Fitc albumin solution

Infection and blood–brain barrier (BBB) disruption were induced as previously reported (35 (link)). Briefly, animals between 6 and 14 wk old were infected intracranially with 2 × 10⁴ PFUs of Daniel strain TMEV in the right hemisphere of the brain in a total volume of 10 μl following anesthetization with 2% isoflurane. Mice were euthanized for flow cytometric analysis, or induced to undergo peptide-induced fatal syndrome (PIFS), at 7, 14, or 28 d postinfection (dpi). The PIFS model employed by our group involves the tail vein injection of 100 μl of a 1 mg/ml solution of VP2_121–130 peptide (FHAGSLLVFM; GenScript, Nanjing, China) in PBS at either 7 or 14 d after the original TMEV infection (33 (link), 36 (link)–40 (link)). Animals were observed until at least one animal in the experiment became hunched and minimally responsive, at which point all animals were injected via tail vein with 100 μl of a 100 mg/ml FITC-albumin solution in PBS (Sigma-Aldrich, St. Louis, MO). PIFS-induced mice were euthanized 1 h after FITC-albumin injection for analysis.

Under anesthesia mice (n = 4 PXR^−/− and n = 4 WT) were injected intracardially with 300 μL of a FITC-Albumin solution (25 mg/ml in PBS; Sigma-Aldrich). Brains were rapidly removed, post-fixed using 4% PFA (48 h) and coronal sections (30 μm) obtained using a vibratome. FITC⁺ vessel quantification (length and number; Fiji skeleton Plug-in) was performed using 10× images (n = 6–10/each) of cortical structures (parietal and temporal), see also (Boussadia et al., 2016 (link)). Neurons were stained using a NeuN primary antibody (1:300; Millipore, MAB377, Fontenay sous Bois, France). Cortical thickness is the distance calculated (at least two measurements of 10× -captured images per sample) between the most external NeuN⁺ cortical cell layer and the white matter border (Fiji). CA hippocampal thickness was similarly determined.