Vascular leakage and severe brainstem pathology during ECM.

(A) Representative confocal images of sagittal brain sections from an uninfected mouse (top) or a symptomatic mouse at d6 p.i. following simultaneous i.v. injection of Evans blue (white) and propidium iodide (red) to visualize vascular leakage and cell death, respectively (n = 5 mice per group). Cell nuclei are shown in blue. Note the presence of severe brainstem pathology (white arrow) in the d6 p.i. mouse. (B) Representative confocal images of brain sections from a naïve (top) or a symptomatic mouse at d6 p.i. following propidium iodide (red) injection and immunohistochemical staining for CD31⁺ ECs (green). Cell nuclei are shown in blue. (C) The bar graph depicts quantification of data shown in (B). Data are represented as the percentage of PI⁺ CD31⁺ ECs of total CD31⁺ ECs per brain region (mean ± SD; n = 5 mice per group; 1 brain section per mouse; 10 images per brain section; 2–3 images per brain region). Asterisks denote statistical significance (*P < 0.05). (D) Representative confocal images of brain sections from an uninfected (top) or a symptomatic mouse at d6 p.i. following propidium iodide (red) injection and immunohistochemical staining for NeuN⁺ neurons (green). Cell nuclei are shown in blue. (E) The bar graph shows quantification of data shown in (D). Each bar represents the total number of PI⁺ cells in each brain region (gray), and within the bar, the red coloration shows the proportion that are also NeuN⁺ (mean ± SD; n = 4 mice per group; 1 brain section per mouse; 12 images per brain section; 3 images per brain region). Asterisks denote a statistically significant difference from all other groups (***P < 0.05).