Evans blue

To evaluate brain edema, the mice were i.v. administered with 10 μl/g of 2% Evans blue (Wako) 2 h before euthanization. The mice were anesthetized with isoflurane and perfused with PBS at 9 ml/min for 1 min. The brains were then removed and minced by scissors. The Evans blue in the minced brains was extracted with 500 μl of 4% paraformaldehyde for 2 days at 38 °C. After Evans blue extraction, the suspensions were centrifuged at 10000 × g for 20 min at room temperature, and the resulting supernatants were collected. The amounts of extracted Evans blue in these supernatants were measured by microplate reader (Thermo Fisher Scientific, Waltham, CA, USA) with a 620-nm wavelength filter against a standard of paraformaldehyde in PBS^{30 (link)}.

Blood–brain barrier breakdown was assessed by Evans blue permeability assay as previously described.²⁹ In brief, mice were intravenously injected with 2% solution of Evans blue (Sigma‐Aldrich) in normal saline (4 ml/kg of body weight) at day 3 post‐surgery. After circulation of Evans blue for 4 h, mice were euthanized and perfused with 40 ml PBS solution. The ipsilateral brains were harvested and weighed. The brain tissue homogenates was incubated with formamide in a 60°C water bath for 72 h to extract the Evans blue. After centrifugation, the Evans blue concentration was quantified photometrically by a microplate reader (Thermo Scientific) at 600 nm. The following formula was used: EB content in brain tissue (μg/g wet brain) = EB concentration (μg/ml) × formamide (ml)/wet weight (g).

DNA extraction from urine, vaginal and preputial fluid samples, and the semi-solid EMJH medium culture was performed using the Dneasy Blood and Tissue Kit (Qiagen, Hilden, Germany) following the recommendations from the manufacturer. In the polymerase chain reaction (PCR), two primers [17 (link)] specific for pathogenic leptospires-LipL3245F (5′AAG CAT TAC CGC TTG TGG TG3′) and LipL32286R (5′GAA CTC CCA TTT CAG CGA TT3′) were used to amplify LipL32 gene. The methodology described by Hamond et al. [18 (link)] was followed, and primers were used in a concentration of 0.6 μM, 1.0 U Taq polymerase, 2.4 μM MgCl₂, and 0.3 mM dNTP in a final volume of 25 μL. One cycle of initial denaturation at 94 °C for two minutes, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing the primers to 53 °C for 30 s and a one-minute extension with 72 °C and final extension cycle at 72 °C for five minutes were used. PCR products were developed by 2% ultrapure agarose gel electrophoresis stained with Evans Blue (Thermo Fisher Scientific, Waltham, MA, USA) and 100 bp ladder, and DNA bands (≅260 bp) were visualized under ultraviolet light. Strain Leptospira interrogans serovar Copenhageni, Fiocruz L1-130 (ATCC BAA-1198) was used as a positive control, and ultrapure water was used as a negative control.

For analysis of BBB permeability, mice were injected with 2% Evans blue (3 mL/kg) (Aladdin, China) intravenously.³⁴ After three hours, mice were anesthetized with 3.5% chloral hydrate and transcardially perfused with 0.1 mol/L cold phosphate‐buffered saline (PBS, pH 7.4). Then, the brains were removed immediately and stored at − 80°C until use. The brains were homogenized in PBS, sonicated, and centrifuged for 30 minutes at 12 000 g and 4°C. The supernatant was collected and equal volume of trichloroacetic acid (TCA) was added, followed by incubation at 4°C for 24 hours. After centrifugation (12 000 g, 4°C, 30 minutes), Evans blue absorbance was measured at 610 nm using a spectrophotometer (Thermo Fisher Scientific) and interpreted based on the standard curve.