Philippe Huber

Transient muscle paralysis disrupts bone homeostasis by rapid degradation of bone morphology

We have previously shown that transient paralysis of murine hindlimb muscles causes profound degradation of both trabecular and cortical bone in the adjacent skeleton within 3 weeks. Morphologically, the acute loss of bone tissue appeared to arise primarily due to osteoclastic bone resorption. Given that the loss of muscle function in this model is transient, we speculated that the stimulus for osteoclastic activation would be rapid and morphologic evidence of bone resorption would appear before 21 days. We therefore utilized high-resolution in vivo serial micro-CT to assess longitudinal alterations in lower hindlimb muscle volume, proximal tibia trabecular, and tibia mid-diaphysis cortical bone morphology in 16-week-old female C57 mice following transient calf paralysis from a single injection of botulinum toxin A (BtA; 2U/100 g body weight). In an acute study, we evaluated muscle and bone alterations at days 0, 3, 5, and 12 following transient calf paralysis. In a chronic study, following day 0 imaging, we assessed the recovery of these tissues following the maximum observed trabecular degradation (day 12) through day 84 post-paralysis. The time course and degree of recovery of muscle, trabecular, and cortical bone varied substantially. Significant atrophy of lower limb muscle was evident by day 5 of paralysis, maximal at day 28 (-34.1+/-0.9%) and partially recovered by day 84. Trabecular degradation within the proximal tibia metaphysis occurred more rapidly, with significant reduction in BV/TV by day 3, maximal loss at day 12 (-76.8+/-2.9%) with only limited recovery by day 84 (-51.7+/-5.1% vs. day 0). Significant cortical bone volume degradation at the tibia mid-diaphysis was first identified at day 12, was maximal at day 28 (-9.6+/-1.2%), but completely recovered by day 84. The timing, magnitude, and morphology of the observed bone erosion induced by transient muscle paralysis were consistent with a rapid recruitment and prolific activation of osteoclastic resorption. In a broader context, understanding how brief paralysis of a single muscle group can precipitate such rapid and profound bone resorption in an adjacent bone is likely to provide new insight into how normal muscle function modulates bone homeostasis.

Metaphyseal and diaphyseal bone loss in the tibia following transient muscle paralysis are spatiotemporally distinct resorption events

When the skeleton is catabolically challenged, there is great variability in the timing and extent of bone resorption observed at cancellous and cortical bone sites. It remains unclear whether this resorptive heterogeneity, which is often evident within a single bone, arises from increased permissiveness of specific sites to bone resorption or localized resorptive events of varied robustness. To explore this question, we used the mouse model of calf paralysis induced bone loss, which results in metaphyseal and diaphyseal bone resorption of different timing and magnitude. Given this phenotypic pattern of resorption, we hypothesized that bone loss in the proximal tibia metaphysis and diaphysis occurs through resorption events that are spatially and temporally distinct. To test this hypothesis, we undertook three complimentary in vivo/μCT imaging studies. Specifically, we defined spatiotemporal variations in endocortical bone resorption during the 3weeks following calf paralysis, applied a novel image registration approach to determine the location where bone resorption initiates within the proximal tibia metaphysis, and explored the role of varied basal osteoclast activity on the magnitude of bone loss initiation in the metaphysis using μCT based bone resorption parameters. A differential response of metaphyseal and diaphyseal bone resorption was observed throughout each study. Acute endocortical bone loss following muscle paralysis occurred almost exclusively within the metaphyseal compartment (96.5% of total endocortical bone loss within 6days). Using our trabecular image registration approach, we further resolved the initiation of metaphyseal bone loss to a focused region of significant basal osteoclast function (0.03mm(3)) adjacent to the growth plate. This correlative observation of paralysis induced bone loss mediated by basal growth plate cell dynamics was supported by the acute metaphyseal osteoclastic response of 5-week vs. 13-month-old mice. Specifically, μCT based bone resorption rates normalized to initial trabecular surface (BRRBS) were 3.7-fold greater in young vs. aged mice (2.27±0.27μm(3)/μm(2)/day vs. 0.60±0.44μm(3)/μm(2)/day). In contrast to the focused bone loss initiation in the metaphysis, diaphyseal bone loss initiated homogeneously throughout the long axis of the tibia predominantly in the second week following paralysis (81.3% of diaphyseal endocortical expansion between days 6 and 13). The timing and homogenous nature are consistent with de novo osteoclastogenesis mediating the diaphyseal resorption. Taken together, our data suggests that tibial metaphyseal and diaphyseal bone loss induced by transient calf paralysis are spatially and temporally discrete events. In a broader context, these findings are an essential first step toward clarifying the timing and origins of multiple resorptive events that would require targeting to fully inhibit bone loss following neuromuscular trauma.

Cortical bone resorption following muscle paralysis is spatially heterogeneous.

Mechanical loading of the skeleton, as induced by muscle function during activity, plays a critical role in maintaining bone homeostasis. It is not understood, however, whether diminished loading (and thus diminished mechanical stimuli) directly mediates the bone resorption that is associated with disuse. Our group has recently developed a murine model in which we have observed rapid and profound bone loss in the tibia following transient paralysis of the calf muscles. As cortical bone loss is achieved via rapid endocortical expansion without alterations in periosteal morphology, we believe this model holds unique potential to explore the spatial relation between altered mechanical stimuli and subsequent bone resorption. Given the available literature, we hypothesized that endocortical resorption following transient muscle paralysis would be spatially homogeneous. To test this hypothesis, we first validated an image registration algorithm that quantified site-specific cortical bone alterations with high precision and accuracy. We then quantified endocortical expansion in the tibial diaphysis within 21 days following transient muscle paralysis and found that, within the analyzed mid-diaphyseal region (3.15 mm), site-specific bone loss was focused on the anterior surface in the proximal region but shifted to the posterior surface at the distal end of the analyzed volume. This site-specific, and highly repeatable biologic response suggests active osteoclast chemotaxis or focal activation of osteoclastic resorption underlies the spatially consistent endocortical resorption induced by transient muscle paralysis. Clarifying this relation holds potential to yield unique insight into how the removal of factors critical for bone homeostasis acutely precipitates local modulation of cellular responses within bone.

A comparison of injury criteria used in evaluating seats for whiplash protection.

A protocol has been proposed for testing seats for whiplash protection, however injury criteria have not yet been chosen. Assuming that whiplash symptoms arise from non-physiological motions of vertebral segments, we determined the ability of proposed criteria to predict peak individual vertebral displacements. Twenty-eight volunteers were subjected to rear impacts while seated in a car seat with head restraint, mounted onto a sled. Accelerometers were used to record head and torso accelerations. The volunteer data was used as a basis for testing post-mortem human specimens (PMHS). The seat was replaced by a platform onto which was mounted each of 11 cervico-thoracic spines. An instrumented headform was mounted to the upper end of the spine. The head restraint, head-to-restraint geometry, sled, and impact pulse remained the same. Head and T1 accelerations were measured and individual vertebral sagittal (XZ) plane rotations and translations were obtained from high speed video. Proposed injury criteria (NIC, Nkm, Nte, Nd) were tested for their ability to predict average, total, and peak intervertebral displacements. PMHS specimens had chest and head X (horizontal) and Z (vertical) linear accelerations similar to volunteers whose heads hit the head restraint. The best predictors were: Nd shear and peak intervertebral posterior translation (r2 = 0.80), Nd extension and peak extension angle (r2 = 0.70), and Nd distraction and peak distraction (r2 = 0.51). Therefore consideration should be given to a displacement based injury criteria such as Nd in assessment of whiplash protection devices.

The role of door orientation on occupant injury in a nearside impact: A CIREN, MADYMO modeling and experimental study

Objective. This study addressed the effects of vehicle height mismatch in side impact crashes. A light truck or SUV tends to strike the door of a passenger car higher causing the upper border to lead into the occupant space. Conversely, an impact centered lower on the door, from a passenger car, causes the lower border to lead. We proposed the hypothesis that the type of injury sustained by the occupant could be related to door orientation during its intrusion into the passenger compartment. Method. Data on door orientation and nearside occupant injuries were collected from 125 side impact crashes reported in the CIREN database. Experimental testing was performed using a pendulum carrying a frame and a vehicle door, impacting against a USDOT SID. The frame allowed the door orientation to be changed. A model was developed in MADYMO (v 6.2) using the more biofidelic dummies, BIOSID, and SIDIIs as well as USDOT SID. Results. In side impact crashes with the lower border of the door leading, 81% of occupants sustained pelvic injury, 42% suffered rib fractures, and the rate of organ injury was 0.84. With the upper border leading, 46% of occupants sustained pelvic injury, 71% sustained rib fracture, and the rate of organ injuries per case increased to 1.13. The differences in the groups with respect to pelvic injury were significant at p = 0.01, rib fracture, p = 0.10, and organ injury, p = 0.001. Experimental testing showed that when the door angle changed from lower to upper border leading, peak T4 acceleration increased by 273% and pelvic acceleration decreased by 44%. The model demonstrated that when the door angle changed from lower to upper border leading, the USDOT SID showed a 29% increase in T4 acceleration and a 57% decrease in pelvic acceleration. The BIOSID dummy demonstrated a 36% increase in T1 acceleration, a 44% increase in abdominal rib 1 deflection, a 91% increase in thoracic rib 1 deflection, and a 33% decrease in pelvic acceleration. Conclusions. These data add more insight to the problem of mismatch during side impacts, where the bumper of the striking vehicle overrides the door beam, causing the upper part of the door to lead the intrusion into the passenger compartment. Even with the same delta V and intrusion, with the upper border of the door leading, more severe chest and organ injuries resulted. This data suggests that door orientation should be considered when testing subsystems for side impact protection.

Differential Suture Loading in an Experimental Rotator Cuff Repair

Background Repairs of large rotator cuff tears often fail to heal. A possible factor in these failures is excessive tension in the repair sutures, causing them to pull through the tendon. Hypothesis Arm positions encountered during early rehabilitation after cuff repair can dramatically increase the relative tension in the different sutures of the cuff repair. Study Design Controlled laboratory study. Methods In a cadaver model, a 4-suture supraspinatus repair was carried out with transosseous sutures. After the repair, the arm was placed in 12 different positions. The tension in each suture was monitored using individual load cells. Results When the arm was externally rotated relative to the plane of the scapula, the tension in the anterior suture was over 10 times that in the posterior suture (P <. 001). When the arm was internally rotated, the tension in the posterior suture was over 10 times that in the anterior suture (P <. 0005). When the arm was in neutral rotation, there was no significant difference in the suture tension. Conclusions This study is the first report of direct suture tension measurement after a model rotator cuff repair. In this model, 30° of either internal or external rotation of the arm in relation to the plane of the scapula created substantial imbalances in the tension between the most anterior and most posterior sutures of a supraspinatus repair, regardless of the position of abduction. Clinical Relevance Avoiding external rotation stretching during the healing of supraspinatus repairs may prevent tension overload in the critical anterior suture.

MicroCT-based phenomics in the zebrafish skeleton reveals virtues of deep phenotyping in a distributed organ system

Phenomics, which ideally involves in-depth phenotyping at the whole-organism scale, may enhance our functional understanding of genetic variation. Here, we demonstrate methods to profile hundreds of phenotypic measures comprised of morphological and densitometric traits at a large number of sites within the axial skeleton of adult zebrafish. We show the potential for vertebral patterns to confer heightened sensitivity, with similar specificity, in discriminating mutant populations compared to analyzing individual vertebrae in isolation. We identify phenotypes associated with human brittle bone disease and thyroid stimulating hormone receptor hyperactivity. Finally, we develop allometric models and show their potential to aid in the discrimination of mutant phenotypes masked by alterations in growth. Our studies demonstrate virtues of deep phenotyping in a spatially distributed organ system. Analyzing phenotypic patterns may increase productivity in genetic screens, and facilitate the study of genetic variants associated with smaller effect sizes, such as those that underlie complex diseases.

Neuron subset-specific Pten deletion induces abnormal skeletal activity in mice

Abstract Individuals with a history of epilepsy are at higher risk for bone fractures compared to the general population. Although clinical studies support an association between low bone mineral density (BMD) and anti‐seizure medications, little is known on whether a history of seizures is linked to altered bone health. Therefore, in this study we tested the hypothesis that bone mass, morphology, and bone mineralization are altered by seizures in genetically epileptic animals and in animals subjected to an episode of status epilepticus. In this study, we used NS‐Pten conditional knockout mice (a well‐studied genetic model of epilepsy). We used microCT analysis to measure BMD, morphology, and mineralization in NS‐Pten+/+ (wildtype) and NS‐Pten−/− (knockout) mice at 4 and 8 weeks, as well as adult Kv4.2+/+ and Kv4.2−/− mice. We measured BMD, bone morphology, and mineralization in adult NS‐Pten+/+ mice that received status epilepticus through kainic acid (20 mg/kg intraperitoneal). Further, we measured locomotion for NS‐Pten+/+ and NS‐Pten−/− mice at 4 and 6 weeks. We found that NS‐Pten−/− mice exhibited low BMD in the tibial metaphysis and midshaft compared to non‐epileptic mice. Morphologically, NS‐Pten−/− mice exhibited decreased trabecular volume fraction, and endocortical expansion in both the metaphyeal and diaphyseal compartments. In the midshaft, NS‐Pten−/− mice exhibited reduced tissue mineral density, indicating impaired mineralization in addition to morphological deficits. NS‐Pten−/− mice exhibited hyperactivity in open field testing, suggesting low bone mass in NS‐Pten−/− mice was not attributable to hypoactivity. Differences in BMD were not observed following kainate‐induced seizures or in the Kv4.2−/− model of seizure susceptibility. Our findings suggest that deletion of Pten in the brain results in impaired bone mass and mineralization, which may contribute to weaker bones and thereby a higher fracture risk. HighlightsNS‐Pten knockout mice show region‐specific changes in bone volume and bone mineral.NS‐Pten knockout mice show age‐dependent bone phenotype.NS‐Pten knockout mice have initially normal activity levels and later hyperactive.NS‐Pten wildtype mice with status epilepticus do not show abnormal bone phenoptype.Kv4.2 knockout mice do not show bone phenotype changes.

microCT-Based Skeletal Phenomics in Zebrafish Reveals Virtues of Deep Phenotyping at the Whole-Organism Scale

Phenomics, which ideally involves in-depth phenotyping at the whole-organism scale, may enhance our functional understanding of genetic variation. Here, we demonstrate methods to profile hundreds of measures comprised of morphological and densitometric traits from a large number sites in the axial skeleton of adult zebrafish. We show the potential for vertebral patterns to confer heightened sensitivity, with similar specificity, in discriminating mutant populations compared to analyzing individual vertebrae in isolation. We identify phenotypes associated with human brittle bone disease and thyroid stimulating hormone receptor hyperactivity. Finally, we develop allometric models and show their potential to aid in the discrimination of mutant phenotypes masked by alterations in growth. Our studies demonstrate virtues of deep phenotyping in a spatially distributed organ. Analyzing phenotypic patterns may increase productivity in genetic screens, and could facilitate the study of genetic variants associated with smaller effect sizes, such as those that underlie complex diseases.Phenomics—in-depth phenotyping at the whole-organism scale—holds promise to enhance our fundamental understanding of genes and genomic variation, yet methods in vertebrates are limited. Here, we demonstrate rapid whole-body profiling of hundreds of traits in the axial skeleton of adult zebrafish. We show the potential for vertebral patterns to confer heightened sensitivity, with similar specificity, in discriminating mutant populations compared to analyzing individual vertebrae in isolation, even when the latter is performed at higher resolution. We identify phenotypes associated with human brittle bone disease and thyroid stimulating hormone receptor hyperactivity. Finally, we develop allometric models and show their potential to discriminate mutant phenotypes masked by growth alterations in growth. Our studies demonstrate virtues of whole-body phenomic pattern analysis in a single organ system. The high sensitivity may increase productivity in genetic screens, and facilitate the study genetic variants of smaller effect size, such as those that underlie complex diseases.