## 4 Discussion  
To date, to the best of our knowledge, a complete characterization of the macromolecular composition of CC has not been available in humans, nor in relation to different regions or to the age of the specimen. Hence, in this study, for the first time, hyperspectral imaging analysis of the genu and splenium regions of this commissure has been performed, by exploiting a multidisciplinary approach that combined histological, FTIRI, and multivariate analyses. FTIR spectroscopy is an analytical technique which studies how chemical bonds can change their vibration due to the interaction with an appropriate infrared frequency. It has been first used to identify the functional groups/bonds in homogeneous organic compounds, both solid and liquid. This IR analysis provided an average information on the chemical nature of the sample. In the early 2000s, IR interferometers were coupled with Vis-IR microscopes; this new setup made it possible to also study non-homogeneous samples, such as biological ones (tissues and cells). Concerning the tissues, the microscope allows an investigation of the morphology of thin sections (7-10 microns) and detection of the areas of interest on which the IR analysis can be performed. In these positions, the FPA detector acquired IR images of 164 \(\times\) 164 \(\upmu\)m constituted of 4096 pixel/spectra with a spatial resolution of 2.56 \(\upmu\)m. Thus, this technique allowed us to perform a morphological analysis of tissues and cells, in order to investigate not only the morphology of the sample but also its chemical composition. Figure 6: Statistical analysis of the protein component of **AY**, **CY**, **AO**, and **CO** samples: ALPHA (helix structures), BETA (\(\beta\) sheet structures), RANDOM (random coil structures), and FOLDED/UNFOLDED (folded to unfolded structures). Asterisks indicate statistically significant differences between **A** and **C** regions within the same group (n.s. \(p\) > 0.05; \(\star\)\(p\) < 0.05; \(\star\)\(p\) < 0.01). Different lowercase letters over histograms were used to indicate significant differences between **Y** and **O** groups (\(p\) < 0.05). Since corpus callosum is mainly made up of myelinated axons, whose sheath contains lipids and proteins in different proportions, the analysis was focused on saturated and unsaturated lipids, fatty acids, and proteins. The multivariate analysis performed on the IR data allowed us to achieve some interesting information on the spectral populations of the genu, body, and splenium regions, also in relation with the age at death of the specimen. In fact, a good segregation was observed only between the spectra collected in the genu and splenium regions, and this was more evident in old than young samples; in these two regions, as expected, the main differences were ascribable to the lipid and protein components. Given the high similarity between the spectra of the body and those of the other two regions, they were not included in further analysis, which was focused then on the genu and splenium regions. The histological images evidenced a regular tissue organization in both the genu and splenium regions, which was also confirmed by the hyperspectral imaging analysis, which displayed, in these two CC areas, a homogeneous distribution of saturated, unsaturated lipids, and fatty acids. In agreement with previous observations, both analyses suggested a greater amount of lipids, and, hence, of myelin in the posterior portion (**C**, splenium) [48]. Notably, while histological images did not pinpoint relevant age-related differences, FTIRI evidenced higher amounts of lipids in younger subjects than in older ones, suggesting an age- and region-related reorganization in the structure and composition of myelin. These results were also confirmed using univariate analysis, which highlighted a higher relative amount of lipids (LIP/TBM, FA/LIP, and LIP/PRT) in splenium with respect to genu, as well as in younger than older subjects.