Brain derived neurotrophic factor (bdnf)

The hippocampal slices from the animal cohorts were fixed in 4% phosphate-buffered saline (PBS) (pH 7.4), and cryosections of 3 μm thickness were made. The sections were washed with PBS and permeabilized with 0.3% Triton X-100 and 0.05% Tween-20 in PBS, followed by treatment with a blocking buffer (5% normal goat serum) for 1 h at room temperature. Subsequently, the sections were incubated overnight at 4℃ with primary antibodies BDNF (1:500; Boster Bio, USA) and HDAC3 (1:500; Boster Bio, USA) in a humidified compartment. The sections prewashed with PBS were incubated with Alexa-Fluor secondary antibodies (1:250; Invitrogen, Oregon, USA). Finally, the tissue sections were washed with PBS and examined the under fluorescence microscope (Olympus FV1000, Japan).

Biochemistry, hematology, and coagulation test were assessed in the central laboratory of each participating hospital. However, the growth factors and SDF-1α determinations of the total patients included in each of 6 hospitals were performed in the Clinical University Hospital of Santiago de Compostela. The GF selected for this study were VEGF, G-CSF, BDNF, Ang-1, and SDF-1α. For these molecular determinations, venous blood samples were collected in Vacutainer tubes (Becton Dickinson, San Jose, CA, USA) at admission, 7 ± 1 days, 3 ± 1, and 12 ± 3 months. After allowing to clot for 60 min, blood samples were centrifuged at 3000×g for 10 min, and the serum was immediately aliquoted, frozen, and stored at − 80 °C until analysis. Serum levels of VEGF, G-CSF, and BDNF (Boster Biological Technology, Encyclopedia Circle Fremont, CA, USA), Ang-1 (Adipo Bioscience Inc., Santa Clara, CA, USA), and SDF-1α (R&D Systems Inc., Minneapolis, MN, USA) were quantified using commercial ELISA kits following instructions provided by commercial houses. The coefficients of intra-assay variation were VEGF, 4.4%; G-CSF, 5.2%; BDNF, 4.8%; Ang-1, 4.7%; and SDF-1α, 3.6%; the coefficients of inter-assay variation were VEGF, 6.9%; G-CSF, 8.3%; BDNF, 7.9%; Ang-1, 8.1%; and SDF-1α, 7.2%. The determinations were performed in an independent laboratory that did not have access to clinical or neuroimaging data.