Fig. 4
EGFL7 treatment ameliorates EAE disease course, preserves BBB and decreases immune cell infiltration into the CNS. a, b 8 to10-week-old wild-type (WT) C57Bl/6 female mice were immunized with MOG35–55 peptide. rmEGFL7 10 µg/ml or vehicle was administered intraperitoneally (i.p.) every other day from days 4–18 (grey box). n = 18 mice per group, from two pooled independent experiments. a EAE score. b EAE prevalence. c 2–3 days after EAE onset, representative mice were lethally anesthetized prior to perfusion (n = 5–6 control mice, from two pooled independent experiments) and leukocyte isolation before analysis of spleens and CNS (brain and spinal cord) by flow cytometry. d In vivo permeability assay in EAE following exposure to rmEGFL7 or vehicle, using intravenous injection of fluorescently-labeled dextrans (3, 20 kDa). Dextran extravasation to the CNS (brain and spinal cord) is expressed as a percentage of blood fluorescence intensity. n = 8 mice/group, d18. e–j BBB integrity as measured by fibrinogen (e–g) or IgG (h–j) leakage was assessed by immunofluorescence on frozen sections from representative EAE mice treated with rmEGFL7 (lower panels) or vehicle (upper panels). Scale bar = 50 μm, spinal cord. f, i Semi-quantitative analysis of fibrinogen or IgG signal (green) in the cerebellum, brainstem, and spinal cord according to mean pixel intensity and g, j leakage of fibrinogen or IgG according to relative pixel intensity (mean pixel intensity × area). n ≥ 5 sections per mouse, from n = 5 mice/group. Statistical analysis performed by two-way repeated measures ANOVA with multiple comparisons test (a), by Mantel-Cox test (b), and by Mann-Whitney U-test (c–j). *p < 0.05, **p < 0.01. All data presented as mean ± SEM
