Density and hydration of fresh and fixed human skeletal muscle

Abstract

The maximum tetanic tension of skeletal muscle (P₀) is often estimated based on calculation of physiological cross-sectional area (PCSA). PCSA depends on muscle volume, pennation angle, and fiber length. Studies documenting PCSA in fixed human muscles usually compute muscle volume by dividing muscle mass by density. These studies use a density value of 1.0597 g/cm³, which was originally based on unfixed rabbit and canine muscle tissue. Due to the dehydration effects of different fixation methods, the variable hydration that occurs when fixed tissue is stored in buffered saline, and the potential for species-specific muscle density, this value may be incorrect and an accurate value for fixed human muscle density is needed. To obtain an accurate density and water content values, 4% formaldehyde-fixed ($n=54$) and 37% formaldehyde-fixed ($n=54$) cadaveric human muscle samples were divided into 6 groups (0, 6, 12, 18, 24, or 30 h) for hydration in phosphate buffered saline (PBS). Measurements of volume, water content, and mass were made enabling calculation of muscle density. Additionally, water content was measured in living muscle ($n=4$) to determine the appropriate hydration time in PBS. Comparisons among groups demonstrated a significant increase in muscle water content and muscle volume over time, reaching living tissue levels after 24 h, but, interestingly, the hydration process did not affect muscle density. These data yield a density value (mean±SE) of 1.112±0.006 g/cm³ in 4% formaldehyde-fixed muscle and 1.055±0.006 g/cm³ in 37% formaldehyde-fixed muscle. These results indicate that the use of inappropriate hydration times or density values can produce PCSA errors of 5–10%.

Introduction

Muscle function is often inferred from its architecture. One key parameter of muscle function is maximum tension (P₀) or maximum force producing capacity. Given the difficulty of directly measuring this value in humans, muscle architecture is often used to estimate P₀. Specifically, physiological cross-sectional area (PCSA) has been shown to be an excellent predictor of P₀ and is calculated using the following equation (Powell et al., 1984; Sacks and Roy, 1982).

$${\mathrm{PCSA}(\mathrm{cm}}^2)=\frac{\mathrm{Muscle}\mathrm{mass}(\mathrm{g})\cos (\theta )}{\rho {(\mathrm{g}/\mathrm{cm}}^3)\mathrm{fiber}\mathrm{length}(\mathrm{cm})},$$

where muscle density (ρ) is 1.0597 g/cm³ (Mendez and Keys, 1960) and θ is fiber pennation angle.

Current biomechanical modeling techniques rely on PCSA to estimate peak muscle force production during a task (Anderson and Pandy, 2003; Buchanan and Shreeve, 1996). However, Brand and colleagues (Brand et al., 1986) demonstrated that muscle force predictions are highly sensitive to changes in PCSA, therefore, it seems apparent that the accuracy of measured values used to compute PCSA is important. Muscle architecture reports typically do not directly measure muscle density (Lieber et al., 1992; Wickiewicz et al., 1983). Instead, most studies use the value 1.0597 g/cm³, which was derived from unfixed rabbit and canine muscle tissue (Mendez and Keys, 1960).

Given the fact that human muscle architecture is often characterized in formaldehyde-fixed tissue, this previously defined value may be inaccurate for several reasons. First, a species effect may exist so that rabbit or canine muscle density may differ from human muscle density. Second, the method and duration of fixation may cause shrinkage and thus dehydration, which may alter muscle density. Finally, the time in which stored muscle samples hydrate in buffered saline may affect volume and thus density. If these variables affect density either separately or in combination, current estimates of muscle PCSA and thus predictions of muscle force may be inaccurate. Thus, the purpose of this study was to measure muscle density directly as a function of fixation method and hydration time in human skeletal muscle.