Form and function of the bulbus arteriosus in yellowfin tuna (Thunnus albacares), bigeye tuna (Thunnus obesus) and blue marlin (Makaira nigricans): static properties
1 Department of Zoology, Cambridge University, Downing Street, Cambridge,
UK, CB2 3EJ
2 Cooperative Marine Education and Research Program, Virginia Institute of
Marine Science, PO Box 1208, Greate Rd, Gloucester Point, Virginia 23062,
USA
3 Department of Zoology, University of British Columbia, Vancouver, BC,
Canada, V6T 1Z4
4 Zoology Animal Care, 6199 South Campus Road, University of British
Columbia, Vancouver, BC, Canada, V6T 1W5
* Author for correspondence (e-mail: mhb31{at}cam.ac.uk)
Accepted 22 June 2003
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Summary |
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Key words: bulbus arteriosus, P-V loop, r-shaped curve, stress, modulus, tuna, marlin, Thunnus, Makaira
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Introduction |
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Whether the bulbus is cardiac or arterial in nature is an open question
(Benjamin et al., 1983).
However, due to some obvious similarities, arterial nomenclature is used to
describe the bulbar morphology. As in arteries, the bulbar wall is composed of
three layers: an intima composed of a single layer of endothelial cells, a
thick media primarily composed of elastin and smooth muscle, and a collagenous
adventitia surrounded by an outer layer of mesothelial cells.
The endothelial cells can be squamous, columnar or cuboidal (Leknes,
1981,
1985
; Benjamin et al.,
1983
,
1984
) and often contain
membrane-bound bodies that stain strongly with periodic acid-Shiff's,
indicating the presence of carbohydrates
(Benjamin et al., 1983
;
Leknes, 1981
). There is
microscopic evidence for the discharge of membrane-bound vesicles into the
lumen (Benjamin et al., 1983
);
however, their contents are unknown. Except for the layer of mesothelial
cells, the adventitia is almost entirely collagen
(Benjamin et al., 1983
;
Bushnell et al., 1992
) and is
thought to limit bulbar strain (Priede,
1976
; Raso, 1993
;
Icardo et al.,
1999a
,b
).
The media forms 90-95% of the bulbus and is composed of smooth muscle sparsely
distributed within an elastin framework
(Licht and Harris, 1973
;
Watson and Cobb, 1979
;
Icardo et al., 2000
).
Exceptions do exist, however, as the bulbus of Pleuronectes platessa
contains no smooth muscle (Santer and
Cobb, 1972
).
The term `J-shaped curve' is used to describe the non-linear mechanical
properties (stress and modulus) of biological materials. While arterial P-V
loops are often sigmoidal, the nonlinear transformation of pressure into
stress, and volume into strain, results in J-shaped stress-strain curves.
Furthermore, over the operational pressure range, most arterial P-V curves are
J-shaped. Within arteries, the J-shaped curve results from elastin and
collagen working in conjunction as a strain-limiting system
(Wainwright et al., 1976). At
low extensions, rubber-like elastin resists deformation, resulting in a low
initial slope. However, at higher extensions, the collagen in the arterial
wall (approximately 1000x stiffer than elastin) is also recruited to
resist deformation, increasing the stiffness of the arterial wall and causing
a sharp increase in the slope. Bulbi also possess elastin, smooth muscle and
collagen, but when a bulbus is inflated
(Licht and Harris, 1973
;
Priede, 1976
;
Bushnell et al., 1992
) the P-V
loops can best be described as `r-shaped' over the physiological pressures,
with the distinguishing feature being a continuously decreasing slope. With
respect to the bulbar inflations, the r-shaped curve describes a sharp initial
rise in pressure followed by a plateau phase in which large changes in volume
result in small changes in pressure.
While both the cause and the significance of the J-shaped arterial P-V loop have been studied extensively and are well understood, similar studies of the unique bulbar inflations do not exist, leaving a fundamental piece of fish physiology unknown. To this end, the analysis presented here addresses the causes of the r-shaped bulbar P-V curve and relates the mechanical properties of the wall material to the many modifications that have occurred within the bulbar wall.
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Materials and methods |
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The gross morphology of the bulbi was compared between species and pictures were taken. Two bulbi and ventral aortae from yellowfin tuna were fixed in buffered formalin and sent to Wax-it Histology Services (Aldergrove, BC, Canada) for sectioning and staining. One bulbus was fixed at physiological pressure (10 kPa), while the other was fixed at zero pressure. The sections were stained with a Verhoeff's elastic stain.
Yellowfin tuna bulbi for use in electron microscopy were prefixed in buffered formalin to prevent structural changes while the tissues were transported to the University of British Columbia Electron Microscopy Facility (Vancouver, BC, Canada). Samples were fixed in 2.5% glutaraldehyde in 0.1 mol l-1 sodium cacodylate (pH 7.2) and, following fixation, were washed in 0.1 mol l-1 sodium cacodylate buffer overnight and post-fixed in 1% osmium tetroxide. After a graded alcohol dehydration, the samples were stained using uranyl acetate and lead and embedded in Epon/Araldite. The blocks were sectioned with a glass knife and viewed with a Zeiss EM 10C (Carl Zeiss Inc., Oberkochen, Germany).
P-V loops
Bulbi from small yellowfin tuna (<3 kg) were obtained from freshly
killed fish held in large outdoor tanks at the National Marine Fisheries
Service Kewalo Research Facility in Honolulu, HI, USA. The water temperature
in the holding tanks was 25°C. After death, an incision slightly posterior
to the gills was made on the ventral surface, exposing the pericardium. The
pericardium was slit in the midline, and the length of the bulbus arteriosus
was measured in situ. The bulbus arteriosus was then removed by
cutting posterior to the bulbo-ventricular junction and anterior to the
bulbo-aortic junction and placed in saline. Bulbi from larger yellowfin tuna
(>10 kg) and blue marlin (>25 kg) came from the fish auction in Honolulu
and were removed through a lateral incision underneath the operculum.
Sections of blue marlin ventral aortae were dissected from fish being sold
at the Honolulu fish auction, while yellowfin tuna ventral aortae were
dissected from freshly killed animals. All yellowfin tuna dorsal aortae were
inflated in situ as they are tightly bound to the spinal column and
our attempts to separate them resulted in leaks. For two yellowfin tuna, the
dorsal aortae were heavily parasitized by the larval cestode Dasrhynchus
talismani (Brill et al.,
1987).
The bulbus was double cannulated using PE tubing with flared ends, and ligatures were placed behind the flare around the bulbo-ventricular and bulbo-arterial junctions. The anterior cannula was attached to a pressure transducer, while the posterior cannula was attached to a syringe filled with saline solution. The bulbi from yellowfin tuna were held in a tissue bath at their in vivo length. The blue marlin bulbi were not held at any specific lengths because we were unable to measure their in vivo size during removal through the lateral incision. The sizes of tubing or syringes were picked to best fit the size of the bulbus. We chose tubing that approximated the diameter of the ventral aorta and syringes that were determined experimentally to be 150% of the respective bulbar volume. The saline solution was left at room temperature (25°C). The arteries were also cannulated in both ends, with one cannula attached to a pressure transducer and the other to an infusion syringe.
A measured volume of fluid was then injected into the bulbus or artery in steps, and the resultant pressure signal was amplified and recorded using DASYLAB software (Dasytec USA, Amherst, NH, USA). To limit the effects of stress-relaxation, cycles of inflation and deflation were performed until consistent results were seen. Preconditioning usually required 5-10 cycles for the bulbus and 3-5 cycles for the arteries. These initial cycles were discarded. Each experiment consisted of 8-15 trials, and results from any trials in which a loss of more than 5% of the injected saline occurred were not used.
Before fluid injections, bulbar volume was assumed to be zero, as the lumen of both the blue marlin and yellowfin tuna bulbi is completely occluded with longitudinal elements at zero pressure. A maximal pressure determined the volume to which the bulbi and arteries were inflated experimentally. Going past this pressure often resulted in failure of the preparation, as evidenced by a fluid leak. To allow comparison among different sized fish or different vessel lengths, injected volume was normalized to the maximum volume of the vessel, resulting in plots of pressure versus a unitless volume with a maximum of one.
Bulbi from freshly killed yellowfin tuna (N=4) were used to study the effects of blocking bulbar smooth muscle and denaturation of bulbar collagen. Bulbi were mounted in a tissue bath full of saline solution. After pre-conditioning the bulbus, inflation-deflation cycles were performed that were considered to be indicative of the normal P-V behaviour. The bulbus was then placed in a 10-5 mol l-1 solution of the Ca2+ channel blocker nicardipine for 10-15 min, while the interior of the bulbus was filled with the same nicardipine solution. The bulbus was again preconditioned, followed by several inflation-deflation cycles. Nicardipine is light sensitive, so these trials were performed in dim light and the bath was covered with a black cloth.
Fish collagen has thermally sensitive inter- and intramolecular crosslinks
that break at relatively low temperatures. Rose et al.
(1988) have shown that the
denaturation temperature of halibut (Hippoglossus stenolepsis)
collagen (a cold-water fish) is 17°C, while the denaturation temperature
of big-eye tuna collagen (Thunnus obesus; a warm-water fish) is
31°C. Our protocol for all fish involved placing the bulbus in water
ranging from 60°C to 100°C for 30-45 min. The bulbus was then cooled
down to room temperatures. The effect of heat treatment is to solubilize the
collagen, reducing its mechanical integrity. Following this, the bulbus was
again preconditioned before performing a series of inflation-deflation loops.
The data from different fish were averaged, and the different P-V loops [(1)
normal; (2) smooth muscle blocked and (3) collagen denatured and smooth muscle
removed] were plotted on the same grid and compared.
The marlin bulbus arteriosus is long and thin. This makes it amenable to a
number of studies not easily performed on bulbi from other fish. Four blue
marlin bulbi were turned inside out and inflated. The normal protocols for
generating P-V loops were followed. An additional five blue marlin bulbi were
turned inside out to dissect away tissue. The order in which the tissues were
removed was: (1) longitudinal elements, (2) remainder of inner layer and (3)
media. The wall was sequentially removed until only a very thin layer of the
outer bulbar wall remained. After each tissue removal, the marlin bulbus was
turned the right way out and inflated. After several bulbar layers had been
removed, the bulbar volume was no longer zero at zero pressure and fluid could
be injected into the empty bulbus with no rise in pressure. Inflation pressure
and volume were recorded from the point where wall elements resisted the
increase in volume. Since we did not count the fluid used to initially fill
the bulbus, the x-axis of the P-V loops is, in fact, the change in
volume (V) of the bulbus after pressure began rising and not
the total volume injected.
Stress-strain experiments
Strips of tissue were dissected from the bulbi of freshly killed yellowfin
tuna and bigeye (Thunnus obesus L.) tuna. The bulbi were obtained
from small (<3 kg) yellowfin and bigeye tuna held in large outdoor tanks at
the National Marine Fisheries Service Kewalo Research Facility in Honolulu,
HI, USA. The tissue was cut into a rectangular shape, and the dimensions were
measured using calipers to an accuracy of ±0.2 mm. One end was glued
transversely to a piece of metal attached to a micromanipulator. The other end
of the tissue was attached to a Grass force transducer (model FT03;
Grass-Telefactor, West Warwick, RI, USA) to record forces produced as the
material was stretched. The internal longitudinal elements were tested as
intact structures. Complete strips of the wall were stretched in both
longitudinal and transverse directions. The outer, adventitial layer of the
wall and the inner, medial layer were dissected free and tested individually.
Extensions of the strips occurred in steps and the length was measured with
calipers. Once force and extension had been recorded, the values of stress,
strain and modulus were calculated as described below.
Transverse loops of large bulbi from yellowfin tuna (N=6) and blue marlin (N=4), obtained from the fish auction in Honolulu, were cut from the anterior, middle and posterior regions of each bulbus. Thickness, width and diameter of each loop were measured with calipers to an accuracy of ±0.2 mm. In addition to the rings consisting of the entire wall, tests were also conducted on rings consisting of the adventitia or outer medial layers of the bulbus. Ventral aortic loops from blue marlin and yellowfin tuna were also tested.
The loops underwent uniaxial force-extension tests. These tests were conducted using a custom-built `stretching' machine. The loops of tissue were mounted over two L-shaped stainless steel bars and placed in a 25°C saline bath. The ends of the stainless steel bars were filed down to a combined diameter of 1.2 mm. One of the bars was attached to the bath while the other was attached to a moveable crosshead through a force transducer. The force-extension tests were performed by slow cyclic stretching via the crosshead at a constant velocity of 6 cm min-1. Preconditioning of the loops was achieved by performing at least five preliminary cycles until the force-extension behaviour of the tissue was consistent.
Strain () is the ratio of the change in length (L) divided
by the initial length (L0) and was calculated by the
formula:
![]() | (1) |
Stress () was expressed as true stress and was calculated assuming
constant volume as:
![]() | (2) |
For bulbar loops, we used a value of thickness that was smaller than the full thickness of the bulbus wall to calculate stress because in a number of force-extension studies, the inner medial layer of the bulbus (with longitudinal elastin fibres) ripped with no change in the slope of the force curve being generated. This suggested that the inner layer of the bulbus was contributing little circumferential strength to the wall. Consequently, including the thickness of the inner medial layer when calculating stress would result in a significant underestimation of the true stress. The inner media is approximately 60% of the wall and, therefore, the value used was approximately 40% of the full thickness of the bulbus wall.
Circumferential stiffness was calculated, according to Bergel
(1961), as the incremental
modulus (Einc):
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The stress-strain curves were smoothed using TABLECURVE (Jandel Scientific, San Rafael, CA, USA), a curve-fitting program that generates a number of different equations describing the curve. Once an appropriate fit was found, the equation was used to generate the exact dependent value of any desired independent variable (the y-value for any input x). This allowed the comparison of different stress-strain curves at the same values of strain.
Statistics
Descriptive statistics (means ± S.E.M.) were calculated
for the P-V curves generated by blocking the smooth muscle and heating the
bulbus, as well as for all the stress-strain curves generated by the various
extension protocols. The treatments in the blocking and heating study were
compared using a twoway repeated measures analysis of variance (ANOVA). For
comparisons between the stress-strain curves for the anterior, middle and
posterior segments of the tuna and marlin bulbi, a two-way ANOVA was
performed. Multiple comparisons between the different treatments for the P-V
and stress-strain curves were performed using a Tukey test. All comparisons
between groups were performed using SIGMASTAT 2.0 (Jandel Scientific).
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Results |
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There are obvious differences between cross-sections from a yellowfin tuna bulbus (Fig. 2A) and ventral aorta (Fig. 2B). Despite the fact that these sections were separated by <1 cm, they show a large difference in wall thickness. It is within the wall, however, where the important differences occur. The ventral aorta has a typical arterial composition. Layers of smooth muscle (sm), sheets of elastin called lamellae (el) and collagen (co) are found in close proximity, forming regular layers throughout the wall. By contrast, the anterior bulbus of yellowfin tuna contains no elastin lamellae. The elastin fibres (ef) are ordered but not joined into large continuous sheets. In the ventral aorta, elastin lamellae alternate with layers of smooth muscle. Despite the same staining technique, no smooth muscle is obvious in this bulbus section (Fig. 2A).
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Bulbar elastin is stained very heavily with Verhoeff's elastin stain, obscuring much of the smooth muscle. Despite this, layers of smooth muscle can still be seen. Fig. 2C is alongitudinal section of the bulbus near the ventricle and shows a block of smooth muscle that is sandwiched between two layers of longitudinal elastin fibres (lf). The smooth muscle layer extends anteriorly just beyond the top of the frame in Fig. 2D and posteriorly until it appears to attach to the collagenous ventriculo-bulbar pocket valve.
The adventitia (a) of the yellowfin bulbar wall is a thin fibrous layer (Fig. 3A,B) containing mesothelial cells and collagen. The bulk of the bulbar collagen is concentrated in this outer layer. The adventitia does contain a small amount of elastin; however, it is not as ordered or in as high a concentration as in the rest of the bulbus. The majority of the bulbus wall is media (m), which is divided into two layers: a dense outer layer containing elastin fibres running in a circumferential manner (om) and a `looser' inner media in which the majority of the elastin fibres are oriented longitudinally (im; Fig. 3A,B). This inner media contains the longitudinal elements (le).
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Wall thickness and internal morphology differ throughout the length of the bulbus. While the size of the collagenous adventitia remains relatively constant in both sections, the outer media at the posterior end (Fig. 3B) is approximately half the thickness of the same layer in the middle bulbus (Fig. 3A). This is accompanied by an increase in the size and complexity of the inner media, specifically the longitudinal elements. The architecture of the longitudinal elements is most elaborate near the ventricle. Their morphology is very irregular, with an overall `spongy' appearance.
The outer portion of the media (Fig. 3C) has a thick layer of circumferential elastin fibres (cf), with the long axes of the fibres obvious in this transverse section. When moving from the adventitia to the lumen, an abrupt transition to longitudinal fibres (lf) occurs, marking the transition between the inner and outer media. The long axes of these elastin fibres cannot be seen; instead, the longitudinal fibres show up as small circles, the result of being sectioned transversely. The longitudinal orientation of elastin fibres is maintained throughout the longitudinal elements (Fig. 3D). However, to the right of the longitudinal element is an attached structure with neither longitudinal nor circumferential fibres. This is a radial element (re) attaching the longitudinal element to the wall.
While the majority of adventitial collagen fibres (Fig. 4A) occur in large longitudinally arranged bundles (lf), there are also smaller bundles that run circumferentially (cf). The wavy circumferential fibre bundles appear to be relatively short. Elastin and smooth muscle make up the majority of the media (Fig. 4B). Very little collagen occurs in the media or along the luminal surface (Fig. 4B). The smooth muscle cells possess a large number of plasmalemmal vesicles (arrows) surrounding much of the cell. While the bulbar elastin is not found in concentric lamellae, the fibrils do show an orientation suggesting an association with the smooth muscle cells.
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Smooth muscle cells in the yellowfin bulbus are not always sparsely
distributed within the elastin (Fig.
4B). Fig. 4C shows
a large number of smooth muscle cells in close proximity. The cells in this
muscle layer are not attached but they do possess projections that may
functionally link the cells. Layers of smooth muscle in the bulbus have been
described as possessing a spiral orientation
(Watson and Cobb, 1979;
Yamauchi, 1980
); however, it
was difficult to establish an exact orientation of the muscle layer within the
yellowfin tuna bulbus.
Endothelial cells modified for a secretory role are common along both the luminal surface and the longitudinal elements (Fig. 4D). These endothelial cells contain the plasmalemmal vesicles found along the margin of the membrane (arrows) as well as much larger vesicles (arrowheads) that are found throughout the cells.
P-V loops
After a small injection of fluid, bulbar P-V loops
(Fig. 5) from both yellowfin
tuna and blue marlin showed a steep initial increase in pressure that was
followed by a compliant plateau phase. The yellowfin tuna bulbi were most
compliant over the mean ventral aortic pressure of 12.08±1.15 kPa
(Jones et al. 1993), while the
J-shaped P-V loops of the yellowfin tuna were most compliant below the mean
ventral aortic pressure (Fig.
5). In fact, most of the pressure increase in the yellowfin tuna
arterial P-V loop occurred over the last 20% of volume injected. The ventral
aorta of the blue marlin did not show a simple J-shaped P-V loop
(Fig. 5). The slope of the
curve fell before rising, resulting in two inflection points. Despite this
more complex behaviour, the marlin ventral aorta possessed inflation
characteristics that were comparable with those of the yellowfin tuna: large
compliance at low pressures, becoming increasingly stiff over the pressure
range of 10.7-21.3 kPa.
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On a P-V loop, the area within the loop as a percentage of the area under the inflation part of the cycle is a measure of hysteresis, or energy loss. Both the yellowfin tuna and blue marlin ventral aortae showed very little hysteresis (10.2% and 9%, respectively), indicating that they were highly resilient elastic structures.
The dorsal aorta of yellowfin tuna exhibited the continuous rise in slope
with increasing volume that is of functional importance for arteries
(Fig. 6). The dorsal aortae of
two yellowfin tuna were parasitized by the larval cestode Dasrynchus
talismani (Brill et al.,
1987). This resulted in the dorsal aortic lumen being occluded at
low pressures. As in the bulbus, the inflation behaviour of these vessels was
distinctly r-shaped over much of the volume range
(Fig. 6). Only at high
pressures did the behaviour of the parasitized and unparasitized vessels
become comparable. At roughly peak systolic pressure for yellowfin tuna
(Jones et al., 1993
),
stiffness in the parasitized aorta rapidly rose, and final pressures in both
the parastized and unparasitized vessels were similar
(Fig. 6).
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When smooth muscle and collagen were removed or inactivated, the P-V curves of the bulbus changed. Control inflations on fresh bulbi from yellowfin tuna showed typical r-shaped inflation curves (Fig. 7). The addition of 10-5 mol l-1 nicardipine (a Ca2+ channel blocker), added to inactivate smooth muscle, decreased the magnitude of the plateau portion of the curve compared with that of the control level. Heating resulted in the bulbus becoming even more compliant, as the curve generated post-heating was lower than either the smooth muscle-blocked bulbi curves or the control bulbi curves. Repeated measures twoway ANOVA showed that the treatments had a significant effect on the P-V curves (P<0.001, N=4).
|
Over the same pressure range, P-V loops for inside-out blue marlin bulbi had the same shaped curves as P-V loops from normal blue marlin bulbar inflations (Fig. 5). After dissecting away the longitudinal elements, the bulbi were turned right way out and retested. Removing the longitudinal elements reduced the pressure level of the plateau (Fig. 8); however, the curve remained r-shaped. When the inner media was also removed and the bulbus was reinflated in its correct morphology, the curve changed drastically, with the initial slope falling 20-fold. The discrepancy in initial slopes resulted in a large difference in the pressures reached at a given volume. An inflation of 4 ml resulted in a pressure increase of approximately 1.33 kPa in the dissected bulbus, while the control curve showed a pressure of almost 10.7 kPa. When the volume in the dissected bulbus was increased from 6 ml to 8 ml of fluid, the slope rose rapidly, resulting in a pressure of 14.7 kPa (Fig. 8). The r-shaped inflation curve of the bulbus became J-shaped. All bulbi tested in this fashion behaved similarly.
|
Stress-strain experiments
Loops of the adventitia and outer media subjected to uniaxial extensions
had J-shaped stress-strain and modulus-strain curves; however, at strains
greater than one, the adventitial layer generated higher stresses
(Fig. 9A) and was much stiffer
(Fig. 9B) than the outer medial
layer. At a strain of 1.2, the adventitial level had a modulus of 1900 kPa,
while the outer medial layer had a modulus of 180 kPa. Despite this large
difference in stress and stiffness at strains above one, at strains below one
the mechanical properties were similar.
|
While the adventitia broke at a strain of 1.25, the outer media reached strains of over 1.7. This large difference in breaking strains is somewhat misleading, because strain is normalized to the initial length of the material being tested. Since the adventitial layer had a larger initial diameter than the outer media, the absolute length at which the adventitial and medial layers broke was much closer than the breaking strains suggest.
The stress-strain curves generated from loops of tissue cut from the bulbi of yellowfin tuna (Fig. 10A) and stretched circumferentially did not have the r-shape characteristics of the bulbar P-V loops. Instead, the stress-strain curves had the normal J-shaped curve of many biological materials. The slope of the curve remained low until a strain of 1.2, at which point the stress rapidly increased. The modulus-strain curve (Fig. 10B) showed that the stiffness of the material rose exponentially, with the major rise beginning at a strain of 1.2. At strains below 0.8, the curves were not significantly different. However, at higher strains, two-way ANOVA showed that the middle portion was significantly different (P<0.001, N=6) from the anterior and posterior portions of the bulbus; the middle section had a higher stiffness and was less extensible than both the posterior and anterior bulbar sections.
|
The levels of stress and modulus measured in all the portions of the
bulbus, despite using the smaller value of thickness, were significantly lower
than the mean value calculated for the ventral aorta
(Fig. 10C). Shadwick
(1999), using vessel
inflations rather than uniaxial extensions, found a similar value for the
elastic modulus of yellowfin tuna ventral aorta at mean blood pressure.
Like yellowfin tuna bulbar rings, blue marlin bulbar rings also reached high levels of strain when stretched circumferentially (Fig. 11). Unlike the yellowfin tuna, however, there was no difference between the different segments with regard to the values of stress or modulus (Fig. 11A,B). At a strain of 1.2 (the largest strain at which there were data for all three segments) there was no significant difference between the values of stress or modulus. The marlin ventral aorta was stiffer than the marlin bulbus (Fig. 11C); modulus values of the marlin ventral aorta were three times higher than those of the marlin bulbus at a strain of one (Fig. 11C).
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Stress-strain data for the extensions of roughly rectangular bulbus segments from yellowfin and bigeye tuna are shown in Fig. 12A. The same data are plotted as modulus-strain data in Fig. 12B. The tissues obviously fell into two general groups: (1) stiff and (2) extensible. At a strain of one, the modulus for most of the tissues fell into a range that lay between 50 kPa and 100 kPa. Values gathered by stretching tissue rings (i.e. middle layer of yellowfin tuna bulbus stretched circumferentially, marked with an asterisk in Fig. 12) also fitted into this range. Despite the variability inherent in dissecting pieces of tissue away from the wall, and irrespective of circumferential or longitudinal extensions, most of the different tissues had similar values of stress and modulus over a wide range of strains. The exceptions were the two longitudinally stretched sections of bulbar outer layer from bigeye tuna. Compared with the other tissues, both pieces became very stiff at a low strain, reaching a modulus of over 400 kPa at a strain of 0.5. These longitudinally stretched outer layers were limited to strains under 0.5; the other tissues all reached strains well over one.
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Discussion |
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While there is no mystery to the differences between the shapes of the bulbar P-V and stress-strain curves, the fact remains that no arterial P-V loop (sigmoid or otherwise) possesses such a steep initial rise in pressure or such a compliant plateau before the reinforcement of the collagen fibres rapidly increases the stiffness of the wall.
Qualifying the descriptions of the inflation curves as r-shaped over the
physiological pressure range is obvious in the case of the yellowfin tuna.
Unfortunately, we do not know the blood pressure in marlin. However, many
aspects of marlin physiology are similar to those of tuna, and the available
evidence suggests that marlin hearts and blood vessels are similar to those of
other teleosts (Davie, 1990).
The fact that tuna bulbi share their special inflation properties with
Cyprinius carpio (Licht and
Harris, 1973
), Oncorhynchus mykiss
(Priede, 1976
; M. H. Braun and
D. R. Jones, unpublished data) and Oncorhynchus kitsuch (M. H. Braun
and D. R. Jones, unpublished data) suggests that, over the physiological
pressure range, the r-shaped P-V loop is a feature of bulbi. For these
reasons, we assumed that the r-shaped marlin inflation curve also occurs over
the physiological pressure range.
The initial rise of the bulbar P-V loop is explained by the Law of Laplace, which states that T=Pr, where T is tension. In essence, a larger pressure is required to inflate a small cylinder than a large cylinder (assuming the two structures have similar wall thicknesses and material properties). While the external diameter of the bulbus is larger than that of the ventral aorta, the thin walls of the ventral aorta result in relatively large internal diameters at all pressures. Therefore, tension rises rapidly as pressure increases. In the bulbus, however, the thick wall, combined with the longitudinal elements, results in a lumen that is relatively small. In fact, when the bulbus is empty, the lumen is nearly occluded by the longitudinal elements (Fig. 13). The small bulbar lumen requires that injected volume be applied at high pressure in order to overcome the large wall tension and allow expansion.
|
As the longitudinal elements are pushed out to the walls and lumen radius grows (Fig. 13), wall tension decreases, and the pressure increments generated by each subsequent volume injection decline, generating the r-shaped curve. Evidence for this interaction was seen when the internal layers were removed from marlin bulbi (Fig. 8). As the effective internal radius increased, the pressure generated by the initial volume increments decreased. This resulted in a drop in the level of the plateau to the point where the inflation became J-shaped.
The yellowfin tuna dorsal aortae occluded by parastic cestodes provided a natural experiment and support our explanation of the role of the Law of Laplace in generation of the r-shaped curve. The P-V loops of normal dorsal aortae were essentially J-shaped, while, over much of the in vivo pressure range, the P-V loops for the parasitized aortae (despite being structurally identical to the unparasitized specimens) were distinctly r-shaped. Just as the bulbus required the initial volume to be injected at a large pressure to push the longitudinal elements against the inner wall, the parasitized dorsal aorta also developed a large pressure that would push the long thin larval cestodes aside, opening the lumen for flow. However, at large volumes, while the bulbar curve remained relatively flat, the parasitized aorta showed the same large rise in pressure as occurred in the unparasitized aorta. The bulbus is more than an occluded artery, and another mechanism is required to explain the compliant plateau phase of the bulbar P-V loop.
The bulbus is composed primarily of elastin, with estimates of the actual
amount of elastin within the bulbus ranging from 70% in salmonids
(Serafini-Fracassini et al.,
1978) to 90% in carp (Licht
and Harris, 1973
). In rainbow trout, the elastin:collagen ratio is
about 14 (Serafini-Fracassini et al.,
1978
), compared with 1.5 in mammalian proximal aorta
(McDonald, 1974
). The higher
the elastin:collagen ratio, the more compliant is the structure. Licht and
Harris (1973
) found the bulbus
to be 32x more distensible than the human thoracic aorta over the same
pressure range. In both yellowfin tuna and blue marlin, the bulbi had larger
breaking strains and much lower moduli than the respective ventral aortae. The
large extensibility and low modulus are key to the extreme compliance of the
bulbus during the plateau of the P-V loop.
Unlike many fish arteries (Leknes,
1986), the design of the yellowfin tuna ventral aorta is very
similar to that of the mammalian artery
(Dobrin, 1978
), with concentric
elastic lamellae separated by layers of smooth muscle and collagen
(Fig. 2B). While this may be an
adaptation to the high blood pressures of tuna, no similar adaptation has
occurred within the yellowfin tuna bulbus (Figs
2A,
4B). In teleosts, bulbar
elastin is found not in lamellae but in fibrils
(Licht and Harris, 1973
;
Serafini-Fracassini et al.,
1978
; Benjamin et al.,
1983
; Icardo et al.,
2000
), and collagen has been largely relegated to the thin
adventitia. The intimate connections between elastin and smooth muscle seen in
mammalian arteries do not exist in the bulbus.
Teleost elastin, unlike its mammalian counterpart, is not found in elastic
fibres composed of an amorphous elastin component and elastin-associated
glycoprotein microfibrils. Elastin-associated microfibrils are extremely rare
in teleost arteries (Isokawa et al.,
1990), and teleost arterial elastin is almost exclusively found in
a fibrillar form, a morphology shared with the bulbus. While the fibrils of
elastin superficially resemble the glycoprotein microfibrils of mammalian
elastic fibres, Serafini-Fracassini et al.
(1978
), Benjamin et al.
(1983
) and Isokawa et al.
(1988
) have demonstrated,
using elastases, elastin stains and molecular analyses, that the fibrils are
elastin.
The benefits of the fibrillar design of bulbar elastin can be seen in the
considerable radial expansion that the bulbus experiences during each heart
beat. Benjamin et al. (1983)
have suggested that lamellar sheets would be unable to manage the large-scale
length changes required. Either the sheets would tear at high strains or the
folding necessary at low strains would disrupt the structure of the bulbar
wall. However, these problems suggested by Benjamin et al.
(1983
) could be remedied by
relatively minor lamellar modifications. Longer lamellae, which would require
a larger extension before becoming taut, would eliminate possible damage at
large strains. Packing the lamellae at low strains would likely be
inconsequential, as the inner bulbar wall is already thrown into numerous
folds and trabeculae. We hypothesize that the fibrillar design of the bulbus
does not combat the dangers of large expansions; it lowers the modulus of the
material.
It is unlikely that the low modulus of the bulbus is due merely to the
gross structure of the elastin fibrils. Teleost elastin is chemically distinct
from other elastin variants
(Serafini-Fracassini et al.,
1978; Spina et al.,
1979
; Sage, 1982
;
Chow et al., 1989
), with a
decreased hydrophobic index due to an increase in polar amino acids. As much
of the recoil in elastin is driven by hydrophobic interactions, this may
result in a lower elastic modulus. Bulbar elastin dissolves under conditions
the ventral aortic elastin can withstand
(Licht and Harris, 1973
),
suggesting that the bulbar variety is even less hydrophobic than ventral
aortic elastin and may be the result of a different gene product.
In most arteries, it is the close association of elastin with collagen that
provides the exponential rise in stiffness as inflation increases. Much of the
bulbar collagen is confined to the adventitia, a loose, fibrous layer that is
not as dense as the outer media (Fig.
4A). Benjamin et al.
(1983), Raso
(1993
) and Icardo et al.
(1999a
,b
,
2000
) have suggested that the
adventitia is primarily responsible for limiting the radial distension of the
bulbus. However, the adventitia has a modulus that is similar to that of the
outer media at strains below one. At these levels, the outer medial layer
actually bears more of the load in the wall than the adventitia does, due to
differences in their respective wall thicknesses.
Very large strains, at which the adventitia would bear the majority of the
load, would result in the third stage of a sigmoid inflation curve: a rapid
increase in pressure with increasing volume. This feature of the bulbus was
suggested by the exponential rise in stiffness as tissue samples encompassing
the entire wall were subjected to large strains (Figs
9,
10,
11,
12). The sharp rise in
pressure was also seen during inflations of bulbi with much of the wall
removed (Fig. 8). Ordinarily,
the thick media is the primary layer resisting bulbar expansion, while the
thin adventitia is only recruited at large strains (>1). By removing the
media, the adventitia was the only layer resisting the increasing strain and
expanded much more than it ordinarily would at any given pressure. This
resulted in the adventitia becoming taut at a low V. While the
tensile tests (Figs 9,
10,
11,
12) and modified inflations
(Fig. 8) show that the bulbus
is capable of a final increase in stiffness, they also show that, for this to
occur in a bulbar inflation, extremely large volumes (and pressures) would be
required. In vivo, the constraints of the stiff pericardium would
limit the amount of bulbar expansion, and the chances of the bulbus reaching a
large enough strain to recruit the adventitia are small. However, by limiting
bulbar expansion, the pericardium itself may cause the final rise in
pressure.
The majority of adventitial collagen is actually longitudinally oriented,
preventing longitudinal rather than radial expansion
(Fig. 4A). Compared with the
other tissues, the adventitia of bigeye tuna bulbus only became very stiff at
a low strain when stretched in the longitudinal direction
(Fig. 12). The outer layer was
limited to a strain of 0.5 when stretched longitudinally, while all other
tissues reached strains greater than one. The maximum in vivo
longitudinal strain reached by yellowfin tuna bulbi has been measured at 0.48
(Braun et al., 2003).
Therefore, the answer to the question about the bulbus' extensibility on the plateau of the curve is multifaceted. The arrangement of the collagen and the structure and chemical nature of the bulbar elastin all combine to allow large volume changes within the bulbus for small pressure changes during the plateau phase of the inflation.
While elastin and collagen are important, smooth muscle function in the
bulbus is not insignificant. It is innervated
(Watson and Cobb, 1979),
responds to a wide variety of pharmacological and environmental stimuli by
increasing or decreasing the bulbar distensibility
(Farrell, 1979
) and causes
large changes in the inflation characteristics of the bulbus when it is
prevented from contracting (Fig.
7): a larger change than that due to the denaturation of the
bulbar collagen. While an extremely compliant vessel would limit the fish's
ability to increase blood pressure when needed, smooth muscle appears able to
adjust the stiffness of bulbi to match the demands of the fish. With smooth
muscle, the bulbar properties can be `tuned' to the situation at hand. While
this role of smooth muscle is in contrast to previous speculation
(Licht and Harris, 1973
) that
bulbar smooth muscle serves merely to produce elastin, the numerous
plasmalemmal vesicles seen in yellowfin tuna smooth muscle
(Fig. 4B,C) do suggest a
secretory role.
The bulbus seems to have been designed to allow maximal expansion, with many of the safeguards against rupture found in arteries either lacking or reduced and with much of the remaining collagen arranged to limit longitudinal rather than circumferential expansion. This suggests that the bulbus is at risk of rupture; however, in vivo, the bulbus is enclosed by the fibrous pericardium, limiting circumferential expansion. The longitudinally arranged collagen is vital as the bulbus is only tethered at its distal end, and unchecked longitudinal expansion would interfere with cardiac function by kinking the ventral aorta and occluding the lumen.
The function of longitudinal elements in teleosts has never been properly
explained. Priede (1976)
contended that they acted as struts, supporting the bulbus wall during
inflation cycles. Due to the thick bulbar wall, the inner layers are subjected
to much larger strains than are the outer layers. Since the longitudinal
elements do not undergo the same circumferential strains, the problem of
strain distribution would be solved. Fig.
12 shows that the longitudinal elements have no greater stiffness
or strength than the other materials in the wall. Thin, extensible, `floppy'
struts would be of little use in a supportive role, and unequal strain
distribution through the thick wall is not a problem because the bulbus is not
made of an isotropic material. Different amounts and orientations of elastin,
collagen and smooth muscle in the adventitia and media generate a host of
material properties. Even though the media experiences a larger absolute
strain than the adventitia during bulbar expansion, the media has a higher
breaking strain than the adventitia (Fig.
9). Designing the bulbus with anisotropic properties, specifically
a higher breaking strain for the media, prevents early failure.
The bulbi of all fish are subject to large expansions, while longitudinal elements seem to be limited to high-performance fish. Any functional explanation of the longitudinal elements would need to take into account the biology of the species possessing them. All these fish (including tuna, marlin, sailfish, trout and salmon) have a suite of adaptations allowing them to maintain aerobic activity for far longer than the average teleost, and the longitudinal elements may also be an adaptation towards a high-performance lifestyle. When the longitudinal elements are removed, the magnitude of the pressure generated on initial inflation drops (Fig. 8). When the longitudinal elements fill up the lumen at the end of diastole (Fig. 13), the bulbus requires a higher pressure for initial inflation. Longitudinal elements may be necessary to ensure that the higher pressure requirements of those fish that possess them are met. During diastole, the bulbus bears down on the contained blood, maintaining flow across the gills. During cardiac ejection, the compliant, easily stretched longitudinal elements are pushed out to the sides of the vessel wall, greatly increasing lumen size and allowing blood to pass freely.
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