Slice Cultures as a Model to Study Neurovascular Coupling and Blood Brain Barrier In Vitro
Figure 4
Mitochondrial free radical formation in pericytes/smooth muscle cells. (a) Representative Z-series of confocal images (1.2 μm) of a vessel double labeled with calcein-AM (green fluorescence channel—upper pictures) and MitSox (red fluorescence channel). MitoSox revealed free radical formation in spindle-shaped contractile cells associated with the wall of a vessel. MitoSox was anti-colocalized with calcein in pericytes/smooth muscle cells indicating low free radical formation in astrocytic endfeet. (b) 3D reconstruction of a vessel double labeled with calcein/MitoSox. The pictures are examples taken at four time points (as marked in (c)) during bolus application of rhodamine-123 into the intraluminal space. Both, calcein and rhodamine-123 fluorescence are represented in the green fluorescence channel (left) whereas the red fluorescence channel (right) corresponds to MitoSox labeling. After penetration of the vessel with the pipette, the lumen becomes slightly fluorescent due to leakage of rhodamine-123. Intraluminal rhodamine-123 fluorescence rapidly increased during bolus application, followed by redistribution of the dye into mitochondria within the vessel. No rise in the rhodamine-123 fluorescence was observed in the surrounding astrocytes. MitoSox almost completely colocalized with rhodamine-123 revealing mitochondrial origin of free radicals in pericytes/smooth muscle cells. Note the contraction of the vessel as a consequence of the increased intraluminal pressure. Scale bars represent 10 μm. (c) Changes in calcein (black traces) and rhodamine-123 (blue traces) fluorescence during bolus application of rhodamine-123 as measured in perivascular astrocytes (calcein, marked with arrowheads in (b)) and within the vessel lumen (rhodamine-123, marked with asterisks in (b)). Note that in spite of the physical contact of the astrocytic endfeet with the vessel wall, no rhodamine-123 appeared in astrocytes, further substantiating the presence of a diffusion barrier related to BBB.
