Histological Assessment of Brain Damage

Sagittal sections from brain tissue were fixed in paraformaldehyde (4%) and embedded in paraffin blocks. After that, blocks were cut into thin slices (5–6 μm) and stained with hematoxylin and eosin staining [24 ]. Different brain regions were examined in each animal by using an Olympus light microscope (BX43, USA) connected to a DP27 digital camera and CellSens dimensions software. The histological damage score was assessed in each animal according to previous studies [25 (link),26 (link)]. Briefly, variant lesions, including neuronal degeneration, neurophagia, gliosis, and necrosis, were scored in different brain regions according to the severity as follows: 0 = absent, 1 = sporadic neuron (<25%), 2 = distributed neurons (25–50%), 3 = distributed neurons (>75), 4 = multifocal (>75). The final score for each animal was obtained by summation of the total score for multiple lesions (4–5 fields/200×).