Ontogeny of baroreflex control in the American alligator Alligator mississippiensis
1 Department of Ecology and Evolutionary Biology, University of California
at Irvine, CA 92697, USA
2 Department of Biology, IFM, University of Linköping, SE-58183,
Sweden
* Author for correspondence (e-mail: dcrossle{at}uci.edu)
Accepted 12 May 2003
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Summary |
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Key words: baroreflex regulation, embryonic development, baroreflex gain, cardiovascular regulation, American alligator, Alligator mississippiensis
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Introduction |
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The two primary species used to study vertebrate cardiovascular
development, sheep and chickens, both possess a functional baroreflex during
fetal or embryonic life (Blanco et al.,
1988; Segar, 1992
;
Altimiras and Crossley, 2000
).
This suggests that either the baroreflex is an important component of
cardiovascular regulation during development or that it simply becomes
functional in anticipation of its neonatal utility. Work in embryonic chickens
suggests the latter, given that barostatic reflexes emerge very late in
development and have a lower gain compared with those in adults
(Altimiras and Crossley, 2000
).
By contrast, fetal sheep possess a baroreflex as early as 60% of gestation,
with a decrease in sensitivity and an increasing set point during development
(Blanco et al., 1988
;
Segar, 1997
;
Segar et al., 1992
). This
apparent contradiction between a mammalian fetus and an avian embryo may be
the result of differing gestational conditions. While the sheep develops
in utero, the chicken develops independent of the mother in an egg
case. The delayed baroreflex maturation in chickens could therefore be a
particular feature of development within an egg case.
To further understand the relevance of an embryonic baroreflex, we chose to study it in embryos of the American alligator. American alligators develop within an egg case, similar to the chicken embryo, thus excluding embryonic maternal physiological interactions. While this is a characteristic shared with birds, reptile eggs exhibit a greater permeability to water flux, possibly subjecting the developing animal to periods of dehydration that might require the earlier onset of a cardiovascular regulatory mechanism. Thus, this study was conducted in embryos of the American alligators to determine the time of activation and the magnitude of the cardiac limb of the baroreflex.
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Materials and methods |
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Surgical procedure in embryos
An egg at the appropriate time of incubation was removed from the
incubator, candled for the location of a tertiary chorioallantoic artery and a
tertiary chorioallantoic vein and placed in a water-jacketed chamber at
30°C. The chamber was open at the top to allow the manipulation of the egg
during surgery. A 1.5 cmx1.5 cm window was cut in the shell, exposing
the underlying chorioallantoic vessels. The previously located artery and vein
were then isolated and catheterized using heat-pulled polyethylene tube
(PE-90), as previously described (Altimiras
and Crossley, 2000). Following catheterization, the egg was placed
in a thermostatically controlled glass chamber that was fitted with a lid with
multiple ports to externalize catheters. The venous catheter was used for the
injection of drugs, while the arterial catheter was used for pressure
measurements.
Surgical procedure in 1-week-old hatchlings
Additional surgical procedures were conducted on six 1-week-old American
alligators. Each animal was placed in a plastic box containing a cloth
saturated with isoflurane to induce anesthesia. Once anesthetized, the glottis
was intubated with PE-90 tubing and ventilated (SAR-830 ventilator) at 2
breaths min1 (tidal volume=1 ml) with a 2% isoflurane/room
air mixture using a FluTec vaporizer (OH, USA) until the surgical plane of
anesthesia was achieved. A 1 cm cut was made in the skin at the midline of the
dorsal surface of a rear limb above the femur, the skin was retracted and
underlying musculature separated to expose the femoral artery and vein. Once
isolated, both vessels were occlusively catheterized using heparinized
saline-filled heat-pulled PE-50 tubing. Both catheters were then tunneled
under the skin, externalized and fixed with a single suture to the back of the
animal. A blind saline-filled injection port was connected to the end of the
arterial catheter, and the venous catheter was heat-sealed. The skin incision
was sutured closed with 4-0 suture and sealed with Vet-bondTM tissue
adhesive. Once surgical procedures were completed, animals were placed in a 30
l glass aquarium covered with cardboard, with 1 cm of water in the bottom and
maintained at 28°C. A saline-filled section of PE-90 tubing fitted with a
21-gauge needle was connected to the catheter port. This section of PE-90
tubing was connected to a pressure transducer (DP6100; Peter von Berg, Sweden)
calibrated against a static column of water. All animals were allowed 24 h to
recover prior to experimentation.
Signal recording and calibration
The arterial catheter was attached to a pressure transducer 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, USA). In all cases, reference zero pressure was set at
the top of the experimental bath, and all values were corrected a
posteriori to take into account the position of the heart as previously
described (Altimiras and Crossley,
2000). In the hatchling alligators, the experimental zero was set
at the level of the heart of the animal.
Experimental protocol
The measured hemodynamic variables were allowed to stabilize for 1 h to
establish control values prior to any experimental manipulation. During this
period, arterial pressure was recorded and heart rate was determined from the
pressure trace with an online software tachograph.
After the control period, a pharmacological assessment of the cardiac limb
of the baroreflex was carried out as previously described
(Altimiras et al., 1998;
Altimiras and Crossley, 2000
).
To assess the baroreflex response to a lowering of systemic pressure
(hypotensive reflex), three sequential doses (25 µg kg1,
50 µg kg1 and 100 µg kg1) of sodium
nitroprusside (SNP) were injected followed by a saline flush. The total volume
injected (75 µl, 105 µl, 105 µl and 150 µl to 60%, 70%, 80% and
90% embryos, respectively) was less than 5% of the embryonic blood volume
estimated from data in chicken embryos. Previous tests showed that saline
injections of up to 5% estimated blood volume did not elicit any pressure
changes. Heart rate and blood pressure were allowed to return to control
levels after each injection. After the third injection of SNP, the baroreflex
response to an increased blood pressure (hypertensive reflex) was studied with
three sequential doses (30 µg kg1, 60 µg
kg1 and 120 µg kg1) of phenylephrine
(PE). Cardiovascular variables were also allowed to return to control levels
between each dose. These protocols were conducted on embryos at 60%
(N=7), 70% (N=8), 80% (N=9) and 90% of incubation
(N=9) and in 1-week-old hatchling animals (N=6). After
completing this protocol, a single bolus of the muscarinic antagonist atropine
(3 mg kg1) was injected to block any contribution of the
parasympathetic autonomic nervous system to baroreflex regulation, and the
baroreceptors were stimulated again with the largest dose of PE (120 µg
kg1). At the conclusion of the experimental protocols,
embryos were euthanized with an intravenous injection of sodium pentobarbital
(50 mg kg1).
Calculations and statistics
The Wilcoxon nonparametric test was used to assess the response to
different drug administrations (atropine, SNP and PE). A MannWhitney
nonparametric U-test was used to determine differences through
development. Since repeated tests were carried out, thereby using the same
data more than once, the fiduciary limit (P=0.05) was corrected
according to the number of times each data set was used, commonly 23
times since the tests between developmental intervals were restricted to
adjacent days (thereby comparing 60% to 70%, 70% to 80%, 70% to 90% and 80% to
90%).
Baroreflex gain in embryos was calculated from the
heart-ratearterial-pressure linear slope as reported previously
(Altimiras and Crossley, 2000).
The hatchling data was fitted to a four-variable sigmoid logistic function as
previously described (Altimiras et al.,
1998
). Data from another study on 1-year-old alligators (B.
Bagatto, D. A. Crossley, J. Altimiras and J. W. Hicks, unpublished) was fitted
to the same type of model for comparison purposes. Although the baroreflex
quantification methods differ between embryos and hatchlings, gain will be
referred to as maximal gain in all cases (G50; see
Altimiras et al., 1998
for a
more extended description).
To establish meaningful comparisons between different developmental stages,
gain was normalized for control mean arterial pressure
(a) and heart rate
(fH) similar to what was previously suggested
(Berger et al., 1980
).
Normalized gain (G50,norm; unitless) was calculated as:
G50,norm=G50(
a/fH).
a where baroreflex gain is maximal
is also obtained from the sigmoid logistic model and will be reported as
aG50 (kPa). Data are shown
as means ± S.E.M. Significant differences were all taken at
the fiduciary level of P<0.05.
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Results |
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Pharmacological pressor responses
SNP induced a pronounced hypotension at all incubation ages
(Fig. 2A). The magnitude of
this response varied with drug dose and developmental age. The intermediate
(50 µg kg1) and maximal (100 µg kg1)
doses produced the greatest reduction in pressure at all incubation ages with
a maximum at 80% of development (a
dropped by 0.52±0.08 kPa).
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PE injection produced a marked hypertensive response that was not dose dependent. While the maximal response to each drug dosage was the same, the effects of PE increased with progressive developmental age (Fig. 2B).
Baroreflex assessment
Increased arterial pressure due to PE injection induced a transient
reciprocal change in fH
(Fig. 3). This response is
indicative of a functional baroreflex at 70% of embryonic alligator
development and later. However, no hypotensive baroreflex response (increase
in fH) was observed after SNP injection at any embryonic
age. Note, however, that the hypertensive heart rate response at 70% and 80%
was brief and subsided even though a
continued to rise (Fig. 3). The
mean time between the maximal heart rate drop elicited by the baroreflex and
the maximal pressure response elicited by PE was 73±16 s at 70% and
47±14 s at 80%. This time was significantly decreased at 90%
(9±7 s; P<0.05) because the heart rate response lasted
longer.
|
The mean baroreflex response in embryonic alligators is shown in
Fig. 4 by plotting the changes
in fH induced after pharmacological alteration of
a. The highest baroreflex gain in
response to increased
a was obtained
at 80% of incubation.
|
In hatchlings, the baroreflex was responsive to hypotension as well as to
hypertension (Fig. 5). The
operational point of the reflex was shifted towards higher blood pressures and
lower heart rates, and this was further shown in 1-year-old alligators
(Fig. 6). For comparative
purposes, Fig. 6 shows the
afH
relationship at all ages studied. The operational point of the baroreflex
together with maximal and normalized baroreflex gains are summarized in
Table 1. As
a rose from 1.2±0.1 kPa (70%)
to 4.3±0.1 kPa (1 year), G50 peaked at 80% of
incubation (41.2 beats kPa1 min1) and fell
during the final 20% of incubation (G50=12.54 beats
kPa1 min1 at 90%;
Table 1). The gain of the
baroreflex continued to fall in hatchlings, reaching a minimal level in
1-year-old animals (G50=9.69 beats kPa1
min1). Due to the different operational values for heart
rate and blood pressure in embryos (high heart rates, low blood pressures)
versus hatchlings (lower heart rates and higher blood pressures),
normalized baroreflex gain (G50,norm) was maximal in
1-year-old alligators (2.12%
fH
%
a1;
Table 1).
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Role of muscarinic receptors in baroreflex regulation
Given that the cardiac baroreflex response in adult crocodiles and all
vertebrate species studied is largely dependent on vagal nerve activity
(Altimiras et al., 1998), the
experimental protocol was extended to verify this dependence by blocking
muscarinic cholinergic receptors pharmacologically with atropine. The
suitability of atropine (3 mg kg1) as a muscarinic
antagonist was first checked by injecting a bolus dose of acetylcholine (100
µg kg1) before and after atropine blockade. The
bradycardia elicited by acetylcholine was abolished by atropine injection in
70% and 90% embryos (Fig.
7).
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After atropine injection, SNP and PE triggered the expected responses in blood pressure in embryos (Fig. 8A) and hatchlings (Fig. 9A), but reflexive fH changes were largely blunted. The hypertensive reflex was abolished in 80% and 90% embryos (Fig. 8B) and also in hatchlings (Fig. 9B).Notice, however, that the hypotensive reflex tested with SNP was not completely abolished in hatchlings after atropine (Fig. 9B).
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Discussion |
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Developmental pattern of heart rate and mean arterial pressure
This study comprised the first assessment of basal blood pressure and heart
rate during embryonic development in a crocodilian species.
a rose consistently during the last
40% of incubation and continued to rise in hatchlings
(Fig. 1), a pattern of pressure
change that is consistent with that of other developing vertebrates
(Blanco et al., 1988
;
Segar et al., 1992
;
Tazawa and Hou, 1997
). By
contrast, fH remained constant during embryonic incubation
and dropped markedly following hatching
(Fig. 1). This sudden reduction
in resting fH is due, in part, to the onset of tonic vagal
depression of heart rate in hatchling alligators (J. Altimiras and D. A.
Crossley, II, unpublished observations). This pattern of
fH change is unique among the egg-laying species that have
been studied thus far. In chicken embryos, for example, fH
is maintained at high levels post-hatching (reviewed in
Tazawa and Hou, 1997
).
Pharmacological manipulation of the vasculature also revealed unique
characteristics during alligator development.
Vascular response to sodium nitroprusside and phenylephrine
Embryonic alligators exhibited the typical `adult' vascular responses to
SNP, a receptor-independent vasodilator, and PE, a receptor-dependent
vasoconstrictor. The vascular response to SNP was dose dependent at all
developmental ages with maximal responses occurring at 80% of incubation
(Fig. 2), which corresponds to
the expected greatest vascular density based on embryonic chicken data
(Romanoff, 1967). In contrast
to the dose-dependent SNP response, PE induced a vasoconstriction that was
independent of dose, indicating that all doses used saturated the number of
functional
-adrenergic receptors. However, this method was sufficient
to illustrate that the maximal vasoconstriction increased with developmental
age, being greatest at 80% and 90%. In chickens, PE produced its greatest
response in vascular rings from femoral and carotid arteries of 90% incubation
embryos, suggesting there is a change in
-adrenergic receptor density
(Le Noble et al., 2000
).
Therefore, while the maximal response pattern could have been related to a
change in vascular density in alligator embryos, it also could have been due
to changes in
-adrenergic receptor density of the vasculature.
Baroreflex features and mechanistic basis
The cardiac limb of the baroreflex was not functional at 60% of incubation
and appeared for the first time at 70% of incubation age. This reflexive
response was present when a
Reductions in
a with SNP only
triggered changes in heart rate in hatchlings and 1-year-old animals. The
bradycardia that followed the hypertension was mediated by vagal activity as
illustrated by the abolished response after atropine pre-treatment in 80% and
90% embryos and in hatchlings (Figs
8,
9). Interestingly, atropine
itself had no effect on heart rate (J. Altimiras and D. A. Crossley, II,
unpublished data), which is similar to what occurs in embryos of the white
leghorn domestic chicken (Crossley and
Altimiras, 2000
). These results indicate that the vagus nerve is
functional at least at 70% of incubation age but it is not tonically active.
This pattern of a functional vagus without tone explains the presence of a
hypertensive baroreflex, dependent on vagal inhibition, and the absence of a
hypotensive baroreflex, which requires in part the withdrawal of vagal tone.
was increased only.
In addition to the withdrawal of vagal tone, sympathetic efferents
constitute a secondary component of the hypotensive baroreflex in adult
amniotic vertebrates (Altimiras et al.,
1998; Korner,
1971
). This mechanism may also be operational in hatchlings, as
seen in animals pre-treated with atropine
(Fig. 9). The slight but
significant tachycardia recorded after the hypotensive bout with SNP (3 beats
min1) is probably due to sympathetic activation, although
the possible activation of other cardiac reflexes cannot be discarded without
further experimentation. However, the late appearance of sympathetic
innervation of the heart in other in ovo developing embryos
(Higgins and Pappano, 1979
)
would indicate that this secondary mechanism is not involved in the embryonic
alligator baroreflex.
The next prominent feature of the cardiac limb of the baroreflex during
alligator development is its apparent rapid resetting at the time of the
initial onset (70% and 80% of incubation time). While the mechanisms involved
in baroreflex resetting cannot be resolved without further experimentation, it
is relevant to discuss the different alternatives that could explain it. Rapid
resetting was observed at 70% and 80% of incubation, as indicated by the
initial pressure-induced bradycardia returning rapidly to control levels while
arterial pressure continued to rise (Fig.
3). At 90% of incubation, the pattern was similar to that found in
hatchlings and 1-year-old alligators, with a sustained bradycardia occurring
until a returned to the control
values (Fig. 3). Several
mechanisms are involved in rapid or acute resetting of the baroreflex. The
best-characterized mechanism relates to an alteration of vessel wall mechanics
where baroreceptive endings are located
(Eckberg, 1977
). At the onset
of a rapid increase in pressure, the tissue of the vessel wall lengthens due
to viscoelastic properties. As a result, the baroreceptors are unloaded even
when the overall vessel diameter or the strain on the vessel wall is increased
(Chapleau et al., 1988
). The
acute resetting observed in the present study could therefore be related to
developmental changes in the anatomic structure of the vessel wall where the
baroreceptive endings are located. In reptiles and birds, the main
baroreceptive area is the proximal truncus and the aortic arch
(Backhouse et al., 1989
;
Berger, 1987
;
Smith, 1994
). In this light,
it is relevant that the development of the aortic wall in other in
ovo developing embryos occurs in two distinct anatomic steps. In
embryonic chickens, until 85% of incubation (day 18) the diameter of the aorta
is increased by deposition of new cell layers
(Arciniegas et al., 1989
).
Later in development the increased aortic diameter is due to an increased
lumen of the vessel (Arciniegas et al.,
1989
). If aortic development occurs in alligators as in chickens,
changes in the mechanical properties of the aortic wall during development may
be linked to changes in baroreflex gain and acute resetting.
These structural changes may also account for the chronic resetting of the
baroreceptors required as a increases
during development (Fig. 2).
Chronic resetting of the baroreflex allows this mechanism to remain
operational within a physiological range of arterial pressures during
vertebrate development (Segar,
1997
). This process was evident by the continuous increase in the
operational point of the baroreflex as alligator development progressed
(
a where baroreflex gain is maximal;
referred to as
aG50 in
Table 1). However, the exact
mechanisms that underlie this process are unknown.
Maturation of baroreflex gain
Change in baroreflex gain is an important quantitative measure of
baroreflex maturation during development. In this context, the experimental
approach used in this study was limited because changes in peripheral
resistance (the peripheral limb of the baroreflex) were not considered.
However, given that sympathetic vascular regulation is absent in the fetal
sheep until term (for a review, see Segar,
1997) and in the domestic chicken until the final 10% of
incubation (Crossley et al.,
2003
; Mulder et al.,
2002
), quantification of the cardiac limb alone was considered a
good approximation to the baroreflex. Absolute baroreflex gain was maximal at
80% development (41.22 beats kPa1 min1),
and dropped thereafter, reaching a minimum in 1-year-old alligators (9.69
beats kPa1 min1;
Table 1). It is possible,
however, that absolute gain cannot be used as a measure of baroreflex
regulation at different developmental ages. This is based on the fact that the
observed changes in baroreflex gain are accompanied by important changes in
resting cardiovascular parameters, as illustrated by the drop in heart rate
seen in hatchling alligators (Fig.
1), which remains low in adult animals
(Fig. 6). Thus, embryonic
alligators have lower arterial pressure and higher heart rates than hatchlings
and adults. If this is accounted for and baroreflex gain is calculated
relative to resting arterial pressure and heart rate
(G50,norm; Table
1), a different picture appears. Among embryonic stages, maximal
relative gain was still seen at 80%, but the normalized maximal gain increases
first in hatchlings and then in 1-year-old alligators. This is due to the fact
that smaller changes in gain have a greater influence on blood pressure
regulation in systems operating at higher pressures and lower heart rates.
This implies that baroreflex regulation is more important as alligator embryos
develop from 80% to hatching.
In summary, baroreflex regulation appears during embryonic development with a substantial gain. This suggests that during embryonic development the baroreflex is necessary for proper cardiovascular maturation. Further differences in the embryonic developmental process between alligators and chickens may require the baroreflex to become functional earlier in embryonic alligators. Therefore, a delayed onset of baroreflex function is not a feature of embryonic development in an egg case.
This work could not have been carried out without the collaboration and competence of Dr Ruth Elsey from the Rockefeller Refuge. J.A. was in receipt of an EU postdoctoral fellowship (TMR Contract# ERBFMBICT982940). The work was supported by National Science Foundation Grant to J.W.H and an American Heart Association training fellowship to D.A.C.
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