweden Telephone: +46 (0)771-975 000

Printed by Mid Sweden University, Sundsvall, Sweden, 2015-05-05 Cover photograph: Stéphane Tourreau (French elite freediver) Arranged from photographs by Morgan Bourc΄his (Dahab, Egypt) and Matt Richardson (Lobouche Peak, Nepal).

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TO EMMA AND LUKAS

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The black star-filled ocean in front of me, full of beauty and mystery, what hides beneath the surface? To find the answers, all you need is to dive in…

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v INITIATION OF SPLEEN CONTRACTION RESULTING IN NATURAL BLOOD BOOSTING IN HUMANS

Angelica Lodin-Sundström

Department of Health Sciences Mid Sweden University, SE-831 25 Östersund, Sweden ISSN 1652-893X, Mid Sweden University Doctoral Thesis 217 ISBN 978-91-88025-10-4

ABSTRACT

The spleen has been shown to contract in apneic situations in humans as well as in other diving mammals, expelling its stored red blood cell content into circulation. This natural blood boosting may increase the circulating hemoglobin (Hb) by up to 10%, which would enhance the carrying capacity and likely increase performance. However, the triggers of this response in humans have not been fully clarified. Study I was therefore focused on the effect of as a trigger of spleen contraction. It was found that 20 min of normobaric hypoxic evoked a substantial reduction in spleen volume showing that hypoxia is an important trigger for spleen contraction. Knowing the role of hypoxia, Study II compared two different hypoxic situations – a 2 min and 20 min normobaric hypoxic breathing – which resulted in the same level of arterial hemoglobin desaturation. Apnea evoked a twice as great spleen volume reduction, implying that variables other than hypoxia were likely involved in triggering spleen contraction. This may be which is present during apnea but not during normobaric hypoxic breathing. Study III therefore investigated the effects of mixtures containing different proportions of CO2 prior to maximal . Pre-breathing mixtures with higher percentages of CO2 resulted in greater spleen contraction, thus demonstrating hypercapnia’s likely role as a trigger in addition to hypoxia. Study IV explored whether an all-or-nothing threshold stimulus for triggering spleen contraction existed, or if contraction was graded in relation to the magnitude of triggering stimuli. Exercise was therefore performed in an already hypoxic state during normobaria. Rest in hypoxia produced a moderate spleen volume reduction, with an enhanced spleen contraction resulting after hypoxic exercise, with a concomitant increase in Hb. This implies that spleen contraction is a graded response related to the magnitude of the stimuli. This could be beneficial in environments with varying oxygen content or work loads. Study V

vi examined the possibility that spleen contraction is part of the acclimatization to altitude, during an expedition to summit Mt Everest. The long-term high altitude exposure, combined with physical work on the mountain, had no effects on resting spleen volume but resulted in a stronger spleen contraction, when provoked by apnea or exercise. This indicates that acclimatization to altitude may enhance the contractile capacity of the spleen, which may be beneficial for the climber. From these studies I concluded that hypoxia is an important trigger for spleen contraction but that hypercapnia also contributes in apneic situations. The spleen contraction likely provides a graded expulsion of erythrocytes in response to these stimuli, causing a temporary increase in gas storage capacity that may facilitate activities such as and climbing. The storage of erythrocytes during rest serves to reduce blood viscosity, which would also be beneficial for the climber or diver. The human spleen contraction appears to become stronger with acclimatization, with beneficial effects at altitude. Such an upgraded response could be beneficial both in sports and diseases involving hypoxia.

Keywords: Acclimatization, altitude, apnea, breath-hold diving, hemoglobin, hypercapnia, hypoxia, triggers.

vii SAMMANDRAG

Mjälten kan kontrahera hos människan och andra dykande däggdjur under apné, vilket temporärt tömmer dess innehåll av lagrade röda blodkroppar. Mjältens kontraktion kan höja den cirkulerande koncentrationen av hemoglobin (Hb) upp till 10 % och därmed öka den syrebärande förmågan och troligen prestationen. Men vad som initierar denna respons har ännu inte klargjorts. Studie I involverar hypoxi som en potentiell utlösare av mjältens kontraktion, där 20 min normobarisk hypoxisk andning reducerade mjältens volym. Som fortsättning på hypoxins roll att initiera denna respons, jämfördes två olika situationer med hypoxi i Studie II. Både en 2 min apné och 20 min hypoxisk andning resulterade i liknande nivå av arteriell desaturation. Apné resulterade i en fördubblad volymreduktion vilket indikerar att andra faktorer än hypoxi är delaktig i att initiera mjältens kontraktion, möjligen av hyperkapni som finns vid apné men inte under normobarisk hypoxisk andning. Studie III fokuserade därför på andning av gasblandningar med olika proportioner av CO2 innan maximala apnéer. Andning av gasblandningar med högre andel CO2 innan apné resulterade i starkare kontraktion av mjälten, vilket demonstrerar att hyperkapni har en roll att trigga denna respons tillsammans med hypoxi. Studie IV utforskar om mjältens kontraktion initieras av ett tröskelvärde som triggar responsen till sitt fullo, eller om den är graderad i relation till graden av stimulus. Därför utfördes ett fysiskt arbete i en redan normobarisk hypoxisk situation. Vila i hypoxi påvisade en reduktion i mjältens volym följt av en förstärkt kontraktion efter arbete i hypoxi samt en ökning av Hb. Detta antyder att mjältens kontraktion är graderad relaterat till magnituden av stimuli, vilket kan vara fördelaktigt i omgivningar med varierande syremängd och arbetsbelastning. Studie V fokuserade därför på att undersöka mjältens potentiella roll att öka Hb under acklimatisering till hög höjd, före och efter en expedition där klättrare besteg Mt Everest. Denna långtidsexponering för hög höjd i kombination med fysiskt arbete på berget gav ingen effekt på mjältens volym i vila, däremot visades en kraftigare kontraktion än innan klättringen. Detta indikerar att acklimatisering till hög höjd kan öka mjältens kontraherande förmåga, något som kan vara till fördel för den aktiva människan på hög höjd. Från dessa studier konkluderas att hypoxi är en viktig faktor som initierar mjältens kontraktion, men att hyperkapni också bidrar i situationer med apné. Mjältens kontraktion tycks ge en graderad uttömning av röda blodkroppar i respons beroende av graden stimuli, vilket ökar den gaslagrande kapaciteten och kan vara gynnsamt under aktiviteter så som fridykning och klättring. Förmågan att lagra röda blodkroppar reducerar blodets viskositet, vilket kan gynna både klättrare och dykare. Människans mjältes kontraktion ser ut att bli starkare med acklimatisering, med fördelaktiga effekter på hög höjd. En förstärkt kontraktionsförmåga kan vara gynnsamt både i sporter samt sjukdommar som involverar hypoxi.

viii ABBREVATIONS AND IMPORTANT TERMS

AMS Acute Mountain Sickness CNS Central Nervous System

CO2 Carbon dioxide COPD Chronic Obstructive Pulmonary Disease DCS Decompresion Sickness EPO Erythropoietin

EtCO2 Expired carbon dioxide percentages HACE High Altitude Cerebral Edema HAPE High Altitude Pulmonary Edema Hb Hemoglobin [g/L] Hb mass Total mass of hemoglobin [g/kg] Hct Hematocrite [%] HIFs Hypoxia Inducible Factors HR MAP Mean Arterial MSNA Muscle sympathetic nerve activity

O2 Oxygen OSAS Obstructive Sleap Apnea Syndrome pCO2 of carbon dioxide pO2 Partial pressure of oxygen

SaO2 Arterial Oxygen Saturation

Oxygen consumption [mL/(kg·min)]

Apnea Cessation of breathing

Asphyxia Pathological changes caused by lack of O2 in respired air, resulting in hypoxia, hypercapnia and acidosis from abnormal breathing Dyspnea Breathlessness or shortness of breath Eupnea Normal breathing Erythrocytes Red blood cells containing Hb that facilitates gas distribution Hyperbaric Greater environmental pressure than at sea level

Hypercapnia Excess of CO2 in the system

Hyperoxia Excess of O2 in the system Hypobaric Less environmental pressure than at sea level

Hypocapnia Deficiency of CO2 in the system Hypoxemia Deficient oxygenation of the blood

Hypoxia Deficiency of O2 in the system Normobaric Environmental pressure at sea level

Normocapnia Normal level of pCO2

Normoxia Normal level of pO2

ix TABLE OF CONTENTS

ABSTRACT ...... VI

SAMMANDRAG ...... VIII

ABBREVATIONS AND IMPORTANT TERMS ...... IX LIST OF STUDIES ...... XII

INTRODUCTION ...... 1

EXPLORING THE EXTREMES ...... 1 CLIMBING HIGH, DIVING DEEP AND WORKING HARD ...... 2 Oxygen transportation and hypoxia ...... 2 High altitude climbing – physiological challenges and known ...... 3 Apneic diving – physiological challenges and known solutions ...... 8 in extreme environments ...... 14 THE SPLEEN ...... 16 History ...... 16 Morphology and functions ...... 17 Spleen contraction ...... 20 MODELING DISEASE ON ATHLETES IN EXTREME ENVIRONMENTS ...... 25 AIMS OF THE THESIS ...... 27

METHODS ...... 28

METHODOLOGICAL CONSIDERATIONS ...... 28 Spleen measurements ...... 28 Measurement of hematological parameters ...... 31 Cardiovascular measurements ...... 32 PARTICIPANTS ...... 32 ETHICAL CONSIDERATIONS ...... 33 EXPERIMENTAL PROCEDURES ...... 33 VARIABLES AND EQUIPMENT ...... 36 Spleen measurements ...... 36 Hematological measurements ...... 37 Cardiovascular measurements ...... 38 Additional parameters ...... 38 ANALYSIS ...... 40

DATA ANALYSIS ...... 40 STATISTICAL ANALYSIS...... 40

x SUMMARY OF RESULTS ...... 42

DISCUSSION ...... 48

MAIN CONCLUSIONS ...... 48 SHORT AND LONG-TERM DEVELOPMENT OF SPLEEN CONTRACTION ...... 48 Short term effects ...... 49 Long term effects ...... 50 HYPOXIA - AN INDEPENDENT TRIGGER OF SPLEEN CONTRACTION ...... 50 Differences between apneic and eupneic hypoxia ...... 51 Spleen contraction during exercise and altitude ...... 53 Early changes in hemoglobin ...... 54 Climbing Mt Everest...... 55 TRAINABILITY OF THE SPLEEN RESPONSE ...... 57 MAY THERE BE SEVERAL MECHANISMS OF SPLEEN CONTRACTION? ...... 59 A combined role of mechanisms ...... 60 SUMMARY OF CONCLUSIONS ...... 61 APPLICATIONS AND FUTURE DICECTIONS OF RESEARCH ...... 62 The spleen at high altitude and in apneic diving ...... 62 The spleen in sports performance ...... 62 The spleen in hemathology ...... 63 The spleen in diseases involving hypoxia ...... 64 ACKNOWLEDGEMENTS ...... 66

REFERENCES ...... 68

xi LIST OF STUDIES

This thesis is mainly based on the following studies, herein referred to by their Roman numbers:

I. Richardson MX, Lodin A, Reimers J, Schagatay E. Short-term effects of normobaric hypoxia on the human spleen. Eur J Appl Physiol. 2008, 104: 395-399.

II. Lodin-Sundström A and Schagatay E. Spleen contraction during 20min normobaric hypoxia and 2min apnea in humans. Aviat Space Environ Med. 2010, 81: 541-549.

III. Richardson MX, Engan HK, Lodin-Sundström A, Schagatay E. Effect of hypercapnia on spleen-related haemoglobin increase during apnea. Diving and Hyperb Med. 2012, 42: 4-9.

IV. Lodin-Sundström A, Ekstam M*, Söderberg D*, Schagatay E. Exercise at simulated altitude enhances spleen contraction. In manuscript. * In alphabetical order.

V. Engan HK, Lodin-Sundström A*, Schagatay F, Schagatay E. The effect of climbing Mount Everest on spleen contraction and increase in hemoglobin concentration during breath holding and exercise. High Alt Med Biol. 2013, 15(1):52-7. * Joint first author.

The published studies are reproduced with the kind permission of the publisher.

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xiii INTRODUCTION

Exploring the Extremes

Human life is limited not only by the common environmental conditions for all biological organisms on Earth, but also by limits specific to homo sapiens that reflect our evolutionary history. Humans nonetheless strive to extend these limits, finding new ways and adaptations in our behavior and physiology, in order to explore the extremes of the Earth, sometimes despite clear risks to survival. Humans travel and colonize a wider range of remote and physiologically inhospitable places, from the high Himalayas and Andes to the freezing Arctic and burning Sahara. The challenges that these extreme environments provide require considerable physiological adaptation for humans to endure and thrive. But in the harsh extreme climates, it is often the difficult conditions for cultivation that are the primary limiting factors for colonization, rather than our physiologically limited ability to adapt to the environment.

Many animal species have adapted to particular extreme environments, reflecting an evolutionary pressure from long periods in such settings. The Weddell seal (Leptonychotes weddellii), for example, is capable of diving down to 600 meters (m) depth for food (Kooyman et al 1980; Qvist et al 1981), and the alpine yak (Bos grunniens) thrives on the Himalayan high plateau at altitudes of up to 5400 m (Schaller and Wulin 1996; Figure 1). Humans, on the other hand, are generalists that can travel between various niches, which also may have been a key to our survival across our evolutionary history. Nonetheless, despite behavioral adaptation and technological innovation there is one environmental factor that imparts more physiological duress than the rest: the lack of oxygen.

Figure 1. Young domestic yak (Bos grunniens) at 3600 m altitude in Rolwaling, Nepal 2013. Photo: Angelica Lodin-Sundström 1 Climbing high, diving deep and working hard

Oxygen transportation and hypoxia

Mainly two environments on Earth provide limited access of usable oxygen (O2) for humans and air breathers in general. These are the vast high altitude plains and mountainous regions, and the even vaster areas of shallow seas of our planet, in which we can find enormous food resources, provided we can reach them (Figure 2). While we now posses technical aids, in human history this had to be reached without breathing.

Figure 2. Topography of the Earth with mountain regions marked in dark colour, the darkest areas roughly representing altitudes of 2000 m and above. Publ. with permission, 123RF.

Mammalian life and our bodies’ inner cellular integrity are fully dependent on the constant supply of O2. In our bodies, the oxygen transport system needs to accommodate to fulfill a wide range of demands for O2 set by e.g. the intensity and duration of an activity. Different cells and organs consume O2 at highly variable rates (Wagner et al 2011) and the total body oxygen consumption varies tenfold from rest to maximal exercise (Wagner 2011). In what is known as the oxygen cascade, the pO2 from the atmospheric air to the mitochondria declines both with time and distance as it is transported through the different steps of the oxygen transportation system. Compensation for lowering of the O2 content of the blood

2 is mainly achieved by regulating cardiac output, either to maintain or augment systemic O2 transport (Biro 2013). When tissue oxygen consumption outweighs oxygen delivery, and oxygen consumption is imbalanced to the negative side, hypoxia occurs (Span and Bussink 2015). The systemic cardiorespiratory responses to hypoxia are initially sensed and propogated by the peripheral chemoreceptors formed by the carotid bodies. These chemosensors are strategically located bilaterally in the bifurcation of the common carotid artery to detect hypoxia affecting the central nervous system (Donnelly 1997; Prabhakar 2006). The sensory firing rate of the carotid sinus nerve-ending within the carotid body is low in normoxia, but is elevated within seconds in a stimulus-dependant manner due to hypoxia. The central nervous system (CNS) then triggers appropriate autonomic changes, including those of breathing and (Prabhakar 2013). The hypoxic afferent nerve activation in the carotid bodies is initiated within seconds, whereas more general cellular responses to hypoxia take more time, with minutes or hours of hypoxic exposure. The carotid bodies also responds to some extent to changes in pH, but changes in partial pressure of carbin dioxide (pCO2) are primarily stimulating the central chemoreceptors in the pontomedullary region of the brain sensing changes in pH caused by cardon dioxide (CO2) into the cerebrospinal fluid. This is a fast sensing and responding system, where changes in alveolar CO2 are reflected in brain extracellular fluid pH on a time course consistent with circulation time (Ahmad and Loeschcke 1982). With sufficient duration and severity, the hypoxia will eventually result in organ dysfunction and death (Treacher and Leach 1998). Yet though our supply of O2 is constantly and naturally shifting, it rarely causes us problems, as our dynamic physiological adaptation enables us to endure shorter periods of decreased O2 supply without serious problem and maintain relative homeostasis in critical organ systems, no matter if we are climbing high, diving deep or working hard.

High altitude climbing – physiological challenges and known solutions

Paul Berts´ seminal work, La Pression Barométricue (Bert 1878) described the deleterious effects of high altitude caused by the physiological lack of oxygen, due to reduced barometric pressure. Messner and Habeler therefore shattered previous biological “truths” when they stood on the summit of Mt Everest on the eighth of May 1978 after having climbed it without supplementary oxygen – a challenge described by many early 20th century physiologists as impossible (West 1998). This showed that well acclimatized humans given favorable physical conditions, in that case a relative high barometric pressure and , can conquer

3 one of the greatest extremes of our planet. With reduced , or reduced total gas pressure with increasing altitude, each partial pressure of each component in the air is also reduced according to Dalton’s law; although the concentration will remain 21 % there will be less available O2 for . In contrast, underwater environments have increasing hydrostatic pressure with depth according to Boyle’s law. Additionally explained by Henry’s law of a gas’s in a liquid, dissolved gases in the blood will increase with increasing hydrostatic pressure. Thus, the apneic diver at depth may experience since O2 diffuses from a very high concentration in the lung to a lower concentration in the blood. Oxygen is consumed by metabolic demands during the cause of the dive. During ascent the pressure of the gas in the lungs decreases, and O2 diffuses from a higher concentration in the blood back towards the lower concentration of the lungs, increasing the risk for hypoxic syncope. The gases dissolved at high concentration in the blood while at depth may furthermore return to gas phase forming bubbles which expand resulting in gas bubbles getting trapped in the systemic circulation, causing sickness (DCS; Paulev 1967; West et al 2013).

ACCLIMATIZATION: Reductions in partial pressure of oxygen (pO2) along the oxygen cascade (Biro 2013) as well as reduced barometric pressure and altitude in relation to the latitude and time of year (West et al 1983) result in reductions in arterial pO2. An altitude of 5500 m appears to be a ceiling for long-term human adaptation within these constraints, which the highest permanent settlements provide evidence for (West 2002). But high altitude settlements are expanding over time, with approximately 140 million persons permanently living above 2500 m altitude in North, Central and South America, East Africa and Asia (Moore 2001). Human physiology adapts to altitude hypoxia by mitigating pO2 reduction via enhanced efficiency of the respiratory (Smith et al 2001), cardiovascular and O2 utilization systems (Hoenhaus et al 1995). This is accomplished through acclimatization, where adjustments to such systems and processes in the body occur over a period of days to weeks (Bärtsch and Saltin 2008; Zafren 2014). While acclimatization occurs within the time frame of an individual’s lifespan, the ability to acclimatize in itself reflects a species´ evolutionary history.

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Figure 3. Altitude climbing in the Himalayas requires good acclimatization and favourable weather conditions. Credit: © . Photo: Emil Sergel.

Acute altitude exposure is characterized by hyperventilation and increased sympathetic nerve activity (West et al 2007; Dempsey et al 2014). The resulting increase in minute ventilation may be the most important adaptation for elevating our alveolar pO2 over the short term. Acute eupneic hypoxia simulates the peripheral chemoreceptors, initiating changes in the chemical control of breathing, hence an important shift in breathing regulation. Hyperventilation is a typical result, and over longer periods this rapid breathing leads to respiratory alkalosis which in turn increases CO2 sensitivity. This is an acclimatization process that shifts the hypercapnic ventilatory response to enhanced ventilation at a lower pCO2. At 2200 m altitude it only takes one day to fully counter- regulate the respiratory alkalosis (Dempsey et al 1972), but as altitude increases the shift requires more time to occur (Lundby et al 2004; Wagner et al 2002). This is due to an increased loss of bicarbonate via renal excretion that reduces the buffering capacity of the plasma (Böning et al 2008). The reduction in bicarbonate concentration in the cerebrospinal fluid, blood and presumably brain extracellular fluid may lower the ventilatory drive set point for CO2 (Crawford and

5 Severinghaus 1978; Dempsey et al 2014). Reduced bicarbonate concentration decreases plasma acidity and shifts the oxygen dissociation curve to the left, thereby increasing hemoglobin’s affinity for O2. Severe respiratory alkalosis therefore contributes to an increased loading of O2 from the alveoli to the blood (West 2000) and slightly reduces the unloading of O2 in the muscles. The hypoxic ventilatory drive will also increase, whereby acclimatization in the peripheral chemoreceptors over days to weeks will lead to responses to a higher pO2 (West et al 2013; Dempsey et al 2014). The phenomenon of periodic breathing, where shorter periods of breathing cessation (apnea) are followed by a series of rapid breaths, decreases with acclimatization but never resolves completely (Anholm et al 1992).

Hypoxia-inducible factors (HIFs) play an important role in the activation of various transcriptional responses in the cell during hypoxia. They are thought of as a “master switch” in the general responses of the body to hypoxia (West 2012a), and are involved in the activation of many different cells responding to the cellular hypoxia, including erythropoietin (EPO) production (Wang and Semenza 1993). An increased production of erythrocytes occurs later during acclimatization due to enhanced EPO production in the hypoxic kidney, resulting in increased Hb concentration (Erslev 1953; Jacobson et al 1957). Hb increases are also observed during acute hypoxic exposure, thought to be due to plasma reduction resulting from the hyperventilation or due to changes in sodium- and water-regulating hormones (Schmidt 2002). Novel erythrocyte production stimulated by EPO increases the oxygen carrying capacity by elevating Hb concentration in the systemic circulation (Douglas et al 1913; Bärtsch and Saltin 2008). This red cell production is a relatively slow process however, and therefore does not meaningfully contribute to increased oxygen carrying capacity until after several days to a few weeks of altitude exposure.

Both the rate of development and the degree of hypoxia determine the body´s response to hypoxia, and for that matter also the rate and degree of acclimatization (West et al 2013; Figure 3). Short-term responses and effects of acute hypoxia include dehydration due to hyperventilation, increased arterial pH, tachycardia, reduced stroke volume, redistribution of blood flow to active muscles, decreased maximal oxygen consumption ( ) as well as headache and fatigue (Bärtsch and Saltin 2008; West et al 2013). Long-term acclimatization responses include polycythemia, increased myoglobin, increased capillarization in muscle tissue, and compensatory alkali loss through the urine (Bärtsch and Saltin 2008; West et al 2013; Bhagi et al 2014). However, altitude acclimatization can only be compensatory to a certain degree, and even when fully developed it is not enough

6 to restore the body functions of physical performance at altitude to its sea level condition (Rahn and Otis 1949; West 2012a). Alveolar pO2 is approx 100 mmHg at sea level (Biro 2013); it is reduced to 45 mmHg after ascent to 4200 m near the equator (Hawaii) and with full acclimatization, it increases on average to only 54 mmHg (West 2012a). Barcroft stated already in 1925 (b) that “All dwellers at high altitude are persons of impaired physical and mental powers”. While this is certainly true of higher altitudes, some individuals may achieve near- or full sea level function at more moderate altitudes, which is desirable for athletes that practice their sports in such environments, such as alpinists and skiers.

ALTITUDE ILLNESS: A faster rate of ascent than the rate of acclimatization to the specific altitude may result in acute mountain sickness (AMS). This condition is characterized by headache, loss of appetite, anorexia, nausea, vomiting, poor judgment, dizziness, fatigue or poor sleep, where headache can coexist with a few or all symptoms (Zafren 2014). The severity of AMS covers a wide range of effects from mild (headache, loss of appetite, fatigue or poor sleep) to life-threatening (cerebral and pulmonary edema). It is the most common health problem amongst people travelling to high altitude (Korzeniweski et al 2014), with an onset typically after 6 – 12 hours (h) following arrival at altitude (Singh et al 1969). And while AMS can affect unprepared altitude tourists it can equally well affect climbers at extreme altitudes, but its presence and effects are highly variable among individuals, with strong responders also among healthy and fit people. Despite much research, no clear predictory risk factors have been identified. On the life- threatening end of the scale, high altitude cerebral edema (HACE) is characterized by confusion, ataxia, psychological changes and disturbance of consciousness that may progress to coma (Hackett and Roach 2004; Zafren 2014). The pathological processes leading to HACE or how AMS generally relates to HACE is under some debate, but one theory is that HACE originates from disruption of the blood-brain barrier (Hackett and Roach 2004). High altitude pulmonary edema (HAPE) is the most common serious altitude illness with the greatest representation in altitude- related death statistics (Bärtsch 1997). This hypoxia-related disorder is thought to arise from vasoconstriction in the lung, which increases pulmonary artery pressure and subsequently microvascular hydrostatic pressure, finally resulting in an accumulation of interstitial fluid in the lung (Bärtsch et al 2004). HAPE can occur alone, although it is often associated with AMS or HACE (Zafren 2014).

7 Apneic diving – physiological challenges and known solutions

Some air-breathing mammals have evolved into diving specialists with remarkable abilities to live in and work under water, at least in comparison to terrestrial mammals. One example is the Weddell seal (Leptonychotes weddellii), with observed diving depths down to 600 m with maximum recorded duration of 82 minutes (min) (Kooyman 1966; Kanatous et al 2002). This species of seal usually dives without elevated post-dive blood lactate (Kooyman et al 1980). The southern elephant seal (Mirounga leonina) has been observed with diving depth to 1653 m (Bennett et al 2001). There is also a range of more moderate mammalian divers, e.g. the sea otter, with the ability to dive repeatedly to 20 - 30 m and perform single dives to 100 m, which is in the range of human diving performance (Fahlman and Schagatay 2014). These divers evolved more effective utilization of a limited supply of O2, via e.g., the diving response, but other modifications as well.

VARIOUS APNEIC DIVING: Even though we now possess equipment and advanced technical aids in favor of foraging under water, there are groups who today still completely rely on apneic diving for fishing, collecting sea molluscs and seaweeds for consumption or trading, such as the , the traditional diving women of Japan (Schagatay et al 2011). Over their 2000 year history according to an ancient reference of Gishi-Wajin-Den published in 268 B.C. (Nukada 1965), these female breath-hold divers have obtained sustenance and economic security from the seafood they collect through diving on shallow, rocky reefs. It was exciting to see this still ongoing ancient profession during research visits 2009 and 2010 (Figure 4). We logged their dives which composed 50 % of their diving time (Schagatay et al 2011). Other sustenance diving populations, such as the marine hunter-gatherer Bajau in South-East Asia, may spend 60 % of their working time submerged for 2 – 9 h per day (Schagatay et al 2011). This proportion of daily life spent under water seems unsurpassed among human natural apneic divers, and remarkable for what is considered a terrestrial mammal. The physiology of both the Ama of Japan and the similarly inclined Hae Nyo of Korea has been relatively well studied (Teruoka 1932; Rahn and Yokoyama 1965; Park et al 1983; Hong et al 1991; Hurford et al 1996; Holm et al 1998; Schagatay et al 2011), providing valuable knowledge of the wide range of features associated with long-term adaptation to breath-hold hypoxia in humans.

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Figure 4. Ama diver collecting seamolluscs, Japan 2010. Photo: Angelica Lodin-Sundström.

In contrast to sustenance freedivers, competitive freedivers or apneists aim to perform one dive of maximal performance of duration, distance and/or depth. All disciplines they partake in depend on apneic duration to some extent, but while the “” discipline is defined solely by resting breath-hold duration, the “dynamic” disciplines additionally require muscle work, and depth disciplines add a hydrostatic pressure dimension to the challenge (Schagatay 2009a, 2009b, 2011). As with all competitive sport, training for improved individual performance is the key. The difference from most other sports is that in competitive apnea ’s main goal is to stay within limits of consciousness when attempting new personal bests, as syncope or inability to perform the proper surface protocol upon completion - both signs of severe hypoxia - results in disqualification (www.aidainternational.com). At the time of writing, (in competitions by the main organization AIDA), the recognized world record for three disciplines are viewed in Table 1. Static apneic performance (STA) measure apneic duration at rest in water, with fins discipline (DYN) measure the greatest horizontal distance traversed in a pool, and constant with fins (CWT), the most common competitive depth discipline, measure water depth (Figure 5).

9 Record Athlete

STA males 11 min 35 sec Stéphane Mifsud, females 9 min 02 sec Natalia Molchanova, Russia DYN males 281 m distance Goran Čolak, females 237 m distance Natalia Molchanova, Russia CWT males 128 m of sea water Alexey Molchanov, Russia females 101 m of sea water Natalia Molchanova, Russia

Table 1. Overview of AIDA world records in three disciplines of apneic performances, STA – static apnea, DYN – dynamic apnea with fins, CWT constant weight with fins.

These performances have confounded physiologists throughout the years, especially considering their high rate of improvement of results over a relatively short period.1 The remarkable accomplishments these records represent need to be physiologically addressed. Changes in pCO2 and pO2 sensed by peripheral and central chemoreceptors regulate respiratory drive (Sapru 1996) to adequately meet the interpreted environmental and physiological challenges imposed on the body. In general, the pCO2 is held relatively constant despite changes in work and rest over an average day, hence small detections of an increase pCO2 demonstrating the regulatory capacity of the ventilatory drive. In subjects untrained in apneic diving pCO2 will be the most important factor in ventilatory control. Although, during hypercapnia the response is slightly magnified if the arterial pO2 is simultaneously reduced below 100 mmHg (West 2012b), which is the case during apnea. This will not occur when pCO2 is normal (West 2012b). Through repeated exposure, i.e. apneic training, freedivers shift the respiratory drive from being primarily regulated by CO2 to being more regulated by hypoxia (Schaefer 1965). During apnea, trained divers have lower arterial pCO2 and higher arterial pO2 compared to subjects untrained in apnea in the same situation. Although, at the end of longer apneas the relationship is the opposite with higher pCO2 and lower pO2 in the divers, demonstrating the divers’ superior ability to tolerate more severe levels of hypoxia and hypercapnia (Ferretti 2001).

1 This was recently reviewed by Schagatay (2009a; 2009b; 2011) in three publications regarding static, dynamic and depth performances.

10 The lack of respiratory exchange during apnea necessitates reliance upon the finite O2 resources already in the blood, lungs and tissues which are under continuously consumption by cells. Restriction on the use of these precious resources becomes a key priority of the body’s regulatory functions. Accumulation of pCO2 also poses a challenge, but the rate of accumulation diminishes with apneic duration (DuBoise et al 1952; Hesser 1965), reflecting a decreased amount of O2 delivered to the tissues including the brain and thus reduced (Ostrowski et al 2012). Cerebral blood flow does however seem to increase to compensate for this (Pan et al 1997; Dujic et al 2009). A shift towards acidosis due to accumulating CO2 and therefore H+, shifts the oxygen dissociation curve to the right. A reduced sensitivity to CO2 was also found in trained escape tank instructors (Schaefer 1965) and in Ama divers (Masuda et al 1981).

DIVING RESPONSE: The diving response conserves O2 during diving in air- breathing mammals, including man (Elsner and Gooden 1983; Kooyman 1989; Gooden 1994; Schagatay and Andersson 1998), and may indeed be the main oxygen-conserving factor in resting apneas (Ferretti 2001; Schagatay 2009a). The response is characterized by bradycardia, decreased cardiac output, peripheral vasoconstriction and at least in humans increased arterial blood pressure (Linér 1993) resulting from simultaneous activation of sympathetic and parasympathetic nervous systems (Finley et al 1979; Fagius and Sundlof 1986; Foster and Sheel 2005). The response is initiated at the onset of apnea and thereby reduces the severity of hypoxia developing across a given duration of apnea (Schagatay et al 2007a). While apnea alone may induce it, face immersion in cold water strengthens the response (Craig 1963; Speck and Bruce 1978; Schuitema and Holm 1988; Andersson and Schagatay 1998). Such a sensory input is clearly beneficial during freediving. The diving bradycardia balances the overall vasoconstriction of the body, and the reduction in blood flow reduces total O2 consumption, with the heart and brain maintaining a higher flow rate.

Other behavioral factors have also been shown to reduce metabolic rate during apnea, such as specific meditation techniques (Telles et al 2000), or prior fasting (Schagatay and Lodin-Sundström 2014). During a voluntary apnea of sufficient duration, the onset of involuntary breathing movements demarcates what is known as the physiological breaking point, which is related to CO2 accumulation. At some point after the onset of involuntary breathing movements, the individual can no longer struggle to maintain the apnea. This is called the breaking point (Mithoefer 1965). The phase before the physiological breaking point is called the “easy phase” and the phase following the physiological breaking point the “struggle phase” (Dejours 1965; Schagatay 1996). The struggle phase

11 has also been shown to be postponed by apneic training (Schagatay et al 2000). The reduction in heart rate (HR) is more pronounced in trained apneic divers, for example Ama divers, compared to untrained subjects (Irwing 1963; Hong and Rahn 1967; Schagatay and Andersson 1998).

Figure 5. The freediver is attached to a line by a safety lanyard when diving in depth. Credit and photo: © Annelie Pompe.

12 OXYGEN STORES: While oxygen conservation can prolong apneic duration, increasing the available oxygen stores in the body is another useful strategy. Large lung volumes characterize apneic divers (Ferretti and Costa 2003), which is beneficial for apneic duration (Schagatay 2009a) and as such partially predictive of apneic performance (Schagatay et al 2012). Recent research shows that lung volume in apneic divers can be increased by stretching exercises and overinflation of the lung (lung packing; Johansson and Schagatay 2012). Increased hemoglobin concentration (Hb) means more O2 available in a closed system, but also improved CO2 buffering capacity. This latter is further enhanced by the Haldane effect when arterial oxygen saturation (SaO2) is low (Schagatay 2009a). Diving mammals have greater blood volumes than other groups (Courtice 1943) and human divers would also likely benefit from an increased O2 storage and buffering capacity. Prommer and colleagues (2007) have, however, shown in a study that of moderate trained apnea divers, scuba divers and triathletes, only triathletes had increased Hb mass and blood volume. In contrast, de Bruijn et al (2004) reported increased baseline Hb in elite freedivers compared to endurance skiers and untrained subjects, suggesting that either apneic training or an advantageous predisposition led to the increase. Increased EPO production appears dependent upon the level of hypoxia (Eckardt et al 1989; Knaupp et al 1992), and falls under the control of HIFs at the cellular level (Wang and Semenza 1993). EPO has been observed to increase due to apneas (Eckardt et al 1989; de Brujin et al 2008), i.e. by 24 % following 15 maximal, serial apneas in non-divers (de Bruijn et al 2008). The effect was considered comparable to EPO elevations seen after 6 h at 1800 m altitude. Another study observed elevated reticulocyte counts following 10 maximal apneas, although Hb concentration remained unchanged; an increase to 15 apneas for two weeks, however, augmented Hb by 3 % (Engan et al 2013).

LONG-TERM EFFECTS: Circulation to the brain increases during longer apneic periods (Pan et al 1997), due to cranial vasodilation from increasing pCO2. This preserves cerebral tissue oxygenation (Palada et al 2007; Dudic et al 2009), along with greater offloading of oxygen to the tissues with the Bohr effect (Ostrowski et al 2012). Still, excessively long apneas will result in unconsciousness when reaching a cerebral hypoxic threshold, effectively leaving the remaining O2 solely for the myocardium and brain to consume. Whether hypoxia-attributed syncope or loss of motor control in apneic athletes are associated with risk of brain damage is unknown. Andersson et al (2009) reported a 37 % increase of the brain damage- associated biochemical marker S100B following mean apneic duration of more than 5 min, they could not conclude whether these findings reflected an increased risk but raised concerns about negative cumulative long-term effects. Other potential threats to the human brain from apneic diving are related to hydrostatic pressure

13 and the aforementioned DCS. In a recent study published about the Ama divers, multiple cerebral infarcts by using brain magnetic resonance imaging (MRI) was found in almost all participants, although their neurological function and general health were unaffected (Koshi et al 2014). The authors concluded that long- term apneic diving increases the risk of brain damage even in the absence of neurological .

Thus, either migrating to high altitude or engaging in breath-hold diving, increases the physiological challenges on O2 transportation processes. The two environments of high altitude and below the surface of the sea have one important factor in common; humans will suffer from hypoxia in both. But due to the high altitude related increase in minute ventilation, humans at altitude will also encounter , and during apneic diving the opposite (hypercapnia). These environments may be hard to sustain during rest. When adding the factor of physical exercise in such environment, our physiological strain is further increased.

in extreme environments

Himalayan Sherpas, well known for their prowess at altitude, have been shown to have a larger vital capacity than both lowlanders and other high altitude residents (Sun et al 1990; Droma et al 1991; Wu and Kayser 2006) and lower metabolic costs while carrying loads, compared to matched Caucasians (Bastien et al 2005). Although in a study of two Sherpa world record holders at the time - one with the most summits of Mt Everest and one with the fastest ascent of Mt Everest - their anthropometrics, blood values, pulmonary and physiological parameters, and response to exercise profiles were similar to those of moderately fit individuals at low altitudes (McIntosh et al 2011). Thus, their seemingly superior abilities, reflecting that their admirably ability of performance perhaps lie in the secret of how to cope with hypoxia.

Hypoxic environments and performance have garnered interest for decades from scientists and athletes alike, in particular during the 1968 Mexico City Olympic Games held at 2300 m altitude (as reviewed by Saunders et al 2013). Several modern regimens of hypoxic training have been developed, with the “live high train low” (LHTL) regimen providing the most evidence for enhancing endurance performance (Bonetti and Hopkins 2009; Millet et al 2010). Historically, tissue responses to decreased O2 delivery have been thought to lead to compensatory increased O2 consumption and thus a greater arterial-venous difference (McLellan and Walsh 2004). More modern research may contradict

14 this view. Forster (1986) showed that working at 4200 m altitude reduced

as well as normal physiological and neuropsychological functions. In a study simulating the ascent of Mt Everest in a hypobaric hypoxic chamber there was no augmentations of oxygen uptake either at rest or during exercise (Sutton et al 1988). This was also confirmed in a hypobaric altitude study with acclimatized subjects at 4559 m altitude where systemic oxygen extraction both during rest and after peak exercise was reduced when compared to sea level, despite the presence of hypoxemia (Martin et al 2015). This phenomenon is still not fully understood but they speculate that reduced oxygen diffusion to the mitochondria in the last step of the oxygen cascade may be responsible.

An individual’s can be limiting during intense exercise, but also increased to a certain degree by training. In aerobic endurance sports the aim is to increase

for high O2 utilization when needed. In apnea sports the goal is instead to reach the minimum O2 uptake ( ; Schagatay 2009a). During resting apnea, a diver’s peripheral vasoconstriction restricts peripheral oxygen use and leaves more for the brain, but dynamic or depth diving involves exercise and therefore increased peripheral oxygen demand (Schagatay 2009a; 2009b; 2011). The prioritization of O2 distribution and blood flow to the working skeletal muscle versus the vasoconstrictive diving response is not fully understood. However, it was suggested by Butler and Woakes (1987) that the peripheral vasoconstriction may only occur efficiently during rest, since any hard working mucles crave their oxygen supply (Butler and Woakes 1987).

Water immersion in itself may affect since the central hypervolemia associated with immersion results in a delayed response to exercise (Hayashi and Yoshida 1999). Measuring , gas and SaO2 is complicated while diving, although it is known that the hydrostatic pressure leads to hyperoxia during descent as alveolar pO2 increases (Linér and Linnarsson 1993). Underperfused tissues (a result of the diving response) become more reliant upon anaerobic metabolism as well, increasing the plasma lactate concentration (Andersson et al 2004). Divers may however possess specific adaptive mechanisms to reduce lactate accumulation, since divers showed lower blood lactate concentration than non-divers after resting apneas (Joulia et al 2002). Hence, regardless if we are climbing high, diving deep or working hard, the need of an oxygen carrying capacity ensuring the sufficient oxygen supply delivered to meet the demands, is critical for performance.

15 One reason why I wanted to study the function of the human spleen in detail, was that it may provide just a means to cope with extreme situations by boosting the blood and increasing the blood´s gas carrying capacity, with many possible benefits in sports, critical situations and common diseases.

The spleen

History

Few organs have had been so widely misunderstood throughout history as the human spleen, described as being without purpose by Aristotle over 2000 years ago, to the 16th-century belief that it produced laughter (cited in Lucas 1991). From a vertebrate evolutionary perspective, the lampreys (Petromyzontiformes) considered the most basal group of the Vertebrata, have a spleen-like lymphiod tissue scattered along the digestive tract. This tissue corresponds both structurally and functionally to the spleen and bone marrow of higher vertebrates, and are believed to represent original spleen functions (Tischendorf 1985). The spleen first appears as an individual organ in bony fishes and sharks. It has been well-preserved throughout evolution of all animal classes possessing the organ, although its red pulp is only seen from bony fishes and on (Tischendorf 1985). The human spleen has long played an important biological role as the largest lymphoid organ in our body, and Henle reported in 1852 the first evidence of spleen emptying in humans via electrical stimulation of the splenic nerve in a decapitated human, as cited in Ayers et al (1972). Morris and Bullock (1919) were first to report in a scientifically-controlled animal model the importance of the spleen in defending against infection, which raised concerns for the vulnerability of splenectomized humans, which had had their spleens removed by surgery. The spleen was first proposed to be a red blood cell storage in mammals by Barcroft in 1925 (a), while studying cats.

Nonetheless, the spleen was considered medically expendable without consequence until relatively recently, with splenectomy occurring routinely also for non-life-threatening conditions. This may have been because during abdominal surgery for trauma or neoplasia, displacement of the spleen is often without immediate consequence (Tarantino et al 2013). Today, however, it is known that splenectomized individuals have 10- to 20-fold higher risk for sepsis than those with intact spleens (Posey and Marks 1983); longer-term studies have demonstrated an increased risk for life-threatening bacterial infections

16 (Brender 2005). Since King and Schumacker (cited in Lucas 1991) reported the effect of post-splenectomy infection in 1952, there has been an increasing recognition of the organ and its importance in the human body. It continued however to be removed in life threatening situations as it has a tendency to bleed after trauma.

Morphology and functions

The spleen is located approximately beneath the 9th to the 11th thoracic rib on the left side of the body and lies between the curving lateral border of the stomach and the diaphragm (Figure 6). The spleen has a deep red color reflecting the contained blood and according to Gray´s Anatomy of the human body (1918) it is between 13 and 15 cm in length, although in a population of 95 healthy people with normal shaped spleens, the average length was calculated to be 11 cm (± SD 1), spanning from 8-15 cm (personal observation). The typical volume is about 200 - 250 cm3 in adults and represents approximately 0.25 - 0.29 % of total body weight (Gray 1918; Rushmer 1972; Koga 1979), although individual spleen size can vary to a great extent (Prassopoulos et al 1997). Reports of correlation between spleen volume and bodyweight, height, gender and/or age are mixed (Spielmann et al 2005; Sonmez et al 2007; Schagatay et al 2012; Prassopoulos et al 1997), and suggest there may be greater individual variations than in most other organs.

The spleen is attached to the stomach and the left kidney by broad bands of the mesentery named the gastrosplenic and lienorenal ligament, also inferiorly supported by the phrenicocolic ligament. The hilius on the visceral surface of the spleen is the point of communication where all vessels and nerves that supply and control the spleen are connected. The abdominal aorta (Aorta abdominalis) branches to the celiac artery (Arteria coeliaca), which in turn braches to the splenic artery (Arteria lienalis) which supplies the spleen with oxygenated blood for its own cells and for storage. The efferent splenic vein and the afferent splenic artery pass through the lienorenal ligament before entering the spleen via six or more independent branches. The blood exits the spleen by the splenic vein (Vena lienalis), which drains into the hepatic portal vein (Vena portae hepatis), an important part of the portal venous system. Finally splenic blood travels to the liver and onward to the heart via the hepatic veins (Cunningham 1918).

17

Figure 6. Location of the spleen in the human body, highlighted in red, Published by permission, 123RF.

The spleen is encased in two capsules, the first being the peritoneum (tunica serosa splenica), which covers the entire spleen except for the hilius. The peritoneum consists of protective and connective tissue with a cavity filled with serous fluid protecting the organ from friction. The second is a fibrous capsule (tunica propria; tunica albuginea), a sponge-like structure that is very strong but highly elastic, mainly composed of fibrous tissue, elastic- and smooth muscle fibres. From the hilium, this tissue passes into the organ and further branches into numerous small fibrous bands that form the trabeculae, the framework of the spleen (Cunningham 1918). The trabeculae are a meshwork composed of a unique structure with formations of red- and white-coloured pulp. The white pulp is related to immunological functions and contains a large number of lymphocytes, with some of the smaller arterial branches covered with lymphoid tissue forming the white pulp. The outer layer is composed of the marginal zone and inside of the T-cell compartments and B-cell follicles resembling the structure of a lymph node (Mebius and Kraal 2005). Since the immune functions are not in focus in this thesis, this part of the spleen functions will not be discussed further.

18 The red pulp is the largest filter of blood in humans and contains a high concentration of erythrocytes. It consists of terminal branches of the splenic artery, which empty blood into splenic cords and further into the venous sinuses (Mebius and Kraal 2005; Figure 7). The small branches of the splenic vein start in these sinuses connecting the arterial and venous circulation (Stewart and McKenzie 2002). How arterial blood is transported into the venous sinuses is still unclear, although two thleories have been presented - the open and closed circulatory theory. The open circulatory theory suggests that the arterial capillaries empty into the cords and then gradually filter into the sinuses, while the closed theory suggests that the arteries empty directly into the venous sinuses (Tischendorf 1985). Both have also been suggested to occur simultaneously (Pinkus et al 1986).

Figure 7. Structure of the spleen showing the afferent artery, efferent vein, venous sinuses and the cords. Reprinted by permission from Macmillan Publishers Ltd: Nature Reviews Immunology, August 2005, doi:10.1038/nri1669. Article: Mebius RE and Kraal G, “Structure and function of the spleen”.

This unique structure makes it possible to filter the blood through these cords, to remove old erythrocytes and recycle the iron. The venous sinuses are composed by long parallel endothelial cells connected by stress fibers, and by contracting these stress fibers, slits are formed between the endothelial cells and the blood can be filtered through (Drenckhahn and Wagner 1986). The structure of the stress fibers is composed by actin- and myocin-like filaments, which may indicate a sliding in the filament to regulate the formation of the slits (Mebius and Kraal

19 2005). This regulates the passage of blood and blood cells into the sinuses and back into circulation. This filtration capacity makes it possible to filter the blood through these cords, removing old erythrocytes with less flexible membranes that cannot pass through the slits (Bratosin et al 1998). Old erythrocytes left in the cords will be phagocytosed by the macrophages therein and recycled for their iron content (Willekens et al 2003). This mechanism also enables the spleen to concentrate erythrocytes and store them as a reservoir. While the circulating arterial hematocrit (Hct) is normally about 40 %, the Hct in the spleen has been suggested to be approximately 78 % (MacDonald et al 1991) or even as high 96 % (Wolski 1998).

Spleen contraction

OTHER SPECIES: While the spleen produces antibodies, lymphocytes and monocytes, as well as filters and dismantles erythrocytes to reuse their iron (Weiss 1988; Tarantino et al 2011), its role as a blood storage site has been largely neglected or even denied (Tarantino et al 2011). This may be due to its comparatively small storage volume in relation to other mammals, such as horses and seals. Horses may store roughly 30 - 50 % of their total erythrocyte volume in their spleens, and seals more than 50 % (Thomas and Fregin 1981; Hurford et al 1996; Qvist et al 1986; Kunugiyma et al 1997) while humans may store closer to 10 % (Stewart et al 2003). It is outermost essensial to understand how other living organisms with physiological specializations function in their own environment. This may broaden the perspectives and contribute in understanding general physiological principles and functions. The spleen expels stored erythrocytes to increase circulating Hct and Hb, which in turn enhances the oxygen carrying capacity and thereby aerobic performance in various species (Vatner et al 1974; Dooley and Williams 1976; Thomas and Fregin 1981; Qvist et al 1986). It also reduces the viscosity of the blood by recollecting excess blood cells, reducing cardiac strain (Stewart and McKenzie 2002; Stewart et al 2003). This reservoir function is also useful in cases of severe hemorrhage or shock and considered important for survival in some animals (Guntheroth and Mullins 1963; Carneiro and Donald 1977). Many airbreathing aquatic mammals are excellent divers, the Weddell seal with extremely large spleens (Figure 8), have the ability to increase arterial Hb of 68 % during diving (Qvist et al 1996), they maintain approximately 15 % of their total body mass as blood cells, compared to 5 - 7 % in humans (Thornton and Hochachka 2004). The question has been raised if the large spleen is an adaptation to diving in these species? Large body sizes has been positively correlated to diving ability, and independant from body mass, so has large spleens in phocid seals (Thornton and Hochachka 2004).

20

Figure 8. The Weddell seal (Leptonychotes weddellii) has outstanding diving capacity, capable of depths down to 600 m and dive durations of over 80 min. They have large spleens and may increase their Hb during diving by more than 60 %. Credit and photo: © , Getty Images.

In a normal physiological state, the spleen rhythmically contracts and dilates as a vague pulsation (Barcroft et al 1932; Grindlay et al 1939). The spleens of dogs at rest show a frequent and spontaneous rhythmical fluctuation in Hb concentration in the splenic vein (Kramer and Luft 1951) suggesting that splenic efferent blood flow is constantly regulated. The stored blood cells in the spleen furthermore appear to be well oxygenated, as shown by the greater O2 saturation level in the splenic vein during hypoxia than in arterial blood. This was also shown by the peak Hb values observed in the splenic vein during strong spleen contraction that was twice as high as in the arterial blood preceding respiratory failure (Kramer and Luft 1951). Hoka and colleagues (1989) confirmed that the spleen contracts in relation to severe hypoxia in dogs, attributing this to sympathetic efferent nervous discharge to the spleen; the same has been observed in rats where α-adrenoreceptors have been implicated (Kuwahira et al 1999).

21 TRIGGERS: The human spleen thus has also been found to have a contractible ability (Hurford et al 1990; Laub et al 1993; Schagatay et al 2001; Bakovic et al 2003), with a possibility to boost the blood and e.g. prolong apneas (Schagatay et al 2001; Bakovic et al 2003). The contractile proteins are found in the walls of the arteries, veins, splenic capsule, and trabeculae of both the white and the red pulp, as well as the reticular cells of the white pulp and the sinuses of the red pulp (Pinkus et al 1986). However, the triggers of contraction and the mechanisms of its initiation are still largely unknown in humans (Richardson et al 2009). The β-adrenoreceptors in the spleen are believed to be associated with the expansive movement and the α-adrenoreceptors with the contractile movement of the spleen (Olsson et al 1976; Kutti et al 1977; Fredén et al 1978). Adrenergic (sympathetic) nerves have been found in mammalian spleens (e.g. dogs, cats and humans) but there is limited information about cholinergic (parasympathetic) nerves (Reilly 1985), however the human spleen is innervated by 98% sympathetic nerve fibers which are associated with the splenic artery (Bakovic et al 2013). The spleen has been found to contract when the body is exposed to stress, with a subsequent release of catecholamines from adrenal glands and the postganglionic fibers of the sympathetic nervous system (Ostrowski et al 2012). Especially stresses in which there are increased oxygen demands and decreased oxygen availability such as during apnea, exercise and in accidents (Flamm et al 1990; Hurford et al 1990; Allsop et al 1992; Laub et al 1993; Haffner et al 1994; Schagatay et al 2001).

ALTITUDE: The effect – if any – of long-term hypoxia has not been well investigated in this regard. Sonmez et al (2007) found that exposure of lowlanders to 1750 m altitude for 6 months resulted in a significant spleen volume reduction parallel with the well known increases in Hb and Hct. Richardson (2008) showed a transient elevation in Hb during apnea that progressively diminished during apnea attempts while ascending to altitude, while a simultaneous polycythemia occurred. During descent from altitude, however, the Hb increase following apnea was enhanced in comparison to measurements taken at the same altitudes while ascending. This may indicate that the splenic reservoir was diminishing during ascent, presumably due to tonic contraction in order to compensate for the experienced hypoxia, but after acclimatization and polycythemia its size and contractile ability began to return to normal. That study was however a field study done on three subjects during a climbing expedition, and the situation deserves further study for making reliable conclusions about spleen function at altitude.

22 CATECHOLAMINES: The rapid contraction of the seal’s large spleen during forced head immersion was measured by MRI and correlated well with increases in circulating catecholamines in the blood (Thornton et al 2001; Thornton and Hochachka 2004). Contraction of the seal spleen has also been observed following epinephrine injection (Cabanac et al 1997; Hurford et al 1996) indicating activation of the sympathetic nervous system, and likely the α-adrenoceptors on the smooth muscle tissue within the spleen. An acute stressful situation is known to increase circulating catecholamines (Cannon 1929), which are mediated via adrenoceptors (α1, α2, β1 and β2) located in the splenic vasculature and capsule (Ayers et al 1972; Stewart and McKenzie 2002). An increased sympathetic tone resulting in reduced splenic inflow and splenic pressure may result in a passive collapse, and could be interpreted as a contraction. However, Bakovic and associates (2003) demonstrated that apneic related spleen contraction occurs with conserved arterial splenic flow indicating an active contraction. During apnea there is an immediate transient increase in HR and a decrease in mean arterial pressure (MAP) (Andersson and Schagatay 1998; Palada et al 2007), related to the inspitation and sessation of breathing, and a rapid spleen contraction after 15 second (sec) apneas was also found to occur alongside these on a similar time scale. This may suggest that neural input from these events, perhaps the unloading of baroreceptors might be involved in initiating the contraction (Bakovic et al 2003; Palada et al 2007). Bakovic and colleagues (2013) observed 40 % spleen contraction following low- dose epinephrine injection, initiated shortly after HR increase and MAP decrease. They also noted increases in monitored muscle sympathetic nerve activity (MSNA), an index of peripheral sympathetic activation, suggesting that a central sympathetic mechanism may trigger early spleen contraction along with unloading of the baroreceptors. Spleen contraction may thus be a part of the sympathetic nervous system response to stressful situations in general.

APNEA: Thus, spleen contraction appears to develop over a series of apneas, with a stronger contraction and concomitant Hb and Hct increase following 3 - 5 apneas than after only one (Schagatay and Andersson 1999; Schagatay et al 2005). The spleen remains contracted during the 2 - 3 min resting periods between apneas, but typically returns to its relaxed volume within 10 min without apnea (Schagatay et al 2001; Espersen et al 2002; Bakovic et al 2003). The concomitant increase in Hb and Hct with spleen contraction does not appear to be a result of either increased or hemoconcentration due to extravasation of plasma volume, as measured values return to baseline concentrations as spleen volume normalizes (Hurford et al 1996; Schagatay et al 2001; Bakovic et al 2005). Splenectomized subjects do not display the characteristic increase in Hb and Hct following apnea (Schagatay et al 2001; Bakovic et al 2005), but elevations are seen

23 in both trained and untrained individuals with intact spleens during apnea (Schagatay et al 2001; Espersen et al 2002). There does, however, appear to be a difference in spleen volume between these two groups. We found that elite freedivers displayed volume reductions of up to 250 ml blood (Schagatay et al 2007b), and about twice the normal elevations of Hct in circulation has been shown during apnea (MacDonald et al 1991). We measured a mean spleen volume of 336 ml (range 215-598 ml) in elite apneic divers during a freediving world championship. It was shown in the same study that the top freedivers with the best results also had the largest spleens, thus spleen volume was correlated with individual apneic diving ability and seemed to be predictive of apneic performance (Schagatay et al 2012). Furthermore, there has been shown to be a close correlation between Hb elevation and spleen contraction (Richardson et al 2006).

It has been claimed that the spleen contraction is an integrated part of the human diving response (Hurford et al 1990; Schagatay et al 2001; Espersen et al 2002; Bakovic et al 2003), but lately there have been indications that it is an entirely separate mechanism (Schagatay et al 2007a). The human diving response characterized by a bradycardia and vasoconstriction develops fast and is enhanced by facial chilling. It has also been shown that the diving response occurred with equal strength in humans irrespectively of arterial hypoxia or hypocapnia (Elsner et al 1971), which not seem to be true for the spleen contraction. Considering that the diving response is instantly initiated at the onset of apnea or by facial chilling (Schagatay 1996), independently from changes in blood gas and peripheral chemoreception whereas the spleen appears to respond to changes in chemoreception (Schagatay et al 2007a), it seems most likely that the responses are of separate origin.

EXERCISE: The human spleen also serves as a reservoir during exercise (Wade et al 1956), releasing stored blood volume into circulation by contraction, similar to other mammals capable of highly aerobic exercise, such as dogs and horses. Froelich et al (1988) found a decrease in spleen volume of 39 % during upright exercise by 99mTc-labelling erythrocytes, and Flamm et al (1990) found a 46 % reduction in spleen volume accompanied by a 4.3 % increase in Hct during a similar protocol. In another study, spleen erythrocyte content diminished by 34 % alongside a concomitant increase in systematically circulating Hct and cathecholamines (Laub et al 1993). Wolski (1998) also studied spleen volume changes to 10 min of exercise at three levels of increasing intensity under normoxic and hypoxic conditions. A stepwise reduction in volume of totally 56 % was observed from continuous cycling at 60 % of up to maximal exercise. This suggested that the spleen regulates its volume during exercise in response to an intensity-dependent signal, in which plasma catecholamines play a role.

24 Exercise-induced hemoconcentration has previously been attributed to a reduction in plasma volume due to e.g. thermal and blood pressure regulation (Stewart and McKenzie 2002). The splanchnic circulation, which is combined of the gastric, small intestinal, colonic, pancreatic, hepatic and splenic circulation (Parks and Jacobson 1985), also serves as a back-up or an “autologous bloodbank” to maintain venous return blood volume or pressure in cases of depleted intravascular volume until hemodilution occurs. The spleen as being part of the splanchnic circulation also unloads the heart and circulation by absorbing excess volumes of erythrocytes at rest (Isbister 1997). Hence, exercise in splenectomized subjects may cause harmful strain on the cardiovascular system due to high blood viscosity (Tarantino et al 2013).

Modeling disease on athletes in extreme environments

The ability to sustain acute and chronic hypoxia is important in many medical conditions, not only in pathological conditions developed in a specific environment such as AMS, HACE and HAPE at altitude or DCS and pulmonary edema in apneic diving. Medical conditions may also give rise to hypoxic states, including respiratory disorders such as chronic obstructive pulmonary disease (COPD) and obstructive sleep apnea syndrome (OSAS). COPD is characterized by progressive, irreversible airflow limitation and abnormal inflammatory response in the lungs (Vestbo et al 2013), and may evoke acute, intermittent and/or chronic hypoxia in subjects (Rabe et al 2007). This results in limited performance (Antonucci et al 2003). OSAS is a prevalent sleep disorder characterized by repetitive cessation of breathing or reduced airflow due to upper airway obstructions, resulting in chronic sleep fragmentation, intermittent hypoxia and hypoxic stress (Gagnon et al 2014; Lavie 2015). This increases the risk of stroke, myocardial infarction, congestive heart failure and hypertension (Mohsenin 2014). In these diseases, there is also a related increase in sympathetic activity (Spicuzza et al 2002) elevating the peripheral resistance and blood pressure (Wolk and Somers 2003).

Applying knowledge of non-pathological performance in hypoxic environments to these pathological conditions may be useful. The similarity between high altitude climbers and COPD patients is as striking as between OSAS patients and apneic divers. Just some examples are that progressive hypoxia and hypercapnia may lead to a reduced chemosensitivity as seen in apneic divers and also in elite endurance athletes (Foster and Sheel 2005); similarly, OSAS leads to a reduced hypoxic ventilatory response and a hypercapnic ventilatory response

25 (Garcia-Rio et al 2002). There may be many other unknown parallels between the natural defense responses occurring in healthy athletes in extreme environments – and subjects to common diseases. Through increased physiological understanding of the common factors in responding to hypoxia among healthy individuals and athletes compared to those occurring in pathological states, we may be able to develop improved treatment for increased survival and enhanced performance.

It should be emphasiced that while many similarities may exist between climbers, divers and different patient groups, there are also certainly differences, and all the experiments done on healthy subjects must be interpreted carefully, before general conclusions about humans can be done. I believe however, that also studies on limited numbers of very fit subjects may lead us to new ideas of which variables and mechanisms to study in the unhealthy subjects – we are all the result of the same evolution.

26 AIMS OF THE THESIS

The overarching aim with this thesis was to identify the stimulus or stimuli that initiate spleen contraction in humans, with the subsequent release of stored red blood cells into circulation, in which environments and on what time scale this occurs. Each of the different studies had the following specific aims:

Study I was aimed to determine whether hypoxia in itself is a specific trigger for spleen contraction, thus if the spleen contracts from the effects of eupneic hypoxia via high altitude exposure, and not only from apneic hypoxia as previously known.

Concluding that a contractile effect results from both types of hypoxic challenges following the first study, Study II was aimed to determine if similar levels of arterial desaturation from apneic hypoxia and normobaric hypoxia, respectively, would lead to the same magnitude of spleen contraction – or if differences were present, suggesting that additional triggers were involved.

Revealing a difference in magnitude of contraction between the two types of hypoxic challenge in the second study, Study III was aimed to determine if hypercapnia initiates or modifies spleen contraction, during apnea or during hypercapnic breathing.

Assuming a contractile effect resulted from hypoxic challenges in the first and second study, Study IV was aimed to determine if spleen contraction during hypoxia is an all-or-nothing response, or a graded response that can be further enhanced by adding an exercise challenge.

Detecting a contractile effect from short-term hypoxic challenges in the first and second study, Study V was aimed to determine if spleen contraction is further enhanced during long-term acclimatization to chronic hypoxic exposure and frequent physical exertion, such as during a high altitude expedition.

27 METHODS

Methodological considerations

This section describes considerations regarding choice of methods and limitations.

Spleen measurements

Ultrasonography is an efficient and well-established method for volume estimations of organs. Spleen volume measurements have previously been performed by ultrasonic imaging, scintigraphy and computed tomography (CT; Koga 1979; Breiman et al 1982; Markisz et al 1987; De Odoriko et al 1999; Espersen et al 2002). Advantageously, ultrasonography does not require ionizing radiation and is accessible, portable, fast and relatively inexpensive. Its accuracy has been confirmed via spleen volume measurements conducted via autopsy (Loftus et al 1999; Koga 1979). Spleen measurements correlated well with the actual weight (Ishibashi et al 1991) and volume (Koga 1979) of resected spleens. Ultrasound measurement can however be difficult to use in estimating spleen volume, due to its variable and irregular shape. It may be difficult to visualize the entire organ with complete contours in one image, due to overlying structures such as the ribs and kidneys, as well as sensitivity to operator experience (Breiman et al 1982). However, the advantages outweigh the disadvantages in the studies included in this thesis.

Various calculations of spleen volume have been presented in the literature. Previous studies employing two-dimensional measurement have calculated spleen volume from length and width (Koga 1979; Bakovic et al 2003). The newer employment of three-dimensional ultrasonic measurement of the spleen has been reported to be more accurate (Gilja et al 1994; Riccabona et al 1996; De Odoriko et al 1999). The measurement is based on “slice by slice” technique where several planar pictures are taken during a sliding movement with the probe, and the volume is calculated by an application in the machine softwear that accounts for the distance between each obtained slice (De Odoriko et al 1999). This approach requires more time to obtain each image, however, and many images are needed to visualize the organ clearly. Studies that require continuous measurements and depending on a time flow are therefore better served by our choosen method described in the next paragraph.

28 When participants serve as their own control, the change in volume during spleen contraction is captured even if the absolute volume accuracy is somewhat lower.

Three-dimensional measurements can also be used to calculate volume by conducting single measurements of the organ along three axes in quick succession: length, width and thickness. One way to calculate spleen volume from three- dimensional measurements is by the “spleen index”, however it has been shown to be larger than true splenic volume (De Odorriko et al 1999). Another option is to calculate spleen volume using the formula length x width x thickness x 0.523, a formula frequently used to estimate irregularly shaped organs, such as the uterus, ovaries and prostate (Breiman et al 1982; Sauerbrei et al 1987; De Odoriko et al 1999; Sonmez et al 2007). However, estimation of volumes becomes sensitive for irregularly shaped organs, so instead of using a general formula applied to variously shaped objects, we used a formula specifically developed for the general splenic shape by S. Pilström: , where (L) is maximal spleen length, (W) is maximal spleen width and (T) is maximal spleen thickness (Schagatay et al 2005; Figure 9; 11 - 12). The formula is based on the averaged shaped spleen and describes the difference between two ellipsoids divided by two, and was used for calculating spleen volume in all studies in this thesis. When comparing this formula with the general formula for irregularly shaped organs in 95 healthy subjects during rest, mean spleen volume differed by less than 4 mL or 1.8 % (NS).

It is known that during physical movements organs may move within the abdominal cavity from the initial chosen site of interest for measurements. By choosing a region of interest for spleen volume measurements in a resting position, and staying with the same site also during or after physical movements such as exercise, may contribute to an increased margin of error in the measurements (Stewart et al 2003). This may also occur in some subjects during apneic situations due to the increased intrathoracic pressure. Some scientists choose to stay at the same site for measurements during all interventions (Bakovic et al 2003) and some choose to change to a new region of interest in relation to spleen movement (Stewart et al 2003). Although, it is unknown how either of these two alternatives affects the results of volume estimations. In the included studies of this thesis, the site of measurement was chosen for best visualization in relation to each intervention, thus in some cases selected a new region of interest to most likely accurately portrait the changes in spleen volume from exercise and apnea. This may represent both a strength and limitation of the measurements. Since the degree of movement of the spleen in the included studies were minimal,

29 it is possible to assume that the effects of influence on volume estimations were small. However, it must be considered that changing the site of measurements may result in a different angle of the organ hence a different size.

Figure 9. Spleen measurement with ultrasonic imaging, Nepal 2013. Photo: Erika Schagatay.

Another aspect worth mentioning is that spleen volume measurements were not blinded, as the measurements must be done directly on the person during a specific intervention, which may be considerate as a limitation. The approach in this thesis was to study potential inputs of spleen contraction, which is why we did not include measurements of neural or hormonal activity, observing spleen contraction from outside the “black box”. This is in fact a limitation, however, it is possible from inputs and outcomes to speculate from inside the box, representing a way of applied physiology.

30 Measurement of hematological parameters

Hb is normally the most important determinant of blood O2 content, and the higher Hb the greater O2 content of the blood at a given pO2. Hb is following expressed as g/L and Hct as % (of blood volume). Representative Hb reference intervals vary between ethnical populations (Ambayya et al 2014), since most participants in this thesis is Caucasian, the reference intervals presented represent 2967 subjects from Germany (Itterman et al 2010) and 700 subjects from UK (Osci-Bimpong et al (2012). In males, Hb ranges between 126 - 164 g/L and for females 113 - 147 g/L in the study by Itterman et al (2010), and between 130 - 170 g/L for males compared to 120 - 150 g/L for females in the study by Osci-Bimpong et al (2012). Hct is a good indication of blood viscosity and normal values ranges between 37 - 48 % for males and 33 - 43 % for females by Itterman and associates (2010), in comparison to 40 - 50 % for males and 35 - 45 % in females by Osci-Bimpong and colleagues (2012).

In study IV we measured blood lactate (La) during an individual anaerobic threshold test (IAT test) in order to establish each subject´s exercise capacity for determination of the load for the main test. The IAT is defined as the metabolic rate where the elimination of La during exercise is both maximal and equal to the rate of diffusion of La into the blood (Stegmann et al 1981). Thus, power outputs above IAT will result in a progressive metabolic acidosis, and exercise time to exhaustion will be inversely related to the amount of exercise work rate exceeding IAT (Stegmann and Kindermann 1982). The procedure in study IV is based on this maximal step-incremental exercise test which has been shown to produce a reliable and reproducible estimate of the IAT for the majority of subjects (McLellan and Jacobs 1993). It is worth mentioning that the IAT test was performed in normoxia and the main test conducted in hypoxia. However, since aerobic performance decreases with altitude, exercise with equal workloads in hypoxia vs. normoxia subsequent offer a higher relative workload in hypoxia (Bätsch and Saltin 2008). However, since all subjects served as their own control, this is not expected to cause a limitation.

Although the capillary and venous Hb measurements have been validated for comparisons, there may be some limitations in comparing changes calculated from capillary and venous blood samples to spleen contraction. The evident delay before splenic blood reaches the periphery has to be accounted for in study V.

31 Cardiovascular measurements

SaO2 via pulse oximetry uses a light sensor containing both red and infrared source of light in different wavelengths, which are absorbed by Hb and transmitted through tissues to a photodetector. The percentage of Hb that is saturated with O2 in the arterial blood is obtained by conversion of the amount of light transmitted through the tissue almost instantly (Barker and Tremper 1987), as well as identifying the arterial flow by its pulsation for HR. It is advantageously noninvasive, using only a clip on the finger, simple to operate and fast responding to changes. It has a good accuracy in comparison to direct blood oxygen saturation measurements, with correlation coefficients from 0.77 - 0.99 when SaO2 is greater than 60 % (Bowes et al 1989). Disadvantages that can affect the accuracy are peripheral vasoconstriction, anemia, a non pulsating vascular bed and , and that its accuracy has been shown to decrease when SaO2 is low (Bowes et al 1989). However, all studies included in this thesis operated in SaO2 range above 60 %. Regarding the potential effect of vasoconstriction, it is unlikely that it caused an influence on the reliability of the measurements. The lowest SaO2 from study I (64.4 %) occurred during hypoxic breathing, and in the studies with apneas where vasoconstriction is a known response, SaO2 only declined to 89 % in study II. In study III where apneas also were performed SaO2 were controlled >98 %, and in study V all apneas around one min were too short (SaO2 >95 %) to cause a major influence on the reliability of the measurements. However, SaO2 was measured on the finger in all studies, which has been shown to be more sensitive to peripheral changes for data collection than data obtained from the forehead in a non controlled environment (Nuhr et al 2004). Although, all studies in this thesis were conducted in a controlled environment where forehead SaO2 has been shown to correlate well with finger SaO2 and arterial blood gas analysis in healthy volunteers (Cheng et al 1988).

Participants

In total, 44 healthy subjects (26 males, 18 females) were studied in the works in this thesis. All were Caucasian except for five male Sherpas. (For details se study). Subjects were recruited via oral information at meetings and e-mail correspondence among healthy students and athletes or climbers. All participants volunteered after oral and written information and did not receive economic compensation. An overview of the studies and participants is presented in Table 2.

32 Study samples were by necessity small, as Ethical rules require that sample size in human experimental work only meets the most limited number necessary for statistics. Therefore any conlusions, especially negative ones, must be interepreted with causion. Also important, in studies requiring extreme performance ( or high climbing) there are only few subjects doing these types of achievements, and we cannot easily increase group sizes. An example of how this was handled: The number of participants in study V was small, and therefore required that Sherpas and Caucasian lowlanders were placed in the same experimental group; comparison by ethnic group was therefore not conducted. It cannot be ruled out that spleen responsiveness to hypoxia may differ among Sherpas due to genetic adaptation. The hypoxic exposure received in Kathmandu for the lowlanders upon arrival may have affected their responses in comparison to the Sherpas. Two of the participants (Sherpas) had been to higher altitudes (3500 and 5400 m respectively) three weeks prior to this study which may have an influence of the results. In study III, the low number of subjects with spleen measurements is a limitation being unable to undergo statistical treatment. Larger number of participating subjects would indeed be beneficial for reliability in all studies.

Ethical considerations

Experimental protocols complied with the 2004 Declaration of Helsinki and had been approved by the Regional Committee for Medical and Health Research Ethics in Sweden and Nepal Health Research Council, Nepal (study V). All participants were informed of the procedures, measurements and potential risks, after which they signed a written informed concent form upon arrival to lab. All subjects were aware they could abort the protocol at any time according to the Declaration of Helsinki. For some subjects, an interpreter was used in Study V.

Experimental procedures

An overview of the methodological setup and experimental procedures is presented in Figure 10. For details in procedures and timing of measurements, see respective study.

33

Overview of the studies and physical characteristics of subjects. the characteristics physical and ofstudies the Overview

. Tabel2

34

Methodological overview of the studies in this thesis. Details in each study.each in thesis. Details this studies in the of overview Methodological

Figure 10.

35 Variables and equipment

Spleen measurements

Spleen size was measured in all studies. The measurements were conducted by a trained specialist using a stationary ultrasonic imaging apparatus (HDI 3000, ATL A, Philips Company, Bothwell, WA, USA) or by portable ultrasonic imaging apparatus (Mindray DP-6600, Shenzhen Mindray Bio-Medical Electronics Co., Ltd., Shenzhen, China; Esaote, MyLab25Gold, Firenze, Italia).

Figure 11. Ultrasonic image of the spleen in the sagittal plane with measurements of length (from tip to tip) and thickness (distance in the centre of the outer surface). Photo: Angelica Lodin-Sundström

36

Figure 12. Ultrasonic image of the spleen in the transverse plane with measurement of the width (anterior to posterior diameter). Photo: Angelica Lodin-Sundström.

Hematological measurements

In study I, III and IV venous blood sampling were obtained via a closed intravenous catheter placed in the anticubital vein (Venflon Pro, Beckton-Dickson AB, Helsingborg, Sweden). No blood samples were taken in study II, and in study V Hb was measured via peripheral capillary puncture. Hct was only measured in study I. Both Hb and Hct were analyzed in triplicate via an automated blood analysis unit (Micros 60 Analyzer, ABX Diagnostics, Montpellier, France). The capillary measurements were collected in microtainer cuvettes and obtained with duplicate samples before analyzation via a portable hemoglobin analyzer (Hemocue AB, Ängelholm, Sweden). The two methods have previously been shown to be consistent (Neufeld et al 2002; Paiva et al 2004). In study IV La was obtained via capillary blood sample from the finger and analyzed immediately (Biosen 5140, EKF Diagnostic, Magdeburg, Germany).

37 Cardiovascular measurements

Pulse oximetry for SaO2 was recorded in all studies. In study I and II SaO2 and HR were logged via a portable pulse oximeter (WristOx 3100, Nonin Medical Inc., Plymouth, Minnesota, USA) placed on the index finger and stored for later analysis. In study III and IV the average window for the raw data logged from the pulse oximeter with the sensor placed on the finger was 2 sec. HR was also simultaneously measured and logged with the same equipment (Biox 3700e, Ohmeda, Madison, WI, USA and). In study V a combined pulse oximeter- capnograph (Medair Lifesense LS1-9R, Nonin Medical Inc, Medair AB, Delsbo, Sweden) was used to continuously record SaO2 and HR every sec and stored in a memory unit (Trendsense, Nonin Medical Inc., Medair AB, Hudiksvall, Sweden) for later analysis.

Blood pressure was taken in study V on the upper right arm via an automated blood pressure monitor (Omron M41, Omron Healthcare, Europe) prior to testing to obtain control values. MAP was continuously recorded on the finger in study III via an automated sphygmanometer (Finapres 2300, Ohmeda, Madison, WI, USA), simultaneously with skin blood flow (SkBF) via laser-Doppler (Periflux System 5000, Perimed, Järfälla, Sweden) on the thumb.

Additional parameters

Vital capacity (VC) was measured in all studies prior to the experimental protocol while standing upright, with the largest of three measurements used. In study I, III and IV a stationary spirometer was used (Compact II, Vitalograph, Buckingham, England), while portable spirometers were used in study II (Spirolite 201 spirometer, Vise Medical Co. Ltd., Chiba, Japan) and study V (Microlab Spirometer, Micro Medical Ltd., Kent, UK).

In study I, III and IV mechanical characteristics of respiratory movements were recorded via a lab-developed chest bellows, placed around the circumference of the thorax just below the pectoralis major muscles. This pneumatic chest bellow produces a pressure change with every chest movement which is amplified and converted to a digital signal for online recording. The respiratory thorax movements were recorded in study III to designate the onset of involuntary breathing movements and the development of struggle phase during apneas, but also to monitor respiratory pattern during eupneic interventions in studies I, III and IV, to provide a gross measure of respiratory rate and depth.

38

The respiratory rate was measured in study V during breath-by-breath recordings via a combined pulse oxymeter-capnograph (Medair Lifesense LS1-9R, Nonin Medical Inc., Medair AB, Delsbo, Sweden) and stored in a memory unit (Trendsense, Nonin Medical Inc., Medair AB, Hudiksvall, Sweden) for later analysis. The same equipment was used to continuously record expired CO2 percentages (EtCO2) during breathing, from the last breath prior to the first breath following each apnea in the same study. EtCO2 was also monitored continuously via a gas analyzer in study III (Normocap OxyTM, Datex-Ohmeda, Helsinki, Finland). The same equipment was used in study IV to monitor breath-by-breath inspiratory and expiratory pCO2 and pO2 via sensors in the non-rebreathable facemask. CO2 was monitored to ensure that hyperventilation did not occur, or in the cases where hyperventilation was instructed that it occurred to a similar level. Post-apnea EtCO2 also provided a gross indication of the amount CO2 produced and transferred to the alveoli during apnea. The level of inspired and expired O2 in study IV was measured to ensure that the hypoxic influence was kept at the same level through the protocol. In studies III and IV all parameters except for blood and spleen volume were stored via a multichannel data acquisition system for later analysis (BioPac Systems Inc., Goleta, CA, USA).

In studies I, II, III and IV participants were exposed to modified inspired gases via a non-rebreathing face mask in order to maintain a specific physiological state. Normobaric hypoxic breathing was altered in studies I, II and IV using a hypoxic generator (Hypoxico, Hypoxico Inc., New York, USA), with a metabolic filter extracting a defined amount of O2 and replacing it with nitrogen without changing the . In study III 100 % inspired O2 was used to remove any potential hypoxic stimulus normally occurring from apnea. This was achieved by administrating medical O2 at a flow rate of 5-10 L/min. In another trial 5 % of inspired CO2 was administrated to elevate the hypercapnic effect during apnea, which was achieved through a mixture of CO2 in medical O2, also with a flow rate of 5-10 L/min. In the same study, a trial with 1 min hyperventilation was used to reduce the stored CO2 prior to apnea, thus reducing the effect of hypercapnia during apnea. This was controlled for by instructing to breath at a specific rate based on feedback from respiratory rate (RR), depth of breathing and EtCO2.

39 ANALYSIS

Data analysis

Each subject served as their own control. All studies employed a crossover design, whereby subjects underwent both control and experimental treatments in the study that they participated in. Changes in both absolute and relative values were calculated and analyzed, with the final statistical outcome comparing mean changes between various treatments or from control. Data was recorded and analyzed via computers equipped with a data acquisition system (AcqKnowledge, Biopac Systems Inc, USA) in studies III and IV. The data was analyzed using spreadsheet software (ExcelTM, Microsoft Corporation, USA) in studies I, II, III and IV, and using GraphPad statistical software (GraphPad Prism version 5.04 for Windows, GraphPad Software, La Jolla, California, USA) in study V.

Statistical analysis

Various statistical approaches are used in the studies included in this thesis. Because studies are in general designed on a paired comparison basis with subjects serving as their own control, or with subject group comparisons, the most commonly applied statistical method used was the Student’s paired t-test, producing p-values where the level of acceptance for statistically significant change was p < 0.05 for both between- and within-group comparisons. Bonferroni corrections were applied for multiple comparisons as required, and the Shapiro-Wilks test was used to ensure normal distribution.

Data were log-transformed when comparing relative values to reduce non-uniformity of error. In study I and III, 90 % confidence intervals for the true value of the calculated parameter were calculated. Pearson r correlations were performed for spleen volume and blood parameters, and an additional statistical method for analysis was included in study I. In study V, both one-way repeated measurements (RM) Analysis of Variance (ANOVA) as well as two-way RM ANOVA were performed.

Cohen´s thresholds for magnitude of change (small (0.2-0.5), moderate (0.5-0.8), large (0.8-1.2) and very large (1.2 and above; Cohen 1988) may not be accepted by some journal reviewers and editors and are therefore added

40 as a complementary measure in some cases. Nonetheless, this method of analysis provides valuable information regarding clinical significance from a functional physiological perspective, where p-value-based significance measures may not be sensitive enough to clinically interesting differences. Cohen’s thresholds can, on the other hand, be extrapolated into a percent probability that the change in the parameter is indeed of clinical significance.

41 SUMMARY OF RESULTS

The results from this thesis show that both hypoxia and hypercapnia are important and independent triggers for spleen contraction. Only apneic hypercapnia (not eupneic hypercapnia) triggered spleen contraction, suggesting that cessation of breathing may be required for hypercapnia to exert its effects. Exercise in a normobaric hypoxic environment enhanced spleen contraction, providing evidence for being a graded response dependant on both the level of stimulus and/or duration of the effect. Long-term exposure to hypobaric hypoxic environments elevated the magnitude of spleen contraction, suggesting the respons to be affected by altitude acclimatization. A summary of the individual study-specific results follows, with numeric results presented as mean percentage change from baseline (standard deviation).

Study I:

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

The study participants’ SaO2 with a baseline of 97 (2) % fell to a nadir of 64 % after exposure to 20 min eupneic breathing 12.8 % O2 in nitrogen, simulating approxemately 4100 m altitude (p > 0.01). From a baseline of 405 (133) mL, spleen volume decreased by 13 (6) % during the first 3 min of exposure (p > 0.01), and had decreased by 18 (9) % by the end of the exposure (p > 0.01). Hb and Hct increased by 2.1 (1) % and 2.1 (1) % respectively (p > 0.05 and p > 0.01) across the hypoxic exposure. Five minutes following return to normoxic breathing, spleen volume was still reduced by 14 (11) % (p < 0.05) from baseline with a corresponding Hb elevation of 3.3 (2) % (p < 0.05). At this point, SaO2 was 2.9 (5) % below baseline value (NS). All variables returned to baseline after 10 min of normoxic breathing.

Main conclusions: Normobaric hypoxia evokes spleen contraction and a concomitant Hb and Hct increase on a short (3 - 20 min) time scale, the response reverses quickly (5 - 10 min) upon return to normoxia. This study using simulated high altitude suggests a splenic contraction likely occurs in the early phase of altitude acclimatization.

42 Study II:

Spleen contraction during 20min normobaric hypoxia and 2min apnea in humans.

During 20 min of hypoxic breathing (14.2 % O2 in Nitrogen), participants´ SaO2 declined gradually to 87 (1) %, a change of 11 % from baseline (p > 0.001), with a spleen volume reduction of 16 % from the baseline at 216 (16) mL over the same period (p > 0.05).

During 2 min of apnea study partcipants’ SaO2 decreased rapidly during the last minute to 89 (2) %, a change of 10 % from baseline (p > 0.001). This was accompanied by a decrease in spleen volume of 35 % from a baseline of 221 (15) mL (p > 0.01).

After both interventions; SaO2 returned to baseline values within 2 min and spleen volume recovered within 10 min after resumed (normoxic) breathing.

Main conclusions: Both 20 min of normobaric hypoxic breathing and 2 min apnea induce spleen contraction but with different speed and magnitude despite similar realized arterial desaturation levels. Apnea triggers a twice as powerful response in a shorter time (Figure 13). This suggests that factors other than hypoxia present only during apnea enhance the spleen contraction.

43

Figure 13. Mean (SE) spleen volume changes (mL) following 20 min hypoxic breathing (HB) and 2 min apnea (A) with similar SaO2. Significant differences denoted by ** for p<0.01 and by *** for p<0.001.

Study III:

Effect of hypercapnia on spleen-related haemoglobin increase during apnea.

Apneic series preceded by pre-breathing of 5 % CO2 in O2 resulted in a 4 % increase in Hb from baseline among study participants (p < 0.002), with a decrease in spleen volume of 33 % (no statistics calculated due to small number of subjects (3)). Two other apneic series preceded by breathing of 100 % O2 and hyperventilation of 100 % O2, respectively, and a eupneic period of breathing 5 % CO2 in O2 did not result in any differences in Hb from baseline; spleen volume was also unaffected except for during eupneic hypercapnia, where a small increase was seen (no statistics calculated due to small number of subjects (3)). Ten min following the last apnea in all series, or alternately the end of hypercapnic breathing in the eupnic series, Hb and spleen volume were at baseline.

Main conclusions: Hypercapnia during apnea causes reversible increases in both Hb and Hct in the absence of hypoxia, which is likely attributable to spleen contraction.

44 Study IV:

Exercise at simulated altitude enhances spleen contraction.

The spleen volume of study participants decreased by 22 % from a baseline of 280 (29) mL after 20 min of breathing 14 % O2 at rest (p > 0.05), simulating approximately 3500 m altitude. Adding 10 min of exercise further decreased the spleen volume to 33 % from the normoxic baseline (p > 0.001). Under the same conditions, Hb did not increase significantly from a baseline of 139 (7) g/L after 20 min hypoxic exposure, but after 10 min exercise it was increased by 7.2 % from baseline (p < 0.001). Spleen volume and Hb returned to baseline after resumed normoxic breathing. SaO2 decreased in a stepwise manner with hypoxia and exercise.

Main conclusions: Spleen contraction is graded in response to varying magnitude of hypoxic stimulus (Figure 14), and results in a concomitant increase in Hb and decrease in SaO2. This may be beneficial in the early phase of acclimatization to altitude.

Figure 14. Mean (SE) changes in spleen volume [mL] and Hb [g/L]. Significant differences indicated by * = p < o.o5 and by *** = p < 0.001.

45 Study V:

The effect of climbing Mount Everest on spleen contraction and increase in hemoglobin concentration during breath holding and exercise.

Before and after an expedition summiting Mt Everest, the spleen volume of participants was studied during apnea and exercise at 1370 m altitude. Prior to the expedition spleen volume did not decrease from the baseline of 213 (101) mL following the three serial apneas or exercise performed. Following the expedition, spleen volume was reduced from a baseline of 206 (52) mL after each of the three serial apneas as well as after exercise, by 39 (17) % after apnea three and by 46 (15) % after exercise (all p > 0.05). When comparing spleen volume following 5 min exercise, posttest volume was 40 % lower compared to the pretest volume (p > 0.05). The volume reduction after apnea and exercise lasted longer post-expedition compared to the development normally seen after contraction involving hypoxic breathing and apneas. At the end of the 10 min recovery period following exercise, the spleen volume was still significantly reduced by 22 (14) % (p < 0.05). Breath-hold durations and cardiovascular responses during apnea and exercise were unchanged after the climb.

Main conclusions: Long-term acclimatization to altitude causes prolonged spleen contraction in response to acute hypoxic challenge at moderate altitude, although spleen volume at rest remains unaffected compared to the period before acclimatization (Figure 15). This may be of benefit while exercising at altitude.

46

Figure 15. Mean percent (SD) change in spleen volume from baseline following each breath-hold (BH1, BH2, BH3), exercise and recovery pre- and post-climbing Mt Everest. Significant differences are denoted by * at p < 0.05 between spleen volume reduction – baseline, and between tests. + denotes significant trend at p < 0.1 for spleen volume between tests.

47 DISCUSSION

Main conclusions

Taken together, the results from the studies in this thesis demonstrate that contraction of the human spleen occurs during apnea, and during both normobaric and hypobaric hypoxic exposure. Hypoxia was the common triggering factor in these situations, but hypercapnia also modulates the contraction during apnea. The spleen contraction did not appear to be an all-or-nothing response but instead occurred in a graded manner in relation to the magnitude of the triggering stimulus. The spleen contraction was closely correlated to elevation of Hb and Hct, possibly increasing oxygen carrying capacity. The spleen contraction in response to apnea and exercise was enhanced after long-term exposure to hypobaric hypoxia; resting spleen volume, however, was not affected by the long-term hypoxic exposure and physical strain of an expedition to Mt Everest.

Short and long-term development of spleen contraction

The results reported in this thesis of spleen contraction lend support to the previously suggested independence of the spleen contraction response from the human diving response during apnea: It was found that the spleen contraction needed 3 - 5 apneas to be fully developed and about 10 min rest for spleen volume to be fully restored after exposure, whereas the diving response was maximized at every apnea. A possible increased oxygen carrying capacity during both apnea and at altitude would be an advantage in reducing the effects of hypoxia in both situations. Thus when traveling to high altitude areas or holding the breath and diving down under the sea surface, our progress is supported by the spleen contracting and boosting our blood, enhancing our gas storage and transportation capacity. Like some specialist animals, we have a capacity to cope with these extreme hypoxic environments in a previously little understood way. The results from this thesis helps us better understand how spleen contraction works.

48 Short term effects

The results reported in study I and II demonstrated that spleen contraction develops more slowly in altitude-related situations than during apnea, as spleen volume decreased in a stepwise pattern during 20 min of hypoxic breathing at rest, thus illustrating that a graded response is present. This was confirmed in study IV that spleen contraction develops as a response to the given magnitude of stimulus. A fine-tuned regulation of the circulating Hb from spleen contraction would indeed be beneficial in any given situation. This enables the regulation of blood viscosity to meet the current demand for oxygen delivery during activity, and unloading the during rest.

Though literature regarding the relationship between hypoxic breathing and spleen responses is sparse, Wolski (1998) did measure the influence of hypoxic exposure during exercise. In that study, hypoxia (fraction of inspired O2 = 0.16) corresponding to approx 2500 m altitude was used in trained and untrained subjects during 30 min exercise, and its effect on spleen volume measured. That study lead to contrasting results to those in the current work. In Wolski’s study, hypoxia had no effect on spleen contraction evoked by exercise in neither trained nor untrained subjects in comparison to the same test in normoxia. The hypoxic stimulus was much shorter (5 min prior to exercise) than in our studies, and spleen measurements took four minutes to perform during this same period. Baseline spleen volume prior to hypoxic breathing was not measured. Thus, the results from Wolski (1998) may be misleading as exposure times were likely too short, magnitude of altitude exposure possibly too low and measurement too uncertain. I therefore think study I and II in this thesis give better indications of human spleen function at altitude, and of factors involved in its elicitation.

Study I was a novel finding of the spleen contraction resulting from eupneic hypoxic breathing, suggesting the presense of a splenic response which may facilitate the early phase of altitude acclimatization, by contributing to increased O2 carrying capacity. How then, does the spleen respond to long-term hypoxic exposure?

49 Long term effects

Data from spleen function after long term exposure to altitude prior to study V is even harder to find. With knowledge from studies on dogs and rats, there is a redistribution of blood flow to vital organs to ensure adequate O2 supply as a response to acute hypoxia (Adachi et al 1976; Kuwahira et al 1993a). If hypoxia is prolonged, this regional blood flow returns to the pattern seen in normoxia, and O2 supply to the tissues and organs is compensated for by a secondary increase in circulating Hb aside from polycythemia (Kuwahira et al 1993b). This has been addressed to spleen contraction, strengthen by that splenectomy abolished this reversible increase in circulating Hb after hypoxia (Kuwahira et al 1999). In humans, Sonmez and associates (2007) studied the effect of altitude exposure at 1750 m for six months, and found a decreased spleen volume after both three and six months. Their study may also have offered a relatively weak stimulus to evoke a clear spleen contraction, in contrast to our long term studies in study V. Also compared to the more acute studies in this thesis; study I had a stimulus equivalent to approximately 4100 m, in study II equivalent to 3300 m, and in study IV equivalent to 3500 m. These levels of stimulation of the spleen may be needed for long term effects to develop. In Sonmez study it is also unclear in which state the spleen volume is measured – and it cannot be ruled out that a temporal response on some stimulus may affect their results.

Hypoxia - an independent trigger of spleen contraction

Previous studies on spleen function have focused on apnea-induced hypoxia, and while it was likely that hypoxia was involved also during apnea, it could not be ruled out that the apnea in itself was necessary to evoke the response. However, a novel finding in study I was that spleen contraction and enhanced Hb and Hct is observed during breathing in a hypoxic environment, simulating altitude exposure. This study further proposed that the Hb elevation occurring during the initial phase of altitude acclimatization before any erythropoietic effects of altitude had time to occur (Bärtsch and Saltin 2008), could in fact be a result of spleen contraction. Previously, any elevation of Hb or Hct seen in the early phase of acclimatization was assigned to plasma volume reduction, although with the misconception of spleen contraction not responding to altitude and hypoxia, early elevations in Hb may be faultily addressed. Studies involving both altitude and exercise demonstrates this early elevation in Hct (Wolski 1998; El-Sayed et al 1999). The lack of measurement, or even consideration of the effects

50 spleen contraction should draw these conclusions into question, along with other methodological discrepancies, such as study protocols, time and duration of exposure (Stewart et al 2003). A greater or lesser influence of spleen contraction on early Hb elevations is suggested by several studies of this thesis, and it is inferred that any study of Hb related to hypoxic conditions should include spleen volume measurements to be able to draw any conclusions.

Differences between apneic and eupneic hypoxia

Study II demonstrated that spleen contraction derived from 20 min of normobaric hypoxic breathing, in comparison to 2 min apnea was about twice the magnitude for the apneic condition, despite similar levels of arterial desaturation. This showed that spleen contraction is triggered not only by the hypoxic stimulus, but also by other factors present during apnea but not eupneic hypoxia. One clear candidate is the accumulation of CO2 leading to a hypercapnic situation; simulated or actual altitude generally results in the opposite – hypocapnia – due to increased ventilation. Thus if the stimulation of chemoreceptors by CO2 or H+ is involved in triggering spleen contraction, these two hypoxic situations would be expected to have opposite effects. The faster desaturation rate during apnea with a drop in SaO2 toward the end of the second minute might in itself be an important stimulus compared to the more moderate reduction during hypoxic breathing. The spleen response generally requires an apneic duration of about 2 min to develop significantly (personal observation), which is also the time at which the SaO2 decline progresses with a steeper slope. In study I the desaturation rate during hypoxic breathing was somewhat faster than that observed in study II which likely reflected the stronger hypoxic stimulus in the former (4100 m versus 3300 m in the latter); the pattern of spleen volume reduction was also similar to the desaturation rate. In comparison, the apneic trial in study II resulted in a considerable SaO2 reduction over just 2 min, and even greater spleen volume reduction than that which resulted from the entire hypoxic breathing period. This not only demonstrates the importance of the hypoxic stimulus in triggering the spleen contraction, but also that its severity, or perhaps sensed immediacy, appears closely related to the development of the contraction. This is supported by previous observations from Balestra and colleagues (2006) in another context; They noted that the rate of change of the stimulus in itself may be of importance as a stimulus for EPO production after an initial phase of hyperoxia which was suddenly reduced to normoxia.

51 Thus there are a number of candidates that may have caused the observed difference in spleen contraction between apnea and hypoxic breathing. Intrathoracic pressure increases during prolonged apnea with large lung volumes (Kobayasi and Sasaki 1967), due to the elastic recoil of the lungs and chest wall (unstimulated stretch receptors) combined with inactive inspiratory muscles and a closed glottis (Colebatch et al 1979). As a potential mechanical compressor of the spleen, this increased pressure may partly explain the differences in spleen contraction between eupneic, hypoxic breathing and apnea, although it is contraindicated by the fact that spleen contraction has been shown to be an active response rather than a passive collapse due to pressure or vasoconstriction (Bakovic 2003; Stewart et al 2003). Another plausible explanation of the stronger spleen contraction from apnea in study II may be the intensified struggle phase at the time near apnea termination. Only non-divers, thus subjects untrained in apnea, participate in the study, which meant that reaching 2 min represented a near maximum apneic duration for many to achieve. A trained apneic diver would not reach the struggle phase in this time, involving the onset of involuntary breathing movements, especially considering their efficient metabolic restrictions and blunted sensitivity to CO2 (Andersson and Schagatay 2009). The involuntary breathing movements associated with the struggle phase have previously been implicated in the normalization of cardiac output. This effect of normalization is achieved by the involuntary breathing movements assisting venous return to increase blood flow through the vena cava, in turn enhancing stroke volume (Palada et al 2008). Indeed, cardiac output and caval flow were almost normalized at the end of the struggle phase in one study, adding further connection to a relation between involuntary breathing movements and splenic contraction (Palada et al 2008).

A main difference between eupneic and apneic hypoxia was of course (as stated above) the capnic situation, the focus of study III. The stepwise influence of CO2 in the various trials was also reflected in the easy phase of the apneas, with the shortest easy phase occurring in hypercapnic trial and the longest in the hypocapnic trial. The capnic status of each trial was confirmed by the same stepwise pattern of EtCO2 upon exhalation. The isolation of hypercapnia from hypoxia as a stimulus in these trials suggested it could be an independent trigger of spleen contraction – and also a magnitude-related one – although it was found to be in effect only in combination with apnea. An independent effect of hypercapnia however cannot be ruled out. It is also possible that during eupneic breathing the hypercapnic stimulus must be greater than 5 % in breathing status in order to elicit a spleen contraction. There are some contrasting results from an earlier study that suggested that hyperventilation

52 might trigger spleen contraction (Stäubli et al 1988), which might logically implicate any rapid change in chemosensor input in itself may act as a trigger. This potential contributor should be further investigated.

Spleen contraction during exercise and altitude

At this point we have concluded that both hypoxia and hypercapnia are important triggers for spleen contraction, and that the magnitudes of these stimuli differ between apnea on one hand and altitude simulation or true altitude on the other. We can therefore try to assess the question of whether spleen contraction is a graded response, thus dependent upon the strength of each independent stimulus, or the sum of the stimuli, or if it is an “all-or-nothing” response initiated after reaching a threshold stimulus. An example of an all or nothing response is the case with the involuntary breathing movements initiated at the physiological breaking point upon reaching a critical level of arterial CO2.

In study IV, the former hypothesis that would see the spleen adjusting the Hb increase with the level of physical activity at high altitude, was indeed the case. Exercise will increase O2 consumption further in an already hypoxic situation (Schagatay 2009a, 2009b, 2011) and may result in spleen contraction even without environmentally induced hypoxia (Sandler et al 1984; Froelich et al 1988; Flamm et al 1990; Laub et al 1993; Wolski 1998). Stewart and colleauges (2003) found no difference in spleen volume following 5, 10 and 15 min of exercise in normoxia with a constant intensity of 60 % , but observed a 56 % reduction of spleen volume in total following maximal exercise. They concluded that the spleen contraction is an active response and contracts in an intensity- dependent manner in relation to heavier exercise.

Study IV is the first to our knowledge to demonstrate this intensity-dependent contraction as a graded response to hypoxia as well. As earlier mentioned, Wolski concluded that normobaric hypoxic breathing had no effect on spleen contraction, and that exercise was the only initiating factor. Possibly, the observed spleen contraction in their study may represent an additive response, with a smaller contractile contribution from the hypoxia “concealed” by the stronger stimulus of exercise on spleen contraction. This may be reflected in study IV, whereas spleen volume first decreased by 22 % during rest at simulated altitude. The minor increase in Hb (+1.4 %; NS) detected may have been due to lack of timing and time lag from spleen contraction to measurements of Hb (Thornton et al 2001). After performing 10 min submaximal exercise the

53 spleen volume had decreased by 33 % from baseline, with a concomitant increase of 7.2 % in Hb. This may be considered as a quite high elevation of circulating Hb, comparable to the upper range of what is typically seen after maximal apneas (Schagatay et al 2001; Schagatay et al 2007a), and since we did not measure plasma volume changes such an influence cannot completely be ruled out. Previous work noted a stable plasma volume reduction of 76 mL after 5 min exercise at 50 % of (Pivarnik et al 1986). However, in study IV the increased Hb concentration was restored to baseline values within 20 min following exercise and so was spleen volume, suggesting that plasma volume reduction could only have had a minor influence on the elevation. Furthermore, the Hb increase found in study IV occurred in close relation to changes in spleen volume, and both also recovered within 10 min after cessation of exercise and resumed normoxic breathing (Figure 14).

Early changes in hemoglobin

Increased ventilation due to hypoxia may also reduce plasma volume, a compensatory effect for respiratory alkalosis by increased renal excretion of bicarbonate (Böning et al 2008). This effect is generally not evident until 24 - 48 h of hypoxic exposure, however (Bärtsch and Saltin 2008) and can therefore be ruled out in studies I, II and IV. Exercise in hypoxia has also been associated with rapid changes in hemodynamics, but this data has been presented with lack of early measurements to trace changes in plasma volume and Hb, and with no consideration of a possible spleen contraction (Grover et al 1986). An early increase in Hb/RCV has been reported during acclimatization to altitudes greater than 4000 m (Reynafarje et al 1959; Pugh 1964; Ryan et al 2014) before the expected increase in erythropoesis is evident after several weeks. This lack of early measurements and consideration of spleen contributions makes previous works’ conclusions regarding the role of plasma volume reduction in increased measured Hb questionable. Ryan and colleagues (2014) recently presented the first full study with data on early alternations of Hb mass in healthy humans with focus on ascent and descent from an altitude above 5000 m. In that study, changes in Hb, plasma volume and Hb mass were not observed until after seven days (although only measured once before), when total Hb mass had increased by 5 % and Hb by 11 %, while plasma volume had decreased by 11 %. This indicates there must have been a contribution of red blood cells from elsewhere. After 16 days, Hb mass had increased further by 4 % while Hb and plasma volume was increased and decreased by 4 %, respectively. In the light of the studies of this thesis, those results indicate that the spleen may be involved

54 in the early phase of altitude acclimatization, before erythropoiesis has occurred. In support of the conclusion from study IV and in line with the study of Ryan et al (2014), Richardson (2008) showed that in three subjects ascending to over 5000 m spleen contraction is most likely progressive with increasing altitude. This was demonstrated by diminishing Hb increases following apnea at progressively higher altitudes, with increasing Hb elevations following apneas upon descent. In the same line, the spleens of lowlanders were recently studied in respons to exercise at different altitudes (1370 to 4200 m) during a 2-week ascent (Lodin-Sundström et al 2014). It was found that resting spleen volume was decreasing and baseline Hb increased at higher altitudes. After exercise spleen volume reductions were proportionally similar at all altitudes, albeit post exercise volumes were progressively smaller with increasing altitude. This is strengthening the suggestion of the spleen serving as a circulatory buffer involved in the acclimatization to altitude, as suggested from the results in this thesis.

The results from the studies mentioned above also support the findings of spleen contraction in study IV, lending further strength to the argument that the spleen responds in a graded manner to the current demands and intensity of the physiological stress. This may also imply that the spleen serves to unload the circulatory system by reducing the blood viscosity during rest. The viscosity of the blood, as a non-Newtonian fluid, is dependent on the flow rate (Kamenewa 1990), and in animal models large spleens have been shown to decrease the impact of viscosity by reducing the red cell volume during periods of resting flow rates, which otherwise would place unnecessary loads on the heart (Fedde and Wood 1993; Elsner and Meiselman 1995). For humans, a similar role for the spleen would be important in situations involving exercise as well as high altitude polycythemia.

Climbing Mt Everest

Having emphasized the importance of hypoxia and hypercapnia in triggering spleen contraction and Hb elevation, and concluded that the combination of these independent stimuli enhances the response, the aspect of acclimatization deserves discussion. The long-duration field study of expedition participants summiting Mt Everest (study V; Figure 16) exposed the participants to a strong, long-term hypoxic stimulus combined with physical work on the mountain. No effects of this extreme exposure on resting spleen volume were seen after the return to lower altitude, however, short episodes of apnea and exercise following descent from the climb resulted in a stronger spleen contraction. This indicates

55 that acclimatization to altitude may enhance the contractile capacity of the spleen. The observation that long-term altitude exposure augments and prolongs spleen contraction in response to acute hypoxic challenges is a novel finding and adds a new dimension to altitude acclimatization. The unchanged baseline spleen volume is contrary to the observation by Sonmez et al (2007) who showed a decrease at three and six months of residence at altitude, as previously discussed. In our studies, the spleen contraction was stronger following exercise than apnea, although apnea also triggered contraction. However, due to lack of breath-holding experience in all participants but one, the apneic durations were too short to reveal the strong spleen contraction present in divers.

Figure 16. Part of the Expedition in 2011 on the summit of Mt Everest. Photo: Tashi Sherpa.

Baseline SaO2 during rest was similar before and after climbing Mt. Everest, suggesting that differences in pO2 may not explain the difference in spleen contraction. Baseline EtCO2 was lower after the climb, which likely reflected the expected shift in hypercapnic ventilatory response and in turn a reduced capnic input on the spleen. Chronic hypoxic exposure has been implicated

56 in increases of circulating catecholamines and therefore decreased spleen volume in relation to high altitude training (Berglund 1992), although further studies are needed of the respective effects of hypoxia and exercise on spleen contraction and the potential effects of an increased sympathetic output. Study IV demonstrates a gradual, stimulus-dependent decrease in spleen volume over the short-term, and this effect appears to be enhanced following a long-term hypoxic exposure as seen in study V. Thus the role of spleen contraction during climbing may previously have been overlooked, and it may serve an important and beneficial function in the early phase of altitude acclimatization. Study V certainly raises the possibility of enhanced contractility through long-term hypoxic exposure. Can one therefore actively train the spleen and thereby enhance its contractile potential?

Trainability of the spleen response

It has been shown that greater apneic proficiency among freedivers primarily depends on three factors: 1) a maximized total gas storage capacity (including the lungs, blood and tissues); 2) tolerance to , represented by low levels of cerebral hypoxia and hypercapnia (the latter through CO2 buffering capacity); and 3) the ability to reduce metabolic rate through improved work economy as well as through an enhanced diving response. Several of these factors have been shown to be trainable for better apneic performance (Schagatay 2009a; 2009b; 2011). Research is limited regarding whether this trainability is applicable to the spleen, but it has been shown that apneic divers have larger spleens than non- divers (Schagatay et al 2007b) with larger elevations of circulating Hb in comparison to endurance athletes and untrained subjects (Richardson et al 2005). The spleen has regenerative ability (Voet et al 1983), reflecting a capacity of adaptation. Furthermore, the splenic volume was positively correlated to diving performance in elite apneic divers during a study in 2006 freediving world championship (Schagatay et al 2012). It is not known if good divers have large spleens with strong contractions due to training induced changes or to genetic diversity and natural selection. Repetitive, periodically-occurring hypoxia may be a powerful enough stimulus to affect the spleen’s contractility in a similar manner as long-term hypoxic exposure. Engan et al (2013) studied the effects of performing 10 maximal effort apneas a day for two weeks in non-divers and found no difference in spleen volume or the contractility following the training period. This training protocol may have been two small a stimulus however, as elite freedivers may engage in up to 20 h a week of apnea-specific training.

57

Figure 17. Exercise at high altitude increases the oxygen deficiency, whereas the spleen may serve beneficial effects for climbers at high altitude in response to hypoxia. Credit: © Annelie Pompe, Photo: Eric Arnold.

Less is known about the spleen contraction from altitude than from apneic diving, but study V is the first to observe a likely training effect on the spleen contraction in any condition. It would be beneficial to further study the rate of initiation and reduction in spleen volume in aerobically and anaerobically trained athletes to learn more about possible differing characteristics such as rate of responsiveness and contractility, which is yet to be revealed. The climbers summiting Everest in study V were all well trained with high aerobic and anaerobic capacities. If physical training would affect spleen contraction in terms of speed and time of initiation, the timing and frequency of spleen size measurement becomes more critical as well; this may explain some of the results in the study by Wolski (1998),

58 where trained individuals displayed smaller resting spleen volume than untrained 2.5 min after the stimulus. On this same topic, study V showed the extremely fit climbers did not increase their resting spleen volume following the expedition, whereas in another study with moderately trained subjects the resting spleen volume increased by 40 % after daily altitude exposure of up to 5200 m (Rodriguez-Zamora et al 2015). However spleen contraction was stronger after altitude exposure during submaximal and maximal exercise tests, elevating Hb concentration more than before the altitude exposure. The increase in spleen contraction provides independent confirmation of study V, of a training effect on contractility from long-term hypoxic exposure in combination with exercise during a high climb (Figure 17).

May there be several mechanisms of spleen contraction?

Even though hypoxia appears to be an initiating trigger of spleen contraction, different situations may employ different triggers, however. Some evidence suggests that spleen contraction during short apneas may be initiated immediately after onset of apnea (Bakovic et al 2003; Palada et al 2007; Bakovic et al 2013). During short apneas with large lung volumes, the immediate increase in HR before a fall in MAP along with a small increase in total peripheral resistance, supports central sympathetic stimulation from the vasomotor center as a trigger. This would most likely be due to a withdrawal of inhibitory baroreceptor activity and reduced cardiac output resulting in a significant fall in MAP. This early spleen contraction has also been suggested to be related to unloading of baroreceptors (Bakovic et al 2003; Palada et al 2007), but if spleen contraction develops fully during these short apneas then chemoreceptor input would logically be ruled out. In such cases, neural input elicited by the unloading of baroreceptors may be involved in initiating the contraction. Low-dose epinephrine injection has also been studied as a stimulator of spleen contraction in which study the spleen contracted by 40 % (Bakovic et al 2013), following the same pattern as in short apneas (Palada et al 2007). Thus, adrenergic stimuli such as circulating or locally-released catecholamines may also be a viable trigger in short apneic situations.

59 A combined role of mechanisms

Chemosensory and adrenergic mechanisms may in fact both play a role, albeit on different time scales. For example, in a study not included in this thesis (Lodin-Sundström et al 2009) three 2 min submaximal apneas followed by one maximal effort apnea was performed by 22 elite freedivers, and spleen volume decreased by 21 % after two submaximal apneas and then leveled out. Following the maximal apnea, contraction increased again by 10 %. This suggests that the level of contraction develops according to the hypoxic challenge at hand. Another study of eight male elite freedivers at a freediving world championship demonstrated that in longer duration apneas (4 min 41(35) sec), the spleen was measured during the apnea and contracted by 21 % in the first 15 sec (similar to observations reported by Palada et al (2007)) then returned to baseline volume after 1 min (Lodin-Sundström and Schagatay 2009). When SaO2 began to decline spleen contraction resumed, in relation to the SaO2 reduction reaching a maximum contraction of 47 % from baseline at the termination of apnea (Figure 18). The spleen contraction during long apneas therefore appears to occur in a biphasic pattern (Lodin-Sundström and Schagatay 2009). This could represent both the neural adrenergic input initiating the contraction through unloading of baroreceptors before hypoxia develops, followed by chemoreceptor input that results from development of hypoxia and/or hypercapnia. Chemoreceptor driven massive contraction at apneic termination may serve as a second line of defense to hypoxia (Lodin-Sundström and Schagatay 2009).

Figure 18. Mean (SE) spleen volume changes during maximal apnea and recovery in elite freedivers. The spleen contracts in a biphasic pattern. From: Lodin-Sundström and Schagatay 2009.

60 It has also been shown that MSNA increases before resumption of breathing during post-hyperventilatory apneas, as a response to chemoreceptor stimulation of gradually accumulating pCO2 during the latter stages of the apnea (St Croix et al 1999). This supports the theory that a stronger spleen contraction at the end of the apnea may occur due to stimulation of chemoreceptors. This relates well to the results from studies II and III involving apneas. In study II the spleen contracted by 34 % accompanied by a reduction in SaO2 by 10 % following normoxic apnea. This reduction in spleen volume is greater than the rapid initiate contraction evident from previously mentioned studies where pO2 and pCO2 were unaffected. In study III, the largest reduction in spleen volume by 33 % was seen after three hypercapnic apneas accompanied by an EtCO2 of 7.6 %. It is known that MAP increases at the end of apnea (Schagatay and Andersson 1998), and it is possible that the spleen contraction in study II and III also occurred in relation to stimulated baroreceptors by activating the sympathetic system. MAP had increased during hypercapnic apneas by 35 % at apneic termination in study III. And so, while the studies in this thesis only provide indirect evidence of the mechanisms of spleen contraction, the results provide substantial building blocks on which to base theoretical explanations and launch further studies aimed at isolating specific triggers.

Summary of conclusions

I conclude from the works in this thesis that hypoxia is an important trigger for spleen contraction, but that hypercapnia also contributes during apneic situations. These studies confirm that repeated apneas trigger spleen contraction and elevate circulating Hb, which would provide a transient increase in gas storage capacity during activities that present a hypoxic challenge. It also originally identifies the prescence of spleen contraction during eupneic hypoxia such as altitude. The spleen releases erythrocytes into circulation upon contraction resulting in natural “blood boosting”, a response that is graded to the stimulus intensity and/or duration. This may facilitate early acclimatization to high altitude, by increasing the O2 carrying capacity of the blood, especially during exercise. It also helps prevent the high blood viscosity resulting from high altitude polycythemia between exercise episodes by spleen reuptake and storage of erythrocytes during rest. The ability of the spleen to contract during exercise apparently increases with acclimatization to altitude, which would be advantageous in hypoxic situations. Long-term hypoxic exposure may thus provide a method to enhance the spleen response with possible benefits for climbers, athletes and in certain disease conditions.

61 Applications and future dicections of research

By studying physiology in healthy subjects and athletes we focus on functions and training effects which are adaptations to maintaining homeostasis in natural environments or situations we have likely experienced in our evolution. To learn how the system works from a healthy perspective may however be essential to understand certain pathological conditions.

The spleen at high altitude and in apneic diving

The relation and possible combinations of mechanisms responsible for spleen contraction in various situations, e.g. hypoxia, need further investigation. It should be clarified in more detail from a short-term perspective how the initiation and early progression of the contraction occurs, but more studies are also needed with long-term perspective regarding chronic hypoxic exposure on the spleen and its contraction.

It would be interesting to follow the persisting sympathoexcitation lasting about double the time of exposure after removal of the stimulus as shown by Xie et al (2000; 2001) after 20 min eupneic hypoxia. This could be studied following hypoxic breathing or after long-term hypoxic exposure. The changes in spleen volume in relation to a decreased sympathetic nerve activity after a chronic hypoxic exposure may reveal underlying mechanisms from long term altitude exposure on the spleen contraction.

Another pursuit would be to further investigate the rate of spleen volume reduction in relation to the rate of changes in pO2 and pCO2. It is implied that the chemical change in itself may be of importance. This could be investigated in an eupneic state with various speed of changes in pO2 and possibly pCO2. In relation to this, it would be beneficial to know if the rate of initiation and the rate of contraction differs in trained versus untrained, in apneic divers, climbers and aerobically trained athletes.

The spleen in sports performance

An obvious direction for further studies is to investigate the potential ability to increase spleen size and its ability to contract by training. Results presented in this thesis make it intriguing to move further down this path. The study

62 performed by Engan et al (2013) with a training protocol of 10 maximal apneas per day for two weeks to study the effects on spleen contraction, was probably not intense and/or long enough to display an effect on spleen contraction. It would be fruitful to extend the training protocol to 15 maximal apneas per day for four to five weeks in order to see whether spleen contraction will be similarly affected, as found with higher altitude exposure.

The role of the spleen in sports performance could be further studied for possible beneficial effects in endurance sports for increased performance. Lately, there have been intense discussions about potential problems with doping in endurance sports (Ekblom et al 2001; Stray-Gundersen et al 2003), and there are varying ways and procedures to test athletes for the use of unethical methods for improved performance. Hb elevations of 10 % after exercise due to spleen contraction may however be natural and can wrongfully place an athlete in suspiscion of doping. To obtain the desired effects by training the spleen must also be a better option than artificially induced blood boosting, a possibility that should be further explored.

The spleen in hemathology

There are many important hematological applications from the spleen contraction, where estimations of plasma volume changes may be one of the most important. Frequently used equations for estimation of plasma volume (Van Beaumont 1972; Dill and Costill 1974) assume that circulating erythrocyte volume is consistent with total erythrocyte volume, and the potential contribution of the spleen as a reservoir is not considered. This is likely causing a substantial error when calculating plasma volume changes from Hb and Hct. Thus, circulating Hb - fine tuned by the spleen - may represent an error of all measurements and calculations based on plasma volume, i.e. aspects of health, research and sports applications.

Cross-country skiers with Hb above 160 and 170 g/L (females and males respectively) are prevented from competition start, whereas the potential influence of spleen contraction is not concidered. Thus, it has been suggested that several Hb tests may be collected during the whole season to eliminate the risk of a peak value in athletes with resting Hb values in the upper range of allowance (Ekblom et al 2001). From this aspect, spleen contraction and its regulation of circulating Hb needs to be taken into concideration and further studied.

63 The spleen in diseases involving hypoxia

Many common diseases involve hypoxic stress. Patients suffering from intermittent hypoxia, such as Obstructive Sleep Apnea Syndrome (OSAS) and chronic hypoxia, e.g. Chronic Obstructive Pulmonary Disease (COPD) may benefit from increased knowledge of our natural defense systems against hypoxia, which may result in better treatments in the future. This may involve increased knowledge of the triggers for this response and the possible training effects of spleen contraction.

COPD was in 1990 the sixth most common cause of death in the world and is expected to be the third most common cause by the year 2020 (Murray and Lopez 1997), yet the potential role of the spleen as a reservoir for increased O2 carrying capacity in these patients has never previously been considered in the literature. We tested spleen function in COPD patients during 6 min of walking, and found a blood boosting effect, which was related to the severity of the disease (Stenfors et al 2009). Generally, spleen volume is recovered to baseline values following 10 min after exercise or apnea, but in these patients it was still reduced at that point. The same prolonged effect in spleen volume recovery was seen after climbing Mt Everest, this possible relationship deserves further investigation. It was also evident from the results that the patients with the most severe hypoxia during rest also had the largest spleens. These responses are most likely protective against hypoxia and may suggest a long term adaptation to hypoxia (Stenfors et al 2009). This suggests that spleen contraction is important for protection in a pathological perspective. Whether these results can be accounted for by genetic predisposition, or training of the spleen´s contractile ability is not known and deserves further study.

Patients with severe OSAS have been found to have high Hct, Hb and EPO levels (Imagawa et al 2001; Choi et al 2006). With three days of treatment reducing the hypoxia, EPO levels were decreased by 38 % (Cahan et al 1995). It is not unlikely to imagine that hypoxia from OSAS may have an effect on spleen contraction together with an already known increased sympathetic activity (Spicuzza et al 2002). An important aspect is that these patients have frequently been exposed to repetitive apneas for a long time. Hence, we compared patients with OSAS to healthy controls to clarify if they have an enhanced Hb response due to spleen contraction or if the diving response was strengthened showing a fortified Hb increase in OSAS patients after face immersion apneas, most likely reflecting an enhanced spleen contraction, possibly due to a training effect from the apneas.

64 Increased physiological understanding from healthy people and athletes in high altitude and below the surface of the sea, may provide us with valuable insights useful to better treat these patient groups suffering from hypoxia. The spleen, this previously misunderstood organ thus has an important role in both health and disease, and it may have provided the generalists we are, with the abilities to explore extreme environments.

65 ACKNOWLEDGEMENTS

It is overwhelming to think about the journey I have made – and travelled - during the years of this PhD research project. It fills my heart with joy and gratefulness, and with the tingling excitement of possible further explorations. From all the places I have been my mind always return to a place that represents a hub in many ways, each time with the sense of repatriation, where many experimental studies were done, and others written. A second home over the years, the sense of Dahab which cannot be put into words - where the mountains meet the sea.

This thesis would not have come to be without the support of so many people I am indebted to.

I would like to express my sincere gratitude to my supervisor Erika Schagatay for your tireless support, enthusiasm and encouragement over these years in so many different ways. I am grateful for the opportunities you gave me and for the inspiration, your patient guidance in times of success and frustration, our stimulating and challenging conversations, and your sharp and constructive reviews of my work in progress. I feel privileged to have you as my supervisor and mentor. Thank you for taking me to places previously only imaginable in my dreams, and for always providing a home far and away.

I also wish to thank my co-supervisor Mats H. Linér for your good advice and guidance in revising this thesis, and for good company in Japan during my first visit to the Ama-divers.

I am also most grateful to Matt Richardson for time spent on revision and valuable feedback on this thesis, and for the warm introduction to research many years ago.

A special thanks to my fellow doctoral student and dear friend Harald Engan for invaluable companionship over the years, your unselfish support, encouragement and laughter. It would not have been the same without you.

I would also like to thank my colleagues Alex Patrician and Lara Rodriguez- Zamora for great company, support and assistance. Also a great thanks to all other co-authors in the different studies and inspiring colleagues not previously mentioned, to all you extraordinary people that have inspired me and been part of my journey.

66 My greatest appreciation also goes to the subjects who volunteered for my studies, none of this could have been done without you. Also a great thank you to competition and expedition organizers and coaches that helped me carry out scientific studies on extreme performances.

Finally, I would like to extend my gratitude to my family, to my children who showed me the highest peak in the world, making everything else worthwhile, and to Markus for your support and understanding. To my sister Elenia Olslind Lodin and Johanna Bergman Lodin for encouragement in so many ways, and to my grandmother Ingeborg Lodin always providing invaluable insights. Christina and (in memory of) Leif Sundström, thank you for everything, I wish both of you could have been here. Last but not least, thank you mum, Anita Mattsson Lodin, for your unconditional love, continuous unselfish support and assistance. Thank you for always believing in me.

67 REFERENCES

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Thank You for jumping in with me, I hope You had a nice dive!

“Happiness is not found in the things you possess, but in what you have the power to release”

Dahab, Egypt. Photo: Angelica Lodin-Sundström Back cover photograph: Sherpa woman, Beding, Nepal. Photo: Angelica Lodin-Sundström

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