Intramuscular fibrous tissue determines muscle echo intensity in amyotrophic lateral sclerosis

Ultrasound is a rapidly evolving technique that can be used in the diagnostic work-up of neuromuscular disorders. Affected muscles show diminished muscle thickness and increased echo intensity (EI). Previous studies have shown that fat and fibrous tissue each can increase EI. However, the exact cause of increased echo intensity is not fully known. We compared the findings of an in vivo quantitative muscle ultrasound study with the postmortem histopathological examination of a 62-year-old woman who had amyotrophic lateral sclerosis (ALS). This patient died 27 months after symptom onset. On autopsy, the diagnosis of ALS was confirmed. Transverse ultrasound scans and total cross-sectional muscle specimens of nine different muscles were taken from the same standardized anatomical sites (Fig. 1). Autopsy was performed with the informed consent of the patient within 12 hours after death. Muscle specimens were fixed in 4% formalin; the fibers were orientated transversely for embedding in paraffin. The largest cross-sectional area of each sample was selected and cut in 4-lm-thick sections, which were stained with Masson trichrome. The percentages of perimysial and endomysial tissue and the percentage of interstitial fat were determined by digital image analysis. Ultrasound examinations were performed 3 months before the patient’s death. Details of the standardized protocol and system settings have been described elsewhere. EI was determined quantitatively using histogram analysis, where the mean gray value of the muscle is expressed as a value between 0 (1⁄4 black) and 255 (1⁄4 white). We found that EI was increased in all muscles (mean 73, range 52–91) to about twice the normal value (range 28–42). Histological examination showed a comparable percentage of intramuscular fat (median 8.6%, range 4.9– 16.2%) and fibrous tissue (12.5%, range 6.7–28.5%; Fig. 1D). Fibrous tissue seemed to be more evenly distributed within the muscle, whereas fat was seen in small collections (Fig. 1C). The sternocleidomastoid muscle was considered an outlier and was not used for calculation of correlations. EI was strongly correlated with fibrous tissue (r 1⁄4 0.86, P 1⁄4 0.007; Fig. 1D), and this correlation remained after correction for fat content (r 1⁄4 0.81, P 1⁄4 0.026). EI and the amount of fatty tissue were not correlated (r 1⁄4 0.46, P 1⁄4 0.248), nor was there a correlation after correction for fibrous tissue (r 1⁄4 0.07, P 1⁄4 0.886). In both this study of a human neurogenic disorder and in a study of canine muscular dystrophy, fibrous tissue was the main contributing factor to increased muscle EI. However, other studies have indicated that intramuscular fat also contributes to increased EI, as shown in patients with inflammatory myopathies, in those with little or no fibrous tissue, and in obese subjects. In this study the amounts of fibrous and fatty tissue were comparable, making it possible to study the influence of both variables on EI simultaneously. The influence of fibrous tissue on EI appeared to be stronger than that of fat, possibly due to differences in their distribution within the muscle. Overall, this suggests that fibrosis is the most important determinant of muscle EI in ALS.