Cardiovascular development in embryos of the American alligator Alligator mississippiensis: effects of chronic and acute hypoxia
1 Department of Ecology and Evolutionary Biology, University of California
Irvine, Irvine, CA 92697, USA
2 Department of Biology, IFM, Linköpings universitet, SE-58183
Linköping Sweden
* Author for correspondence at present address: Oregon Health and Science University, Department of Physiology and Pharmacology/L334, 3181 SW Sam Jackson Park Road, Portland, OR 97201-3098, USA (e-mail: dcrossle{at}uci.edu)
Accepted 22 October 2004
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Summary |
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Key words: cardiovascular, embryonic, American alligator, Alligator mississippiensis, development, hypoxia.
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Introduction |
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To date two studies have examined the impact of hypoxic incubation on the
morphologic and physiological development of reptiles. In embryonic turtles
Pseudemys nelsoni, chronic hypoxic incubation results in a depression
of metabolic rate, an increase in hematocrit, and an increase in ventricular
mass (Kam, 1993). Hypoxic
incubation in the American alligator embryo also results in an increase in
hematocrit (Warburton et al.,
1995
), but little is known about the effects on the cardiovascular
physiology of these embryonic animals.
Given the limited understanding of the system, the first objective of the study was to determine the impact of chronic hypoxic incubation on embryonic growth and cardiovascular function in the American alligator. The second objective was to test the hypothesis that incubation under hypoxia would decrease the embryonic response to acute hypoxic stress.
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Materials and methods |
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Upon arrival eggs were numbered, weighed, and five eggs from each clutch
randomly assigned to one of three experimental groups, which differed in the
oxygen content of the incubation environment: normoxia, i.e. 21%O2
(group N21), hypoxia 15%O2 (group H15), and hypoxia
10%O2 (group H10). Eggs within a given group were further divided
and placed in one of three plastic boxes (volume9 liters) containing
vermiculite mixed with water at a 2:1 ratio. The water content of vermiculite,
determined by mass at the beginning of the study, was maintained by weighing
the box twice weekly, with water added as needed.
Control of ambient oxygen during incubation
Following the distribution of eggs between the experimental groups, all
boxes were placed inside large plastic bags that were sealed with duct tape.
Two holes in the bags allowed the connection of each box in parallel to inflow
and outflow gas lines made from Tygon tubing. The inflow gas-line was then
connected to a gas reservoir that was supplied with 21%, 15%, or
10%O2. The 10%O2 and 15%O2 gas mixtures were
set using two gas flow rotameters (Cole Parmer, IL, USA) for air and nitrogen.
All gas mixtures were passed through a H2O bubbler to ensure water
saturation and then into the gas reservoir. An outflow gas-line was connected
to each box and placed inside each plastic bag to allow gas to escape the
boxes, first into the bag and then vent via the outflow hole. Gas
flow in the egg boxes was maintained at 750 ml min-1 and gas
composition in each box was checked twice daily with an oxygen analyzer (S-3A;
Applied Electrochemistry Technologies Inc., IL, USA). All egg boxes were
maintained at 30°C in an environmental chamber during the course of the
study.
Surgical procedures
At 60%, 70%, 80% and 90% of a 72 day total incubation period, eggs were
taken from the incubation boxes, candled to locate a chorioallantoic artery
and placed in a temperature-controlled chamber at 30±0.5°C. A
portion of the eggshell was removed and the previously located artery was
occlusively catheterized under a dissection microscope (M3Z; Wild, IL, USA)
using heat-pulled saline-filled polyethylene tube (PE-90; Becton Dickson, NJ,
USA) as previously described (Crossley and
Altimiras, 2000). Once catheterization was completed the catheter
was fixed to the shell with cyanoacrylic glue and the egg was placed in an
experimental chamber. Each chamber consisted of a water jacketed glass
container and a glass lid with three ports, which provided an avenue for
externalizing the arterial catheter as well as routes for the inflow and
outflow of different gas mixtures. During the period of experimentation eggs
were maintained at 30±0.5°C.
Signal recording and calibration
The catheter was attached to a pressure transducer (DP6100; Peter von Berg
Medizintechnik GmbH, Eglharting, Germany) connected to a 4CHAMP amplifier
(Somedic AB, Sweden) and the pressure trace stored in a DELL Latitude computer
using a custom-made data acquisition program (LabView, National Instruments
Corp., TX, USA). In all cases, reference zero pressure was set at the top of
the experimental bath, and all values were corrected after the experiment as
previously described (Altimiras and
Crossley, 2000).
Experimental protocol
Prior to experimental manipulation a control period of 45 min post-surgery
in normoxia was given. During this period, blood pressure and heart rate
reached stable values. Embryos that failed to do so were eliminated from the
study.
The experimental protocol included two acute exposures of the normoxic incubated embryos (group N21) to 15% and 10%O2 for 5 min. The other experimental groups (H15 and H10) were acutely exposed to 10%O2 only. After acute hypoxia, a recovery period of 30-60 min was given to allow the return of cardiovascular variables to pre-hypoxic values. This experimental manipulation was conducted for the purpose of comparison of the three groups. The total number of embryos tested at each developmental age (60%, 70%, 80% and 90% of incubation) is equal to the number used to determine heart mass in Table 1.
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After completing the experimental protocol embryos were euthanized with an
arterial injection of pentobarbital (50 mg kg-1) and saturated KCl.
The embryo was then removed from the egg to determine the stage of embryonic
development, embryonic wet mass, yolk wet mass, heart wet mass and liver wet
mass. Embryo staging was conducted by one of the authors (D.A.C.) in a blinded
manner to limit biases in accordance with Ferguson
(1985).
Statistical analysis
A two-way analysis of variance (ANOVA), with incubation age (from 60% to
90%) and incubation environment (normoxic, 15%O2 and
10%O2) as independent variables, was used to determine statistical
differences (P<0.05) in all mass parameters (embryo, yolk, liver
and heart mass) and also in normoxic control cardiovascular values
(a and fH). In
the normoxic incubated alligator embryos, a repeated-measures (RM)-ANOVA was
used to assess statistical differences (P<0.05) in heart rate and
blood pressure between the three phases of the acute hypoxic response;
control, during hypoxia and after hypoxia. A one-way ANOVA model was used to
determine the statistical differences (P<0.05) in the heart rate
and blood pressure change during acute exposure to 10%O2 at each
developmental age between incubation conditions (groups N21, H15 and H10). A
Tukey's significant difference for unequal sample sizes was used for
post-hoc comparisons in all cases. All data are presented as mean
± S.E.M. All statistical tests were
conducted with the software package Statistica (Statsoft version 5).
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Results |
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Mass changes during development
Embryonic wet mass rose from 10.4±1.0 g at 60% to 38.0±2.0 g
at 90% under normoxic conditions (Fig.
1A) with significant increases in the two last age groups studied.
Embryonic alligators from H10 were significantly smaller than those from N21
and H15 at 80% and 90% of incubation (Fig.
1A). In the normoxic group, egg yolk wet mass fell from an initial
level of 33.0±1.0 g to 12.5±1.0 g at the end of the study
(Fig. 1B) with group H15
exhibiting similar changes. The amount of yolk in H10, however, was
significantly higher than the other groups at 90% (8.5 g more yolk,
Fig. 1B).
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Wet heart mass in the normoxic embryos rose from 60.0±8.0 mg at 60% to 176.0±11.0 mg at 90% of incubation with significant changes occurring at 80% and 90% of incubation (Table 1). Heart mass was similar in N21, H15 and H10 at all incubation ages (Table 1). The embryonic heart mass to body mass ratio revealed that incubation at 10%O2 resulted in a relative heart mass that was significantly larger than N21 at 80% and 90% of incubation (Table 1). Wet liver mass in the normoxic embryos increased approximately tenfold, with significant increases occurring at 80% and 90% of incubation (Table 1). Embryos from H15 showed the same growth pattern. However, embryos from H10 had a significantly smaller liver when compared to N21 at 90% of incubation but the liver to body mass ratio was not different (Table 1).
Cardiovascular parameters
Control cardiovascular parameters for N21 were similar to those determined
in a prior study (Crossley et al.,
2003b). Mean arterial pressure
(
a) increased during incubation (from
0.44±0.06 kPa to 2.3±0.18 kPa), with significant increases in
pressure over the last 20% of development studied
(Fig. 2A). Hypoxic incubation
at both 15% and 10%O2 (H15 and H10 respectively) resulted in a
significantly lower
a (
0.90 kPa
lower) at 90% of incubation than the N21 group
(Fig. 2A).
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Heart rate was unchanged between incubation ages and experimental groups with the exception of H10 embryos, which had significantly lower heart rate (17 min-1 lower) at 90% of incubation (Fig. 2B).
Cardiovascular responses to hypoxia in normoxic embryos (Group N21)
Embryonic alligators incubated under normoxic conditions exhibited a
biphasic hypoxic response that was typified by the trace in
Fig. 3. The general pattern
included a drop in heart rate during the hypoxic exposure with little change
in arterial pressure. This was followed by a longer lasting post-hypoxic
tachycardic hypertension, which peaked once the embryo was returned to
normoxic levels. The intensity of the hypoxic bradycardia, post-hypoxic
tachycardia, or post-hypoxic hypertension varied between embryonic ages as
shown in Figs 4 and
5. Only N21 embryos at 60% of
development displayed a mild change in arterial pressure simultaneous with the
hypoxic bradycardia (Figs 4A,
5A). The post-hypoxic
hypertensive response in N21 embryos was mild after acute exposure to
15%O2 (only significant at 80% of incubation,
Fig. 4A) and more intense
following 10%O2 acute exposure (pressure increases of 0.09, 0.31
and 0.37 kPa from 70% to 90%, respectively;
Fig. 5A).
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During the exposure to 15%O2, hypoxic bradycardia was found at 60% and 70% of incubation only (Fig. 4B), but was characteristic at all incubation ages when embryos were acutely exposed to 10%O2 (decreases of 14 min-1, 10 min-1, 9 min-1 and 13 min-1 at 60%, 70%, 80% and 90% respectively; Fig. 5B). Post-hypoxic tachycardia occurred following the return to 21%O2 at 70%, 80% and 90% of incubation following acute exposure to both 15% and 10%O2. The average tachycardia was 3.4 min-1 and 3.5 min-1 at 15% and 10%, respectively (Figs 4B, 5B).
Cardiovascular responses to acute hypoxia in embryos incubated under chronic hypoxia (H15 and H10)
H15 and H10 embryos also exhibited a bimodal response to 10%O2,
but there was a trend towards a reduction in the hypoxic bradycardia and
post-hypoxic hypertension as shown at older incubation ages (80% and 90%
embryos). A comparison of the acute a
responses to 10%O2 revealed no differences in the pressure response
from 60% to 90% of incubation (Fig.
6A). However, H10 embryos displayed a reduced post-hypoxic
hypertension that was significant at 80% and 90% of incubation
(Fig. 7A).
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The hypoxic bradycardia was reduced in H10 embryos at 80% and was reversed to a hypoxic tachycardia (+3 min-1) in 70% embryos (Fig. 6B). The post-hypoxic heart rate response differed between the three groups at two points of development: H10 embryos at 70% of incubation showed an accentuated post-hypoxic tachycardia (+6 min-1 vs +3 min-1, Fig. 7B) and this pattern was reversed at 80% of incubation with H10 differing from H15 (+2 min-1 vs +4 min-1 respectively, Fig. 7B).
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Discussion |
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Alteration in growth patterns
Under normoxic conditions embryonic wet mass increased
(Fig. 1A) in a similar manner
to that previously reported in American alligators during incubation
(Deeming and Ferguson, 1989).
Yolk mass changed in the opposite manner due to its utilization in embryonic
growth, organogenesis and metabolism (Fig.
1B; Deeming and Ferguson,
1989
). Embryonic heart mass in the normoxic group changed in a
pattern similar to that of body mass in order to increase perfusion to the
growing embryo. However, the growth was not isometric because heart mass
increased 2.9-fold when embryonic mass increased 3.7. The calculated
allometric mass exponent is 0.82, a value very similar to the exponent of 0.85
determined in chickens (J.A., unpublished). Liver mass exhibited an
accelerated growth rate (10 times increase) in the developmental ages studied.
The allometric mass exponent of 1.45 (Table
1) indicates that the liver, unlike the heart, grows relatively
more than the whole embryo at the end of incubation. Whether such growth is
required or not for the proper function of the embryo cannot be resolved
without further research.
Not unexpectedly, incubation under hypoxic conditions (10%O2)
produced important changes in the growth patterns. Embryonic mass was
significantly reduced at 80% and 90% (Fig.
1A) and this was accompanied by an increased amount of yolk
present at 90%, thus there was a reduced conversion of yolk to tissue due to
hypoxia (Fig. 1B). Such changes
have been previously observed in embryonic turtles and chickens incubated
under hypoxic conditions (Metcalfe et al.,
1984; Handrich and Girard,
1985
; Kam, 1993
)
as well as embryonic alligators with altered eggshell conductance
(Deeming and Ferguson, 1991
).
Indeed, hypoxic incubation is routinely used in chickens as a model of
impaired fetal growth (Ruijtenbeek et al.,
2000
; Rouwet et al.,
2002
). Stunting of embryonic growth under hypoxic conditions in
reptilian and avian embryos has been associated with metabolic depression due
to a reduction in tissue O2 delivery
(Metcalfe et al., 1984
;
Kam, 1993
). This undoubtedly
contributed to the reduced embryonic size and increase yolk mass in this
study. Interestingly, there was no difference in developmental stage between
normoxic and hypoxic embryos at any developmental age
(Table 2). Thus, although
tissue growth was compromised, normal differentiation was not.
The absence of a difference in total incubation length under all conditions
in this study (Table 2)
contrasts the findings of a prior study
(Warburton et al., 1995). In
that study hatching was defined as the time of external pipping. However, it
is well known that embryonic reptiles spend extended periods of time inside
the eggshell following external pipping
(Packard and Packard, 2002
).
This pipping period has, in fact, been shown to depend on incubation
conditions and is doubled in snapping turtles incubated in a dry substrate
(1.6 days vs 0.8 days in turtles incubated in a wet substrate;
Packard and Packard, 2002
).
Thus, this may account for the difference between our study and the
aforementioned study. Incidentally, a constant incubation length under hypoxic
conditions in Florida red-bellied turtles Pseudyms nelsoni has also
been reported (Kam, 1993
).
While embryonic mass was lowered in the H10 group, the effects of this
level of hypoxia were manifested differently among the organ systems. Heart
mass in H10 embryos was similar to that of the N21 embryos at 80% and 90%
(Table 1) of incubation. Since
overall embryonic mass in H10 was lower
(Fig. 1A) than the N21 group,
the heart to body mass ratio was higher in the H10 group. Cardiac hypertrophy
under hypoxic incubation has been shown previously in hatchling alligators
(Warburton et al., 1995) and
also in Florida red-bellied turtles (Kam,
1993
), chickens (Handrich and
Girard, 1985
; Asson-Batres et
al., 1989
; Dzialowski et al.,
2002
; Miller et al.,
2002
) and sheep (Gagnon et
al., 1997
). This relative change in heart mass could be partially
attributed to an increase in cardiac function in an attempt to deliver
adequate O2 to developing tissues. Hypertrophy could also result
from an increased workload imposed on the heart associated to increased
hematocrit and blood viscosity (Warburton
et al., 1995
).
Unlike the heart, liver growth was impaired in H10 embryos, but relative to
body mass was not significantly different from N21. In chickens, stunting of
the liver growth under hypoxic incubation has been previously reported in some
studies (McCutcheon et al.,
1982) but not in others
(Miller et al., 2002
;
Lindgren, 2004
). Acute anoxic
exposure decreases the fraction of cardiac output delivered to the liver
(Mulder et al., 2001
) in a
systemic response mediated by
-adrenergic receptors in embryonic
chickens (Mulder et al., 2001
;
Crossley et al., 2003a
). In
sheep, elevation of circulating catecholamines, due to hypoxia, can shift
liver blood flow towards the ductus venosus due to a greater constrictor
effect of catecholamines on intra-hepatic veins
(Tchirikov et al., 2003
). It
is possible, however, that liver blood flow is restored under chronic hypoxia
based on a yet-uncharacterized mechanism, such as adrenergic desensitization.
This mechanism would subsequently maintain liver perfusion and allow the rapid
growth of this organ in the last third of development.
Alteration in normoxic control cardiovascular variables
H15 and H10 embryos had a significantly lowered
a (77% and 61%, respectively) in
comparison to N21 embryos at 90% (Fig.
2), a finding that is probably the result of increased
vascularization coupled to altered vascular reactivity. Chronic hypoxia
triggers angiogenesis of the extra-embryonic circulation, resulting in an
increased vascularization of the chorioallantoic membrane of 10% in alligators
(Corona and Warburton, 2000
)
and 54% in chickens (Dusseau and Hutchins,
1989
). Thus, the addition of parallel vascular beds will decrease
the resistance of the chorioallantoic circulation and lower systemic blood
pressure. However, in light of the substantial decrease in
a found in this study other
mechanisms might be in operation. Chronic hypoxia is also known to decrease
the adrenergic sensitivity of peripheral systemic vasculature in embryonic
chickens (Ruijtenbeek et al.,
2000
) due to constant adrenergic stimulation caused by elevated
catecholamine titers (Mulder et al.,
2000
). Such receptor desensitization is compensated by an
increased periarterial sympathetic innervation
(Ruijtenbeek et al., 2000
),
but it is unknown at present if such compensation suffices to maintain
embryonic vascular resistance. If not, and provided that similar mechanisms
are operating in alligator embryos, it may contribute to the hypotension in
H15 and H10 found in this study.
To directly relate changes in systemic resistance to changes in systemic
pressure, cardiac output () must
remain relatively constant. No direct chronic measurements of
in in ovo developing
embryonic animals exist, but oxygen consumption, a close indicator of
, is similar between chronically mild
hypoxic and normoxic incubated embryonic alligators
(Warburton et al., 1995
).
Similarly, differences in metabolic rate
O2 smaller than
10% have been reported under different regimes of hypoxic incubation in
chicken embryos (Dzialowski et al.,
2002
), suggesting that
may be maintained in embryonic animals subjected to hypoxic incubation.
Like blood pressure, normoxic control heart rate in H10 alligator embryos
at 90% of development was 20% lower than controls. A putative mechanism for
such hypoxia-induced bradycardia would be the hyperpolarization of pacemaker
cells caused by opening of ATP-sensitive K+ channels
(Han et al., 1996). It is
unlikely though that the bradycardia itself had a major impact on oxygen
delivery because the 20% increase in relative cardiac mass and the 25%
increase in filling time could support a compensatory increase in stroke
volume that would maintain
.
Altered responses to an acute hypoxic challenge
Acute hypoxic exposure (10%O2) in N21 embryos triggered a
biphasic cardiovascular response: an initial hypoxic bradycardia followed by a
post-hypoxic hypertension and tachycardia (Figs
3,
5). During acute exposure to
15%O2 only the post-hypoxic tachycardia was consistently seen
(Fig. 4). Two non-exclusive
mechanisms could account for the hypoxic bradycardia: (1) reflexive vagal
bradycardia and (2) hypoxia acting directly on cardiac tissue. In adult
crocodiles a bradycardia is elicited by increased vagal activity
(Altimiras et al., 1998).
Alligator embryos lack a vagal tone during development but the vagus is able
to elicit a baroreflexive bradycardia in the last third of development
(Crossley et al., 2003b
). Such
a mechanism is also present in fetal sheep, which exhibit a clear vagal,
mediated hypoxic bradycardia (Giussani et al.,
1993
,
1994
). While other reflexive
mechanisms may account for the reduction in heart rate, hypoxia could also
directly induce acetylcholine release from parasympathetic nerve terminals as
it occurs in chickens (Crossley et al.,
2003a
).
The existence of the post-hypoxic response to 10%O2 implies that
hypoxia triggered a systemic response that carried over into the recovery
period, affecting heart rate and a.
Of the several regulatory mechanisms that could be responsible for these
effects, increased levels of catecholamines are probably a major component.
Catecholamine levels increase during periods of hypoxic exposure in embryonic
chickens (Crossley and Altimiras,
2000
; Mulder et al.,
2000
) partly mediated by the direct stimulation of chromaffin
tissue (Crossley et al.,
2003a
). Significant levels of plasma catecholamines have also been
measured in alligator embryos (J.A., unpublished), so it is feasible that
these levels are increased by bouts of hypoxia, as occurs in chickens. The
relative maintenance of
a during the
hypoxic exposure may also be attributed to the release of catecholamines,
which could effectively buffer the direct depressive action of hypoxia.
Embryos incubated under chronic hypoxia (H15 and H10) showed an attenuated
response to the acute hypoxic challenge (Figs
6,
7). First, the hypoxic
bradycardia was decreased (even reversed to a tachycardia at 70%) with heart
rate not going below 75 min-1. Since normoxic control heart rate in
the H10 group was lower at 90% (75 min-1 vs 86
min-1) in than the N21 group, the scope of any hypoxic vagal reflex
would be drastically limited when compared to the normoxic group.
Interestingly, despite the reduced normoxic control heart rate at 90% in H10
embryos, acute hypoxia did cause the heart rate to fall by 4 min-1
to 63 min-1, which could be interpreted as a more mature vagal
reflex in agreement with the maturation of baroreflex regulation previously
published (Crossley et al.,
2003b). Second, post-hypoxic hypertension was lower late in
incubation (80% and 90%) in the H15 and H10 groups, possibly because
adrenergic sensitivity was lowered, related to either a reduced release of
catecholamines or receptor desensitization and/or downregulation.
Collectively, the results confirm the hypothesis that embryos incubated under hypoxia are less responsive to acute hypoxic stress than control embryos. The impact of such a reduction in responsiveness is, however, difficult to evaluate without further experimentation. The main responses elicited by chronic incubation, namely, cardiac enlargement, blunted hypoxic response and systemic vasodilation, may provide H10 embryos with a new physiological repertoire for responding to hypoxia that is independent of acute activation of other systems.
Conclusions
The American alligator demonstrated a substantial tolerance to hypoxia
during embryonic development relative to other developing terrestrial
vertebrates. Chronic hypoxic incubation induced significant changes in embryo
and organ mass, as expected, but it also changed normoxic control
cardiovascular variables (heart rate and
a) and blunted the cardiovascular
response to acute hypoxic challenge. It is of interest that the earliest
differences between normoxic and hypoxic embryos were the physiological
responses to acute hypoxic challenges (70%). Not until 90% of incubation did
gross morphological differences appear in the form of impaired growth and
cardiac enlargement.
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Acknowledgments |
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