The Optimization of the Isolation and Immunocytochemical Analysis of Primary Neurons
Landis, Rory W.
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The general decline in physiological function during the normal aging process can be attributed to several factors including oxidative stress. The increased dysfunction of the mitochondrial electron transport chain (mETC) with age is thought to be the main contributor in the generation of oxidative stress via the production of reactive oxygen species (ROSs). Iron is necessary for the proper functioning of the mETC and accumulates in mitochondria with age. Astrocytic and other components of the central nervous system have shown increased susceptibility to the toxicant 1,3-dinitrobenzene (1,3-DNB), a chemical intermediate shown to target several mETC components. The present study focuses on developing and optimizing a model system for the indirect, qualitative measurement of iron via immunocytochemistry (ICC) of iron-regulatory proteins, transferrin and frataxin, in primary neuron cultures of varying age cohorts exposed to 1,3-DNB. Cells were isolated from Fischer 344 male rats of ages 1 m.o., 3 m.o., and 18 m.o., indicative of preadolescent, young adult, and older adult organisms, respectively. The morphological similarity of isolated cells to that of neurons coupled with the ambiguous ICC results suggests that while culture conditions may be sufficient, future optimization steps and follow up studies will be required for more conclusive mechanistic studies that will elucidate variations in the expression of iron-regulatory proteins as a function of age (time in culture), anatomic localization and/or increased susceptibility to 1,3-DNB exposure.