(24) Developing a Novel Primary Neonatal Mouse Astrocyte Cell Culture Model of Iron Deficiency

Emma Tripp, University of Minnesota Medical School, Eden Prairie, MN, United States; Daniel Mickelson, University of Minnesota Medical School, United States; Thomas Bastian, University of Minnesota Medical School, United States

Background: Iron deficiency (ID) is one of the most common nutrient deficiencies worldwide. It affects ~40-50% of women of reproductive age globally and 80% of pregnant women in low-resource countries, negatively influencing the developing fetus. ID causes impaired neural development and hindered energy metabolism. These cellular effects in early development translate to deficits in learning and memory and underdeveloped brain structures.
Glial cells (e.g microglia, oligodendrocytes and astrocytes) play a critical role in brain development through myelination, regulating ion and neurotransmitter concentrations, and neuronal support. The absence of astrocyte support of these processes would be detrimental to the neonatal brain and cause long term impairments. However, compared to neurons, little is known about the cellular structure and function of astrocytes under physiological, neonatal ID.

Objectives: Our overall objective is to test the hypothesis that ID-induced impairments to astrocyte cell development and function contribute to the persistent neurobehavioral deficits following early-life ID. The aim of this study was to develop a novel culture model of primary neonatal mouse glial cells under developmental ID.

Design/Methods: Primary mixed glial cell cultures were prepared from the postnatal day 1 mouse cerebral cortex. Once the astrocyte layer reached confluency, the iron chelator, deferoxamine (DFO), was added to the iron-sufficient culture medium to create iron deficient conditions. DFO was added in increasing concentrations from 0μM to 100μM. Astrocyte cell morphology and viability were assessed using phase contrast light microscopy after one week of DFO treatment. Cellular ID was assessed by quantifying Tfrc mRNA levels using qPCR.

Results: DFO concentrations from 2 and 5μM increased Tfrc mRNA levels by ~2.5x, confirming cellular ID. This degree of ID did not alter cell morphology and health compared to 0μM DFO iron-sufficient cultures. At 9μM DFO, the glia were disformed with extensive vacuole formation. At 15µM to 100μM DFO, there was extensive cell death with a ~50% reduction in cell density.

Conclusion: Our findings support our hypothesis that developing neonatal astrocytes are sensitive to ID. We are currently assessing the effect of ID on key astrocyte specific genes and proteins involved in cell iron homeostasis, metabolism, and support of neuronal development/function. By further understanding the role of iron in astrocyte cellular development and function, we can more fully reveal how ID impacts the brain.