FIGURE 5.

Cells escaping from the ischemic explants induce glial scar formation in vitro, and IDA colonize the cell-free areas. (A,B) Brain cortical ischemic explants (IE) obtained from eGFP+ animals subjected to ischemia after 3 DPL, seeded on primary astrocytic confluent monolayers during 3 DIV induced the formation of glial scar-like structures (arrow) that clustered and segregated cells escaping from explants, as easily seen in bright field (A) and escaping eGFP+ cells (B) observed under UV illumination, bar = 500 μm. (C,D) Representative images of cells escaping from ischemic explants showing that the majority of cells were clustered in the astrocytic-free areas (C) compared with the area with astrocytes limited by the scar-like structure (arrow) (D). (E) High magnification detail of eGFP cells escaping from ischemic explants showing the different phenotypes, going from macrophage-like rounded phenotype (thin arrow) to the polygonal fusiform IDA (thick arrow). (F,G) IDA growing within the limits imposed by the corral glial scar-like structure by 5 days after seeding the eGFP+ ischemic explants on primary astrocytic monolayers. The mesh-like in vitro scar is clearly seen when stained with GFAP (red; arrow). (H,H′,H′′) Images of eGFP-IDA differentiated to stellated GFAP+ astrocytes in the vicinity of the astrocytic monolayer and scar-like structures stained with GFAP (red). (I) Several eGFP-IDA morphologically differentiated to stellated astrocytes (I) when seeded on top of a confluent primary astrocytic monolayer that is visible under phase contrast (I′).