## 3 Results
Assignments of the most relevant bands were performed according to relevant literature [39, 41, 42, 43, 44, 45] and taking into account the biological nature of the samples: in this light, bands were not merely related to chemical groups, but also the most represented biomolecules within tissue samples were taken into account, leading us to divide, as commonly accepted, the biological infrared spectrum into regions assigned to specific groups of molecules [30, 39]. The most significant bands are as follows: \(\sim\)3010 cm\({}^{-1}\) (stretching vibration of =CH moiety in lipid alkyl chains); \(\sim\)2956 cm\({}^{-1}\) and \(\sim\)2870 cm\({}^{-1}\) (asymmetric and symmetric stretching vibrations of CH\({}_{3}\) groups in lipid alkyl chains); \(\sim\)2921 cm\({}^{-1}\) and \(\sim\)2850 cm\({}^{-1}\) (asymmetric and symmetric stretching vibrations of CH\({}_{2}\) groups in lipid alkyl chains); \(\sim\)1735 cm\({}^{-1}\) (stretching vibration of C=O ester moiety in fatty acids), and \(\sim\)1650 cm\({}^{-1}\) and \(\sim\)1560 cm\({}^{-1}\) (Amide I and II band of proteins). Figure 2: Imaging analysis of representative **A** and **C** sections of **Y** and **O** experimental groups. (**a**) Histological images stained with the Luxol Fast Blue sec. Klüwer-Barrera kit (myelin is colored in turquoise color, while neurons and glial nuclei are violet; scale bar 50 \(\upmu\)m, magnification 20\(\times\)). (**b**) Microphotographs and corresponding false-color images showing the topographical distribution of (**c**) saturated (SAT) and (**d**) unsaturated (UNSAT) lipids, (**e**) fatty acids (FA), and (**f**) proteins (PRT). False-color images were composed of 4096 pixel/spectra with a spatial resolution of 2.56 \(\upmu\)m; the dimensions were 164 \(\times\) 164 \(\upmu\)m. Different scales were adopted to highlight the different macromolecular compounds (blue color indicates areas with the lowest absorbance values, while pink/white colors the highest ones). The macromolecular composition of the genu (**A**) and splenium (**C**) regions was better explored, also in relation with the age of donor subjects, by statistically analyzing the following band area ratios (Figure 4): LIP/TBM (calculated as the ratio between the integrated areas of the range 3030-2800 cm\({}^{-1}\) and the sum of the ranges 3030-2800 cm\({}^{-1}\) and 1780-900 cm\({}^{-1}\), considered as the Total BioMass, TBM); FA/LIP (calculated as the ratio between the integrated areas of the 1780-1700 cm\({}^{-1}\) and 3030-2800 cm\({}^{-1}\) ranges); PRT/TBM (calculated as the ratio between the integrated areas of the 1700-1480 cm\({}^{-1}\) and TBM, already defined); and LIP/PRT (calculated as the ratio between the integrated areas of the 3030-2800 cm\({}^{-1}\) and 1700-1480 cm\({}^{-1}\) ranges). In particular, (i) by comparing **AY** and **AO** samples, significantly lower levels of lipids and fatty acids were found in **AO** ones, as suggested by the LIP/TBM (total amount of lipids), FA/LIP (representative of the fatty acids fraction), and LIP/PRT (relative amount of lipids to proteins) ratios (\(p<0.05\)). Conversely, no different amounts of proteins were found (PRT/TBM; \(p>0.05\)). (ii) A similar trend was observed by comparing **CY** and **CO** samples, with a lower amount of lipids and fatty acids in **CO** ones (LIP/TBM, FA/LIP, and LIP/PRT; \(p<0.05\)); in this case, no statistically significant difference was found in the protein component (PRT/TBM; \(p>0.05\)). (iii) With regard to the composition of the two regions, higher amounts of lipids and fatty acids were found in **CY** than **AY**, and in **CO** than **AO** (LIP/TBM, FA/LIP, and LIP/PRT; \(p<0.05\)); no statistically significant difference was found between **AY** and **CY**, nor between **AO** and **CO** with regard to the protein component (PRT/TBM; \(p>0.05\)). Figure 3: Average IR spectra of genu (**A**, blue line) and splenium (**C**, red line) regions of **Y** and **O** CC samples.