To assess the time course of astrocyte lesion development, AQP4 Abs and human complement were injected intracortically and animals were perfused after 3 hours, 6 hours, 24 hours, 3 days, and 7 days. Controls were injected with an irrelevant Ab in the presence of human complement. Three hours after Ab and complement injection, GFAP-positive astrocytes were still observed at the injection site (A). Monastral blue marks the injection site. However, dying GFAP-positive cells with retracting processes were also found (A, insert). Twenty-four hours after injection, large, well-demarcated areas with loss of GFAP (B) and AQP4 immunoreactivity (C; serial sections of the same lesion, astrocyte loss marked by dotted line) were detected. Quantification of GFAP immunoreactivity revealed initial loss 6 hours after lesion induction, peaking between 24 hours and 3 days, with a subsequent repopulation of GFAP-positive cells. No astrocyte loss is observed after injection of control Ab (ctrl-Ab) together with human complement. Number of lesions: 3 hours, n = 3; 6 hours and 24 hours, n = 10; 3 days, AQP4/2B4, n = 6/4; 7 days, n = 5 (D). Simultaneously, with the loss of astrocytes, a prominent extravasation of the injected tracers FITC-albumin (60 kDa) (E) and Texas Red cadaverine (0,69 kDa) (F) into the brain parenchyma was observed 6 hours after focal injection. No vascular leakage of either molecule was detected 24 hours after stereotactic injection (G, FITC-albumin; H, Texas Red cadaverine), which is confirmed by quantification (I). Number of lesions: FITC-albumin: 3 hours, n = 3; 6 hours, n = 8; 24 hours, n = 9; 3 days, n = 4. (J) Texas Red cadaverine, n = 3. (D, I, and J) Kruskal-Wallis test followed by Dunn’s multiple comparison test. *P < 0.05; **P < 0.01; ***P < 0.001. Graphs are shown as mean ± SEM. Scale bars: 100 μm (A–C); 500 μm (E–H).