FIGURE 2.

Ischemia-derived astrocytes (IDAs) are present in ischemic explants and proliferate in vitro. (A) Representative images of cortical explants obtained from 3 DPL ischemic lesions, contralateral hemisphere, LPS-injected animals (5 mg/kg, i.p.) or sham animals showing a large number of cells that spontaneously escape from ischemic tissue. Only cells derived from ischemic explants successfully survive in vitro. Quantitative results show the total number of cells escaping from ischemic explants in a 0.5 mm² area sectioned from the microscopic field. Values are represented as mean ± SEM and statistical significance was confirmed with ANOVA and Student Newman Keuls post-test (***p < 0.001). (B) Time lapse images acquired every 12 h of the same 3 DPL cortical ischemic explants from eGFP transgenic rat strain [Wistar-TgN(CAG-GFP)184ys] showing the rapid escape of cells from ischemic explants observed from 1 DIV (DIV, days in vitro) to 4 DIV. Insets show the detail of different cell morphologies and the abundance of fusiform cells by 4 DIV. Morphology of cells escaping from ischemic explants are different after 1, 2, 3, or 4 DIV. Polygonal or fusiform IDA are the predominant cells after 3–4 DIV. (C) Immunocytochemistry images showing the Iba-1 and S100B expression in the cells escaping from ischemic explants. From 1 to 7 DIV the intense Iba-1 expression is reduced and S100B expression increased. Quantitative results show the area occupied by the S100B(+)/Iba-1(-) (S100B-only) expressing cells. Values are represented as mean ± SEM and statistical significance was confirmed with the non-parametric Kruskal–Wallis test and Dunn’s Multiple Comparison Test (**p < 0.01). (D) Different representative images of IDA escaping away from the 3 DPL ischemic explants after 3 DIV. Rats have previously been injected with eGFP+ BMMC and then were subjected to ischemia by cortical devascularization. Note that eGFP+ cells have not fusiform IDA phenotype. Bar = 15 μm.