Eb dye

As reported, BBB permeability was evaluated by EB dye extravasation assay.¹⁵ Briefly, 4% EB dye (4 mL/kg, Sigma‐Aldrich) was injected intraperitoneally at 3 h before sacrifice evaluation, and mice were sacrificed 24 h after ICH. The right hemisphere was homogenized and sonicated within 1100 μL PBS and then centrifuged for 30 min at 15,000 g. 500 μL supernatant from each sample was mixed with 500 μL 50% trichloroacetic acid overnight at 4°C. After centrifugation, the supernatant was collected, and EB dye extravasation was evaluated at 610 nm and quantified according to a standard curve.

The evaluation of BSCB integrity was performed with quantitative and qualitative measurements of EB extravasation and spinal cord edema. The content and fluorescence of EB were examined. Briefly, 60 min after the injection of EB dye (20 g/L; Sigma) at a dose of 10 ml/kg via the tail vein, the animals were anesthetized and then transcardially perfused with 0.9% saline containing 10 IU/ml heparin, until colorless perfusion fluid was obtained from the right atrium, to remove all EB dye that had not leaked into the interstitium. The L4–6 spinal cord tissue was weighed, then soaked with methanamide for 24 h (60 °C) and centrifuged. The fluorescent absorption of the supernatant was detected at 632 nm with a microplate reader (BioTek, Winooski, USA) and presented as EB amount per tissue weight (μg/g) with standardized curve. Furthermore, the spinal cord tissue was fixed with 4% paraformaldehyde. The transverse sections (10 μm) were visualized under a BX-61 (Olympus, JP) fluorescence microscope. EB dye was visualized as red under the green fluorescence excitation mode. The integrated optical density (IOD) of the positive fluorescence labeling in the lumbar spinal cord tissue was analyzed to quantify the EB extravasation (Image Pro Plus 6.0). The percentage of water content in the spinal cord tissue was calculated with a wet-dry method: (wet weight − dry weight)/wet weight × 100.

For BBB permeability analysis active PDGF-CC core protein (3 μM) or PBS as control was ICV injected into the dorsal 3rd ventricle (bregma-0.94, mediolateral 0 and dorsoventral-2.45) in mice pre-treated with either imatinib (or PBS as control) or 6B3 antibody (or BM4 as control) 2-5 h before ICV injection. Directly after ICV injection mice were intravenously injected with 0.1 ml of 4% EB dye (Sigma-Aldrich). 2 h later, animals were perfused with HBSS for 8 min and the brains were removed and photographed with a Samsung s4mini mobile phone (Samsung, South Korea). Each brain was then homogenized in N,N-dimethylformamide (Sigma-Aldrich) and centrifuged for 45 min at 25,000 rcf (Eppendorf centrifuge, model 5417R). The supernatants were collected and quantitation of EB extravasation was performed as described^{65 (link)}. EB levels in each brain were determined from the formula: (A₆₂₀ nm − ((A₅₀₀ nm + A₇₄₀ nm) ∕ 2)) ∕ mg wet weight. One-way ANOVA with Fisher´s LSD (*P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001) was used.