Effects of continuous mycophenolate mofetil (MMF) treatment from different time points after injury on neuronal survival and microglial activation in organotypic hippocampal slice cultures (OHSCs).
(A) Treatment protocols. (B) Confocal laser scanning microscopy images in overview and higher magnification, double-labeled with propidium iodide (PI) (degenerating neurons, red) and IB4 (microglial cells, green). Untreated control (CTL) OHSCs had very good preservation of the hippocampal formation with almost no PI+ pyknotic nuclei and only a few ramified IB4
+ microglial cells. Slices treated with NMDA 50 μmol/l for 4 hours had massive accumulation of amoeboid IB4
+ microglial cells and a dramatic increase in PI+ neuronal nuclei. Delayed treatment (until 12 hours after injury) with MMF resulted in a significant reduction of PI+ degenerating neurons. Numbers of microglial cells were reduced even with late administration of MMF (after 48 hours post-injury). (C-D) Quantitative analyses of the effects of MMF on NMDA-lesioned OHSCs. The mean numbers of (C) PI+ degenerating neurons and (D) IB4
+ microglial cells were calculated for each experimental group and compared with OHSCs treated with NMDA alone (*P < 0.05 vs. NMDA). DG, dentate gyrus; Hi, hilus. Scale bars = 50 μm in overviews; 20 μm in insets