How does tissue preparation affect skeletal muscle transverse isotropy?

Abstract

The passive tensile properties of skeletal muscle play a key role in its physiological function. Previous research has identified conflicting reports of muscle transverse isotropy, with some data suggesting the longitudinal direction is stiffest, while others show the transverse direction is stiffest. Accurate constitutive models of skeletal muscle must be employed to provide correct recommendations for and observations of clinical methods. The goal of this work was to identify transversely isotropic tensile muscle properties as a function of post mortem handling. Six pairs of tibialis anterior muscles were harvested from Giant Flemish rabbits and split into two groups: fresh testing (within four hours post mortem), and non-fresh testing (subject to delayed testing and a freeze/thaw cycle). Longitudinal and transverse samples were removed from each muscle and tested to identify tensile modulus and relaxation behavior. Longitudinal non-fresh samples exhibited a higher initial modulus value and faster relaxation than longitudinal fresh, transverse fresh, and transverse rigor samples (p<0.05), while longitudinal fresh samples were less stiff at lower strain levels than longitudinal non-fresh, transverse fresh, and transverse non-fresh samples (p<0.05), but exhibited more nonlinear behavior. While fresh skeletal muscle exhibits a higher transverse modulus than longitudinal modulus, discrepancies in previously published data may be the result of a number of differences in experimental protocol. Constitutive modeling of fresh muscle should reflect these data by identifying the material as truly transversely isotropic and not as an isotropic matrix reinforced with fibers.

Keywords: Constitutive modeling; Material testing; Mechanical properties; Rigor mortis.

Figure 1

A) Specimen groupings and testing timeline, showing the fresh testing group and the group subject to non-fresh conditions and a freeze/thaw cycle. Each of these groups yielded longitudinal and transverse samples for a total of four groups. (B) Dissection orientations show that each muscle yielded two samples, one in the longitudinal direction and one in the transverse direction.

Figure 2

Specimen testing procedures. A: Experimental setup showing speckled sample with gauge length (black arrow), three width measurement locations (white arrows), and digital image correlation region of interest (DIC ROI – light blue dotted box). B: Testing outline (not to scale) highlighting initial ramp phase, relaxation phase, and final ramp phase to failure (strain of 0.3 given as an example), with representative stress shown as solid black line and strain in dotted gray line.

Figure 3

A) Raw data results showing stress relaxation step stress-time data with standard deviation. The loading phase and initial relaxation are highlighted for clarity on left with no standard deviation. (B) Quasi-static testing stress-strain data with standard deviation. (C) Moduli values with standard deviation for all four groups at the peak of the stress relaxation step, at equilibrium of the stress relaxation step, and at 20% strain of the quasi-static testing phase (* denotes statistically different from longitudinal fresh samples and # denotes different from longitudinal non-fresh samples, p<0.05).

Figure 4

A) Logarithmic plot of the mean relaxation behavior for the four experimental groups. (B) Mean and standard deviation power law b values for all four groups (# denotes significantly different than longitudinal non-fresh samples, p<0.05). (C) Mean and standard deviation relaxation ratio for all four groups between 0–5 seconds, 5–50 seconds, and 50–300 seconds (# denotes significantly different than longitudinal non-fresh samples, p<0.05).