Histopathological examination, neural, and myelin staining

Four brain tissue samples were taken from different groups and fixed in 10% neutral buffered formalin for 72 h. Samples were trimmed and processed in serial grades of alcohols, and cleared in Xylene. Subsequently, samples were infiltrated and embedded into paraplast tissue embedding media where paraffin tissue blocks were thus prepared. Four-micrometer-thick sagittal brain sections were cut by rotatory microtome for demonstration of hippocampal regions in different samples. The obtained tissue sections were collected on glass slides and stained by hematoxylin and eosin stains as a general morphological examination staining method and examined by a light microscope (Leica Microsystems GmbH, Wetzlar, Germany) as previously described [40].

As previously described, tissues were also stained by toluidine blue stain for demonstration of damaged and intact neurons and examined by using a light microscope (Leica Microsystems GmbH, Wetzlar, Germany) [41]. Six non-overlapping fields were randomly selected and scanned by experienced histologist from CA1 and CA3 hippocampal subregions per tissue section to determine the numbers of intact neurons counts in Toluidine blue-stained tissue sections in each region in four samples in each group.

Additionally, tissues were also stained by Luxol fast blue stain for demonstrating the myelinated nerve fibers in corpus callosum regions and examined by using a light microscope (Leica Microsystems GmbH, Wetzlar, Germany). Six non-overlapping fields were randomly selected and scanned from corpus callosum regions for quantification of positively stained mylinated nerve fibers to calculate the total mean expression levels of four samples in each group.