The influence of environmental PO2 on hemoglobin oxygen saturation in developing zebrafish Danio rerio
1 Institute for Zoology and Limnology, University of Innsbruck,
Austria
2 Center for Molecular Biosciences, Innsbruck, Austria
* Author for correspondence (e-mail: bernd.pelster{at}uibk.ac.at)
Accepted 24 November 2004
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Summary |
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Key words: ontogeny, oxygen exchange, circulatory system, heart, zebrafish, Danio rerio
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Introduction |
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Collectively, these results indicate that up to a certain body size of the larvae the oxygen supply of the tissues can be met by bulk diffusion (Territo and Altimiras, 2001). Nevertheless, it is quite obvious that beyond a certain body mass convective oxygen transport must come into play, but data on the oxygenation status of the larvae during the time of cutaneous respiration is still missing. If convective oxygen transport contributes to the oxygen supply to tissues, loading and unloading of the blood must be detectable, i.e. partly deoxygenated blood and hemoglobin must be found in central parts of the body. Blood vessels underneath the skin, in turn, should carry mostly oxygenated hemoglobin. Based on these considerations we hypothesized that the oxygenation state of hemoglobin in central blood vessels would be an indication of loading and unloading of hemoglobin, and thus reflect the contribution of convective oxygen transport to the oxygen supply of the tissues. The presence of partly deoxygenated hemoglobin in central parts of the body, however, could indicate hypoxic areas (tissues) in the body. In this case, incubation under hyperoxic conditions should improve oxygenation of the hemoglobin and thus of the tissues. The aim of the present study was therefore to test whether tissue oxygenation of zebrafish larvae can be improved by hyperoxic exposure. Tissue oxygenation was assessed from hemoglobin oxygen saturation in vivo by combining video imaging techniques with spectrophotometrical analysis of hemoglobin light absorption.
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Materials and methods |
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Rearing
The experimental design of the present study required culturing zebrafish
larvae under constant normoxic conditions. Therefore, a new rearing system was
developed, which guaranteed an equivalent oxygen supply for all animals
investigated. Before water was introduced into the system, it was vigorously
aerated. Fresh water at a temperature of 28°C was pumped into a water
reservoir above the rearing system. In a gravity-fed system water dropped from
the reservoir into the rearing beakers, which had a bottom of fine plastic
meshwork, ensuring a permanent normoxic, downward water flow. To reduce the
danger of infection, a UV-light (Selzle UV-C Keimfilter, Typ UV500, TC 5 Watt,
Selzle Technik GmbH, 63110 Rodgau, Germany) was included into the water
circuit.
Respirometry
For measuring oxygen consumption of zebrafish larvae, fertilized eggs of
`brass' mutants were placed into the examination chamber of a `Twin-flow'
respirometer (Cyclobios, Innsbruck, Austria), equipped with Clark oxygen
electrodes at the inflow and outflow of the chamber
(Gnaiger, 1983). The oxygen
consumption of seven groups of eggs could be continuously measured from the
egg (1 d.p.f.) until day 9 of development, when the experiment had to be
terminated because too many larvae died in the respirometer. Microbial
respiration and background respiration of the respirometer system (including
oxygen uptake of the Clark oxygen electrode) were measured daily
(Dalla Via, 1983
) and
subtracted from fish oxygen consumption values. For determination of
background respiration, recalibration and cleaning of the system, measurements
were interrupted for about 3 h every day.
Preparation of the animals for video recordings
Preliminary experiments revealed that the oxygenation of larvae embedded
into low melting agarose (see Schwerte and
Pelster, 2000) was not always reproducible. Therefore a modified
incubation chamber was developed, in which the slightly anesthetized fish
larvae (0.1 g l1 MS222; Sigma Aldrich Chemie GmbH, 89552
Steinheim, Germany) were kept between polytetrafluoroethylene (PTFE; Teflon)
membranes (YSI Membrane Kit STANDARD; PTFE membranes for oxygen electrodes,
Yellow Springs Instrument Co., Inc., Yellow Springs, Ohio, USA). The larva was
placed in a drop of anesthetic (0.1 g l1 MS222) onto the
lower PTFE membrane (Fig. 1)
and a cylindrical top containing a second PTFE membrane lowered on top,
enclosing the zebrafish larva between two membranes in a sandwich-like
position. Aeration of the larva was carried out by directing temperature
controlled and humidified air (or gas) from a gas-mixing device directly onto
the two PTFE membranes. The high oxygen permeability of PTFE membranes ensured
that the oxygen reached the surface of the animal virtually without delay.
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Measurement of hemoglobin oxygen saturation
Maximum absorption peaks of fully oxygenated blood (413 nm), and fully
deoxygenated blood (431 nm), and the position of the isosbestic point (421
nm), were ascertained by spectrophotometrical analysis using zebrafish whole
blood. The absorption of zebrafish blood in the ventricle was visualized by
successively irradiating the embedded larva with light of the three specified
wavelengths (413 nm, 421 nm and 431 nm), provided by a monochromator
(Polychrome IV, Till Photonics, 82152 Martinsried, Germany) connected to an
inverted microscope (Axiovert 200M, Carl Zeiss GmbH, 37030 Göttingen,
Germany) via a light guide. With an imaging system (Optimas 6.5,
Media Cybernetics, Inc. Silver Spring, MD, USA) 100 consecutive pictures of
the ventricle at each wavelength, beginning at 431nm, were recorded.
Wavelengths were switched manually. The acquisition of 100 absorption images
and their storage onto the computer's hard drive required approximately 30 s
at each wavelength.
By using Optimas 6.5, two templates were drawn into a single recorded image of a larva's diastolic ventricle. One template confined the boundaries of the diastolic blood volume, while the second was used for standardization and therefore set into the nearby background. By using a macro, the imaging system calculated the template's mean pixel values for each of the 100 images and exported them for further examination into an Excel-file. Images in which absorption peaked were taken to represent the end diastolic stage of the ventricle (i.e. maximum filling with blood). Only those images were used for subsequent calculations. The changing dimensions of the ventricle caused a changing path length of light and resulted in a changing light absorption. It was assumed that end-diastolic volume was similar between subsequent contractions of the ventricle and therefore path length was constant. By using only values recorded during end-diastole, the path length was thus eliminated as a possible source of error. The background-template always had very high pixel values due to the low absorption of PTFE membranes and of the fluid film surrounding the larva.
Ventricular blood absorption values (as % of light emitted from the light source) were determined using the equation: % absorption = 100 (mean of the five lowest pixel values of 100 images / mean background pixel values of 100 images) x100.
Finally, graphs were made for the three absorption values obtained under
the varying oxygen saturation conditions. Absorption values of the isosbestic
points differed slightly between the three graphs due to a varying amount of
blood within the template during end diastolic stage. Standardization was
therefore carried out using a method that normalized the absorption values at
413 nm and 431 nm to the mean isosbestic point of all three measurements
(Tateishi et al., 1992). The
wavelength for deoxygenated blood, 431 nm, turned out to be the most sensitive
value for visualizing differences in blood oxygen saturation.
Measurement of blood absorption values under varied oxygen partial pressures
The oxygenation status of larval zebrafish blood was analyzed by comparing
the absorption pixel values of the blood at different levels of oxygenation.
Absorption values obtained from larvae under hyperoxic conditions were set to
100% oxygenation. It was assumed that data obtained under normoxic conditions
revealed `complete oxygenation' if the values were within the standard
deviation of those obtained under hyperoxic conditions. `Normoxic' data,
significantly exceeding the absorption ratios obtained from the same animal
under hyperoxic conditions, would be due to a greater absorption at 431 nm,
the specific wavelength for deoxygenated hemoglobin. Accordingly, these values
indicated a partial deoxygenation of the hemoglobin under normoxic
conditions.
In order to relate the measured absorption values of the blood to oxygen partial pressure, red blood cell (RBC) absorption values were determined in relation to the oxygen partial pressure in the fluid. After connecting the embedding chamber with the aeration device, a zebrafish larva was superfused with varying oxygen partial pressures. The experimental series was started with air (PO2=20 kPa). By mixing of air and nitrogen the oxygen partial pressure was then successively lowered to 16 kPa, 12 kPa, 8 kPa, 4 kPa, 0 kPa and in a final step again increased to 20 kPa. Incubation of the larvae at 0 kPa oxygen also provided the absorption value for complete deoxygenation of the hemoglobin. To verify the gas partial pressures in the embedding device an oxygen electrode (FOXY-18G probe with FOXY-AF-MG overcoat; Ocean Optics, 6921 RK Duiven, The Netherlands) was inserted into the lower gas chamber during the experiments. With the high gas permeability of the PTFE membranes and the relatively small tissue barrier of a fish larva in early developmental stages a stable oxygenation signal of blood in the ventricle was observed within a few minutes. No difference in oxygenation of the larvae's blood between 10 and 20 min after changing gas partial pressure of the incubation medium was detected. Therefore, for each step an equilibration period of 10 min was used.
Hyperoxygenation of larval zebrafish blood and tissue was achieved by superfusing larvae with pure oxygen (100 kPa) for a period of 10 min. Under these conditions ventricular blood was completely oxygenated, and hemoglobin oxygen saturation was 100%.
In a final step the internal calibration for the experiment was determined by measuring the maximum difference in absorption values between full saturation and deoxygenation of the hemoglobin for each individual larva. This step was necessary because optical conditions for spectrophotometric recording and analysis of blood oxygen saturation differed, depending on the developmental status of the zebrafish larvae (tissue thickness and blood cell content). Because of a decrease in transparency the change in light absorption observed between full oxygenation and deoxygenation significantly decreased from 11.25±0.61% at 4 d.p.f. to 4.06±1.47% at 12 d.p.f. (Fig. 2). Accordingly, in later developmental stages our method for measuring hemoglobin oxygen saturation in vivo was not applicable. Actual absorption values of blood were then expressed as percentage of maximum RBC saturation.
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Recording of the absorption images and determination of heart rate was performed 10 min after adjusting the appropriate oxygen partial pressure. The mean value of three consecutive measurements at each applied gas tension was used.
Heart rate
Heart rate was obtained from zebrafish larvae from 2 to 12 d.p.f. Heart
rate was determined by measuring the time interval for 30 heart beats. The
average value obtained from triplicate measurements was extrapolated to get
the number of beats per minute for each individual fish.
Statistics
For comparison of two means, statistical significance was evaluated by
unpaired Student's t-test. For multiple comparisons, one-way analysis
of variance (ANOVA) followed by StudentNewmanKeuls multiple
comparison test was used. Differences were considered significant at
P<0.05.
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Results |
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As to be expected, a reduction in PO2 of the incubation water (progressive hypoxia) resulted in a progressive reduction in hemoglobin saturation in the ventricle of animals at all stages. As an example, Fig. 4 presents the data for 2 and 8 d.p.f. animals, in which a complete oxygen saturation of the hemoglobin was observed under normoxic conditions. At 2 d.p.f. oxygen saturation decreased under hypoxia and at a PO2 of 4 kPa oxygen saturation was significantly reduced compared to normoxic conditions. After switching back to normoxia hemoglobin oxygen saturation was restored. At 8 d.p.f. a significant deoxygenation of the hemoglobin was first observed at a PO2 of 8 kPa. In all other stages the blood in the ventricle was already hypoxic under normoxic conditions (see Fig. 3), and with progressive hypoxia the oxygen saturation of the blood continuously decreased down to zero at a PO2 of 0 kPa.
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Heart rate under hyperoxic and hypoxic conditions
Heart rate was measured in order to test whether the changing oxygen
partial pressure not only affected hemoglobin oxygen saturation, but also
cardiac activity. At 2 d.p.f. and 3 d.p.f., heart rate under normoxic and
hyperoxic conditions did not differ significantly
(Fig. 5). Beginning at 4
d.p.f.hyperoxia significantly lowered heart rate in all stages until 12 d.p.f.
At 9 d.p.f. and 12 d.p.f., however, this difference was not significant.
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Heart rate at 4 d.p.f. and 5 d.p.f. was also modified in response to acute hypoxia, but the actual oxygen partial pressure at which this response was observed was dependent on the developmental stage (Fig. 6). At 4 d.p.f. a significant increase in heart rate was observed only at a PO2 of 8 and 4 kPa, 5 d.p.f. larvae responded at 8, 4 and 0 kPa. At 8 d.p.f., zebrafish larvae showed no significant response to hypoxia, although at 8 and 12 d.p.f. a minor decrease in heart rate was observed at the onset of hypoxia, but this bradycardia was not significant. Under more pronounced hypoxia, however, heart rate increased at 12 d.p.f., as was seen in earlier stages.
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Oxygen consumption
Oxygen consumption rates of developing zebrafish significantly increased
from day 1 (egg) until day 5 (swim-up larvae, digesting external food).
Starting at 3.8±0.135 nmol h1 per individual at 1
d.p.f., oxygen consumption increased more or less continuously to
15±0.46 nmol h1 per individual at 5 d.p.f.. No
significant difference was found between 5 d.p.f. and 6 d.p.f., whereas a
significant decrease in oxygen consumption rate was detected from 6 d.p.f. to
7 d.p.f., followed by another significant elevation of oxygen consumption rate
from 7 d.p.f. to 8 d.p.f. and 9 d.p.f., respectively
(Fig. 7). Oxygen consumption of
zebrafish larvae in later stages has been measured in several studies
(Barrionuevo and Burggren,
1999; Bagatto et al.,
2001
), and these values are in line with our data. Adjusting the
data of Bagatto et al. (2001
)
to the conditions of our study revealed that the increase in oxygen
consumption as development progressed continued as expected due to the
increase in body mass (Fig.
7).
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Discussion |
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Immobilization of the larvae between the two PTFE membranes was mainly achieved by regulating the volume of the liquid surrounding the larvae. The aeration chamber itself provided an excellent device for experiments using different gas mixtures, as the gas flow along the PTFE membranes above and below the zebrafish larvae guaranteed constant gas tensions.
Data acquisition and analysis
The templates drawn within the diastolic ventricle of the zebrafish for the
determination of the mean pixel values were identical throughout the
measurements in an individual larva. However, in some cases the larva moved
slightly between the normoxic and hyperoxic or hypoxic measurements. If this
happened, another template was drawn at about the same position, ensuring
comparable values. Particularly in later developmental stages some graphs,
obtained under normoxic and hyperoxic conditions, showed too little contrast
in gray values. This appeared to be due to an increase of ventricular and body
cell mass of the zebrafish larvae, which reduced the visibility of the
erythrocytes in the vascular system (see
Fig. 2). These data were not
used for further analysis. Beyond 12 d.p.f. our method for the determination
of hemoglobin oxygen saturation in vivo was no longer applicable,
because of decreased transparency.
Oxygenation status of venous blood
Several studies have shown that bulk diffusion appears to be sufficient to
fuel aerobic metabolism during early development of the zebrafish. The present
results indicate, however, that this does not prevent partial deoxygenation of
the blood in the ventricle, which contains venous blood. At 4 d.p.f., 5 d.p.f.
and also at 12 d.p.f. the significantly different blood absorption values
observed under normoxic and hyperoxic conditions clearly showed that venous
return into the ventricle was partially deoxygenated. During these periods,
acute hyperoxia significantly increased oxygen saturation. Nevertheless,
measurements of oxygen consumption at this time do not provide any indication
of a switch to anaerobic metabolism
(Pelster and Burggren, 1996;
Barrionuevo and Burggren, 1999
;
Bagatto et al., 2001
).
At 45 d.p.f. we observed a nearly complete reduction of the yolk
extensions and the yolk sac itself (see also
Kimmel et al., 1995). Rombough
(1998
), investigating the
impact of the yolk sac as a possible site of gas exchange in rainbow trout
larvae, concluded that the blood vessel density in the richly vascularized
yolk sac had no significant effect on gas exchange. In zebrafish we observed
the lowest blood oxygen saturation at 4 d.p.f. and 5 d.p.f., a stage when yolk
sac degradation was largely completed. A reduction of the gas exchange area
would probably affect the balance between oxygen supply and oxygen consumption
and thus could be responsible for a partial deoxygenation of the blood. This
coincidence therefore suggested that in zebrafish larvae the yolk sac does
contribute to oxygen uptake and thus to gas exchange. Danio rerio is
a tropical fish that typically experiences higher temperatures
(2530°C) than trout larvae, which are typically exposed to
temperatures of 515°C. Given the effect of temperature on
metabolism, zebrafish larvae should thus have a significantly higher metabolic
rate. Metabolic rate typically increases by a factor of two for a temperature
increase of 10°C (Q10=2), and in fish larvae Q10
values of up to 5 and 6 have been reported
(Pelster, 1999
;
Barrionuevo and Burggren,
1999
). It thus appears possible that the higher metabolic rate in
zebrafish larvae requires additional sites of oxygen uptake, whereas in trout
bulk diffusion through the body surface without the support of the yolk sac is
sufficient. Besides, protuberances on the surface of the zebrafish yolk sac
were observed, which may increase the surface area in order to support oxygen
uptake (T. Schwerte, unpublished observation). Accordingly, the yolk sac of
zebrafish larvae could substantially contribute to the diffusive O2
uptake.
Similar to the putative role of the zebrafish yolk sac in gas exchange, the
highly vascularized chorioallantoic membrane (CAM) of chick embryos
contributes to gas exchange and even adapts to different environmental oxygen
pressures. Richards et al.
(1991) reported that
chorioallantoic membrane wet mass increased under hypoxic conditions, and was
reduced under hyperoxic conditions.
The improved oxygenation of the blood observed in our study beyond day 5 coincided with a decrease in oxygen consumption. It was not obvious why oxygen uptake decreased between day 5 and 7, but with this lower rate of oxygen uptake bulk diffusion apparently again ensured a better oxygenation of the whole animal. In spite of the continuing development of the animal, oxygen consumption at 8 d.p.f. was slightly lower than at 5 d.p.f., and at 8 d.p.f. venous blood in the ventricle was almost completely oxygenated.
A second period in which partially deoxygenated blood was detected in the
ventricle of the zebrafish occurred at 12 d.p.f. At this relatively late stage
in larval development the incomplete oxygen saturation of the tissues could be
attributed to an increase in diffusional resistance, to an increased internal
oxygen consumption, or perhaps to both factors. Between 6 d.p.f. and 13
d.p.f., zebrafish wet and dry mass increased from 0.35±0.01 mg to
0.46±0.02 mg, and from 0.057±0.002 mg to 0.069±0.007 mg,
respectively (Bagatto et al.,
2001). Respiration data of zebrafish larvae reveal a significant
increase in oxygen consumption from day 7 to day 15. An increase in oxygen
demand requires an increase in oxygen supply, and the secondary lamellae, the
actual site of gas exchange in fish, start to form between days 12 and 14
(Rombough, 2002
). Our data
clearly show a partial deoxygenation of blood at 12 d.p.f. It can therefore be
assumed that this is mainly caused by an increase in the rate of oxygen
consumption, while the secondary lamellae, mainly responsible for the gas
exchange, are only just starting to develop
(Rombough, 2002
).
Thus, the data available so far fit together nicely. Early developmental
stages of the zebrafish up to 1214 d.p.f. do not need a circulatory
system for oxygen supply to tissues under normal `resting' conditions. At
about 12 d.p.f. the partial deoxygenation of hemoglobin in the central
circulation indicates that oxygen is not only taken up by bulk diffusion, but
also removed from the blood. This is also the time (1214 d.p.f.) where
chronic hypoxemia is no longer compatible with proper development
(Jacob et al., 2002) and
confirms the conclusion that at this point in development hemoglobin becomes
necessary for oxygen transport. Thus, at about 124 d.p.f. the
cardiovascular system of the zebrafish takes over responsibility for the
oxygen supply of tissues.
This may also be the time when cardiac activity and metabolic demand of the
tissues become coupled, as in adult vertebrates. The control system is
necessary to achieve such a coupling is established much earlier. Hypoxic
incubations (Jacob et al.,
2002; Schwerte et al.,
2003
) and studies in which metabolic activity was enhanced by
swimming movements (Pelster et al.,
2003
) revealed that oxygen receptors sensing hypoxic conditions
are present at 3 or 4 d.p.f., and the control loop modifying cardiac activity
in response to the afferent information of oxygen receptors is also operating
at this time in development.
Implications of hyperoxia and acute hypoxia on heart rate
In a previous study it was demonstrated that heart rate represents a
sensitive parameter for identifying cardiovascular responses to chronic
hypoxia. Therefore it appeared interesting to see whether the presence of
partially deoxygenated hemoglobin would somehow correlate with cardiac
activity. Partially deoxygenated blood was present at a
PO2 of 20 kPa at 4 and 5 d.p.f.; nevertheless,
a significant elevation of heart rate during progressive hypoxia was not
observed down to a PO2 of 8 kPa at any of the
stages analyzed. Thus, the presence of partially deoxygenated blood in the
ventricle did not provoke significant changes in heart rate. This result is in
line with the data of Jacob et al.
(2002), who reported that
hypoxemia (i.e. a reduced oxygen-carrying capacity of the blood) does not
modify cardiac activity in early developmental stages.
Acute hyperoxia had no effect on larval zebrafish's heart rate at 2 d.p.f.
and 3 d.p.f., but led to a significant bradycardia in all stages from 4 d.p.f.
until 16 d.p.f.. Zebrafish raised under chronic hypoxia have an increased
heart rate at about hatching time or shortly thereafter
(Jacob et al., 2002), which
indicates that at this time oxygen receptors are present, sensing hypoxic
conditions and using the information for a suitable response, i.e. to increase
cardiac activity in order to stimulate convective oxygen transport. Our study
complements the work of Jacob et al.
(2002
). Oxygen sensors in the
larval zebrafish are working in both directions. Hypoxia induces a stimulation
of cardiac activity, while hyperoxia reduces cardiac activity, and these
responses are observed at a stage when the circulatory system is apparently
not yet essential to ensure oxygen supply to the tissues.
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Acknowledgments |
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