J Appl Physiol
89: 1046–1054, 2000.
Alterations in skeletal perfusion with simulated
microgravity: a possible mechanism for bone remodeling
PATRICK N. COLLERAN,1 M. KEITH WILKERSON,1 SUSAN A. BLOOMFIELD,1
LARRY J. SUVA,3 RUSSELL T. TURNER,4 AND MICHAEL D. DELP1,2
Departments of 1Health and Kinesiology and 2Medical Physiology, and Cardiovascular Research
Institute, Texas A&M University, College Station, Texas 77843; 3Department of Bone and Cartilage
Biology, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406;
and 4Department of Orthopaedic Surgery, Mayo Clinic, Rochester, Minnesota 55905
Received 25 April 2000; accepted in final form 17 May 2000
that occur with weightlessness include cardiovascular deconditioning and musculoskeletal atrophy. The osteopenic effect of spaceflight first manifested itself when increased urinary
calcium excretion was observed in cosmonauts after
the Vostok 2 and 3 missions (17). Significant loss of
calcaneus mass was detected in astronauts after Gemini, Apollo, and Skylab flights (17). Evaluation of cosmonauts occupying the Mir space station has provided
the opportunity to study the effects of long-term exposure to microgravity. Prolonged spaceflight resulted in
a marked loss of cancellous and cortical bone of the
tibia (7) and a reduced bone mineral density of the
femoral neck and trochanter, despite the employment
of exercise countermeasures (39).
Extensive analysis of rats sent into orbit on board
the Soviet Cosmos Biosatellites and American
Spacelab 3 has contributed greatly to the understanding of mechanisms underlying human skeletal adaptations to weightlessness. These rats were characterized
by a reduced trabecular mass in the tibial metaphysis
(49) and a slowing of periosteal bone formation of the
tibial and humeral shafts (48). In spite of these spaceflight experiments, the paucity of animals exposed to
weightlessness during spaceflight has led to the development of ground-based models. Hindlimb unloading
(HU) provides a model of mechanical unloading of the
hindlimb while producing a headward fluid shift similar to that induced by microgravity. Skeletal adaptations in HU rats are similar to those observed in rats
exposed to spaceflight, including a loss of cancellous
bone mass in the proximal metaphysis of the tibia (47)
and reduced periosteal bone formation in the tibia and
femur (25).
The cephalic fluid shift that accompanies simulated
and actual weightlessness may affect bone blood flow
and potentially alter interstitial fluid pressures and
flows. For example, previous work has demonstrated
that HU-mediated alterations in perfusion pressure
and blood flow induce a remodeling of the arterial
resistance vasculature in skeletal muscle (9) and cerebral tissue (46). If these alterations were also to occur
in the hindlimb bones, then the mechanical environment of osteoprogenitor cells, osteoblasts, and osteoclasts would be compromised. Such a perturbation
would presumably result in the altered regulation of
these cells and subsequently in changes in the balance
between bone formation and bone resorption (4, 33).
Original submission in response to a special call for papers
on “Physiology of a Microgravity Environment.”
Address for reprint requests and other correspondence: M. D. Delp,
Dept. of Health and Kinesiology, Texas A&M Univ., College Station,
TX 77843 (E-mail: [email protected]).
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.
bone blood flow; hindlimb unloading; vascular resistance
PHYSIOLOGICAL ADAPTATIONS
1046
8750-7587/00 $5.00 Copyright © 2000 the American Physiological Society
http://www.jap.org
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Colleran, Patrick N., M. Keith Wilkerson, Susan A.
Bloomfield, Larry J. Suva, Russell T. Turner, and Michael D. Delp. Alterations in skeletal perfusion with simulated microgravity: a possible mechanism for bone remodeling. J Appl Physiol 89: 1046–1054, 2000.—Bone loss occurs
as a consequence of exposure to microgravity. Using the
hindlimb-unloaded rat to model spaceflight, this study had as
its purpose to determine whether skeletal unloading and
cephalic fluid shifts alter bone blood flow. We hypothesized
that perfusion would be diminished in the hindlimb bones
and increased in skeletal structures of the forelimbs and
head. Using radiolabeled microspheres, we measured skeletal perfusion during control standing and after 10 min, 7
days, and 28 days of hindlimb unloading (HU). Femoral and
tibial perfusion were reduced with 10 min of HU, and blood
flow to the femoral shaft and marrow were further diminished with 28 days of HU. Correspondingly, the mass of
femora (⫺11%, P ⬍ 0.05) and tibiae (⫺6%, P ⬍ 0.1) was
lowered with 28 days of HU. In contrast, blood flow to the
skull, mandible, clavicle, and humerus was increased with 10
min HU but returned to control levels with 7 days HU.
Mandibular (⫹10%, P ⬍ 0.05), clavicular (⫹18%, P ⬍ 0.05),
and humeral (⫹8%, P ⬍ 0.1) mass was increased with chronic
HU. The data demonstrate that simulated microgravity alters bone perfusion and that such alterations correspond to
unloading-induced changes in bone mass. These results support the hypothesis that alterations in bone blood flow provide a stimulus for bone remodeling during periods of microgravity.
SKELETAL PERFUSION DURING SIMULATED MICROGRAVITY
Therefore, the purpose of this study was to determine
whether the unloading of hindlimb bones and the corresponding cephalic fluid shift alter bone perfusion
rates. We hypothesized that perfusion would be diminished in the hindlimb bones and increased in skeletal
structures of the forelimb and head. Furthermore, we
also predicted that reductions in blood flow to the
hindlimb bones would occur in regions where unloading-induced bone loss has been reported to occur.
METHODS
7-day and 28-day HU rats, blood flow was measured with the
animals in the unloaded position. After the final microsphere
infusion, euthanasia solution (0.22 ml/kg; Euthanasia-5 Solution, Henry Schein) was infused through the carotid catheter. Bones and muscles were then removed from the carcass.
The femora from both hindlimbs were sectioned into three
regions, the proximal and distal metaphyses and diaphysis;
the femoral marrow was removed from the diaphysis and was
counted as a fourth region (Fig. 1). The corresponding femoral sections from the left and right hindlimbs were combined
for each animal to ensure sufficient microspheres in the
marrow sample. The fibula was cut from the tibia and
counted as a single sample (Fig. 1). The tibia was sectioned
into the proximal and distal metaphyses and diaphysis. The
marrow in the tibial shaft was not isolated or counted separately. Likewise, the humerus was sectioned into three regions with the marrow remaining in the humeral diaphysis
(Fig. 1). Tissue samples were weighed and placed in counting
vials for flow determination. The weight of the femoral marrow was determined by weighing the shaft before and after
the marrow was removed.
Blood flow measurements. Radiolabeled (85Sr, 113Sn, and
46
Sc) microspheres (New England Nuclear) with a 15-␮m
diameter were used for blood flow measurements as previously described (24). Radioactivity of the samples was
measured with a gamma counter (Packard AutoGamma
5780), and flows were computed (PC-GERDA V2.9 Software) from counts per minute and tissue wet weights. To
ensure microsphere-blood mixing, flows to head and forelimb were taken only when the carotid catheter tip was
confirmed to be in the left ventricle. Head and forelimb
blood flows from two control and two 7-day HU rats were
excluded when the carotid catheter tip was found to be in
the ascending aorta. Microsphere mixing was considered
adequate for hindlimb tissue flows when right and left
kidney blood flows were within 15% of each other. On the
basis of this criterion, no hindlimb tissue flows were excluded from the study.
Central hemodynamics and vascular resistance. Electronically averaged mean arterial pressure and heart rate (pulse
pressure rate) were recorded from the caudal catheter immediately before and after the microsphere infusion and were
Fig. 1. The sampling pattern for determining regional blood flows in
the femur, tibia-fibula, and humerus.
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Animals. All procedures performed in this study were
approved by the Texas A&M University Institutional Animal
Care Committee and conformed to the National Institutes of
Health (NIH) Guide for the Care and Use of Laboratory
Animals [DHEW Publication No. (NIH) 85–23, Revised 1985,
Office of Science and Health Reports, DRR/NIH, Bethesda,
MD 20892].
Six-month-old male Sprague-Dawley rats were obtained
from Harlan (Houston, TX) and individually housed in a
temperature-controlled (23 ⫾ 2°C) room with a 12:12-h lightdark cycle. Water and rat chow were provided ad libitum.
The animals were randomly assigned to either a normal
weight-bearing control group (n ⫽ 11), a 7-day HU group
(HU, n ⫽ 11), or a 28-day HU group (n ⫽ 7). The hindlimbs
of the HU groups were elevated to an approximate spinal
angle of 40–45° via orthopedic traction tape placed around
the proximal two-thirds of the tail in a modification of techniques previously described (9, 46, 47). The height of hindlimb elevation was adjusted to prevent the hindlimbs from
touching supportive surfaces while the forelimbs maintained
contact with the cage floor. This allowed free range of movement around the cage while achieving the desired experimental results. HU animals were kept in this position for a period
of either 7 or 28 days. Control animals were individually
maintained in their normal cage environment. The unloading
of rats within the 7-day and 28-day HU groups was staggered
so that only one blood flow experiment would be performed on
any single day. Experiments with control rats were interspersed among those with the HU animal.
Surgical procedures. HU and control rats were anesthetized with pentobarbital sodium (30 mg/kg ip). The HU rats
were anesthetized while remaining in the unloaded position
to avoid any weight-bearing activity. A catheter (Dow Corning, Silastic; ID 0.6 mm, OD 1.0 mm) filled with heparinized
(200 U/ml) saline and connected to a pressure transducer and
chart recorder was advanced into the left ventricle of the
heart via the right carotid artery as previously described
(11). This catheter was subsequently used for the infusion of
radiolabeled microspheres to measure tissue blood flow and
record arterial pressure. A second polyurethane catheter
(Braintree Scientific, Micro-renathane; ID 0.36 mm, OD 0.84
mm), used for the withdrawal of a reference blood sample and
recording arterial pressure, was implanted in the caudal
artery of the tail and filled with heparinized saline as previously described (11). Both catheters were externalized and
secured on the dorsal cervical region.
Experimental protocol. After 24 h of recovery from the
surgical procedure, control and HU animals were instrumented for blood flow determination. Blood flow in the control group was first measured while the animals were in a
normal standing position. The hindlimbs of control rats were
then elevated via tail suspension similar to that of the chronically unloaded rats. After a 10-min HU period, a second
blood flow measurement was made in this position. In the
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SKELETAL PERFUSION DURING SIMULATED MICROGRAVITY
RESULTS
averaged. In addition, pressure recordings were made with
the transducer at the level of the heart and hindlimb to
estimate thoracic aorta and hindlimb (caudal) arterial pressures, respectively. Aortic pressure estimates were confirmed
in the two control and two 7-day HU rats having the carotid
catheter tip in the ascending aorta. Arterial pressure was
recorded in control rats during normal standing and after 10
min of HU and in HU rats while they remained with elevated
hindlimbs. Caudal arterial pressures with the pressure
transducer at the level of the midfemur were used to calculate vascular resistance in hindlimb bones and muscles.
Arterial aortic pressures (with the pressure transducer at the
level of the heart) were used to calculate vascular resistance
in head and forelimb bones and muscles. Thus vascular
resistance for specific tissues was calculated as the quotient
of arterial pressure (caudal or aortic) divided by the tissue
blood flow.
Data analysis. A one-way ANOVA was used to compare
heart rate, mean arterial pressure, tissue mass, blood flow,
and vascular resistance across conditions. The Student-Newman-Keuls method was used as a post hoc test to determine
the significance of differences among means. All values are
presented as means ⫾ SE. A P ⬍ 0.05 was required for
significance, although differences among means having a P ⬍
0.1 are acknowledged.
Table 1. Hemodynamic responses to acute
and chronic hindlimb unloading
n
Heart rate, beats/min
Aortic arterial pressure,
mmHg
Caudal arterial
pressure, mmHg
Control
Standing
10 Min
HU
7 Days
HU
28 Days
HU
11
432 ⫾ 12
11
434 ⫾ 15
11
436 ⫾ 3
7
430 ⫾ 12
127 ⫾ 4
142 ⫾ 3*
136 ⫾ 3
127 ⫾ 3†
127 ⫾ 4
131 ⫾ 3
126 ⫾ 3
116 ⫾ 4*†
Values are means ⫾ SE. HU, hindlimb unloading. * Mean value
significantly different from that of control standing condition (P ⬍
0.05); † mean value significantly different from that of 10 min HU
condition (P ⬍ 0.05).
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Fig. 2. Effect of hindlimb unloading on regional femoral blood flow
(A) and vascular resistance (B). Values are means ⫾ SE; n ⫽ 11 (for
control standing and 10 min and 7 day unloaded) and n ⫽ 7 (for 28
day unloaded). * Mean is different from control standing mean (P ⬍
0.05); † mean is different from 10-min unloaded mean (P ⬍ 0.05);
∧ mean is different from 7-day unloaded mean (P ⬍ 0.05).
Body and soleus muscle mass. The body masses at
death of control (458 ⫾ 9 g), 7-day HU (432 ⫾ 10 g), and
28-day HU (447 ⫾ 13 g) rats were not different. HU
reduced soleus muscle mass from 7-day HU (206 ⫾ 9
mg) and 28-day HU (148 ⫾ 15 mg) rats relative to that
of control animals (286 ⫾ 34 mg). Soleus muscle mass
was not different between the two HU groups. These
alterations in soleus muscle mass are similar to the
relative changes previously reported in HU rats (8, 9,
30, 46). Furthermore, soleus muscle atrophy, which is
characteristic of reduced skeletal muscle weight-bearing activity, confirms the effectiveness of the HU intervention.
Hindlimb tissue blood flow and vascular resistance.
Ten minutes of HU significantly reduced blood flow to
the proximal femur, the femoral shaft, and the femoral
shaft marrow (Fig. 2). After 7 days HU, blood flow to all
portions of the femur, including the distal metaphysis,
was significantly less than blood flow to femurs of
control rats; there were no differences in femoral blood
flows between 10 min and 7 days HU. After 28 days of
unloading, femoral blood flow continued to be lower
than that of control rats. However, flow to the femoral
shaft and marrow after 28 days of tail suspension was
less than that with 7 days HU. The reductions in
femoral flow with 10 min and 7 days of HU resulted
from an increase in vascular resistance (Fig. 2), because tissue blood flow is primarily the result of arterial perfusion pressure and vascular resistance, and
caudal arterial pressure was not different at these
times (Table 1). The further decrease in flow to the
femoral shaft with 28 days HU was accompanied by a
continued increase in resistance. In contrast, the further reduction in shaft marrow perfusion with 28 days
HU did not result from a continued increase in vascular resistance but resulted from a decreased hindlimb
perfusion pressure (Table 1).
Perfusion of the proximal metaphysis and shaft plus
marrow of the tibia was reduced by 10 min, 7 day, and
28 day HU; there were no differences in blood flow to
these regions of the tibia among the three HU conditions (Table 2). These reductions in perfusion appear to
result primarily from increases in vascular resistance
SKELETAL PERFUSION DURING SIMULATED MICROGRAVITY
Table 2. Skeletal blood flow and vascular resistance
following acute and chronic hindlimb unloading
Control
Standing
Tibia
Proximal end
7 Days
HU
28 Days
HU
23 ⫾ 4*
6.7 ⫾ 1.3*
10 ⫾ 2*
13 ⫾ 2
9⫾3
23 ⫾ 5
17 ⫾ 3
9⫾1
17 ⫾ 4
11 ⫾ 2
9⫾1
17 ⫾ 2
14 ⫾ 2
12 ⫾ 2
18 ⫾ 3*
9⫾2
8 ⫾ 2*
20 ⫾ 3*
12 ⫾ 1*
12 ⫾ 1
19 ⫾ 1*
6.8 ⫾ 0.4*
8 ⫾ 1*
18 ⫾ 1*
10 ⫾ 2
19 ⫾ 4
14 ⫾ 2
10 ⫾ 1
11 ⫾ 2
16 ⫾ 2
7⫾2
29 ⫾ 5
10 ⫾ 2
17 ⫾ 3
9 ⫾ 3†
28 ⫾ 6
4 ⫾ 1†
43 ⫾ 8
9⫾1
18 ⫾ 4
20 ⫾ 3*
6.4 ⫾ 0.7*
8 ⫾ 1*
17 ⫾ 2*
5⫾2
36 ⫾ 8
10 ⫾ 1*†
13 ⫾ 2*
12 ⫾ 1
12 ⫾ 2
6⫾1
22 ⫾ 3
10 ⫾ 2
16 ⫾ 4
6 ⫾ 2†
26 ⫾ 5
3 ⫾ 1†
57 ⫾ 8†
8⫾1
18 ⫾ 4
normalization of central arterial perfusion pressure.
An almost identical pattern of blood flow response to
unloading occurred in the mandible and clavicle (Table
2). The skull similarly demonstrated an increase in
blood flow with acute HU. However, unlike in the
humerus, mandible, and clavicle, this increased perfusion was associated with a decreased vascular resistance.
Skeletal muscles of the forelimb (triceps brachii) and
face (masseter) did not exhibit changes in perfusion
with either acute or chronic HU (Fig. 5). Unlike in
many of the forelimb, shoulder, and head bones, blood
flow during 10 min HU was unaltered because the
increased perfusion pressure was countered by an increase in vascular resistance.
Skeletal mass. Femoral mass was reduced by 28 days
HU (Table 3). In addition, there was a tendency for
tibial and fibular mass to be reduced with prolonged
HU (P ⬍ 0.1). In contrast, there was an increased
clavicular and mandibular mass with 28 days HU and
a tendency for a greater humeral mass (P ⬍ 0.1). Mass
Values are means ⫾ SE. BF, blood flow; R, vascular resistance.
Units for BF are ml⫺1 䡠 min⫺1 䡠 100 g and R are mmHg 䡠 ml⫺1 䡠 min䡠100
g. * Mean value significantly different from that of control standing
condition (P ⬍ 0.05); † mean value significantly different from that
of 10 min HU condition (P ⬍ 0.05).
(Table 2). Blood flow and vascular resistance in the
distal tibial metaphysis were unaltered by HU.
Fibular blood flow was unaffected by 10 min and 7
days HU (Table 2). However, fibular perfusion at 28
days HU was lower than that during control standing
and after 10 min HU. The reduction in flow with 28
days HU appears to be the result of an increase in
vascular resistance (vs. control standing) and a decreased perfusion pressure (vs. 10 min HU).
Blood flow to vastus intermedius and the red portion
of vastus lateralis muscles, which are predominantly
composed of type I fibers and type IIA and IIX fibers
(10), respectively, and which lie adjacent to the femur,
was reduced by 10 min, 7 days, and 28 days HU (Fig.
3). These reductions in flow resulted from increases in
vascular resistance. In contrast, perfusion of the white
portion of vastus lateralis muscle, which is composed
exclusively of type IIB fibers (10), was elevated with 7
days and 28 days HU. In this muscle section, vascular
resistance was diminished with 10 min, 7 days, and 28
days HU (Fig. 3).
Forelimb and head tissue blood flow and vascular
resistance. Perfusion of the humerus was acutely elevated with 10 min HU but returned to control levels by
7 days HU (Fig. 4). The acute increase in humeral
blood flow was not the result of a decrease in vascular
resistance (Fig. 4) but resulted from an elevated perfusion pressure (Table 1). The subsequent return of
blood flow to control levels with 7 days HU resulted
from an elevation in vascular resistance. In contrast,
the normalized humeral blood flow with 28 days HU
was not the result of a heightened resistance but of a
Fig. 3. Effect of hindlimb unloading on hindlimb muscle blood flow
(A) and vascular resistance (B). Values are means ⫾ SE; n ⫽ 11 (for
control standing and 10 min and 7 day unloaded) and n ⫽ 7 (for 28
day unloaded). * Mean is different from control standing mean (P ⬍
0.05); † mean is different from 10-min unloaded mean (P ⬍ 0.05);
∧ mean is different from 7-day unloaded mean (P ⬍ 0.05).
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BF 35 ⫾ 3
R 3.9 ⫾ 0.3
Shaft and marrow BF 15 ⫾ 2
R
9⫾1
Distal end
BF
8⫾1
R
19 ⫾ 2
Fibula
BF 19 ⫾ 2
R
7⫾1
Pelvis
BF 13 ⫾ 1
R
10 ⫾ 1
Radius/ulna
BF
6⫾1
R
23 ⫾ 4
Scapula
BF 13 ⫾ 3
R
12 ⫾ 2
Clavicle
BF
9⫾1
R
16 ⫾ 3
Skull
BF
3⫾1
R
51 ⫾ 7
Mandible
BF
7⫾1
R
19 ⫾ 2
10 Min
HU
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SKELETAL PERFUSION DURING SIMULATED MICROGRAVITY
of the pelvis, radius/ulna, scapula, and skull did not
change with HU.
DISCUSSION
The primary purpose of this study was to test the
hypothesis that mechanical unloading of hindlimb bones
and the decreased arterial perfusion pressure to the hindlimbs decreases skeletal perfusion. In addition, we
sought to determine whether the unloading-induced cephalic fluid shift alters skeletal blood flow in forelimb,
shoulder, and head bones. The results indicate that HU
rapidly diminishes blood flow to the femoral and tibial
metaphysis (cancellous bone), diaphysis (cortical bone),
and marrow, and that prolonged unloading further decreases perfusion of the femoral shaft and marrow.
Chronic unloading was also necessary to decrease fibular
perfusion rate. In contrast, HU acutely elevates blood
flow to the humerus, clavicle, skull, and mandible. The
decline in blood flow to the hindlimb bones appears to
coincide with a diminished mineral apposition rate, density, and mass of both cortical and cancellous bone observed in HU rats (present study, 5, 8, 16, 25, 31, 38, 44).
Fig. 5. Effect of hindlimb unloading on forelimb and facial muscle blood
flow (A) and vascular resistance (B). Values are means ⫾ SE; n ⫽ 9 (for
control standing and 10 min and 7 day unloaded) and n ⫽ 7 (for 28 day
unloaded). *Mean is different from control standing mean (P ⬍ 0.05).
Correspondingly, the acute increase in blood flow to forelimb, shoulder, and head bones appears to coincide with
some reports of increased bone mass in HU rats (present
study, 38). Therefore, our data support the hypothesis
Table 3. Bone mass in control and hindlimb
unloaded rats
n
Femur
Tibia
Fibula
Pelvis
Humerus
Radius/ulna
Scapula
Clavicle
Skull
Mandible
Control
7 Days HU
28 Days HU
11
1,490 ⫾ 50
1,025 ⫾ 25
124 ⫾ 9
2,330 ⫾ 89
596 ⫾ 21
509 ⫾ 21
466 ⫾ 41
74 ⫾ 4
3,550 ⫾ 167
1,480 ⫾ 44
11
1,410 ⫾ 31
1,052 ⫾ 30
107 ⫾ 9
2,500 ⫾ 87
601 ⫾ 19
515 ⫾ 31
515 ⫾ 41
77 ⫾ 4
3,890 ⫾ 166
1,540 ⫾ 39
7
1,330 ⫾ 20*
962 ⫾ 15†
95 ⫾ 8†
2,320 ⫾ 80
648 ⫾ 16†
517 ⫾ 20
502 ⫾ 46
87 ⫾ 6*
3,930 ⫾ 164
1,640 ⫾ 30*
Values are means ⫾ SE given in mg. * Mean value significantly
different from that of control rats (P ⬍ 0.05); † mean value different
from that of control animals (P ⬍ 0.1).
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Fig. 4. Effect of hindlimb unloading on regional humeral blood flow
(A) and vascular resistance (B). Values are means ⫾ SE; n ⫽ 9 (for
control standing and 10 min and 7 day unloaded) and n ⫽ 7 (for 28
day unloaded). * Mean is different from control standing mean (P ⬍
0.05); † mean is different from 10-min unloaded mean (P ⬍ 0.05).
SKELETAL PERFUSION DURING SIMULATED MICROGRAVITY
osteoblast mitogen (18, 37) with an inhibitory effect on
osteoclast bone resorption (22, 27, 28, 43). PGE2 stimulates bone formation and attenuates bone loss with
immobilization in vivo (1), presumably via stimulation
of osteoblast mitosis, reduction of osteoclast number
(20), and inhibition of osteoclast activity (6, 15). Results from the present study of an increased blood flow
and perfusion pressure in the forelimb, shoulder, and
head are consistent with the hypothesis of an elevated
transmural interstitial fluid pressure gradient between the endosteal vasculature and the periosteal
lymphatic drainage system. Although blood flow to the
skull and fore bones of the HU rat was only acutely
elevated, this does not necessarily imply that the stimulus for bone remodeling was brief. For instance, the
rise in head and forelimb arterial perfusion pressure
would increase vascular transmural pressure in bone,
thereby promoting capillary/sinusoid fluid filtration
and elevating interstitial fluid pressure. The elevation
of interstitial fluid pressure would serve to normalize
vascular transmural pressure and return blood flow to
control levels. However, the maintenance of a high
interstitial fluid pressure, which serves to offset the
chronically elevated arterial perfusion pressure (30,
present study), may provide a chronic stimulus to increase bone formation through a NO (21, 42) or PGE2
(34, 36) signaling mechanism.
Fig. 6. Hypothetical sequence of events linking the
unloading of hindlimb bones and the reduction in skeletal blood flow to a remodeling imbalance that leads to
a net loss of bone. Two possible mechanisms are illustrated, that mediated through a diminished interstitial
shear stress and that mediated through a reduced vascular shear stress (see text for details). PGE2, prostaglandin E2; NO, nitric oxide; ED-NO, endotheliumderived nitric oxide; ED-PGI2, endothelium-derived
prostacyclin.
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that HU-induced changes in bone perfusion lead to alterations in the balance between bone resorption and bone
formation.
Several mechanisms could serve to link changes in
bone perfusion with skeletal remodeling (Fig. 6). The
first is the effect that alterations in blood flow may
have on interstitial fluid flow. For example, it has been
proposed that alterations in bone interstitial fluid flow
may influence bone remodeling (41). Interstitial fluid
flows radially through cortical bone, driven by a transmural pressure gradient between the vasculature of
the endosteal surface and the periosteal lymphatics
(36). Mechanical loading of the skeletal system causes
fluid flow through cancellous bone and the lacunacanalicular network of cortical bone, exacerbating flowinduced shear stress imposed on surface bone cells
(40). The shear stresses generated by bone interstitial
fluid flow appear to be of similar magnitude to those
occurring at the blood-vascular endothelium interface
(37). Bone cells respond to fluid shear forces in a
manner similar to vascular endothelial cells, by generating autocrine/paracrine signals that modulate remodeling activity (18, 21, 36, 40). For example, cultured osteoblasts increase production of nitric oxide
(NO) and PGE2 when exposed to elevated flow-induced
shear stress, but mechanical strain does not elicit a
similar release (40). NO has been shown to be an
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SKELETAL PERFUSION DURING SIMULATED MICROGRAVITY
pathetic neural mechanism, because cephalic fluid
shifts engage cardiopulmonary receptors and elicit reflexive decreases in efferent sympathetic nerve activity
(45). More likely, the decreased loading of the hindlimb
bones diminished the net metabolic rate of the bone
cells in these male animals. Metabolic control of vascular resistance is the classic regulatory mechanism
whereby tissue metabolism is coupled to oxygen delivery. In addition to increases in vascular resistance, a
decrease in hindlimb perfusion pressure also appears
to contribute to the decreased blood flow. For example,
blood flow to the femoral marrow is lower with 28 days
HU than with 10 min or 7 days HU. This decreased
flow is not the result of a further increase in vascular
resistance, indicating that the lower caudal arterial
pressure is responsible for the diminished perfusion.
Similarly, fibular perfusion at 28 days HU is lower
than at 10 min HU without a corresponding increase in
vascular resistance, indicating that the lower flow with
prolonged unloading is the result of a diminished perfusion pressure. It is interesting to note that changes
in blood flow to hindlimb skeletal muscles consistently
correspond to changes in vascular resistance (Fig. 3).
These data indicate that autoregulation of vascular
resistance, rather than the lower perfusion pressure,
appears to be entirely responsible for determining
hindlimb muscle perfusion.
Unlike with the hindlimb bones, blood flow to the
skull, humerus, clavicle, and mandible increased with
acute unloading. The transient increase in skull perfusion resulted from a lower vascular resistance. It is
possible that the lower resistance may have resulted
from an increased loading of the skull, a consequence of
a transient increase in intracranial pressure (29). In
contrast, increased perfusion of the humerus, clavicle,
and mandible were not accompanied by changes in
vascular resistance, indicating that the elevated flow to
these bones was a result of the increased thoracic
arterial perfusion pressure. With 7 days HU, however,
vascular resistance was elevated in these fore bones.
Interestingly, blood flow to facial and forelimb muscles
did not increase with acute or chronic unloading, owing
to the fact that elevations in vascular resistance offset
elevations in perfusion pressure. These data suggest
that the resistance vasculature in both the fore and aft
skeleton is not as adept at compensating for fluid
pressure shifts as the skeletal muscle resistance vasculature. Although this apparent inability to precisely
regulate blood flow appears to make bone more “susceptible” to fluid shifts, this could serve a functionally
important role as a stimulus or signal for skeletal
remodeling.
Rat HU is a popular ground-based model to study the
effects of microgravity on the musculoskeletal and cardiovascular systems (9, 30). To date, there are no bone
blood flow data obtained in a weightless environment
to support or refute the findings of an increased and
decreased blood flow to the skeleton of the HU rat.
However, there are data from space-flown rats that
provide indirect evidence of a diminished perfusion of
hindlimb bones in microgravity. Doty and colleagues
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 17, 2017
The increases in bone mass with HU (present study,
38) are not unique to this model of microgravity. Increases in skull bone density have been reported to
occur in humans during bed rest (3, 26) and in cosmonauts after long-duration spaceflight (39). The observations of an increased head bone density in humans
are consistent with the hypothesis that headward fluid
shifts may alter normal bone remodeling.
Results from the present study also demonstrate a
reduced blood flow to hindlimb bones with mechanical
unloading. Diminished bone perfusion may have direct
effects on interstitial fluid flow and, consequently, bone
formation by reducing capillary and sinusoid fluid filtration (Fig. 6). Reductions in interstitial fluid movement have been reported in the rat femur with HU
(12). Furthermore, Bergula and collaborators (4) used
femoral vein ligation to increase intramedullary fluid
pressure and presumably interstitial fluid flow in HU
rats. Ligation increased femoral bone mineral content
and trabecular density over sham-operated control
limbs. The authors suggested that the protective effect
of ligation on bone mass was apparently due to increased interstitial fluid flow and the corresponding
release of NO and PGE2. These studies collectively
provide support for the notion that hindlimb bone loss
in HU rats is associated with reduced interstitial fluid
flow.
Altered bone perfusion may also be linked to skeletal
remodeling via a vascular mechanism (33). On the
basis of the juxtaposition of blood vessels and bone
cells, it was first proposed in 1930 that the bone’s
vascular network might be an active mediator of skeletal adaptation (19). More recently, it has been demonstrated that blood vessels are located in the basic
multicellular units containing osteoclasts and osteoblasts that carry out bone remodeling (cf. 33). In addition, these vessels are located no more than 100 ␮m
from the site of each unit’s remodeling activity (cf. 33).
Vascular endothelial cells release substances in response to changes in fluid flow and shear stress that
may act directly on bone cell populations or serve as
paracrine modulators of bone cell activity within the
basic multicellular unit (2, 14, 22, 28, 33). Specifically,
NO and PGI2 are potent vasodilators that are, in many
tissues, released by the vascular endothelium in response to blood flow and, correspondingly, intravascular shear stress (32). The effects of NO on bone formation and resorption are well established (18, 22, 27, 28,
37, 43). PGI2 has been shown to exert a direct inhibitory effect on osteoclasts (6, 35). This raises the possibility that HU-induced increases and decreases in
blood flow and shear stress alter vascular endothelial
cell release of these agents, which could subsequently
modify the focal balance between osteoblast and osteoclast activity (Fig. 6).
There appear to be several mechanisms responsible
for the increased and decreased blood flow to the fore
and aft skeleton of the HU rat, respectively. The initial
decreases in flow to the femora and tibiae were accompanied by increases in vascular resistance. Presumably
this increase in resistance is not mediated by a sym-
SKELETAL PERFUSION DURING SIMULATED MICROGRAVITY
This study was supported by National Space and Biomedical
Research Institute Grant NCC9-58-H (to S. A. Bloomfield, L. J. Suva,
R. T. Turner, and M. D. Delp) and National Aeronautics and Space
Administration Grants NAGW-4842 and NAG5-3754 (to M. D. Delp).
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