Midi fl

Immuno uorescence staining for cleaved-caspase 3 were performed as we previously described [21] . Brie y, the brain sections were incubated in 3% hydrogen peroxide for 0.5 h and then blocked with normal bovine serum for 0.5 h. The brain sections were washed three times and incubated with rabbit polyclonal anti-cleaved caspase-3 (1:400, CST, #9664), overnight at 4°C. The slices were then washed with PBS and incubated with Alexa Fluor 647-conjugated donkey anti-rabbit IgG at room temperature for 2 h. Finally, the samples were counterstained with DAPI (Sigma-Aldrich, St. Louis, MO) for 10 min. Digital images of the whole brain sections were obtained by a MIDI FL (3D Histech, Hungary) system.

The rats (n=8 per group) were sacri ced by decapitation on day 3 after CCI, and then 5 μm-thick coronal para n sections at the level of the hippocampus were made according to the Paxinos atlas of the rat brain [24] . To assess the survival apoptosis of neurons in the hippocampus, H&E staining and TUNEL assay (Roche, Germany) were carried out as we previously described [19] . Brie y, after rehydration, tissue sections were incubated with proteinase K working solution (20 mg/ml in 10 mM Tris-HCl buffer, pH 7.5-8.0) for 10 min at 37°C. They were rinsed twice with 0.01 M PBS (pH 7.4) and subsequently incubated at 37°C with the TUNEL reaction mixture for 1 h. Finally, color development was conducted for 10 min using 3, 3-diaminobenzidine (DAB) substrate. A MIDI FL (3D Histech, Hungary) system was used to obtain the digital images of the brain sections. A digital image analysis system (3D Histech, Hungary) was used to count the number of neurons and apoptotic cells in the ipsilateral hippocampus (three sections) as previously described [16] . Data are presented as the number of hippocampal neurons per mm and the total number of TUNEL-positive cells. All analysis was performed in a blinded fashion.