Elsevier

Neurobiology of Disease

Volume 126, June 2019, Pages 76-84
Neurobiology of Disease

Review
Minocycline attenuates brain injury and iron overload after intracerebral hemorrhage in aged female rats

https://doi.org/10.1016/j.nbd.2018.06.001Get rights and content

Highlights

  • ICH caused brain iron overload in aged female rats.

  • ICH resulted in reproducible brain swelling and brain atrophy in aged female rats.

  • Minocycline reduced ICH-induced brain iron overload and brain damage in aged female rats.

Abstract

Brain iron overload is involved in brain injury after intracerebral hemorrhage (ICH). There is evidence that systemic administration of minocycline reduces brain iron level and improves neurological outcome in experimental models of hemorrhagic and ischemic stroke. However, there is evidence in cerebral ischemia that minocycline is not protective in aged female animals. Since most ICH research has used male models, this study was designed to provide an overall view of ICH-induced iron deposits at different time points (1 to 28 days) in aged (18-month old) female Fischer 344 rat ICH model and to investigate the neuroprotective effects of minocycline in those rats. According to our previous studies, we used the following dosing regimen (20 mg/kg, i.p. at 2 and 12 h after ICH onset followed by 10 mg/kg, i.p., twice a day up to 7 days). T2-, T2-weighted and T2 array MRI was performed at 1, 3, 7 and 28 days to measure brain iron content, ventricle volume, lesion volume and brain swelling. Immunohistochemistry was used to examine changes in iron handling proteins, neuronal loss and microglial activation. Behavioral testing was used to assess neurological deficits. In aged female rats, ICH induced long-term perihematomal iron overload with upregulated iron handling proteins, neuroinflammation, brain atrophy, neuronal loss and neurological deficits. Minocycline significantly reduced ICH-induced perihematomal iron overload and iron handling proteins. It further reduced brain swelling, neuroinflammation, neuronal loss, delayed brain atrophy and neurological deficits. These effects may be linked to the role of minocycline as an iron chelator as well as an inhibitor of neuroinflammation.

Introduction

Intracerebral hemorrhage (ICH) is a devastating stroke subtype with high mortality and morbidity rate. Lysis of red bold cells (RBCs) in the hematoma results in increased perihematomal iron content. Iron may trigger a cascade of deleterious events, which can lead to secondary brain injuries and irreversible neurological deficits years after ICH (Keep et al., 2012). The cytotoxicity of hemolysis-generated iron is related to oxidative stress and an inflammatory response (Xi et al., 2006). During the past decade, studies have found that preventing iron-mediated toxicity is a promising therapeutic strategy for ICH (Keep et al., 2012; Xi et al., 2006).

Sex and age are two major modifiers of brain injury after ICH. Studies have shown that ICH-induced brain damage is less in young female animals, which may be related to normal circulating estrogen (Xi et al., 2006). ICH-induced brain damage is also age dependent. ICH results in more severe brain injury and neurological deficits in aged animals than in young ones (Xi et al., 2006).

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). However, the effect of minocycline in female rats remains unknown. According to the initial Stroke Therapy Academic Industry Roundtable (STAIR) recommendations, further studies should be performed in females, aged animals and animals with comorbid conditions (Fisher et al., 2009). It should be noted that minocycline reduces ischemic brain infarcts in male rats but not female rats (Li and McCullough, 2009).

The current study used an ICH model in aged female rats to investigate changes in perihematomal iron content and iron handling proteins as well as brain injury at different time points after ICH. It next examined whether or not minocycline can reduce iron overload and attenuate brain injury after ICH in those aged female rats.

Section snippets

Animals and establishment of ICH model

All experiments followed the animal procedure protocols approved by the University Committee on Use and Care of Animals, University of Michigan. Seventy-two 18-month old female Fischer 344 rats (National Institutes of Health, Bethesda, MD, USA) were used in the experiments. The rats had free access to food and water before and after surgery and were housed in a 12 h light/dark cycle. The study complies with the ARRIVE guidelines for reporting in vivo experiments. Randomization was carried out

ICH induces iron overload in perihematomal area in aged female rats

ICH resulted in T2 lesions in the brain (reflecting hematoma at day 1 and brain iron overload at day 28). T2 lesions remained till 28 days although it decreased over time (Fig. 1A). Brain iron content was measured using T2 array scanning and shown as R2 (1/T2) values. The operation and injection of saline did not cause variations in R2 values (Fig. 1A). Compared to the contralateral hemisphere, R2 values increased significantly at day 1 (p < 0.01, Fig. 1A) after ICH onset in

Discussion

The major findings of this study are: (1) ICH caused a significant increase in perihematomal brain iron content, iron handling proteins and microglia activation and in aged female rats; (2) the iron overload lasts at least 28 days after ICH; (3) ICH leads to significant short-term brain swelling and long-term brain tissue loss at 28 days in aged female rats; (4) minocycline significantly reduced ICH-induced brain iron overload, attenuated brain injury and improved neurological functional

Conflict of interest

None.

Acknowledgements

This study was supported by grants NS-091545, NS-090925, NS-096917, and NS-106746 from the National Institutes of Health (NIH).

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