## 4 Discussion
### Extracranial Sites  
The human arm is a heterogeneous structure composed of layers with distinct optical properties. It is covered by skin and appendages, has a subcutaneous adipose layer between the skin and muscles, and contains an underlying bone, the humerus, all of which are variably vascularized. Due to its low absorption, NIR light can penetrate several centimeters into biological tissues, allowing for non-invasive extraction of information from deeper structures. Previous studies have already demonstrated that NIR reflectance spectra acquired from the ventral and dorsal aspect of the arm mainly reflect the current biology of the biceps and triceps, and that the ability to distinguish between muscles is based on features like muscle architecture, metabolic enzyme content, and contractile protein expression [3; 4]. Spectral differences observed in biceps and triceps between patients with MS and healthy controls highlight significant biochemical and structural changes linked to the disease. These alterations at specific wavelengths (i.e., 1000 nm, 1800-1950 nm, and 2200-2300 nm) shed light on systemic manifestations of MS extending beyond CNS pathology.  
Spectral features at 1000 nm reflect changes in lipid and protein composition within muscle tissue. This wavelength corresponds to second overtone C-H stretching vibrations in fatty acids and methyl/methylene groups in various lipids. In MS, muscle atrophy and inflammatory stress alter lipid metabolism and protein density [39]. Muscle atrophy arises from decreased mobility [40], leading to reduced muscle mass and altered storage of lipids, while systemic inflammation promotes lipolysis and protein breakdown. These changes contribute to the observed spectral shifts at 1000 nm, providing a window into the systemic effects of MS on muscle tissue.  
In 1800-1950 nm, the absorption is primarily due to elevated water content and collagen remodeling. The first overtone of O-H stretching vibrations in this range is sensitive to changes in muscle edema, which is enhanced by systemic inflammatory cytokines in MS [41]. Concurrently, chronic inflammation leads to increased collagen deposition, affecting the scattering properties of muscle tissue. This dual effect (edema and fibrosis) underscores how systemic inflammation in MS impacts muscle integrity, contributing to fatigue and weakness [42].  
The spectral changes at 2200-2300 nm highlight alterations in lipid peroxidation and oxidative stress within muscle tissue. Lipid peroxidation, a hallmark of oxidative damage, leads to the formation of lipid peroxides absorbing in this range. Moreover, the loss or alteration of long-chain fatty acids due to oxidative stress affects the absorption peak. MS-related mitochondrial dysfunction in muscle cells [43] enhances reactive oxygen species (ROS) production, exacerbating lipid peroxidation. This oxidative stress is central to muscle fatigue and weakness in MS patients [44], illustrating how NIR spectroscopy can capture systemic metabolic dysregulation.  
Overall, these findings suggest that NIR spectroscopy-derived biomarkers could complement CNS-centered diagnostic tools by offering insights into systemic disease activity. Muscle weakness in MS is a result of the central neural changes from the primary disease process [45], but over time, patients experience accelerated muscle loss and dysfunction because of the interaction between weakness, fatigue, decreased mobility of paretic limbs [46], and age-related decline. An NIR spectroscopy approach might facilitate the monitoring of systemic MS effects and therapeutic responses beyond traditional CNS measures.