Laser ablation-inductively coupled plasma-mass spectrometry imaging of white and gray matter iron distribution in Alzheimer's disease frontal cortex
Graphical abstract
Introduction
Disrupted iron metabolism appears to be a pathological hallmark in the Alzheimer's disease brain (Roberts et al., 2011). Numerous studies have identified abnormal increases in the iron concentration within a range of affected brain regions (Belaidi and Bush, 2015), including accumulation on the β-amyloid senile plaques that are characteristic of AD (Lovell et al., 1998). While a 2011 meta-analysis suggested a possible citation bias has overstated the significance of iron elevation (Schrag et al., 2011), it should not be ignored that disrupted iron homeostasis without a measurable increase still has the potential to promote oxidative stress through improper redox-silencing of this highly reactive species. Changes in chemical properties of brain iron have been observed dating back over half a century (Hallgren and Sourander, 1960), and contemporary biotechnology has identified a range of genetic and metabolic factors that support iron dyshomeostasis as playing an important role in AD pathology (Crespo et al., 2014).
Important iron regulatory proteins, including ferritin and transferrin appear to be both dysfunctional and abnormally distributed in the AD brain (Connor, J.R., et al., 1992a, Connor, J.R., et al., 1992b, Connor, J.R., et al., 1995), potentially contributing to the reactive ‘labile iron pool’ through mismanagement of normal metabolic pathways. Neuroinflammation, where glial cells promote the deposition of iron, contributes to elevated oxidative stress and mitochondrial dysfunction, and may also promote the aggregation of the β-amyloid peptide and tau protein, forming the plaques and tangles characteristic of the disease (Ong and Farooqui, 2005). Combined with the natural accumulation of iron in the aging brain, endogenous response to elevated cortical iron (such as heme oxygenase-1, which degrades heme and can release free, reactive ferrous [Fe2 +] iron) may represent an important biochemical mechanism preceding neuronal damage in AD (Ward et al., 2014).
In vivo imaging of the AD brain using magnetic resonance imaging (MRI) has provided useful insight into both structural changes (Bartzokis et al., 2003) and iron deposition (Bartzokis, G., et al., 2000, Langkammer, C., et al., 2014), using techniques such as R2 and R2* relaxometry (Langkammer et al., 2010) and phase imaging (Zhu et al., 2009). However, differentiation between white and gray matter iron distribution in the neocortex using MRI is challenging, as typical MRI approaches are not absolutely quantitative; there are multiple contributions to tissue contrast (including myelin, iron and CSF); and most MRI methods have a spatial resolution that precludes fine detail definition of brain iron distribution at micrometer scales. Because of these many limitations, MR imaging of brain iron has been largely constrained to deep brain nuclei, such as the basal ganglia, which contain the highest iron content throughout the brain.
In this study we employed quantitative iron imaging by laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) to compare the distribution of iron in white and gray matter regions of postmortem AD and healthy control (HC) frontal cortex tissue which are primarily affected by AD pathology. LA-ICP-MS employs a focused beam (typically in the ultra-violet range) that ablates particles from the tissue sample surface, which are then carried to the ICP-MS and measured on the basis of mass-to-charge (m/z) ratio (Hare et al., 2015). LA-ICP-MS is highly specific and sensitive to iron, with detection limits well below the typical biological concentrations found in neurological tissue (O'Reilly et al., 2014). With appropriate signal normalization and periodic sampling of standards with comparable matrix composition, LA-ICP-MS can provide absolute quantitative information at the low micrometer scale (1–100 + μm) (Hare, D.J., et al., 2012a, Miliszkiewicz, N., et al., 2015). As an element-specific detector, LA-ICP-MS also permits simultaneous detection of multiple analytes and generation of hyperspectral images. We exploited this capability here by using phosphorus distribution as a proxy for white and gray matter, which was then applied to differentiating iron distribution in the two regions of frontal cortex tissue from both AD and HC brains.
Section snippets
Human brain samples
Formalin fixed and paraffin embedded AD (n = 4) and HC (n = 5) cortical tissue from the superior frontal gyrus was obtained from the Victorian Brain Bank Network at the Florey Institute of Neuroscience and Mental Health. All procedures were conducted in accordance with the Australian National Health and Medical Research Council's National Statement on Ethical Conduct in Human Research (2007), the Victorian Human Tissue Act (1982), the National Code of Ethical Autopsy Practice (2002) and the
Results
Quantitative images of iron in the AD and HC sections are presented in Fig. 1a (shown here on the same scale, see Supplementary Fig. S3 for individually scaled images). In AD tissue, mean iron concentration in the entire scanned section was elevated compared to controls (mean iron concentration AD = 18.80 ± 2.23 μg g− 1; HC = 12.80 ± 1.17 μg g− 1; p < 0.05, Student's two-tailed t-test; Fig. 1b). Perls staining with DAB enhancement (Fig. 1a) revealed only minor non-heme iron deposition within white matter. Iron
Considerations for postmortem artifact
Previously reported iron levels in digests of formalin-fixed frontal white matter, measured using solution nebulization ICP-MS, were markedly higher than our results; iron concentrations were ~ 50% of those reported in fixed frontal lobe tissue reported by others (Hallgren, B. and Sourander, P., 1960, Langkammer, C., et al., 2012a, Langkammer, C., et al., 2012b). However, leaching did not appear to be specific to cortical tissue; the iron concentration in the caudate nucleus excluded from our
Conclusions
We have demonstrated that postmortem analysis of frontal cortex tissue from AD and HC subjects displays a marked change in cortical gray matter iron distribution in this degenerating region of the brain. Although this method is only possible using postmortem tissue, we present important supporting evidence for existing MRI studies that have focused on discerning white and gray matter iron distributions in vivo using a highly sensitive and quantitative imaging approach. Results from this study
Acknowledgements
The authors would like to thank Dr. Ian Birchall and Dr. Jeff Duyn for their helpful advice, and Ms. Fairlie Hilton of the Victorian Brain Bank Network for her assistance with case notes. D.J.H. and P.A.D. are supported by funds from Australian Research Council Linkage Project (LP120200081) in conjunction with ESI Ltd. and Agilent Technologies. D.J.H. and B.R.R. are additionally supported through Australian Research Council Linkage Project (LP140100095) with Agilent Technologies. E.P.R. is
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These authors contributed equally.