2P-based live-imaging of brain capillaries reveals blood-brain barrier alterations in Alzheimer’s disease mouse model

Amira Hanafy1, Pia Steinlein1, Alf Lamprecht2, Dirk Dietrich3

1 Department of Pharmaceutics and Department of Neurosurgery (UKB),University of Bonn, Germany
2 Department of Pharmaceutics, University of Bonn, Germany
3 Department of Neurosurgery, University Clinic Bonn (UKB), Germany

Ever since the identification of Alzheimer’s disease (AD) a century ago, the blood-brain barrier (BBB) remains a resilient hurdle against developing an effective treatment. Disruptive tight junctions (TJ), altered expression of Pglycoprtien (P-gp) and multidrug resistance-1 receptor (MRP1) seem linked to AD pathogenesis. However, the reported data are inconsistent and based on in vitro or in vivo BBB models that oversimplify the microenvironment or allow other biological factors to interfere, respectively.

A newly established in situ BBB model is used to study transporter alterations in AD. Acute cortical brain slices were isolated from wildtype and transgenic Tg(APPSWE)2576Kha AD mice, and placed in a submerged chamber of a twophoton (2P) microscope under constant sACSF perfusion. A blood vessel is pierced with a glass pipette filled with the test compound and slight pressure was applied to eject the compound in the vessel system for 30 min. Calcein-AM
accumulation in the endothelial cells was monitored using its green fluorescence in order to assess P-gp and MRP1 function upon inhibition by 1 μM Elacridar (ELA) and 0.6 mM Probenecid (PRO), respectively. The TJ integrity was assessed via monitoring the leakage of Tetramethylrhodamine (TMR) into the capillaries’ vicinity and the diffusion of FM 1-43 dye from the luminal to the abluminal side of endothelial cell membranes.

Compared to wildtype mice, AD capillaries exhibited a significantly higher Calcein accumulation under no transporter inhibition. Under sole MRP1 inhibition, insignificant difference was found between wildtype and AD capillaries, insinuating the downregulation of MRP1 in the latter.