Exercise mitigates the stunting effect of cold temperature on limb elongation in mice by increasing solute delivery to the growth plate

Abstract

Ambient temperature and physical activity modulate bone elongation in mammals, but mechanisms underlying this plasticity are a century-old enigma. Longitudinal bone growth occurs in cartilaginous plates, which receive nutritional support via delivery of solutes from the vasculature. We tested the hypothesis that chronic exercise and warm temperature promote bone lengthening by increasing solute delivery to the growth plate, measured in real time using in vivo multiphoton microscopy. We housed 68 weanling female mice at cold (16°C) or warm (25°C) temperatures and allowed some groups voluntary access to a running wheel. We show that exercise mitigates the stunting effect of cold temperature on limb elongation after 11 days of wheel running. All runners had significantly lengthened limbs, regardless of temperature, while nonrunning mice had shorter limbs that correlated with housing temperature. Tail length was impacted only by temperature, indicating that the exercise effect was localized to limb bones and was not a systemic endocrine reaction. In vivo multiphoton imaging of fluoresceinated tracers revealed enhanced solute delivery to tibial growth plates in wheel-running mice, measured under anesthesia at rest. There was a minimal effect of rearing temperature on solute delivery when measured at an intermediate room temperature (20°C), suggesting that a lasting increase in solute delivery is an important factor in exercise-mediated limb lengthening but may not play a role in temperature-mediated limb lengthening. These results are relevant to the study of skeletal evolution in mammals from varying environments and have the potential to fundamentally advance our understanding of bone elongation processes.

Fig. 1.

Temperature effects on femur length in wheel-running and non-wheel-running mice housed at warm (25°C) and cold (16°C) temperatures. Cold housing without an exercise wheel decreased bone length, but wheel exercise increased limb length at both temperatures. All long bone lengths measured from digital images were, in general, as follows: WW ≈ CW > WN > CN, where WW is warm wheel, CW is cold wheel, WN is warm no wheel, and CN is cold no wheel. Scale bar, 1 mm.

Fig. 2.

Temperature effects on tail and femur length in wheel-running and non-wheel-running mice. A: tail lengths confirmed previous findings that extremities are shorter at cold temperatures. There was a significant and sizable temperature effect on tail elongation (2-way ANOVA: F = 216.3, P < 0.001) but no effect of wheel-running exercise. Change in tail length in the cold mice measured only half of that in the warm groups, regardless of exercise. B: femur length varied with temperature and exercise. Conclusion is that the exercise effect was localized to limb bones and was not a systemic or endocrine reaction. Femur is plotted as change in length from a common baseline starting point, determined by average femur length of 8 mice at 24 days age. Tail is plotted as change in length from the start, measured for each individual mouse. Values are group means ± SE. Statistically significant (P < 0.05) by 1-way ANOVA and Tukey's test for multiple independent comparisons: *WW and WN vs. CW and CN in pairwise comparisons; +WW > WN and WW > CN; ++CW > CN.

Fig. 3.

Timed accumulation curves of fluorescein (FL) tracer in the growth plate and metaphyseal vasculature. A: exercise significantly increased FL arrival and entry into the growth plate. Within 5 min after tracer injection, absolute FL concentrations were ∼1.5-fold greater in the growth plates of the wheel-running than nonrunning groups, independent of temperature. B: there was also more tracer in the adjacent metaphyseal vasculature of the wheel-running groups: >1.3 times more FL had accumulated in the metaphyseal region of the runners by 5 min (temperatures pooled), indicating enhanced tracer delivery into and around the growth plates of exercised mice. These differences in absolute values, however, were not statistically significant because of within-group variation. Group means are shown without error bars to facilitate viewing.

Fig. 4.

Plot of relative growth plate fluorescence measured 5-min after fluorescein tracer injection. Fluorescein levels in the growth plate were standardized to peak concentrations in the metaphyseal bone region to account for variation in imaging conditions between mice (36, 82). Two-way ANOVA applied to these standardized data revealed a significant exercise effect (F = 4.3, P = 0.02) and confirmed the apparent absence of a temperature effect on relative fluorescein accumulation in the growth plate. Values are group means ± SE. +Statistical significance (P < 0.05) by 1-way ANOVA and Tukey's test for multiple independent comparisons: WW > WN and WW > CN.

Fig. 5.

Relationship between femur length and wheel-running distance. A: voluntary wheel usage varied from 5 to 25 km/day, but there was no significant difference in average distance between cold and warm groups. There was a slight positive correlation between distance run and femur length (Pearson's r = 0.3, P = 0.03, outliers excluded as denoted by # and ##), suggesting that the amount of wheel running could be related to degree of limb lengthening. This 5-fold range in average daily distance may also account for increased variation in other data collected (see Figs. 2 and 4). #Mice with unusually long femurs; ##mouse with extremely high running activity.