(a) Schematic of a mouse after preparative microsurgery for two-photon imaging, with (b) craniotomy over the somatosensory cortex. (c) Three-dimensional reconstruction of the brain microvasculature from a single Z-stack after i.v. injection of NaFluo. Each Z-stack corresponds to a single time point and underwent dimensionality reduction by maximum intensity projection along the Z-axis (depth). (d) Relative fluorescence change over the baseline (first projected Z-stack in the recording). Contours delineate the main cerebral microvasculature. Compared to WT, the Apom-/- animals had increased accumulation of NaFluo in the brain parenchyma over time, which was ameliorated by treatment with S1PR1 agonist SEW2871. (e) Bolus injections of fluorophore were separated by 30 min time-lapse imaging intervals. The panels show fluorescent molecule accumulation in the brain parenchyma for all tested fluorophores at 30 min post-injection. (f) Changes in fluorescence intensity over time in the brain parenchyma and the blood circulation expressed as a fraction of the baseline. (g) Quantitative assessment of the total effect 30 min post-injection by area under the curve (AUC) calculation. Left bar plot: increased paracellular permeability of the BBB in Apom-/- mice towards sodium fluorescein (0.365 kDa) and Alexa Fluor488 (0.643 kDa), but not large molecule FITC-dextran (10 kDa). Right bar plot: no differences in the signal decrease of blood-circulating fluorophores among all experimental groups. (h) The mean arterial blood pressure during imaging was the same in all experimental groups. All data are presented as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001.