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Histomorphometric and Densitometric Changes in the Femora of Spinal Cord Transected Mice.

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THE ANATOMICAL RECORD 291:303–307 (2008)
Histomorphometric and Densitometric
Changes in the Femora of Spinal Cord
Transected Mice
SYLVAIN PICARD,1,2 NICOLAS P. LAPOINTE,1
JACQUES P. BROWN,1 AND PIERRE A. GUERTIN1,2*
1
Laval University Medical Center (CHUL), Faculty of Medicine,
Laval University, Quebec City, Quebec, Canada
2
Department of Anatomy and Physiology, Faculty of Medicine,
Laval University, Quebec City, Quebec, Canada
ABSTRACT
Spinal cord injury (SCI) leads generally to significant bone tissue loss
within a few months to a few years post–trauma. Although, increasing
data from rat models are available to study the underlying mechanisms
of SCI-associated bone loss, little is known about the extent and rapidity
of bone tissue changes in mouse models of SCI. The objectives are to
characterize and describe quantitatively femoral bone tissue changes during 1 month in adult paraplegic mice. Histomorphometric and densitometric measurements were performed in 3- to 4-month-old CD1 mice spinal cord transected at the low-thoracic level (Th9/10). We found a general
decrease in bone volume (222%), trabecular thickness (210%), and trabecular number (214%) within 30 days post-transection. Dual-energy Xray absorptiometric measurements revealed no change in bone mineral
density but a significant reduction (214%) in bone mineral content. These
results show large structural changes occurring within only a few weeks
post–spinal cord transection in the femora of adult mice. Given the
increasing availability of genetic and molecular research tools for
research in mice, this murine model may be useful to study further the
cellular and molecular mechanisms of demineralization associated with
SCI. Anat Rec, 291:303–307, 2008. Ó 2008 Wiley-Liss, Inc.
Key words: parapalegic; rodent; secondary complications; chronic
injury; immobilization
Spinal cord injury (SCI) generally leads to an immediate and irreversible loss of both sensory and voluntary
motor functions below the level of injury. The state of
paralysis and chronic immobilization caused by SCI has
been associated also with several serious clinical concerns such as cardiovascular problems, muscular atrophy, obesity, immune system deficiency, bladder infection, hormonal dysregulation, chronic pain, and osteoporosis (Cruse et al., 1996; Kocina, 1997; Bauman et al.,
2000; Cavigeli and Dietz, 2000). In fact, nearly all complete SCI individuals experience a significant loss of
bone mineral tissue (up to 30% in the femora) leading to
a marked increase of fracture incidence within 1 yr of
injury (Ragnarsson and Sell, 1981; Garland et al., 1992;
Wilmet et al., 1995; Lazo et al., 2001; Sabo et al., 2001).
Although, the basic mechanisms underlying osteoporosis in postmenopausal women have been extensively
Ó 2008 WILEY-LISS, INC.
studied (i.e., slow progression of osteoclastic activity),
those mechanisms involved in chronic immobilization
and disuse have received considerably less attention. In
animal models of disuse, traditionally in rats, hindlimb
immobilization has been found to induce a drastic and
sudden loss of femoral bone tissue, suggesting that difGrant sponsor: Fonds de la Recherche en Santé du Québec.
*Correspondence to: Pierre A. Guertin, Laval University Medical Centre, Neuroscience Unit, RC-9800, 2705 Laurier Boulevard, Quebec City (Quebec), Canada G1V 4G2. Fax: 418-6542753. E-mail: pierre.guertin@crchul.ulaval.ca
Received 18 September 2007; Accepted 19 November 2007
DOI 10.1002/ar.20645
Published online 29 January 2008 in Wiley InterScience (www.
interscience.wiley.com).
304
PICARD ET AL.
ferent mechanisms may be involved in disuse-related
(fast bone loss) vs. age-related osteoporosis (slow bone
loss; Bagi and Miller, 1994). For instance, a 10–30% (up
to 50% after 18 months) decrease of cancellous bone was
found within a few weeks in the ipsilateral femur of rats
that had their hindlimbs immobilized with a cast or an
elastic bandage (Li et al., 1990; Chen et al., 1992; Maeda
et al., 1993). Some of the changes associated with disuse
are believed to be mediated by both an increase of osteoclastic bone resorption and a decrease of osteoblastic
bone formation (Rantakokko et al., 1999; Kingery et al.,
2003).
On the other hand, growing evidence suggests that
several factors, other than mechanical unloading per se,
can influence the sets of cellular and molecular mechanisms underlying disuse-related bone loss (Uebelhart
et al., 1995). For instance, in the case of disuse induced
by sciatic nerve lesion in rats, bone tissue loss was found
to be partly caused by a disruption of bone marrow
innervation (neurogenic; Zeng et al., 1996; Kingery
et al., 2003). Moreover, differential tissue- and biomarker-specific changes have been reported between the
tail-suspension and the sciatic nerve-lesion models
(Hanson et al., 2005). In the case of microgravity, bone
tissue changes have been attributed mainly to a marked
decrease of osteoblast formation in young adult rats
(Matsumoto et al., 1998). Taken together, this suggests
that each model and condition of disuse may be associated, to some extent, with different sets of cellular and
molecular demineralizing mechanisms.
Here, the aim was to characterize some of the main
structural changes occurring within a few weeks in
adult mice (3–4 months old) spinal cord transected (Tx)
at the low-thoracic level (Th9/10). Although, increasing
data are available from spinal Tx rats (Sugawara et al.,
1998; Minematsu et al., 2003), little is known in spinal
Tx mice. Recent experiments in mouse models of disuse
have already led to meaningful insights into bone turnover processes and plasticity after immobilization
(Priemel et al., 2002; Judex et al., 2004). Given the
increasing availability of genetic and molecular research
tools for murine models, the study of demineralization
in SCI mice may contribute in the next few years to a
detailed characterization of the cellular and molecular
events underlying osteoporosis after SCI.
by CO2 asphyxia. Complete Tx was confirmed by (1) full
paralysis of the hindlimbs, (2) postmortem microscopic
examination of the spinal cord lesion, or (3) histological
examination of coronal or midsagittal spinal cord sections stained with luxol fast blue/cresyl violet.
All mice received preoperative care involving administration of 1 ml of lactate-Ringer’s solution, 5 mg/kg
Baytril (Bayer, Toronto, ON), and 0.1 mg/kg buprenorphine (Schering-Plough, Pointe-Claire, QC). Postoperative care consisted of lactate-Ringer’s solution (2 ml/day,
SC), buprenorphine (0.2 mg/kg/day, SC), and Baytril
(5 mg/kg/day, SC) administration for 4 days. Bladders
were expressed manually twice daily for 4 days or until
a spontaneous return of micturition. Only data from
animals with complete spinal Tx was used for further
analyses.
Densitometry
Dual-energy X-ray absortiometry (DEXA) measurements (PIXImus 2, Lunar Corp., Madison, WI; Kolta
et al., 2003) were performed on the femora of control
(nontransected, n 5 24) and Tx mice (n 5 14, 7 tested at
15–20 and 7 tested at 30 days post-Tx). Calibration of
the apparatus was conducted according to the manufacturer’s protocol. Bone mineral density (BMD) values
(g/cm2) were measured within a predetermined metaphyseal common region of interest (ROI, see Fig. 1) in
the metaphyseal area for all specimens. In contrast, the
entire femora were used for bone mineral content (BMC,
MATERIALS AND METHODS
Animal Model
All experimental procedures were conducted in accordance with the Canadian Council for Animal Care guidelines and accepted by the Laval University Animal Care
and Use Committee. Sixty-four adult mice (3- to 4month-old male CD1, Charles River Canada, St-Constant, QC) initially weighing 35–40 g were used for this
study. In brief, a complete Tx of the spinal cord was performed using microscissors inserted between the 9th and
10th thoracic vertebrae in mice completely anesthetized
with 2.5% isoflurane (Guertin, 2004a,b; Guertin and
Steuer, 2005). To ensure that complete Tx was achieved,
the inner vertebral walls were explored and entirely
scraped with small scissor tips. The opened skin area
was sutured, and animals were placed for a few hours
on heating pads. Animals were left in their cage
with food and water ad libitum until the day of killing
Fig. 1. Representative histological sections. A,B: Results showing
the femoral metaphyseal area of a control (non–spinal transected [Tx],
A) and a 7-day Tx mouse (B). Staining using slight red fuschin acid
was performed. Measurements for all specimens were made within
the common region of interest (ROI).
305
BONE LOSS IN PARAPLEGIC MICE
in grams) assessment. Proximal metaphyseal diameter
and bone length were also measured using a calibrated
digital caliper (Traceable1 Model no. 62379-531, precision of 0.01 mm). These experiments were performed by
the McGill’s Centre for Bone and Periodontal Research.
were pooled to increase statistical power and statistical
analyses using a Student’s t-test (SAS/STAT, SAS International, Heidelberg) were done between control and Tx
groups. P values < 0.05 were considered statistically
significant.
Tissue Preparation for Histomorphometry
Two groups of animals consisting of control non-Tx (n
5 14) and spinal Tx mice (n 5 12, 4 in each subgroup,
i.e., killed at 10, 15–20, or 30 days post-Tx) were used.
Immediately after killing, at 10, 15–20, or 30 days postTx, mice were weighed and the femoral bones were dissected and cleaned of soft tissue. The femoral bones
were wrapped in saline-soaked gauze and frozen at
2208C in sealed vials until testing. On the day of testing, the femoral bones were slowly (overnight) thawed
at 48C.
Histomorphometry
The femoral bones were fixed for 24 hr in 0.1 M phosphate-buffered (PB) solution containing 4% paraformaldehyde (pH 7.4). They were then decalcified in a 10%
ethylenediaminetetraacetic acid and 0.5 M PB solution
(pH 7.4) and dehydrated using a series of increasing
ethanol concentrations. The bones were washed in three
separate baths of toluene, embedded in paraffin using a
Miles Tissue-Tek VIP processor (Sakura/Miles Finetek,
Torrance, CA), sectioned (5 mm) through the ROI (microtome RM 2135, Leica Microsystems, Mississauga, ON),
and stained with slight red fuschin acid for static histomorphometry (Fig. 1).
Histomorphometric measurements were performed
using a semiautomated image analyzer (Bioquant Nova;
R&M Biometrics Inc., Nashville, TN) and a SummaSketch III professional digitalizing tablet (Summagraphics, Anaheim, CA) in conjunction with a Leitz Aristoplan microscope (Leica Microsystems) equipped with a
Dage MTI black-and-white cooled camera. System calibration and quality control for accurate measurements
were performed periodically with a 20-mm-spacing calibration scale bar (Leica Microsystems). Measurements
excluded the outer region (1 mm) at the growth plate–
metaphyseal junction to include only data from the secondary spongiosa cancellous bone region (Kimmel and
Jee, 1980). Data represented two-dimensional measurements including bone volume (BV, surface area in mm2),
cancellous bone area and bone surface (BS; mm2; Parfitt
et al., 1987). In brief (1) trabecular bone volume (TBV)
was defined as the percentage of trabecular cancellous
bone within the spongiosa space: TBV 5 (BV/TV) 3 100,
where BV is cancellous bone area (mm2) and TV is tissue
area (mm2). (2) Trabecular bone thickness (TbTh, mm)
corresponded to the mean trabecular thickness: TbTh 5
2/(BS/BV). (3) Trabecular number (TbN, number/mm)
was calculated according to the parallel plate model:
TbN 5 [(BV/TV) 3 10]/TbTh. (4) Trabecular bone separation (TbSp, mm) was defined as (1000/TbN) 2 TbTh.
Statistical Analyses
Comparisons between time points post-Tx were performed initially with a nonparametric Kruskal-Wallis H
test (several independent samples). Given that no significant difference was found between time points, data
RESULTS
Histomorphometry
All histomorphometric measurements were made from
the metaphyseal area of the femora (see ROI, Fig. 1).
Cancellous bone volume in Tx mice was found to
decrease significantly by 22.4% (P 5 0.02) compared
with control. Indeed, average values of 6.64 6 2.29 and
8.55 6 2.43% were found in the Tx (n 5 12) and the
non-Tx groups (n 5 14), respectively (Fig. 2A). Although
volumes as low as 5.37 6 2.28 (37.2% decrease) were
found at 15–20 days post-Tx, results were the same (not
dissimilar) between the 10, 15–20, and 30 days post-Tx
groups (not shown). In turn, average trabecular bone
thickness was found to significantly (P 5 0.04) decrease
by 10.65% (Fig. 2B). Average values of 22.83 6 3.79 and
25.15 6 3.35 mm were found in the Tx and the non-Tx
groups, respectively (Fig. 2B). As for bone volume, most
bone thickness changes were already achieved by 10
Fig. 2. Average histomorphometric and densitometric data. A: Trabecular bone volume (TBV 5 (BV/TV) 3 100). B: Trabecular bone
thickness (TbTh 5 2/(BS/BV)). C: Trabecular number (TbN 5 (BV/TV)
3 10/TbTh). D: Trabecular bone separation (TbSp 5 (1000/TbN) 2
TbTh). Bone mineral density (BMD, E) and bone mineral content
(BMC, F) values were measured using dual-energy X-ray absorptiometry (DEXA).
306
PICARD ET AL.
days post-Tx (85.6% of control). Trabecular number (i.e.,
number of trabecular bone areas) appeared to decrease
(214.5%) after injury but did not reach significance (P 5
0.09). Measurements of 3.38 6 0.74 and 2.89 6 0.84 nbr/
mm2 were found in the Tx and the non-Tx groups (Fig.
2C). As expected, an inverse relationship was found
with trabecular separation (defined as the space between
trabecular bone areas). In fact, as with trabecular number, trabecular separation appeared to change (124.03%)
but did not reach significance (P 5 0.07), with values of
353.9 6 120.0 mm and of 285.3 6 74.4 mm in the Tx and
the control groups, respectively (Fig. 2D).
Densitometry
BMD measured by DEXA revealed no significant
change after injury. Figure 2E shows BMD values of
0.073 6 0.001 and 0.073 6 0.001 g/cm2 in Tx (n 5 14)
and control (n 5 24) groups, respectively. Nonsignificant
differences were found at 15–20 or 30 days post-Tx with
0.075 6 0.006 and 0.072 6 0.007 g/cm2 (not shown).
Note that measurements at 10 days post-Tx were not
performed. No significant change in bone length and
proximal metaphyseal diameter values were found
between Tx and control animals (length, 15.13 6 0.13
vs. 15.09 6 0.11, P 5 0.81; diameter, 2.59 6 0.07 vs.
2.59 6 0.08 P 5 0.98). In contrast, bone mineral content
(BMC) values decreased after injury. Indeed, measurements of 0.032 6 0.003 and 0.037 6 0.002 g were found
in the Tx and the non-Tx groups representing a significant (P < 0.001) 13.5% decrease (Fig. 2F).
DISCUSSION
The results of this study mainly showed that the femoral bone of early chronic paraplegic mice undergo relatively large changes soon after trauma. Indeed, within
30 days post-Tx, large decreases in volume (222%, P 5
0.02), trabecular thickness (210%, P 5 0.04), trabecular
number (214% P 5 0.09), and BMC (214%, P < 0.001)
were found, whereas a 24% increase (P 5 0.07) in trabecular separation was detected (Fig. 2).
Other models of disuse have also reported comparable
bone losses. Researchers have shown a 10–30% decrease
in femoral cancellous tissue within a few weeks (up to
50% after 18 weeks) of unilateral hindlimb immobilization using an elastic band in 6- to 9-month-old female
rats (Li et al., 1990; Maeda et al., 1993). Similar levels
but faster bone losses (within 2–8 weeks of immobilization) have been detected with a comparable model of
disuse but in younger rats (2–3 months old, Chen et al.,
1992). Because the extent of bone loss is comparable in
age-matched immobilized rats and Tx mice (results of
this study) but not in older rats (slower progression),
this may suggest that ‘‘age’’ rather than ‘‘species’’ (i.e.,
rats vs. mice) is a determinant factor in the rate of bone
loss. This is supported also by results from young adult
(4 months old) mice showing a 20% loss of trabecular
bone after only 15–21 days of hindlimb suspension
(Judex et al., 2004). Significant differences between
male or female animals are unlikely, because hindlimb
unloading using the tail suspension model has induced
similar effects in young male and female rats (6-weekold animals showing a 20–30% decrease in trabecular
volume, thickness, and number; Chen et al., 1992; Basso
et al., 2005).
Although similar bone loss is apparently found across
all models of disuse, a close comparison has revealed
some differences both at the structural and mechanical
levels. In adult mice, greater mechanical property (i.e.,
femur stiffness, elastic and maximum forces) losses but
smaller muscle size losses have been measured after
tail-suspension compared with sciatic nerve-crush, providing evidence of tissue-specific and model-dependent
changes (Hanson et al., 2005). Site-specific changes have
also been found in tail-suspended mice from three different strains suggesting that genetics can influence bone
morphology and define bone response to mechanical
unloading (Squire et al., 2004).
Some of the mechanisms that may underlie model-specific changes could involve differences in the neural
innervation of bones. Indeed, adult rats that have had
their sciatic nerve completely transected have displayed
rapid femoral bone loss that was partially prevented by
daily administration of substance P, a well-recognized
neurotransmitter involved in the neural control of bone
remodeling (Zeng et al., 1996; Lundberg and Lerner,
2002). However, it remains unclear to date whether a
complete spinal Tx could also modulate, in some ways,
the neural activation and control of bone remodeling.
Although not examined in this study, changes after Tx
are likely to lead to functional changes in bone mechanical properties. Sugawara and colleagues have shown in
adult Tx rats, a 50% reduction of femoral bone strength
(compressive load to fracture) using a three-point bending system 24 weeks after trauma (Sugawara et al.,
1998). Of interest, similar changes in a comparable
model were reported as early as 2 weeks post-Tx (Minematsu et al., 2003), strongly suggesting that the results
presented here in Tx mice were likely associated also
with changes in femoral bone strength.
We also found that BMC values significantly changed
unlike BMD values. It is in contrast with results from
another model where 7–14 days of tail-suspension has
induced an 18–22% loss of naturally occurring BMD
increase in 5-week-old growing rats (Matsumoto et al.,
1998). This apparent discrepancy may be due to agespecific changes (i.e., faster bone remodeling processes in
younger animals) or to model-dependent differences but
not to a concomitant decrease in bone surface area,
because bone length and diameter values remained
unchanged after Tx (see end of the Results section). However, although reasons for such differences are unclear,
they may involve technical limitations associated with the
measurement device (Traceable1 digital caliper, resolution
0.01 mm), especially in a small model such as the mouse
(Kolta et al., 2003). This said, a lack of proportional
changes between BMD and BMC is not uncommon
because it has been reported also in other conditions and
models (e.g., Soon et al., 2006; Antolic et al., 2007).
It is important to specify also that most of the data
reported here were taken from the cancellous bone structure. However, the BMD and BMC were conducted on the
whole femora, which makes it difficult to directly compare
morphometric changes with densitometric ones. Also, note
that, although bone remodeling is mainly discussed here,
we cannot exclude the possibility that reduced bone
growth was a critical factor that contributed to some of
these adaptive bone changes post-Tx in mice.
BONE LOSS IN PARAPLEGIC MICE
Clear differences in bone loss progression can also be
observed between most models of disuse and age/
hormone-related models (e.g., ovariectomized mice; Bagi
and Miller, 1994), suggesting that differences in bone
remodeling mechanisms may exist in elderly vs. in
young immobilized rats. In fact, it has been clearly
established that osteoporosis in menopausal women, for
instance, is caused essentially by a slow increase of
osteoclastic activity. However, preliminary data from
SCI patients suggest that both a decrease of osteoblastic
activity and an increase of osteoclastic activity contribute to bone loss after trauma (Roberts et al., 1998). This
finding closely reflects some results obtained in a mouse
model of disuse (hindlimb immobilization with a cast)
showing a rapid decrease of osteocalcin and a sharp
increase of acid phosphatase (i.e., markers of osteoblastic
and osteoclastic activities respectively) within a few
days to a few weeks postimmobilization (Rantakokko
et al., 1999). It will be of interest in future experiments
to determine whether comparable changes in biomarker
levels can be found in SCI mice. Overall, results of this
study show that this mouse model is a valuable tool to
investigate bone remodeling processes specifically associated with SCI.
ACKNOWLEDGMENTS
We thank Eric Landry for surgical interventions,
Sonia Jean for statistical analyses, and the graphics
service of the CRCHUL for figures.
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