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BEHAVIORAL PHARMACOLOGY

Dose Effects of Propranolol on Cancellous and Cortical Bone in Ovariectomized Adult Rats

Institut National de la Santé et de la Recherche Médicale U658, Caracterisation du Tissu Osseux par Imagerie, Techniques, et Applications, Hopital Regional d'Orléans, Université d'Orléans, Orleans, France (N.B., E.D., C.L.B., D.C.); Institut National de la Santé et de la Recherche Médicale E366, St. Etienne, France; Laboratoire de Biologie du Tissu Osseux, Université Jean Monnet, St. Etienne, France; and IFR62 Laennec, Lyon, France (N.L., L.V.)

Received March 29, 2006; accepted May 31, 2006.

	Abstract

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Animal studies suggest that bone remodeling is under -adrenergic control via the sympathetic nervous system. The purpose of this study was to examine the preventive effect of different doses of nonspecific -blockers (propranolol) on trabecular and cortical bone envelopes in ovariectomized rats. Six-month-old female Wistar rats were ovariectomized (OVX, n = 60) or sham-operated (n = 15). Then, OVX rats were subcutaneously injected with 0.1 (n = 15), 5 (n = 15), or 20 (n = 15) mg/kg propranolol or vehicle (n = 15) for 10 weeks. Tibial and femoral bone mineral density (BMD) were analyzed longitudinally by dual-energy X-ray absorptiometry. At death, the left tibial metaphysis and L₄ vertebrae were removed, and microcomputed tomography (Skyscan 1072; Skyscan, Aartselaar, Belgium) was performed for trabecular bone structure investigation. Histomorphometry analysis was performed on the right proximal tibia to assess bone cell activities. After 10 weeks, OVX rats had decreased BMD and trabecular parameters and increased bone turnover, as well as cortical porosity compared with the sham group (p < 0.001). Bone architecture alteration was preserved by 0.1 mg/kg propranolol due to higher trabecular number and thickness (+50.35 and +6.81%, respectively, than OVX; p < 0.001) and lower cortical pore number (–52.38% than OVX; p < 0.001). Animals treated by 0.1 mg/kg propranolol had a lower osteoclast surface and a higher osteoblast activity compared with OVX. Animals treated by 20 mg of propranolol did not significantly differ from OVX rats. Animals treated by 5 mg of propranolol have been partially preserved from the ovariectomy. These results showed a dose effect of -blockers. The lower the dose of propranolol breeding, the better the preventive effect against ovariectomy.

It is estimated that up to 45% of adult and ageing people suffer from cardiovascular disease and osteoporosis. Therefore, the interest of a dual-benefit effect of only one treatment on both heart and skeletal systems seems important.

However, serious issues have been raised regarding the prescription of propranolol for bone treatment. As the absence of leptin in Ob/Ob mice, the effects of altered sympathetic nervous system signaling on bone vary throughout the skeleton according to local factors (Hamrick et al., 2004; Warden et al., 2005). A decrease of muscle tissue mass has been observed in leptin knockout mice (Warmington et al., 2000), where noradrenalin showed low values, suggesting a decrease in muscle mass when the sympathetic nervous activity impairs or when the effect of noradrenalin is inhibited. Because muscle mass correlates positively with bone mass (Banu et al., 2003), this observation suggests a negative effect of an alteration of the sympathetic nervous activity on bone mass.

Furthermore, different studies moderate the beneficial effect of the sympathetic nervous system inhibition on bone. Dhillon et al. (2004) did not observe any protection from ovariectomy-induced bone loss in 12-adrenergic receptor KO mice. Preliminary data from Pierroz et al. (2005) described a decrease of cortical bone mass in 12-adrenergic receptor KO mice. Whether data from mouse models are relevant to humans remains to be addressed because phenotypes of mouse and human are quite different.

Whether -blocker may improve bone quantity and quality in ovariectomized models has yet be investigated. Drug doses and forms of administration described in the literature are different. To our knowledge, there is no specific information on low-, middle-, and high-dose effects of propranolol on axial and appendicular bone characteristics. The aim of this work was to investigate the dose effect of propranolol on bone of ovariectomized rats and to further elucidate its role on trabecular and cortical bone compartments.

	Materials and Methods

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Animals and Treatment. Ninety female Wistar rats (Animal Production Center, Olivet, France) were acclimatized for 2 weeks and maintained under constant temperature (21 ± 2°C) and under 12-/12-h light/dark cycles during the experience. The rats were housed by three in standard cages and provided with a commercial standard diet. At 34 weeks of age, animals were either ovariectomized (OVX, n = 60) or sham-operated (SHAM, n = 30). Bilateral ovariectomies were performed under anesthesia with pentobarbital. Sham operations were performed by exteriorizing the ovaries. One group of 15 rats (sham-operated at 34 weeks), chosen at random, was sacrificed at a 36 weeks age for baseline histomorphometry and microarchitecture evaluation. In the remaining rats, the whole-body BMD was determined by dual-energy X-ray absorptiometry (DXA). These animals were then divided into five groups of 15 rats matched for the whole-body BMD. At 36 weeks of age, treatment on three OVX groups (OVX PRO) was initiated with propranolol (Sigma-Aldrich Chimie, St. Quentin Fallavier, France) at doses of 0.1, 5, and 20 mg/kg, the last group of OVX being treated with sterile saline, injected subcutaneously 5 days per week, for 10 weeks. The SHAM group received saline injections at an identical dosage regimen. Dose and treatment protocol were based upon those described by Kondo and Togari (2003) and Minkowitz et al. (1991): therapeutic regime (range, 0.1–5 mg/kg) and doping doses (20 mg/kg). Propranolol (20 mg/kg) has been used previously for doping purpose in rats to study the effect on muscle, and it corresponds to a 10-fold higher dose than those typically used to treat hypertension in humans (Kondo and Togari, 2003). Food consumption was recorded weekly for the SHAM group, and this amount was then fed to the OVX rats over the following week.

Bone labeling of rats by an i.p. injection of tetracycline (30 mg/kg body mass) was performed 14 and 4 days before death. At the end of the study, all groups were sacrificed by an overdose of pentobarbital. Soon after death, the weights of hindlimb muscle (soleus, gastrocnemius, and extensor digitorum longus), uterus, and the heart were recorded. In all rats, femurs, tibiae and lumbar vertebrae (L₂, L₃, and L₄) were excised and cleared of fat and connective tissues. The right tibia and L₂–L₃ vertebrae were immediately fixed in 10% formaldehyde for 48 h at +4°C. The other bones were placed in plastic tubes and frozen at –20°C for the microarchitectural and biomechanical tests. The procedure for 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 Institut National de la Santé et de la Recherche Médicale 45-001).

Body Mass, Fat Mass, and Lean Mass. Body mass was recorded at weekly intervals throughout the study. At baseline and 3, 6, and 9 weeks, lean and fat masses were measured by DXA using a specific rat body composition mode (line spacing, 1.5 mm; resolution, 0.7 mm). Because muscle mass represents 94 to 96% of lean mass, it is generally accepted to extrapolate from lean to muscle mass. The coefficients of variation (CV; S.D./mean) were determined for these parameters from seven repeated measures with repositioning on one cadaverous animal. The CV was 4.76 and 1.64%, respectively, for fat and muscle masses.

Bone Mineral Content, Area, and Bone Mineral Density Measurements. In vivo bone mineral content (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 (Hologic, Waltham, MA) adapted for 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 L₄ vertebrae was bathed in saline water 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 described previously by Pastoureau et al. (1995). The first subregion corresponds to the femoral distal metaphysis and to the tibia proximal metaphysis, which is rich in cancellous bone. The second region is the diaphysis, mainly represented by cortical bone. The coefficients of variation were determined by seven repeated measures on one femur, one tibia, and one vertebrae over several days, with repositioning for each scan. The CV 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. Microarchitecture of the femoral, tibial, and L₄ vertebrae trabecular bone was investigated using a microcomputed tomograph (µCT; Skyscan 1072; Skyscan, Aartselaar, Belgium). The characteristics and methods have already been described elsewhere (McLaughlin et al., 2002). The X-ray source was set at 75 kV and 100 µA, with a pixel size at 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 (Skyscan). The registered data sets were segmented into binary images. Because of a low noise and the relative good resolution of the data sets, we used simple global thresholding methods. The trabecular bone was extracted by drawing ellipsoid contours with the CT analyzer software (Skyscan). Trabecular bone volume (BV/TV; percentage), trabecular number (Tb.N), and trabecular separation (Tb.Sp; micrometers) were calculated by the mean intercept length method. Trabecular thickness (Tb.Th; micrometers) was calculated according to the method of Hildebrand and Ruegsegger (1997). The structure model index (SMI) was measured for the prevalence of plate- or rod-like trabecular structures, where 0 represents "plates," and 3 represents "rods" (Hildebrand and Ruegsegger, 1997). The degree of anisotropy (DA) was calculated by superimposing parallel test lines in various directions on the three-dimensional image. DA defines the magnitude of the preferred orientation of the trabeculae. The higher the DA, the more trabeculae are preferentially oriented (Ulrich et al., 1999).

The L₄ microarchitecture analysis was performed on the middle region of L₄ defined as 35 to 65% of the total height, which corresponds to 200 slices. On the femur, 250 slices were selected from the distal growth plate to the shaft proximally. On the tibia, 250 slices were selected from the proximal growth plate to the shaft distally.

Cortical Scanning Electron Microscopy. A proximal-diaphysis section of one tibia in each group was rendered anorganic by a 5% sodium hypochlorite treatment. The sections were then rinsed in water, dehydrated in acetone, and dried. Bones were examined in a scanning electron microscope (Hitachi S-4500; Hitachi, Tokyo, Japan) with a 1-kV energy. We observed two levels of pores. Small pores characterized by a small diameter (<10 µm) present in all groups, and large pores were characterized by a diameter higher than 40 µm. By the spot size of the X-ray source, microcomputed tomography allows only large pore analysis higher than 11 µm.

Morphological Characteristics of the Cortical Bone. Cortical bone has been described in the femoral and tibial mid-diaphysis using a microcomputed tomograph. The characteristics and methods have already been described elsewhere (Lotinun et al., 2004). We used the same acquisition characteristics as for trabecular bone. After reconstruction, the cortical bone was extracted by drawing polygon contours with the CT analyzer software. Before inversion of the image, we applied simple global thresholding methods, and the algorithms developed for trabecular bone analysis were used to characterize the network of the porosity. The porosity (BV/TV equivalent) was labeled Ct.Po. Pore number (TbN equivalent) was measured by the mean intercept length method. Pore diameter (TbTh equivalent) and pore spacing (TbSp equivalent) were derived from the Hildebrand method and pore surface on volume (BS/BV equivalent) from the triangulation method (Hildebrand and Ruegsegger, 1997). For the femur, 100 slices were selected starting 12 mm far from the distal growth plate on the shaft proximally for cortical femur analysis, corresponding to the distal diaphysis region. One hundred slices were selected starting 12 mm far from the proximal growth plate on the shaft distally for cortical tibia analysis, corresponding to the proximal diaphysis region.

Bone Histomorphometry. After scanning electron microscopy and after 48 h of fixation, the right tibia was dehydrated in absolute acetone and embedded in methylmethacrylate at low temperature according to the method described by Chappard et al. (1987). 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. They were used for measurement in secondary spongiosa of several parameters according to the American Society for Bone and Mineral Research histomorphometry nomenclature (Parfitt et al., 1987) using an automatic image analyzer (BIOCOM, Lyon, France): BV/TV, Tb.Th, Tb.N, Tb.Sp, osteoid surface (percentage), and osteoid thickness. Five 8-µm-thick sections were stained with tartrate-resistant acid phosphatase activity to measure active osteoclastic surfaces (Oc.S/BS) and osteoclast number. Histodynamic parameters were determined on five unstained, 12-µm-thick sections under UV light: mineral apposition rate (MAR; micrometers per day), single-labeled surface (percentage), and double-labeled surface (percentage). Mineralizing surface per bone surface (MS/BS; percentage) was calculated by adding double-labeled surface and one-half single-labeled surface. Bone formation rate (micrometers cuber per micrometer squared per day) was calculated as the product of MS/BS and MAR. Five 8-µm-thick sections were stained with 4,6-diamidino-2-phenylindole dihydrochl to measure adipocyte number (number of cells per millimeter squared) and relative volume of fat in the marrow cavity (adipocyte volume/marrow volume).

The aforementioned parameters of bone resorption and formation were measured with a semiautomatic system made of a digitizing table (Summasketch-Summagraphics, Paris, France) connected to a personal computer and to a Reichert Polyvar microscope equipped with a drawing system (Camera Lucida; Reichert-Jung Polyvar).

Bone Geometric Characteristics. Due to the asymmetric shape of the femoral and tibial shaft, two-dimensional bone slice at middiaphysis obtained by microcomputed tomography can be characterized by an ellipsoid shape. An ellipse yields two diameters, a large one corresponding to the mediolateral (ML) direction and a small one corresponding to the anteroposterior (AP) direction. These two diameters were assessed at the mid-diaphysis (50% of the femur or tibia length) of the left femur and left tibia. Cortical width of the long bone is an average of the cortical width measured in ML and AP direction.

Inner and outer cortical width was measured on five slices located at 50% of the total height. The results represent an average of those five slices. Geometric measurements are illustrated in Fig. 1.

View larger version (23K): [in this window] [in a new window]   Fig. 1. Geometric measurement of long bone and vertebrae. Inner and outer cortical widths were measured by microcomputed tomography on five slices located at 50% of the total height. On long bone, two-dimensional bone slice at mid-diaphysis obtained by microcomputed tomography can be characterized by an ellipsoid shape. An ellipse yields two diameters, a large one corresponding to the ML direction and a small one corresponding to the AP direction. These two diameters were assessed at the mid-diaphysis (50% of the femur or tibia length) of the left femur and left tibia.

	Results

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General Observations
For all groups, the body mass of rats increased from baseline to the end of the treatment. Despite receiving a similar amount of food as the SHAM group, OVX and 20 mg/kg OVX PRO groups were 5% heavier at the end of the treatment. Animals of 0.1- and 5-mg OVX PRO groups increased their body mass similar that of SHAM group (Table 1). At necropsy, the uterine mass in all OVX animals was reduced by more than 75% compared with the SHAM, indicating a successful ovariectomy (Table 1). We did not observe significant difference between groups for the gastrocnemius and extensor digitorum longus, but we noticed a lower soleus mass in the 20-mg OVX PRO compared with OVX groups (Table 1).

Tibial, Femoral, and Vertebral BMD
Longitudinal BMD measurement at the tibia and femur revealed a significantly higher BMD gain in the 0.1-mg OVX PRO group compared with the OVX group. At the tibia, animals treated with 5 mg of propanolol revealed a higher BMD increase compared with the OVX group (not noticed in the femur). Femoral BMD decreased more significantly in the 20-mg OVX PRO group than in OVX animals (not noticed in the tibia) (Fig. 2). At the end of the study, BMD measurements at tibial, femoral, and vertebral sites revealed a significantly lower BMD in OVX and 20-mg OVX PRO groups compared with SHAM and 5- and 0.1-mg OVX PRO groups (Table 2). For each bone site, no significant difference was observed between SHAM and 5- and 0.1-mg OVX PRO groups.

View larger version (19K): [in this window] [in a new window]   Fig. 2. Time course of total BMD change in tibia and femurs. , OVX; , SHAM; , 20 mg/kg OVX PRO; , 5 mg/kg OVX PRO; X, 0.1 mg/kg OVX PRO. The notes a to e express significantly statistical comparisons between groups: a, comparison with SHAM group (p < 0.05); b, comparison with OVX group (p < 0.05); c, comparison with 0.1 mg/kg OVX (p < 0.05); d, comparison with 5 mg/kg OVX (p < 0.05); and e, comparison with 20 mg/kg OVX (p < 0.05). Means ± S.E.M.

Tibial and femoral metaphysis of 0.1-mg OVX PRO group had similar BMD than the SHAM group, both were significantly higher than OVX and 20-mg OVX PRO groups. Animals of the 5-mg OVX PRO group presented a significantly lower tibial metaphysis BMD compared with the SHAM group, whereas there was nonsignificant difference on the femur metaphysis BMD. Tibial and femoral metaphysis BMD in the 20-mg OVX PRO group did not differ from the OVX group. With regard to the diaphysis BMD, there was nonsignificant difference between groups in the tibia. We observed lower femoral diaphysis BMD in the 20-mg OVX PRO group compared with 5- and 0.1-mg OVX PRO groups.

Trabecular Bone Microarchitecture
Proximal Tibia. At the end of the treatment, compared with the SHAM animals, three-dimensional trabecular structure of the OVX rats revealed a 14.65% loss of trabecular thickness and a 50.90% loss of trabecular number. OVX animals yielded an overall reduction in trabecular bone volume fraction of 54.65% (Fig. 3). Trabecular bone proportion in 0.1-mg OVX PRO group had increased by +50.35% compared with the OVX group, and BV/TV finally was similar to that of the SHAM group. We observed comparable differences concerning trabecular number (Fig. 3). Trabecular thickness increased by +6.81% in the 0.1-mg OVX PRO group compared with the OVX group. The SMI increase from baseline was significantly higher in the OVX group (+77.45%, p < 0.001) compared with the SHAM group (+10.72%). SMI in the 0.1-mg OVX PRO group was 54% lower than that of the OVX group and was similar compared with the SHAM group. The DA increased significantly in the OVX group (+48,98%, p < 0.01) compared with 20, 5, and 0.1-mg OVX PRO groups where the values were similar compared with the SHAM group. For all other tibial microarchitecture parameters, the 20-mg OVX PRO group did not differ significantly from the OVX group (Fig. 3). Microarchitecture parameters of 5-mg OVX PRO group were comparable with 0.1-mg OVX PRO group except for the Tb.Th, which was not significantly different compared with the OVX group (Fig. 3).

View larger version (38K): [in this window] [in a new window]   Fig. 3. Trabecular bone structure parameters of the proximal tibia assessed by microcomputed tomography. The notes a to f express significantly statistical comparisons between groups: a, comparison with SHAM group (p < 0.05); b, comparison with OVX group (p < 0.05); c, comparison with 0.1 mg/kg OVX (p < 0.05); d, comparison with 5 mg/kg OVX (p < 0.05); e, comparison with 20 mg/kg OVX (p < 0.05); and f, comparison with baseline (p < 0.05). Tb.Sp, micrometers; Tb.Th, micrometers; SMI, 0 plate to 3 rod. Histograms, means ± S.E.M.

L₄ Vertebrae. The OVX group displayed lower BV/TV, Tb.N, Tb.Th (–31.20, –24.83, and –8.26%; p < 0.001, respectively) but also 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. No significant difference was found between the SHAM and 20-mg OVX PRO groups, with the exception of BV/TV, which was significantly lower in the 20-mg OVX PRO group (–17.53%, p < 0.01). However, the 20-mg OVX PRO group had significantly higher BV/TV than the OVX group (+16.67%, p < 0.01). Animals of 0.1-mg OVX PRO group had higher BV/TV (+32.26%, p < 0.01) compared with the OVX group. Structural model index in the 0.1-mg OVX PRO group was significantly lower (–30.18%, p < 0.01) than in the OVX group. Microarchitecture parameters of the 5-mg OVX PRO group were comparable with 0.1-mg OVX PRO for BV/TV and Tb.N but not for SMI and Tb.Th, which were not significantly different compared with the OVX group (Table 3).

Cortical Parameters
Tibia. µCT analyses revealed that the OVX group significantly decreased the cortical width compared with SHAM (–6.21%, p < 0.01) (Table 4). Animals of 0.1-mg OVX PRO and SHAM groups increased their cortical width, respectively, by +3.33 and +4.01% compared with baseline values (nonsignificant). OVX and 20-mg OVX PRO groups had significantly decreased their cortical width by –3 and –9% compared with baseline values (p < 0.01).

At the end of the treatment, compared with the SHAM group, the cortical structure revealed a higher pore diameter (+9.67%, nonsignificant) and a higher pore number (+47.62%, p < 0.01) in the OVX group, which yielded an overall higher cortical porosity of +51.66% (p < 0.05). Cortical porosity tended to be lower in 0.1-mg OVX PRO (–63.63% of Ct.Po, compared with OVX) than in 5-mg OVX PRO (–11.00% of Ct.Po, compared with OVX). Pore number was significantly lower in 0.1-mg OVX PRO group compared with OVX and 5- and 20-mg OVX PRO groups. Animals of 20-mg OVX PRO group had similar cortical parameters than the OVX group. These results were confirmed by those obtained from the scanning electron micrographs (Fig. 4).

View larger version (98K): [in this window] [in a new window]   Fig. 4. Scanning electron micrographs taken from tibial proximal-diaphysis. Pore geometry image represents the two levels of pore diameter: large pore, >40 µm; small pore, <10 µm.

L₄ Vertebrae. Inner cortical width in the OVX group (215.8 µm) was significantly lower than in the SHAM group (240.0 µm). Animals of 20-mg OVX PRO group (194.3 µm) did not significantly differ from the OVX group. Inner cortical thicknesses of 5- (248.1 µm) and 0.1-mg (263.3 µm) OVX PRO groups were significantly higher than OVX animals. The same statistical difference was observed between groups for the outer cortical width and for the cortical area.

Histomorphometry Measurement
Trabecular Bone Volume and Structure in the Proximal Tibial Metaphyses. Static histomorphometry results were close to the morphometric parameters obtained by µCT (data not shown). Correlations of BV/TV, Tb.N, Tb.Sp, and Tb.Th between histomorphometry and µCT were all significant (r ranged between 0.67 and 0.85, p < 0.001).

Bone Formation and Resorption. At the end of the experiment, the mineralized surface of the OVX group had significantly increased by 61% compared with baseline. Animals of 5- and 0.1-mg OVX PRO groups had lower MS/BS than OVX and 20-mg OVX PRO groups, but MS/BS were significantly higher than in the SHAM group. After 10 weeks, the osteoid surface was significantly higher in OVX and 0.1- and 20-mg OVX PRO groups compared with SHAM and 5-mg OVX PRO groups (p < 0.001).

There was no difference between MAR of the OVX and SHAM groups. Animals of 0.1-, 5-, and 20-mg OVX PRO groups had significantly higher MAR compared with SHAM and OVX groups. MAR was significantly higher (p < 0.001) in 0.1-mg OVX PRO (+35.92%, compared with OVX) than in 5-mg OVX PRO (+22.00%, compared with OVX).

From baseline, osteoclast surface and osteoclast number increased by 70 and 73.2%, respectively, in the OVX group and remained high in the 20-mg OVX PRO group. Animals of the 5-mg OVX PRO group had significantly lower osteoclast surface compared with the 20-mg OVX PRO group but significantly higher Oc.S/BS than the SHAM group. Animals of 0.1-mg OVX PRO had similar Oc.S/BS and Oc.N than the SHAM group (Fig. 5).

View larger version (27K): [in this window] [in a new window]   Fig. 5. Histomorphometry parameters of the proximal tibia after 10 weeks of propranolol treatment. The notes a to e express significantly statistical comparisons between groups: a, compared with SHAM group (p < 0.05); b, compared with OVX group (p < 0.05); c, compared with 0.1 mg/kg OVX (p < 0.05); d, compared with 5 mg/kg OVX (p < 0.05); e, compared with 20 mg/kg OVX (p < 0.05). Baseline group have the same difference than SHAM group. N.At, adipocyte number (number of cells per millimeters squared). Histograms represent means ± S.E.M.

Discussion
The main findings of this study were that propranolol effects on the skeleton are dose- and site-dependent in rats models. Propranolol (0.1 mg) bred better preventive effects on trabecular and cortical bone in ovariectomized rats. The dose of 5 mg of propranolol prevents the effects of ovariectomy on architecture but not on the femoral BMD and the tibial osteoclast surface. Globally, the high-dose treatments (20-mg OVX PRO group) did not induce any effect, except, for tibial BMD and osteoclast surface, which are lower and higher, respectively, than in OVX. The low BMDs observed in the tibia of the group treated by 20 mg of propranolol compared with the OVX group are associated with a lower gastrocnemius muscle mass. The lower muscle mass is probably due to a decreased activity of the animals, which presented a lower heart rate and lower motricity than the other groups (analysis were made with an electrocardiogram and an open field test; N. Bonnet, D. Courteix, L. Vico, V. Eder, and C. L. Benhamou, unpublished data). The fact that patients with muscular dystrophy are osteopenic provides further evidence for a functional association between decreased muscle mass and low bone mass (Aparicio et al., 2002). Our results with animals treated by 20 mg of propranolol caution the findings obtained on 12-adrenergic receptor KO mice in appendicular bone (tibia, femur), where no preventive effects of propranolol on BMD and in metaphysis architecture were shown (Elefteriou et al., 2005a; Pierroz et al., 2005). However, we noticed an increase of BMD L₄ and a higher trabecular bone volume in the axial bone (vertebrae L₄) of 20-mg OVX PRO group compared with the OVX group. These results on the vertebrae are in accordance with the results of Takeda et al. (2002) and Levasseur et al. (2003). This suggests that bone cell of the spine responds to 20 mg of propranolol in a fundamentally different manner than cells of the appendicular bone. Another possible explanation is the potentially osteogenic effects of propranolol on long bone are overcome by the consequences of the muscle mass and physical activity decrease. In fact, the preventive effect of propranolol on axial bone cannot be effective if there is a decrease of muscle mass and activity. Regardless tentative explanations, it is clear that propranolol at a dose of 20 mg has different effects on axial and appendicular skeleton.

It was shown that 5 or 0.1 mg of propranolol prevents the deterioration of the cancellous bone due to ovariectomies. However, the general positive effects observed in OVX rats treated with 5 or 0.1 mg of propranolol differ for several parameters, particularly in the cellular activity. Animals treated by .1 mg of propranolol marked a similar mineralized surface than OVX and 5-mg OVX PRO groups but a higher osteoblast activity compared with SHAM, OVX, and 5-mg OVX PRO groups. (MAR + 40%, compared with OVX; +18%, compared with 5-mg OVX PRO group). Animals treated with 5 mg of propranolol have similar osteoclast surface than OVX, whereas, 0.1 mg of propranolol treatment prevents the increase in osteoclast surface. Other drugs classically used in therapeutics act only on bone resorption or on bone formation. Propranolol (0.1 mg) effect on osteoblast and osteoclast is close to the decoupling effect described for the strontium ranelate (Marie, 2005), a new molecule that has been announced to play a main role in the future management of osteoporosis (Reginster et al., 2005). However, the effect of propranolol on the osteoblast did not indicate if there is an increase of osteoblast number or a decrease of apoptosis of this cell.

The net results of these cellular changes were a tibial BMD decrease in the OVX group, whereas SHAM BMD did not change during the experiment. Animals treated with 5 and 0.1 mg of propranolol had the same tibial BMD evolution as SHAM, whereas the 20-mg OVX PRO group had a higher tibial BMD decrease than the OVX group. On the proximal metaphysis BMD, only the animals treated by 0.1 mg of propranolol had higher values than the OVX group. Nonsignificant difference was observed on the diaphysis BMD between groups. Total vertebrae BMD difference between groups was similar to total femur.

Previous reports (Levasseur et al., 2003) did not include bone microarchitecture analysis. In the present study, microarchitectural parameters revealed a severe microarchitectural alteration in OVX and 20-mg OVX PRO groups in proximal tibia, as demonstrated by lower Tb.N and Tb.Th (–51 and –15%, compared with SHAM, respectively, in both groups). Perforation of trabeculae and subsequent cavity enlargement by osteoclasts have been shown to be responsible for this loss of cancellous structural integrity (Abe et al., 1999). We observed the same alterations on the vertebrae but only in the OVX group and with less severity. Propranolol at 5- and 0.1-mg doses totally prevents the effects of OVX on all of the microarchitectural parameters in proximal tibia and distal femur. In vertebrae, it was possible to separate the microarchitectural effect of 0.1 and 5 mg of propranolol. Animals treated by 5 mg of propranolol had Tb.Th and SMI in the same range as the OVX group, whereas 0.1 mg of propranolol treatment had significant higher Tb.Th and higher proportion of plate shapes than the OVX group.

Propranolol (5 or 0.1 mg) treatment had also a preventive effect on adipocyte proliferation observed in OVX rats. Propranolol is suggested to inhibit the differentiation of the adipocyte lineage and therefore to contribute by this mechanism to the inhibition of the bone deterioration induced by estrogen deficiency (Vicennati et al., 2002). The high adiposity observed in the 20-mg OVX PRO group contrasts the last suggestion of Vicennati et al. (2002). However, bone marrow adipogenesis is known to increase with hindlimb unloading (Justesen et al., 2001; Ahdjoudj et al., 2002). Thus, the high adiposity observed in the tibia of 20-mg OVX PRO group was not surprising, given the decrease of physical activity and therefore gastrocnemius muscle mass.

Scanning electron microscopy and µCT analysis revealed that cortical porosity was primarily located close to the endocortical surface at mid-diaphysis, whereas they were more present in periosteal surface at the proximal diaphysis. Animals treated with 0.1 mg of propranolol had lower cortical porosity, pore number, and higher space between pores compared with the OVX group. This observation responds to Reid et al. (2005) questioners who suggested a hypothetic effect of propranolol on cortical properties.

The usual contradictory results reported in the literature concerning the relationship between -blockers and human bone might be explained by different doses of treatment as was reported by De Vries et al. (2005) in their preliminary data regarding the use of -blockers and the potential risk of hip and vertebral fracture. The studies of Pasco et al. (2004) and Reid et al. (2005) are limited by a lack of information with respect to the duration and doses of -blocker treatment. Furthermore, based upon the study of Kondo and Togari (2003), it was suggested (Reid et al., 2005) that the dose of propranolol necessary to block the effects of sympathetic activation on bone must be doses reaching 10-fold higher than for a therapeutical treatment for hypertension (20 mg/kg). Accordingly to Reid et al. (2005), the present results suggest a dose effect of propranolol showing a better effect with the lowest dose. In parallel to the bone investigation, we have made an evaluation of the cardiac hemodynamic functions. As expected, the various parameters of cardiac functions were affected in rats treated with 20 mg/kg/day propranolol; few of them were affected by 5 mg/kg/day propranolol, but none were changed with the dose of 0.1 mg/kg/day (N. Bonnet, D. Courteix, L. Vico, V. Eder, and C. L. Benhamou, unpublished data). These cardiac investigations are in accordance with the main study achieve on heart and -blockers (Yaoita et al., 2002). These results, consistent with the dominant nature of the adrenergique-2 receptor-deficient mice bone and cardiac phenotype, suggested that low doses of -blockers affecting only the 2-receptor may be an efficacious osteoporosis treatment. Our results are comparable with those reported by Minkowitz et al. (1991) suggesting a preventive effect of -blockers with a dose of 0.1 mg/kg body mass. In addition, our results describe the specific impact of -blockers propranolol on bone architecture and cellular activity, showing its effect on the cortical features of ovariectomized rats. These data must be confirmed by a clinical study taking into account the dose and duration of such treatment.

	Acknowledgements

 
We thank H. Beaupied and A. Basillais for kind technical assistance and for critical reading of the manuscript. We are very grateful to Professor Eddie Van Praagh who kindly revised in detail the English grammar of this article.

	Footnotes

 
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.106.105437.

ABBREVIATIONS: SNS, sympathetic nervous system; BMD, bone mineral density; KO, knockout; OVX, ovariectomized; SHAM, sham-operated; DXA, dual-energy X-ray absorptiometry; OVX PRO, OVX with propranolol; CV, coefficient(s) of variation; BMC, bone mineral content; µCT, microcomputed tomograph; BV/TV, trabecular bone volume; Tb.N, trabecular number; Tb.Sp, trabecular separation; Tb.Th, trabecular thickness; SMI, structure model index; DA, degree of anisotropy; Ct.Po, porosity; Oc.S/BS, osteoclastic surfaces; MAR, mineral apposition rate; MS/BS, mineralizing surface per bone surface; ML, mediolateral; AP, anteroposterior.

Address correspondence to: Dr. Nicolas Bonnet, Institut National de la Santé et de la Recherche Médicale U658, CHR Orleans, 1 rue Porte Madeleine, 45000 Orleans, France. E-mail: nicolas.bonnet15{at}wanadoo.fr

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