Figure 6

Replenishment of microglia-depleted slice cultures with primary mouse microglia reduces excitotoxicity-induced neuronal cell death. After 9 days in vitro, cultured primary mouse microglia were carefully pipetted onto depleted slice cultures at a density of 400 cells per slice culture. 12 days later, slice cultures were immuno-stained for NeuN (grey) and Iba1 (yellow) revealing that exogenously applied microglia showed equal distribution and a ramified morphology (A,D) and were integrated into the tissue (B). The cells (yellow arrows) had distributed themselves throughout the total depth of the slice cultures as examined by confocal microscopy (B: orthoview of a z-stack). 3D reconstructions of microglia filaments, created by IMARIS filament tracer software from Iba1 fluorescently stained cells in z-stacks of slice cultures, were used to analyse the morphology of endogenous microglia and replenished primary mouse microglia. Figure C and D show examples for reconstructions of endogenous microglia (endo microglia) and replenished primary microglia (primary microglia), respectively. The starting point of the filaments was set at the cell soma (blue). Analysis of the morphologic parameters total dendritic length (E) and number of branch points (F) revealed significantly shorter dendritic length and less branching points in replenished primary microglia compared to endogenous microglia (*** p < 0.001). NMDA (25 μM)-induced neuronal cell death in the dentate gyrus was significantly reduced in slice cultures replenished with primary mouse microglia (21.6%) compared to microglia-free slice cultures (53.6%), as determined by total PI uptake (G). Data are provided as mean ± SEM. N = 25 cells per group for E and F and N = 4 for G. Scale bars: Scale bars indicate 100 μm (A-B; shown in A) and 10 μm (C-D).