Functional analysis of limb recovery following autograft treatment of volumetric muscle loss in the quadriceps femoris

Abstract

Severe injuries to the extremities often result in muscle trauma and, in some cases, significant volumetric muscle loss (VML). These injuries continue to be challenging to treat, with few available clinical options, a high rate of complications, and often persistent loss of limb function. To facilitate the testing of regenerative strategies for skeletal muscle, we developed a novel quadriceps VML model in the rat, specifically addressing functional recovery of the limb. Our outcome measures included muscle contractility measurements to assess muscle function and gait analysis for evaluation of overall limb function. We also investigated treatment with muscle autografts, whole or minced, to promote regeneration of the defect area. Our defect model resulted in a loss of muscle function, with injured legs generating less than 55% of muscle strength from the contralateral uninjured control legs, even at 4 weeks post-injury. The autograft treatments did not result in significant recovery of muscle function. Measures of static and dynamic gait were significantly decreased in the untreated, empty defect group, indicating a decrease in limb function. Histological sections of the affected muscles showed extensive fibrosis, suggesting that this scarring of the muscle may be in part the cause of the loss of muscle function in this VML model. Taken together, these data are consistent with clinical findings of reduced muscle function in large VML injuries. This new model with quantitative functional outcome measures offers a platform on which to evaluate treatment strategies designed to regenerate muscle tissue volume and restore limb function.

Keywords: Gait analysis; Muscle autograft; Muscle functional testing; Preclinical model; Volumetric muscle loss.

Figure 1. Methods

A. Representative images showing the muscle defect surgery (top) and excised 8-mm diameter muscle (bottom). B. Representative diagram of the top-down view of muscle strength measurements. C. Representative diagram of the side view of muscle strength measurements. D. Timeline of longitudinal in vivo (MRI, Catwalk) and ex vivo (muscle torque, muscle mass, histology) measurements, with samples sizes, for the whole study.

Figure 2. MRI Visualization of the Extent of Muscle Injury

A. Representative selection of region of interest using T1-weighted image. B. Representative superposition of ROI on thresholded T2-weighted image. Dark pixels indicate pixels with intensities above threshold. C. Total muscle volume, measured using axial T₁-weighted MRI images. D. Percentage of the left leg muscle that was inflamed, as determined by edema in the quadriceps area visualized by T₂-weighted images. E-H. Representative T₂-weighted images at days 2 (E), 4 (F), 7 (G), and 14 (H) days are shown, with inflamed areas indicated by white arrows. *p<0.05, n=3, ANOVA with Bonferroni correction.

Figure 3. Quantitative Measures of Muscle Function

A. Representative graph of tetanic muscle torque measurement for empty defect (dotted), morselized autograft (dash), whole autograft (dash-dot-dash), and unoperated control (solid) groups. B. Maximum isometric tetanic torque of the quadriceps muscle in the injured left leg (I) and contralateral control right leg (C). C. Muscle torque expressed as a ratio of the injured leg to contralateral control (I/C). *p<0.05, n=6 per group per time point.

Figure 4. Muscle Mass and Representative Cross-Sectional Images of Muscle

A. Post-mortem muscle mass of the quadriceps muscle from the injured left leg (I) and contralateral control right leg (C). B. Post-mortem muscle mass expressed as a ratio of the injured leg to contralateral control (I/C). C. Representative H&E stained transverse (cross-sections) images of muscles from all treatment groups and control. *p<0.05, n=6 per group per time point.

Figure 5. Quantitative Measures of Gait and Limb Recovery

A. Print area, a static gait parameter, at baseline (measured prior to surgery), 2 weeks, and 4 weeks post injury. Representative paw prints are shown on the right. B. Duty cycle, a dynamic gait parameter that measures the time a paw is in contact with the ground as compared to stride duration. *p<0.05, n=12 at 2 weeks, n=6 at 4 weeks

Figure 6. Fibrosis and regenerating fibers, visualized in histology

Fibrosis can be seen in Masson’s Trichrome stained sections (collagen fibers stain blue). Regenerating muscle fibers, in which nuclei are centrally located, can be seen in H&E stained sections (indicated by black arrows). Images were taken at 10x magnification, scale bars: 100μm.