Biochemical and Biophysical Research Communications
Minocycline attenuates iron neurotoxicity in cortical cell cultures
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.
References (36)
- et al.
Heme oxygenase-2 knockout neurons are less vulnerable to hemoglobin toxicity
Free Rad. Biol. Med.
(2003) - et al.
Minocycline protects the blood–brain barrier and reduces edema following intracerebral hemorrhage in the rat
Exp. Neurol.
(2007) Absorption of minocycline hydrochloride and tetracycline hydrochloride. Effect of food, milk, and iron
J. Am. Acad. Dermatol.
(1985)- et al.
Iron-catalyzed hydroxyl radical formation. Stringent requirement for free iron coordination site
J. Biol. Chem.
(1984) - et al.
Mechanism of heme degradation by heme oxygenase
J. Inorg. Biochem.
(2000) - et al.
Heme oxygenase-2 gene deletion increases astrocyte vulnerability to hemin
Biochem. Biophys. Res. Commun.
(2004) - et al.
Effect of heme oxygenase-1 on the vulnerability of astrocytes and neurons to hemoglobin
Biochem. Biophys. Res. Commun.
(2006) - et al.
Regulation of ferritin genes and protein
Blood
(2002) - et al.
Iron regulatory protein 2 as iron sensor. Iron-dependent oxidative modification of cysteine
J. Biol. Chem.
(2003) - et al.
Neurons lacking iron regulatory protein-2 are highly resistant to the toxicity of hemoglobin
Neurobiol. Dis.
(2008)
Mechanisms of brain injury after intracerebral haemorrhage
Lancet Neurol.
Iron and iron-handling proteins in the brain after intracerebral hemorrhage
Stroke
Long-term effects of experimental intracerebral hemorrhage: the role of iron
J. Neurosurg.
Systemic zinc protoporphyrin administration reduces intracerebral hemorrhage-induced brain injury
Acta Neurochir. Suppl.
Attenuation of oxidative injury after induction of experimental intracerebral hemorrhage in heme oxygenase-2 knockout mice
J. Neurosurg.
Deferoxamine-induced attenuation of brain edema and neurological deficits in a rat model of intracerebral hemorrhage
J. Neurosurg.
Inflammation after intracerebral hemorrhage
J. Cereb. Blood Flow Metab.
Intracerebral hemorrhage induces macrophage activation and matrix metalloproteinases
Ann. Neurol.
Cited by (57)
Association of serum iron and hepcidin levels with stroke from 1990 to 2022: A systematic review and meta-analysis
2023, Journal of Functional FoodsIron toxicity in intracerebral hemorrhage: Physiopathological and therapeutic implications
2022, Brain Research BulletinMinocycline attenuates brain injury and iron overload after intracerebral hemorrhage in aged female rats
2019, Neurobiology of DiseaseCitation Excerpt :Minocycline, a broad-spectrum tetracycline, can protect against neurological impairment in animal models of traumatic brain injury, stroke and neurodegenerative disease via its anti-inflammatory properties including inhibiting microglia activation and matrix metalloproteinases (Murata et al., 2008; Wang and Dore, 2007). However, a study has also demonstrated that minocycline can chelate iron in cortical neuronal cultures (Chen-Roetling et al., 2009). Our previous studies have shown that systemic administration of minocycline attenuates brain injury and improves functional deficits after ICH by reducing iron overload in both young and aged male rats (Cao et al., 2018; Zhao et al., 2011).
Protective effect of vitreous against hemoglobin neurotoxicity
2018, Biochemical and Biophysical Research CommunicationsIron released from reactive microglia by noggin improves myelin repair in the ischemic brain
2018, Neuropharmacology