Effect of aestivation on long bone mechanical properties in the green-striped burrowing frog, Cyclorana alboguttata
1 School of Life Sciences, The University of Queensland, Brisbane,
Queensland 4072, Australia
2 School of Biomedical Sciences, The University of Queensland, Brisbane,
Queensland 4072, Australia
* Author for correspondence (e-mail: cfranklin{at}zen.uq.edu.au)
Accepted 7 November 2003
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Summary |
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Key words: osteoporosis, disuse, immobilisation, anuran, aestivation, Cyclorana alboguttata
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Introduction |
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In addition to skeletal disuse resulting from clinical or otherwise
unnatural circumstances, many animals experience extended periods of
immobilisation in nature that could also affect bone properties or size. Two
prominent examples of natural immobilisation are hibernation and aestivation.
Hibernation in endotherms is a dormancy strategy characterised by a reduction
in body temperature near to ambient temperature, a markedly reduced metabolic
rate and spontaneous arousals by activation of major heat-producing mechanisms
(Bartholomew and Hudson, 1960;
Kayser, 1961
;
Lyman et al., 1982
).
Similarly, aestivation is a state of reduced metabolism seen most commonly in
organisms, such as anurans, inhabiting periodically dry habitats
(Pinder et al., 1992
). The
green-striped burrowing frog, Cyclorana alboguttata, survives
droughts through the excavation of an underground chamber, the formation of a
waterproof cocoon and the storage of water in the bladder
(Flanigan et al., 1993
;
Withers, 1993
). The inactivity
of hibernation is known to be correlated with decreases in bone size and
strength (Doty and Nunez,
1985
; Whalen et al.,
1971
) although the response varies between species. However,
whether aestivation also induces such changes in bone properties is
unknown.
Limb immobilisation related to aestivation can be extreme. In such a
capacity, the long bones of the hindlimbs can be effectively immobilised for
several years at a time. C. alboguttata induced to aestivate on top
of pressure-sensitive piezoelectric film in glass jars showed no limb
movements in a 12-week experimental period (N. Hudson and C. Franklin,
unpublished data). However, it has become apparent that the morphology of the
semimembranosus capillary beds is unaffected by aestivation
(Hudson and Franklin, 2003).
Moreover, following nine months of aestivation in a laboratory setting, C.
alboguttata have the immediate capability to resume muscle performance as
measured by burst swimming and in vitro gastrocnemius force
production (Hudson and Franklin,
2002a
).
Taken together, these data strongly suggest that C. alboguttata
emerge from aestivation in the field with a fully competent locomotor system,
and therefore within hours of surfacing can transmit forces through the long
bones that are representative of an active specimen. This contrasts with the
situation in hibernating mammals where there is some impairment in muscle
performance. For example, Harlow et al.
(2001) found a 23% deficit in
the contractile performance of the tibialis anterior in the overwintering
black bear Ursus americanus. Although overall locomotor performance
has never been measured in a hibernator, it is likely to parallel the deficit
in isolated muscle performance and thereby reduce the demands on the skeletal
system until the muscle regains its prehibernation performance. Hibernation
also induces loss of bone in bats (Whalen
et al., 1971
), hamsters
(Kayser and Frank 1963
) and
squirrels (Mayer and Bernick,
1959
). In light of the empirically determined difference in muscle
performance between emerging hibernators and aestivators, it seems possible
that, in contrast to hibernators, aestivating frogs may preserve long bone
size and strength in order to withstand the forces associated with jumping
upon emergence.
To assess the impact of three and nine months of aestivation on the
skeletal system of C. alboguttata, we tested the ex vivo
mechanical properties of the femur and tibiofibula using three-point bending
and compared these results with those of frogs kept active in the laboratory.
The femur and tibiofibula are the main skeletal elements in the hind limbs and
are typically sensitive to usage-dependent remodelling in vertebrates.
Although bending has never been experimentally demonstrated in frogs
(Calow and Alexander, 1973), it
is predicted on the grounds that the long bones (particularly the femur)
display some curvature and are subject to compression and because the
predominant direction of muscle and reaction forces rarely coincides with the
bones' longitudinal axes (Biewener,
1983
).
We estimated an appropriate in vivo loading rate to apply to the
long bones of C. alboguttata using force plate records of ground
reaction forces developed during jumping. The mechanical data were
supplemented by histological analysis to assess osteocyte activity, as it is
known that in hibernating bats osteocytic osteolysis accounts for much of the
associated bone loss (Whalen et al.,
1971).
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Materials and methods |
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Estimation of in vivo bone loading rate during jumping
To determine a biologically relevant loading rate for bending tests, we
used force platform records of jumps to evaluate an in vivo rate of
bone loading. Ground reaction forces represent one of the major forces
experienced by the long bones during locomotion; therefore, the time to peak
force was judged to be the most biologically relevant duration of loading to
apply during tests of bone mechanical properties. Control frogs
(N=35) of various masses were encouraged to leap off a custom-built
force platform at 25°C (platform based on design of
Katz and Gosline, 1993; see
Wilson and Franklin, 2000
for
details) that simultaneously measured the vertical, horizontal and lateral
ground reaction forces exerted during a single jump. Net ground reaction force
was calculated as the vector sum of the three force components. Changes in
force in the three dimensions were detected with 5-mm aluminium foil strain
gauges attached to the outer side of each spring blade. Each strain gauge,
which corresponded to a separate dimension, formed a quarter of a bridge
circuit that fed a signal directly into a Maclab bridge amplifier. Data were
collected by a Maclab 4e (AD Instruments, Castle Hill, Australia)
analog-todigital data acquisition system that sampled at 1000 Hz. Data were
recorded and analysed using Chart 3.5 software. As it has previously been
shown in swimming C. alboguttata that locomotor performance after
nine months aestivation is indistinguishable from control levels
(Hudson and Franklin, 2002a
),
it was assumed that the same would be true for jumping; thus, force traces
from post-aestivating frogs were not obtained. For each individual, the
longest of three jumps was considered to represent a maximal performance.
However, we cannot exclude the possibility that variations in takeoff angle
could confound the results, as Marsh
(1994
) has previously pointed
out.
Ex vivo bone bending
The femur and tibiofibula of control (N=8; mass=19.75±3.86
g; snoutvent length=5.60±0.22 cm, mean
±S.E.M.), 3-month-aestivating (N=11;
mass=23.70±4.94 g; snoutvent length=5.53±0.24 cm, data
collected immediately post aestivation for both aestivation treatment groups)
and 9-month-aestivating frogs (N=6; mass=19.2±5.06 g;
snoutvent length=5.38±0.35 cm) were removed by disarticulation
at the hip joint. The variation in sample size was a simple function of the
availability of animals. The overlying soft tissues were left intact. The
samples were wrapped in 0.9% salinemoistened tissue, placed in plastic vials
within sealed polystyrene bags and stored frozen at 20°C. Prior to
mechanical testing, the bones were thawed to room temperature (22°C),
cleared of surrounding soft tissue and measured.
Each bone was rested (metaphysis to metaphysis) on rigid brass supports
19.5 mm apart and broken by three-point bending using an Instron 8722
servo-hydraulic materials testing machine (Instron, High Wycombe, UK).
Three-point bending was achieved using a hydraulically driven actuator with a
single point of load application equidistant between the supports. All bones
were loaded at approximately mid-shaft with the posterior surface experiencing
compressive loading (Fig. 1).
This loading regime was chosen for practical reasons as the bones were stable
in this orientation throughout the whole loading event. In vivo, the
tibiofibula is most probably loaded in this way, with the femur experiencing
anterior compressive loading primarily by the cruralis and gluteus magnus,
coupled with a bending moment caused by the resultant ground reaction force.
However, the femur is relatively cylindrical with little variation in cortical
thickness; as such, the second moment of area is not influenced by the bone's
orientation, assuming a neutral axis passing through the centre of the bone's
cross-section. Consequently, the effect of orientation on femoral bending
strength was considered minimal; either way, the orientation was the same in
all tests. An actuator speed of 20 mm s1, used as a
preliminary experiment, showed that it led to bone fracture after
approximately 50 ms, comparable with the time-to-maximum force recorded for
in vivo jumping of C. alboguttata. This loading rate is
somewhat faster than has been used on organisms of comparable size.
Correspondingly, values for stress and modulus of elasticity will be higher
than those generated at less realistic loading rates
(Currey, 1975).
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The load cell output was pre-amplified (QUANTEC, Brisbane, Australia) and subsequently collected at 1000 Hz on a Maclab data acquisition system interfaced with a Macintosh 4e computer running Chart software (version 3.5). Load-displacement records were re-plotted using Sigmaplot 5.0 software (SPSS, Chicago, IL, USA). Values for the load and displacement at the yield and failure points were determined from these plots. The point of yield was defined as the intersection of tangents drawn to the linear elastic and plastic deformation portions of each curve. Areas under the curves, indicative of the energies of yield and of failure, were also determined from the plots.
Following mechanical testing, each bone was sectioned transversely
approximately 1 mm distal to the site of fracture with a diamond saw microtome
(Leitz 1600, Oberkochen, Germany). Each bone was then mounted under a
dissection microscope (Leica M26, Witzlar, Germany) and the cross-section
photographed (Panasonic KR-222 digital camera, Osaka, Japan) at known
magnification. Images were rotated to the same orientation (Corel Draw 7.0;
Corel Corporation) and then printed. The endosteal and periosteal perimeters
were traced on these printouts using a digitising tablet attached to a
Macintosh computer running NIH image analysis software. The cross-sectional
area and second moment of area about the assumed bending axis were calculated
using a custom-written macro. These data were used in conjunction with the
force data to calculate femoral and tibiofibular elastic moduli (a measure of
stiffness) and ultimate strengths in accordance with the following formulae
(Carrier and Leon, 1990):
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The cross-sectional areas of the femur and tibiofibula were normalised to snoutvent length (cross-section/snoutvent length; mm2 cm1) for the tabulated data to determine if there were any gross changes in tissue size with aestivation.
Histology
The tibiofibula and femur from two control frogs and two 9-month
aestivators were fixed in 10% neutral buffered formalin, embedded in methyl
methacrylate and mounted on glass slides. Serial sections (65 µm thick)
were cut from the undecalcified midpoints of bones using a Leitz 1600 diamond
saw microtome. For each bone, a representative section was qualitatively
examined and photographed using an Olympus DP10 digital camera attached to an
Olympus BX60 microscope.
Statistical analysis
Data are presented as means ± S.E.M. of N
measurements. The comparisons of cross-sectional area were made using analysis
of covariance (ANCOVA) on the original (non-normalised) data with
snoutvent length as the covariate. Comparisons of ultimate strength,
moduli, second moment of area, energy of yield, energy of failure and yield
displacement between the treatment groups were made using 1-way analysis of
variance (ANOVA). Data were log transformed if they initially failed to meet
the assumption of normality. All analyses were performed using Sigmastat and
SPSS software. P<0.05 was considered significant.
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Results |
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Ex vivo bone bending
During a typical load-displacement on a control femur and tibiofibula,
strain initially varied with stress linearly
(Fig. 2B). When the bones
yielded (after 53±3 ms for the femur and 54±1 ms for the
tibiofibula), increments in strain led to smaller increases in stress. Soon
thereafter (64±3 ms for the femur and 66±2 ms for the
tibiofibula) the bone would fracture and stress dropped abruptly.
The ultimate strength and moduli of the tibiofibula and femur of C.
alboguttata after 3-months and 9-months aestivation were compared with
those of active frogs. Aestivation had no significant effect on these bending
mechanical properties in the long bones
(Table 1), with
9-month-aestivating frogs displaying the same resistance to bending forces as
the controls (femoral ultimate strength, P=0.45; femoral modulus,
P=0.32; tibiofibular ultimate strength, P=0.09; tibiofibular
modulus, P=0.21). Energy of yield (femoral, P=0.91;
tibiofibular, P=0.42) and failure (femoral, P=0.73;
tibiofibular, P=0.59) and yield displacement (femoral,
P=0.80; tibiofibular, P=0.96) were also compared, and again
aestivation had no significant effect on these parameters
(Table 1). Admittedly, the data
do approach significance at the 0.05 level in some of the comparisons, and a
higher sample size may have produced a significant difference. Having said
this, previous studies in which differences following dormancy were found
(e.g. Krook et al., 1977) used
smaller sample sizes than the present study.
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Bone size
Second moment of area was compared, and aestivation had no significant
effect (femoral, P=0.53; tibiofibular, P=0.68). Neither
femoral nor tibiofibular cross-sectional area (normalised to snoutvent
length) changed following the disuse associated with either 3- or 9-months
aestivation (femoral, P=0.108; tibiofibular, P=0.063;
Table 1).
Histology
Qualitative examination of the histological parameters indicated the
absence of disuse osteoporosis despite 9 months of limb immobilisation. For
example, the bone margins of aestivators had no ruffle border, remaining
relatively smooth and bearing no evidence of osteoclastic activity
(Fig. 4).Osteocytes were
distributed similarly through the cortex in both controls and aestivators and
there was no intracortical remodelling in any bone. Finally, the osteocytes in
the aestivators were not enlarged compared with those of the controls and did
not possess either basophilia or metachromasia.
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Of further note was the presence of arrest/growth lines running parallel to the endosteal perimeter in the middle of the cortex in some of the specimens. This feature was evident in both the tibiofibula and femur of one of the aestivators and in the femur of one of the controls.
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Discussion |
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The major findings of this study are consistent with the preservation of
other components of the locomotor system in C. alboguttata, namely
hind limb muscle masses (Hudson and
Franklin, 2002a), capillary tortuosity
(Hudson and Franklin 2003
),
in vitro contractile properties of the gastrocnemius and burst
swimming performance (Hudson and Franklin,
2002a
).
Mechanical properties
The present study presents a comprehensive analysis of bone mechanical
properties in an amphibian. The ultimate strength of the femur and tibiofibula
in C. alboguttata (328 MPa and 253 MPa in the controls) corresponds
favourably with values calculated for small mammals. For example, Biewener
(1982) found that the chipmunk
(Tamais striatus) femur and tibia had ultimate strengths of 263 MPa
and 303 MPa, respectively, while the rat (Rattus norvegicus) femur
and tibia showed values of 253 MPa and 233 MPa. Equally, the bending strengths
of whole bones in birds are also broadly comparable, although in these cases
there seems to be a greater disparity between femoral and tibiofibular data.
Analysis of three-point bending in the painted quail (Excalfatoria
chinensis) gave values of 311 MPa and 170 MPa for the femur and tibia,
respectively, while those of the bobwhite quail (Colinus virginianus)
were 193 MPa and 294 MPa (Biewener,
1982
).
In the present study, no unequivocal pattern emerges when comparing the
strength of the femur with respect to the tibiofibula, which is consistent
with some prior data on small mammals and birds
(Biewener, 1982). However,
greater femoral strength has previously been noted for alligators, iguana
(Blob and Biewener, 1999
) and
horses (Currey, 1984
). In
these cases, one possible explanation was given in terms of the bone's
position. The more distal the bone, the greater contribution it makes to the
leg's moment of inertia and the more energy is required to move it
(Currey, 1984
). As such, in
some cases the energy saved by the animal in having a lighter tibia (which is
distal to the femur) might offset the `riskier' safety factor that follows the
reduction in strength. This general pattern may be evident in the elongated
metatarsals of frogs that are distal to the tibiofibula but were not examined
in this study of C. alboguttata. Alternatively, the advantages of
reducing distal limb segments may scale with mass and the lack of a pattern in
these frogs may be a simple function of small body size.
Blob and Biewener (1999)
have shown that magnitudes of peak ground reaction force acting on the limbs,
relative to body weight, are lower in green iguanas (Iguana iguana)
and American alligators (Alligator mississippiensis) than in mammals
and birds. Force platform recordings indicate that peak ground reaction force
magnitudes acting on a single limb range between 1.3x and 2.4x
body weight for quadrupedal mammals but only 1.1x for iguanas and
alligators. The peak ground reaction force during jumping for C.
alboguttata is about 1.4x body weight (each limb supports half the
0.49 N force exerted during a jump; i.e. 0.49/2=0.245 N, the mass of the
frog=0.018 kg or 0.17 N; thus, 0.245/0.17=1.4), falling well into the range
for the ectotherms but very much at the lower end of the scale for the
endotherms. Fundamentally, the ground reaction force data for C.
alboguttata are in line with those found in the American alligator and
green iguana, species that have high safety factors
(Blob and Biewener, 2001
).
Moreover, a relatively high safety factor was shown experimentally by Calow
and Alexander (1973
) for a
different species of anuran, the European common frog Rana
temporaria. Accordingly, it seems probable that C. alboguttata
have a high safety factor, although one cannot be accurately calculated from
the present data without detailed strain gauge data or frog bone strains
in vivo. The fact that bone strength could presumably be lost in
these frogs without undue risk of fracture, but is not, points to a simple,
inherent preservation mechanism.
Mechanism underpinning the preservation of bone size and strength
Bradymetabolic organisms are relatively quiescent, which means prior to
immobilisation their bones have only a moderate loading history. Assuming that
the rate of remodelling depends on the extent of unloading (sensu
Jee and Ma, 1999), bone tissue
of relatively inactive organisms (such as amphibians) should be intrinsically
more resistant to disuse osteoporosis than more active, tachymetabolic
organisms (such as mammals) because the change in stimulus is that much
weaker. In a sense, it could be argued that burrowing frogs are predisposed to
withstand lengthy periods of disuse without losing bone mass. We have
previously invoked this argument to partly explain the preservation of
skeletal muscle structure in aestivating C. alboguttata
(Hudson and Franklin, 2002b
).
In light of this predisposition, the pertinence of C. alboguttata as
a model system for biomedical studies of disuse osteoporosis is reduced. This
logic points clearly to the ideal biomedical system: a tachymetabolic mammal
that suffers no disuse osteoporosis during natural disuse.
Nevertheless, in mammals, a period of disuse produces a fairly rapid
response involving the loss of bone and there may be compensatory cellular
mechanisms in operation in aestivating frogs to prevent bone loss. In
hibernating mammals, parafollicular cells in the thyroid gland are enlarged
and active compared with those in non-hibernators
(Whalen et al., 1971).
Parafollicular cells secrete calcitonin, an inhibitor of bone resorption. In
hibernating bats, the parafollicular cells show striking seasonal changes in
structure. During the first half of hibernation, the amount of granular
endoplasmic reticulum is reduced and they lose their solid dense core,
suggesting that calcitonin may be functionally inactive during hibernation
(Whalen et al., 1971
). This is
consistent with the bone resorption observed in these animals. Intriguingly,
Krook et al. (1977
) showed
that injections of calcitonin could prevent this bone loss in hibernating bats
and argued that this was reflected in smaller femoral osteocytes compared with
those in untreated hibernators. Unfortunately, the endocrinology of
aestivating frogs has been neglected and its study may provide us with a
better understanding of the effects of aestivation.
Ecological implications
Aestivating C. alboguttata can emerge within 24 h of the first
heavy summer rainfall (N.J.H., personal observation). Indeed, the emergence
process itself is presumably demanding on the skeletal system as it involves
digging through wet clay. The frogs immediately engage in predator avoidance
strategies that require a fully competent locomotor system such as burst swims
and powerful jumps. The latter transmits considerable forces through the long
bones of the hindlimbs during the leg extension phase. Given that
post-aestivation locomotor performance in swimming matches control levels
(Hudson and Franklin, 2002a)
and that frogs may actually gain mass during aestivation (N.J.H., unpublished
data), the transmission of forces through the hindlimbs in frogs after
dormancy is almost certainly equivalent to that in control frogs. The
maintenance of bone mechanical properties throughout aestivation facilitates
performance of these activities by ensuring that an adequate capacity for load
bearing is maintained.
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Acknowledgments |
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