Maria A. Serrat

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

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.

Temperature alters solute transport in growth plate cartilage measured by in vivo multiphoton microscopy

Solute delivery to avascular cartilaginous plates is critical to bone elongation, and impaired transport of nutrients and growth factors in cartilage matrix could underlie many skeletal abnormalities. Advances in imaging technology have revolutionized our ability to visualize growth plates in vivo, but quantitative methods are still needed. We developed analytical standards for measuring solute delivery, defined by amount and rate of intravenous tracer entry, in murine growth plates using multiphoton microscopy. We employed an acute temperature model because of its well-established impact on bone circulation and tested the hypothesis that solute delivery changes positively with limb temperature when body core and respiration are held constant (36 degrees C, 120 breaths/min). Tibial growth plates were surgically exposed in anesthetized 5-wk-old mice, and their hindlimbs were immersed in warm (36 degrees C) or cool (23 degrees C) saline (n = 6/group). After 30 min of thermal equilibration, we administered an intracardiac injection of fluorescein (50 microl, 0.5%) and captured sequentially timed growth plate images spanning 10 min at standardized depth. Absolute growth plate fluorescence was normalized to vascular concentrations for interanimal comparisons. As predicted, more fluorescein infiltrated growth plates at 36 degrees C, with standardized values nearly double those at 23 degrees C. Changing initial limb temperature did not alter baseline values, suggesting a sustained response period. These data validate the sensitivity of our system and have relevance to strategies for enhancing localized delivery of therapeutic agents to growth plates of children. Applications of this technique include assessment of solute transport in models of growth plate dysfunction, particularly chondrodysplasias with matrix irregularities.

Allen's Rule Revisited: Temperature Influences Bone Elongation During a Critical Period of Postnatal Development

Limbs of animals raised at warm ambient temperature are significantly and permanently longer than those of siblings housed in the cold. These highly reproducible lab results closely parallel the ecogeographical tenet described by Allens extremity size rule, which states that appendage length correlates with temperature and latitude. It is unclear what mechanisms underlie these differences and in what pattern they emerge, since the morphology is traditionally thought to reflect naturally selected genomic adaptations for thermoregulation. This study tests the a posteriori hypothesis that adult extremity length is subject to substantial modification by temperature during a brief but critical period of early postnatal development. Weanling mice (N = 28) were divided into three groups and housed at 7°C, 21°C, or 27°C for eight weeks. Tail lengths and body mass were measured weekly. Mass did not differ at any age. Analysis of tail elongation curves revealed two distinct phases: an initial period of rapid temperature‐sensitive growth in which elongation rate was directly impacted by temperature; and a second phase of continued growth in which rates were identical among groups. Comparable growth reactions occur in response to other environmental variables such as exercise, suggesting that the skeleton is most responsive to external stimuli during a window of heightened sensitivity when growth occurs most rapidly. Knowledge of the timing and degree to which growth plasticity permits mammals to immediately adjust to novel temperature conditions will be important for analyzing skeletal variation in fluctuating climates, particularly for assessing factors that may accelerate skeletal evolution at temperature extremes. Anat Rec, 296:1534–1545, 2013.

Independent learning modules enhance student performance and understanding of anatomy

Didactic lessons are only one part of the multimodal teaching strategies used in gross anatomy courses today. Increased emphasis is placed on providing more opportunities for students to develop lifelong learning and critical thinking skills during medical training. In a pilot program designed to promote more engaged and independent learning in anatomy, self‐study modules were introduced to supplement human gross anatomy instruction at Joan C. Edwards School of Medicine at Marshall University. Modules use three‐dimensional constructs to help students understand complex anatomical regions. Resources are self‐contained in portable bins and are accessible at any time. Students use modules individually or in groups in a structured self‐study format that augments material presented in lecture and laboratory. Pilot outcome data, measured by feedback surveys and examination performance statistics, suggest that the activity may be improving learning in gross anatomy. Positive feedback on both pre‐ and post‐examination surveys showed that students felt the activity helped to increase their understanding of the topic. In concordance with student perception, average examination scores on module‐related laboratory and lecture questions were higher in the two years of the pilot program compared with the year before its initiation. Modules can be fabricated on a modest budget using minimal resources, making implementation practical for smaller institutions. Upper level medical students assist in module design and upkeep, enabling continuous opportunities for vertical integration across the curriculum. This resource offers a feasible mechanism for enhancing independent and lifelong learning competencies, which could be a valuable complement to any gross anatomy curriculum. Anat Sci Educ 7: 406–416.

Unilateral heat accelerates bone elongation and lengthens extremities of growing mice.

Linear growth failure results from a broad spectrum of systemic and local disorders that can generate chronic musculoskeletal disability. Current bone lengthening protocols involve invasive surgeries or drug regimens, which are only partially effective. Exposure to warm ambient temperature during growth increases limb length, suggesting that targeted heat could noninvasively enhance bone elongation. We tested the hypothesis that daily heat exposure on one side of the body unilaterally increases femoral and tibial lengths. Mice (N = 20) were treated with 40 °C unilateral heat for 40 min/day for 14 days post‐weaning. Non‐treated mice (N = 6) served as controls. Unilateral increases in ear (8.8%), hindfoot (3.5%), femoral (1.3%), and tibial (1.5%) lengths were obtained. Tibial elongation rate was > 12% greater (15 μm/day) on the heat‐treated side. Extremity lengthening correlated with temperature during treatment. Body mass and humeral length were unaffected. To test whether differences persisted in adults, mice were examined 7‐weeks post‐treatment. Ear area, hindfoot, femoral, and tibial lengths were still significantly increased ∼6%, 3.5%, 1%, and 1%, respectively, on the heat‐treated side. Left‐right differences were absent in non‐treated controls, ruling out inherent side asymmetry. This model is important for designing noninvasive heat‐based therapies to potentially combat a range of debilitating growth impediments in children.

Imaging IGF-I uptake in growth plate cartilage using in vivo multiphoton microscopy

Bones elongate through endochondral ossification in cartilaginous growth plates located at ends of primary long bones. Linear growth ensues from a cascade of biochemical signals initiated by actions of systemic and local regulators on growth plate chondrocytes. Although cellular processes are well defined, there is a fundamental gap in understanding how growth regulators are physically transported from surrounding blood vessels into and through dense, avascular cartilage matrix. Intravital imaging using in vivo multiphoton microscopy is one promising strategy to overcome this barrier by quantitatively tracking molecular delivery to cartilage from the vasculature in real time. We previously used in vivo multiphoton imaging to show that hindlimb heating increases vascular access of large molecules to growth plates using 10-, 40-, and 70-kDa dextran tracers. To comparatively evaluate transport of similarly sized physiological regulators, we developed and validated methods for measuring uptake of biologically active IGF-I into proximal tibial growth plates of live 5-wk-old mice. We demonstrate that fluorescently labeled IGF-I (8.2 kDa) is readily taken up in the growth plate and localizes to chondrocytes. Bioactivity tests performed on cultured metatarsal bones confirmed that the labeled protein is functional, assessed by phosphorylation of its signaling kinase, Akt. This methodology, which can be broadly applied to many different proteins and tissues, is relevant for understanding factors that affect delivery of biologically relevant molecules to the skeleton in real time. Results may lead to the development of drug-targeting strategies to treat a wide range of bone and cartilage pathologies.NEW & NOTEWORTHY This paper describes and validates a novel method for imaging transport of biologically active, fluorescently labeled IGF-I into skeletal growth plates of live mice using multiphoton microscopy. Cellular patterns of fluorescence in the growth plate were completely distinct from our prior publications using biologically inert probes, demonstrating for the first time in vivo localization of IGF-I in chondrocytes and perichondrium. These results form important groundwork for future studies aimed at targeting therapeutics into growth plates.

Heat-Induced Limb Length Asymmetry Has Functional Impact on Weight Bearing in Mouse Hindlimbs

Limb length inequality results from many types of musculoskeletal disorders. Asymmetric weight bearing from a limb length discrepancy of less than 2% can have debilitating consequences such as back problems and early-onset osteoarthritis. Existing treatments include invasive surgeries and/or drug regimens that are often only partially effective. As a noninvasive alternative, we previously developed a once daily limb-heating model using targeted heat on one side of the body for 2 weeks to unilaterally increase bone length by up to 1.5% in growing mice. In this study, we applied heat for 1 week to determine whether these small differences in limb length are functionally significant, assessed by changes in hindlimb weight bearing. We tested the hypothesis that heat-induced limb length asymmetry has a functional impact on weight bearing in mouse hindlimbs. Female 3-week-old C57BL/6 mice (N = 12 total) were treated with targeted intermittent heat for 7 days (40 C for 40 min/day). High-resolution x-ray (N = 6) and hindlimb weight bearing data (N = 8) were acquired at the start and end of the experiments. There were no significant left-right differences in starting tibial length or hindlimb weight bearing. After 1-week heat exposure, tibiae (t = 7.7, p < 0.001) and femora (t = 11.5, p < 0.001) were ~1 and 1.4% longer, respectively, on the heat-treated sides (40 C) compared to the non-treated contralateral sides (30 C). Tibial elongation rate was over 6% greater (t = 5.19, p < 0.001). Hindlimb weight bearing was nearly 20% greater (t = 11.9, p < 0.001) and significantly correlated with the increase in tibial elongation rate on the heat-treated side (R2 = 0.82, p < 0.01). These results support the hypothesis that even a small limb length discrepancy can cause imbalanced weight distribution in healthy mice. The increase in bone elongation rate generated by localized heat could be a way to equalize limb length and weight bearing asymmetry caused by disease or trauma, leading to new approaches with better outcomes by using heat to lengthen limbs and reduce costly side effects of more invasive interventions.