Osteoporos Int DOI 10.1007/s00198-014-2748-8
ORIGINAL ARTICLE
Effects of low intensity vibration on bone and muscle in rats with spinal cord injury H. M. Bramlett & W. D. Dietrich & A. Marcillo & L. J. Mawhinney & O. Furones-Alonso & A. Bregy & Y. Peng & Y. Wu & J. Pan & J. Wang & X. E. Guo & W. A. Bauman & C. Cardozo & W. Qin
Received: 27 December 2013 / Accepted: 12 May 2014 # International Osteoporosis Foundation and National Osteoporosis Foundation 2014
Abstract Summary Spinal cord injury (SCI) causes rapid and marked bone loss. The present study demonstrates that low-intensity vibration (LIV) improves selected biomarkers of bone turnover and gene expression and reduces osteoclastogenesis, suggesting that LIV may be expected to benefit to bone mass, resorption, and formation after SCI. Introduction Sublesional bone is rapidly and extensively lost following spinal cord injury (SCI). Low-intensity vibration (LIV) has been suggested to reduce loss of bone in children with disabilities and osteoporotic women, but its efficacy in SCI-related bone loss has not been tested. The purpose of this study was to characterize effects of LIV on bone and bone cells in an animal model of SCI.
Electronic supplementary material The online version of this article (doi:10.1007/s00198-014-2748-8) contains supplementary material, which is available to authorized users. H. M. Bramlett : W. D. Dietrich : A. Marcillo : L. J. Mawhinney : O. Furones-Alonso : A. Bregy Miami Project to Cure Paralysis, Department of Neurological Surgery, University of Miami Miller School of Medicine, Miami, FL, USA
Methods The effects of LIV initiated 28 days after SCI and provided for 15 min twice daily 5 days each week for 35 days were examined in female rats with moderate severity contusion injury of the mid-thoracic spinal cord. Results Bone mineral density (BMD) of the distal femur and proximal tibia declined by 5 % and was not altered by LIV. Serum osteocalcin was reduced after SCI by 20 % and was increased by LIV to a level similar to that of control animals. The osteoclastogenic potential of bone marrow precursors was increased after SCI by twofold and associated with 30 % elevation in serum CTX. LIV reduced the osteoclastogenic potential of marrow precursors by 70 % but did not alter serum CTX. LIV completely reversed the twofold elevation in messenger RNA (mRNA) levels for SOST and the 40 % reduction in Runx2 mRNA in bone marrow stromal cells resulting from SCI. Conclusion The findings demonstrate an ability of LIV to improve selected biomarkers of bone turnover and gene expression and to reduce osteoclastogenesis. The study indicates a possibility that LIV initiated earlier after SCI and/or continued for a longer duration would increase bone mass. Keywords Bone loss . Bone marrow cells . Low intensity vibration . Spinal cord injury
H. M. Bramlett Bruce W. Carter Miami VA Medical Center, Miami, FL, USA Y. Peng : Y. Wu : J. Pan : W. A. Bauman : C. Cardozo : W. Qin (*) National Center of Excellence for the Medical Consequences of Spinal Cord Injury, James J. Peters VA Medical Center, 130 West Kingsbridge Road, Bronx, NY 10468, USA e-mail:
[email protected] J. Wang : X. E. Guo Department of Biomedical Engineering, Columbia University, New York, NY, USA W. A. Bauman : C. Cardozo : W. Qin Mount Sinai School of Medicine, New York, NY, USA
Introduction Immobilization due to bed rest, space flight, or paralysis results in atrophy of skeletal muscle and loss of bone in immobilized body regions. In most cases, immobilizationrelated bone loss results from the combined effects of increased resorption and reduced formation. Spinal cord injury (SCI) results in an extreme form of immobilization that results in substantial muscle atrophy and bone loss. Muscle atrophy after SCI is associated with diminished strength, power, and
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muscle fatigue resistance [1–3]. Alterations in bone metabolism after SCI include a reduced bone formation rate, as well as marked acceleration of bone resorption associated with increased osteoclast surface and increased potential of bone marrow progenitors for osteoclastic differentiation [1–3]. Bone loss after SCI is relatively unique in its localization to the sublesional regions with the greatest declines observed at the distal femur and proximal tibia, where bone mineral density (BMD) may be reduced by more than 50 % within a few years after SCI [1, 2, 4–7]. Consequently, fractures of the distal femur and proximal tibia are the most common fractures in individuals with SCI [8–11]. In humans with SCI, BMD declines rapidly at all sublesional boney sites at rates as high as 1 % per week for the first year after injury [1, 2, 4]. In studies of SCI rats, loss of trabecular bone in the proximal tibia approaches 70 % at 3 weeks after a complete spinal cord transection [12] and over 60 % at the distal femoral metaphysis at 10 days after severe spinal cord contusion [13]. The observation that low intensity high-frequency mechanical vibration (LIV) increases bone mass and bone formation in healthy sheep [14] and mice [15] has generated interest in the possibility that LIV might also reduce or reverse pathological remodeling of bone. A growing body of evidence in laboratory animals and humans supports this concept. In clinical studies, LIV has been found to improve BMD in children with disabilities [16, 17] and post-menopausal women [18–21]. Anabolic effects of LIV do not appear to require gravitational loading [22, 23]. LIV has also been found to exert beneficial effects on the pool of bone marrow mesenchymal stem cells capable of osteoblastic differentiation. Numbers of bone marrow cells with the potential to differentiate into osteoblast-like cells when cultured in vitro are reduced by immobilization and normalized by LIV [24]. In some studies, muscle has also been suggested to be beneficially affected by LIV, as exemplified by finding that in normal mice, whole body LIV increased soleus muscle fiber cross sectional area [15]. Clinical studies have demonstrated beneficial effects of LIV for individuals with SCI including improvements in walking function [25], muscle blood flow [26], and spasticity [27, 28]. LIV may be expected to slow or reverse bone loss after severe immobilization due to conditions such as SCI [2, 4, 5, 29] and can be administered through the supine body in a manner that would permit significant skeletal loading [30]. The possible benefits of LIV to individuals with SCI are supported by findings of a case report of a single subject with motor-incomplete SCI in whom vibration appeared to improve BMD [31]. However, it remains uncertain whether LIV can reverse of attenuate structural and functional changes in bone and muscle after SCI. The objectives of this study were to characterize effects of whole body LIVon muscle and bone in a rat model of SCI. We reasoned that effects of LIV might be enhanced by skeletal
loading and thus conducted these investigations using an animal model of moderate severity contusion SCI in which sufficient motor function had returned by 28 days after the injury that animals were capable of weight bearing. We examined the effects of the application of LIV after an SCI on BMD, three-dimensional architecture of metaphysical trabecular bone, serum markers of bone metabolism, and differentiation potential of bone marrow progenitors, and on mass of paralyzed skeletal muscle and biomarkers of neuromuscular activation. Because little is known about the effects of a moderate severity contusion SCI on bone, we also examined the changes in bone, bone metabolic markers, and bone marrow progenitors resulting from this form of SCI. We expected that LIV would reduce bone resorption, stimulate bone formation, and correct abnormalities in osteoblastogenic or osteoclastogenic potential of marrow precursors in animals with SCI. We also expected that LIV would increase the mass of skeletal muscle and increase biomarkers of neuromuscular activity in skeletal muscle in animals with SCI.
Methods Animals Female Sprague Dawley rats 3 months of age (Harlan Laboratories) were housed in temperature and humidity controlled rooms and provided with a 12:12-h day to night cycle. Animals were fed standard rat chow ad libitum. All procedures with experimental animals were approved by the Institutional Animal Care and Use Committee of the University of Miami and were in conformance with the National Institutes of Health Guide. Experimental design Rats were randomly assigned to one of the following groups: SCI without whole body LIV (SCI, N=14), SCI with whole body LIV (LIV, N = 13), or sham SCI consisting of a laminectomy only (Sham, N=8). A moderate severity SCI at the interspace between the ninth and tenth thoracic vertebra was produced using a New York University (MASCIS) impactor by dropping a 10 g weight from a height of 12.5 mm as described in detail elsewhere [32]. Control animals underwent only a laminectomy. LIV was initiated 28 days after SCI. Starting with the LIV at 28 days post-injury was chosen based on following considerations: (i) we believed that gravitational loading might enhance any benefits of LIV, and animals have regained the ability to weight bear at 28 days as reflected by the mean Basso, Beattie, and Bresnahan (BBB) scores at this time for SCI animals (SCI-LIV 9.42±0.31; SCI-no vibration 9.46± 0.25). This type of partial functional recovery in moderate
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severity SCI animals at 28 days post-injury has similarly demonstrated by other studies [33, 34]; (ii) This lag time prior to starting vibration may be somewhat comparable to that which occurs in a rehabilitation setting, where several weeks may pass before starting rehabilitation therapy after injury; and (iii) We recently demonstrated that when begun on day 29 after SCI and continued for 28 days, anabolic steroid nandrolone was able to reduce bone loss after SCI through the regulation of Wnt signaling pathway [35]. Animals randomized to the LIV group underwent 35 days of treatment performed twice daily for 15 min each session for 5 days each week (Fig. 1a). Animals with SCI were divided among groups that received LIV and a group that was placed directly on the vibrating platform (Soloflex®) for the same period of time without activating it (Fig. 1b). The vibration device was programmed in order to achieve frequency of vibration within a range of about 40 Hz (0.3 g) because these parameters were similar to those used in studies by Field-Fote and coworkers [27] and others [36, 37]. In particular, Xie et al. showed that whole body vibration at 45 Hz (0.3 g) for 15 min a day inhibited trabecular bone resorption and increased bone formation in the skeleton of grown mice [36]. Thirty-minute sessions were chosen based on the hope that these would be tolerable and of a duration that might be clinically feasible. Twice daily sessions were chosen because of information in animals and cells that response to LIV was greater with twice daily dosing in work from Rubin and coworkers, and we reasoned that this paradigm might enhance any beneficial effect in our animal model [38]. Characteristics of the vibration provided were determined with an iPad and the Vibration
app from Diffraction Limited Design, LLC. The iPad was placed in each chamber with the plate activated. The observed frequency of vibration was approximately 37 Hz, and the mean acceleration across the four chambers was 0.238 g 0 to peak (range 0.161 to 0.273 g 0 to peak). The control animals post laminectomy were also placed on the platform with activation in a manner identical to the SCI animals. The SCI animals were assigned to groups based on BBB score [39] at 4 weeks to prevent any confounding influence that might arise from unequal distributions of function among the groups (SCI-LIV 9.42±0.31; SCI-no vibration 9.46±0.25). To provide LIV intervention, animals were placed in chambers of a plexiglass box; a paper towel was placed on the bottom of each chamber. The box contained four chambers, and one rat was placed into each chamber. Rats were placed in chambers in a random order from one session to the next. Animals were anesthetized (3 % isoflurane, 70/30 % N2O/ O2) at 65 days post-SCI for blood collection, muscle, and bone harvesting. Blood was collected by intraventricular puncture, allowed to clot at room temperature, and centrifuged; serum was then removed and stored at −20 °C. Muscles were isolated by careful dissection, weighed, and flash frozen in liquid nitrogen. Hindlimbs were freed from the pelvis by cutting ligaments and connective tissues at the hip. Left hindlimbs were placed into sterile tubes containing ice-cold Minimum Essential Alpha Medium and kept at 4 °C until processing for isolation of bone marrow cells. Right hindlimbs were immersed in 4 % paraformaldehyde overnight after which fixative was drained and replaced with 70 % ethanol in water. Dual energy X-ray absorptiometry Areal bone mineral density (BMD) was measured using a small animal dual energy X-ray absorptiometer (DXA) (Lunar Piximus, Inside Out Sales, Fitchburg, WI) as previously described [40, 41]. Hindlimbs were positioned on the DXA platform with the knee flexed at an angle of 135°, and DXA images were acquired with Lunar Piximus software. The instrument was calibrated with a phantom following the procedures recommended by the manufacturer on each day of use. The metaphysis of the distal femur and proximal tibia were selected as regions of interest (ROI). The coefficient of variation for the repeated measurements for the ROI was approximately 1.5 %. MicroCT
Fig. 1 a Scheme of the experimental design. b A picture that illustrates the setup of a SOLOFLEX device. Animal images are superimposed into the pictures to depict the arrangement of animals used
To evaluate trabecular architecture of the distal femur, microCT was performed on fixed bones, as described previously [40] using a Scanco μCT scanner and a 16-μm voxel size. Image reconstruction and 3D quantitative analysis were performed using software provided by Scanco. Scans were
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initiated at the growth plate and moved proximally for a total of approximately 300 slices. A region of interest consisting of 100 slices beginning 0.5 mm proximal to the growth plate and continuing in a proximal direction were included in the analysis. Standard nomenclature and methods for bone morphometric analysis were employed [42]. Culture and differentiation of bone marrow progenitors Culture and differentiation of bone marrow progenitors was assessed as previously described [40]. Briefly, cells were flushed from the marrow cavity with α-MEM and seeded into tissue culture wells in this medium. To culture osteoclasts (OC), cells were cultured for 2 days in α-MEM supplemented with macrophage colony-stimulating factor (M-CSF, 5 ng/ml), after which non-adherent cells were collected, purified by centrifugation in Ficoll-Plus (GE Life Sciences), then seeded into wells and cultured in α-MEM supplemented with M-CSF (30 ng/ml) and RANKL (60 ng/ml) for 4–6 days. Osteoclasts were identified by staining for tartrate-resistant acid phosphatase (TRAP) using a kit (Sigma-Aldrich, St. Louis, MO). Recruitment of marrow stromal cells to the osteoblast lineage was assessed at 10 days of culture by staining for alkaline phosphatase (AP) using a kit (SigmaAldrich); cells were cultured in α-MEM supplemented with15 % preselected fetal calf serum (Hyclone, Logan, UT) and ascorbic acid-3-phosphoate (1 mM). Isolation of total RNA and real-time PCR (qPCR) Procedures for qPCR were performed as previously described [43]. qPCR was performed with an ABI Via7 thermal cycler using ABI Taqman 2X PCR mix and ABI Assay on Demand qPCR primers, except for regulator of calcineurin (RCAN) 1.4 which was detected using an ABI Assay by Design primer and probe set. The real time PCR strategy for RCAN1.4 focused on detecting the unique boundary between exons 4 and 5 in RCAN 1.4. Changes in expression were calculated using the 2−ΔΔCt method [44] using 18S RNA as the internal control. Serum levels of CTX and osteocalcin Serum CTX and osteocalcin were determined by EIA using commercially available kits according the manufacturers recommended procedures. CTX was assayed using a RatLaps kit from Immunodiagnostic Systems (Fountain Hills, AZ). For osteocalcin, an Osteocalcin EIA kit from Biomedical Technologies Inc. (Stoughton, MA) was used. All samples were assayed in duplicate.
Statistics Data were expressed as mean ± SD. The number N for each group is noted in the legend of each figure. The statistical significance of differences among means was tested using one-way ANOVA followed by a Newman–Keuls test post hoc to examine the significant differences between individual pairs of means. Differences were considered significant at P
