Minocycline attenuates iron neurotoxicity in cortical cell cultures

https://doi.org/10.1016/j.bbrc.2009.06.026Get rights and content

Abstract

Iron neurotoxicity may contribute to the pathogenesis of intracerebral hemorrhage (ICH). The tetracycline derivative minocycline is protective in ICH models, due putatively to inhibition of microglial activation. Although minocycline also chelates iron, its effect on iron neurotoxicity has not been reported, and was examined in this study. Cortical cultures treated with 10 μM ferrous sulfate for 24 h sustained loss of most neurons and an increase in malondialdehyde. Minocycline prevented this injury, with near-complete protection at 30 μM. Two other inhibitors of microglial activation, doxycycline and macrophage/microglia inhibitory factor, were ineffective. Oxidation of isolated culture membranes by iron was also inhibited by minocycline. Consistent with prior observations, minocycline chelated iron in a siderophore colorometric assay; at concentrations less than 100 μM, its activity exceeded that of deferoxamine. These results suggest that attenuation of iron neurotoxicity may contribute to the beneficial effect of minocycline in hemorrhagic stroke and other CNS injury models.

Introduction

Tissue iron is increased within 1 day in the vicinity of an experimental intracerebral hemorrhage [1], and persists for at least 3 months [2]. A growing body of experimental evidence suggests that this iron may contribute to cell injury. Reducing heme breakdown and iron release with heme oxygenase (HO) inhibitors or HO gene knockout is beneficial in animal models [3], [4], and protects neurons from hemoglobin toxicity in cell culture [5]. More specifically, post-hemorrhage treatment with the iron chelator deferoxamine reduces edema, oxidative injury markers, and neuronal loss, and also improves behavioral outcome [2], [6].

A peri-hematomal inflammatory infiltrate, consisting of leukocytes and activated microglia, is observed within 24 h of experimental intracerebral hemorrhage and also may contribute to secondary injury [7]. This inflammation hypothesis has recently been tested using the tetracycline derivative minocycline [8], [9], [10], which inhibits microglial activation and is beneficial in several ischemic stroke models [11], presumably due to its anti-inflammatory effect. However, as described by Grenier et al. [12], minocycline has strong iron-chelating activity, which has been of some clinical relevance. Its absorption after oral administration is greatly reduced when administered with iron or calcium supplements, consistent with its affinity for metal cations [13]. Skin hyperpigmentation, an adverse effect of long-term minocycline therapy, is a consequence of dermal precipitation of a minocycline–iron complex [14]. By depriving bacteria of an essential nutrient, iron chelation may also account in part for its antibiotic effect [12], although evidence supporting the physiologic relevance of this mechanism is limited.

The redox activity of iron is altered in a highly variable manner by chelator binding. Catalysis of hydroxyl radical generation via the Fenton reaction requires at least one of six iron coordination sites to be available, or occupied by a low-affinity ligand such as water [15]. A chelator that occupies fewer than six sites may not prevent oxidative injury, and may even increase it if it mobilizes iron from protein binding sites in a redox-active state [16]. Despite the recent interest in minocycline therapy for hemorrhagic stroke, its effect on iron-mediated oxidative neuronal injury has never been reported. The present study tested that hypothesis that minocycline attenuates the oxidative neurotoxicity of iron in primary cortical cell cultures.

Section snippets

Materials and methods

Cortical cell cultures. All procedures on animals were reviewed and approved by the Thomas Jefferson University Institutional Animal Care and Use Committee (IACUC). Mixed cortical cell cultures, containing both neurons and glia were prepared from fetal B6129 mice (gestational age 13–15 days), as previously described [5]. The dissociated cell suspension was plated on glial feeder cultures (>90% GFAP+, approximately 2% microglia by tomato lectin staining [17]) in 24-well plates (Falcon, Becton

Minocycline protects cortical neurons from iron

Consistent with prior observations using this model [5], [23], cultures treated with 10 μM FeSO4 for 24 h sustained widespread neuronal death, without injury to the feeder glial monolayer. Concomitant treatment with equimolar minocycline reduced neuronal death, as measured by LDH release assay, by approximately one-third at 24 h (Fig. 1A). Increasing the minocycline concentration to 30 μM reduced LDH release by 87.4 ± 1.8%, which was similar to the neuroprotection provided by 30 μM deferoxamine (91.0 ± 

Discussion

These results demonstrate that minocycline potently inhibits iron neurotoxicity in cortical cell cultures and iron-catalyzed lipid oxidation in isolated cell membranes. Furthermore, two other inhibitors of microglial activation, doxycycline and macrophage/microglia inhibitory factor (MIF) [24], [25], provide no cytoprotection. Since iron neurotoxicity contributes to neuronal loss after experimental ICH [2], [6], these data suggest that any therapeutic benefit of minocycline may be due at least

Acknowledgments

Funding for this study was provided by grants from the National Institutes of Health (NS042273) and the Great Rivers Affiliate of the American Heart Association.

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