Muscle does not drive persistent posttraumatic elbow contracture in a rat model

Abstract

Introduction: Posttraumatic elbow contracture is clinically challenging because injury often disrupts multiple periarticular soft tissues. Tissue specific contribution to contracture, particularly muscle, remains poorly understood.

Methods: In this study we used a previously developed animal model of elbow contracture. After surgically inducing a unilateral soft tissue injury, injured limbs were immobilized for 3, 7, 21, and 42 days (IM) or for 42 IM with 42 days of free mobilization (42/42 IM-FM). Biceps brachii active/passive mechanics and morphology were evaluated at 42 IM and 42/42 IM-FM, whereas biceps brachii and brachialis gene expression was evaluated at all time points.

Results: Injured limb muscle exhibited significantly altered active/passive mechanics and decreased fiber area at 42 IM but returned to control levels by 42/42 IM-FM. Gene expression suggested muscle growth rather than a fibrotic response at 42/42 IM-FM.

Discussion: Muscle is a transient contributor to motion loss in our rat model of posttraumatic elbow contracture. Muscle Nerve 58:843-851, 2018.

Keywords: contracture; elbow; fibrosis; gene expression; mechanics; muscle.

Figure 1.

(A) Active stress of injured limbs biceps brachii at 42 IM was significantly decreased compared to control (average ± std; * p < 0.05). (B) Peak and (C) equilibrium passive stress-strain curves of the biceps brachii for control (squares) and injured limbs (circles); only includes strains at which all samples have data. Evaluation was completed at 42 IM and 42/42 IM-FM. Only 42 IM injured limbs exhibited significantly different stress compared to control; 42/42 IM-FM was not different compared to control (average ± std; * p < 0.05 and ** p < 0.01 for 42 IM only). (D) Peak and (E) equilibrium passive stress-muscle length curves of the biceps brachii for control and injured limbs qualitatively represent the effect of muscle shortening.

Figure 2.

Biceps brachii (A) muscle length at maximum tetanic contraction was only significantly decreased at 42 IM compared to control, but (B) muscle weight was not significantly different at either time point (average ± std; * p < 0.05, ** p < 0.01).

Figure 3.

(A) Representative transverse histology sections of the biceps brachii (hematoxylin and eosin) from control and injured limbs at 42 IM and 42/42 IM-FM (scale bar = 200μm). Quantitative measures computed from fluorescent muscle sections: (B) fiber area (μm²) and (C) extracellular matrix (ECM) area fraction for injured and control limbs at 42 IM and 42/42 IM-FM. While there were no significant changes in ECM area fraction at either time point, fiber area was significantly decreased at 42 IM compared to its respective control (average ± std; * p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 4.

qPCR: Fold change in the (A) biceps brachii and (B) brachialis muscle of injured limbs evaluating expression of genes for fibrosis, extracellular matrix (ECM) regulators, collagen, myogenesis, and proteoglycans (yellow and blue correspond to increased or decreased expression, respectively, while white represents no change relative to control). (C) Left: One-way ANOVA during immobilization only, including a heatmap representing p values. Right: Results of unpaired t-tests that compared injured limbs at 42 IM and 42/42 IM-FM. White boxes represent when the p value was not significant (BI = biceps brachii, BR = brachialis).

Figure 5.

qPCR: Fold change in the (A-E) biceps brachii and (F-J) brachialis muscle of injured limbs showing expression of (A,F) transforming growth factor beta-3 (TGFβ3), (B,G) connective tissue growth factor (CTGF), (C,H) collagen type I (Col I), (D,I) collagen type IV (Col IV), and (E,J) myostatin (MSTN). (average ± std; *p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).