Fig. 3: MCAO-induced BBB disruption is reduced by treatment with SKI-1 inhibitor PF-429242.

a, LSM imaging and corresponding quantification of LSM dye leakage in the ipsilateral cortex versus contralateral control in vehicle-treated (n = 8) and PF-429242-treated (n = 7) mice 48 hours after MCAO. b, Pericyte coverage of endothelial cells was done by staining pericytes with a desmin antibody (Y66) and an endothelial cell marker (IB4). Quantification shows that PF-429242 treatment significantly restores coverage. Each point represents the coverage for one brain. c–f, Confocal imaging and quantification of markers in peri-infarct area of vehicle-treated and PF-429242-treated mice 7 days after MCAO. c, NeuN⁺ neurons (n = 7 for each group). d, FJC⁺ degenerating cells (n = 7 for each group). e, NEFH⁺ cells (n = 7 for each group). f, GFAP⁺ astrocytes (n = 6 for each group). g, Open field assessment for total activity and distance traveled. h,i, NSS and forelimb placing scores, respectively, evaluated until 7 days after MCAO. j,k, Neurological deficit and seizure activity after stroke assessed using Bederson and Racine scoring systems, respectively. Neurological scores h,j improved in PF-429242 3–7 days after MCAO, relative to their respective controls. The Racine scores k improved in 24 hours and 72 hours after MCAO. l, Confocal imaging of SKI-1 in isolectin (IB4)-positive endothelial cells. m, Concentration of PF-429242 in the plasma and brain 30 minutes and 150 minutes after intravenous injection of PF-429242, indicating that PF-429242 does not cross the BBB. n, Difference of PF-429242 found in MCAO hemisphere relative to the contralateral hemisphere shows that MCAO does not change PF-429242 levels in the brain. Therefore, the drug action is likely mediated through endothelial cells (that is, not initiated within brain). All graphs indicate mean ± s.e.m. Significance of b–f was assessed with two-tailed unpaired t-tests. Each dot represents an independent experiment. Time series g–j were assessed with repeated-measures factorial ANOVAs (between-group P values in the legends), and two-tailed unpaired t-tests were applied to individual timepoints. Each dot represents an independent experiment for a given timepoint. Time series k was assessed with a two-tailed unequal-variance t-test applied to cumulative subject scores (P value in the legend) and similarly for individual timepoint scores. Each dot represents a subject with non-zero score at a given timepoint. m and n do not assess significance.

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