## 4 Discussion
### Cranial Sites  
The NIR reflectance spectra acquired from cerebral cortical regions reveal distinct wavelength-specific differences between patients with MS and healthy controls, offering novel insights into the biochemical and microstructural alterations underpinning MS pathology. These spectral variations, observed at 1050, 1500, 1900-2000, and 2200-2250 nm, align with known disease mechanisms and provide a non-invasive means to probe both focal and diffuse tissue damage. Between-group spectral differences emerge prominently at 1050 nm. This wavelength is associated with aromatic (ArCH) or primary amine (RNH2) vibrations and reflects the distribution of proteins and lipids, essential components for the structural integrity of neural tissue. The finding likely reflects demyelination-driven lipid depletion, particularly the loss of myelin-specific galactocerebrosides and sulfatides [26], that exhibit strong absorption due to second overtone C-H stretching vibrations in their methyl (-CH3) and methylene (-CH2-) groups. This finding corroborates histopathological studies demonstrating a 20-30% reduction in myelin lipids within chronic active lesions [27]. Concurrently, elevated scattering coefficients at this wavelength may arise from neurofilament accumulation [28], a hallmark of axonal injury in MS. These observations align with prior work linking neurofilament light chain levels in cerebrospinal fluid to disease activity [29], suggesting NIR spectroscopy could serve as a surrogate for tracking axonal degeneration. Spectral differences found at 1300 nm collected from the cranial brainstem site reflect lipid loss but are mediated by distinct molecular mechanisms and spectral contributions due to a more complex interplay of lipid loss and water accumulation. Third overtone C-H vibrations in long-chain fatty acids (e.g., C24:0 sulfatides) dominate this region, but the signal is confounded by overlapping O-H stretching vibrations from water molecules. In MS, lipid depletion reduces absorption, while concurrent vasogenic edema, driven by blood-brain barrier disruption, increases water content [30], resulting in a flattened or shifted spectral profile. This duality complicates interpretation but provides critical \begin{table}
\begin{tabular}{c c c c c} \hline
**PLS Model** & **Biceps FSS** & **Triceps FSS** & **Brainstem FSS** & **Cortical FSS** \\ \hline
**LVs** & 6 & 3 & 3 & 7 \\ \hline
**RMSEC** & 7.233 & 8.7686 & 4.6228 & 3.4493 \\
**RMSECV** & **7.295** & **8.7293** & **4.6745** & **3.5037** \\ \hline
**Bias C** & 0 & 0 & 0 & 0 \\ \hline
**Bias CV** & **-0.006235** & **-0.000297** & **0.033647** & **-0.000959** \\ \hline
**R\({}^{2}\)c** & 0.737 & 0.618 & 0.894 & 0.941 \\
**R\({}^{2}\)cv** & 0.735 & 0.617 & 0.891 & 0.939 \\ \hline
**Cohen’s \(I^{2}\)** & 2.774 & 1.611 & 8.174 & 15.393 \\ \hline \end{tabular}  
* LVs: latent variable; RMSEC: root mean square error in calibration; RMSECV: root mean square error in cross-validation; Bias C: calibration bias; Bias CV: cross-validation bias; R\({}^{2}\)C: coefficient of determination in calibration; R\({}^{2}\)CV: coefficient of determination in cross-validation.  
\end{table} 
Table 5: Performance parameters for the FSS-based PLS regression models of the spectra collected on biceps, triceps, brainstem, and cortical sites. complementary information; the 1300 nm band captures both structural demyelination and dynamic inflammatory processes. The combined analysis of these wavelengths may enhance diagnostic specificity. For instance, a reduced 1050/1300 nm absorption ratio could potentially distinguish chronic demyelination (predominantly lipid loss) from acute inflammation (predominant edema). This differentiation would have clinical relevance, possibly aiding in the identification of lesion subtypes that might require distinct therapeutic approaches.