Analysis of ischemic muscle in patients with peripheral artery disease using X-ray spectroscopy

Abstract

Background: Peripheral artery disease (PAD) is a vascular disease caused by atherosclerosis, resulting in decreased blood flow to the lower extremities. The ankle-brachial index (ABI) is a standard PAD diagnostic test but only identifies reduced blood flow based on blood pressure differences. The early signs of PAD manifest themselves not only at a clinical level but also at an elemental and biochemical level. However, the biochemical and elemental alterations to PAD muscle are not well understood. The objective of this study was to compare fundamental changes in intracellular elemental compositions between control, claudicating, and critical limb ischemia muscle tissue.

Materials and methods: Gastrocnemius biopsies from three subjects including one control (ABI ≥ 0.9), one claudicating (0.4 ≤ ABI < 0.9), and one critical limb ischemia patient (ABI < 0.4) were evaluated using a scanning electron microscope and energy dispersive X-ray spectroscopy to quantify differences in elemental compositions. Spectra were collected for five myofibers per specimen. An analysis of variance was performed to identify significant differences in muscle elemental compositions.

Results: This study revealed that intracellular magnesium and calcium were lower in PAD compared with control myofibers, whereas sulfur was higher. Magnesium and calcium are antagonistic, meaning, if magnesium concentrations go down calcium concentrations should go up. However, our findings do not support this antagonism in PAD. Our analysis found decreases in sodium and potassium, in PAD myofibers.

Conclusions: These findings may provide insight into the pathologic mechanisms that may operate in ischemic muscle and aid in the development of specialized preventive and rehabilitative treatment plans for PAD patients.

Keywords: Atherosclerosis; Muscle damage; Scanning electron microscopy; Vascular disease; X-ray microanalysis.

Figure 1

Morphological changes that occur in PAD muscle as the disease progresses. Normal muscle morphometry consists of uniform myofiber size and shape. A) Representative myosin labeled images of control, claudicating, and CLI muscle with myofiber outlines are overlaid in green. B) Segmented binary image of the myofibers. In claudicating and CLI muscle, myofibers become more irregular, the endomysium and perimysium thicken, and atrophy is observed. In CLI muscle, there is an increase in muscle features that have been associated with stage of PAD. Specifically, key changes such as increased variation in size and shape and clustering of irregular smaller myofibers can be observed. C) Overlay image of the sarcolemma combined with the outlined myofibers.

Figure 2

Scanning electron microscope (SEM) images of CLI (A–B) and claudicating (C–D) PAD myofiber cross-sections. A) Presents a section at the fascicle level, B–C) presents muscle tissue at the myofiber level, and D) an image at the myofibril level.

Figure 3

A) Elemental maps of Carbon (C), Oxygen (O), Calcium (Ca), Potassium (K), Sodium (Na), Magnesium (Mg), and Sulfur (S) of PAD muscle at the fascicle level. B) Energy dispersive X-ray spectroscopy (EDS) micro-analysis spectral profile from the intracellular elemental contents from a region of interest inside the myofiber. The region of interest that was measured is shown within the magenta box displayed in the scanning electron micrograph.

Figure 4

An analysis of variance (ANOVA) identified significant differences in elemental composition between the muscle fibers analyzed from the critical limb ischemia patient, the claudicating patient, and the control patient. Decreases in A) Calcium (p=0.003), B) Magnesium (p=0.0001), C) Potassium (p=0.0094), and D) Sodium (p=0.0001), and an increase in E) Sulfur (p=0.004) were observed. (** represents a P ≤ 0.01, *** represent a P ≤ 0.001)