A novel murine muscle loading model to investigate Achilles musculotendinous adaptation

Abstract

Achilles tendinopathy is a debilitating condition affecting the entire spectrum of society and a condition that increases the risk of tendon rupture. Effective therapies remain elusive, as anti-inflammatory drugs and surgical interventions show poor long-term outcomes. Eccentric loading of the Achilles muscle-tendon unit is an effective physical therapy for treatment of symptomatic human tendinopathy. Here, we introduce a novel mouse model of hindlimb muscle loading designed to achieve a tissue-targeted therapeutic exercise. This model includes the application of tissue (muscle and tendon)-loading "doses," coupled with ankle dorsiflexion and plantarflexion, inspired by human clinical protocols. Under computer control, the foot was rotated through the entire ankle joint range of motion while the plantar flexors simultaneously contracted to simulate body mass loading, consistent with human therapeutic exercises. This approach achieved two key components of the heel drop and raise movement: ankle range of motion coupled with body mass loading. Model development entailed the tuning of parameters such as footplate speed, number of repetitions, number of sets of repetitions, treatment frequency, treatment duration, and treatment timing. Initial model development was carried out on uninjured mice to define a protocol that was well tolerated and nondeleterious to tendon biomechanical function. When applied to a murine Achilles tendinopathy model, muscle loading led to a significant improvement in biomechanical outcome measures, with a decrease in cross-sectional area and an increase in material properties, compared with untreated animals. Our model facilitates the future investigation of mechanisms whereby rehabilitative muscle loading promotes healing of Achilles tendon injuries.NEW & NOTEWORTHY We introduce a novel mouse model of hindlimb muscle loading designed to achieve a tissue-targeted therapeutic exercise. This innovative model allows for application of muscle loading "doses," coupled with ankle dorsiflexion and plantarflexion, inspired by human loading clinical treatment. Our model facilitates future investigation of mechanisms whereby rehabilitative muscle loading promotes healing of Achilles tendon injuries.

Keywords: Achilles; eccentric; muscle loading; preclinical; tendinopathy.

Figure 1.

A: image of the human exercise treatment modeled by the murine protocol showing dorsiflexion (“heel drop,” left) and plantarflexion (“heel raise,” right). B: murine heel drop/raise experimental setup: a: footplate to rotate ankle through range of motion, b: electrodes to control nerve stimulation. C: overview of the foot plate movement and electrode stimulation profiles. Electrodes were stimulated in conjunction with the foot plate movement to replicate body weight loading treatment. “Length” corresponds to the movement of the footplate, with a negative value denoting dorsiflexion and positive denoting plantarflexion. Profile shown illustrates one repetition of the exercise protocol.

Figure 2.

Tendinopathy induction and treatment timeline. Each treatment session consisted of 3 sets of 10 repetitions (100 Hz stimulation) and was administered twice weekly.

Figure 3.

Biomechanical outcome measures following 3 wk of muscle loading treatment (twice weekly). Treatments showed no statistically significant changes in structural, geometric, or material properties compared with naïve animals. Error bars denote means ± SD. Each scatterplot symbol denotes data from an individual mouse Achilles tendon. P < 0.05 was deemed to be statistically significant.

Figure 4.

A: biomechanical outcome measures for 2 stimulation frequencies (50 vs. 100 Hz, applied to uninjured tendon). No significant differences between either treatment group compared with naïve untreated animals. B: hematoxylin-eosin (H&E) images of the myotendinous region. Top: sham control. Middle: 50-Hz stimulation. Bottom: 100-Hz stimulation. Left panel: 10x; middle panel: 40x; right panel: higher magnification insets of area in box in middle panel. M, muscle; T, tendon. Scale bars, 100 µm. C: muscle strength outcomes following 2 wk of treatment. *Significant differences from other groups. Errors bars denote means ± SD. Each scatterplot symbol denotes data from an individual mouse Achilles tendon. P < 0.05 was deemed to be statistically significant.

Figure 5.

A: comparison of tendon biomechanical properties following early and delayed treatment of tendinopathy. Symbols above groups denote statistically significant differences (P < 0.05) compared with all other groups. Horizontal lines denote significant differences between 2 individual groups. Inj, injury; E, early; D, delayed; Trt, treatment. Early muscle-loading treatment significantly decreased cross-sectional area (CSA) and increased material properties relative to untreated injury. No significant differences were observed for structural properties, stiffness, and maximum load (data not shown). B: muscle strength outcomes following 2 wk of early treatment. Errors bars denote means ± SD. Each scatterplot symbol denotes data from an individual mouse Achilles tendon. P < 0.05 was deemed to be statistically significant. *,#Significant differences from all other groups.