Atypical antipsychotic drugs inhibit trabecular bone accrual in C57BL/6J mice

Abstract

Objective

To investigate the effects of the atypical antipsychotics drugs (AADs), olanzapine and risperidone, on femoral bone characteristics in female C57BL/6J mice.

Methods

Mice were treated with placebo or AADs (olanzapine or risperidone) for 3–4 weeks. Femoral cortical and trabecular bone characteristics were determined using micro-computed tomography.

Results

Olanzapine-treated mice tended to have lower trabecular bone volume (P = 0.088) and connectivity (P = 0.057) but no significant differences in bone density (P = 0.521) relative to controls. Risperidone-treated mice had significantly lower trabecular bone density (P = 0.001) and volume (P = 0.008), bone volume/total volume (P = 0.001), connectivity (P = 0.007), and trabecular number (P = 0.003) relative to controls. Cortical bone was not significantly affected by olanzapine or risperidone treatment.

Conclusion

AADs inhibited trabecular bone accrual in C57BL/6J mice suggesting that alternative treatment options may need to be considered for the schizophrenia patient with potential osteoporosis risk.

Introduction

Atypical antipsychotic drugs (AADs), such as olanzapine and risperidone, are currently being prescribed to treat psychotic disorders and aggressive behavior [1, 2]. Side effects of these drugs, such as weight gain, have been observed in both clinical applications [1] and animal research [2, 3]. Recently, several clinical studies reported that treatment with AADs was associated with lower bone mineral density (BMD) and might cause high bone fracture risks in schizophrenia patients [4–7], although some studies showed no such effects [8, 9]. Additional studies are needed to investigate the effects of AADs on BMD and its biological mechanisms [10, 11].

The purpose of the present study was to investigate the effect of olanzapine and risperidone on bone characteristics in a AADs mouse model and compare the differential effect between olanzapine and risperidone.

Materials and methods

Mice

Eight-week-old female C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME, USA) were maintained under standard laboratory conditions on a 12/12 h light/dark cycle (light on at 0600h) in a temperature (22.0 ± 1.0 °C) controlled room. Animals were group housed for one week and then housed individually to decrease inter-individual variation [12]. Standard mouse chow (Teklad Global 16% Protein Rodent Diet, Harlan, Madision, WI, USA) and autoclaved water were provided ad libitum for all mice during the experiment. Ten-week-old mice were acclimated to plain peanut butter pill administration (placebo; twice a day; 0900 and 1500 h) for two weeks as previously described [3, 13]. On the second week of peanut butter pill training, mice were weighed and assigned to one of two groups matched for body weight. In OLZ-experiment, mice were divided into OLZ or PLA groups (n=8/group), and RISP or PLA groups (n=12/group) in RISP-experiment. AAD treatment started when the mice were 13 weeks old and lasted for 3 (RISP) or 4 (OLZ) weeks. All procedures were approved by the University of Alabama at Birmingham Institutional Animal Care and Use Committee.

Drug treatment

Mice were treated orally twice a day (0900 and 1500 h) with peanut butter pills containing olanzapine (Zyprexa®, Eli Lilly and Company, Indianapolis, IN, USA, total daily dose, ~9mg/kg body weight) and risperidone (Cat. 193714, Sigma, St Louis, MO, USA, total daily dose, ~4mg/kg body weight;). Each pill contained ½ the daily dose for both drugs or placebo as previously described [3, 13]. The dose was based on the concentration that induced the greatest weight gain in an optimal dosing experiment with C57BL/6J mice [3, 13].

Micro-computed tomography (uCT)

Micro-architecture of the distal trabecular bone and midshaft cortical bone of the femur were measured using a Scanco µCT 40 (Scanco Medical AG, Brüttisellen, Switzerland). Bones were placed vertically in 12 mm diameter scanning holders and scanned at the following settings: 12 µm resolution, 55 kVp energy, 145 µA intensity, and an integration time of 200 ms.

µCT scanning and analysis followed the guideline proposed by Bouxsein et al. [14]. The trabecular bone scan was performed from the growth plate, and consisted of 200 slices (each slice was 12 µm in thickness). Scans were automatically reconstructed into 2-D slices, and 100 slices were analyzed using the µCT Evaluation Program (v5.0A, Scanco Medical). The region of interest (ROI) was drawn on each of the 100 slices just inside the cortical bone to include only the trabecular bone and marrow. Trabecular bone was thresholded at 235, to distinguish it from the marrow. The 3-D reconstruction was performed using all the outlined slices. No cortical bone was included in this analysis. Data were obtained on bone volume (BV), total volume (TV), BV/TV, bone density, trabecular number and connectivity.

The cortical bone scan was performed at the midshaft of the femur and consisted of 25 slices (each slice was 12 µm in thickness). Fewer slices were needed for the cortical scan as the cortical bone at the midshaft is very uniform. Scans were reconstructed as for the trabecular scans and the region of interest was drawn on all 25 slices. For the cortical scan the ROI was drawn at the outside of the cortical bone and included all the cortical bone and marrow. There was no trabecular bone in these images at the midshaft. Cortical bone was thresholded at 294, and the 3-D reconstruction was performed on all 25 slices. Data were obtained on BV, TV, BV/TV, bone density, and cortical thickness.

Statistical analyses

All statistical analyses were performed using SAS (Version 9.1; SAS Institute Cary, NC). Independent t-test was used to determine if there were significant differences in trabecular and cortical bone characteristics between placebo and AAD-treated mice. Data are reported as mean ± standard error of the mean (SEM). The criterion for statistical significance was P < 0.05 (two-tailed).

Results

Femoral cortical and trabecular bone

OLZ-treated mice tended to have less trabecular bone volume (P = 0.088, Fig 1b) and connectivity (P = 0.057, Fig 1d), but there was no significant effect on trabecular bone density (P = 0.521, Fig 1a), BV/TV (P = 0.101, Fig 1c) and trabecular bone number (P = 0.324, Fig 1e) when compared with PLA-treated mice. OLZ-treated mice tended to have lower cortical bone density (P = 0.082), but there was no significant differences in cortical bone volume (P = 0.117), BV/TV (P = 0.522), and bone thickness (P = 0.762, data not shown). Similar, RISP-treatment had no significant effect on cortical bone density (P = 0.497), bone volume (P = 0.852), BV/TV (P = 0.880), and thickness (P = 0.924, data not shown). In contrast, RISP-treated mice had a significantly lower trabecular bone volume (P = 0.001, Fig 2b), BV/TV (P = 0.001, Fig 2c), bone connectivity (P = 0.007, Fig 2d), and bone number (P = 0.003, Fig 2e) than PLA-treated mice, while no significant effect on trabecular bone density was observed (P = 0.161, Fig 2a).

Figure 1.

Effect of olanzapine (OLZ) on trabecular bone density (a), volume (b), BV/TV (c), connectivity (d), and number (e) in female C57BL/6J mice treated for 4 weeks (n=8 for OLZ, and n=7 for PLA group). Representative three dimensional uCT images of distal femur trabecular bone from OLZ- and PLA-treated mice (f). Data are shown as mean ± SEM.

Figure 2.

Effect of risperidone (RISP) on trabecular bone density (a), volume (b), BV/TV (c), connectivity (d), and number (e) in female C57BL/6J mice treated for 3 weeks (n=11 for RISP, and n=12 for PLA group). Representative three dimensional uCT images of distal femur trabecular bone from OLZ- and PLA-treated mice (f). Data are shown as mean ± SEM.

Discussion

In the present study, we showed that both olanzapine and risperidone caused a reduction in trabecular bone in female C57BL/6J mice, with risperidone having a greater effect than olanzapine. However, neither olanzapine nor risperidone demonstrated effects on cortical bone in female C57BL/6J mice.

Clinical studies have shown that AADs such as olanzapine, clozapine, and risperidone induce a reduction in BMD after one year of monotherapy, with increased bone fracture risk, in both male and female schizophrenia patients [4, 6, 15]. To date, there have been two studies that have investigated the effects of AADs on bone directly using rodent models [11, 10]. In growing rats, clozapine treatment reduced whole body BMD, as well as trabecular and cortical bone volume [10]. Similarly, risperidone administration caused a significant lower trabecular bone BV/TV and trabecular number in both male and female C57BL/6J mice with the measurement of uCT, associated with weight loss and reduced body fat mass [11]. In contrast, our previous [3, 13] and present study (unpublished data) showed that olanzapine and risperidone induced weight gain and hyperphagia associated with trabecular bone loss in female C57BL/6J mice. The consistent results of AADs on trabecular bone provided further evidence that AADs’ effect on bone is independent of body weight changes [11]. One possible mechanism to explain AADs induced bone loss is that AADs induce an imbalance of bone metabolism by inhibition of osteoblast differentiation or activation of osteoclasts in bone remodeling areas [10, 11]. AADs induced hyperprolactinemia, by inhibition of the hypothalamo-pituitary-gonadal axis, may be another mechanism for the reduction in bone [5]. Taken together, clinical and basic research results suggest that administration of AADs reduce trabecular bone by direct or indirect pathways, and lead to a high risk of bone fracture or osteoporosis.

Interestingly, clinical studies as well as animal model studies indicate that antipsychotic drugs might have differential side effects on bone density [4, 10]. For example, risperidone, in contrast to olanzapine, decreased BMD in female premenopausal schizophrenia patients [4]. The effect of risperidone on bone loss was confirmed in animal models including mouse and rats [10, 11], but not with a typical anti-psychotic drug haloperidol [10]. In the present study, we found risperidone induced a significant reduction of trabecular bone compared with placebo mice. In contrasts, the bone reduced degree of olanzapine on trabecular bone is less than risperidone, which was similar with previous clinical study [4]. One possible explanation is risperidone and olanzapine differ in their hyperprolactinemic properties related to high dopamine-2 receptor occupancy by risperidone, which may affect BMD differently [4, 16]. To our knowledge, no studies have tested drug-differential effects on bone characteristics in animal models to determine which AADs have potential clinical implications in patients with high risk of osteoporosis. The observed differential effect of AADs on bone suggests that alternative treatment options may be considered with schizophrenia patients with potential osteoporosis risk.