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 Sante´ du Que´bec. *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:
[email protected] 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).
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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).
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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).
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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.
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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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