Figure 1. Spatio-temporal characterization of functional BRB formation, See also Figure S1, S2 and S5.

Intravenous injections of tracers in neonatal mice reveal that functional BRB is formed gradually from proximal to distal in the primary plexus. (A-B) Spatio-temporal development of the mouse retina primary plexus. (A)Vessel ingression at the optic nerve head (ONH) occurs at P1. Vessels then expand from the ONH to the retinal periphery during postnatal development. (B) Vessels reach the peripheral edge of the retina at P8. A spatio-temporal gradient of vessel maturation is observed: the vessels proximal to the ONH are more mature whereas the vessels distal to the ONH are nascent. (C) In P1 retinas, when blood vessels (isolectin; green) first enter the retina, Sulfo-NHS-Biotin (left) and 10-kDa dextran (right) tracer (red) leakage is observed around budding vessels (arrowheads). (D-G) In P3 (D) and P5 (E-G) retinas, a gradient of barrier functionality is observed. More mature vessels proximal to the ONH confine tracer (arrows) whereas nascent vessels distal to the ONH leak tracer (arrowheads). Ticks in (E-G) represent distance in microns from the ONH. 0 is slightly before the ONH. (H) Permeability index of P5 retinas from tracer-injected pups reveals the functional BRB is gradually formed in a proximal to distal fashion. The permeability index is measured by the ratio of tracer-positive area over isolectin-positive area. A ratio greater than 1 indicates that the BRB is permeable and immature. A ratio of 1 indicates that the BRB is impermeable and hence mature. 250 μm² field of views were sampled and tiled starting from 0 to the angiogenic front. The average distance from the ONH to the angiogenic front at P5 is 1651 ± 288 μm. Data are mean ± s.e.m. (n = 5-6). (I) Higher magnification of distal vessels of the primary plexus show Sulfo-NHS-Biotin (top) and 10-kDa Dextran (bottom) tracer leakage at P8 and P9 (arrowheads). At P10, both tracers are confined in distal vessels (arrows). (J) Quantification of distal vessel permeability for Sulfo-NHS-Biotin and 10-kDa dextran throughout BRB development. Data are mean ± s.e.m. (n = 5-6 mice per age per tracer group, from 3 different litters). Statistical significance was determined by oneway ANOVA with a post-hoc Bonferroni multiple comparison adjustment, comparing the various neonatal ages with the adult in the respective Sulfo-NHS-Biotin and 10-kDa Dextran group. *, P < 0.05, ***, P < 0.001. Scale bar represents 100 μm for all panels.