A convolutional neural network to characterize mouse hindlimb foot strikes during voluntary wheel running

Voluntary wheel running (VWR) is widely used to study how exercise impacts a variety of physiologies and pathologies in rodents. The primary activity readout of VWR is aggregated wheel turns over a given time interval (most often, days). Given the typical running frequency of mice (∼4 Hz) and the intermittency of voluntary running, aggregate wheel turn counts, therefore, provide minimal insight into the heterogeneity of voluntary activity. To overcome this limitation, we developed a six-layer convolutional neural network (CNN) to determine the hindlimb foot strike frequency of mice exposed to VWR. Aged female C57BL/6 mice (22 months, n = 6) were first exposed to wireless angled running wheels for 2 h/d, 5 days/wk for 3 weeks with all VWR activities recorded at 30 frames/s. To validate the CNN, we manually classified foot strikes within 4800 1-s videos (800 randomly chosen for each mouse) and converted those values to frequency. Upon iterative optimization of model architecture and training on a subset of classified videos (4400), the CNN model achieved an overall training set accuracy of 94%. Once trained, the CNN was validated on the remaining 400 videos (accuracy: 81%). We then applied transfer learning to the CNN to predict the foot strike frequency of young adult female C57BL6 mice (4 months, n = 6) whose activity and gait differed from old mice during VWR (accuracy: 68%). In summary, we have developed a novel quantitative tool that non-invasively characterizes VWR activity at a much greater resolution than was previously accessible. This enhanced resolution holds potential to overcome a primary barrier to relating intermittent and heterogeneous VWR activity to induced physiological responses

Rescuing Loading Induced Bone Formation at Senescence

The increasing incidence of osteoporosis worldwide requires anabolic treatments that are safe, effective, and, critically, inexpensive given the prevailing overburdened health care systems. While vigorous skeletal loading is anabolic and holds promise, deficits in mechanotransduction accrued with age markedly diminish the efficacy of readily complied, exercise-based strategies to combat osteoporosis in the elderly. Our approach to explore and counteract these age-related deficits was guided by cellular signaling patterns across hierarchical scales and by the insight that cell responses initiated during transient, rare events hold potential to exert high-fidelity control over temporally and spatially distant tissue adaptation. Here, we present an agent-based model of real-time Ca2+/NFAT signaling amongst bone cells that fully described periosteal bone formation induced by a wide variety of loading stimuli in young and aged animals. The model predicted age-related pathway alterations underlying the diminished bone formation at senescence, and hence identified critical deficits that were promising targets for therapy. Based upon model predictions, we implemented an in vivo intervention and show for the first time that supplementing mechanical stimuli with low-dose Cyclosporin A can completely rescue loading induced bone formation in the senescent skeleton. These pre-clinical data provide the rationale to consider this approved pharmaceutical alongside mild physical exercise as an inexpensive, yet potent therapy to augment bone mass in the elderly. Our analyses suggested that real-time cellular signaling strongly influences downstream bone adaptation to mechanical stimuli, and quantification of these otherwise inaccessible, transient events in silico yielded a novel intervention with clinical potential

A microCT-based platform to quantify drug targeting

Abstract Background Heterotopic ossification (HO) is a frequent and debilitating complication of traumatic musculoskeletal injuries and orthopedic procedures. Prophylactic dosing of botulinum toxin type A (BTxA) holds potential as a novel treatment option if accurately distributed throughout soft-tissue volumes where protection is clinically desired. We developed a high-resolution, microcomputed tomography (microCT)-based imaging strategy to assess drug distribution and validated this platform by quantifying distribution achieved via a prototype delivery system versus a single-bolus injection. Methods We injected an iodine-containing contrast agent (iodixanol 320 mg I/mL) into dissected rabbit musculature followed by microCT imaging and analysis. To contrast the performance of distributed versus bolus injections, a three-dimensional (3D) 64-cm3-printed soft-tissue holder was developed. A centered 2-cm3 volume of interest (VOI) was targeted with a single-bolus injection or an equal volume distributed injection delivered via a 3D-printed prototype. VOI drug coverage was quantified as a percentage of the VOI volume that was < 1.0 mm from the injected fluid. Results The microCT-based approach enabled high-resolution quantification of injection distribution within soft tissue. The distributed dosing prototype provided significantly greater tissue coverage of the targeted VOI (72 ± 3%, mean ± standard deviation) when compared to an equal volume bolus dose (43 ± 5%, p = 0.031) while also enhancing the precision of injection targeting. Conclusions A microCT-based imaging technique precisely quantifies drug distribution within a soft-tissue VOI, providing a path to overcome a barrier for clinical translation of prophylactic inhibition of HO by BTxA. Relevance statement This platform will facilitate rapid optimization of injection parameters for clinical devices used to effectively and safely inhibit the formation of heterotopic ossification. Key points • MicroCT provides high-resolution quantification of soft-tissue drug distribution. • Distributed dosing is required to maximize soft-tissue drug coverage. • Imaging platform will enable rapid screening of 3D-printed drug distribution prototypes. Graphical Abstrac

Botulinum toxin A-induced muscle paralysis stimulates Hdac4 and differential miRNA expression.

At sufficient dose, intramuscular injection of Botulinum toxin A causes muscle wasting that is physiologically consistent with surgical denervation and other types of neuromuscular dysfunction. The aim of this study was to clarify early molecular and micro-RNA alterations in skeletal muscle following Botulinum toxin A-induced muscle paralysis. Quadriceps were analyzed for changes in expression of micro- and messenger RNA and protein levels after a single injection of 0.4, 2 or 4U Botulinum toxin A (/100g body weight). After injection with 2.0U Botulinum toxin A, quadriceps exhibited significant reduction in muscle weight and increased levels of ubiquitin ligase proteins at 7, 14 and 28 days. Muscle miR-1 and miR-133a/b levels were decreased at these time points, whereas a dose-responsive increase in miR-206 expression at day 14 was observed. Expression of the miR-133a/b target genes RhoA, Tgfb1 and Ctfg, and the miR-1/206 target genes Igf-1 and Hdac4, were upregulated at 28 days after Botulinum toxin A injection. Increased levels of Hdac4 protein were observed after injection, consistent with anticipated expression changes in direct and indirect Hdac4 target genes, such as Myog. Our results suggest Botulinum toxin A-induced denervation of muscle shares molecular characteristics with surgical denervation and other types of neuromuscular dysfunction, and implicates miR-133/Tgf-β1/Ctfg and miR-1/Hdac4/Myog signaling during the resultant muscle atrophy

Systems-Based Identification of Temporal Processing Pathways during Bone Cell Mechanotransduction

<div><p>Bone has long been established to be a highly mechanosensitive tissue. When subjected to mechanical loading, bone exhibits profoundly different anabolic responses depending on the temporal pattern in which the stimulus is applied. This phenomenon has been termed temporal processing, and involves complex signal amplification mechanisms that are largely unidentified. In this study, our goal was to characterize transcriptomic perturbations arising from the insertion of intermittent rest periods (a temporal variation with profound effects on bone anabolism) in osteoblastic cells subjected to fluid flow, and assess the utility of these perturbations to identify signaling pathways that are differentially activated by this temporal variation. At the level of the genome, we found that the common and differential alterations in gene expression arising from the two flow conditions were distributionally distinct, with the differential alterations characterized by many small changes in a large number of genes. Using bioinformatics analysis, we identified distinct up- and down-regulation transcriptomic signatures associated with the insertion of rest intervals, and found that the up-regulation signature was significantly associated with MAPK signaling. Confirming the involvement of the MAPK pathway, we found that the insertion of rest intervals significantly elevated flow-induced p-ERK1/2 levels by enabling a second spike in activity that was not observed in response to continuous flow. Collectively, these studies are the first to characterize distinct transcriptomic perturbations in bone cells subjected to continuous and intermittent stimulation, and directly demonstrate the utility of systems-based transcriptomic analysis to identify novel acute signaling pathways underlying temporal processing in bone cells.</p></div

Rest intervals enhance flow-induced ERK1/2 activation by enabling recurring spikes in activity.

<p>(A) Western blot for p-ERK1/2 and ERK1/2 for cells subjected to no flow (NF), rest-inserted flow (RF), and continuous flow (CF) following cessation of each of the four flow bouts. (B) Quantification of Western blots from three separate experiments. Cells exposed to continuous flow exhibited a single spike in p-ERK1/2. In contrast, cells exposed to rest-inserted flow exhibited multiple spikes in p-ERK1/2 following each flow bout. We observed only modest increases in ERK1/2 activity after the second and fourth bouts but a significant increase after the third bout, suggesting that ERK1/2 may possess a refractory period of longer than 15 min but shorter than 45 min. *,**p<0.05, p<0.01 when compared to no flow at same time point, respectively. #p<0.05 when compared to continuous flow at same time point.</p

Pathways analysis of transcriptomic perturbations implicates MAPK signaling.

<p>(A–B): Results from KEGG analysis for Groups A and B. Group A was significantly associated with MAPK signaling (an intracellular signaling pathway) as well as two pathways associated with extracellular signal transduction: focal adhesion and cytokine-cytokine receptor interactions. Group B was significantly associated with cytokine-cytokine receptor interactions only. *Bayes Factor >6 (C–D): Results from SPEED analysis for Groups A and B. SPEED analysis revealed a significant overlap in Group A genes with the MAPK gene expression signature, and a significant overlap of Group B genes with the TNF-alpha pathway. *FDR <0.05.</p

Continuous and rest-inserted fluid flow give rise to acute differences in gene expression that are highly transient.

<p>(A) Panel of 20 genes screened for differential expression immediately following cessation of continuous or rest-inserted flow. Though most of the genes assessed did not exhibit significant alterations by the insertion of rest intervals, we observed two sub-clusters (highlighted in blue and yellow) that exhibited detectable differences in expression following rest-inserted and continuous flow. Of these six genes, four were found to be significantly different under the two flow conditions: OPG, RUNX2, DSCR1, and OPN. (B–D) Time course of gene expression for these four genes following exposure to one hour of continuous or rest-inserted flow. Differences in expression observed immediately following continuous or rest-inserted flow were highly transient and tended to be focused within the 0–1 h period follow cessation of the flow. *p<0.05, **p<0.01, ***p<0.001 for rest-inserted flow vs. continuous flow.</p

Lower-dose CsA supplementation was required to amplify bone response to rest-inserted loading.

<p>Periosteal MS/BS (a), MAR (b), and BFR/BS (c; mean+s.e.) were significantly increased in experimentally loaded (X) versus contralateral controls (C; ‘+’). Supplementing rest-inserted loading, but not cyclic loading, with low-dose CsA (at 0.1 mg/kg) significantly enhanced MAR and BFR/BS compared with that induced by rest-inserted loading alone (‘#’). Additionally, CsA supplementation enhanced MAR (at 0.1, 0.3 mg/kg) and BFR/BS (at 0.1 mg/kg) in animals subject to rest-inserted versus cyclic loading (‘$’).</p