Aberrant iron deposition in the brain is associated with neurodegenerative disorders including Multiple Sclerosis, Alzheimer's disease and Parkinson's disease. To study the collective response to iron loading, we have used hippocampal organotypic slices as a platform to develop a novel ex vivo model of iron accumulation. We demonstrated differential uptake and toxicity of iron after 12 h exposure to 10 μM ferrous ammonium sulphate, ferric citrate or ferrocene. Having established the supremacy of ferrocene in this model, the cultures were then loaded with 0.1-100 μM ferrocene for 12 h. One μM ferrocene exposure produced the maximal 1.6-fold increase in iron compared with vehicle. This was accompanied by a 1.4-fold increase in ferritin transcripts and mild toxicity. Using dual-immunohistochemistry, we detected ferritin in oligodendrocytes, microglia, but rarely in astrocytes and never in neurons in iron-loaded slice cultures. Moreover, iron loading led to a 15% loss of olig2-positive cells and a 16% increase in number and greater activation of microglia compared with vehicle. However, there was no appreciable effect of iron loading on astrocytes. In what we believe is a significant advance on traditional mono- or dual-cultures, our novel ex vivo slice-culture model allows characterization of the collective response of brain cells to iron-loading.