Download THE VENOUS CIRCULATION IN TELEOST FISH

THE VENOUS CIRCULATION IN
TELEOST FISH
RESPONSES TO EXERCISE,
TEMPERATURE AND HYPOXIA
AKADEMISK AVHANDLING
för filosofie doktorsexamen i zoofysiologi som enligt naturvetenskapliga
fakultetens beslut kommer att försvaras offentligt fredagen den 27 april 2007,
kl. 10.00 i föreläsningssalen, Zoologiska institutionen, Medicinaregatan 18,
Göteborg
ERIK SANDBLOM
Department of
Zoology/Zoophysiology
2007
Published by the Department of Zoology/Zoophysiology
Göteborg University, Sweden
Published papers are used with permission from the publisher:
I. The Company of Biologists
II. and IV. Elsevier
III. and V. The American Physiological Society
Printed by Vasastadens Bokbinderi AB, Göteborg 2007
© Erik Sandblom 2007
ISBN 978-91-628-7131-4
DISSERTATION ABSTRACT
Sandblom, Erik (2007). The venous circulation in teleost fish -Responses to exercise,
temperature and hypoxia. Department of Zoology/Zoophysiology, Göteborg University,
Box 463, SE-405 30 Göteborg, Sweden.
In fish and other vertebrates, venous capacitance changes have important implications on
venous return and cardiac filling pressure. The main objective of this thesis was to gather
information on venous haemodynamic responses and neurohumoral control mechanisms in
two teleost species; the sea bass, Dicentrarchus labrax, and the rainbow trout, Oncorhynchus
mykiss. As previous studies of venous function in fish have primarily focused on the
pharmacology of the venous vasculature, special attention was paid to venous responses
elicited by exercise, acute temperature changes and environmental hypoxia, which represent
natural cardiovascular challenges in aquatic environments.
Methods: Cardiac output (Q), central venous (Pven) and dorsal aortic (Pda) blood
pressures were recorded in vivo. The mean circulatory filling pressure (MCFP), a measure of
vascular capacitance, was measured as the venous plateau pressure during ventral aortic
occlusion. In one study, vascular capacitance curves were also constructed by measuring
MCFP at different blood volumes (between 80-120% of the assumed blood volume), to
investigate changes in vascular compliance (C) and unstressed blood volume (USBV) during
normoxia and hypoxia. In another study, blood volume was measured using dilution of 51Crlabelled red blood cells. Drugs were administered systemically to elucidate the role of
adrenergic control systems and the renin-angiotensin system (RAS) in the observed
cardiovascular responses.
Results and conclusions: Exercise, in both sea bass and rainbow trout, results in
increased Q and increased MCFP. Although Pven increases during exercise in both species,
cardiac stroke volume (SV) only increases in rainbow trout, whereas increased heart rate (fH) is
exclusively responsible for the increased blood flow in sea bass. When ambient temperature
was raised acutely from 10 to 13 and 16°C, rainbow trout respond with a significantly elevated
Q which, in contrast to the exercise response, is exclusively mediated by tachycardia with an
unchanged Pven and SV. Similarly, however, MCFP increases which indicates an actively
reduced vascular capacitance, especially since the blood volume does not change between 10
and 16°C. In both species, blockade of α-adrenoceptors delays the increase in Pven during
exercise, and in rainbow trout, additional blockade of angiotensin converting enzyme
abolishes all venous exercise responses. Environmental hypoxia typically elicits bradycardia
that is associated with reduced vascular capacitance and an increased Pven. Q is unchanged or
increased during hypoxia due to an increased SV. The capacitance responses during hypoxia
are mainly due to changes in USBV that are mediated by both nervous and humoral αadrenergic mechanisms.
In summary, it is shown that vascular capacitance decreases during exercise, acute
temperature increase and hypoxia. This mobilizes blood to the central venous compartment
which, depending on the heart rate response, results in maintained or increased Pven, SV and
Q. It is also suggested that the decrease in capacitance during exercise and acute temperature
increase prevents blood from passively pooling in the venous periphery as blood flow
increases. RAS is activated during exercise after α-blockade to increase Pven and MCFP. Thus,
RAS affects venous capacitance in fish and not only arterial tone as previously suggested.
Keywords: blood volume, catecholamines, central venous pressure, exercise, hypoxia, mean
circulatory filling pressure, preload, renin-angiotensin, temperature, vascular capacitance.
LIST OF PAPERS
This thesis is based on the following papers which are referred to in the text
by their Roman numbers:
I.
Sandblom E., Farrell A. P., Altimiras J., Axelsson M. and
Claireaux G. (2005). Cardiac preload and venous return in swimming
sea bass (Dicentrarchus labrax). J Exp Biol 208, 1927-1935.
II.
Sandblom E., Axelsson M. and McKenzie D. J. (2006) Venous
responses during exercise in rainbow trout, Oncorhynchus mykiss: αadrenergic control and the antihypotensive function of the reninangiotensin system. Comp Biochem Physiol 144, 401-409.
III.
Sandblom E. and Axelsson M. (2007) Venous hemodynamic
responses to acute temperature increase in the rainbow trout
(Oncorhynchus mykiss). Am J Physiol, (In Press).
IV.
Sandblom E. and Axelsson M. (2005) Effects of hypoxia on the
venous circulation in rainbow trout (Oncorhynchus mykiss). Comp Biochem
Physiol 140, 233-239.
V.
Sandblom E. and Axelsson M. (2006) Adrenergic control of venous
capacitance during moderate hypoxia in the rainbow trout
(Oncorhynchus mykiss): role of neural and circulating catecholamines. Am
J Physiol 291, 711-718.
TABLE OF CONTENTS
ABBREVIATIONS ..................................................................................2
1. INTRODUCTION ...............................................................................4
1.1 GENERAL OVERVIEW OF FISH CIRCULATORY SYSTEMS ........5
1.1.1 Adrenergic and cholinergic control of the heart ................................................6
1.1.2 Adrenergic and cholinergic control of the vasculature .......................................7
1.1.3 The renin-angiotensin system..........................................................................8
1.2 THE VENOUS CIRCULATION.................................................................9
1.2.1 Gravitational forces in an aquatic environment...............................................9
1.2.2 Central venous pressure and the Frank-Starling mechanism .........................10
1.2.3 Mean circulatory filling pressure and venous capacitance................................13
1.2.4 Coupling of cardiac output and venous return ...............................................16
1.2.5 Passive and active responses .........................................................................17
1.2.6 Control of the venous circulation...................................................................18
1.3 INTEGRATED CARDIOVASCULAR RESPONSES ..........................21
1.3.1 Exercise......................................................................................................21
1.3.2 Temperature................................................................................................23
1.3.3 Hypoxia .....................................................................................................24
2. AIM ..................................................................................................... 28
3. RESULTS AND DISCUSSION ......................................................... 30
3.1 METHODOLOGICAL CONSIDERATIONS.......................................30
3.1.1 Mean circulatory filling pressure...................................................................30
3.1.2 Stressed blood volume and vascular compliance .............................................35
3.1.3 Blood volume...............................................................................................37
3.1.4 Future perspectives.......................................................................................37
3.2 INTEGRATED CARDIOVASCULAR RESPONSES ..........................38
3.2.1 Exercise......................................................................................................38
3.2.2 Temperature................................................................................................41
3.2.3 Hypoxia .....................................................................................................43
4. CONCLUSIONS ................................................................................ 46
5. ACKNOWLEDGEMENTS ............................................................... 48
6. REFERENCES .................................................................................. 50
1
ABBREVIATIONS
ACE
Ang I
Ang II
APP
BL s-1
C
CaO2
CvO2
fH
Mb
MCFP
MO2
Pda or PDA
PO2
Pven or PCV
ΔPven or ΔPV
Q
RAS
Rsys
Rven or Rv
SBV
SV or Vs
VPP
VR
Ucrit
USBV
angiotensin converting enzyme
angiotensin I
angiotensin II
arterial plateau pressure
body lengths per second
vascular compliance
arterial oxygen content
mixed venous oxygen content
heart rate
body mass
mean circulatory filling pressure
oxygen consumption
dorsal aortic blood pressure
oxygen partial pressure
central venous blood pressure
venous pressure difference
cardiac output
renin-angiotensin system
systemic vascular resistance
venous vascular resistance
stressed blood volume
cardiac stroke volume
venous plateau pressure
venous return
critical swimming speed
unstressed blood volume
2
3
1. INTRODUCTION
The cardiovascular system is one of the most central components in the
physiological machinery which maintains homeostasis of the animal body. The
role of the cardiovascular system is diverse and ranges from being a carrier of
information in the form of circulating hormones, to being involved in the
regulation of temperature and hydromineral balance. However, its most
fundamental role is possibly to supply O2 and nutrients and to remove CO2
and metabolic waste products produced by cellular metabolic processes
throughout the body. The main driving force to circulate the blood is
provided by the arterial blood pressure which is generated by the beating
heart, while local blood flow is regulated by changes in arteriolar resistance. In
most vertebrates, the rate and force of cardiac contraction is directly
controlled by the autonomic nervous system and various humoral control
systems. However, secondary factors like arterial and venous blood pressure
also affect cardiac performance. The venous circulation, which contains a large
portion of the total blood volume, and is responsible for carrying oxygendepleted blood back to the heart, is particularly interesting in this regard. An
early conclusion made by the Danish Nobel prize-winning zoophysiologist
August Krogh may serve to emphasize the importance of the venous
vasculature for cardiac performance:
“The heart cannot do more than send out what it gets”
(Krogh, 1912)
In the large and diverse vertebrate group represented by the fishes, the
importance and function of the cardiovascular system for the exchange of
gases, nutrients and metabolites does not represent any major exceptions from
the general picture outlined above. However, as fish are ectothermic, breathe
water and spend their entire life in water which has a density (very) similar to
their own body fluids, the aquatic lifestyle presents a number of cardiovascular
challenges which differ considerably from those experienced by terrestrial
animals. Thus, before land was colonized by vertebrate life, the selection
pressures behind the evolution of the vertebrate cardiovascular system were
those of an aquatic environment. Understanding the form and function of the
cardiovascular system of our evolutionary predecessors, including the fishes, is
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therefore ultimately of fundamental importance in order to fully understand
and appreciate the form and function of the human cardiovascular system.
In general, this thesis focuses on the cardiovascular responses of teleost
fish to exercise, acute temperature changes and environmental hypoxia, which
all represent naturally occurring events in their aquatic environment. More
specifically, the neurohumoral control of the venous circulation during these
cardiovascular challenges is the central theme.
1.1 GENERAL OVERVIEW OF FISH CIRCULATORY SYSTEMS
The typical teleost circulation is comprised of a single heart connected in
series with the gills. The heart is a four-chambered structure with the sinus
venosus, atrium, ventricle and bulbus arteriosus serially connected and
enclosed in a more or less rigid pericardial cavity (Farrell, 1991; Farrell and
Jones, 1992). Venous blood returns from the periphery and enters the sinus
venosus, via the paired ducts of Cuvier before entering the atrium. The
ventricle, which is the main pressure generating component of the heart, is
filled by atrial contraction, but also by direct inflow of blood from the central
veins during diastole (Lai et al., 1998). Both the atrio-ventricular and sino-atrial
junctions are guarded by valves, whereas the connections between the sinus
venosus and ducts of Cuvier are not (Farrell and Jones, 1992). The ventricle
pumps blood via the highly compliant bulbus arteriosus into the ventral aorta,
which splits into four pairs of afferent branchial arteries that perfuse the gills
where gas exchange takes place. There are two major pathways in the gills. In
the arterio-venous pathway, blood flows directly over to the central venous
compartment and supplies the gill tissues with oxygen and nutrients, whereas
oxygenated blood in the arterio-arterial pathway leaves the gills via four pairs
of efferent branchial arteries (Nilsson and Sundin, 1998). The main portion of
this blood enters the dorsal aorta, but some is also directed to the cephalic
region via the carotid arteries. In unfed fish, 30-40 percent of cardiac output
(Q) is typically diverted to the gut circulation (stomach, intestine and liver) via
the coeliaco-mesenteric artery which in many species branches directly from
the dorsal aorta (Farrell et al., 2001; Thorarensen et al., 1991). After having
passed through the capillary beds, blood is returned to the heart via the
venous circulation. Blood from the stomach and intestine is collected by the
hepatic portal vein carrying blood to the liver, which in turn is drained by the
hepatic veins directly into the sinus venosus. The hepatic veins are typically
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short and differ in number among species. It has been suggested that active
control of sphincters in these veins is a mechanism by which blood can be
rapidly mobilized from the splanchnic circulation to the central venous
circulation (Johansen and Hanson, 1967). Paired posterior and anterior
cardinal veins drain the caudal and cranial portions of the body, respectively.
They fuse with the ducts of Cuvier dorsal to the heart. Dorsal, lateral and
ventral cutaneous veins primarily drain the skin, but also the buccal and
opercular cavities (Satchell, 1991; Satchell, 1992). Valves are present in fish
veins, but only at the junction of tributary vessels (ostial valves), and not along
the length of the vessels (parietal valves) as in mammals (Fig. 1). A more
detailed description on the control and function of the venous circulation is
given in section 1.2.
i.
ii.
Figure 1. Schematic illustration of the location of valves in a venous segment. Mammals have
both ostial (i.) and parietal (ii.) valves, whereas only the former seem to be present in fish
veins. Arrows indicate direction of blood flow. After Satchell, 1991.
1.1.1 Adrenergic and cholinergic control of the heart
The teleost heart receives a dual autonomic innervation from exitatory
adrenergic fibers, as well as inhibitory cholinergic fibers (Nilsson, 1983, 1997;
Taylor, 1985; Taylor et al., 1999). One notable exception to this pattern among
the teleosts is the Pleuronectids, which lack adrenergic innervation (Donald and
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Campbell, 1982; Santer, 1972). The cholinergic cardiac fibres are carried in
cranial nerve X, the vagus, and travels along the ducts of Cuvier to the sinus
venosus, where the pacemaker tissue is believed to be located. In many
species, the atrium, but not the ventricle, also receives cholinergic innervation.
The inhibitory action of these fibres is due to release of acetylcholine which
binds to muscarinic receptors associated with the pacemaker tissue.
Adrenergic fibres have been found in all parts of the teleost heart and reach
the heart either via the vagus (“vago-sympathetic trunk”) and/or along
coronary arteries and the anterior spinal nerves (Holmgren, 1977; Nilsson,
1983). In contrast to mammals for example, where noradrenaline is the
dominating neuronal catecholamine, both adrenaline and noradrenaline are
released from adrenergic neurons in teleosts, with the relative importance of
the respective transmitter differing among species. Catecholamines bind to βadrenoceptors associated with the myocardium and pacemaker tissue, and
exert both an inotropic as well as a chronotropic stimulatory effect on the
heart (Nilsson, 1983). There is also the possibility that cardiac β-adrenoceptors
may be stimulated by catecholamines released into the circulation from
chromaffin cells located in the walls of the cardinal veins in the head kidney
(Holmgren, 1977; Reid et al., 1998). In teleosts, plasma catecholamine levels
increase during stressful stimuli such as hypoxia and severe exercise (Butler et
al., 1989; Perry and Bernier, 1999; Perry et al., 1991; Perry and Reid, 1992;
Randall and Perry, 1992; Reid et al., 1998; Wendelaar Bonga, 1997). The
teleost heart is clearly under the tonic influence of both adrenergic and
cholinergic control systems. During routine conditions, treatment with βadrenoceptor antagonists reduces heart rate, whereas muscarinic blockade
with atropine or bilateral vagotomy, increases heart rate and strongly
attenuates beat to beat variation (Altimiras et al., 1997; Axelsson et al., 1987;
Campbell et al., 2004; Priede, 1974).
1.1.2 Adrenergic and cholinergic control of the vasculature
The systemic vasculature is well innervated by adrenergic nerves in teleosts,
and both vasoconstrictory α-adrenoceptors as well as β-adrenoceptors causing
vasodilation, are present (Nilsson, 1983, 1994). However, the α-adrenergic
response dominates and adrenaline injection typically results in increased
vascular resistance and arterial blood pressure (Axelsson and Farrell, 1993;
Stevens et al., 1972; Wood and Shelton, 1980a). Experiments with the
adrenergic nerve blocking agent bretylium in Atlantic cod (Gadus morhua) and
rainbow trout (Oncorhynchus mykiss) show that the systemic adrenergic tonus at
7
rest, as well as during moderate exercise and hypoxia, is primarily mediated by
neural mechanisms and not by circulating catecholamines (Axelsson and
Fritsche, 1991; Axelsson and Nilsson, 1986; Fritsche and Nilsson, 1990;
Smith, 1978; Smith et al., 1985). It is possible, however, that in response to
severely stressful stimuli plasma catecholamine levels may reach levels high
enough to increase systemic vascular resistance and arterial blood pressure
(Bernier and Perry, 1999; Perry and Bernier, 1999; Randall and Perry, 1992). In
addition, circulating catecholamines have been suggested to be an important
source for neuronal uptake, which may be a prerequisite for sustained
neuronally mediated vasoconstriction (Xu and Olson, 1993).
The control of the branchial vasculature is complex and involves neural
and circulating catecholamines, as well as cholinergic nerves (Nilsson and
Sundin, 1998; Sundin and Nilsson, 2002). A relatively sparse adrenergic
innervation projects to the arterio-venous pathways where α-adrenoceptors
dominate, but also to the afferent filamental artery and the sphincter region of
the efferent filamental artery where β-adrenoceptors dominate. It seems that
circulating catecholamines may be of greater importance for the control of the
gill circulation than the systemic circulation (Nilsson and Sundin, 1998; Sundin
and Nilsson, 2002). Injected catecholamines (adrenaline) typically reduce
overall branchial resistance (Nilsson, 1983). Cholinergic efferents constrict the
efferent filamental artery sphincter by stimulation of muscarinic receptors
(Nilsson and Sundin, 1998; Sundin and Nilsson, 2002).
1.1.3 The renin-angiotensin system
The renin-angiotensin system (RAS) is often regarded as an endocrine “antidrop” factor for arterial blood pressure in fish (Olson et al., 1994; Olson,
1992; Platzack, 1995; Platzack et al., 1993; Russell et al., 2001; Zhang et al.,
1995). During hypotension and/or hypovolemia, the proteolytic enzyme renin
is released into the circulation, mainly from juxtaglomerular cells in the kidney
(Nishimura, 1978; Nishimura et al., 1979; Olson, 1992). Renin converts the α2globulin angiotensinogen to the decapeptide angiotensin I (Ang I), which is
further hydrolyzed to the octapeptide angiotensin II (Ang II) by angiotensin
converting enzyme (ACE) (Olson, 1992). The gills are believed to be the
primary site for ACE activity in teleosts (Olson, 1998; Olson et al., 1989). Ang
II is the biologically active molecule in the RAS and has intrinsic properties,
but also exerts some of its vasoactive properties through activation of
adrenergic control systems (Bernier et al., 1999; Bernier and Perry, 1999;
Carroll and Opdyke, 1982; Oudit and Butler, 1995). At present, the general
8
consensus is that the primary vasoactive site for RAS in fish is the systemic
resistance vasculature (Olson et al., 1994; Olson, 1992; Russell et al., 2001;
Zhang et al., 1995).
1.2 THE VENOUS CIRCULATION
Our knowledge regarding the control and function of the venous circulation in
fish, and all non-mammalian animals for that matter, is still fragmentary. The
following account is an attempt to summarize the literature on venous
haemodynamics and its control in fish. In many places, specific information
for fish is still missing, and here, references to the more abundant mammalian
literature are made. It should be kept in mind, however, that extrapolations
from studies on mammals to other animals need to be done cautiously.
Despite the general paucity of data for fish in many places, it is my hope that
the following sections will serve as a useful background for future, more
precise, studies on the venous circulation in fish.
1.2.1 Gravitational forces in an aquatic environment
The cardiovascular systems of land-living animals are constantly challenged by
gravitational forces. This implies that blood tends to pool in the lower parts of
the body (i.e. below the heart), and the taller the animal, the greater is the
gravitational impact. In mammals, a number of homeostatic mechanisms have
evolved to prevent orthostatic blood pooling. Compression of veins by the
surrounding skeletal muscles (“the muscle pump”), in combination with active
and passive changes in venous capacitance, are the most important
mechanisms by which this is achieved (Pang, 2001). These mechanisms serve
to prevent formation of oedema and ensure that blood is returned to the
heart.
In water-living animals such as fish, the gravitational impact on the
cardiovascular system is small due to the fact that blood has a density similar
to water and the hydrostatic water pressure counteracts the gravitational forces
acting on the blood in the circulatory system (Fig. 2). Thus, orthostatic blood
pooling is unlikely to be a major concern for fish (Satchell, 1991, 1992).
9
Nevertheless, some teleosts have been found to tolerate gravitational stress
surprisingly well when exposed to gravitational forces in air (Ogilvy and
DuBois, 1982; Ogilvy et al., 1989). The differences between air and water in
terms of gravitational impact is probably also an important selection pressure
behind the evolution of both ostial and parietal valves in mammalian veins,
whereas only the former are present in fish (Fig. 1).
air
water
4
4
8
12
4
8 8
12 12
Figure 2. The effect of gravity on a
theoretical cardiovascular system represented
by a blood-filled compliant tube in air and
water. In air, pressure increases with the
height of the blood column and fluid tends
to pool and distend the lower portion of the
column. In water, the potential increase in
pressure with increasing height of the blood
column is counteracted by an increased
hydrostatic water pressure with increasing
depth. Modified from Satchell (1991).
However, despite the small gravitational impact upon the cardiovascular
system in water, it has become increasingly clear that active control of the
venous circulation is still highly important in fish. This will be addressed in the
following sections.
1.2.2 Central venous pressure and the Frank-Starling mechanism
Venous blood pressure in fish is low with the extremes being found in
elasmobranchs which display strongly sub-ambient pressures in the great veins
proximal to the heart (Table 1). Central venous pressure (Pven) is the ultimate
determinant of the ventricular end-diastolic volume (cardiac preload), although
filling time, and to a lesser degree, myocardial compliance and atrio-ventricular
valvular resistance will affect cardiac filling as well (Olson and Farrell, 2006).
Cardiac preload affects cardiac performance via the Frank-Starling
mechanism which implies that stroke volume and myocardial force of
contraction increases with increasing myocardial stretch (Olson and Farrell,
2006). In fact, cardiac filling pressure is likely even more important for
10
determining SV in fish than in mammals, because the ejection fraction for fish
hearts is high (80-100 %). This leaves little scope for increasing SV by
reducing the end-systolic volume (Coucelo et al., 2000; Farrell and Jones,
1992; Forster and Farrell, 1994; Franklin and Davie, 1992; Lai et al., 1990).
However, this conclusion is so far based on experiments on relatively few
species and it is possible that exceptions from this pattern may emerge. Both
atrial and ventricular muscle in fish responds in accordance with the FrankStarling mechanism (Farrell and Jones, 1992).
Table 1. Summary of literature values for routine venous pressures in fish
Species
Cyclostomes
Lampetra tridentata
Myxine glutinosa
Eptatretus cirrhatus
Elasmobranchs
Scyliorhinus canicula
Squalus acanthias
Triakis semifasciata
Heterodontus portusjacksoni
Squalus acanthias
Cephaloscyllium isabella
Dipnoans
Protopterus aethiopicus
Teleosts
Anguilla anguilla
Dicentrarchus labrax
Oncorhynchus mykiss
Synbranchus marmoratus
Anguilla japonica
Oncorhynchus mykiss
Pseudopleuronectes americanus
Platichtys stellatus
Oncorhynchus mykiss
SV/DC
PCS/VC*
PCV
CV
HPV/SIV
PV*
-0.4 - -0.1
0.10
0.01 - 0.07
-0.45
-0.08 - -0.07
-0.15
0.02 - 0.16
0.04
0.21
0.1 - 0.4
0.21
~0
-0.53 - 0.66
0.11
~0.40
0.27
0.47
0.40 - 0.47
~0.20
0.40
-0.06
~0.02
0.01
0.06 - 0.07
0.06
~0.25 - 0.47
0.13
0.12
0.19
0.74
0.40 - 0.53
0.40
0.28
Johansen et al., 1973
Johnsson et al., 1996
Foster and Forster., 2007
Johnsson et al., 1996
Short et al., 1977
Sandblom et al., 2006
Johansen and Hanson., 1967
Lai et al., 1990
Birch et al., 1969
Capra and Satchell., 1977
Satchell and Weber.., 1987
-0.10 - 0.00
0.20 - 0.26
-0.28
Source
~0.25 - 0.5
Johansen et al., 1968
1.06 - 1.20
Mott., 1951
Paper I
Zhang et al., 1995
Conklin et al., 1997
Olson et al., 1997
Zhang et al., 1998
Perry et al., 1999
Hoagland et al., 2000
Altimiras and Axelsson., 2004
Paper II
Paper III
Paper IV
Paper V
Skals et al., 2006
Chan and Chow., 1976
Kiceniuk and Jones., 1977
Wood and Shelton., 1980
Cech et al., 1976
Cech et al., 1977
Wood et al., 1979
Stevens and Randall., 1967
Values are obtained from both anaesthetized and unanaesthetized animals. SV (sinus
venosus); DC (duct of Cuvier); PCS (posterior cardinal sinus); VC (vena cava); PCV
(posterior cardinal vein); CV (caudal vein); HPV (hepatic portal vein); SIV (supraintestinal
vein) and PV (pulmonary vein) * only in Protopterus.
11
Thus, an increased filling pressure may therefore have substantial effects on Q
by increasing atrial stroke volume and contraction force. As a large portion of
the ventricular filling is mediated by atrial contraction in fish, this will in turn
significantly affect filling and performance of the ventricle (Farrell, 1984, 1991;
Farrell and Jones, 1992). Atrial contraction is not the only mechanism by
which the ventricle fills. Similar to the situation in mammals, direct inflow to
the ventricle from the central veins occurs during diastole, but likely to a much
lesser extent than in mammals (Lai et al., 1998; Olson and Farrell, 2006). The
pressure in the central veins will therefore to some extent directly influence
ventricular filling and performance.
The cardiac responses to filling pressure can conveniently be studied using
in situ perfused heart preparations. This has been done on several groups of
fish including teleosts (Blank et al., 2002, 2004; Davie et al., 1992; Farrell et al.,
1982, 1983, 1989; Icardo et al., 2005; Stuart et al., 1983), elasmobranchs (Davie
and Farrell, 1991; Franklin and Davie, 1993) and hagfish (Forster, 1989;
Forster et al., 1991; Johnsson et al., 1996). It seems that all hearts examined
respond to increased filling pressure with an increased Q in accordance with
the Frank-Starling mechanism.
Cardiac filling patterns
The term vis a tergo is used to describe the pressure that fills the heart from
behind, while the opposite, vis a fronte, describes the cardiac suction force
created by the contracting heart inside a more or less rigid pericardial cavity.
The vis a fronte mechanism allows the heart to generate flow even at subambient filling pressures (Farrell, 1984, 1991; Farrell and Jones, 1992; Olson
and Farrell, 2006). In the intact animal, vis a tergo is equivalent to the central
venous pressure (filling pressure) and both cardiac as well as vascular factors
and blood volume determine this pressure. The cardiac effect on filling
pressure is primarily related to heart rate changes, such that Pven and SV are
inversely proportional to fH (Altimiras and Axelsson, 2004; Farrell et al., 1989;
Short et al., 1977; Taylor et al., 1977). In other words, when fH decreases,
blood tends to pool in the central veins and Pven and SV increases. In contrast,
if fH increases, the diastolic filling time is shortened and Pven and SV are
reduced. In rainbow trout, this mechanically coupled mechanism keeps Q
more or less constant if heart rate is pharmacologically manipulated over a
relatively broad range of heart rates (Altimiras and Axelsson, 2004). The
vascular factors dictating cardiac filling pressure are related to capacitance
changes of the venous vasculature as will be discussed in detail below.
A rigid pericardium is undoubtedly an important prerequisite for vis a fronte
filling (Farrell and Jones, 1992; Johansen, 1971). In elasmobranchs, where the
12
pericardium is particularly rigid, the vis a fronte mechanism is pronounced and
most likely explains the strongly sub-ambient central venous pressures
frequently observed in this group (Table 1). However, also teleosts, such as
the rainbow trout, can generate routine cardiac outputs at sub-ambient filling
pressures (Farrell et al., 1988) and Pven can be negative in rainbow trout in vivo
(Altimiras and Axelsson, 2004; Erik Sandblom, unpublished observation). In
situ perfused rainbow trout hearts can generate up to around 50% of
maximum Q at sub-ambient filling pressures but, positive filling pressures are
required to raise Q further (Farrell et al., 1988; Farrell and Jones, 1992). This
strongly indicates that the vis a fronte mechanism is important in teleosts as
well, and it has been suggested that a switch from vis a fronte to vis a tergo occurs
when the circulatory system is challenged, such as during exercise (Farrell and
Jones, 1992).
1.2.3 Mean circulatory filling pressure and venous capacitance
The vascular factors dictating venous return and cardiac filling pressure are
primarily determined by the capacitance of the venous vasculature. The mean
circulatory filling pressure (MCFP) is often used as an index of venous
capacitance (Pang, 2001; Rothe, 1993). MCFP is the pressure in the circulation
when blood flow is zero. It is dependent on vascular tone, vascular
compliance (C) and blood volume. MCFP is much lower than arterial
pressure, lower than capillary pressure and higher than central venous pressure
(Pang, 2001; Rothe, 1993). Given that blood volume is not altered, MCFP
measurements can be used to provide an estimate of active changes in the
venous capacitance vasculature. This has been done for mammals, fish and
reptiles (Conklin et al., 1997; Hoagland et al., 2000; Olson et al., 1997; Pang,
2001; Rothe, 1993; Sandblom and Axelsson, 2005; Sandblom et al., 2006 Skals
et al., 2005; Skals et al., 2006; Zhang et al., 1995; Zhang et al., 1998). In
mammals, it is generally assumed that MCFP is equivalent to the peripheral
venous pressure at the level of the small veins and venules and MCFP may
thus provide an estimate of the upstream driving pressure for venous return
(VR; Guyton, 1955; Guyton et al., 1955; Pang, 2001; Rothe, 1993). Hence, the
pressure difference that drives VR (ΔPven) is described by the equation:
(1)
ΔPven= MCFP-Pven
As VR equals Q at steady state, the resistance to venous return (Rven) is
calculated as:
13
(2)
Rven= (MCFP-Pven)/Q
By using the above equations and assuming that MCFP correctly estimates
venular blood pressure, Rven has been estimated to account for 2% of the total
systemic vascular resistance in rainbow trout (Zhang et al., 1995).
Compliance and stressed blood volume
From a change in MCFP it is not possible to directly distinguish if a response
results from changes in compliance and/or unstressed blood volume. This
information can be obtained by measuring MCFP at different blood volumes
and construct vascular capacitance curves (Fig. 3).
blood
volume (%)
routine
140
increased smooth
muscle tone
120
decreased
compliance
100
SBV
80
USBV
60
0
0.0
0.2
0.4
0.6
0.8
MCFP
(kPa)
Figure 3. Schematic illustration of vascular capacitance curves. The slope of the curves
represents vascular compliance (C) and the intercept of the y-axis at zero MCFP is the
unstressed blood volume (USBV) which does not create pressure by stretching the
vasculature. The remainder of the blood volume, which creates pressure, is the stressed blood
volume (SBV). An increased vascular tone produces a right-ward parallel displacement of the
curve and consequently a reduction in USBV. A decreased C rotates the curve clockwise
without changing USBV. Thus, both increased tone and decreased C can result in an identical
change in MCFP at 100% blood volume (vertical arrows), but through entirely different
mechanisms.
14
These curves describe the relationship between contained blood volume and
transmural pressure in the circulation. As vascular capacitance is the
relationship between pressure and contained volume, it cannot be described
by a single number, but rather in terms of vascular capacitance curves (Pang,
2001; Rothe, 1993). Two vascular factors determine vascular capacitance
namely: unstressed blood volume (USBV) and C. USBV is the blood volume
which is required to fill the residual vascular space up to the point where
pressure starts to increase. Thus, USBV can be thought of as being
haemodynamically inert. The remaining part of the blood volume, which
stretches the vasculature and creates pressure, is the stressed blood volume
(SBV). The USBV is illustrated on the vascular capacitance curve as the
extrapolated intercept of the y-axis, i.e. the blood volume at zero MCFP (Fig.
3). The slope of the vascular capacitance curve equals C. As compliance is a
measure of vascular elasticity it is described by the ratio of a change in
distending pressure (ΔP) to the resultant change in volume (ΔV) according to
the equation:
(3)
C= (ΔV)/ (ΔP)
Several studies have determined vascular capacitance in vivo for rainbow
trout by measuring MCFP at different blood volumes. Unstressed blood
volumes in the range of 13.3 to 26.0 ml kg body mass-1 (Mb-1) and compliances
of 12.8 to 25.5 ml kPa-1 kg Mb-1 have been obtained (Conklin et al., 1997;
Hoagland et al., 2000; Olson et al., 1997; Zhang et al., 1995, 1998).
Although the in vivo vascular capacitance curve is in fact a measure of the
entire circulatory capacitance, it is generally assumed to primarily reflect
venous capacitance (Pang, 2001; Rothe, 1993). In mammals, this assumption is
based on the fact that approximately 70% of the total blood volume is
contained within the venous circulation, with the major portion being
restricted to small veins and venules. Furthermore, the compliance of the
venous compartment is significantly much higher compared with the arterial
compliance (Greenway and Lautt, 1986; Hainsworth, 1986; Pang, 2001; Rothe,
1993).
15
1.2.4 Coupling of cardiac output and venous return
The relationship between cardiac output and venous return can be described
by cardiac and vascular function curves (Fig. 4). This concept was first
developed by Arthur C. Guyton and co-workers in the early 1950’s (Guyton,
1955; Guyton et al., 1954). The cardiac function curve is determined by the
cardiac filling pressure, such that Q increases with increasing Pven in
accordance with the Frank-Starling mechanism. Venous return depends on the
pressure difference between the venous periphery (MCFP) and the central
venous pressure at the level of the heart (Pven). In Figure 4, MCFP equals Pven
at zero flow and during steady state conditions, Q and VR are equal and
equilibrium at a specific Pven will be reached. During conditions when Q
increases, such as exercise, both the cardiac and the vascular curves are
presumably affected.
cardiac output / venous return
adrenergic stimulation
routine cardiac output
2.
3.
4.
1.
ad
re
ne
ro
rg
ut
ic
in
st
e
im
ve
ul
no
at
us
io
n
re
tu
rn
central venous pressure
=MCFP
Figure 4. Schematic illustration of cardiac and vascular function curves. Cardiac output
increases with increasing filling pressure (central venous pressure), whereas venous return
decreases. The intersection of the curves indicates where steady-state equilibrium between
cardiac output and venous return is established (1). General adrenergic stimulation of heart
rotates the cardiac function curve counter-clockwise as heart rate increases and the heart
becomes more sensitive to filling pressure. Adrenergic stimulation of the vasculature results in
a right-shift of the vascular function curve due to a decreased vascular capacitance. In this
example, a new equilibrium is attained at a higher flow and a lower vascular capacitance, but at
the same central venous pressure (2). Point (3) demonstrates a hypothetical equilibrium when
only the vasculature is stimulated and (4) demonstrates a hypothetical equilibrium when only
the heart is stimulated. The intercept of the vascular function curve at the x-axis (i.e. at zero
flow), is the mean circulatory filling pressure. Modified from Guyton (1963).
16
For example, adrenergic stimulation of the heart increases the sensitivity to
changes in filling pressure and rotates the curve counter-clockwise.
Furthermore, increased vascular adrenergic tone increases MCFP and the
pressure gradient for VR becomes steeper. This is reflected as a right shift of
the vascular function curve. Thus, from Figure 4 it is clear that an increase in
venous tone need not necessarily result in any significant increase in Pven if Q
increases concomitantly (i.e. point 2). This may seem paradoxical, but serves to
emphasize the complex interrelationship between the venous circulation and
the heart. It also illustrates that measurement of only Pven does not provide
enough information to draw detailed conclusions about venous responses in
vivo. Although Guyton’s curves nicely illustrate the complex interrelationship
between the heart and the peripheral venous circulation, they have been
criticised for presenting an over-simplified view of the circulatory system
where the possible effects of heart rate and afterload on cardiac output are not
fully taken into account (Rothe, 1993).
As previously mentioned, the effects of changes in cardiac filling pressure
and adrenergic stimulation on the cardiac function curve have been
investigated in detail in fish by using perfused heart preparations. The vascular
function curve is somewhat more technically difficult to study and this has not
been done for fish, although the individual factors that affect vascular
capacitance, such as USBV and C, have been studied in some detail as outlined
above. In both Guyton’s model and when the driving pressure for venous
return and venous resistance is calculated (equations (1) and (2)), MCFP is
assumed to equal the pressure in the small veins and venules. This assumption
may seem plausible for fish as well, but direct experimental evidence
supporting this is lacking (see also Results and Discussion).
1.2.5 Passive and active responses
If a change in contained blood volume of a vascular bed is mediated by
changes in vascular, and in particular venous, compliance and/or unstressed
blood volume, it is generally referred to as an active change. However, blood
volume can also change passively when the flow rate through the vasculature
changes (Hainsworth, 1986; Rothe, 1993). This occurs because the pressure
drop along a vascular segment decreases when flow decreases, according to
Pouiseille’s law
(4)
Q= πΔPr4/8 μl
17
where π = 3.14, ΔP = the pressure difference, r = vessel radius, μ = viscosity
and l = vessel length. Hence, if the arterial resistance to a specific vascular bed
increases (r becomes smaller), inflow (Q) to the venous circulation
downstream of the arterioles will decrease. Given that all other variables
remain unchanged, ΔP along the downstream vasculature will then decrease.
Primarily, this is the result of a decreased upstream distending pressure which
causes the venous vessels to passively recoil and transfer blood away from that
tissue (Hainsworth, 1986; Rothe, 1986; Rothe et al., 2006). This mechanism
makes it difficult to determine if changes in contained blood volume are due
to altered upstream arterial resistance or from active changes in the
capacitance vasculature, especially as venous and arterial tone often change
simultaneously. In mammals, it seems that the relative importance of passive
and active responses for blood volume mobilization differs considerably
among different organs and vascular beds (Hainsworth, 1986). In fact, the
relative importance of active and passive blood volume changes in overall
haemodynamics is still a matter of considerable debate (Mitzner et al., 2006;
Rothe et al., 2006). Clearly, this represents an area in cardiovascular physiology
where our present knowledge is limited and will be a challenging future
research task.
1.2.6 Control of the venous circulation
In this thesis, the role of neural and humoral adrenergic control systems and
the renin-angiotensin system during exercise, acute temperature changes and
environmental hypoxia have received special attention. The following account
summarizes what is known about these two control systems with regard to the
venous circulation in fish.
Catecholamines
Venous vascular control by means of adrenergic mechanisms is probably the
most extensively studied and best understood control system to date. Early
investigators reported that adrenaline increases venous blood pressure in fish.
Capra and Satchell (1977) injected boluses of various adrenergic agonists in
dogfish (Squalus acanthias) and noted that central and caudal venous pressures
increase in response to adrenaline and decrease in response to noradrenaline.
The β-adrenoceptor agonist isoprenaline decreases central venous pressure,
but has a variable effect on caudal venous pressure. In the Japanese eel
(Anguilla japonica), pressures in the cardinal vein and sinus venosus increase in a
dose-dependent manner after both adrenaline and noradrenaline, whereas
18
isoprenaline decreases the same variables (Chan and Chow, 1976). Similarly, in
rainbow trout caudal venous pressure increase dose-dependently in response
to boluses of both adrenaline and noradrenaline, whereas isoproterenol and
phenylephrine has no significant effect (Wood and Shelton, 1980a).
Unfortunately, no attempts were made to estimate venous capacitance
responses in these early studies which makes it difficult to separate the relative
contribution of cardiac effects, passive flow effects and active veno-specific
events.
Later studies have more specifically sought to investigate the
catecholaminergic control of venous capacitance in fish. Both adrenaline and
noradrenaline dose-dependently increase tension, but do not affect C, in
isolated rainbow trout vascular segments from the anterior cardinal vein,
ductus of Cuvier and intestinal vein. Posterior cardinal vein segments appear
refractory to both agonists (Conklin and Olson, 1994b). The responses are
blocked by phentolamine, but unaffected by propranolol, revealing an αadrenoceptor mediated mechanism.
In rainbow trout in vivo, infusion of adrenaline at 3.3 nmol min-1 kg Mb-1
increases Pven and ventral and dorsal aortic pressures, whereas noradrenaline at
3.3 nmol min-1 kg Mb-1 only increases the arterial pressures (Zhang et al., 1998).
The adrenaline-induced change in Pven is associated with a significant reduction
of venous capacitance as infusion of adrenaline (1.0 nmol min-1 kg Mb-1) results
in reductions of both C and USBV. Interestingly, noradrenaline infusion at 2.6
and 10.4 nmol min-1 kg Mb-1 has no effect on C or USBV in rainbow trout
(Zhang et al., 1998). In the air-breathing teleost, Synbranchus marmoratus,
injection of adrenaline and the specific α-adrenoceptor agonist phenylephrine
increases Pven and MCFP, whereas the β-adrenoceptor agonist isoproterenol
has the opposite effect (Skals et al., 2006). Elasmobranchs also have the
capability to alter venous capacitance by means of adrenergic mechanisms.
Bolus injections of adrenaline and phenylephrine increase Pven and MCFP in
dogfish, whereas isoproterenol decreases the same variables (Sandblom et al.,
2006).
The relative importance of humoral and neural catecholamines in venous
control has not been investigated. An immunohistochemical examination of
the innervation pattern of various large veins from Atlantic cod and rainbow
trout failed to demonstrate adrenergic nerves in any of the vessels examined
and it was suggested that this may have been due to a non-functional antibody
(Johnsson et al., 2001). I am not aware of any imunohistochemical studies
were the innervation pattern of the venous microcirculation (where vascular
capacitance primarily is believed to be regulated) has been examined in fish.
However, the notion that the venous vasculature rapidly constricts within 8-10
19
s due to baroreflex stimulation during transient stoppage of cardiac output in
rainbow trout (Sandblom and Axelsson, 2005; Zhang et al., 1995), but not in
dogfish (Sandblom et al., 2006), may suggest that adrenergic nervous reflex
control of venous capacitance is well developed in some teleosts, but not in
elasmobranchs.
The renin-angiotensin system
Available, albeit limited, data suggest that routine venous tone from the reninangiotensin system is limited in fish. Blockade of ACE with lisinopril
decreases Pda, but does not affect routine venous capacitance in rainbow trout
as measured by vascular capacitance curves (Olson et al., 1997; Zhang et al.,
1995). Furthermore, isolated segments of the intestinal vein and the posterior
cardinal vein from rainbow trout only contract modestly in response to Ang
II. The anterior cardinal vein and the ductus of Cuvier are refractory to the
peptide and compliance is not affected in any of the vessels (Conklin and
Olson, 1994b). Paradoxically, precontracted strips from the anterior cardinal
vein and ductus of Cuvier relax in response to Ang II. This response seems to
be mediated via an endothelium-dependent prostanoid-mediated mechanism,
of which the physiological function in vivo is uncertain (Conklin and Olson,
1994a).
The above findings have lead to the suggestion that RAS in fish primarily
regulate arterial/arteriolar resistance and not venous capacitance (Olson et al.,
1994; Russell et al., 2001). However, it is possible that the apparent lack of
response in routine venous capacitance to ACE inhibitors in vivo could be
explained by concomitant changes in blood volume that mask the vascular
effect of the antagonist. It is also possible that the pharmacological responses
of segments of large isolated veins do not accurately reflect the whole-body
capacitance response in vivo. In mammals, angiotensins are potent
venopressors (Pang and Tabrizchi, 1986; Rothe and Maass-Moreno, 2000;
Tabrizchi et al., 1992; Tabrizchi and Pang, 1992) and in American eel (Anguilla
rostrata) and Antarctic borch (Pagothenia borchgrevinki), injection of Ang II
increases Q through an increased SV, which has been suggested to be
mediated by an increased cardiac filling pressure (Axelsson et al., 1994; Oudit
and Butler, 1995). These responses are attenuated, but not blocked, by αadrenoceptor blockade which could indicate active constriction of the venous
capacitance vasculature from Ang II. Clearly, more work is required to resolve
the importance of the RAS for venous control in fish.
20
1.3 INTEGRATED CARDIOVASCULAR RESPONSES
Catching prey, escaping predators, migrating to spawning grounds or
undertaking vertical migrations, are examples of naturally occurring events
that are associated with exercise in fish. In fact, some pelagic species such as
the tunas are obligate ram-ventilators and thus need to swim constantly in
order to ventilate their gills (Graham and Dickson, 2004). Furthermore, many
aquatic environments are highly heterogeneous in terms of temperature and
oxygen availability. Fish may frequently encounter large variations in ambient
temperature. This occurs both on a long term (seasonal) scale, but also on a
much shorter scale, for example, in salmonids that may swim through
thermoclines when foraging in surface waters. Hypoxia is much more
common in water than air, because less oxygen can be dissolved in water and
the diffusion rate for oxygen in water is only a fraction of that in air, and the
solubility for oxygen decreases with increasing temperature and salinity
(Dejours, 1975). In other words, water contains relatively little oxygen and
when it is consumed it is slowly replaced. Hypoxic conditions therefore occur
naturally, or due to anthropogenic impact, on a regular basis in many aquatic
environments. (Brauner and Val, 2006; Nilsson and Renshaw, 2004; Val et al.,
2006; Wu, 2002).
The following account summarizes what is known about the
cardiovascular responses in fish to exercise, acute temperature changes and
environmental hypoxia, which all represent naturally occurring events in
aquatic environments.
1.3.1 Exercise
Swimming can be broadly classified into burst, prolonged or sustained,
depending on the duration of the swimming period (Beamish, 1978). Burst
swimming is a rapid and mainly anaerobic event, whereas the other two
require an increased oxygen uptake and delivery to match the increased tissue
oxygen demand. Different variables can be altered to ensure adequate supply
of oxygen to the working muscles. This is summarized by the Fick equation:
(5)
MO2= fH * SV (CaO2-CaO2)
where MO2 is oxygen consumption per unit time and CaO2 and CvO2 are
arterial and mixed venous oxygen contents, respectively. Hence, an increased
21
oxygen demand can be met either by an increased Q through an increased fH
or SV, or by an increased blood oxygen extraction such that the difference in
arterio-venous oxygen content (CaO2-CvO2) increases. The relative
contribution of these different factors seems to vary among species and type
of cardiovascular challenge. In this thesis, the hearts ability to increase tissue
oxygen delivery by increasing fH or SV has been the primary focus.
Cardiovascular responses
Previously it was generally thought that fish primarily increase SV to increase
Q during exercise (Butler, 1985, 1986; Farrell, 1991; Farrell and Jones, 1992;
Jones and Randall, 1978; Kiceniuk and Jones, 1977; Randall and Daxboeck,
1982; Stevens and Randall, 1967b). These assumptions, however, were largely
based on studies of salmonids, more or less heavily instrumented. It has since
become increasingly clear that changes in fH may be equally or even more
important in salmonids, as well as in other species (Altimiras and Larsen, 2000;
Axelsson et al., 1992; Axelsson and Nilsson, 1986; Chatelier et al., 2005; Clark
et al., 2005; Cooke et al., 2003; Joaquim et al., 2004; Kolok et al., 1993;
Korsmeyer et al., 1997). The cardiac chronotropic and inotropic responses
during exercise are mediated by various combinations of both intrinsic
mechanisms, as well as by the influence of various neurohumoral control
systems (Farrell and Jones, 1992).
The changes in arterial blood pressure and vascular resistance associated
with exercise are quite variable and result from opposing effects of metaboliteinduced vasodilation and increased vasomotor tone in the somatic,
gastrointestinal and branchial circuits (Bushnell et al., 1992). In Atlantic cod
and rainbow trout, the increased systemic vasomotor tone during exercise is
mediated by an increased adrenergic nervous tone (Axelsson and Fritsche,
1991; Axelsson and Nilsson, 1986; Smith, 1978). In fact, it is doubtful whether
plasma catecholamine levels normally increase at all during non-exhaustive
exercise in teleosts (Axelsson and Nilsson, 1986; Butler, 1986; Butler et al.,
1986; Primmett et al., 1986). A metabolite- or β-adrenoceptor-mediated
relaxation of the systemic circulation is typically unmasked after treatment
with the adrenergic nerve-blocking agent bretylium and/or α-adrenoceptor
antagonists. In Atlantic cod, RAS is activated during swimming after αadrenoceptor blockade and counteracts the typical hypotension and results in
a post-exercise hypertension (Platzack et al., 1993). At least in unfed fish,
blood flow to the gastrointestinal circulation decreases during exercise to
prioritize perfusion of the swimming musculature (Axelsson et al., 1989;
Axelsson and Fritsche, 1991; Farrell et al., 2001; Thorarensen et al., 1993).
22
Information about venous blood pressure changes during exercise is
scarce for fish. Kiceniuk and Jones (1977) recorded pressure in the right
common cardinal vein of swimming rainbow trout. Despite an almost
doubling of SV and relatively small changes in fH at the critical swimming
speed (Ucrit), no significant increase in venous pressure was observed.
Conversely, in the leopard shark (Triakis semifasciata) pressure in the cardinal
sinus increases significantly from 0.20-0.26 to 0.32-0.49 kPa (min-max values)
during swimming at 0.3-0.7 BL s-1 (Lai et al., 1990). Overall, the mechanisms
controlling venous function during exercise are poorly understood in fish.
1.3.2 Temperature
Many aquatic environments display a significant spatial thermal heterogeneity
(Clark et al., 2005; Levy, 1990; Rodnick et al., 2004). In this thesis, special
attention has been paid to the cardiovascular responses to short-term (acute)
variations in temperature that can be expected to occur in fish that swims
through thermoclines.
Cardiovascular responses
Most fish are ectothermic water-breathers and gas exchange takes place at a
highly efficient counter-current arrangement between water and blood at the
gills. This also means that metabolically produced heat is effectively dissipated
to the surrounding water and changes in ambient temperature are also rapidly
mirrored by the body temperature of the fish (Crawshaw, 1976; Reynolds,
1977; Taylor et al., 1997). Metabolic rate is directly related to temperature in
fish (Brett, 1973; Farrell, 1997; Lee et al., 2003) and acute environmental
temperature changes are therefore associated with a number of cardiovascular
responses to meet the changes in metabolic demand. Cardiac output often
increases with temperature (Brodeur et al., 2001; Cech et al., 1976; Farrell,
1984, 1997; Gollock et al., 2006; Korsmeyer et al., 1997; Lannig et al., 2004;
Mark et al., 2002; Stevens et al., 1972), although an increased blood oxygen
extraction, with unaltered or only slightly increased Q, may be important as
well. The relative importance of changes in Q and increased blood oxygen
extraction seems to vary depending on where in the animal’s “thermal
window” the temperature change takes place (Cech et al., 1976; Lannig et al.,
2004; Mark et al., 2002). However, when Q increases, the mechanism by
which this is accomplished also vary among species. In Atlantic cod, lingcod
(Ophiodon elongatus) and winter flounder (Pseudopleuronectes americanus); the
increased cardiac output with increasing temperature is mediated by
23
tachycardia whereas SV is unchanged (Cech et al., 1976; Gollock et al., 2006;
Stevens et al., 1972). Yet, in other species such as rainbow trout, the Antarctic
bernach (Trematomus bernacchii) and yellowfin tuna (Thunnus albacares), Q also
increase through tachychardia, but SV tends to drop as fH increases (Axelsson
et al., 1992; Brodeur et al., 2001; Korsmeyer et al., 1997). The reduced SV in
these species could be the effect of decreased cardiac filling time, reduced
cardiac filling pressure, reduced cardiac contractility or a combination of these
factors (Altimiras and Axelsson, 2004; Farrell et al., 1989; Shiels et al., 2002). It
could be speculated that species which maintain SV at high temperatures do
so by increasing cardiac filling pressure. However, even if SV is sometimes
maintained in the winter flounder when temperature and heart rate increases,
there is no pressure increase in the caudal vein (Cech et al., 1976). The venous
capacitance response to temperature has not been investigated in fish.
The arterial blood pressure response to acute temperature increase varies
among species as well (and likely experimental protocols). In rainbow trout
and Japanese eel, Pda increases (Heath and Hughes, 1973; Takei and Tsukada,
2001), whereas caudal artery pressure in winter flounder is unchanged (Cech et
al., 1976). According to Pouiseille’s law (equation (4)), blood pressure is not
only affected by vascular resistance and cardiac output, but will also be
affected by a change in the viscosity of the blood. Blood viscosity decreases
with increasing temperature and shear rate (Bushnell et al., 1992; Fletcher and
Haedrich, 1987; Graham and Fletcher, 1983, 1985; Graham et al., 1985;
Macdonald and Wells, 1991). This implies that arterial blood pressure during
an acute temperature increase will be affected by an apparently complex
interaction of changes in vascular resistance, blood viscosity and cardiac
output.
1.3.3 Hypoxia
A number of behavioural and physiological strategies have evolved in fish to
cope with changes in ambient oxygen levels. The behavioural responses often
involve escape behaviour which can be regarded as a “first line of defence” to
hypoxia (Brauner and Val, 2006). Furthermore, many tropical fish species have
evolved the ability to breathe air through more or less refined behavioural,
morphological and physiological mechanisms (Brauner and Val, 2006; Fritsche
and Nilsson, 1993; Reid et al., 2006). However, for fishes which are obligate
water breathers, and when the hypoxic conditions cannot be avoided, it is
essential for the fish to be able to rapidly adjust its physiology to the reduced
24
oxygen availability. The following account is a description of the general
trends of the cardiovascular responses to rapidly induced environmental
hypoxia in water-breathing teleosts.
Oxygen sensing
Due to the high solubility for CO2 in water, most water-breathing animals
monitor O2-, rather than CO2-levels, to regulate breathing and cardiovascular
function (Milsom, 1998). Receptors that monitor environmental O2 levels
(external), as well as the O2 levels of the blood (internal), are essential for the
fish to rapidly respond to altered oxygen conditions. Oxygen chemoreceptors
are primarily located in the gills, including the pseudobranch, with the afferent
fibres travelling in cranial nerves IX (the glossopharyngeal) and X (the vagus).
Oxygen-sensitive receptors innervated by cranial nerves V and VII in the
orobuccal cavity have also been demonstrated in some species (Fritsche and
Nilsson, 1993; Perry and Gilmour, 2002; Sundin and Nilsson, 2002; Taylor et
al., 1999). The support for centrally-located oxygen receptors in fish seems to
be rather weak (Jones and Milsom, 1982; Sundin and Nilsson, 2002; Taylor et
al., 1999). Recent patch-clamp studies of neuroepithelial cells from the gills of
zebrafish (Danio rerio) and channel catfish (Ictalurus punctatus) have verified that
these cells give rise to the afferent signal during environmental hypoxia. The
cellular mechanism by which this is achieved closely resembles that of the
mammalian O2-chemoreceptors in the lungs and aortic arch (Burleson et al.,
2006; Jonz et al., 2004).
Cardiovascular responses
Both ventilation frequency and amplitude increase in most species during
hypoxia, but if water PO2 becomes too low and the cost of ventilation exceeds
the gain of the O2 uptake, ventilation may decrease (Fritsche and Nilsson,
1993). In contrast to exercise, hypoxia is a potent stimulus for catecholamine
release in teleosts (Perry and Bernier, 1999; Randall and Perry, 1992; Reid et
al., 1998; Wendelaar Bonga, 1997). It has been suggested that the hypoxic
threshold for catecholamine release is set by the P50 value for the O2haemoglobin saturation curve, as release typically occurs when haemoglobinO2 saturation drops below 50% (Randall and Perry, 1992; Reid et al., 1998).
This seems to be a highly species-dependent level, with species like tunas and
salmonids having high P50 values, whereas more hypoxia tolerant species have
lower P50 values.
The cardiovascular responses to hypoxia have been studied in some detail
and it is clear that many of the responses vary among species and depending
on experimental conditions. In most teleosts, rapidly induced hypoxia results
25
in reduced heart rate (hypoxic bradycardia) due to an increased cholinergic
tone on the heart (Burleson and Smatresk, 1990; Farrell, 1982; Fritsche, 1990;
Fritsche and Nilsson, 1989, 1990, 1993; Holeton and Randall, 1967; Perry et
al., 1999; Randall, 1982; Smith and Jones, 1978; Wood and Shelton, 1980b).
However, in the Antarctic fish Trematomus bernachii, average fH increases slightly
(Axelsson et al., 1992) and in Sea raven (Hemitripterus americanus) and fivebearded rockling (Ciliata mustela) heart rate is more or less irresponsive to
environmental hypoxia (Fritsche, 1990; Saunders and Sutterlin, 1971).
Depending on the magnitude of the bradycardia, Q may drop slightly or
remain unchanged because SV often increases and compensates for the
reduced heart rate (Farrell, 1982; Fritsche and Nilsson, 1989; Perry and
Desforges, 2006; Wood and Shelton, 1980b).
The vascular responses to hypoxia are also variable and depend on the
interaction between altered neurohumoral vasomotor tone and on possible
direct effects of the chemical composition of the blood. Therefore, arterial
hypertension, hypotension, as well as unchanged arterial blood pressure, have
been reported for teleosts during hypoxia (Bushnell et al., 1992). However, it
seems that overall vasomotor tone generally increases. For example, when
Atlantic cod is exposed to acute hypoxia (water PO2=4-5.3 kPa), both ventral
and dorsal aortic blood pressure increase and this response can be blocked
with bretylium, an adrenergic nerve blocking agent. Additional treatment with
phentolamine further reduces the hypotensive response. This suggests that
adrenergic nerves and, to a lesser extent, circulating catecholamines are
responsible for the hypoxic hypertension in Atlantic cod (Fritsche and
Nilsson, 1990). However, the relative importance of circulating and neural
catecholamines is likely characterized by large inter-specific differences (Perry
and Bernier, 1999).
Resistance of the coeliac and mesenteric arteries increases and reduces
blood flow to the gut during hypoxia. Again, the response can be partly
blocked with bretylium, and more or less completely with phentolamine,
suggesting that both adrenergic nerves as well as circulating catecholamines
mediate the response (Axelsson and Fritsche, 1991). Gut blood flow also
decreases in unfed sea bass (Dicentrarchus labrax) during hypoxia (Axelsson et
al., 2002). The compromised perfusion of the gastrointestinal tract likely
occurs to favour perfusion of more vital organ systems.
Gill resistance in rainbow trout increases during severe hypoxia (water
PO2=1.1-8.6 kPa) (Perry et al., 1999; Sundin and Nilsson, 1997). This is mainly
due to an increased cholinergic tone, presumably on the efferent filamental
artery sphincter (Sundin and Nilsson, 1997). In Atlantic cod, arterio-arterial
resistance is unaffected by hypoxia (water PO2 = 5.3-6.5 kPa), whereas arterio26
venous resistance increases due to an increased α-adrenergic tone (Sundin,
1995). The resistance in perfused gills from rainbow trout and Atlantic cod
increases in response to hypoxia (Pettersson and Johansen, 1982; Ristori and
Laurent, 1977; Smith et al., 2001).
Reports on venous responses to hypoxia are few. Pven increases in rainbow
trout during graded hypoxia (Perry et al., 1999), whereas pressure in the caudal
vein does not change in winter flounder after approximately two hours of
exposure to hypoxic water (Cech et al., 1977). An increase in Pven during
hypoxia may well be explained by the accompanying bradycardia. It is
unknown whether active changes in venous vascular tone and/or compliance
also contribute in fish. In anaesthetized dogs, hypoxic stimulation of internal
chemoreceptors results in increased venous smooth muscle tone (Rothe et al.,
1990a, 1990b).
Overall, there is no general consensus regarding the physiological
significance of the cardiovascular responses to hypoxia in fish. It has been
suggested that increased blood pressure and reduced heart rate may enhance
branchial gas transfer during hypoxia, but in a recent study the authors failed
to detect any beneficial effect of either bradycardia or arterial hypertension on
arterial blood gas levels (Perry and Desforges, 2006). As oxygenation of the
heart in fish to a varying degree relies on the oxygen left in venous blood, it
can be speculated that bradycardia may enhance oxygenation of the
myocardium during environmental hypoxia (A. P. Farrell, personal
communication).
27
2. AIM
Prior to this thesis, very few studies have addressed the venous haemodynamic
responses that likely occur during various natural cardiovascular challenges in
fish. The general objective of this thesis is to gather basic information about
control mechanisms and overall function of the venous circulation in teleost
fish in vivo. More specifically, the role of the venous circulation during various
natural cardiovascular challenges has been investigated and the following
aspects have received special attention:
1.
Control of central venous blood pressure and venous capacitance by
adrenergic mechanisms and the renin-angiotensin system during
sustained exercise.
2.
Venous capacitance and blood volume responses to acute temperature
changes.
3.
Nervous and humoral catecholaminergic control of central venous
blood pressure and venous capacitance during environmental hypoxia.
28
29
3. RESULTS AND DISCUSSION
3.1 METHODOLOGICAL CONSIDERATIONS
In this thesis, venous responses have been measured in vivo in teleost fish
during various natural cardiovascular challenges. Four of the studies were
conducted on freshwater adapted rainbow trout, Oncorhynchus mykiss (i.e. Papers
II, III, IV and V), while one study was conducted on the European sea bass,
Dicentrarchus labrax, adapted to seawater (Paper I). With around 30 000 extant
species of fish, that can be found in extremely diverse habitats ranging from
polar seas to hot springs, and from several thousand meters depth to dry land,
any extrapolation from these studies to a general picture for “fish”, must off
course be done wisely and with a bit of caution.
Some of the experimental approaches used to quantify vascular
capacitance changes in these studies are relatively novel in fish cardiovascular
research. Therefore, before moving on to discuss the main findings regarding
venous control and function in fish, a more comprehensive discussion will be
made on the overall applicability of these methods in comparative
cardiovascular research. For more detailed discussions and methodological
descriptions, the reader is referred to the individual papers (Papers I-V).
3.1.1 Mean circulatory filling pressure
MCFP is measured as the central venous blood pressure during zero flow.
Stoppage of cardiac output was therefore accomplished by mechanical
occlusion of the ventral aorta (i.e. Papers I, II, III and V). In rainbow trout,
the surgically accessible portion of the ventral aorta, between the pericardium
and first pair of afferent branchial arteries, is rather short (Fig. 5). A combined
Doppler flow/occlusion probe was therefore custom-made to enable
recordings of MCFP and Q in the same fish (i.e. studies II, IV and V). In
similarly sized sea bass, however, the accessible portion of the ventral aorta is
considerably longer and in these fish it is possible to place a Doppler flow
probe adjacent to a separate vascular occluder on the same vessel (Fig. 5, see
also Paper I).
30
Length of occlusion
In Papers I, II, III and V, MCFP is measured as an average of the venous
plateau pressure between the 5th and the 7th second during a ~8-10 s ventral
aortic occlusion (Fig. 6). The length of this period is a compromise between
two opposing factors, namely barostatic reflexes triggered by the reduced
arterial/branchial blood pressure, and possible inequalities in venous and
arterial plateau pressures at the end of the occlusion.
i.
ii.
iii.
VO + Q
VO
Q
Figure 5. Schematic illustrations of rainbow trout (upper) and sea bass (lower) showing
placement of vascular occluder (VO) and flow probe (Q). Given the limited access to the
ventral aorta in trout, a custom-made combined flow probe and vascular occluder was used.
A cross-section of the probe used on trout is magnified to illustrate PE-50 catheter connected
to latex-baloon (i.); inflatable latex-baloon (ii.) and doppler crystal with lead (iii.). In sea bass, a
separate occluder and flow probe was placed on the same vessel. In both cases, care was
taken not to damage the pericardium. Modified from Sandblom and Axelsson, 2005 and
Paper I.
When the length of the ventral aortic occlusion exceeds ~8-10 s, barostatic
reflexes are initiated in both mammals and rainbow trout (Rothe, 1993;
Sandblom and Axelsson, 2005; Zhang et al., 1995). This leads to a reflex
mediated constriction of the capacitance vasculature and MCFP is
consequently overestimated. However, in in vivo studies of venous function in
dogfish (Sandblom et al., 2006) and South American rattlesnake (Crotalus
durissus) (Skals et al., 2005), occlusion times beyond 20 s have been used
without any apparent baroreflex responses.
31
Pda (kPa)
5
4
3
2
1
Pven (kPa)
0
0.3
0.2
Figure 6. Original recordings of
dorsal aortic pressure (Pda),
central venous pressure (Pven)
and cardiac output (Q) in a 680g
rainbow trout during MCFP
measurement. Zero flow was
induced by ventral aortic occlusion between vertical arrows.
The horizontal bar show the 2 s
period where MCFP is taken.
Note the slight arterial and
venous hypertension following
the occlusion.
0.1
0.0
Q (arbitrary units)
-0.1
0
10 s
This either indicates that reflex control of the vasculature is poorly developed
in these animals, or that the vasculature was constricted prior to the occlusion
leaving little scope for further changes. It should be kept in mind that in most
of these experiments the pericardium had to be opened to place a perivascular
occluder around the heart’s outflow tract(s), and pericardioectomy may affect
vascular tone as discussed below. In elasmobranchs, nervous control of the
vasculature is limited (Butler and Metcalfe, 1988; Holcombe et al., 1980;
Nilsson and Holmgren, 1988; Opdyke et al., 1972; Satchell, 1992). This may
allow the use of longer occlusion times in elasmobranchs than in teleosts.
The opposing problem with short occlusion times is that arterial and
venous plateau pressures do not equilibrate during the occlusion. However,
even with longer occlusions, arterio-venous pressure equilibrium is generally
not achieved, as the capillary beds collapse before full pressure equilibrium is
reached (see Fig. 6). In Guyton’s pioneering studies of MCFP in dogs, an
attempt was made to solve this problem by using an arterio-venous shunt to
pump blood from the arterial circulation to the central venous compartment.
MCFP was then taken at the point of intersection where arterial and venous
pressures were equal (Guyton et al., 1954). This method is technically
challenging for a number of reasons, especially if experiments are conducted
32
on unanaesthetized animals. Attempts to mathematically compensate for
remaining arterio-venous pressure differences have also been made using the
following equation
(6)
MCFP=VPP + K(APP – VPP)
where K is the ratio of arterial to venous compliance, VPP is the venous
plateau pressure and APP is the arterial plateau pressure during zero flow
(Pang, 2000).
All MCFP measurements on fish to date have assumed that the effects of
a lack of full pressure equilibrium are negligible. This assumption is based on
the supposedly large compliance difference between the venous and arterial
compartments. For example, according to equation (6) in a hypothetical
circulatory system with a venous to arterial compliance ratio of 1/25 and
where the arterial and venous vascular volumes for simplicity are assumed to
be equal, a remaining arterio-venous pressure difference of 1 kPa would only
account for an underestimation of the measured MCFP by 0.04 kPa.
Furthermore, small differences in the arterio-venous pressure difference with
different treatments may thus be assumed to have a minimal effect on MCFP.
For example, during exercise Rsys sometimes decreases (Papers I and II). This
may lower the arterio-venous pressure difference somewhat, but it is unlikely
that this could explain only but a very small fraction of the measured increase
in MCFP with exercise.
The arterio-venous compliance ratio of isolated, large conducting vessels
from rainbow trout has been investigated in vitro. The arterial (efferent
branchial artery) to venous (anterior cardinal vein) compliance ratio is 1/21
and 1/32 for hatchery reared rainbow trout and wild steelhead trout,
respectively (Conklin and Olson, 1994b). However, it is possible that the
compliance of the small veins and venules may be even higher than the large
conducting veins. Nevertheless, these findings render support for the
assumption that any underestimation of MCFP due to remaining blood in the
arterial circulation during vascular occlusion is small also in fish.
As the concept of MCFP assumes that all pressures equalize in the
circulation during zero flow, not only remaining pressure differences between
the arterial and the central venous circulation may affect interpretation of the
measured value of MCFP. Also, remaining pressure differences within various
parts of the venous compartment will affect this. In mammals, the central
venous and portal venous plateau pressures have been compared during
transient cardiac arrest (Gaddis et al., 1986; Tabrizchi et al., 1993).
Theoretically, these pressures should be the same during the MCFP
33
manoeuvre, but following infusion with vasoactive agents (Tabrizchi et al.,
1993) and blood volume depletion (Gaddis et al., 1986), the portal venous
plateau pressure typically exceeds MCFP as measured in the central veins. This
suggests that the central venous plateau pressure may underestimate MCFP at
certain physiological states. These types of experiments have not yet been
conducted for fish, but should clearly be addressed in the future to validate the
accuracy of MCFP measurements for fish.
In conclusion, exercise (Papers I and II), acute temperature increase
(Paper III) and environmental hypoxia (Paper V) resulted in increased MCFP
(i.e. a decreased vascular capacitance). The relatively short occlusion times
used in these studies may, if anything, have resulted in small underestimations
of MCFP, and consequently the magnitude of the decreases in capacitance.
Integrity of the pericardium
Previous studies on venous pressure and capacitance in rainbow trout have
involved surgical opening of the pericardium to place flow probes and
vascular occluders around the bulbus arteriosus and/or electrodes for
ventricular fibrillation (Conklin et al., 1997; Hoagland et al., 2000; Olson et al.,
1997; Zhang et al., 1995, 1998). When MCFP was measured in the present
studies, a vascular occluder was placed around the ventral aorta without
opening the pericardium (see Fig. 5). Table 2 compares recorded and
calculated venous variables from Paper V with previous studies of the same
variables in rainbow trout. It appears that Pven is considerably higher in fish
where the pericardium has been opened (see also Table 1). It can also be
suggested that this may be due to an increased venous tone, as USBV is
typically lower in most studies using fish with opened pericardia (Table 2). In
elasmobranchs, which normally have a negative central venous pressure,
pericardioectomy increases Pven to slightly positive values and MCFP is
elevated (Sandblom et al., 2006; Sudak, 1965).
Experiments with perfused hearts have demonstrated that the Starling
curve is right-shifted when the pericardium is cut in both teleosts (Farrell et
al., 1988) and elasmobranchs (Franklin and Davie, 1993). This demonstrates
that a higher filling pressure is required to produce the same cardiac output
after the pericardium has been opened. Taken together, this suggests that the
fish is forced to switch from vis a fronte to vis a tergo filling to maintain Q when
the intrapericardial pressure is made ambient, and this is likely achieved by an
increased venous smooth muscle tone. Minerick et al. (2003) estimated C in
rainbow trout in vivo, using a ramp-infusion protocol, and reported that there
was no difference between fish with cut and intact pericardia. USBV was not
determined.
34
Table 2. Summary of previous studies on vascular capacitance in rainbow trout (Oncorhynchus mykiss).
Reference
USBV
C
Pven
(ml kg Mb-1) (ml kPa-1 kg Mb-1) (kPa)
Temp
(ºC)
Catheter used for
blood volume
manipulation
Pericardium
opened?
Zhang et al., (1995)
13.3
25.5
~0.40
12
dorsal aortic catheter
yes
Conklin et al., (1997)
20.7+0.6
12.8+1.5
0.27+0.08
12
-
yes
Olson et al., (1997)
18.8+0.5
19.6+0.8
20.6+0.6
17.2+0.7
18.0+3.7
18.0+0.7
0.47+0.03
12
-
yes
Zhang et al., (1998)
18.3+0.7
22.0+0.5
23.0+0.9
22.5+1.5
15.0+0.7
16.5+1.5
0.40+0.03
0.40+0.03
0.47+0.03
15
-
yes
Hoagland et al., (2001)
26.0+0.5
19.5+0.7
0.40+0.03
12
dorsal aortic
and venous
catheter
yes
Paper V
25.2+2.8
21.2+4.4
0.06+0.07
15
venous
catheter
no
Summary of data for untreated Oncorhynchus mykiss including mean values (+S.E.M. or S.D. In Paper
V) of routine unstressed blood volume (USBV) and vascular compliance (C) at 90-110% blood
volume and routine central venous pressure (Pven). In cases where absolute values were not reported,
Pven was estimated from mean pressure traces. Note, in Olson et al., (1997) and Zhang et al., (1998),
cardiovascular data was reported for 3 individual series.
3.1.2 Stressed blood volume and vascular compliance
In Paper V, vascular capacitance curves were constructed (i.e. Fig. 2 and Table
1) for normoxic and hypoxic rainbow trout by measuring MCFP at different
blood volumes (80-120% of the assumed blood volume). By extrapolating the
curve back to zero MCFP it can be determined whether the capacitance
changes are mediated by changes in unstressed blood volume (the intercept of
the blood volume axis) and/or vascular compliance (slope of the line). Since
the relationship is not necessarily linear over the full blood volume range,
USBV and C are calculated for 80-100, 90-110 and 100-120% of the assumed
blood volume (Rothe, 1993; Zhang et al., 1998). Thus, from Table 1 in Paper
V, it is clear that absolute values for routine USBV and C may differ
depending on what blood volume range they are calculated from. However,
35
the qualitative response to hypoxia is essentially the same, regardless of what
interval is being used.
In order to be able to compare USBV and C statistically, these variables
need to be calculated for individual fish (i.e. Table 1, Paper V). Thus, the
capacitance curves presented in Paper V are constructed using mean values for
USBV and C which are derived from individual values of USBV and C. In
other studies on fish and snakes, it seems that mean MCFP values have been
used for vascular capacitance curves (Conklin et al., 1997; Hoagland et al.,
2000; Skals et al., 2005; Zhang et al., 1998). However, when capacitance curves
were calculated from mean MCFP values in Paper V, this produced slightly
different values of mean USBV and C. Therefore, I suggest that in order to
construct vascular capacitance curves and to enable statistical comparisons,
mean values of USBV and C should be used, rather than mean MCFP values.
Another concern when constructing capacitance curves is that blood
volume needs to be manipulated. When blood volume is decreased, this will
trigger baroreflexes and lead to an overestimate of MCFP as discussed above.
On the other hand, when blood volume is increased, this may lead to stretchinduced release of atrial natriuretic peptide which increases C in rainbow trout
(Cousins and Farrell, 1996; Cousins et al., 1997; Farrell and Olson, 2000;
Olson et al., 1997). In Paper V, blood volume was altered via a large-bore (ID,
1 mm) venous catheter to change blood volume as quickly as possible
(typically 3-15 s) to minimize the effect of baroreflex stimulation. However, a
potential overestimate of routine C due to atrial natriuretic peptide release, as
blood is injected into the sinus venosus, can clearly not be ignored.
For both fish and mammals, it seems that both USBV and C are
controlled and can change independently. Stressed blood volume is believed to
be determined by vascular smooth muscle tone, whereas the mechanistic basis
for compliance changes is poorly understood, even in mammals (Pang, 2001).
However, for fish, it has been suggested that changes in capacitance through
modulation of C and USBV have different physiological functions, with
changes in compliance being more important in hypervolemic states and
changes in stressed volume being more important in hypovolemic states
(Conklin et al., 1997; Olson et al., 1997). For example, atrial natriuretic
peptide, which is released in response to atrial stretch caused by hypervolemia
(Cousins and Farrell, 1996; Cousins et al., 1997; Farrell and Olson, 2000),
reduces Pven by increasing C in rainbow trout (Farrell and Olson, 2000; Olson
et al., 1997). Conversely, the neurohypophyseal hormone arginine vasotocin,
which is released in response to hypovolemic stress, decreases USBV whereas
C is unchanged in rainbow trout (Conklin et al., 1997).
36
3.1.3 Blood volume
During the course of the studies presented in this thesis, it has become
increasingly clear that blood volume is an important variable to monitor
during these types of experiments. In Paper V, blood volume was directly
measured by calculating the dilution of 51Cr-labeled red blood cells (Duff et al.,
1987; Gingerich et al., 1987). This method was used as other indicators, such
as plasma dyes and radiolabelled albumins, tend to overestimate blood volume
due to leakage into extra-vascular spaces (Olson, 1992). However, working
with radiolabelled red blood cells is awkward in many ways, and simpler
methods for reliable estimates of blood volume in fish are desirable.
In several of the studies, prazosin was used to block α-adrenoceptors (i.e.
Papers I, II, IV and V). This antagonist clearly abolishes many of the venous
responses to exercise and hypoxia as will be discussed below, but routine Pven
increases (Papers I, II, IV and V) and routine MCFP is unchanged (Papers I,
II and V). This somewhat paradoxical response may be the result of an
increased blood volume due to a reduced capillary filtration pressure after the
blocker. Thus, possible blood volume changes should always be considered
when using blockers that will affect both arterial and venous tone.
Interestingly, in another study on rainbow trout, prazosin lowered Pven and
occasionally increased USBV (Zhang et al., 1998). However, in the study by
Zhang and co-workers (1998), venous variables were measured approximately
20-40 min after administration of the blocker, whereas at least 1.5 hours was
allowed before recordings began in the present studies. This could indicate
that 20-40 minutes is not enough time for a new pressure-volume steady state
to be established.
3.1.4 Future perspectives
As previously pointed out, the calculation of the pressure gradient for venous
return and venous resistance (i.e. equations (1) and (2) in Papers I and III) and
the concept of vascular function curves (Fig. 4), rests on the assumption that
MCFP gives an accurate estimate of the pressure in the small veins and
venules. Although this assumption may seem plausible for fish as well, it needs
to be emphasised again that I am not aware of any experimental evidence
verifying the accuracy of this assumption for fish. Another open question is
what regions in the circulatory systems have the most important capacitance
function. In mammals, the splanchnic (liver, spleen and small and large
intestine) venous circulation is highly compliant and contains a large portion
37
of the total blood volume (around one-fourth). Thus, the splanchnic
circulation is considerer the most important blood volume reservoir in
mammals (Pang, 2001). The amount of blood contained in the splanchnic
circulation of fishes also seems to be quite large, although it has been
suggested that a comparatively larger portion of the total blood volume is
located in large conducting vessels, respiratory organs (i.e. the gills) and the
heart in fish (Olson, 1992). Regional vascular capacitance control and function
in fish clearly represents an important area to address in the future.
In mammals, the pressure-diameter relationships of intestinal venules have
been determined using micro pressure recording devices (i.e. servo-null
systems) in combination with video microscopy techniques. From these
studies it has become clear that the venules of the splanchnic circulation are
highly reactive to vasoactive substances and baroreflex stimulation (Haase and
Shoukas, 1991, 1992; Shoukas and Bohlen, 1990). Applying these methods to
fish could provide useful clues to help answer some of the questions outlined
above.
The mechanistic basis for vascular compliance modulation, and its
potential interaction with changes in stressed blood volume, clearly also needs
to be investigated. This is a fundamental question that, to my knowledge, has
not been resolved for any animal group.
3.2 INTEGRATED CARDIOVASCULAR RESPONSES
3.2.1 Exercise
The swimming speeds in the two exercise experiments were 1 and 2 BL s-1 for
sea bass (Paper I) and 2/3 BL s-1 for rainbow trout (Paper II). These
swimming speeds were chosen as they resulted in reasonably stable and clear
cardiovascular responses and allowed us to swim fish continuously for up to
30 min, even after pharmacological treatment. However, when interpreting
these results it should be kept in mind that swimming at other velocities
and/or durations could produce qualitatively, as well as quantitatively,
different responses.
Exercise in both sea bass and rainbow trout is associated with significant
increases in Q and Pven, and a significant reduction of venous capacitance as
38
indicated by the increase in MCFP. Despite these similarities, the mechanism
by which Q increases differs significantly between the two studies. The
increase in sea bass is entirely mediated by tachycardia (Paper I, Fig. 3 and
Table 1), whereas mainly SV increases in rainbow trout (Paper II, Fig. 3).
Although SV does not increase in sea bass, the increase in Pven observed in this
species is likely important to maintain SV as the cardiac filling time decreases
when fH increases. Other studies on sea bass have also reported that fH
modulation is the primary means of increasing Q in this species (Axelsson et
al., 2002; Chatelier et al., 2005, 2006). Similarly, the SV increase in rainbow
trout (Paper II) is also in agreement with previous studies of exercise
performance in rainbow trout, at least under similar experimental conditions
(Claireaux et al., 2005; Farrell, 1991; Farrell and Jones, 1992; Kiceniuk and
Jones, 1977; Randall and Daxboeck, 1982; Taylor et al., 1996). However,
especially for the rainbow trout study (Paper II), it should be noted that postsurgical stress and the experimental protocol as such, may increase resting fH
and therefore reduce the available scope for increasing this variable during
exercise. When fH is monitored in swimming rainbow trout using a noninvasive wireless ECG recording system and after a recovery time of at least 3
days in the swim tunnel, resting heart rate is notably lower. The scope for
changes in heart rate is therefore significantly higher in these rainbow trout
compared with more invasive studies (Altimiras and Larsen, 2000). Most
studies of blood pressure and blood flow in unanaesthetized animals have up
to recently involved surgical instrumentation and more or less rigorous
confinement of the animal to make the necessary recordings. However, newly
developed bio-telemetry technology will allow future studies of cardiovascular
variables, including multiple pressures and flows, to be made in free-ranging
animals with minimal effects of surgery and confinement stress (Axelsson et
al., 2007).
Blockade of α-adrenergic receptors with prazosin has a marked effect on
the haemodynamic response to exercise. The nearly instantaneous increase in
Pven observed during exercise in both sea bass (Paper I, Fig. 3) and rainbow
trout (Paper II, Fig. 1) is abolished after α-adrenoceptor blockade. The
rapidity of these responses, even at relatively low swim speeds, may suggest
that adrenergic nervous constriction of the venous capacitance vasculature is
responsible for this response. Contribution from the “muscle pump” in raising
Pven during swimming can, of course, not be ignored. Although this
mechanism may have contributed to the increased Pven in untreated fish, it is
clearly not functioning after prazosin treatment at 1 BL s-1 in sea bass (Paper I)
and during the first ~10 min of swimming at 2/3 BL s-1 in rainbow trout
(Paper II). Again, this finding rather points towards venous capacitance
39
changes being responsible for the increase in Pven during exercise. MCFP
increases at the end of the exercise period in both Paper I and II. Although it
cannot be claimed that this is also the case early in the swim period, this clearly
seems reasonable to assume. Measurements of MCFP at the onset of exercise
are needed to fully resolve this possibility.
The finding that Pven and MCFP also increases significantly during
exercise after prazosin in sea bass at 2 BL s-1 (Paper I), led to the suggestion
that other vasoactive systems may be activated during exercise to maintain
venous (and arterial) tone. Previous studies have demonstrated that the reninangiotensin system is activated in swimming fish after α-adrenoceptor
blockade and this serves to restore arterial blood pressure (Platzack et al.,
1993). Part of the objective of the study on rainbow trout in Paper II was
therefore to investigate in more detail any potential influence from RAS in the
control of venous function during exercise. In these experiments, a slowly
developing increase in Pven, that is significant after about 13 min, was observed
during swimming after prazosin treatment (Paper II, Fig. 1). Similarly to the
sea bass, MCFP is significantly increased at the end of the exercise period
suggesting a reduced venous capacitance. After an additional dosage of
enalapril to block the formation of Ang II, no changes in Pven are observed
during exercise and MCFP is unchanged at the end of the exercise period
(Paper II, Figs. 1 and 2). These findings suggest that RAS affects venous
capacitance in fish as well, and not only systemic arterial resistance as
previously suggested (Conklin and Olson, 1994a, 1994b; Olson et al., 1994;
Olson, 1992; Russell et al., 2001; Zhang et al., 1995). However, it remains to
be elucidated what role (if any) the RAS has on venous function in fish with
an intact α-adrenergic control system.
An argument can be made that blood volume may change during exercise
in fish and this would in turn affect MCFP. Depending on the blood volume
response, this would either overestimate (increased blood volume), or
underestimate (reduced blood volume) MCFP. Gill lamellar recruitment
during exercise could theoretically favour uptake of fluid in freshwater and
loss of intravascular fluid in seawater (Stevens, 1968; Wood and Randall,
1973). However, exercise typically results in reduced plasma volume in both
freshwater and saltwater species, presumably due to increased capillary
filtration and/or accumulation of intracellular metabolites which leads to
osmotic fluid shifts (Olson, 1992; Pearson and Stevens, 1991; Stevens, 1968;
Wang et al., 1994; Wood and Randall, 1973; Yamamoto and Itazawa, 1989;
Yamamoto et al., 1980). The exercise studies in Paper I and II were
performed on a saltwater species and a freshwater species, respectively, and
yet MCFP increases in both. Taken together, it seems reasonable that, if
40
anything, the venous capacitance responses in the two exercise studies may
represent small underestimations of the vascular capacitance response due to a
potential decrease in plasma volume. More importantly, this means that the
changes in MCFP that were observed in Paper I and II represent true vascular
responses and not secondary effects due to an increased blood volume.
In Paper V, the blood volume response to acute temperature increase was
measured directly in rainbow trout. An acute increase in ambient temperature
from 10 to 16°C results in an increase in Q by 31% and a small increase in Pda.
This response likely resulted in an increase of the perfused gill area as well, but
no significant effect on blood volume was found (Paper V, Fig. 3). However,
to fully appreciate the magnitude of the venous exercise responses, future
studies should also monitor changes in blood volume.
The benefit of the reduction in venous capacitance during exercise is
probably at least twofold. When blood flow to the swimming musculature
increases during exercise it may be important to make the venous capacitance
vasculature “stiffer” to prevent passive flow induced pooling of blood in the
venous circulation. Furthermore, the decreased capacitance is probably also a
reflection of an active blood transfer from areas such as the splanchnic
circulation. This serves to increase Pven to maintain or increase stroke volume
and increases the blood volume available for oxygen delivery to the swimming
musculature.
3.2.2 Temperature
Relatively few studies have investigated the cardiovascular responses to acute
temperature changes in fish. This is surprising given the highly heterogeneous
thermal environment which many fishes inhabit. As all fishes are largely
ectothermic, changes in ambient temperature will rapidly be reflected by the
body temperature and the overall metabolism. When the ambient temperature
is acutely elevated, cardiac output often increases to meet the increased
metabolic demand (Brodeur et al., 2001; Cech et al., 1976; Farrell, 1984;
Farrell, 1997; Gollock et al., 2006; Korsmeyer et al., 1997; Lannig et al., 2004;
Mark et al., 2002; Stevens et al., 1972).
In Paper III, the venous responses to an acute temperature increase was
investigated in rainbow trout. Increasing temperature from 10 to 13 and 16°C
results in an increased Q by 20 and 31%, respectively (Paper III, Fig. 1). The
presumed increase in oxygen consumption is additionally met by an increased
oxygen carrying capacity, as indicated by the fact that splenic release of
41
erythrocytes results in a significantly elevated hematocrit from a routine of
21% at 10°C to 27% at 16°C. The increased hematocrit is not an effect of
erythrocyte swelling, as no haemoconcentration is observed in splenectomised
fish (Paper III, Fig. 3).
In contrast to the exercise response in rainbow trout, the increase in Q
during acute temperature elevation is entirely the result of tachycardia. If
anything, SV drops, possibly due to the associated reduction in cardiac filling
time. It is worth noting that, as far as I know, no fish species examined so far
seem to increase SV during acute temperature increase. This clearly contrasts
with the typical cardiac response to exercise in many species.
The cardiac filling pressure, as indicated by Pven, does not change in the
rainbow trout when temperature is increased (Paper III, Fig. 2). Since MCFP
increases, the maintained Pven with increasing heart rate at higher temperatures
is most likely mediated by a decreased venous capacitance, which mobilizes
blood to the central venous compartment. It is reasonable to speculate that
without changes in venous capacitance, Pven will drop as fH increases (Altimiras
and Axelsson, 2004) and this will clearly compromise the heart’s ability to
increase Q. In some species, such as Atlantic cod and lingcod, SV is not
compromised as in the rainbow trout, but rather maintained when
temperature is acutely increased over a broad range of temperatures (Gollock
et al., 2006; Stevens et al., 1972). It can be speculated that this is achieved by
an increased cardiac filling pressure, but at present there are no data to either
support or refute this idea. Thus, inter-specific comparisons of the venous
haemodynamic response to acute temperature changes represent an interesting
area to explore in the future.
In mammals, vascular compliance increases passively with temperature
(Green and Jackman, 1979; Rubini, 2005; Shoukas and Brunner, 1980),
although compliance of isolated frog mesenteric venules appears rather
insensitive to temperature (Neal and Michel, 2000). In fish, it is unknown if
temperature has a direct effect on vascular compliance. However, if
compliance does increase with temperature, there will be a conflict between
vascular factors that dictate venous return and cardiac filling pressure (i.e.
venous capacitance) and the need to increase cardiac output with increasing
temperature. In other words, an increased venous compliance at high
temperatures would likely reduce cardiac filling pressure and consequently
compromise the heart’s ability to increase Q. This suggests that compensatory
changes in venous tone and/or compliance are necessary to offset any passive
effects of temperature on vascular compliance in ectothermic animals. Studies
of temperature effects on compliance in isolated vessels/organs from various
42
ectotherms, in combination with studies of intact animals, would likely
provide information about these possibilities.
3.2.3 Hypoxia
The involvement of the venous circulation in the cardiovascular responses to
environmental hypoxia was studied in Paper IV and V. The typical response to
rapidly induced hypoxia in most teleosts is a reflex bradycardia due to an
increased cholinergic tone on the heart (Burleson and Smatresk, 1990; Farrell,
1982; Fritsche, 1990; Fritsche and Nilsson, 1989, 1990, 1993; Holeton and
Randall, 1967; Perry et al., 1999; Randall, 1982; Smith and Jones, 1978; Wood
and Shelton, 1980b). A bradycardic response was observed during severe
hypoxia (water PO2 = 7.3 kPa) in Paper IV and at a water PO2 of ~9 kPa in
Paper V as well. However, the mild hypoxia (water PO2 = 11.5 kPa) in Paper
IV, does not evoke a bradycardic response suggesting that this level of
hypoxia is above the threshold necessary to elicit this response. In both
studies, the observed bradycardia is offset by an increased SV, such that Q
does not change. Regardless of the heart rate response, however, SV always
increased during hypoxia in the present studies. Thus, with the mild hypoxia in
Paper IV, where no bradycardia occurs, this results in a significantly increased
Q. In both studies, hypoxia is associated with a significantly increased cardiac
filling pressure as indicated by the increase in Pven (Paper I, Figs. 1 and 2;
Paper II, Fig. 3). The increased central venous pressure in response to hypoxia
in the present studies is in agreement with one previous study on rainbow
trout (Perry et al., 1999).
It is not possible to directly interpret an increased Pven as a response
mediated by increased venous tone or decreased compliance. The bradycardia
observed in Paper IV and V, and possibly also the reduction in systemic
resistance observed during severe hypoxia in Paper IV, could both explain this
response without an active reduction of venous capacitance. However, the
increased Pven during mild hypoxia in Paper IV, without changes in fH or
vascular resistance, certainly argues in favour of an active mobilization of
blood to the central venous compartment. The study presented in Paper V
was designed to further investigate the venous responses to hypoxia in a more
detailed mechanistic manner. Vascular capacitance curves were constructed
during normoxia and hypoxia (water PO2 = ~9 kPa) in one group of untreated
fish, and in two additional groups of fish that had received pretreatment with
either prazosin to block α-adrenoceptors or bretylium to block transmitter
43
release from adrenergic nerves. This experimental approach allowed us to
address two fundamental questions. (1) Is the increase in Pven observed during
hypoxia associated with an adrenergically mediated decrease in venous
capacitance? (2) If so, is that response mediated by adrenergic nerves and/or
catecholamines released into the blood stream?
It is clear from Table 1 in Paper V that rapidly induced hypoxia does result
in an increased α-adrenergic venous tone; because USBV decreases
significantly whereas C is unchanged at all blood volume intervals and
prazosin treatment blocks these capacitance changes. The response to hypoxia
after bretylium treatment is somewhat more equivocal as the reduction in
USBV is abolished at the 90-110 and 100-120% blood volume intervals, but
not at 80-100% and C decreases significantly during hypoxia at 90-110%
blood volume with bretylium, a response not observed with the other two
treatments. Furthermore, when mean values for MCFP at the different blood
volumes are compared, there does not seem to be any major difference in
response between untreated and bretylium treated rainbow trout, as MCFP
increases significantly during hypoxia in both treatments (Paper V, Fig. 1).
Although it is safe to conclude that an increased α-adrenergic tone can explain
most (if not all) of the capacitance responses to the level of hypoxia used in
Paper V, the relative involvement of circulating and neural catecholamines to
these responses is less certain. As bretylium only partially blocks the
capacitance response to hypoxia, it can be speculated that both circulating as
well as neural catecholamines are involved.
One shortcoming of the study presented in Paper V is that plasma
catecholamines were not measured. However, the level of hypoxia used has
previously been shown to elicit catecholamine release in rainbow trout under
similar experimental conditions (Perry and Reid, 1994; Perry and Bernier,
1999; Ristori and Laurent, 1989). It would be interesting and likely informative
to repeat the study presented in Paper V at milder levels of hypoxia, where
release of circulating catecholamines is less likely to occur, such as the mild
hypoxia used in Paper IV (water PO2 = 11.5 kPa) where Pven increases without
changes in fH or Rsys. It can be suggested that the outcome of such
experiments would be that venous capacitance is mainly controlled by
adrenergic nervous mechanisms at milder levels of hypoxia, while the
importance of circulating catecholamines increases with deeper hypoxia.
Although major inter-specific differences in the overall hypoxic
cardiovascular response exist, it has been debated whether circulating
catecholamines reach levels high enough to affect systemic resistance in
hypoxic teleosts in vivo (Nilsson, 1994; Perry and Bernier, 1999). The study on
rainbow trout in Paper V indicates that circulating catecholamines do not
44
compensate for the hypoxia-induced reduction in vascular resistance, as Rsys
and Pda decrease during hypoxia after both prazosin, as well as bretylium
treatment (Paper V, Fig. 3). The decrease in venous capacitance, however, is
not markedly affected by adrenergic nerve blockade, while it is almost
completely blocked by general α-adrenoceptor blockade (Paper V, Fig. 1 and
Table 1) Thus, it may be speculated that the venous capacitance vasculature in
teleosts is a more important target for circulating catecholamines than the
resistance vessels of the arterial vasculature.
Although it is clear from the above observations that venous capacitance
decreases in rainbow trout during hypoxia, the most fundamental question
regarding the overall benefits of the cardiovascular responses to hypoxia still
remains unanswered. It seems, however, that venous capacitance decreases in
order to increase cardiac filling pressure and SV. The decreased capacitance
probably also reflects a mobilization of blood from less oxygen demanding
tissues (presumably the gastrointestinal tract) to favour perfusion of more vital
organ systems.
45
4. CONCLUSIONS
Prior to this thesis, few studies have addressed the control and function of the
venous circulation in fish. Although some have investigated the
pharmacological responses to various vasoactive substances in vivo, very few, if
any, have examined the putative venous responses that occur in fish during
various natural cardiovascular challenges. The studies presented in this thesis
are therefore an important contribution to the field of comparative
cardiovascular physiology as they, for the first time, demonstrate that vascular
capacitance is actively controlled in teleosts when their cardiovascular system
is challenged by events that occur on a more or less regular basis in their
natural environment.
When Q increases in sea bass and rainbow trout during exercise, a rapid
increase in central venous blood pressure and MCFP is observed. This
suggests that the vascular capacitance decreases and blood is mobilized to the
central venous compartment. Blockade of α-adrenoceptors with prazosin
abolishes the rise in Pven and MCFP in sea bass at 1, but not at 2 BL s-1. In
rainbow trout, the increase in Pven develops much slower during exercise after
prazosin treatment. Additional blockade of angiotensin converting enzyme
with enalapril completely abolishes all changes in Pven and MCFP in rainbow
trout, suggesting that activation of the renin-angiotensin system is responsible
for the slowly developing responses after prazosin. Hence, this study suggests
that the venous circulation in teleost fish is controlled by the RAS, a finding in
agreement with previous studies on mammals, but different from other studies
on fish. Despite the similar venous haemodynamic responses to exercise in sea
bass and rainbow trout, the two species use completely different mechanisms
for increasing Q. Sea bass only increase heart rate, while mainly stroke volume
increases in the rainbow trout during swimming. However, when rainbow
trout is exposed to an acute elevation of the ambient temperature, they also
respond with increased Q and a decreased vascular capacitance, but in contrast
to the exercise response in this species, Q only increases through tachycardia
whereas SV is reduced at the high temperature.
Taken together, it may be suggested that a decreased vascular capacitance
is important when Q increases in order to mobilize blood from less oxygen
demanding tissues, such as the gastrointestinal tract, to the central venous
compartment. However, depending on the heart rate response, Pven and SV
may increase, decrease or remain unchanged. An increased venous tone or
decreased compliance may also be important as a means of preventing blood
46
from passively pooling in the peripheral venous vasculature when blood flow
increases.
Environmental hypoxia typically elicits bradycardia in rainbow trout,
whereas SV increases and Q is unchanged which is in agreement with
numerous previous studies on teleosts. These responses are associated with a
significant increase in Pven and a reduced vascular capacitance. At least with the
present experimental protocol, this is the result of an α-adrenoceptormediated reduction of the unstressed blood volume, involving both neural and
humoral components.
Future studies on venous function in fish can preferably be directed at
regional capacitance changes. For example, the splanchnic venous vasculature
is probably the most important blood volume reservoir in mammals. More
detailed studies of this part of the circulation in fishes would likely provide a
more complete understanding of the importance and function of the venous
capacitance vasculature in fish. Furthermore, direct studies of the small veins
and venules in different vascular beds are required to resolve their role in
overall circulatory homeostasis in fish.
47
5. ACKNOWLEDGEMENTS
There are many people that have contributed to this thesis in different ways. I would like to
thank the following people:
First of all, my dear supervisor Michael Axelsson, for your positive, generous, wise and
helpful personality. Working under your supervision has made my PhD-studies extremely
inspiring and a true pleasure.
Tony Farrell, for being my “external mentor”. Your impact on my training to be a
scientist cannot be overestimated. While working at West Vancouver lab, you and your wife
Miriam have accommodated me in your home for several weeks; I have enjoyed and learned
tremendously from those stays.
Susanne Holmgren, for always backing up the PhD-students at the department and for
keeping a high and professional standard on the PhD-education.
Guy Claireaux, for inviting us to do some productive work on sea bass in La Rochelle.
Thank you for all the nice dinners in the backyard and for lending your bikes so Karin and I
could see Ile de Ré. Jordi Altimiras, also part of this research trip, for many interesting
discussions during late nights in the lab. Thank you for always showing such encouragement
and genuine interest in my work. David McKenzie, for kindly lending me the swim-tunnel
used in the sea bass work and for inviting me to Hirtshals to use the same swim-tunnel for
some more work on rainbow trout. Thank you all for excellent and interesting collaboration.
Lena Sundin, my assistant supervisor, for always taking time to help and discuss whatever
crossed my mind.
Thrandur Björnsson, for helping me with all the bureaucracy towards the end of my
period as a PhD-student and for linguistic improvement of this thesis.
In addition, I have been fortunate to be surrounded by people, who have not been directly
involved in the work with this thesis, but have facilitated or brightened up my time at the
department in various ways:
The rest of the COCA-group, including: Jenny Turesson, who took really good care of
me and let me stay in “her” room when I was a fresh PhD-student. Henrik Seth, who joined
the group after me and was supposed to learn fish surgery from me (now I learn from him),
it’s great with someone who actually thinks that you know what you’re doing… Albin Gräns,
who recently joined the group and who I already have enjoyed a fair number of good beers
with. By the way, Albin, I really look forward to future “pike adventures”! Thank you all for
friendship, inspiring collaboration and great company at conferences and other occasions.
Gunilla Rydgren, for helping me with odds and ends, from surgery to ordering
equipment.
Stefan Nilsson and Kristina “Snuttan” Sundell for being great sources of inspiration. In
the early years, your lectures opened up my eyes for the beauty of comparative physiology.
Stefan, thank you for reading and providing valuable input to this thesis.
The GENI group, Jenny, Monika, Anna, Catharina, Fatemeh, Susanne and Christina, for
tasty cakes and for reading and commenting on this thesis.
Snuttan and Barbro, for help with scary isotopes.
The professional “TA-personnel”, Bernth, Lena, Ann-Sofie, Agneta, Erika, Lillioth, LarsÅke, Birgit, Mattias, Kalle, Birgitta, Peter and Bengt, for helping me with all the difficult
things. Particularly, I would like to thank Bernth for bringing home all the fish used in my
studies.
48
The boys in the “whisky-club”: Jon, Daniel, Andreas, Henrik and Albin, for well-deserved
breaks and interesting, but not always so “scientifically relevant”, discussions.
David and Åsa, for occasionally visiting Göteborg and for accommodating me in Umeå.
It’s just a pity you live so close to the polar circle.
And, of course, all other past and present PhD-students at Zoologen, from first to fourth
floor (you know who you are). Thank you for all good times!
During my time at Zoologen I have had the opportunity to work on various other exciting
projects, not included in this thesis. I am indebted to:
Tobias Wang, for great hospitality and dinner-parties while working on turtles in Århus,
for agreeing to be my opponent and for being such an energetic advocate of “old-fashion
physiology”. We should definitely publish together some day!
Maria Faldborg Steinhausen, a Danish “iron lady”, who I spent weeks together with in a
tent swimming sockeye salmon. Your energy and positive attitude is just beyond imagination.
I wish we can work together again sometime.
Joakim Larsson, for interesting collaboration on β-blockers.
Christopher Wilson, for excellent practical assistance and food (i.e. donuts, pizza and
Asian junk food) delivery while working on dogfish in Vancouver.
I also would like to thank some old and very dear friends:
Stig Jonasson, for being like no one else and for reminding me about the (other) real
world outside Zoologen.
Fredrik Jutfelt and Henrik Sundh, for long-lasting friendship and many memorable
moments, both academically, but perhaps even more important, under “significantly nonacademic circumstances”. Fredrik, I will never forget our first scientific and non-scientific
experiments back in the “good old days” in Kalmar. They shaped me forever and you can, to
a large extent, be held responsible for where I am today.
Ida Dahlberg (and Fredrik J), for friendship and a particularly welcoming and open home.
Behind successful scientists there is often a supportive family that rarely receives the
appreciation it deserves. Although I do not claim to be a successful scientist, I would
nevertheless like use this opportunity to thank my family, which is the most supportive and
caring one could wish for.
My second family, Ingrid and Roger, Anna and Fredrik. Thank you for spoiling us in
Dalsjöfors.
My first family, Mum, Dad, Anna, Karin and Fredrik and the rest of the “Sandblom clan”
in Sandhult. Mum and Dad, I don’t know whether it’s the genes or the environment, but I‘m
sure you made me understand that anything is possible with hard work and a curious and
positive mind. Thank you for always letting me follow my dreams and “keep on playing with
my fishes”, instead of trying to force me into something more conventional.
Finally, unarguably the most important person:
Karin, my love and wise companion through life. I do not think you fully realize how
important your contribution to this thesis is. Your company, endless support and
understanding means everything to me.
My research has been financially supported by: The faculty of Science, Göteborg University and grants from
Vetenskapsrådet, Helge Ax:son Johnsons stiftelse, Paul och Marie Berghaus donationsfond, Kungliga
Vetenskapsakademien, Hierta-Retzius fond, Knut och Alice Wallenbergs stiftelse, Jubileumsfonden, Viktor Rydbergs
minne and Society for experimental biology.
49
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