(F-232) Male and Female mouse bone respond differently to disuse, independent of Endocannabinoid Receptor 1 activation.

Introduction

With the rising use of cannabinoids in clinical practice and increasing number of patients experiencing disuse-related bone loss, understanding the influence of the endocannabinoid system (eCBS) on bone metabolism during mechanical unloading becomes imperative. The eCBS has been shown to play an important role in postnatal bone metabolism and bone mass determination. Data from our lab suggests that male C57BL/6J globally CB1 deficient mice (CB1KO) are more sensitive than wildtype (WT) mice to bone loss during single limb immobilization (SLI)¹. The increased sensitivity of male CB1KO mice to disuse-induced bone loss indicates that the activation of the CB1 might offer protection against disuse-induced bone loss. Therefore, we tested the efficacy of Arachidonyl-2’-chloroethylamide (ACEA), a highly selective CB1 agonist, in mitigating bone loss induced by single limb immobilization in both male and female C57BL/6J mice.

Methods

20-week-old male and female C57BL/6J WT mice were assigned to control (n=7) or ACEA treatment groups (n=7) and placed in casts to immobilize the left hindlimb. The right hindlimb served as a contralateral control. Experimental mice received a daily ACEA (10mg/kg; i.p.) and control mice received a daily vehicle (90% saline, 5% EtOH, and 5% corn oil; i.p.). After 21 days of SLI, mice were sacrificed, and femurs harvested. The skeletal phenotype was assessed using in vivo micro-CT scans (7um) at day 0 and day 21. Cortical bone at the mid-diaphysis and cancellous bone at epiphyseal and metaphyseal regions were analyzed. Data were analyzed using repeated measures 3-way ANOVA with Sidak post-hoc test (p< 0.05).

  Results

In the metaphyseal region, there was a significant main effect of casting, sex, and a significant interaction between casting and sex (figure 1). In particular, only vehicle treated female mice experienced disuse induced bone loss due to casting. In addition, changes in the female mice control limbs were significantly lower from the corresponding male groups, regardless of treatment. In the epiphyseal region, there was an effect of both casting and sex, with female mice in both vehicle and agonist groups experiencing greater changes in BV/TV from the control limb compared to males due to casting. In the diaphyseal region, there was a significant main effect of agonist treatment and casting, however, we did not see any interaction effects. For example, agonist treated mice showed greater bone loss independent of casting or mouse sex.

Conclusion

This study demonstrates that CB1 activation by ACEA contributed to bone loss in the diaphyseal regardless of casting or mouse sex. Therefore, contrary to our hypothesis, CB1 activation didn’t attenuate bone loss from casting. In addition, female mice experienced significantly greater epiphyseal and metaphyseal bone loss compared to the control limb, regardless of ACEA treatment, compared to male mice. This was likely due to the finding that male mice experienced greater bone loss in the control limbs from baseline than female mice. In contrast to earlier studies, where CB1 deletion influenced disuse induced bone loss, we saw no effect of CB1 activation via agonist treatment on disuse induced bone loss.  Our results suggest that male and female mice react differently to disuse induced bone loss, however this bone loss is not affected by agonist treatment in either sex.

References: [1] DeNapoli, R.C. et al. Journal of Biomechanics (2023).