## Materials and Methods
### FTIR Data Processing and Analysis  
Spectra acquired from the border between a brain tissue and a substrate, affected by resonance Mie scattering (commonly present in biological samples) and spectra recorded from a substrate (outside the brain tissue area) were eliminated from further analysis [18]. FTIR spectra were vector normalized between 3500-900 cm\({}^{-1}\) and the baseline correction was performed on the full range of wavelengths for the spectra. The relative amount of the specific molecular content was quantified from the original absorbance spectra [2]. The approximate values were determined from the brain samples of different animals, based on their bio-chemical variations. Functional group images were obtained by calculating the ratios of the baseline corrected areas, under some peaks, based on Table 1 (see Figure 1). The total lipid content was represented by the C-H stretching region at 2994-2800 cm\({}^{-1}\)[19; 20]. The protein concentration was represented by the amide II band at 1555-1535 [21; 22]. For more insight and deep lipid analysis, the ratios of some specific lipid spectral bands (v\({}_{\text{as}}\)(CH\({}_{3}\)) asymmetric, v\({}_{\text{s}}\)(CH\({}_{2}\)) symmetric, v\({}_{\text{as}}\)(CH\({}_{2}\)) asymmetric stretching, carbonyl ester v(C=O) ester, and olefinic =CH bands), to the total lipid (C-H stretching region), were calculated. For each animal, a mean and standard deviation was calculated and the average (\(n=6\)) was generated. Mann Whitney-U test was performed on all data to test for significant differences (\(p<0.05\)). All \(t\)-tests were two-tailed and the 95% confidence limit was used to test for significance.  
Principal component analyses (PCA) were performed using \"prcomp\" function in the \"stats\" package in R. The PCA analysis was performed on the spectral second derivatives, in the range of 4000-700 cm\({}^{-1}\) (the range of 2500-2000 cm\({}^{-1}\) removed). PCA analysis was performed on a total of  
\begin{table}
\begin{tabular}{c c} \hline
**Infrared Band** & **Spectral Rage (cm\({}^{-1}\))** \\ \hline
**Lipid components** & \\ CH\({}_{2}\) symmetric stretching & 2852-2800 \\ CH\({}_{2}\) asymmetric stretching & 2915-2930 \\ CH\({}_{3}\) asymmetric stretching & 2950–2960 \\
**\({}^{*}\) C–H stretching** & 2994–2800 \\ Olefin=CH & 3000–3027 \\ Carbonyl ester (C=O) stretching & 1745–1731 \\
**Protein components** & \\ Amide I & 1700–1600 \\ Amide II & 1555–1535 \\ \hline \end{tabular} \({}^{*}\) Total lipid region.  
\end{table}
Table 1: The spectral regions used for the analytical purposes.  
1270 wavenumbers, in the range of 4000-700 cm\({}^{-1}\), from the FTIR spectra obtained from the different brain regions, and PCA was performed, based on the covariance of these features. Furthermore, during PCA we randomly divided the spectral collection into 80% training and 20% test. The norm of the reconstruction error for each test spectra was 0.05, on average, while using the first three principal components. The 3D scores plot of these components enabled the spectra to be grouped according to the chemical information they contained. Hierarchical cluster analysis (HCA) was applied, using the \"A2 Rplot\", to compare the seven brain regions, based on their PCA projections in the 3D score plot. HCA was used to group spectra that displayed the same degree of similarity. The result was visualized in a dendrogram and the grouping of the seven brain regions were presented in clusters, according to a heterogeneity scale [23; 24].