The removed brain was fixed in 10% neutrally buffered formalin (Sigma-Aldrich) followed by embedding in optimal cutting temperature compound (Tissue-Tek OCT, Sakura Finetek Inc., Torrance, CA, USA) and storage at −50°C. The brain samples were serially sectioned with a slice thickness of 15 µm. The direction of these slices was the same to FUS sonication direction. The bluest section was identified and subjected to hematoxylin and eosin (H&E) staining to confirm the presence of erythrocyte extravasations. In addition, terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL, ApopTag kit, Intergen Co., Purchase, NY, USA) was used to detect apoptotic neurons [29] (link). Histology evaluations were performed using light microscopy by a person who was blinded to the ultrasound parameters but was informed of sonication location of the brain.
The extent of brain damage was rated according to a four-point scale as follows [3] (link), [4] (link): grade 0 = no tissue damage or erythrocyte extravasation; grade 1 = presence of few extravasated erythrocytes, but no neuronal loss; grade 2 = presence of extensive erythrocyte extravasations; and grade 3 = extensive erythrocyte extravasations along with apoptotic neuronal death.
The extent of brain damage was rated according to a four-point scale as follows [3] (link), [4] (link): grade 0 = no tissue damage or erythrocyte extravasation; grade 1 = presence of few extravasated erythrocytes, but no neuronal loss; grade 2 = presence of extensive erythrocyte extravasations; and grade 3 = extensive erythrocyte extravasations along with apoptotic neuronal death.