Fig. 7

Tracer inflow occurs around venules, arterioles and the central canal. A An example of an anterior spinal artery under confocal microscopy. In all animals where extramedullary arterial fluorescent ovalbumin (AFO-647) deposition was evident, tracer was detected not only external to (right arrowhead) the smooth muscle layer (*) but also within, and internal to the tunica media (left arrow). Left pointing arrowhead denotes the endothelium. A 3D video of an anterior spinal artery (Additional file 7: Video S7) demonstrates this more clearly. B, C AFO-647 was detected within the ependymal layer of the central canal. There was an uninterrupted serpiginous trail of tracer (arrowhead) between subependymal microvessels and the central canal (right arrow marking venule, while c denotes the lumen of the central canal in B and C). This suggests privileged pathways may exist between the central canal and the subependymal perivascular spaces. AFO-647 tended to concentrate on the luminal aspect of the ependymal cells. Tracer was occasionally observed within the lumen of the central canal. Tracer unevenly deposited on the luminal side of the ependymal layer (note speckled appearance in C). The nuclei of ependymal cells were demarcated by tracer (marked by e), suggesting uptake by these cells. D–G Depicts multiple distinct layers of AFO-647 tracer within and external to the smooth muscle layer of an intramedullary grey matter arteriole (AFO-647, SMA, RECA-1 and merged channels are shown in D, E, F and G respectively). H AFO-647 accumulated around the outside of the endothelium of a large intramedullary venule (* marks the lumen). I A venule at the pial surface had similar deposition of AFO-647 around the endothelium (arrow). In I, * marks transpial migration of tracer, depositing in a convoluted, lattice pattern which reflects the spinal extracellular space architecture