Slice Cultures as a Model to Study Neurovascular Coupling and Blood Brain Barrier In Vitro
Figure 3
Diffusion barrier around the vessel lumen in slice cultures. (a) Z-series of confocal images (1.2 μm) from a vessel after long-term (60 min) bulk staing with calcein-AM in the incubator. Note that calcein fluorescence below the astrocytic endfeet is almost absent, indicating a diffusion barrier and/or powerful extrusion mechanisms in endothelial cells. (b) Z-series of confocal images (1.2 μm) from a vessel after bolus application of calcein-AM into the lumen of a vessel. Endothelial cells showed bright calcein fluorescence, whereas no fluorescence was observed in astrocytes outside of the vessel. The asterisks on the consecutive images represent the application pipette. (c) Bolus application of calcein-AM into the stratum pyramidale resulted in a neuronal/astrocytic/microglial labeling up to 80 μm distance from the application place (left). Arrowhead marks a microglial cell containing calcein in vesicles. Calcein within neuronal processes can travel for several 100 μm (right). Scale bars represent 10 μm.
