Eb solution

Evans blue staining was applied to evaluate the blood–brain barrier integrity. 2% EB solution (8 mL/kg, Sigma–Aldrich) was intra-peritoneally injected after anesthetization. After 24 h, the rats received trans-cardiac perfusion with 0.1M PBS. Next, the brain was removed and homogenized in 50% trichloroacetic acid. The sample was incubated in a water bath (50°C) for 48 h and centrifuged at 15,000 × g for 30 min. Afterward, the supernatant was detected through spectro-fluorophotometry at 620 nm (Zhao et al., 2016 (link)).

For quantitative and qualitative analysis of the BSCB dysfunction severity, the contents of the extravasating EB and fluorescence were detected. The optical density (OD) value was measured by a spectrophotometer at 620 nm wavelength. The standard curve was drawn and the corresponding linear regression equation was calculated according to the testing result of EB solution at different concentrations (6, 3, 1.5, 0.75, 0.35, 0.175 μg/mL). A 2% EB solution (10 mL/kg; Sigma) was slowly intravenously administered through the vena caudalis. The solution was allowed 24 h for microcirculation and sufficient extravasation into the spinal cord parenchyma in vivo. After satisfactory anesthesia, the rats were transcardially perfused with 500 mL/kg saline. The intact cervical spinal cord was harvested and weighed, then kept in 3 mL methanamide solution for 72 h (37 °C). The cord tissues were centrifuged at 1500 r/min for 15 min. The supernatant was obtained to measure the OD value at 620 nm and the EB concentration can be calculated according to the standard cure. The EB content was calculated based on the following equation:
On the other side, the spinal cord tissue was frozen in liquid nitrogen and then embedded in OCT. Slices at 20 μm were sectioned and visualized using a confocal laser scan microscope system (Germany Leica, TCS SPS II; Olympus OLS-3100).

EB solution (2% given at 4 mL/kg, dissolved in PBS; Sigma-Aldrich) was infused into mice through the tail vein and allowed to circulate for 2 h, as previously described [28 (link)]. The mice were euthanized and transcardially perfused with ice-cold PBS to remove the intravascular EB dye. For fluorescence images, brain cryosections were sectioned, fixed in precooled 4% PFA, stained with DAPI, and observed under an IX81 fluorescence microscope (Olympus). For quantitative analysis, the brains were harvested, weighed, homogenized in formamide (1:20 w/v), and incubated at 60 °C for 72 h. The brain homogenate was centrifuged at 14,000 rpm for 30 min to collect the supernatant, in which EB was determined at OD 620 nm using a SpectraMax M5 plate-reader (Molecular Devices, Sunnyvale, CA), quantified using a linear standard, and expressed as μg/g brain tissue.

Blood-spinal cord barrier leakage was assessed by EB extravasation (Li et al., 2014 (link); Zhou et al., 2017 (link)). EB solution (3%, 45 mg/kg, Sigma-Aldrich) was administrated through the femoral vein after anesthetization. One hour later, spinal cord samples of 1.0-cm long, where the injured site was centered, were obtained after transcardial perfusion with 0.1M PBS. The sample was homogenized in 3 mL 50% trichloroacetic acid and centrifuged at 12,000 × g at 4°C for 20 min. Next, 1 mL of the supernatant was mixed with 1 mL of ethanol and trichloroacetic acid solution (1:3) and incubated at 4°C overnight. The sample was then centrifuged at 15,000 × g at 4°C for 30 min. The supernatant was examined at an emission wavelength of 680 nm and excitation wavelength of 620 nm with a microplate reader (Bio-Tek Instruments, Winooski, VT, United States). The fluorescence value was converted to an EB content (μg) according to the standard curve. The researcher was blinded to the group assignments of the rats. Each test was conducted twice, and the average value was taken as the result. The results were expressed as the EB content (μg) in unit tissue (g).

EB extravasation assays were performed 1 d after cerebral I/R injury to evaluate Blood-Brain Barrier (BBB) permeability. Briefly, after the rats were tranquillized, an EB solution (2%, 4 ml/kg, Sigma-Aldrich) was injected into the femoral vein, where it was circulated for over 60 min. Phosphate-buffered saline (PBS) was then perfused through the heart to remove blood cells. The ischemic lateral hemisphere was weighed and equalized with 3 mL/600 mg of N, N-dimethylformamide (Sigma-Aldrich). were allowed to culture at 55 °C for 24 h and incubated, followed by centrifugation (13,000 rpm for 20 min). Using a microplate reader, EB dye in the supernatant was measured at 632 nm.

Blood–brain barrier leakage was assessed via EB staining at 24 h after ICH. Two percent EB solution (8 mL/kg, Sigma–Aldrich) was applied through femoral vein after anesthetization. Two hours later, the rats received transcardial perfusion with 0.1M PBS. Next, injured brain hemisphere was collected and homogenized in N, N-dimethylformamide. The sample was incubated in water bath (50°C) for 48 h and centrifuged at 12,000 × g for 30 min. Finally, the supernatant was collected and measured at 620 nm with a spectrophotometer (2,000°C, Thermo Fisher) (Zhao et al., 2016 (link)).

On day 7, mice treated with DSS were euthanized with CO₂ and then the luminal contents were cleaned with sterile, cold PBS before the two ends of the luminal tube were ligated to prepare the colorectal intestinal sac described as previously.²⁵ The colorectal intestinal sac was then injected with 100 μl of Evans blue (EB) solution (Cat No E2129, Sigma‐Aldrich) [1.5% (w/v) in PBS] and placed in 20 ml of Krebs solution (Sigma‐Aldrich). After 30 min, the colorectal intestinal sac was washed with PBS until the flushing solution became clear, dried for 24 h at 37°C, and obtained a dry weight. It was then cultured at 55°C for another 24 h with 1 ml of formamide (Sigma‐Aldrich). Finally, the intestinal colorectal sac was removed, and the supernatant was centrifuged. The absorbance was measured at 655 nm.