The authors state that they have no conflicts of interest.
The bone response to physical exercise may be under control of the SNS. Using a running session in rats, we confirmed that exercise improved trabecular and cortical properties. SNS blockade by propranolol did not affect this response on cortical bone but surprisingly inhibited the trabecular response. This suggests that the SNS is involved in the trabecular response to exercise but not in the cortical response.
Introduction: Animal studies have suggested that bone remodeling is under β−adrenergic control through the sympathetic nervous system (SNS). However, the SNS contribution to bone response under mechanical loading remains unclear. The purpose of this study was to examine the preventive effect of exercise coupled with propranolol on cancellous and cortical bone compartments in ovariectomized rats.
Materials and Methods: Six-month-old female Wistar rats were ovariectomized (OVX, n = 44) or sham-operated (n = 24). OVX rats received subcutaneous injections of propranolol 0.1 mg/kg/day or vehicle and were submitted or not submitted to treadmill exercise (13 m/minute, 60 minutes/day, 5 days/week) for 10 weeks. Tibial and femoral BMD was analyzed longitudinally by DXA. At death, the left tibial metaphysis and L4 vertebrae were removed, and μCT was performed to study trabecular and cortical bone structure. Histomorphometric analysis was performed on the right proximal tibia.
Results: After 10 weeks, BMD and trabecular strength decreased in OVX rats, whereas bone turnover rate and cortical porosity increased compared with the Sham group (p < 0.001). Either propranolol or exercise allowed preservation of bone architecture by increasing trabecular number (+50.35% versus OVX; p < 0.001) and thickness (+16.8% versus OVX; p < 0.001). An additive effect of propranolol and exercise was observed on cortical porosity but not on trabecular microarchitecture or cortical width. Biomechanical properties indicated a higher ultimate force in the OVX-propranolol-exercise group compared with the OVX group (+9.9%; p < 0.05), whereas propranolol and exercise alone did not have any significant effect on bone strength.
Conclusions: Our data confirm a contribution of the SNS to the determinants of bone mass and quality and show a antagonistic effect of exercise and a β-antagonist on trabecular bone structure.
Current strategies for the prevention of osteoporotic fractures in women focus on increasing peak bone mass at skeletal maturity and minimizing bone loss after menopause. Exercise is known to improve bone quality and decrease the fracture risk in postmenopausal women.(1–3)
In animal studies, exercise is generally considered to positively influence peak bone mass. However, only a few studies have analyzed the training effect on ovariectomized (OVX) adult rats.(4–6) OVX rats exhibit decreased BMD, poor trabecular microarchitecture and bone strength, and increased bone turnover rate. These phenomena are considered to be consequences of osteoporosis in postmenopausal women.(7–9) Yeh et al.(4) showed that training in adult female rats induced a 34% increase in trabecular and cortical bone volumes. Barengolts et al.(5,6) showed an increase in bone formation parameters (such as mineral apposition rate [MAR] and bone formation rate [BFR]) and a decrease in bone resorption, as indicated by lower labeled perimeters. Peng et al.(10) confirmed these results: treadmill-trained OVX rats exhibited a reduction of trabecular bone loss ranging from –51.7% to –32.2% and an increase in maximal load ranging from 77.1N to 86.5N compared with OVX alone.
Mechanical loading generated by physical activity has been reported to be the main way of preserving and improving bone strength.(12) Treadmill exercise also induces several systemic variations of calciotropic hormones: Iwamoto et al.(13) observed an increase of 1,25-dihydroxyvitamin D3 and a decrease of PTH level after 11 weeks of treadmill exercise, but the response of PTH to exercise was not consistent in other experiments.(14,15) Evaluating the interactions between exercise and the sympathetic nervous system (SNS) in a treadmill protocol, Galbo et al.(16) showed that an increase in plasma adrenalin was related to exercise period and power in humans. In 2004, Desgorces et al.(17) showed an increase in catecholamine sensitivity after a long exercise period. Independently of exercise, catecholamines have a direct impact on bone metabolism by increasing resorption activity that induces harmful effects on BMD and microarchitecture.(18,19) Bourrin et al.(20) suggested that the increase in bone resorption during vigorous exercise (>80% Vo2max) was mediated by an increase in catecholamines. The first hypothesis of this study was that, during moderate exercise (defined as 50–70% of Vo2max), the beneficial effect of mechanical loading overruns the harmful effect of the catecholamine excess induced by exercise.
In addition, propranolol, a β-adrenergic antagonist, has been shown to prevent the bone symptoms caused by the intracerebral effects of leptin(18) and to suspension or immobilization.(21,22) Elefteriou et al.(23) reported that mice lacking only one copy of adrenergic β2 receptor (Adrβ2R) displayed a higher bone mass phenotype than the wild lineage. Furthermore, they showed that the bone phenotype of OVX Adrβ2R knockout (KO) mice remained high, suggesting that the effect of estrogen on bone could be mediated through the SNS. Based on these studies, we previously analyzed the effect of propranolol on OVX rats and showed a preventive effect of propranolol on BMD and microarchitecture (+50.35% of BV/TV versus placebo) by increasing MAR (+35.9% versus placebo) and decreasing osteoclast surface on bone surface (−46.2% versus placebo).(24) In this previous study, the beneficial effect of propranolol was more significant on trabecular bone than on cortical bone.(24)
Consequently, the combination of exercise and a β-antagonist may be of interest, because (1) moderate physical exercise is known to be beneficial on bone through mechanical loading despite exercise-induced catecholamine secretion, and (2) β-antagonist administration in this case could theoretically increase the beneficial effect of exercise by suppressing this catecholamine effect.
However, serious issues have contradicted the beneficial effect of the sympathetic nervous system inhibition on bone. Dhillon et al.(25) did not observe any protection against OVX-induced bone loss in Adrβ1β2R KO mice, and Pierroz et al.(26) described a decrease in cortical bone mass for Adrβ1β2R KO mice. The aim of this work was to assess the separate and combined effects of treadmill exercise and propranolol treatment in the OVX rat model of induced bone loss.
MATERIALS AND METHODS
Sixty-eight female Wistar rats (Animal Production Center, Olivet, France) were acclimatized for 2 weeks and maintained under constant temperature (21 ± 2°C) and under 12-h/12-h light-dark cycles during the experiment. Rats were housed in groups of three in standard cages and provided with a commercial standard diet. At 34 weeks of age, animals were either ovariectomized (OVX, n = 44) or sham-operated (SHAM, n = 24). Bilateral ovariectomies were performed under pentobarbital anesthesia. Sham operations were performed by exteriorizing the ovaries. One group of 12 rats (sham-operated at 34 weeks), chosen at random, was killed at 36 weeks of age for baseline histomorphometric and microarchitectural evaluation. In the remaining rats (one SHAM group and four OVX groups), whole body BMD was determined by DXA. OVX rats were divided into four groups as follows: physiological saline-treated group (OVX); propranolol-treated (0.1 mg/kg/day; Sigma-Aldrich Chimie, St. Quentin Fallavier, France) group (OVX PRO); physiological saline-treated and exercise (OVX EXE); and propranolol treatment combined with exercise (OVX PRO EXE). The groups were treated by subcutaneous injections 5 days a week for 10 weeks. The SHAM group received saline injections at an identical dosing regimen. Dose and treatment protocols were based on those described by Minkowitz et al.(27) and Bonnet et al.(24) Food consumption was recorded weekly for the SHAM group, and this amount was fed to the OVX rats over the following week.
Bone labeling of rats by an intraperitoneal injection of tetracycline (30 mg/kg of body weight) was performed 14 and 4 days before death. At the end of the study, all groups were killed by an overdose of pentobarbital sodium. Soon after death, the weights of hindlimb muscles (soleus, gastrocnemius, and extensor digitorum longus), the uterus, and the heart were measured. The femurs, tibias, and lumbar vertebrae (L2, L3, and L4) were excised and cleared of fat and connective tissues. The right tibia was immediately fixed in 10% formaldehyde for 48 h at +4°C. Other bones were placed in plastic tubes and frozen at –20°C for the microarchitectural and biomechanical tests. The procedure for the care and killing of the animals was in accordance with the European Community standards on the care and use of the laboratory animals (Ministère de l'agriculture, France, Authorization INSERM45–001).
The rats were trained 5 days a week for 10 weeks. During the first week, the treadmill speed and the duration of each running session were gradually increased from 8 m/minute for 15 minutes to 13 m/minute for 60 minutes. During the last 9 weeks, the running session consisted of 13 m/minute for 60 minutes, which corresponds to moderate exercise for the age of these rats.(28) Control resting (SHAM, OVX, OVX PRO) rats were handled twice daily at 1-h intervals to mimic the stress induced by handling before and after running. Maximal aerobic speed (MAS), defined by Leger and Boucher(29) to be a good marker of physical capacity, was determined to confirm that our treadmill training corresponded to moderate exercise. MAS was measured three times after 3, 6, and 9 weeks of treadmill exercise. The measurement protocol has been described previously by Cavalie et al.(30)
Body weight, fat mass, and lean mass
Body weight was measured at weekly intervals throughout the study. At baseline and 3, 6, and 9 weeks, lean and fat masses were measured by DXA (Hologic QDR-1000W) using a specific rat body composition mode (line spacing, 1.5 mm; resolution, 0.7 mm). Because muscle mass represents 94–96% of lean mass, it is generally accepted to extrapolate muscle mass from lean mass. The CVs (CV = SD/mean) were determined for these parameters from seven repeated measures with repositioning on one animal cadaver. The CVs were 4.76% and 1.64% for fat and muscle masses, respectively.
BMC, bone area, and BMD measurements
In vivo BMC and BMD of the left tibia and femur were measured at baseline and 3, 6, and 9 weeks by DXA using a Hologic QDR-1000W apparatus adapted to small animals. An ultra-high-resolution mode (line spacing: 0.254 mm; resolution: 0.127 mm) was used with a 0.9-mm-diameter collimator.
Ex vivo, the left femur, left tibia, and the L4 vertebra were bathed in saline solution during DXA measurements (2.5 cm height for all experiments). BMC and BMD of the total femur and total tibia and two subregions were determined ex vivo, as previously described by Pastoureau et al.(31) The first subregion corresponds to the femoral distal metaphysis and to the tibial proximal metaphysis, which are rich in cancellous bone. The second region is the diaphysis, mainly composed of cortical bone. The CVs were determined by seven repeated measures on one femur, one tibia, and one vertebra over several days, with repositioning for each scan. The CVs for BMC and BMD measurements ranged from 0.33% to 4.64%, depending on the bone site.
Morphological and topological characteristics of the trabecular bone
The microarchitecture of the femoral, tibial, and L4 vertebra trabecular bone was studied using μCT (Skyscan 1072; Skyscan, Aartselaar, Belgium). The characteristics and methods have already been described elsewhere.(32) The X-ray source was set at 75 kV and 100μA, with a pixel size of 11 μm. Four hundred projections were acquired over an angular range of 180° (angular step of 0.45°). The image slices were reconstructed using the cone-beam reconstruction software version 2.6 based on the Feldkamp algorithm. The registered data sets were segmented into binary images. Because of the low noise and relatively good resolution of the data sets, simple global thresholding methods were used. The trabecular bone was extracted by drawing ellipsoid contours with “CT analyzer” software (Skyscan). Trabecular bone volume (BV/TV, %), trabecular number (Tb.N), and trabecular separation (Tb.Sp, μm) were calculated by the mean intercept length (MIL) method. Trabecular thickness (Tb.Th, μm) was calculated according to the method of Hildebrand and Ruegsegger.(33) The structure model index (SMI) was measured to determine the prevalence of plate-like or rod-like trabecular structures, where 0 represents plates and 3 represents rods.(33) Trabecular bone pattern factor (TBPf) was calculated: the higher the TBPf, the more trabecular bone is organized in the form of rod-like structures. The degree of anisotropy (DA) was calculated by superimposing parallel test lines in various directions on the 3D image. DA defines the magnitude of the preferred orientation of the trabeculae: the lower the DA, the more trabeculae are preferentially oriented.(34)
The microarchitecture of L4 was analyzed on the middle region of L4 defined as 35–65% of the total height, which corresponds to 200 slices. Two hundred fifty slices were selected from the distal growth plate to the shaft of the femur, and 250 slices were selected from the proximal growth plate to the shaft of the tibia.
Morphological characteristics of cortical bone
Cortical bone was described in the femoral and tibial mid-diaphysis using μCT. The characteristics and methods have already been described elsewhere.(35,36) The acquisition characteristics were the same as those used for trabecular bone. After reconstruction, cortical bone was extracted by drawing polygonal contours with “CT analyzer” software. Before inversion of the image, simple global thresholding methods were applied, and the algorithms developed for trabecular bone analysis were used to characterize the porosity network. Porosity (BV/TV equivalent) was labeled Ct.Po and PoN (pore number, TbN equivalent) was measured by the MIL method. PoDm (pore diameter, TbTh equivalent) and PoSp (pore spacing, TbSp equivalent) were derived from the Hildebrand method and PoS/PoV (pore surface on volume, BS/BV equivalent) was derived from the triangulation method.(33) For analysis of the femoral cortex, 100 slices were selected starting 12 mm from the distal growth plate and proximally along the shaft, corresponding to the distal diaphysis region.
For analysis of the tibial cortex, 100 slices were selected starting 12 mm from the proximal growth plate and distally along the shaft, corresponding to the proximal diaphysis region.
Because of the asymmetric shape of the femoral and tibial shafts, a 2D bone slice at the mid-diaphysis obtained by μCT was characterized by an ellipsoid shape. An ellipse yields two diameters: a large diameter corresponding to the mediolateral (ML) direction and a small diameter corresponding to the anteroposterior (AP) direction. These two diameters were assessed at the mid-diaphysis (=50% of the length of the femur or tibia) of the left femur and left tibia. From these measurements, the cortical width of the long bone was calculated as the mean cortical width in the ML and AP directions. The inner and outer cortical width of the L4 vertebra was measured on five slices located at 50% of the total height. The results are expressed as the mean of these five slices.
After 48 h of fixation, the right tibia was dehydrated in absolute acetone and embedded in methylmethacrylate at a low temperature according to the method developed by Chappard et al.(37) The central plane of the proximal part of the tibia was sliced frontally with a microtome (Reichert-Jung Polycut, Heidelberg, Germany). Five 8-μm-thick sections were stained with Goldern's trichrome and were used for measurements of the following parameters in the secondary spongiosa according to the ASBMR histomorphometry nomenclature,(38) using an automatic image analyzer (BIOCOM, Lyon, France): BV/TV, Tb.Th, Tb.N, Tb.Sp, osteoid surface (OS/BS, %), and osteoid thickness (O.Th). Five 8-μm-thick sections were stained with TRACP to measure active osteoclastic surfaces (Oc.S/BS) and osteoclast number (N.Oc/BS). Histodynamic parameters were determined on five unstained, 12-μm-thick sections under UV light: mineral apposition rate (MAR, μm/day), single labeled surface (sLS/BS, %), and double-labeled surface (dLS/BS, %). Mineralizing surface per bone surface (MS/BS, %) was calculated by adding dLS/BS and one-half sLS/BS. Bone formation rate (BFR/BS, μm3/μm2/day) was calculated as the product of MS/BS and MAR.
The aforementioned parameters of bone resorption and formation were measured with a semiautomatic system consisting of a digitizing table (Summasketch-Summagraphics, Paris, France) connected to a personal computer and a Reichert Polyvar microscope equipped with a drawing system (Camera Lucida; Reichert-Jung Polyvar).
Bone mechanical tests
Mechanical properties of the left femur were assessed by three-point bending tests. Four hours before mechanical testing, the bones were thawed at room temperature. Each bone was secured on the two lower supports of the anvil of an Universal Testing Machine (Instron 4501; Instron, Canton, MA, USA). The diameter of these supports was 4 mm, and the distance between the two supports was 20 mm. The cross-head speed for all tests was 1 mm/minute. Load-displacement curves were recorded using specialized software (Instron 4501 software). Biomechanical properties were calculated from these curves: ultimate force (the maximum force supported by the bone before fracture, Fult, N); energy to ultimate force (work energy required to fracture the bone, U, N.m); and stiffness (extrinsic rigidity of the femur, S, N.m). Because of the irregular shape of the femoral diaphysis, the femoral diameter used in the calculation was the mean of mediolateral and the antero posterior femoral mid-diaphysis diameters. Ultimate stress (σu, MPa) and Young's modulus (E, MPa, modulus of elasticity) were determined by the equations previously described by Turner and Burr.(39) To ensure good reproducibility between measurements, the femur was always mounted so that the cross-head could be applied just in the middle of the bone.(39)
The distal metaphysis of the right femur was tested in compression using the same material testing system used for the bending test. To extract specimens at the same relative position of each femur, the distal metaphysis was cut to a length of 2.5 mm using a standardized procedure: the location of each specimen was standardized from image analysis of the μCT to extract the same region of interest used to evaluate bone microarchitecture. The location of the point at which the primary spongiosa below the epiphyseal growth plate transitioned to fully cancellous bone was determined from the image. The ratio of the distance measured between this point and the distal end of the femur over the total length of the femur was calculated for each bone. These data were averaged for all bones. The overall average ratio was multiplied by the length of each femur to define the value of “s” for each bone. The first cut was made at that point, and the more proximal cut was made to produce specimens nominally 2.5 mm long. Both cuts were made perpendicular to the long axis of the bone using a low-speed diamond blade wafering saw under continuous irrigation (Buehler Isomet 4000).
The specimens were loaded between flat parallel plates by uniform compression. The load was applied in the craniocaudal direction using a steel disk (5 cm) at a nominal deformation rate of 0.5 mm/minute.(40,41) Load-displacement curves were recorded during testing. Extrinsic parameters were measured directly from the force-displacement curve: ultimate force (Fult, N), displacement at ultimate force (dult, mm), energy to ultimate force (U, N.m), and stiffness (S, N.m). Extrinsic properties reflected the combined effects of bone size and shape and tissue material properties. Intrinsic properties were calculated from the strenght-deformation data. Intrinsic properties referred to the tissue material behavior and were derived by adjusting the extrinsic properties to the size and shape of the specimen. Because the metaphysis used in this study contained both trabecular and cortical parts, the intrinsic properties represented the combined contributions of these two components. Therefore, the area used to calculate intrinsic parameters was the total cross-sectional area determined from μCT images. The following intrinsic properties were calculated assuming purely uniaxial loading: ultimate stress, ultimate strain, intrinsic energy to ultimate strain, and Young's modulus.
Osteocalcin (a marker of bone formation) and C-terminal collagen cross-links (CTX, a marker of bone resorption) were assayed in duplicate by ELISA (Nordic Bioscience Diagnostics, Herlev Hovedgade, Denmark). The within-assay and between-assay CVs were <10% in our laboratory.
The Gaussian distribution of the data were confirmed by a χ2 test. Results are expressed as mean ± SE. Body composition, BMD, geometric data, architectural parameters, biochemical analyses, and femoral mechanical properties were analyzed by one-way ANOVA at baseline. One-way ANOVA with repeated measurements was used to compare baseline versus end of treatment. Statistical analysis was also applied to all groups by two-way ANOVA. When necessary, posthoc differences were determined by the Newman-Keuls test.
Correlations were performed using Pearson's test.
In all groups of rats, body weight increased from baseline to the end of treatment. Despite receiving a similar amount of food as the SHAM group, OVX (+16.4%) and OVX EXE (+17.5%) rats had a higher body weight gain compared with the SHAM group (9.7%). Animals of the OVX PRO and OVX PRO EXE groups presented a similar body weight gain to that observed in the SHAM group (Fig. 1).
A higher total muscle mass gain was observed in the OVX EXE group (+13.2%) than in the OVX group (+8.6%, p < 0.05), and a lower total muscle mass gain was observed in the OVX PRO group (+4.9%, p < 0.01) than in the other groups. No significant difference in total muscle mass gain was observed between the OVX PRO EXE, OVX, OVX EXE, and SHAM groups.
However, a lower fat mass gain was observed in the OVX EXE group (+13.3%) compared with the OVX (+39.9%, p < 0.01) and OVX PRO (+40.3%, p < 0.01) groups. The OVX PRO EXE group (−22.9%, p < 0.01) also displayed a lower fat mass than all other groups (Fig. 1).
At necropsy, the uterine weight in all OVX animals was >75% lower than that of the SHAM group, indicating successful ovariectomy. No significant difference in skeletal muscle mass was observed between the OVX EXE and OVX groups, but a higher soleus mass was observed in the OVX EXE and OVX PRO EXE groups compared with the OVX PRO group (+25% and +31%, respectively).
Tibial, femoral, and vertebral BMD
Longitudinal BMD measurements at the tibia and femur revealed a significantly higher BMD gain in the OVX EXE and OVX PRO EXE groups compared with the OVX group (Fig. 2). No difference in BMD gain was observed between the OVX EXE, OVX PRO, and OVX PRO EXE groups for either the tibia or the femur.
At the end of the study, vertebral BMD measurements revealed significantly higher values in the SHAM (242.6 mg/cm2), OVX PRO (238.6 mg/cm2), and OVX PRO EXE (234.2 mg/cm2) groups compared with the OVX (216 mg/cm2) and OVX EXE (217.4 mg/cm2) groups, and similar L4 BMD values were observed in the OVX and OVX EXE groups.
Trabecular bone microarchitecture
At the end of treatment, OVX rats had a 14.6% loss of trabecular thickness and a 50.9% loss of trabecular number compared with SHAM animals. OVX animals showed a 54.6% overall reduction of trabecular bone volume fraction, whereas trabecular bone volume fraction was increased by +50.3% and +38.6% in the OVX PRO and OVX EXE groups compared with the OVX group, respectively. BV/TV were similar for the OVX PRO and SHAM groups. Trabecular thickness increased by +6.81% in the OVX PRO group and +16.8% in the OVX EXE group compared with the OVX group. The increase in TbTh for the OVX EXE group was significantly higher than in the OVX PRO group (p < 0.01).
No significant positive interaction was observed between propranolol and exercise: the combination of propranolol and exercise had a significantly lower effect on BV/TV than propranolol treatment or exercise alone (Table 1). Propranolol inhibited the effect of exercise on trabecular bone parameters. We observed lower BV/TV (−17%) and Tb.Th (−10.9%) and higher bone surface on bone volume (+4.3%) in the OVX PRO EXE group compared with the OVX EXE group (Table 1).
Table Table 1.. Effects of Propranolol (PRO) Treatment and Exercise Training (EXE) on Trabecular Bone Microarchitecture of the Proximal Tibial Metaphysis in OVX Rats
At the end of treatment, between-group differences in femoral microarchitecture parameters indicated the same trend as that observed in the tibia (Table 2). For example, the OVX PRO EXE group had a lower Tb.Th (−9.3%, p < 0.05) and a higher SMI (49.6%, p < 0.05) compared with OVX EXE group. However, exercise induced a lower effect on the femur than on the tibia. For example, a +10.1% increase of femoral TbTh was observed in the OVX EXE group compared with the OVX group, whereas a +16.8% increase was observed in the tibia.
The OVX group displayed lower BV/TV, Tb.N, and Tb.Th (−31.20%, –24.83%, and −8.26%, respectively; p < 0.001) and higher Tb.Sp (+22.92, p < 0.001) and SMI (+29.51%, p < 0.001) than the SHAM group, indicating a loss of architectural integrity. The OVX PRO group had higher BV/TV (+32.26%, p < 0.01) compared with the OVX group. The SMI in the OVX PRO group was significantly lower (−30.18%, p < 0.01) than in the OVX group. Microarchitectural parameters of the OVX EXE group were comparable with those of the OVX group except for DA, which was significantly lower in the OVX EXE group (Table 2). The OVX PRO EXE group was not significantly different from the OVX group for any parameter of bone microarchitecture.
No significant difference for femoral mid-diaphysis cortical width was observed between the OVX EXE (432.5 μm) and OVX PRO EXE (443.7 μm) groups. Conversely, the cortical widths in the OVX EXE and OVX PRO EXE groups were significantly higher than in the OVX PRO group (374.3 μm, p < 0.05).
No significant difference for tibial mid-diaphysis cortical width was observed between the SHAM (575.2 μm), OVX EXE (567.4 μm), and OVX PRO EXE (576.1 μm) groups, but these values were significantly higher than those observed in the OVX (536.5 μm, p < 0.05) and OVX PRO (507.8 μm, p < 0.05) groups.
No significant difference in cortical porosity was observed between the OVX EXE, OVX PRO, and OVX PRO EXE groups in either the tibia or the femur (Fig. 3). However, the OVX PRO EXE group had a lower porosity than the OVX group (−46.4%, p < 0.05), whereas the OVX PRO and OVX EXE groups were not significantly different from the OVX group.
Inner cortical width in the OVX group (215.8 μm) was significantly lower than in the SHAM (240.0 μm), OVX EXE (251.7 μm), OVX PRO (263.3 μm), and OVX PRO EXE (256.9 μm) groups. However, no significant difference was observed between the OVX, OVX EXE, and OVX PRO EXE groups for outer cortical width.
The histomorphometric results showed a significant effect of exercise, propranolol, and exercise combined with propranolol on bone formation parameters and osteoclast surface. The osteoclast surface in the OVX EXE, OVX PRO, and OVX PRO EXE groups was decreased by –39%, −46%, and –33%, respectively, compared with the OVX group (Table 3).
Table Table 2.. Effects of Propranolol (PRO) Treatment and Exercise Training (EXE) on Trabecular Bone Microarchitecture of the Distal Femur and Vertebral Body of OVX Rats
Despite a similar MAR between the OVX EXE, OVX PRO, and OVX PRO EXE groups, a higher MS/BS was observed in the OVX PRO EXE group compared with the OVX PRO group (+54.3%, p < 0.01).
The bending test showed a significantly higher ultimate force in the OVX PRO EXE group than in the OVX group, whereas the OVX PRO and OVX EXE groups were not significantly different from the OVX group. A higher energy to ultimate force was observed in the OVX PRO EXE group compared with the OVX PRO group, and a higher Young modulus was observed in the OVX PRO, OVX EXE, and OVX PRO EXE groups compared with the OVX group (+32.6%, 34.9%, and +40.2%, respectively; p < 0.05; Table 4). Significant correlation was shown between ultimate force and total BMD (r = 0.48, p < 0.001), cortical width (r = 0.51, p < 0.001), and cortical porosity (r = −0.4, p < 0.01).
Results obtained from the compression test showed a significantly higher stress, intrinsic energy to ultimate force, and Young modulus in the OVX PRO and OVX PRO EXE groups compared with the OVX group. The exercise group did not differ from OVX except for ultimate force and Young modulus, which were significantly higher (+23% and +26%, respectively; p < 0.05; Table 5). Significant correlation was shown between ultimate force and TBPf (r = −0.53, p < 0.001) and between intrinsic energy and TbTh or TBPf (r = 0.33 p < 0.01; r = −0.65, p < 0.001).
At the end of the experiment, the CTX level was 26.6% lower in the OVX EXE group (18.7 ng/ml) than in the OVX group (25.47 ng/ml, p < 0.12), and the osteocalcin level was 17.6% higher in the OVX EXE group (251.22 ng/ml) than in the OVX group (206.91 ng/ml, p < 0.22), but these differences were not statistically significant. Osteocalcin was significantly higher in the OVX PRO (301.21 ng/ml) and OVX PRO EXE (274.30 ng/ml) groups than in the OVX group (+31% and +25%, respectively; p < 0.05). Furthermore, the OVX PRO group displayed a higher osteocalcin level than the OVX EXE group (251.22 ng/ml, p < 0.05). The CTX level was not significantly different between the OVX, OVX PRO, OVX EXE, and OVX PRO EXE groups.
This study confirmed that the use of propranolol or the practice of physical exercise has a preventive effect on bone loss after OVX. We showed that propranolol had a synergistic effect to that of exercise on cortical porosity without any complementary effect on cortical width but an antagonist effect on trabecular bone structure. In the same way, we showed that exercise reduces the trabecular microarchitecture effect of propranolol, probably because of the fact that exercise rats had a SNS activity higher than sedentary and a different energetic metabolism. Propranolol combined with exercise globally increased bone strength. This suggests a beneficial effect of β blockade plus exercise on bone material properties rather than on trabecular microstructure.
Table Table 3.. Histomorphometry Parameters of the Proximal Tibia After 10 Weeks of Exercise Training and Propranolol
Table Table 4.. Influence of Exercise and Propranolol on Biomechanical Properties of the Femur in Three-Point Bending Test
Table Table 5.. Effect of Exercise and Propranolol on Biomechanical Properties of the Distal Metaphysis Femur in OVX Rats
As anticipated, bone mass as measured by the BV/TV ratio was decreased by 30–50% in long bone metaphyses and vertebral bodies in placebo-treated OVX rats compared with SHAM rats. In addition, osteoclast number was increased, whereas trabecular number and thickness were decreased in this OVX placebo group. These results are consistent with the notion that OVX decreases bone mass by increasing bone resorption parameters.(42,43)
In this study, treadmill exercise positively influenced long bone BMD (tibia and femur) and their trabecular microarchitecture, cortical porosity, and biomechanical intrinsic properties by decreasing bone resorption (CTX) and increasing bone formation (osteocalcin). According to the linear regression established by Cavalie et al.(30) between maximal aerobic speed (MAS) and oxygen uptake (Vo2) for adult female Wistar rats, our treadmill protocol corresponded to a Vo2max of 44.7 ml/minute/kg with a maximal oxygen uptake (Vo2max) of 67.9 ml/minute/kg at the beginning of exercise, 76.8 ml/minute/kg at one-half time, and 78.6 ml/minute/kg at the end of exercise. These results confirm that our treadmill protocol corresponded to moderate exercise ranging between 57% and 66% of Vo2max. This estimated exercise intensity was in accordance with the results of Bourrin et al.,(20) showing a beneficial effect of exercise on trabecular bone morphometry and cell activity. According to Turner and Robling,(12) mechanical stresses caused by weight loading and muscle contractions strongly affect both bone formation and resorption. It is noteworthy that our results were clearly observed on the tibia and less on the femur. During running exercise in rats, the tibia is more exposed to mechanical loading (with more impacts) than the femur. One explanation is that tibia is located more distally than the femur.(13) On the other hand, no significant effect of the exercise program was observed on the vertebrae (a non–weight-bearing bone in rats), suggesting no effect of exercise on the endocrine status in adult rats, in contrast with the effect of exercise in young rats described by Yeh et al.(44)
In agreement with Elefteriou et al.(23) and Takeda et al.,(18) we observed a beneficial effect of propranolol on BMD and trabecular microarchitecture for both appendicular and axial bones. In addition, propranolol had a weaker effect on the architectural deterioration caused by OVX in the vertebrae compared with the effect on the tibia. Kinney et al.(45) previously observed a similar result with estrogen therapy on these bone sites. They showed that a higher dose of this treatment was necessary to improve vertebral architecture in the same range as in the tibia. We have previously discussed this dose-effect of propranolol according to bone site.(24) Furthermore, we found in the literature several experiments that showed that low doses of propranolol is efficient. Silke et al.(46) showed in a human study that propranolol at a low dose (2–16 mg) induced significant dose-related reductions in heart rate and cardiac output. In dogs, Sharma(47) showed that 0.3 mg/kg completely prevented ventricular arrhythmias produced by adrenaline and noradrenaline. The dose of 0.1 mg/kg has been previously used in vivo by Minkowitz et al.,(27) who showed a beneficial effect on bone mechanical properties with a 33% higher ultimate force in torsion compared with placebo. Furthermore, they showed a higher MAR at the femoral metaphysis (saline group: 0.43 μm/day versus propranolol group: 0.88 μm/day) and an increase of midshaft periosteal and endosteal bone formation. Similarly Yirmiya et al.(48) showed that a low dose of 0.4 mg/day induced an increased of 16% of the vertebral BV/TV in chronic mild stress mice.
Both cortical and mixed trabecular-cortical sites were analyzed concerning (1) extrinsic mechanical properties that reflected the combined effects of bone size and shape and tissue material properties and (2) intrinsic properties corresponding exclusively to the tissue material behavior. In the cortical site (mid-diaphysis), propranolol induced higher values of intrinsic parameters, whereas extrinsic parameters were not changed. These data suggest a preferential effect of propranolol on bone material rather than on its geometry. This hypothesis was confirmed by the absence of significant effect of propranolol on cortical width. In compression, biomechanical properties measured at the metaphysis indicated a preferential effect of propranolol on intrinsic parameters. These results are in accordance with the correlation reported in this study (TBPf and ultimate force, r = −0.53, p < 0.001) and those observed by Muller et al.(49) Furthermore, Minkowitz et al.(27) showed, in a torsion test, that 0.1 mg/kg PRO increased by 33% the ultimate force of femoral rats compared with placebo.
To provide a better understanding of the influence of propranolol and physical training on bone tissue, we studied the efficacy of the combination of these two interventions. A lower tibial BV/TV and vertebral TbTh were observed in the group treated with both PRO and EXE compared with groups treated with PRO or EXE alone. BMD results were similar between these three groups. These results suggest that propranolol inhibited the effect of exercise and that exercise inhibited the effect of propranolol on trabecular bone.
Yao et al.(50) showed that the benefit of treadmill exercise on trabecular bone morphometric parameters in rats was antagonized by vascular endothelial growth factor (VEGF) blocking antibodies. They suggested that the effect of exercise on long bones was mainly caused by an increase in both angiogenesis and vascular diameter. Interestingly, Weil et al.(51) and Fredriksson et al.(52) showed that propranolol treatment of cardiac myocytes and adipocytes reduced the level of mRNA VEGF. This may suggest that, in our study, propranolol treatment antagonized the beneficial effect of exercise, particularly on trabecular bone proportion and trabecular thickness, by decreasing VEGF levels and that exercise slowed down the effect of propranolol (particularly on trabecular number) by increasing SNS activity, but we have no direct evidence of such interactions. One hypothesis would be that with a higher dose of propranolol exercise would have not slowed down PRO bone effect, suggesting that the efficient dose of PRO in sedentary and exercise rats could be different.
Long bone cortical widths were similar between the OVX PRO EXE and OVX EXE groups, and these groups had higher cortical widths than the OVX or OVX PRO groups. Furthermore, the lowest cortical porosity was observed in the OVX PRO EXE group.
A first explanation for the differential effect on cortical and trabecular bone is a greater influence of mechanical loading (caused by exercise) on cortical bone than on trabecular bone.(53) Second, the effect of exercise on bone by increasing VEGF level and vascularization would be higher in trabecular bone than cortical bone because of the compactness of the cortical bone structure. Third, our results suggest a differential influence of SNS on these two types of bone compartments, with a lower impact of SNS on cortical bone compared with trabecular bone. To confirm this hypothesis, it would be useful to show lower innervation in cortical bone compared with trabecular bone or a lower number of β-receptors in cortical bone.
Biomechanical properties derived from the three-point bending test indicated that only the OVX PRO EXE group had a higher ultimate force than the OVX group. Correlation tests suggested that the combined influence of exercise and propranolol on cortical porosity could partially explain this finding and that other intrinsic material properties were probably influenced by the combination of PRO and EXE. The results seem to indicate that the OVX PRO EXE group reflected the effects of exercise on bone geometry and the effects of propranolol on bone material properties.
Compression tests showed that the biomechanical properties of the metaphysis in the OVX PRO EXE group did not differ from those of the OVX PRO group. The ultimate force was higher in the OVX EXE group than the OVX group, suggesting that the antagonistic effect of propranolol and exercise on trabecular bone microarchitecture dampened a possible synergistic effect on the biomechanical properties of the metaphysis.
We are aware of some limitations of this study. First, we have partial histomorphometric data on the metaphysis. The count of osteoblast number would be relevant, because Fu et al.(54) previously showed that propranolol affects osteoblastic proliferation through the clock gene, but we showed that PRO increased osteocalcin levels. Second, we did not provide histomorphometry information on cortical bone.
In conclusion, the bone mass increase was mainly associated with an increased trabecular number in the PRO treatment group but was induced by an increase of trabecular thickness in the exercise group. Biomechanical analysis indicated a significant effect of propranolol on intrinsic properties, whereas exercise preferentially influenced extrinsic properties. Despite lower microarchitecture properties, the effect of the propranolol and exercise combination on biomechanical properties was as intense as that of propranolol or exercise alone. Our results concerning the bone response to treadmill exercise suggest a lower contribution of the SNS in cortical bone than in trabecular bone. However, further studies are necessary to determine the direct influence of the SNS on β-adrenergic bone receptors or its indirect influence, for example through an effect on angiogenesis. This study suggests that more attention should be paid in patients treated by propranolol and practicing exercise particularly on trabecular bone sites.
This work was supported by the World Anti-Doping Agency.