High-Resolution Visualization of Blood–Brain Barrier Permeability in Acute Brain Slices

Hanafy AS, Steinlein P, Pitsch J, Silva MH, Vana N, Becker AJ, Graham ME, Schoch S, Lamprecht A, Dietrich D. Subcellular analysis of blood-brain barrier function by micro-impalement of vessels in acute brain slices. Nat Commun 2023;14(1):481. doi: 10.1038/s41467-023-36070-6.

Background: The blood–brain barrier (BBB) regulates molecular exchange between blood and brain and changes dynamically with physiological and disease states. Technical limitations in maintaining tissue integrity and imaging resolution have hindered detailed study of BBB transport. This study aimed to overcome these challenges by creating a high-resolution model that enables direct visualization and quantification of BBB permeability and cellular mechanisms in real time.

Hypothesis: This study hypothesized that direct micro-impalement and perfusion of brain capillaries in acute slices, combined with live fluorescence imaging, would enable precise, subcellular analysis of BBB permeability, transporter function and structural integrity under both physiological and pathological conditions.

Methods: The authors prepared acute brain slices from mouse and human hippocampus and micro-perfused fluorescent tracers and dyes into individual vessels. Imaging was conducted with two-photon microscopy using ScanImage. Endothelial cell morphology, tracer diffusion and transporter activity were assessed using fluorescent markers including TMR, FM1-43, calcein, rhodamine123 and BSA-Alexa488. Proteomic analyses quantified transporter proteins in pilocarpine-treated animals.

Results: Perfusion of tracers showed that small molecules like 7-hydroxycoumarin diffused beyond vessel walls, while larger tracers remained intravascular. FM1-43 crossed endothelial junctions by membrane diffusion. ABC transporters actively extruded rhodamine123 and calcein, effects reversed by inhibitors. Chemical (DMSO, mannitol) and epileptic conditions caused selective leakage to small but not large molecules, indicating partial BBB compromise.

Conclusions: This study established a reproducible live-slice assay for quantifying BBB function at subcellular resolution, demonstrating active transporter-mediated exclusion, differential permeability by molecular size and localized barrier disruption under pathological conditions.