1 Departments of Medicine and
Physiology, To determine whether the rat hindlimb elevation model can be
used to study the effects of spaceflight and loss of gravitational loading on bone in the adult animal, and to examine the effects of age
on bone responsiveness to mechanical loading, we studied 6-mo-old rats
subjected to hindlimb elevation for up to 5 wk. Loss of weight bearing
in the adult induced a mild hypercalcemia, diminished serum
1,25-dihydroxyvitamin D, decreased vertebral bone mass, and blunted the
otherwise normal increase in femoral mass associated with bone
maturation. Unloading decreased osteoblast numbers and reduced
periosteal and cancellous bone formation but had no effect on bone
resorption. Mineralizing surface, mineral apposition rate, and bone
formation rate decreased during unloading. Our results demonstrate the
utility of the adult rat hindlimb elevation model as a means of
simulating the loss of gravitational loading on the skeleton, and they
show that the effects of nonweight bearing are prolonged and have a
greater relative effect on bone formation in the adult than in the
young growing animal.
aging; disuse osteoporosis; mechanical loading of bone
SPACEFLIGHT is accompanied by a decrease in bone mass
in humans (25, 32, 35, 37). Urinary calcium increases early and tends
to remain elevated throughout flight (29, 39). The serum concentration
of 1,25-dihydroxyvitamin D
[1,25(OH)2D], although elevated on the first day, decreases during flight (24). Serum parathyroid hormone (PTH) remains unchanged (24). On long-duration missions, calcium balance becomes negative and bone mineral deficits can reach as high as 1.4%/mo in the calcaneus (37).
Ground-based bed-rest studies in humans mimic these findings (1, 17,
18, 22, 26, 35). Loss of gravitational loading as effected by head-down
antiorthostatic (to induce the cephalad fluid shift encountered during
spaceflight) or horizontal bed rest in normal adult volunteers induces
selective loss of bone mineral in those segments of the skeleton that
normally experience the greatest physical loads. Mineral apposition
rate decreases and resorption surfaces increase (35). Intestinal
calcium absorption decreases, and urinary and fecal calcium increases,
resulting in a negative calcium balance (18). Serum ionized calcium
tends to increase, PTH decreases or remains unchanged, and serum
1,25(OH)2D decreases (1, 18).
To study the influence of gravitational loading on bone and mineral
metabolism, we developed a ground-based animal model in which the
hindlimbs of young growing rats are unweighted (5, 7, 9, 42). This
reduces mechanical loading on the rear limbs and produces a cephalad
fluid shift similar to that encountered during spaceflight. In the
young growing animal, hindlimb unloading decreases serum
1,25(OH)2D, reduces bone formation
in the tibia, and induces a bone mineral deficit in unloaded regions of
the skeleton, thus mimicking the effects of spaceflight on mineral metabolism. This model uses young growing rats, however, and its relevance for the study of gravitational loading in the adult is not
clear. To examine the appropriateness of hindlimb elevation as a model
for diminished mechanical loading in adults, and to determine the
influence of age on bone responsiveness to skeletal unloading, we
studied the effects of hindlimb unloading in adult (6-mo-old) rats and
compared our results with previous skeletal unloading studies in young
growing animals. The results suggest that hindlimb unloading in the
adult rat closely resembles the effects of diminished gravitational
loading and spaceflight on mineral metabolism in adult humans. The data
also illustrate unique age-related differences in the responsiveness of
bone and mineral metabolism to skeletal unloading.
Animal protocols.
Fifty-six 6-mo-old virgin male Sprague-Dawley rats weighing
400-450 g (Simonsen Laboratories, Gilroy, CA) were fed standard laboratory rat chow (Purina Rodent Diet 5012) containing 1.01% calcium
and 0.74% phosphorus and were maintained on a 12:12-h light-dark
cycle. After a 6-day equilibration period in the Animal Care Facility,
the rats were divided into seven groups of eight animals each: a
baseline control group and groups unloaded or pair-fed for 1, 3, and 5 wk. The study was conducted twice over the course of 12 mo to ensure
that the results were reproducible.
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
Bone histomorphometry. Tibial and humeral diaphyseal segments were dehydrated, defatted in acetone followed by ether, and embedded undecalcified in polyester casting resin (Chemco, San Leandro, CA). Cross sections (80 µm) of the embedded bones were cut using a Gillings Hamco thin sectioning machine (Rochester, NY), mounted on slides, and examined using fluorescence microscopy. The first section proximal to the complete detachment of the fibula from the tibia was analyzed, and this site is referred to as the tibiofibular junction (TFJ). Additional sections from a site 4 mm proximal to the TFJ (midshaft) and sections from the midshaft of the humerus were also cut and analyzed. The area of bone between the calcein and demeclocycline labels was determined using a modification of the NIH Image program and was divided by the time interval between administration of the labels to determine the periosteal bone formation rate (12).
The proximal tibia was shaved across the anterior face (to expose the marrow cavity and permit penetration of fixative) and placed in 10% neutral phosphate-buffered Formalin for 24 h. The bones were dehydrated in ethanol and embedded undecalcified in methyl methacrylate (3). Longitudinal sections (4 and 8 µm) were cut with an AO Autocut/Jung 1150 microtome and either stained (4 µm sections) according to the Von Kossa method with a tetrachrome counterstain (Polysciences, Warrington, PA) or left unstained (8 µm sections) for fluorochrome-based measurements. Cancellous measurements were performed in an area beginning 1.0 mm distal to the growth plate-metaphyseal junction to exclude primary spongiosa and ending 4 mm distal to the growth plate. Two sections were examined from each animal, resulting in histomorphometric measurements along 30-40 mm of cancellous bone perimeter. All cancellous bone measurements were performed using the Bioquant Bone Morphometry System (R & M Biometrics, Nashville, TN) as previously described (40, 41). Cancellous bone volume as a percentage of bone tissue area and osteoblast and osteoclast surfaces as percentages of total cancellous perimeter were measured in 4-µm-thick stained sections. Fluorochrome-based indexes of bone formation, including percentages of cancellous bone surfaces with double fluorochrome labels (mineralizing surfaces, MS) and mineral apposition rate (MAR) were measured in 8-µm-thick, unstained sections from the animals euthanized after 3 wk of skeletal unloading only. Labels in the animals euthanized at 1 wk were insufficiently separated, and the calcein label (given at the time of unloading) in the animals euthanized at 5 wk was too diffuse to permit measurement of cancellous MS or MAR in these groups of animals. Bone formation rate (total surface referent, BFR/BS) was calculated by multiplying MS by MAR (uncorrected for obliquity of the plane of section) (4).Bone fat-free weight. The right femur, right humerus, and lumbar vertebrae (L2+L3) were extracted in ethanol followed by diethyl ether by use of a Soxlet apparatus, dried overnight at 100°C, and weighed to determine the fat-free weight.
Clinical laboratory analyses.
The serum concentration of PTH was determined in duplicate using a
commercially available immunoradiometric assay kit (Nichols Institute
Diagnostics/Immunotopics, San Juan Capistrano, CA), which measures
intact rat PTH-(184). Intra-assay and interassay coefficients of
variation for this assay are 6.9 and 12.4%, respectively, at a serum
concentration of 19.6 pg/ml. The minimum detectable concentration
(B/Bo = 0.8) is 6 pg/ml. The serum
concentration of 1,25(OH)2D was
measured using the method of Reinhardt et al. (30).
Statistical analyses. For consistency, the results of experiment 1 are presented unless otherwise indicated. Data are reported as means ± SD. Statistical analysis was performed using Student's t-test, the Mann-Whitney rank-sum test for nonparametric populations, and two-way analysis of variance and the Newman-Keuls test where appropriate (Sigma-Stat, Jandel Scientific, San Rafael, CA). Linear regression analysis was used to estimate the slope of the relationship between bone fat-free weight and time of unloading for both the control and unloaded rats.
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RESULTS |
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Results of the two experiments were virtually identical. Body weight
was below basal levels (5.7%,
P < 0.05) after 1 wk in both control
and unloaded animals (Fig. 1). By 3 and 5 wk, body weight had returned to the basal level in control but not
unloaded animals. At 1, 3, and 5 wk, body weights tended to be lower in unloaded compared with normally loaded rats (448 ± 18 vs. 463 ± 18 g, unloaded vs. loaded, P < 0.05).
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Thymus weight was not different in normally loaded and unloaded animals and did not change during the experiment (Fig. 2). Ca2+ was also not different in normally loaded and unloaded animals at all time points (Fig. 3). Whole blood pH was unaffected by skeletal unloading.
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The serum concentration of 1,25(OH)2D remained unchanged in normally loaded animals but decreased sharply from 31 ± 8 to 13 ± 4 pg/ml (P < 0.001) after 1 wk of skeletal unloading (Fig. 4). By 3 and 5 wk, the serum concentration of 1,25(OH)2D in hindlimb-elevated animals had increased from its nadir toward normal but tended to remain below control levels even after 5 wk. The serum concentration of PTH remained constant and did not differ between loaded and unloaded animals at any time during the 5-wk experiment (data not shown). Overall mean serum PTH levels in control and unloaded animals were 36 ± 10 and 31 ± 4 pg/ml, respectively.
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Muscle weights in the hindlimbs decreased significantly during skeletal
unloading (Fig. 5). After 1 wk of hindlimb
elevation, the weights of the soleus and gastrocnemius muscles were
reduced by 35 and 16%, and by 5 wk by 48 and 27%, respectively, from
control animals (P < 0.001).
Plantaris weight was also significantly lower (10%,
P < 0.002) in unloaded animals (data
not shown).
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Cortical bone changes in the skeletally unloaded animals were striking.
Mean periosteal MARs between the time of unloading and 2 and 4 wk were
reduced by 85 and 81%, respectively, in the unloaded compared with the
normally loaded animals (data not shown). Mean periosteal BFR at the
TFJ, tibial midshaft, and humeral midshaft are summarized in Fig.
6. Mean formation rates, measured between 0 and 2 wk of unloading in the animals hindlimb elevated for 3 wk and
between 0 and 4 wk of unloading in the animals hindlimb elevated for 5 wk at both the TFJ and tibial midshaft, were reduced by ~80%
(P < 0.001). No change in mean
periosteal BFR was observed in the humerus, a normally loaded bone in
our model. The results from experiment 2 were similar. Mean BFR at the TFJ during unloading decreased by
72 ± 6%.
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Cancellous bone measurements from the proximal tibia are summarized in
Fig. 7,
A-C,
and Table 1. The percent surface of
cancellous bone occupied by osteoblasts decreased by 66%
(P < 0.05) within 1 wk of skeletal
unloading and remained below control levels for the duration of the
experiment. In the second experiment, osteoblast numbers were decreased
by 50,
28, and
48% at 1, 3, and 5 wk, respectively. Neither osteoclast surface nor cancellous bone volume, however, was significantly altered by skeletal unloading. Cancellous MS, MAR, and BFR decreased by 54, 33, and 69%
(P < 0.05), respectively, during
unloading (weeks 0-2) (Table 1).
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|
Femoral, vertebral, and humeral fat-free weights were compared in
normally loaded and unloaded animals using regression analysis (Fig.
8). Significant differences in the
regression slopes between loaded and unloaded animals were observed for
the femur and vertebrae but not the humerus. The deficits in bone mass
induced by unloading, although small, were reproducible. At 5 wk,
unloading produced deficits in vertebral mass of 13 ± 10 and
9.3 ± 4.3% (P < 0.05) in
experiments 1 and
2, respectively. Hindlimb elevation
decreased mass (vertebrae) or blunted the otherwise normal increase in
mass (femur) associated with bone maturation in the rat.
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DISCUSSION |
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Our data demonstrate that skeletal unloading by use of the adult rat hindlimb elevation model closely mimics the effects of spaceflight on bone and mineral metabolism in the human. The small but significant decrease in body weight (5.7%) during unloading is consistent with observations that some but not all astronauts lose weight during flight (34). The loss of weight, which occurs acutely after unloading and then stabilizes, probably represents a combination of diminished appetite and loss of fluids as a consequence of the cephalad fluid shift induced by head-down tilt in our model (10). The reason for the acute loss of weight in the control animals after 1 wk is not clear but may reflect the influence of pair-feeding and diminished food intake in the unloaded animals.
Glucocorticoid excess is known to induce thymic atrophy in rats (14). Because thymus weight did not change in unloaded rats, stress and excessive glucocorticoid secretion are not likely to play a major role in the bone and mineral changes induced by hindlimb elevation. That stress and increased circulating corticosterone are not factors in the bone changes induced by hindlimb elevation is further supported by the observation that normally loaded bones in our model, such as the humerus, show no evidence of glucocorticoid excess, and direct measurements of serum corticosterone throughout the day in young hindlimb-elevated animals show no elevation in hormone levels (7).
Ca2+ was modestly elevated in hindlimb-unloaded animals, a finding consistent with previous spaceflight and ground-based immobilization studies in humans (1, 11, 15, 23, 24, 31). The mild hypercalcemia associated with loss of weight bearing frequently does not reach significance, may be transient, and probably arises from the abrupt decrease in bone formation and/or increase in bone resorption induced by unloading.
Spaceflight, as well as disuse induced by bed rest or immobilization, is routinely accompanied by a decrease in the serum concentration of 1,25(OH)2D (1, 8, 9, 23, 24, 31). In our adult rat model, hindlimb elevation induced a 52% decrease in serum 1,25(OH)2D after 1 wk of unloading. Thereafter, serum 1,25(OH)2D increased, reaching an apparent steady state by 3 wk. At 3 and 5 wk, serum 1,25(OH)2D remained below control levels, but the difference did not reach significance. The pattern in serum 1,25(OH)2D after hindlimb elevation in the adult rat is virtually identical to that seen in other models of skeletal unloading and is similar to that previously observed in the young growing hindlimb-elevated animal (8, 9) between 0 and 2 wk of unloading. Previous studies in the young rat indicate that the decrease in serum 1,25(OH)2D induced by skeletal unloading is a consequence of a decrease in synthesis of the hormone and not an increase in metabolic clearance rate (8). Whether the decrease in synthesis is linked to changes in renal hemodynamics (27), a decrease in the demand for calcium by the bone, the mild hypercalcemia associated with skeletal unloading, or other metabolic changes, is not clear.
Serum PTH did not change during unloading. This is consistent with the findings during spaceflight (24) but differs from the results in bed-rest studies (1, 31), in which serum PTH tends to decrease. Previous studies with the hindlimb elevation model in young growing animals also did not reveal a change in serum PTH (8). The absence of a consistent decrease in serum PTH in our model despite the modest increase in serum calcium is not clear. It is possible that basal serum PTH is almost maximally suppressed to begin with and cannot be further reduced, or that our assays are not sufficiently sensitive to detect the magnitude of change associated with unloading.
The decrements in soleus, gastrocnemius, and plantaris muscle weights after 5 wk of hindlimb elevation in the adult rat (48, 27, and 10%, respectively) are consistent with those reported by other investigators (20, 33). Changes in muscle mass in response to decreased use appear to occur more rapidly and reach a new steady state before changes in bone. Conceivably, part of the change in bone mass is due to the reduction in muscle mass.
MS, MAR, and BFR on the periosteal surface decreased by as much as 80% during unloading. These same parameters in the humerus, a normally loaded bone in our model, were unaffected by hindlimb elevation, suggesting that the mechanisms effecting bone loss are primarily local in origin. The decreases in MS and MAR are consistent with the observed decrease in osteoblast number and also suggest a decrease in bone-forming activity per cell. Similar changes in cancellous bone were observed, along with a decrease in fluorochrome-labeled surface. These data clearly demonstrate that skeletal unloading effected by hindlimb elevation can decrease both osteoblast number and activity.
Osteoclast surface did not change significantly during unloading, suggesting that the decrease in bone mass induced by hindlimb elevation is primarily a consequence of a decrease in bone formation. However, it is possible that unloading induced an increase in osteoclastic activity (without a change in cell number). Indeed, previous studies using the taped-hindlimb rat model to effect skeletal disuse indicate that eroded surfaces can increase during unloading (20). Furthermore, spaceflight data indicate that urinary hydroxyproline is increased during flight, suggesting that bone resorption can increase during loss of gravitational loading (16, 28).
That cancellous bone volume did not decrease significantly during 5 wk of unloading despite a decrease in bone formation may be a consequence of the relatively large bone mass to begin with and the relatively short duration of our experiment. In long-term studies of hindlimb taping, cancellous bone volume has been shown to decrease and reach a new steady state after ~20 wk (20). The total deficit approaches 60%. Although cancellous bone volume did not decrease, total bone mass (fat-free weight) in unloaded skeletal regions (femur, lumbar vertebrae) was less in the hindlimb-elevated animals, a consequence presumably of the dramatic decrease in cortical bone formation.
The bone changes induced by hindlimb elevation in our adult rat model
of skeletal unloading are similar to the bone changes associated with
bed rest in adult humans (1, 22, 26, 35) and limb immobilization in
adult animals (2, 13, 19-21). They also resemble the bone changes
induced by hindlimb elevation in young growing rats but with a few
notable exceptions (5-7, 42). In the adult, unloading is
associated with a dramatic decrease in periosteal bone formation rate
(80%), a sustained decrease in the cancellous osteoblast
population, and a continued loss of bone through
5 wk. Indeed, the
bone deficit probably continues to accumulate through 20 wk before
reaching a new steady state, after which bone histomorphometric
parameters return to normal (20). In the young growing rat, the
decrease in periosteal formation is less (
40%). Osteoblast
surface decreases initially in the young growing rat but by 2 wk
returns to normal. MS, MAR, and BFR also return to normal by 2 wk in
the young rat, and the deficit in bone mineral reaches a steady state.
In the growing animal, bone formation appears to be driven largely by
growth of the animal, whereas in the adult, the relative rate of bone
formation is more dependent on mechanical loading. The effects of
unloading on bone in the adult are also prolonged. In both aged models,
however, osteoclast surfaces are not significantly affected by
unloading, suggesting that bone resorption in the rat is relatively
insensitive to weight bearing, at least under the experimental
conditions used in our studies.
In summary, our results demonstrate the utility of the adult rat hindlimb elevation model as a means of simulating both the loss of gravitational loading on the skeleton and the cephalad fluid shift induced in adult humans by spaceflight. They also show that the effects of nonweight bearing are prolonged and have a greater relative effect on bone formation in the adult than in the young growing animal.
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ACKNOWLEDGEMENTS |
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This study was supported by National Aeronautics and Space Administration Grant NCC-2-668.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: B. Halloran, 111N Endocrine Research Unit, VA Medical Center, San Francisco, CA 94121.
Received 2 June 1998; accepted in final form 28 August 1998.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Arnaud, S.,
D. Sherrard,
N. Maloney,
R. Whalen,
and
P. Fung.
Effects of 1-week head-down tilt bed rest on bone formation and the calcium endocrine system.
Aviat. Space Environ. Med.
63:
14-20,
1992[Medline].
2.
Bagi, C. M.,
M. Mecham,
J. Weiss,
and
S. C. Miller.
Comparative morphometric changes in rat cortical bone following ovariectomy and/or immobilization.
Bone
14:
877-883,
1993[Medline].
3.
Baron, R.,
A. Vignery,
L. Neff,
A. Silvergate,
and
A. Santa Maria.
Processing of undecalcified bone specimens for bone histomorphometry.
In: Bone Histomorphometry: Techniques and Interpretation, edited by R. R. Recker. Boca Raton, FL: CRC, 1983, p. 13-35.
4.
Frost, H. M.
Bone histomorphometry: analysis of trabecular bone dynamics.
In: Bone Histomorphometry: Techniques and Interpretation, edited by R. R. Recker. Boca Raton, FL: CRC, 1983, p. 109-139.
5.
Globus, R. K.,
D. D. Bikle,
and
R. R. Morey-Holton.
Effects of simulated weightlessness on bone mineral metabolism.
Endocrinology
114:
2264-2269,
1984[Abstract].
6.
Globus, R. K.,
D. D. Bikle,
and
E. R. Morey-Holton.
The temporal response of bone to unloading.
Endocrinology
118:
733-742,
1986[Abstract].
7.
Halloran, B. P.,
D. D. Bikle,
C. M. Cone,
and
E. Morey-Holton.
Glucocorticoids and the inhibition of bone formation induced by skeletal unloading,
Am. J. Physiol.
255 (Endocrinol. Metab. 18):
E875-E879,
1988
8.
Halloran, B. P.,
D. D. Bikle,
J. Harris,
H. C. Foskett,
and
E. Morey-Holton.
Skeletal unloading decreases the production of 1,25-dihydroxyvitamin D.
Am. J. Physiol.
264 (Endocrinol. Metab. 27):
E712-E716,
1993
9.
Halloran, B. P.,
D. D. Bikle,
T. J. Wronski,
R. K. Globus,
M. J. Levens,
and
E. Morey-Holton.
The role of 1,25-dihydroxyvitamin D in the inhibition of bone formation induced by skeletal unloading.
Endocrinology
118:
948-954,
1986[Abstract].
10.
Hargens, A. R.,
J. Steskal,
C. Johansson,
and
C. M. Tipton.
Tissue fluid shift, forelimb loading, and tail tension in tail-suspended rats.
The Physiologist
27:
S37-S38,
1984.
11.
Heath, H., III,
J. M. Earll,
M. Schaaf,
J. T. Piechocki,
and
T. K. Li.
Serum ionized calcium during bedrest in fracture patients and normal men.
Metabolism
21:
633-640,
1972[Medline].
12.
Hill, E. L.,
R. B. Martin,
E. Gunther,
E. Morey-Holton,
and
V. R. Holets.
Changes in bone in a model of spinal cord injury.
J. Orthop. Res.
11:
537-547,
1993[Medline].
13.
Ijiri, K., Y. F. Ma, W. S. Jee, T. Akamine,
and X. Liang. Adaptation of former nongrowing epiphyseal
trabecular bones to aging and immobilization in the rat.
Bone 17, Suppl. 4:
207S-212S, 1995.
14.
Ingle, D. J.
Effect of 2 steroid components on weight of thymus of adrenalectomized rats.
Proc. Natl. Acad. Sci. USA
44:
174-175,
1940.
15.
Leach, C. S.
Medical results from STS1-4: analysis of body fluids.
Aviat. Space Environ. Med.
54:
50-54,
1983.
16.
Leach, C. S.,
and
P. C. Rambaut.
Biological responses of the Skylab crewmen: an overview.
In: Biomedical Results from Skylab, edited by R. S. Johnson,
and L. Dietlein. Washington, DC: NASA, 1977, p. 204-216. (SP-377)
17.
LeBlanc, A.,
V. S. Schneider,
H. J. Evans,
D. A. Engelbretson,
and
J. M. Krebs.
Bone mineral loss and recovery after 17 weeks of bed rest.
J. Bone Min. Res.
5:
843-850,
1990[Medline].
18.
LeBlanc, A., V. Schneider, E. Spector, H. Evans, R. Rowe, H. Lane, L. Demers, and A. Lipton. Calcium absorption, endogenous
excretion and endocrine changes during and after long-term bed rest.
Bone 16, Suppl. 4:
301S-304S, 1995.
19.
Li, X. J.,
and
W. S. Jee.
Adaptation of diaphyseal structure to aging and decreased mechanical loading in the adult rat: a densitometric and histomorphometric study.
Anat. Rec.
229:
291-297,
1991[Medline].
20.
Li, X. J.,
W. S. Jee,
S. Y. Chow,
and
D. M. Woodbury.
Adaptation of cancellous bone to aging and immobilization in the rat: a single photon absorptiometry and histomorphometry study.
Anat. Rec.
227:
12-24,
1990[Medline].
21.
Ma, Y.,
W. S. Jee,
Y. Chen,
J. Gasser,
K. Z. Ke,
X. J. Li,
and
D. B. Kimmel.
Partial maintenance of cancellous bone mass by antiresorptive agents after discontinuation of human PTH in right limb immobilized rats.
J. Bone Min. Res.
10:
1726-1734,
1995[Medline].
22.
Minaire, P.,
P. Meunier,
C. Edouard,
J. Bernard,
P. Courpron,
and
J. Bourret.
Quantitative histological data on disuse osteoporosis.
Calcif. Tiss. Int.
17:
57-73,
1974.
23.
Morey-Holton, E. R.,
and
S. B. Arnaud.
Skeletal responses to spaceflight.
In: Advances in Space Biology and Medicine, edited by S. L. Bonting. Greenwich, CT: JA1 Press, 1991, vol. 1, p. 37-69.
24.
Morey-Holton, E. R.,
H. K. Schnoes,
H. F. DeLuca,
M. E. Phelps,
R. F. Klein,
R. F. Nissenson,
and
C. D. Arnaud.
Vitamin D metabolites and bioactive parathyroid hormone levels during Spacelab 2.
Aviat. Space Environ. Med.
59:
1038-1041,
1988[Medline].
25.
Oganov, V. S.,
A. S. Rakhmanov,
and
V. E. Novikov.
The state of human bone tissue during spaceflight.
Acta Astronaut.
23:
129-133,
1991.[Medline]
26.
Palle, S.,
L. Vico,
S. Bourrin,
and
C. Alexandre.
Bone tissue response to four month antiorthostatic bed rest: a bone histomorphometric study.
Calcif. Tiss. Int.
51:
189-194,
1992[Medline].
27.
Provost, S. B.,
and
B. J. Tucker.
Effect of 14 day head down tilt on renal function and vascular and extracellular fluid volumes in the conscious rat.
Physiologist
35:
S105-S106,
1992[Medline].
28.
Rambaut, P. C.,
and
R. S. Johnston.
Prolonged weightlessness and calcium loss in man.
Acta Astronaut.
4:
1113-1122,
1979.
29.
Rambaut, P. C.,
C. S. Leach,
and
G. D. Whedon.
A study of metabolic balance in crewmembers of Skylab IV.
Acta Astronaut.
6:
1313-1322,
1979.[Medline]
30.
Reinhardt, T. A.,
R. L. Horst,
J. W. Orf,
and
B. W. Hollis.
A microassay for 1,25-dihydroxyvitamin D not requiring high performance liquid chromatography: application to clinical studies.
J. Clin. Endocrinol. Metab.
58:
91-97,
1984[Abstract].
31.
Stewart, A. F.,
M. Adler,
C. M. Byers,
G. V. Segre,
and
A. E. Broadus.
Calcium homeostasis in immobilization: an example of resorptive hypercalciuria.
N. Engl. J. Med.
306:
1136-1140,
1982[Abstract].
32.
Stupakov, G. P.,
V. S. Kazeykin,
A. P. Kozlovsky,
and
V. V. Korolev.
Evaluation of changes in human axial skeletal bone structures during long-term spaceflights.
Space Biol. Med.
18:
42-47,
1984.
33.
Thomason, D. B.,
R. E. Herrick,
D. Surdyka,
and
K. M. Baldwin.
Time course of soleus muscle myosin expression during hindlimb suspension and recovery.
J. Appl. Physiol.
63:
130-137,
1987
34.
Thornton, W. E.,
and
J. Ord.
Physiological mass measurements in Skylab.
In: Biomedical Results from Skylab, edited by R. S. Johnson,
and L. Dietlein. Washington, DC: NASA, 1977, p. 175-182. (SP-377)
35.
Vico, L.,
D. Chappard,
C. Alexandre,
S. Palle,
P. Minaire,
G. Riffat,
B. Morukov,
and
S. Rakhmanov.
Effects of a 120-day period of bedrest on bone mass and bone cell activities in man: attempts at countermeasure.
Bone Miner.
2:
383-394,
1987[Medline].
36.
Vogel, J. M.,
and
M. W. Whittle.
Bone mineral changes: the second manned Skylab mission.
Aviat. Space Environ. Med.
47:
396-400,
1976[Medline].
37.
Vogel, J. M.,
M. W. Whittle,
M. C. Smith,
and
P. C. Rambaut.
Bone mineral measurement experiment M078.
In: Biomedical Results from Skylab, edited by R. S. Johnson,
and L. Dietlein. Washington, DC: NASA, 1977, p. 183-190. (SP-377)
38.
Vorobyov, E. L.,
O. G. Gazenk,
A. M. Genin,
and
A. D. Egorov.
Medical results of Solyut 6 manned spaceflights.
Aviat. Space Environ. Med.
54:
S31-S40,
1983[Medline].
39.
Whedon, G. D.,
L. Lutwak,
J. Reid,
M. W. Whittle,
M. C. Smith,
J. Reid,
C. Leach,
C. R. Stadler,
and
D. D. Sanford.
Mineral and nitrogen metabolic studies on Skylab orbital spaceflights.
Trans. Assoc. Am. Physicians
87:
95-110,
1974[Medline].
40.
Wronski, T. J.,
L. M. Dann,
and
S. L. Horner.
Time course of vertebral osteopenia in ovariectomized rats.
Bone
10:
295-301,
1989[Medline].
41.
Wronski, T. J.,
L. M. Dann,
K. S. Scott,
and
M. Cintron.
Long term effects of ovariectomy and aging on the rat skelton.
Calcif. Tissue Int.
45:
360-366,
1989[Medline].
42.
Wronski, T. J.,
and
E. R. Morey.
Skeletal abnormalities in rats induced by simulated weightlessness.
Metab. Bone Dis. Relat. Res.
4:
69-75,
1982[Medline].