Ferric iron chelation lowers brain iron levels after intracerebral hemorrhage in rats but does not improve outcome
Highlights
► Brain iron levels are persistently high after collagenase-induced ICH in rats. ► X-ray florescence (XRF) measured several elements, including iron, after ICH. ► Deferoxamine (DFX) lowered total brain iron levels measured with XRF after ICH. ► DFX did not reduce injury or behavioral deficits in the collagenase model of ICH.
Introduction
Brain injury after intracerebral hemorrhage (ICH) starts with direct mechanical trauma as blood rapidly enters the parenchyma, and progresses because of numerous deleterious processes causing secondary degeneration. Well-known among these mechanisms are the toxic effects of blood that cause cell death, inflammation, blood brain barrier disruption and cerebral edema. The ever-expanding knowledge of ICH pathophysiology has lead to the development and testing of many neuroprotective treatments in animal models including some drugs that have undergone or are currently in clinical trials (Aronowski and Zhao, 2011, Frantzias et al., 2011, MacLellan et al., 2009).
Among many deleterious processes occurring after ICH, iron is perhaps the most widely studied contributor to secondary degeneration (Aronowski and Zhao, 2011, Xi et al., 2006), and iron is widely thought to have a central role in other neurodegenerative diseases (Kell, 2010). Following ICH, iron is liberated by heme metabolism through heme oxygenase-1 activity, but it is important to note that this does not occur immediately after ICH. Instead, hemolysis only begins about 24 h after ICH occurs and is complete after several days. During this time, activated microglia and macrophages protect the brain by removing intact and damaged erythrocytes from the parenchyma. They also remove released hemoglobin and its component, heme, via uptake of haptoglobin-hemoglobin and hemopexin-heme complexes, respectively (Aronowski and Zhao, 2011). However, these protective measures along with increases in iron binding proteins, ferritin and transferrin, are not sufficient to prevent a substantial increase in oxidative stress, which, for instance, damages DNA after ICH (Aronowski and Zhao, 2011, Nakamura et al., 2005, Wagner et al., 2002, Wu et al., 2012, Wu et al., 2002). Post-ICH oxidative stress originates in part from ‘free’ or loosely bound ferrous iron that promotes the generation of highly reactive free radicals (e.g., OH·) through Fenton chemistry.
Use of animal models shows substantial elevations in iron after ICH. For example, non-heme iron was 2–4 × higher in brain tissue samples from 3 to 28 days after a striatal infusion of whole blood (Wu et al., 2003), a common rodent model of ICH. Likewise, iron histochemistry (Perls stain) shows prominent ferric iron concentration within and near the hematoma in rodents (Wu et al., 2003) as does magnetic resonance imaging (MRI) that detects paramagnetic ferritin iron (Wu et al., 2010). Several lines of evidence strongly suggest that excessive iron mediates secondary damage, although not all data concur. First, intracerebral FeCl2 infusion leads to edema and rapid cell death (Huang et al., 2002, Nakamura et al., 2005). We have also observed neuronal death occurring over months after FeCl2 infusion along with marked dendritic atrophy (Caliaperumal, Ma and Colbourne, unpublished data). Thus, iron alone appears to causes injury similar to that seen after ICH, but these data do not prove that this is a key mechanism of secondary degeneration after ICH. Further evidence comes from numerous studies showing that free radical scavengers improve outcome in animal models of ICH. However, these drugs often did not reduce lesion size (Peeling et al., 1998, Peeling et al., 2001) and a clinical trial with NXY-059, a free radical scavenger, was also negative (Lyden et al., 2007).
The strongest data to support the hypothesis that iron mediates secondary degeneration comes from the use of iron chelators in animal models. The most widely studied drug is deferoxamine (DFX), a Fe3 + chelator used in patients with iron overload and currently in clinical trials for ICH (Selim et al., 2011). Post-ICH treatment with DFX improves behavioral scores, reduces edema and blood brain barrier dysfunction, and lessens cell death in the whole blood model in rats (Hua et al., 2006, Nakamura et al., 2003, Okauchi et al., 2009, Okauchi et al., 2010, Song et al., 2008), mice (Wu et al., 2012) and pigs (Gu et al., 2009). However, results in the collagenase model of ICH are less encouraging (Warkentin et al., 2010, Wu et al., 2012). Collagenase is an enzyme that breaks down the basal lamina of vessels resulting in spontaneous hemorrhaging (Rosenberg et al., 1990). It is widely used to model ICH in rodents, but only a few studies have tested whether DFX reduces injury in this model. In one, DFX treatments like those used successfully in whole blood studies failed to lessen edema, behavioral impairments or tissue loss in the collagenase model (Warkentin et al., 2010). Another collagenase study also found that DFX did not lessen edema or lesion volume, but there was a reduction in peri-hematoma cell death and modest functional benefits (Wu et al., 2012). Studies in the whole blood model show that DFX lessens iron levels in cerebrospinal fluid, but interestingly not in the brain (Wan et al., 2006, Wan et al., 2009). Others report that there are fewer iron positive cells, including many macrophages and microglia, as identified with the Perls stain after collagenase-induced ICH (Wu et al., 2012).
There are numerous ways to determine iron levels in biological tissues (McRae et al., 2009). In the ICH field, studies have relied upon the Perls histochemical stain for ferric iron and the non-heme iron assay. The former identifies iron-rich cells, such as macrophages, and hemosiderin, but staining is not easily quantified nor does it reflect total iron. The latter is suitable for quantifying non-heme iron levels (Wu et al., 2008) in homogenized tissue samples but not in histological slides. These two methods cannot quantitatively determine the spread of iron (e.g., relative to the hematoma) nor relate the distribution of neuronal death with iron levels. In contrast, synchrotron rapid scanning X-ray fluorescence (RS-XRF) can both map and quantify elements in tissue slides (Habib et al., 2010, Popescu et al., 2009, Popescu and Nichol, 2011). While RS-XRF has been applied to human stroke (Zheng, Haacke, Webb and Nichol, unpublished data), it has not been used in rodent ICH models. Thus, we used RS-XRF to simultaneously map several elements at moderate resolution (e.g., 50 μm) in coronal sections of rat brain at 3 survival times after collagenase-induced ICH. We also evaluated the effect of DFX treatment on total iron levels as measured with RS-XRF. Tissue loss and behavioral deficits were evaluated too. Finally, for comparative purposes we used a Ferrozine-based non-heme iron assay at 3 times post-ICH.
Section snippets
Animals
Sixty-nine male Sprague–Dawley rats were obtained from the Biosciences breeding colony at the University of Alberta. They were approximately 11 weeks of age when they were entered into this study. Procedures were in accordance with the Canadian Council on Animal Care and were approved by the Biosciences Animal Care and Use Committee at the University of Alberta. Rats were given free access to food and water throughout the study.
Sixteen rats were used in the first experiment that quantified
Experiment 1: non-heme iron levels following ICH
There were no exclusions or unexpected mortality in experiment 1.
One-way ANOVAs showed significant main effects for the non-heme iron levels (Fig. 1) within the injured (p < 0.001) and non-injured hemispheres (p = 0.026), but not the cerebellum (p = 0.533) or liver controls (p = 0.642). For the injured hemisphere, the day 3 and 28 groups had significantly more iron than naïve or day 1 ICH groups (p ≤ 0.013) and there was more iron at 28 than 3 days post-ICH (p = 0.017). For the uninjured side, only day 3
Discussion
To our knowledge, this is the first experiment using RS-XRF to map and quantify iron, and other elements, after collagenase-induced ICH in rats. Total iron in the damaged hemisphere was significantly and persistently elevated after ICH, but elevations were localized to the hematoma and immediate peri-hematoma regions. Other studies using Perls histochemistry and non-heme spectrophotometry assays (Wu et al., 2003) have drawn similar conclusions. Similarly, we demonstrated a persistent increase
Acknowledgments
This work is supported by a joint Canadian Institutes of Health Research (CIHR)/Heart and Stroke Foundation of Canada team grant: Synchrotron Medical Imaging (#CIF 99472) awarded to H. Nichol, P. Paterson, F. Colbourne and others. A. Auriat is a CIHR-fellow in Health Research Using Synchrotron Techniques and also supported by a McCormick Fellowship. G. Silasi received funding from a focus on stroke doctoral scholarship. F. Colbourne is a senior medical scholar of Alberta Innovates — Health
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