Fiber optic Raman spectroscopy for the evaluation of disease state in Duchenne muscular dystrophy: An assessment using the mdx model and human muscle

Abstract

Introduction/aims: Raman spectroscopy is an emerging technique for the evaluation of muscle disease. In this study we evaluate the ability of in vivo intramuscular Raman spectroscopy to detect the effects of voluntary running in the mdx model of Duchenne muscular dystrophy (DMD). We also compare mdx data with muscle spectra from human DMD patients.

Methods: Thirty 90-day-old mdx mice were randomly allocated to an exercised group (48-hour access to a running wheel) and an unexercised group (n = 15 per group). In vivo Raman spectra were collected from both gastrocnemius muscles and histopathological assessment subsequently performed. Raman data were analyzed using principal component analysis-fed linear discriminant analysis (PCA-LDA). Exercised and unexercised mdx muscle spectra were compared with human DMD samples using cosine similarity.

Results: Exercised mice ran an average of 6.5 km over 48 hours, which induced a significant increase in muscle necrosis (P = .03). PCA-LDA scores were significantly different between the exercised and unexercised groups (P < .0001) and correlated significantly with distance run (P = .01). Raman spectra from exercised mice more closely resembled human spectra than those from unexercised mice.

Discussion: Raman spectroscopy provides a readout of the biochemical alterations in muscle in both the mdx mouse and human DMD muscle.

Keywords: Duchenne muscular dystrophy; Raman spectroscopy; biomarker; exercise; mdx mouse; muscle necrosis.

FIGURE 1

Effect of exercise on mdx muscle pathology. A, Distance run by each mdx mouse in the study. B, There was a significant difference in the percentage of necrosis between exercised and unexercised mice (P = .03). C, Scatterplot shows the relationship between percentage of necrosis and distance run on the running wheel. Although not significant, a clear trend can be observed. D, Low‐magnification image shows regions of necrosis with inflammatory cells (arrows). Relatively spared areas can be seen to the right of the image. E, Higher magnification image of the region shown in C. Small, darkly stained (basophilic) inflammatory cells can be seen (notched arrows), together with hypercontracted and degenerating necrotic myofibers with fragmented sarcoplasm (chevrons). Regenerated fibers with central nuclei can also be observed (arrowheads).

FIGURE 2

In vivo intramuscular Raman spectra in unexercised and exercised mice. A, Average spectra (with standard deviation) of unexercised and exercised mdx mice. B, Linear discriminant function (LDF) loading plot for the unexercised vs exercised comparison. Tentative peak and pathology assignments are shown. C, Nested LDF scores for each mdx mouse. D, Scatterplot shows the relationship between average LDF score for each mdx mouse and distance run on the running wheel.

FIGURE 3

mdx and human ex vivo Raman spectra. A, Average Raman spectra (with standard deviation) for ex vivo mdx gastrocnemius muscle (unexercised and exercised) and human quadriceps muscle. Spectra are vertically offset for clarity. B, Cosine similarity of exercised/unexercised mdx spectra and human muscle. The exercised spectra exhibit a slightly higher degree of similarity with human spectra in the lower wavenumber regions. C, Average Raman spectra (with standard deviation) for ex vivo and in vivo mdx gastrocnemius muscle and human quadriceps muscle. Spectra are vertically offset for clarity. D, Comparison of ex vivo mdx and ex vivo human spectra (plus sign) consistently demonstrates a greater degree of similarity than the comparison between in vivo mdx spectra and ex vivo human spectra (asterisk). This is most apparent in the low‐ to mid‐range wavenumbers (ie, 900 to 1400 cm⁻¹).