Application module for tissue section analysis

An intact cell count was performed using toluidine blue staining. Multiple fields per section were used for intact cell quantitation in the midbrains, striata, and substantia nigra in different groups. Glass slides were visualized using a Full HD microscopic camera operated by a Leica application module for tissue section analysis (Leica Microsystems GmbH, Wetzlar, Germany) (Ibrahim et al. 2021 (link)).

The brains of the first subset were promptly fixed in 10% formalin after scarification for 24 h. After washing with ice-cold saline, the brains were dehydrated in serial dilutions of alcohol and embedded in paraffin blocks. Five-micron slices were sectioned and stained with hematoxylin and eosin (H & E) to examine any morphological changes and signs of inflammatory cell infiltration in the hippocampal CA3 region and the corpus callosum. The corpus callosum is a dense myelinated fiber bundle in the central nervous system and its demyelination occurs early in MS (Rimkus Cde et al. 2011 (link)). Herein, the severity of the hippocampus damage was further assessed via toluidine blue staining, where the number of intact neurons was determined. Moreover, the corpus callosum’s demyelination was assessed using luxol fast blue (LFB) staining. The percentage of corpus callosum’s myelinated nerve fibers and the number of intact neurons in the CA3 of the hippocampus were quantified using a full HD microscope camera and the Leica application module for tissue section analysis (Leica Microsystems GmbH, Wetzlar, Germany) (Abd El Aziz et al. 2021 (link)). The histopathological examination was performed by an expert investigator who was blinded to the identity of the samples.

Skin samples were collected from three rats per group, that were embedded in paraffin wax for sectioning and staining with hematoxylin and eosin (H&E) using the Leica application module for tissue section analysis (Leica Microsystems GmbH, Wetzlar, Germany).