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Background/aim: Altered iron metabolism is one of the pathophysiological mechanisms occurring during hypoxic injuries in the central nervous system. Proper homeostasis of cellular iron is regulated by iron import, storage, and export proteins that prevent excess iron overload or iron starvation in cells. Therapeutic hypothermia is an approved treatment for hypoxic ischemia in newborns, but the underlying molecular mechanism is still unknown. We studied the effects of hypoxia, preceded with preconditioning, on the iron homeostasis of glial cells, known as a major actor in the inflammatory process during perinatal brain injury.Materials and methods: Primary microglia and astrocytes in culture were exposed to 12 h of hypoxia with or without mild hypothermic preconditioning. The mRNA expression was assessed using qPCR. Iron accumulation was visualized via modified Perl's histochemistry. Cytokine levels in cell cultures were measured using ELISA.Results: Hypothermic preconditioning enhanced microglial viability, which previously was decreased in both cell types due to hypoxia. Hypoxia increased iron accumulation in the mixed glial cells and in ferritin expression in both microglia and astrocytes. Hypotermic preconditioning decreased the elevated ferritin-light chain expression significantly in microglia. Iron importer proteins, DMT1 and TfR1, both increased their mRNA expression after hypoxia, and hypothermic preconditioning continued to support the elevation of DMT1 in both glial cell types. Ferroportin expression increased as a survival factor of the glial cell following hypoxia. Hypothermic preconditioning supported this increase in both cell types and was especially significant in astrocytes. IL-10 levels were prominently increased in cell culture after hypothermic preconditioning. Conclusion:The data suggest that hypothermic preconditioning affects cellular iron homeostasis by regulating the storage and transfer proteins of iron. Regulation of the cellular iron traffic may prevent glial cells from experiencing the detrimental effects of hypoxia-related inflammation.

A well-balanced intracellular iron trafficking in glial cells plays a role in homeostatic processes. Elevated intracellular iron triggers oxidative stress and cell damage in many neurological disorders, including migraine. This study aimed to investigate the effects of glyceryl trinitrate (GTN), on cellular iron homeostasis, matrixmetalloproteinase (MMP)-9, and calcitonin gene related peptide (CGRP) receptor (CRLR/CGRPR1) production in microglia, astrocyte, and meningeal cell cultures. Primary glial and meningeal cells in culture were exposed to GTN for 24 h. Messenger RNA expression was assessed using qPCR. Iron accumulation was visualized via modified Perl’s histochemistry. MMP-9 levels in cell culture supernatants were measured using ELISA. Ferritin and CRLR/CGRPR1 proteins were visualized via immunofluorescence staining. Nitric oxide production increased significantly with GTN in meningeal and glial cells. GTN significantly increased the expression of the storage protein ferritin for all three cell types, but ferritin-L for meningeal cells and microglia. Iron trafficking associated with the efflux protein ferroportin and influx protein divalent metal transporter (DMT)1 was affected differently in all three cell types. MMP-9 expression was increased in astrocytes. GTN stimulation increased both CRLR/CGRPR1 expression, and immunostaining was apparent in microglia and meningeal cells. This study showed for the first time that GTN modulates intracellular iron trafficking regulated by storage and transport proteins expressed in meningeal cells and glia. CRLR/CGRPR1 expression might be related to altered iron homeostasis and they both may stimulate nociceptive pathways activated in migraine. These molecules expressed differently in glial and meningeal cells in response to GTN may bring not only new targets forward in treatment but also prevention in migraine.

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