Ex vivo investigation of iron handling in the brain

Abstract

Aberrant iron deposition in the brain is associated with aging and neurodegenerative disorders including multiple sclerosis, Alzheimer’s disease and Parkinson’s disease. It is unclear whether metabolic dyshomeostasis of iron is causative or is a consequence of brain pathology. An improved understanding of the mechanisms underlying iron handling might yield critical information relevant to neurodegenerative disease. The main focus of this work was to establish, validate and characterise a novel ex vivo model of iron loading. Having characterized and validated the iron homeostasis system in ex vivo cultures, compared with postnatal and adult tissue, 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. At a transcript level, we also detected modest increases in the expression of several import molecules while the export molecules remained unchanged. 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. The development of such a model, which enables the study of inter- and intra-cellular iron movement and the cellular basis of iron-induced pathology within the complex brain milieu equips researchers with an improved platform to comprehensively study the intricate iron handling system. Indeed, we used this model to preliminarily study the putative intersection between iron metabolism and the unfolded protein response in the CNS. In the iron-loaded slice cultures there were modest increases in transcripts of chaperones and transcription factors associated with the UPR. Moreover, we found that concurrent activation of the unfolded protein response leads to the amelioration of iron accumulation and the resulting toxicity and glial perturbations seen following iron-loading. Finally, given that critical role of image analysis is this work, we critically appraised image segmentation algorithms to the develop, optimize and validate an image analysis workflow for handling the complex micrographs of glial immunohistochemistry acquired from thick 3D ex vivo hippocampal slice cultures.