J Bone Miner Metab
DOI 10.1007/s00774-017-0830-y
ORIGINAL ARTICLE
Disuse osteopenia induced by botulinum toxin is similar
in skeletally mature young and aged female C57BL/6J mice
Jens Bay Vegger1   · Annemarie Brüel1 · Mikkel Bo Brent1 ·
Jesper Skovhus Thomsen1 
Received: 30 December 2016 / Accepted: 19 February 2017
© The Japanese Society for Bone and Mineral Research and Springer Japan 2017
Abstract Osteopenia and osteoporosis predominately
occur in the fully grown skeleton. However, it is unknown
whether disuse osteopenia in skeletally mature, but grow-
ing, mice resembles that of fully grown mice. Twenty-four
16-week-old (young) and eighteen 44-week-old (aged)
female C57BL/6J mice were investigated. Twelve young
and nine aged mice were injected with botulinum toxin in
one hind limb; the remaining mice served as controls. The
mice were euthanized after 3 weeks of disuse. The femora
were scanned by micro-computed tomography (µCT) and
bone strength was determined by mechanically testing the
femoral mid-diaphysis and neck. At the distal femoral met-
aphysis, the loss of trabecular bone volume fraction (BV/
TV) differed between the young and aged mice. However,
at the distal femoral epiphysis, no age-dependent differ-
ences were observed. Thinning of the trabeculae was not
affected by the age of the mice at either the distal femoral
metaphysis or the epiphysis. Furthermore, the aged mice
lost more bone strength at the femoral mid-diaphysis, but
not at the femoral neck, compared to the young mice. In
general, the bone loss induced by botulinum toxin did not
differ substantially between young and aged mice. There-
fore, the loss of bone in young mice resembles that of aged
mice, even though they are not fully grown.
Keywords  Immobilization · Mice · Aging · Micro-CT ·
Mechanical testing
* Jens Bay Vegger
jbve@biomed.au.dk
1
Department of Biomedicine, Aarhus University, Wilhelm
Meyers Allé 3, 8000 Aarhus C, Denmark
Introduction
In life sciences, the motivation for using laboratory animals
is to understand the physiology and pathology that are not
readily studied in humans. Therefore, when selecting an
animal model for an experiment it is important to consider
the consistency and comparability of that particular model
with the life science question at hand [1].
Bone loss in humans has different causes, and it has
been shown that patients undergoing strict bed rest lose
approximately 1% spinal areal bone mineral density
(aBMD) weekly [2], whereas lactating and postmenopau-
sal women lose approximately 1% monthly [3] and 0.75%
yearly [4], respectively. In mice, disuse- and immobiliza-
tion-induced osteopenia has traditionally been studied in
relatively young animals because they are inexpensive and
easily obtained [5–9]. However, osteopenia and osteopo-
rosis are foremost an aging phenomenon in humans. Sec-
ondary osteoporosis resulting from immobilization gener-
ally affects the mature skeleton, and as young mice have a
higher growth rate than older mice [10], the younger mice
might also be influenced differently by unloading than aged
mice. Consequently, the younger mice may not be so good
a model for disuse osteoporosis as the older mice.
Several mice models have been used to study disuse
osteopenia, e.g., plaster casting [11], elastic bandaging
[12], surgical denervation [13], tail suspension [14], or, as
in the present study, injection of botulinum toxin (BTX) [5,
8]. In mice, after the injection of BTX in one hind limb, the
maximum loss of bone mass and strength is attained after
3–4 weeks of disuse [5–9].
In our laboratory, we usually perform immobilization
studies in skeletally mature, but growing, 16-week-old
mice. However, at the age of 44 weeks, skeletal growth of
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 J Bone Miner Metab

the mice has ceased and therefore more closely resembles performed by scans of the two solid-state phantoms pro-
that of an adult human skeleton [15–18]. Therefore, the vided with the scanner.
aim of the study was to investigate whether the bone loss
after 3 weeks of immobilization differed between skeletally
mature young, but growing, and fully grown aged mice. Micro‑computed tomography (µCT)

The mid-diaphyses and the distal femora were scanned in

a desktop µCT scanner (Scanco µCT 35; Scanco Medi-
Materials and methods
cal, Brüttiselen, Switzerland). The mid-diaphyses were
scanned with an isotropic voxel size of 7 µm, X-ray volt-
Animals
age of 55 kV, current of 145 µA, and an integration time
of 300 ms; the distal femora were scanned with an iso-
Forty-two 16-week-old female C57BL/6J mice (Taconic,
tropic voxel size of 3.5 µm, X-ray voltage of 55 kV, cur-
Ry, Denmark) with a body weight (BW) of 22.4 ± 1.2 g
rent of 145 µA, and an integration time of 800 ms.
were housed at 20 °C with a 12:12 h light/dark cycle. The
The mid-diaphyses were analyzed by semi-automati-
animals had free access to tap water and standard mice
cally drawing an 819-µm-high volume of interest (VOI)
chow (1324; Altromin, Lage, Germany).
including cortical bone only with the software provided
At the age of 15 weeks, 1 week before the study start,
with µCT scanner (version 6.5) (Fig. 1a). The three-
the animals were randomized according to their BW into
dimensional (3D) data sets were low-pass filtered using
four groups: two groups with 12 mice in each group and
a Gaussian filter (σ  = 0.8, support = 1) and segmented
two groups with 9 mice in each group. At study start, 12
with a fixed threshold filter (556.4 mg HA/cm3). The
mice were injected i.m. with 2 IU BTX (Botox; Allergan,
minimum point between the marrow and the bone peak in
Dublin, Ireland) per 100 g BW, distributed equally into
the attenuation histograms was automatically determined
the quadriceps and calf muscles of the right hind limb; the
using IPL (version 5.11), and the median of these thresh-
other 12 mice were injected in a similar way with saline to
olds was used as the threshold for all segmentations. The
serve as age-matched controls (Ctrl). The two groups with
9 mice in each group were housed until they were 44 weeks
old, whereafter one group was injected with BTX and the
other group was injected with saline, as already described.
Three weeks after the injections, the mice were euthanized
by anesthesia (IsoFlo Vet; Orion Pharma Animal Health,
Copenhagen, Denmark), followed by removal of the heart.
One mouse destined to serve as a 44-week-old Ctrl ani-
mal was euthanized prematurely because of an ileus-like
condition.
Immediately after euthanasia, the rectus femoris muscles
were isolated and the wet weight determined. The femora
were isolated, cleaned from soft tissue, and stored in Ring-
er’s solution at −20 °C. Before dual-energy X-ray absorpti-
ometry (DEXA) scanning, femoral lengths were measured
with a digital sliding caliper.
The experiment complied with the EU Directive
2010/63/EU for animal experiments, and all proce-
dures were approved by the Danish Animal Experiments
Inspectorate.

Dual‑energy X‑ray absorptiometry (DEXA)

The femora were placed in a DEXA scanner (Sabre XL;
Norland Stratec, Pfortzheim, Germany) and scanned with
an isotropic pixel size of 0.1 mm. Bone mineral content
(BMC) and areal bone mineral density (aBMD) were
determined for the whole femur. Quality assurance was
Fig. 1  Micro-computed tomography (µCT) scan of a mouse femur.
a Green area represents analyzed volume of interest (VOI) of the
femoral mid-diaphysis consisting of cortical bone only. b Blue area
represents analyzed VOI of the trabecular bone in the distal femoral
metaphysis; red area represents analyzed VOI of the trabecular bone
in the distal femoral epiphysis (color figure online)

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analyses included bone area, tissue area, marrow area,
polar moment of inertia (pMOI), and bone material den-
sity (ρ).
The distal femoral metaphyses were analyzed by semi-
manually drawing a 1-mm-high VOI including trabecular
bone, but excluding cortical bone, placed 0.2 mm proximal
to the growth plate (Fig. 1b). The 3D data sets were low-
pass filtered using a Gaussian filter (σ  = 1, support = 2)
and segmented with a fixed threshold filter (527.6 mg HA/
cm3). The distal femoral epiphyses were analyzed by semi-
manually drawing a VOI placed distally to the growth
plate and including trabecular bone only until the inter-
condylar fossa (Fig. 1b). The 3D data sets were low-pass
filtered using a Gaussian filter (σ  = 1, support = 2) and
segmented with a fixed threshold filter (550.2 mg HA/cm3).
The thresholds were determined in the same way as for the
mid-diaphyses. Analyses included bone volume fraction
(BV/TV), trabecular thickness (Tb.Th), trabecular number
(Tb.N), trabecular separation (Tb.Sp), connectivity density
(CD), structure model index (SMI), and bone material den-
sity (ρ).
The analyzes were performed according to the cur-
rent guidelines [19]. Quality assurance was performed
by weekly (density) and monthly (geometry) scans of the
solid-state calibration phantom provided with the scanner.
3D visualization and imaging were performed using Amira
(version 5.6.0; FEI, Mérignac, France).
Mechanical testing
The fracture strength of the femoral mid-diaphyses was
determined with a three-point bending test. The femora
were placed on a custom-made testing jig, supporting the
proximal and distal part of the femoral shaft. The distance
between the two supporting rods was 7.1 mm. Load was
applied in an anterior-posterior direction at the midpoint of
the femur at a constant deflection rate of 2 mm/min until
fracture using a materials testing machine (5566; Instron,
High Wycombe, United Kingdom).
Subsequently, the proximal femora were placed in
another custom-made fixation jig supporting the shaft
under the neck. Load was applied to the top of the femoral
head with a constant deflection rate of 2 mm/min until frac-
ture of the femoral neck.
Statistics
Differences inflicted by BTX were analyzed by a two-
way analysis of variance (ANOVA). If significant vari-
ance between the BTX- and saline-injected mice was
found, a multiple comparison with the Student–Newman–
Keuls method was performed to differentiate between the
effect on the BTX-injected hind limb, the noninjected
contralateral hind limb of the BTX-injected mice, and the
saline-injected hind limb of the Ctrl mice. Furthermore,
the two-way ANOVA is capable of detecting interactions,
wherein a significant interaction indicates a difference in
the effect of BTX between the 19- and 47-week-old mice,
i.e., that the effect of BTX is dependent of the age of the
mice. Data are given as mean ± SD, and results were
defined as statistically significant at the two-tailed p < 0.05.
The statistical analyses and graphing were carried out
using SigmaPlot (version 13.0; Systat Software, San Jose,
CA, USA).
Results
Animals
At euthanasia, both the 19- and 47-week-old BTX mice
had lost BW compared to their respective Ctrl groups, and
the loss of BW was independent of the age of the mice
(Table 1).
BTX induced a pronounced atrophy of the injected rec-
tus femoris muscle, but also affected the contralateral non-
injected rectus femoris muscle compared to the respective
Ctrl groups. The BTX-induced muscle atrophy was more
pronounced in the 47-week-old mice than in the 19-week-
old mice because an interaction was detected (Table 1). The
injection of BTX did not affect the length of the femora
(Table 1).
Dual‑energy X‑ray absorptiometry (DEXA)
The BMC of the whole femur was lower in the BTX-
injected hind limb compared to that of the Ctrl group and
the contralateral noninjected hind limb (Table 2). After
areal adjustment, the aBMD of the whole femur was lower
in the 19-week-old BTX-injected mice compared to that of
the Ctrl group and the contralateral noninjected hind limb;
this was not the case for the 47-week-old mice, wherein
the contralateral noninjected hind limb had a higher aBMD
than both the BTX and the Ctrl groups (Table 2). Further-
more, an age-dependent interaction of the BTX-induced
alterations in aBMD was found.
Micro‑computed tomography (µCT)
At the femoral mid-diaphysis, the loss of bone area in the
BTX-injected hind limb was caused by endosteal bone
resorption because the marrow area was larger in the
BTX-injected groups than in the respective Ctrl groups.
The expansion of the marrow cavity was not affected by
the age of the mice (Fig. 2; Table 3). Moreover, the pMOI
was lower in the BTX-injected hind limb compared to the
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Table 1  Body weight, femur length, and rectus femoris muscle mass in mice
Body weight at injection (g) Body weight at euthanasia (g) Femur length (mm) Rectus femoris muscle mass (mg)

Mice 19 weeks old

 Ctrl 22.3 ± 0.6 22.1 ± 0.8 15.48 ± 0.15 63.4 ± 3.4
 BTX NI – – 15.37 ± 0.25 55.7 ± 3.2a
 BTX 22.1 ± 0.8 20.7 ± 0.7a 15.37 ± 0.38 33.9 ± 5.1a,b
Mice 47 weeks old
 Ctrl 28.1 ± 3.2 25.8 ± 1.8 15.96 ± 0.24 76.3 ± 6.1
 BTX NI – – 15.89 ± 0.28 57.9 ± 2.4a
 BTX 27.4 ± 2.5 23.5 ± 1.6a 15.82 ± 0.21 32.7 ± 4.1a,b
Interaction No No No Yes

Female C57BL/6J mice, 19 or 47 weeks old, injected with botulinum toxin (BTX) and compared with age-matched controls (Ctrl). BTX NI refers
to the contralateral noninjected hind limb of the BTX-injected mice
a
  Significant difference from the respective Ctrl group
b
 Significant difference from the respective BTX NI group. Interaction indicates an age-dependent difference of BTX between 19- and
47-week-old mice. Mean ± SD

Table 2  Dual-energy X-ray absorptiometry (DEXA) data of whole contralateral noninjected hind limb. Moreover, the loss
femur of BV/TV was affected by the age of the mice. Notably,
BMC (g) aBMD (g/cm2) the trabecular bone ρ was lower in the BTX-injected hind
limb (Table 4). For values of Tb.N, Tb.Sp, CD, SMI, and
Mice 19 weeks old
ρ, see Table 4.
 Ctrl 0.016 ± 0.001 0.048 ± 0.002
At the distal femoral epiphysis, the BTX injections also
 BTX NI 0.015 ± 0.001a 0.047 ± 0.002
resulted in a loss of BV/TV and Tb.Th, which was not
 BTX 0.011 ± 0.001a,b 0.036 ± 0.002a,b
affected by the age of the mice (Figs. 3b, d, 5). The bone
Mice 47 weeks old
loss was accompanied by an increase in SMI, indicating a
 Ctrl 0.016 ± 0.001 0.035 ± 0.005 shift toward more rod-like trabeculae (Table 4). Further-
 BTX NI 0.015 ± 0.001 0.038 ± 0.005a more, the trabecular bone ρ was lower in the BTX-injected
 BTX 0.011 ± 0.001a,b 0.031 ± 0.003b hind limb compared to both the contralateral noninjected
Interaction No Yes hind limb and Ctrl mice (Table 4). For values of Tb.N,
Female C57BL/6J mice, 19 or 47 weeks old, injected with botulinum Tb.Sp, CD, SMI, and ρ, see Table 4.
toxin (BTX) and compared with age-matched controls (Ctrl). BTX NI
refers to the contralateral noninjected hind limb of the BTX-injected
mice. BMC bone mineral content, aBMD areal bone mineral density Mechanical testing
a
  Significant difference from the respective Ctrl group
b
 Significant difference from the respective BTX NI. Interaction The three-point bending test at the femoral mid-diaphysis
indicates an age-dependent difference of BTX between 19- and
47-week-old mice. Mean ± SD
revealed that the BTX injections resulted in both lower
fracture strength and maximum stiffness compared to the
Ctrl groups (Fig. 6a, c). Moreover, the femur of BTX-
hind limb of Ctrl mice (Table 3). In the 19-week-old mice, injected hind limb group had a lower fracture strength and
the BTX-injections resulted in a lower cortical bone ρ in maximum stiffness compared to the contralateral nonin-
both the BTX-injected and contralateral noninjected hind jected hind limb. Notably, an age-dependent interaction
limb compared to that of Ctrl mice. In contrast, the ρ in the was detected in the BTX-induced lower bone strength and
47-week-old mice was lower only in the BTX-injected hind stiffness.
limb, but not in the contralateral noninjected hind limb, At the femoral neck, only the BTX-injected hind limb
compared to the Ctrl mice. Furthermore, age of the mice had a lower fracture strength and maximum stiffness than
affected the loss of ρ (Table 3). in the Ctrl mice, whereas the fracture strength of the con-
At the distal femoral metaphysis, the BTX injections tralateral noninjected hind limb did not differ from that of
resulted in a loss of both BV/TV and Tb.Th (Figs. 3a, the Ctrl mice (Fig. 6b, d). In addition, the magnitude of the
c, 4). Unexpectedly, the BTX-injected 47-week-old BTX-induced lower maximum stiffness was dependent on
mice did not have a lower BV/TV compared to the the age of the mice.

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Fig. 2  Representative illustrations of the femoral mid-diaphysis acquired from the µCT scans and chosen according to mean values. BTX NI
refers to the contralateral noninjected hind limb of the BTX-injected mice

Table 3  Micro-computed Bone area ­(mm2) Tissue area ­(mm2) Marrow area ­(mm2) pMOI ­(mm4) ρ (mg/cm3)
tomography (µCT) data of
cortical bone obtained from Mice 19 weeks old
femoral mid-diaphyses
 Ctrl 0.78 ± 0.04 1.78 ± 0.11 1.00 ± 0.07 0.37 ± 0.04 1141 ± 5
 BTX NI 0.73 ± 0.05a 1.76 ± 0.07 1.02 ± 0.05 0.35 ± 0.03 1132 ± 7a
 BTX 0.64 ± 0.03a,b 1.72 ± 0.05 1.07 ± 0.06a,b 0.30 ± 0.02a,b 1130 ± 9a
Mice 47 weeks old
 Ctrl 0.85 ± 0.04 1.95 ± 0.06 1.10 ± 0.05 0.43 ± 0.03 1222 ± 6
 BTX NI 0.80 ± 0.05a 1.92 ± 0.08 1.11 ± 0.07 0.40 ± 0.04 1219 ± 6
 BTX 0.68 ± 0.06a,b 1.93 ± 0.10 1.25 ± 0.07a,b 0.36 ± 0.05a,b 1204 ± 10a,b
Interaction No No No No Yes

Female C57BL/6J mice, 19 or 47 weeks old, injected with botulinum toxin (BTX) and compared with
age-matched controls (Ctrl). BTX NI refers to the contralateral noninjected hind limb of the BTX-injected
mice. pMOI polar moment of inertia
a
  Significant difference from the respective Ctrl group
b
  Significant difference from the respective BTX NI. Interaction indicates an age-dependent difference of
BTX between 19- and 47-week-old mice. Mean ± SD

Discussion However, DEXA is a relatively rough measure that includes

The present study showed that both young and aged skel-
etally mature female C57BL/6J mice lose bone mass and
strength when subjected to BTX-induced immobilization
of one hind limb.
Age‑related differences in BTX‑induced immobilization
compared to age‑matched, vehicle‑treated controls
At euthanasia, the old mice had lost more muscle mass
than the young mice compared to the respective Ctrl
groups because an interaction was seen.
The BTX-induced loss of BMC was similar in the young
and aged mice, but when corrected for the 2D bone size,
the loss of aBMD was statistically significant for the young
mice only, and an age-dependent interaction was observed.
cortical bone, trabecular bone, and calcified cartilage in
a collective calculation. When separating the cortical and
trabecular bone with µCT, the loss of cortical bone, from
endosteal bone resorption, did not differ between the young
and aged mice. The young mice seemed to have a small,
although not significant, BTX-induced reduction of peri-
osteal bone, i.e., a reduced tissue area. Loading is known
to influence tissue area [20], and therefore the nonsignifi-
cant reduction in tissue area might be explained as a BTX-
induced decrease in modeling drift as the young mice are
still growing. Interestingly, the loss of cortical bone had
a larger impact on the bone strength and stiffness of the
femoral mid-diaphyses in the aged than in the young mice.
The age-related interaction of BTX-induced osteopenia on
mechanical properties might be explained by a age-related
decline in bone quality [21], i.e., the interplay between the

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Fig.  3  a Bone volume/trabecular volume (BV/TV) of the distal
femoral metaphysis. b BV/TV of the distal femoral epiphysis. c Tra-
becular thickness (Tb.Th) of the distal femoral metaphysis. d Tb.Th
of the distal femoral epiphysis. BTX NI refers to the contralateral non-
injected hind limb of the BTX-injected mice. a denotes a significant
difference from the respective Ctrl; b denotes a significant difference
from the respective BTX NI. Interaction indicates an age-dependent
difference of BTX between the 19- and 47-week-old mice. Mean ±
SD

Fig. 4  Representative three-dimensional (3D) images of the distal femoral metaphysis acquired from the µCT scans and chosen according to
mean values. BTX NI refers to the contralateral noninjected hind limb of the BTX-injected mice

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Table 4  µCT data of trabecular Tb.N (1/mm) Tb.Sp (µm) CD (1/mm3) SMI (–) ρ (mg/cm3)
bone obtained from distal
femoral metaphyses and distal Distal femoral metaphysis
femoral epiphyses
Mice 19 weeks old
 Ctrl 3.37 ± 0.15 294 ± 14 159 ± 33 2.22 ± 0.17 884 ± 14
 BTX NI 3.29 ± 0.19 302 ± 19 144 ± 30 2.33 ± 0.20 875 ± 8
 BTX 3.16 ± 0.13a 313 ± 13 136 ± 25 2.33 ± 0.13 837 ± 11a,b
Mice 47 weeks old
 Ctrl 1.99 ± 0.21 508 ± 54 22 ± 11 2.79 ± 0.38 914 ± 17
 BTX NI 1.97 ± 0.22 519 ± 63 26 ± 12 2.70 ± 0.23 893 ± 21a
 BTX 1.85 ± 0.15 547 ± 42 26 ± 14 2.51 ± 0.19a 855 ± 16a,b
Interaction No No No Yes No
Distal femoral epiphysis
Mice 19 weeks old
 Ctrl 4.93 ± 0.46 224 ± 18 205 ± 23 0.12 ± 0.11 970 ± 14
 BTX NI 4.90 ± 0.33 219 ± 10 228 ± 39 0.33 ± 0.15a 963 ± 13
 BTX 4.21 ± 0.35a,b 246 ± 21a,b 329 ± 52a,b 0.97 ± 0.13a,b 908 ± 14a,b
Mice 47 weeks old
 Ctrl 4.63 ± 0.43 253 ± 19 107 ± 32 0.19 ± 0.12 1004 ± 12
 BTX NI 4.77 ± 0.70 254 ± 32 130 ± 30 0.35 ± 0.11a 994 ± 8
 BTX 3.97 ± 0.36a,b 267 ± 24 281 ± 66a,b 0.91 ± 0.15a,b 936 ± 17a,b
Interaction No No No No No

Female C57BL/6J mice, 19 or 47 weeks old, injected with botulinum toxin (BTX) and compared with age-
matched controls (Ctrl). BTX NI refers to the contralateral noninjected hind limb of the BTX-injected mice
a
  Significant difference from the respective Ctrl group
b
  Significant difference from the respective BTX NI. Interaction indicates an age-dependent difference of
BTX between 19- and 47-week-old mice. Mean ± SD

Fig. 5  Representative 3D images of the distal femoral epiphysis acquired from the µCT scans and chosen according to mean values. BTX NI
refers to the contralateral noninjected hind limb of the BTX-injected mice

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Fig.  6  a Fracture strength of femoral mid-diaphysis. b Fracture
strength of femoral neck. c Maximum stiffness of femoral mid-diaph-
ysis. d Maximum stiffness of femoral neck. BTX NI refers to the con-
tralateral noninjected hind limb of the BTX-injected mice. a denotes
macroscopic bone structure and material properties, such as
cortical porosity, micro-damage, mineralization, and colla-
gen cross-linkage [22].
At both the distal femoral metaphyses and epiphyses,
the mice lost BV/TV and trabecular thickness compared
to the respective Ctrl groups. However, the BTX-induced
loss of BV/TV at the distal femoral metaphysis was
dependent on the age of the mice. The explanation for
this is not especially clear, but the loss might be the result
of the sparse amount of trabecular bone at the distal fem-
oral metaphysis in the aged mice, thereby limiting the
comparable amount of bone or even the amount of bone
that is responsive and lost consequent to BTX-induced
disuse. The strength of the femoral neck was similarly
affected by disuse in both young and aged mice, although
the aged mice had a larger loss of bone stiffness.
To the best of our knowledge, the age-related differences
and susceptibility to disuse-induced osteopenia have not
previously been investigated in mice, and the present study
is consequently the first to compare BTX-induced bone
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a significant difference from the respective Ctrl; b denotes a signifi-
cant difference from the respective BTX NI. Interaction indicates an
age-dependent difference of BTX between the 19- and 47-week-old
mice. Mean ± SD
loss in skeletally mature young and aged female C57BL/6J
mice.
Age‑related differences in the systemic effects
of localized BTX injections
The BTX injections resulted in a reduction in BW, which
has previously been shown by us and others [5, 8]. Three
animal models are frequently used to study disuse osteo-
penia: tail suspension (TS), sciatic neurectomy (SN), and
injection of BTX. In mice, loss of BW is also seen with
TS, but not with SN, although injection of BTX seems to
be particularly likely to affect BW [5, 14, 23–25]. One can
only speculate why mice undergoing SN do not lose BW,
and the loss of BW seen with TS- and BTX-induced immo-
bilization might be caused by the mice still having a func-
tioning autonomic and sensory nervous system. The muscle
mass of the contralateral noninjected rectus femoris mus-
cle also underwent some atrophy, although not to the same
extent as the BTX-injected muscle. Muscle atrophy in the
J Bone Miner Metab
contralateral noninjected hind limb of immobilized animals
has previously been observed [5, 26], but the data are con-
flicting [7, 8]. Interestingly, we have previously shown that
the loss of muscle mass in the sternocleidomastoid muscle
of BTX-injected rats points toward a stress response from
suddenly having one hind limb paralyzed rather than a
direct systemic effect of the localized, intramuscular hind
limb BTX injections [27].
At the distal femora of the contralateral noninjected
hind limb of both 19- and 47-week-old mice we observed
a slight, but significant, loss of trabecular thickness and
bone mass compared to the Ctrl mice. Previous studies
have reported inconsistent results regarding the systemic
effect of BTX [7, 8, 28], and the effects vary [5, 26]. Fur-
thermore, at the femoral mid-diaphyses the BTX injections
resulted in loss of bone strength and stiffness of the con-
tralateral noninjected hind limb. In contrast, no significant
loss of bone strength was seen in the femoral neck, and this
difference is not easily explained because the contralateral
noninjected hind limb seems to lose trabecular bone, which
is present in the femoral neck, rather than cortical bone.
Limitations and future prospects
The present study did not find any major differences in
BTX-induced osteopenia between young and aged female
mice. However, although the included analyses only
focused on tissue level properties, age-related differences
might exist at a cellular or molecular level. Furthermore,
we have previously shown that it is important to perform
cellular and molecular analyses after 1 week of disuse to
detect relevant differences [9]. Therefore, future studies are
needed to elucidate any age-related differences at a cellular
or molecular level.
In the present study, female mice were included as these
are widely used for studying disuse osteopenia. Never-
theless, sex differences might exist, which consequently
calls for future studies in male mice. Although the skele-
tal effects of BTX did not differ between young and aged
female mice, the skeletal effects of disuse induced by either
TS or SN might differ between young and aged mice.
Conclusion
In general, the loss of bone and mechanical properties
seen following injections of BTX does not differ substan-
tially between young and aged mice. Therefore, the bone
loss observed in skeletally mature 16-week-old female
C57BL/6J mice resembles that seen in aged mice, even
though they are not fully grown.
Acknowledgments The authors are grateful for the hard work and
excellent technical assistance of Jytte Utoft. We thank Visiopharm
for the contribution to the newCAST stereology software system and
the VELUX Foundation for the donation of the µCT scanner. The
study was kindly supported by the Health at Aarhus University, Oda
and Hans Svennings Foundation, The Vanføre Foundation, Frimodt-
Heineke Foundation, Helga and Peter Kornings Foundation, Læge
Søren Segel and hustru Johanne Wiibroe Segels Reseachfoundation,
Inge og Per Refshalls Researchfoundation, Direktør Jacob Madsen og
hustru Olga Madsens Foundation, and the Novo Nordisk Foundation.
Compliance with ethical standards 
Conflict of interest  All authors have no conflict of interest.
References
1. Shanks N, Greek R, Greek J (2009) Are animal models predic-
tive for humans? Philos Ethics Humanit Med 4:2
2. Krølner B, Toft B (1983) Vertebral bone loss: an unheeded side
effect of therapeutic bed rest. Clin Sci (Lond) 64:537–540
3. Kovacs CS, Kronenberg HM (1997) Maternal-fetal calcium and
bone metabolism during pregnancy, puerperium, and lactation.
Endocr Rev 18:832–872
4. Ebbesen EN, Thomsen JS, Beck-Nielsen H, Nepper-Rasmussen
HJ, Mosekilde L (1999) Age- and gender-related differences in
vertebral bone mass, density, and strength. J Bone Miner Res
14:1394–1403
5. Warner SE, Sanford DA, Becker BA, Bain SD, Srinivasan
S, Gross TS (2006) Botox-induced muscle paralysis rapidly
degrades bone. Bone 38:257–264
6. Grimston SK, Silva MJ, Civitelli R (2007) Bone loss after tem-
porarily induced muscle paralysis by botox is not fully recovered
after 12 weeks. Ann N Y Acad Sci 1116:444–460
7. Manske SL, Boyd SK, Zernicke RF (2010) Muscle and bone fol-
low similar temporal patterns of recovery from muscle-induced
disuse due to botulinum toxin injection. Bone 46:24–31
8. Lodberg A, Vegger JB, Jensen MV, Larsen CM, Thomsen JS,
Brüel A (2015) Immobilization induced bone loss is strain spe-
cific in mice. Bone Rep 2:59–67
9. Vegger JB, Brüel A, Dahlgaard AF, Thomsen JS (2016) Altera-
tions in gene expression precede sarcopenia and osteopenia in
botulinum toxin immobilized mice. J Musculoskelet Neuronal
Interact 16:355–368
10. Gall GAE, Kyle WH (1968) Growth of the laboratory mouse.
Theor Appl Genet 38:304–308
11. Rantakokko J, Uusitalo H, Jämsä T, Tuukkanen J, Aro HT, Vuo-
rio E (1999) Expression profiles of mRNAs for osteoblast and
osteoclast proteins as indicators of bone loss in mouse immobili-
zation osteopenia model. J Bone Miner Res 14:1934–1942
12. Jämsä T, Koivukangas A, Ryhänen J, Jalovaara P, Tuuk-
kanen J (1999) Femoral neck is a sensitive indicator of bone
loss in immobilized hind limb of mouse. J Bone Miner Res
14:1708–1713
13. Sakai A, Nakamura T, Tsurukami H, Okazaki R, Nishida S,
Tanaka Y, Norimura T, Suzuki K (1996) Bone marrow capacity
for bone cells and trabecular bone turnover in immobilized tibia
after sciatic neurectomy in mice. Bone 18:479–486
14. Simske SJ, Greenberg AR, Luttges MW (1991) Effects of sus-
pension-induced osteopenia on the mechanical behaviour of
mouse long bones. J Mater Sci Mater Med 2:43–50
15. Buie HR, Moore CP, Boyd SK (2008) Postpubertal architec-
tural developmental patterns differ between the L3 vertebra and
proximal tibia in three inbred strains of mice. J Bone Miner Res
23:2048–2059
13

16. Willinghamm MD, Brodt MD, Lee KL, Stephens AL, Ye J, Silva
MJ (2010) Age-related changes in bone structure and strength in
female and male BALB/c mice. Calcif Tissue Int 86:470–483
17. Glatt V, Canalis E, Stadmeyer L, Bouxsein ML (2007) Age-
related changes in trabecular architecture differ in female and
male C57BL/6J mice. J Bone Miner Res 22:1197–1207
18. Beamer WG, Donahue LR, Rosen CJ, Baylink DJ (1996)
Genetic variability in adult bone density among inbred strains of
mice. Bone 18:397–403
19. Bouxsein ML, Boyd SK, Christiansen BA, Guldberg RE, Jepsen
KJ, Müller R (2010) Guidelines for assessment of bone micro-
structure in rodents using micro-computed tomography. J Bone
Miner Res 25:1468–1486
20. Galea GL, Hannuna S, Meakin LB, Delisser PJ, Lanyon LE,
Price JS (2015) Quantification of alterations in cortical bone
geometry using site specificity software in mouse models of
aging and the responses to ovariectomy and altered loading.
Front Endocrinol (Lausanne) 6:52
21. Jilka RL (2013) The relevance of mouse models for investigating
age-related bone loss in humans. J Gerontol Ser A Biol Sci Med
Sci 68:1209–1217
22. Donnelly E (2011) Methods for assessing bone quality: a review.
Clin Orthop Relat Res 469:2128–2138
13
J Bone Miner Metab
23. Kodama Y, Dimai H, Wergedal J, Sheng M, Malpe R, Kutilek S,
Beamer W, Donahue LR, Rosen C, Baylink DJ, Farley J (1999)
Cortical tibial bone volume in two strains of mice: effects of
sciatic neurectomy and genetic regulation of bone response to
mechanical loading. Bone 25:183–190
24. Lescale C, Schenten V, Djeghloul D, Bennabi M, Gaignier F,
Vandamme K, Strazielle C, Kuzniak I, Petite H, Dosquet C, Frip-
piat JP, Goodhardt M (2015) Hind limb unloading, a model of
spaceflight conditions, leads to decreased B lymphopoiesis simi-
lar to aging. FASEB J 29:455–463
25. Meakin LB, Delisser PJ, Galea GL, Lanyon LE, Price JS (2015)
Disuse rescues the age-impaired adaptive response to external
loading in mice. Osteoporos Int 26:2703–2708
26. Ellman R, Grasso DJ, van Vliet M, Brooks DJ, Spatz JM, Conlon
C, Bouxsein ML (2014) Combined effects of botulinum toxin
injection and hind limb unloading on bone and muscle. Calcif
Tissue Int 94:327–337
27. Vegger JB, Brüel A, Thomsen JS (2015) Vertical trabeculae are
thinned more than horizontal trabeculae in skeletal-unloaded
rats. Calcif Tissue Int 97:516–526
28. Poliachik SL, Bain SD, Threet D, Huber P, Gross TS (2010)
Transient muscle paralysis disrupts bone homeostasis by rapid
degradation of bone morphology. Bone 46:18–23