## 3 Results  
Selected IR spectra of all the experimental groups were submitted to principal component analysis (PCA) in the 3050-900 cm\({}^{-1}\) range. In Figure 1, the PCA scores plots obtained for the **A**, **B**, and **C** spectral populations of both the **Y** and **O** groups and the corresponding PC1 loadings are displayed. With regard to the CC samples of young specimens (**Y**), only a poor segregation was observed between **AY** and **CY** (explained variance 48.2% along PC1 axis), with **BY** spectra in an intermediate position and overlapping **AY** ones (Figure 1a). A better separation was displayed between **AO** and **CO** (explained variance 15.8% along PC2 axis); in this case, even if more grouped, the **BO** spectra were in the middle, although they were closer to the **AO** ones (Figure 1b). The analysis of PC1 loadings evidenced that the main spectral differences were observed in both **Y** and **O** experimental groups in the 3030-2800 cm\({}^{-1}\) region, which was related to t he vibrational transitions of CH, CH\({}_{2}\), and CH\({}_{3}\) moieties in lipid alkyl chains (grey boxes); to a minor extent, spectral differences were observed in the 1780-1480 cm\({}^{-1}\) region, which was attributable to the vibrational modes of peptide linkage in proteins (pink boxes) (Figure 1c,d). The lack of segregation between the spectral population of CC portions, collected in the body region (**B** spectra) and the genu and splenium ones (**A** and **C** spectra, respectively), observed in the PCA scores plots of both **Y** and **O** experimental groups could be ascribable to the nature of the samples: in fact, there was an objective lack of a definite separation among the three regions within the tissue, and, hence, it is difficult to precisely collect CC portions in the intermediate region. Hence, to obtain reliable and reproducible data, we decided to continue the study focusing only on the anterior (genu, **A**) and posterior (splenium, **C**) regions of young (**Y**) and old (**O**) experimental groups. In Figure 2, the imaging analysis combining histological and spectral images of the genu (**A**) and splenium (**C**) regions of the CC of **Y** and **O** groups is reported. The histological images, obtained using Luxol Fast Blue sec. Kluwer-Barrera staining, which allowed us to highlight the presence of both myelin (turquoise) and neurons and glial nuclei (violet), evidenced a homogeneous turquoise stain in all the analyzed sections, suggesting a ubiquitous presence of myelin. In particular, higher amounts of myelin were observed in the **CY** sections than the **AY** ones, and in **CO** with respect to **AO**; no evident difference was highlighted between **AY** and **AO**, nor between **CY** and **CO**. The analysis of IR images provided additional information regarding the chemical composition and topographical distribution of meaningful macromolecular compounds, such as unsaturated and saturated lipids, fatty acids, and proteins (Figure 2c-f). As expected, the main component of all the analyzed sections was represented by lipids, including the saturated (SAT images, Figure 2c) and unsaturated (UNSAT images, Figure 2d) fractions, as well as fatty acids (FA images, Figure 2e). Interestingly, higher levels of lipids were found in **AY** samples than **AO** ones, and in **CY** samples with respect to **CO** (SAT, UNSAT, and FA images, Figure 2c-e). Moreover, within each group, all the lipid components displayed an increasing gradient from the genu (**A**) to the splenium region (**C**). No meaningful differences were found in the protein content (PRT images, Figure 2f). Figure 1: Principal component analysis of **A**, **B**, and **C** spectral populations of **Y** and **O** CC samples: (**a,b**) PCA scores plots in the 3050–900 cm\({}^{-1}\) range and (**c,d**) corresponding PC1 loadings. In Figure 3, the IR spectra of representative regions of **AY**, **CY**, **AO**, and **CO** are displayed.