Histopathological, biomechanical, and behavioral pain findings of Achilles tendinopathy using an animal model of overuse injury

Abstract

Animal models of forced running are used to study overuse tendinopathy, a common health problem for which clear evidence for effective and accessible treatments is still lacking. In these models, pain evaluation is necessary to better understand the disease, help design and evaluate therapies, and ensure humane treatment of the animals. Therefore, the main objective of this study was to evaluate pain and pathologic findings in an animal model of moderate Achilles tendinopathy induced by treadmill running. Air puffs, instead of electrical shocks, were used to stimulate running so that pain associated with stimulation would be avoided. Pressure pain sensitivity was evaluated in vivo using a new instrumented plier, whereas spinal cord peptides were analyzed ex vivo with high-performance liquid chromatography tandem mass spectrometry. Tendon histologic slides were semiquantitatively evaluated, using the Bonar score technique and biomechanical properties, using the traction test. After 8 weeks of treadmill running (2 weeks for adaptation and 6 weeks for the lesion protocol), the protocol was stopped because the air puffs became ineffective to stimulate running. We, nevertheless, observed some histologic changes characteristic of overuse tendinopathy as well as decreased mechanical properties, increased Substance P and dynorphin A peptides but without pressure pain sensitivity. These results suggest that air-puffs stimulation is sufficient to induce an early stage tendinopathy to study new therapeutic drugs without inducing unnecessary pain. They also indicate that pain-associated peptides could be related with movement evoked pain and with the sharp breakdown of the running performance.

Keywords: Air‐puff stimulation; Bonar score; Randall–Selitto test; calcitonin gene‐related peptide; dynorphin A; instrumented plier; mechanical properties; substance P; treadmill.

Figure 1.

Custom treadmill. Five rats run on a polyurethane belt in separate corridors. To stimulate running, resting rats are detected by infrared sensors which trigger the injection of air puffs via the opening of solenoid valves connected to a cylinder of compressed air.

Figure 2.

Instrumented plier for the evaluation of pressure pain sensitivity in the Achilles tendon in vivo. The pressure on the rat tendon induces the bending of the measuring beam. The resulting strain is measured with two strain gages in half bridge.

Figure 3.

Schematic representation of a longitudinal tendon section showing the position of the two ROIs for histologic analysis.

Figure 4.

Progression of the running experiment including 2 weeks of adaptation and 6 weeks of running. Rats ran, on the average, a total of 36.6 km. The experiment was stopped because the stimulation became too high. The number of air puffs followed an exponential curve and reached 15 air puffs/min (900 air puffs/h). Data are shown as mean ± SEM.

Figure 5.

Micrographs of the proximal ROI of Achilles tendons (A–H; Bar = 200 μm) and micrographs of knee sections (I–J; Bar = 500 μm; Tib tibia, m meniscus, f femur) for a control rat and a runner. Arrows indicate blood vessels, asterisks indicate collagen disorganization, and arrow head indicate round nucleus and abundant cytoplasm. In the example shown, the Bonar score for the runner tendon is: Nucleus morphology: 3, Cytoplasm morphology: 2, Cellular arrangement: 2, Collagen arrangement: 2, Vascularity: 2, Ground substance: 3, Cell density: 64 and the Bonar score for the control tendon is: Nucleus morphology: 0, Cytoplasm morphology: 1, Cellular arrangement: 0.5, Collagen arrangement: 1.5, Vascularity: 2, Ground substance: 1, Cell density: 39. The scores for the control (I) and runner (J) knee histopathology are zero (details of parameters evaluated found in text). Images were subjected to color balance and brightness/contrast adjustments.

Figure 6.

Histologic results for the Achilles tendons of runner and control rats. A–B: Bonar scores. C–D: Cell density. Columns represent the median, and symbols represent data. Encircled symbol represents an outlier removed from the analysis. Horizontal bars stand for significant differences between groups (P < 0.05)

Figure 7.

Biomechanical results for the Achilles tendons of runner and control rats. Columns represent the median, and symbols represent data. Encircled symbols represent outliers removed from the analysis. Horizontal bars stand for significant differences between groups (P < 0.05).

Figure 8.

Pain threshold measured with the instrumented plier for the treadmill and collagenase injection studies. Columns represent the median, and symbols represent data. Horizontal bars stand for significant differences between groups (P < 0.05).

Figure 9.

Spinal cord peptide concentrations for the treadmill and collagenase injection studies. Columns represent the median, and symbols represent data. Horizontal bars stand for significant differences between groups (P < 0.05).