Download Repeated immobilization stress reduces rat vertebral bone growth

Am J Physiol Regulatory Integrative Comp Physiol
280: R79–R86, 2001.
Repeated immobilization stress reduces rat vertebral
bone growth and osteocalcin
PATRICIA PATTERSON-BUCKENDAHL,1 MILAN RUSNÁK,2
KOKI FUKUHARA,3 AND RICHARD KVETNAN̆SKÝ2,3
1
Center of Alcohol Studies, Rutgers University, Piscataway, New Jersey 08854;
3
National Institutes of Health, National Institute of Neurological Disease and Stroke,
Bethesda, Maryland 20892; and 2Institute of Experimental Endocrinology, Slovak Academy
of Sciences, Vlarska 3, 833 06 Bratislava, The Slovak Republic
Received 27 December 1999; accepted in final form 7 September 2000
THERE IS A GROWING AWARENESS that anxiety and other
mental or emotional stimuli can affect bone and its
metabolic products. Most striking is the fact that melancholic depression is often accompanied by osteoporosis (22). Although this could be due to inactivity, which
has profound effects on bone often leading to osteoporosis, the authors found that the primary factor was a
greatly elevated endogenous glucocorticoid level. Both
osteoblast function and bone formation rate are suppressed by high endogenous and pharmacological
doses of glucocorticoids (15–17).
Osteocalcin (OC, also called bone Gla-protein or
BGP) is a bone-specific protein that binds to the surface
of the bone mineral crystal. Small amounts of newly
synthesized OC are released into circulation (34) and
have been well correlated with histological indices of
osteoblastic activity and bone formation rate (6).
Plasma OC (pOC) was negatively correlated with histomorphometric measures of bone formation in patients on corticosteroid therapy (6). In vitro experiments demonstrated a dose-dependent corticosteroidinduced suppression of OC production by osteoblasts
(1). Glucocorticoid repression has been found to act via
the promoter region for OC gene expression, suggesting that some of the effects of physiological or pharmacological levels of glucocorticoids on bone formation
involve the suppression of OC synthesis and/or secretion (24).
We previously showed that mental/emotional stressors alter pOC. Stressor effects differ significantly depending on type and severity of the stimulus. Under
conditions of relatively mild stress that produce an
anxiety response in which the major hormonal effect is
elevated glucocorticoid levels, rat pOC levels were significantly decreased within 1–1.5 h of imposition of
stressor (32). pOC levels were inversely correlated with
corticosterone levels (the major glucocorticoid in rodents). This effect could also be induced in a time- and
dose-dependent manner by injection of amounts of corticosterone within the range of physiological stress
response. A similar response has been seen in human
patients experiencing stressful conditions related to
surgery, trauma, or coronary disease independent from
accompanying disuse and bed rest (25). These data
suggest caution should be taken in interpretation of
serum OC in experimental subjects, which may not
reflect bone formation and/or turnover under these
circumstances.
A very different effect was seen in rats subjected to 2
h of foot-restraint immobilization (Immo), a more severe, well-characterized model of stress that induces
the classic “fight-or-flight” response (4, 5). The hormonal response to this stressor is rapid elevations of
corticosterone and the catecholamines epinephrine
Address for reprint requests and other correspondence: P. Patterson-Buckendahl, Center of Alcohol Studies, 607 Allison Road,
Rutgers Univ., Piscataway, NJ 08854 (E-mail: buckendp@rci.
rutgers.edu).
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. Section 1734
solely to indicate this fact.
restraint immobilization; bone mineral; corticosterone
http://www.ajpregu.org
0363-6119/01 $5.00 Copyright © 2001 the American Physiological Society
R79
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Patterson-Buckendahl, Patricia, Milan Rusnák,
Koki Fukuhara, and Richard Kvetn̆anský. Repeated immobilization stress reduces rat vertebral bone growth and
osteocalcin. Am J Physiol Regulatory Integrative Comp
Physiol 280: R79–R86, 2001.—We previously showed that
psychological stressors alter plasma levels of osteocalcin
(pOC), a bone-specific mineral binding protein, in ways that
differ with the type of stressor. To determine effects of
chronic stress, we examined vertebrae, pOC, and corticosterone levels from conscious rats subjected to foot-restraint
immobilization (Immo) daily for 1–42 times. After 40–42
Immo, basal pOC was decreased by 25% compared with
unstressed rats, and the subsequent rise in pOC during
Immo was blunted. Corticosterone was elevated 10-fold during Immo. Immo for seven times did not change vertebral OC
concentration, but caused a slight decrease in calcium and
phosphorous concentrations in younger rats. Rats Immo for
42 times exhibited reduced body weight, vertebral weight,
and vertebral OC concentration but no significant differences
in vertebral mineral concentrations. Body fat content was
visibly decreased. We do not know the source of or the
stimulus for the initial rise in pOC. We conclude that both
decreased growth and bone OC concentration are due to
repeatedly elevated stress hormones.
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IMMOBILIZATION REDUCES BONE GROWTH AND OSTEOCALCIN
METHODS
Immobilization stress was carried out by methods described by Kvetnansky and Mikulaj (18). The stress procedure was reviewed and approved by the Animal Care Committees of the National Institutes of Health-National
Institute of Neurological Diseases and Stroke (experiments 1
and 2) and the Institute for Experimental Endocrinology,
Slovak Academy of Sciences (experiment 3) before the current
series of experiments. Briefly, each animal was restrained in
a prone position on an immobilization board for periods not
exceeding 120 min. Its head was restricted from movement
by a metal loop over the nose, and its feet were taped to
raised supports with bandage tape. This type of immobilization is a standardized procedure, which results in well-characterized catecholamine and corticosteroid responses. Male
Sprague-Dawley-derived rats were obtained from Taconic
Farms, Germantown, NY (experiments 1 and 2), or Charles
River Laboratories, Wiga, Germany (experiment 3). Rats
were housed four to a cage under vivarium conditions of a 6
AM to 6 PM light cycle, with food and water provided ad
libitum. Experiments began at least 7 days after arrival, and
animals in each experimental group were weight matched
(range 280–320 g) at the beginning of the experiment.
For repeated Immo, rats were removed daily from their
cages, immobilized for 2 h, and returned to their cages. Immo
took place between 7 AM and 12 noon. For experiments 1 and
3, Immo was repeated 5 days a week with 2 days’ rest in
between until the final week of the experiments, when Immo
took place every day. Maximum number of Immo was 40
(experiment 1) or 42 times (experiment 3).
Experiment 1 provided blood samples to compare 40 times
Immo rats (40⫻, n ⫽ 10) to 1 time Immo (1⫻, n ⫽ 12). To
allow repeated blood sampling during Immo, each rat had a
cannula placed into the tail artery under pentobarbital sodium anesthesia 24 h before the last Immo. Blood was collected from the cannula before Immo and after 5, 20, 60, and
120 min of Immo and replaced with equal volumes of isotonic
saline containing 50 units heparin/ml. Trunk blood was obtained from all other rats after decapitation.
Experiment 2 compared absolute (never stressed) controls
with one time (1⫻), six times with 24 h recovery (6⫻), and
seven times (7⫻) Immo rats; all groups consisted of seven
rats each. 6⫻ rats served as previously stressed, adapted
controls for 7⫻ rats for evaluation of recovery from Immo.
Absolute control and 6⫻ adapted control rats were killed by
decapitation on removal from their cages; 1⫻ and 7⫻ Immo
rats were killed immediately on removal from the Immo
board.
Experiment 3 was similar to experiment 2, but also included a group of 1⫻ Immo allowed to recover for 24 h (1⫻
adapted), one group Immo for 41 times and allowed to recover
for 24 h (41⫻ adapted), and one group Immo for 42 times
(42⫻). The animals that were allowed to recover for 24 h after
the last Immo served as previously stressed controls for acute
1⫻, short-term (7⫻), and long-term (42⫻) Immo killed immediately after 120 min Immo for evaluation of recovery
from Immo. Animals were weighed twice a week to monitor
growth; all animals were killed on the same day.
Third lumbar vertebrae were dissected from rats in experiments 2 and 3 after decapitation, cleaned of soft tissue, and
freeze-dried to constant weight. Bones were then ground to a
fine powder in a liquid nitrogen-cooled mill (Spex Industries,
Edison, NJ). Duplicate aliquots of each bone powder were
extracted for 24 h with 0.5 M ammonium EDTA, pH 8,
containing protease inhibitors, in Eppendorf tubes turned
end over end at 4°C to solubilize OC (12). Mineral concentrations were determined on additional duplicate samples (1–
1.5 mg) of bone powder extracted for 24 h with 100 ␮l
concentrated HCl.
Plasma and bone OC were assayed by the RIA method of
Patterson-Allen et al. (28), with guinea pig anti-rat OC antibody and purified rat OC for standard and 125I-labeled
tracer. Inter- and intra-assay variations were 8.6 and 6.2%,
respectively. Sensitivity for rat OC is 0.3 ng/ml of sample.
The average variation of OC determination in duplicate bone
samples was 3.4%. Calcium concentrations were determined
by atomic absorption spectrophotometer (Perkin-Elmer) and
inorganic phosphorus by the ammonium molybdate method
(Sigma reagents). The average variation of duplicate bone
samples was 1.9% for calcium and 2.2% for phosphorous.
Plasma corticosterone was determined with commercial RIA
reagents from ICN. Intra-assay variation was 3.4%. All samples from one experiment were analyzed in the same assay.
Data are presented as means ⫾ SE. Plasma and weight
gain data were analyzed for significant effects using SAS for
Macintosh General Linear Models procedure for repeatedmeasures ANOVA. Other data were analyzed by one-way
ANOVA. Duncan’s multiple-range test with least squared
means comparisons was used for post hoc analysis to determine significant differences between groups. Significance
level was set at P ⬍ 0.05.
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(Epi) and norepinephrine (NE), which persist for ⬎1 h
(18). These hormones normally prepare the body to
react to a threat of danger by heightening arousal,
blood flow, and/or muscular activity. The Immo model
caused a rapid and marked increase in pOC, significant
within 5 min and maximally 50–100% above basal
levels after 20–30 min (33). Selective elimination of the
hormonal influences by surgical removal of the adrenal
medulla (elimination of adrenal catecholamines), the
entire adrenal (elimination of adrenal catecholamines
and corticosteroids), or by chemical blockade of release
of hormones at the level of sympathetic ganglia (NE or
others) showed that both corticosterone and sympathetic neural function, but not adrenomedullary hormones, were required for pOC to return to basal levels.
Immobilization repeated daily for 7 days blunted but
did not eliminate the pOC response. The consistency of
the acute elevation of pOC during Immo suggests that
it may be a very important component of an animal’s
response to severe stress.
Because repeated imposition of Immo appeared to
blunt the pOC response, we therefore wished to determine whether there would be a corresponding effect on
bone. Previous studies had indicated a correlation between pOC and bone OC concentrations. For example,
both microgravity of spaceflight and ground-based simulation studies of microgravity decreased bone growth
rate as well as OC concentration in the third lumbar
vertebrae (30, 31). On the other hand, supplementation
with vitamin D, which stimulates OC synthesis, resulted in an increased accumulation of vertebral bone
OC, but no change in bone weight (29). The current
study describes the effects of 2-h Immo repeated daily
for up to 42 times on pOC and on bone growth and
accumulation of OC and mineral in the third lumbar
vertebra.
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IMMOBILIZATION REDUCES BONE GROWTH AND OSTEOCALCIN
RESULTS
Experiment 1. Figure 1 is a comparison of plasma OC
levels in 1⫻ Immo rats compared with those of rats
that were restrained 2 h daily for a total of 40 times
(40⫻) and killed after the last Immo. These data show
that elevation of pOC during Immo is blunted, but not
abolished, by long-term repetition. Analysis of between-subjects effects indicated highly significant differences in the response of the two groups (P ⬍ 0.001).
Duncan’s multiple-range test showed that 1⫻ rats
were significantly different from 40⫻ rats at each of
the time points (P ⬍ 0.01), with basal pOC level in
repeatedly stressed rats being ⬃25% below that of the
1⫻ rats before the onset of Immo (P ⬍ 0.01). After 20
min Immo, the maximal increase in 1⫻ rat pOC was
⬃50% above basal value (P ⬍ 0.001), whereas 40⫻ rats
increased by only 40% (P ⬍ 0.01). After 120 min, both
groups were ⬃40% lower than their respective levels at
the beginning of Immo (P ⬍ 0.01 for both groups).
Experiment 2. Table 1 gives data for pOC and vertebral weight and composition from animals that were
subjected to Immo repeated daily for up to seven times.
(6⫻ rats that had been allowed to recover for 24 h after
their last Immo served as previously stressed controls
for 7⫻ rats for evaluation of recovery from and possible
adaptation to Immo.) Duncan’s multiple-range test
showed that pOC levels of both 6⫻ and 1⫻ rats killed
immediately after first Immo were not different from
absolute (never stressed) controls. However, pOC in
rats killed after 120 min of a seventh Immo was significantly different from all other groups (P ⬍ 0.01).
They were 30% below that of absolute controls and 33%
lower than 6⫻ adapted controls (P ⬍ 0.001). There
were no differences in the vertebral weights among the
four groups of rats, nor did bone OC concentrations
differ. Mineral analysis did show slight but significant
differences between control and Immo rats. Calcium
concentrations in all Immo groups were lower by ⬃5%
in controls (P ⬍ 0.001), and phosphorous concentration
was lower in the 6⫻ adapted controls and 7⫻ groups
compared with absolute controls (P ⬍ 0.05 and 0.001,
respectively) and was also different between 6⫻ and
7⫻ (P ⬍ 0.05). Calcium-to-phosphorous ratios in Immo
groups were also significantly lower than in controls.
Total bone content of calcium in 1⫻ vertebrae was
Table 1. Plasma OC, weight, and concentration and
total content of calcium, phosphorus, and OC in third
lumbar vertebrae of rats subjected to 2 h foot restraint
Immo daily for 1–7 times compared with unstressed
(absolute control) or 6 times Immo rats allowed to
recover for 24 h (6⫻ adapted control)
Absolute
Control
Plasma OC, ng/ml
Vertebral wt, mg
209 ⫾ 5
135 ⫾ 4
1⫻ Immo
210 ⫾ 23
129 ⫾ 4
6⫻ Adapted
Control
216 ⫾ 8
132 ⫾ 5
7⫻ Immo
145 ⫾ 8b,e
132 ⫾ 2
Concentration of osteocalcin and minerals in vertebrae
OC, ␮g/mg
Ca, ␮g/mg
Pi, ␮g/mg
Ca/Pi ratio
2.73 ⫾ 0.11
254 ⫾ 2
113 ⫾ .5
2.24 ⫾ .01
2.84 ⫾ 0.06
243 ⫾ 2c
113 ⫾ 0.5
2.15 ⫾ .01c
2.67 ⫾ 0.04
243 ⫾ 2b
111 ⫾ 0.6a
2.19 ⫾ .02a
2.69 ⫾ 0.09
238 ⫾ 2c,d
109 ⫾ 0.6c,d
2.18 ⫾ .01a
Content of osteocalcin and minerals in vertebrae
OC, ␮g
Ca, mg
Pi, mg
370 ⫾ 25
34.1 ⫾ 1.2
15.2 ⫾ 0.5
366 ⫾ 11
352 ⫾ 13
31.3 ⫾ 0.8a 32.2 ⫾ 0.8
14.6 ⫾ 0.4 14.7 ⫾ 0.4
351 ⫾ 13
31.6 ⫾ 0.7
14.3 ⫾ 0.3
All values are means ⫾ SE. Significant difference from absolute
controls is indicated as follows: a P ⬍ 0.05; b P ⬍ 0.01; c P ⬍ 0.001.
d
and e indicate significant differences of P ⬍ 0.05 and P ⬍ 0.001
between adapted control (6⫻) and 7⫻ foot restraint immobilization
(Immo). OC, osteocalcin.
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Fig. 1. Time course of plasma osteocalcin (pOC) response to foot-restraint immobilization (Immo) of rats
for 1 time (1⫻, n ⫽ 12) or the last of 40 times repeated
Immo (40⫻, n ⫽ 10). All animals were the same age at
the time of blood sampling. pOC is expressed as mean
percent ⫾ SE of baseline levels of 1⫻ rats. Where no
error bars are visible, error was within the space occupied by the plot symbol. #Statistically significant differences of P ⬍ 0.01 at each time point between 1⫻ and
40⫻. ** And *** indicate significant differences of P ⬍
0.01 and P ⬍ 0.001, respectively, between Immo rats and
their own values at time 0.
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IMMOBILIZATION REDUCES BONE GROWTH AND OSTEOCALCIN
significantly less than in absolute controls (P ⬍ 0.05),
but 6⫻ and 7⫻ vertebrae did not differ from absolute
controls or from each other. There were no differences
in total phosphorus or OC contents of these bones.
Experiment 3. This experiment compared effects of
both short-term repetition (7⫻) and long-term repetition of Immo (42⫻) with acute (1⫻) and untreated
(absolute) controls. As in experiment 2, rats immobilized for the same number of times but allowed to
recover for 24 h before being killed served as previously
stressed controls for evaluation of recovery from and
possible adaptation to Immo. It should be noted that
these rats were 7 wk older at the end of the experiment
than those in experiment 2, when both growth rate and
bone formation rate have begun to decrease. Figure 2
depicts weight gained by the respective groups over the
56-day experimental period, with black bars indicating
the entire period of Immo for each repeatedly Immo
group. Absolute controls and both 1⫻ groups were
identically treated until the last day of the experiment,
and there were no statistical differences in body
weights at any time; therefore, data for these three
Fig. 3. OC (A) and corticosterone
(B) levels in plasma of rats subjected
to 2-h daily Immo from 1 (1⫻), 7
(7⫻), or 42 (42⫻) times, compared
with absolute (unstressed) control
or adapted control animals. Adapted
controls were Immo for 1, 6, or 41
times and allowed to recover for 24 h
before being killed. All animals were
the same age and killed on the same
day. Each group contained 8 rats,
except for 42⫻ (n ⫽ 9), and 41⫻ ⫹
24 h (n ⫽ 10). * And *** indicate
significant differences from unstressed controls of P ⬍ 0.05 and
P ⬍ 0.001, respectively.
groups were combined. Duncan’s multiple-range test
showed no significant differences in weight among control and 1⫻ or 7⫻ rat groups until the 7⫻ group was
Immo toward the end of the experimental period. By
the seventh Immo of this group, the animals had actually lost weight and were 11% lighter than control and
1⫻ Immo rats (P ⬍ 0.001). Rats in the 42⫻ Immo group
had diverged from the other animals by the 10th experimental day (P ⬍ 0.001) and weighed an average of
18% less than controls at the end of the experiment
(P ⬍ 0.001). We attribute most of this difference to a
visibly decreased body fat in the repeatedly Immo rats
noted but not quantified during necropsy.
Figure 3 shows pOC and corticosterone levels at the
end of experiment 3. Rats that were immobilized one
time had pOC equal to absolute control rats immediately after 120 min Immo or after 24 h recovery. Duncan’s multiple-range test showed that rats killed immediately after 120 min of the seventh Immo had
significantly lower pOC than absolute controls (P ⬍
0.05), but did not differ from short-term adapted controls that were 6⫻ Immo and had a 24-h recovery
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Fig. 2. Mean body weight vs. experiment day for rats
that were subjected daily to 2-h Immo for 0, 1, 6, 7, 41, or
42 times. Control and 1⫻ Immo rats (total n ⫽ 24) were
combined because they were treated identically until the
last experimental day. Black bars indicate schedule of
Immo for 7⫻ and 42⫻ rats. Weights of 6⫻ and 7⫻ rats,
which were Immo beginning on the last 7 days of the
experiment, did not differ significantly and were combined (n ⫽ 16). Weights of 41⫻ and 42⫻ groups, which
were on a schedule of 5 days Immo, 2 days rest, until the
last 8 days of experiment when Immo was imposed daily
also did not differ significantly and were combined (n ⫽
18). Error bars indicate SE. Where no bars are visible,
error was within the space occupied by the plot symbol.
***Significant differences from control and 1⫻ rats of
P ⬍ 0.001.
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IMMOBILIZATION REDUCES BONE GROWTH AND OSTEOCALCIN
Table 2. Body weight, freeze-dried weight, OC, calcium and phosphorous composition of third lumbar
vertebrae of rats subjected to 2 h Immo daily for up to 42 times, compared with previously unstressed
(absolute) controls and with adapted controls allowed to recover for 24 h after 1, 6, or 41 daily Immo
Absolute
Control
(n ⫽ 8)
Body wt, g
Vertebral wt, mg
Vertebral wt,
mg/g body wt
1⫻ Adapted
Control
(n ⫽ 8)
1⫻ Immo
(n ⫽ 8)
6⫻ Adapted
Control (n ⫽ 8)
506 ⫾ 15
239 ⫾ 6
509 ⫾ 13
245 ⫾ 6
521 ⫾ 14
245 ⫾ 7
443 ⫾ 16†
233 ⫾ 0.010
0.473 ⫹ 0.008
0.483 ⫾ 0.001
0.472 ⫾ 0.013
0.526 ⫾ 0.011†
2.43 ⫹ 0.12
235 ⫾ 2
103 ⫾ 1
2.39 ⫾ 0.06
238 ⫾ 2
102 ⫾ 2
7⫻ Immo
(n ⫽ 8)
473 ⫾ 7
259 ⫾ 8
0.547 ⫾ 0.011‡
41⫻ Adapted
Control (n ⫽ 9)
42⫻ Immo
(n ⫽ 10)
410 ⫾ 13‡
214 ⫾ 10*
424 ⫾ 10‡
216 ⫾ 6*
0.521 ⫾ 0.003†
0.510 ⫾ 0.004*
1.98 ⫾ 0.07‡
232 ⫾ 2
102 ⫾ 1
2.00 ⫾ 0.02‡
236 ⫾ 4
99 ⫾ 2
420 ⫾ 14‡
49.8 ⫾ 2.6*
21.8 ⫾ 1.1*
430 ⫾ 14‡
50.8 ⫾ 1.8*
21.4 ⫾ 0.8†
Concentration of osteocalcin and minerals in vertebrae
OC, ␮g/mg
Ca, ␮g/mg
Pi, ␮g/mg
2.64 ⫾ 0.07§
236 ⫾ 2
104 ⫾ 1
2.44 ⫾ 0.07
232 ⫾ 2
103 ⫾ 2
2.51 ⫾ 0.06
239 ⫾ 2
103 ⫾ 1
Content of osteocalcin and minerals in vertebrae
580 ⫹ 31
56.1 ⫾ 1.4
24.6 ⫾ 0.6
585 ⫾ 20
58.3 ⫾ 1.6
25.0 ⫾ 0.9
648 ⫾ 22§
58.0 ⫾ 1.6
25.6 ⫾ 0.8
568 ⫾ 26
54.0 ⫾ 2.1
23.9 ⫾ 0.9
650 ⫾ 25*§
61.8 ⫾ 1.8*§
26.6 ⫾ 0.9§
All values are means ⫾ SE. Vertebral weight is also shown relative to body weight. OC and minerals are given as concentration and total
bone content. Significant difference from absolute controls is indicated as follows: * P ⬍ 0.05; † P ⬍ 0.01; ‡ P ⬍ 0.001. § indicates P ⬍ 0.05
between Immo and the respective adapted control allowed to recover 24 h.
period before being killed. Animals killed after 42
Immo had pOC ⬃16% below absolute controls (P ⬍
0.05), whereas rats killed 24 h after the 41st Immo had
pOC 25% below absolute controls (P ⬍ 0.05). This
confirms observations in experiment 1 that repeated
Immo impairs the ability to restore normal levels of
pOC. Corticosterone levels in all rats killed immediately after Immo were elevated at least 10-fold over
absolute controls (P ⬍ 0.001), but had returned to
relatively normal levels in all rats killed after 24 h
recovery from Immo.
Table 2 shows body and bone weights and OC, calcium, and phosphorus concentration and content in
third lumbar vertebrae from rats in experiment 3. Combined data for vertebral weight from the two groups of
long-term Immo rats (41⫻ and 42⫻, which did not
differ significantly from each other) indicated a 12%
lower bone weight compared with control and 1⫻ Immo
rats (P ⬍ 0.001). When adjusted for body weight, vertebrae of both short-term and long-term repeatedly
Immo rats were heavier than absolute controls, indicating a greater deficit in body than in skeletal weight
gain.
Concentrations of OC but not mineral in bones of
long-term Immo rats differed from controls. OC concentrations in these vertebrae were decreased by 18%
compared with absolute controls. Least squares means
analysis yielded significance of P ⬍ 0.001 versus all
other groups. There was a significant difference in OC
between 1⫻ Immo and 1⫻ adapted control vertebrae
(P ⬍ 0.05). Total vertebral bone OC was 27% lower in
42⫻ Immo rats than in absolute controls (P ⬍ 0.001).
Significantly greater content of OC was found in both
1⫻ and 7⫻ Immo relative to their adapted controls
killed 24 h after Immo (P ⬍ 0.05). There were no
differences in mineral concentrations in these bones.
Total content of calcium and phosphorus in the vertebrae was decreased in 7⫻ and in 41⫻ and 42⫻ rats
(P ⬍ 0.05) compared with absolute controls in proportion to their lower bone weight. There was a greater
content of both minerals in 7⫻ than in 6⫻ adapted
controls (P ⬍ 0.05).
DISCUSSION
Foot-restraint Immo of conscious rats causes a rapid
increase in plasma levels of stress hormones and is
accompanied by an equally rapid increase in pOC (33).
As shown in Fig. 1, the acute increase of pOC in
animals repeatedly subjected to this stressor is
blunted, but not eliminated. We have now analyzed
samples from more than 15 experiments comparing a
variety of treatments, including surgical ablation of
hormones, sympathetic blockade, cold exposure, and
repeated Immo, and in all cases to date, pOC was
acutely increased within the first 20 min of Immo (Ref.
33 and unpublished observations). Furthermore, we
have detected significantly increased pOC within the
first 3 min of Immo (Patterson-Buckendahl et al. unpublished observations). Repeated sampling over a
120-min period of rats similarly cannulated but not
subjected to Immo showed no change from baseline
levels (Ref. 33 and unpublished observations). pOC is
clearly affected by the physiological response to stress
and may be an integral part of that response.
We do not as yet know whether Immo-elevated pOC
results from increased osteoblastic secretion or
whether it might be coming from the bone surface by a
noncellular mechanism. Hypocalcemia induced by citrate or EDTA infusion was accompanied acutely by
both increased parathyroid hormone and pOC in a time
course similar to what we observed with Immo (10, 11,
37). A similar response was seen in monkeys anesthetized with isoflurane, but not with ketamine or ketamine plus atropine (13). It has been suggested that
the acute rise in OC in these induced hypocalcemias
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OC, ␮g/bone
Ca, mg/bone
Pi, mg/bone
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IMMOBILIZATION REDUCES BONE GROWTH AND OSTEOCALCIN
with severe osteoporosis in humans. Although most
studies of osteoporotic effects involve pharmacological
doses of steroids far higher than those reached even
under severely stressful conditions, chronic low doses
administered by inhalation have also been shown to
decrease bone density (14). Moderate to high endogenous cortisol levels in humans (within the range of
corticosterone in our rats) with Cushing’s disease or
with major depression also result in osteoporosis (22).
Our data are the first we are aware of to document the
effects of chronic immobilization stress and the associated endogenous elevations of stress hormones on skeletal growth.
The biochemical makeup of the third lumbar vertebrae was also altered. In younger rats (weight range
280–300 g) subjected to Immo for seven times (experiment 2, Fig. 2), the mineral makeup of bone was altered, but not the OC concentration. At this age, OC
synthesis rate and osteoblastic activity are high and
decrease markedly during the next 2 mo (12) and may
override the effects of Immo. The magnitude of the
mineral decrease is consistent with the percentage of
bone mineral believed to be readily exchangeable. This
could result from decreased dietary intake (20) as well
as impairment of intestinal calcium absorption. Other
investigators have reported decreased plasma calcium
in response to foot-restraint Immo (23). It seems probable that this mineral, which is crucial to muscle and
nerve function, would be replaced from a ready supply
in bone that is not dependent on cellular activation.
We cannot explain the ⬃10% greater concentration
of OC in 1⫻ Immo rats killed immediately compared
with those killed after 24 h of recovery (adapted controls). Neither group was significantly different from
the absolute controls that had never been subjected to
Immo. Both sets of animals were housed and handled
identically until 1⫻ rats were subjected to Immo at the
end of the experimental period. Other investigators
have estimated a 5-min half-life for pOC clearance (35).
It seems unlikely that all of the acutely elevated pOC
could be scavenged at that rate by the one vertebra
analyzed. Even if it were, it would still not account for
the differences between rats killed immediately after
120 min Immo and their adapted control Immo rats on
the previous day. On the assumption of an elevation of
pOC from the control level of ⬃70 ng/ml and a plasma
volume of 6% of body weight, there should have been
⬃2 ␮g pOC in circulation. Given an acute twofold
elevation such as we have seen in previous experiments (33) and a 5-min half-life, ⬃30 ␮g would be
cleared during 120 min Immo. This is still less than the
63 ␮g difference between 1⫻ Immo rats and their
adapted or absolute controls. It is also unlikely that
synthesis was increased during Immo, given the high
corticosterone levels of the rats. The nonsignificant
increase in vertebral weight of 7⫻ Immo compared
with 6⫻ adapted or absolute control rats could account
for the differences in total bone content of OC, calcium,
and phosphorous in those animals.
In experiment 3, elevated pOC levels in 1⫻ and 7⫻
Immo rats were followed by recovery to control levels
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originates from the bone surface rather than from new
synthesis or secretion (11). Induction of de novo synthesis is much less likely, due to the extreme rapidity
of the response. It has also been suggested that the
elevated OC may somehow be mediating the hypocalcemia that results from these treatments (2).
Repeated foot-restraint Immo markedly altered the
rate of increase in body weight. Animals that were 7⫻
Immo toward the end of experiment 3, when growth
rate in controls had actually slowed, exhibited an 11%
decrease in body weight over the 7-day period, but no
change in vertebral weight. The two groups whose
Immo began 8 wk earlier gained weight, but at a
decreased rate, and by the end of the experiment had
gained 18% less than 1⫻ and control rats. A major
factor in these differences in body weight is the decreased amount of body fat in the repeatedly Immo
animals (personal observations). It has been long established that elevated glucocorticoids as well as Epi
mobilize energy stores to enable animals to respond to
acute severe stressors. Furthermore, Marti et al. (20)
reported decreased food intake in repeatedly Immo
animals. Michajlovskij et al. (21) reported reduced food
and water intake by rats Immo for 150 min daily for 7
days. The greatest change in consumption followed the
first Immo, and there was a gradual increase over the
next 6 days. When normalized to body weight, consumption of food and water after a sixth Immo did not
differ from the control pre-Immo period, suggesting
adaptation of subsequent food consumption to the
Immo stress procedure (21). The combined effects of
decreased food intake and increased fat mobilization
could explain the decreased weight gain of Immo rats.
Our animals were housed in group cages to avoid the
added stressful influence of isolation, thus we could not
directly evaluate individual food intake.
A further effect of repeated Immo was seen in the
lower weight of the third lumbar vertebrae. Some of
this decrease may be attributed to decreased body
weight, which is well known to affect ultimate bone
weight. The physiological responses to decreased food
intake, resulting in decreased body weight, are complex and include changes in growth hormone, insulin,
thyroid hormone, leptin, and many other factors. Many
of these factors also directly influence bone metabolism. Recently, leptin (a hormone produced by adipose
tissue) was shown to affect bone mass via a hypothalamic pathway (7). We have not measured leptin in
Immo rats, but would expect it to be altered if food
intake was decreased.
It is also likely that repeated high corticosterone
levels were a major factor in the decreased bone growth
as well as in decreased weight gain. The myriad direct
and indirect effects of glucocorticoids on bone were well
summarized by Lukert and Kream (19). Among the
specific bone parameters affected are collagen and OC
synthesis, as well as differentiation of bone-forming
osteoblast cells. Furthermore, intestinal uptake of calcium is diminished, thus reducing the supply of mineral for deposition into bone in growing animals.
Chronic exogenous glucocorticoids are often associated
IMMOBILIZATION REDUCES BONE GROWTH AND OSTEOCALCIN
The authors thank Slavka Gorcikova for assistance with immobilization and Dale Buckendahl for assistance with bone preparation
and editing of the manuscript. We thank Larissa Pohorecky, Arthur
Tomie, and Greg Blakley for helpful discussion of the manuscript.
Work was supported in part by National Institute on Alcohol
Abuse and Alcoholism R21-AA-12705–01 (to P. Patterson-Buckendahl) and Slovak Grant Agency for Science 2–610999 (to R. Kvetnansky).
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