One could argue that the dismetabolism of iron, whether throughout the whole body or only in the brain, is a recurring theme in neurodegenerative disease. Evidence of such has been available for several decades and has taken many forms, including techniques allied to quantitative determinations of biogenic iron associated with pathogenic structures such as proteinaceous plaques in AD and MS and Lewy bodies in PD.

Iron is no stranger to biochemical evolution, and its deposition in the brain may well be an early adaptation to excess free iron, for example, leading to the essential function of biogenic magnetite in magnetotactic bacteria (see Scheffel et al., 2006). We do not know why iron is deposited in nervous tissue in neurodegenerative diseases, nor do we know very much about the forms of iron that are deposited. The latter has only recently been the subject of intense investigation. Jon Dobson (a colleague of mine at Keele), Mark Davidson, and co-workers are leading exponents of this field.

Their application of high...  Read more

One could argue that the dismetabolism of iron, whether throughout the whole body or only in the brain, is a recurring theme in neurodegenerative disease. Evidence of such has been available for several decades and has taken many forms, including techniques allied to quantitative determinations of biogenic iron associated with pathogenic structures such as proteinaceous plaques in AD and MS and Lewy bodies in PD.

Iron is no stranger to biochemical evolution, and its deposition in the brain may well be an early adaptation to excess free iron, for example, leading to the essential function of biogenic magnetite in magnetotactic bacteria (see Scheffel et al., 2006). We do not know why iron is deposited in nervous tissue in neurodegenerative diseases, nor do we know very much about the forms of iron that are deposited. The latter has only recently been the subject of intense investigation. Jon Dobson (a colleague of mine at Keele), Mark Davidson, and co-workers are leading exponents of this field.

Their application of high resolution synchrotron techniques (Collingwood et al., 2005), as well as highly sensitive interference magnetometry, SQUID (Hautot et al., 2003), has moved our understanding of the structures of brain iron deposits into the twenty-first century. Only now are we beginning to differentiate these deposits based on their primary biogenic structures. The latter is important for a number of reasons. For example, we may be able to use early deposition of iron in a particular form, for example, magnetite, as a signature for an ongoing disease process. The ability to "see" iron spectroscopically with noninvasive in-vivo imaging techniques could, potentially at least, revolutionize diagnosis of chronic neurodegenerative conditions such as AD and MS.

Equally important is that knowing the form of deposited iron in a particular condition could give an important insight into disease etiology. I am convinced that the form of iron in, for example, senile plaques in AD, is dictated by the "protein" environment that "templates" its deposition. For example, the protein amyloid-β is only involved in the deposition; it is not part of the biogenic product (though some could conceivably be occluded within the matrix of the final product). The biogenic product is extremely insoluble in comparison to any putative protein/peptide-iron complexes.

Thus, the disease process dictates the form of deposited iron. If this were the end of the story, then probably the brain would be sufficiently robust to cope with the excess iron using evolutionarily conserved mechanisms. However, the additional presence of aluminum in the brain upsets the apple-cart (see Khan et al., 2006). Aluminum, most likely as an aluminum superoxide semi-reduced radical ion (AlO2•2+), is able to reduce Fe3+ and thereby delay its deposition as biogenic iron. It is perhaps by this route that iron remains active as an oxidant in the vicinity of senile plaques, etc., and Fe2+ promotes the oxidative damage that is characteristic of neurodegenerative diseases.

View all comments by Chris Exley

Imagine Imaging Iron in Alzheimer Disease
The early detection and diagnosis of Alzheimer disease (AD) is an extremely active area since it is likely to provide better therapeutic opportunities for patients both in the very earliest stages of disease as well as those at risk of developing disease. To date, the majority of studies have focused on structural changes (MRI) or metabolic analysis (PET) that likely represent downstream consequences of neuronal atrophy rather than initiators of disease. More recently, a great deal of attention has been given to the imaging of amyloid-β deposits using the Pittsburgh compound (PIB). However, while amyloid-β deposits are pathognomonic for AD, their high prevalence in normal aged individuals makes diagnosis problematic in the absence of clinical symptoms. On the other hand, oxidative stress, which is known to predate amyloid-β deposits (Odetti et al., 1998; Nunomura et al., 2001; Pratico et al., 2001; Pratico et al., 2002), may represent a superior diagnostic target. Since imbalances in iron homeostasis appear to be intimately...  Read more

Imagine Imaging Iron in Alzheimer Disease
The early detection and diagnosis of Alzheimer disease (AD) is an extremely active area since it is likely to provide better therapeutic opportunities for patients both in the very earliest stages of disease as well as those at risk of developing disease. To date, the majority of studies have focused on structural changes (MRI) or metabolic analysis (PET) that likely represent downstream consequences of neuronal atrophy rather than initiators of disease. More recently, a great deal of attention has been given to the imaging of amyloid-β deposits using the Pittsburgh compound (PIB). However, while amyloid-β deposits are pathognomonic for AD, their high prevalence in normal aged individuals makes diagnosis problematic in the absence of clinical symptoms. On the other hand, oxidative stress, which is known to predate amyloid-β deposits (Odetti et al., 1998; Nunomura et al., 2001; Pratico et al., 2001; Pratico et al., 2002), may represent a superior diagnostic target. Since imbalances in iron homeostasis appear to be intimately related to oxidative stress (Smith et al., 1997; Sayre et al., 2000), the studies by Collingwood and Dobson (Collingwood et al., 2005; Collingwood and Dobson, 2006) are likely to be of paramount importance for future imaging studies to capture individuals at greatest risk of progressing to occult disease.

References:
Collingwood J, Dobson J (2006) Mapping and characterization of iron compounds in Alzheimer's tissue. J Alzheimers Dis 10, in press.

Collingwood JF, Mikhaylova A, Davidson M, Batich C, Streit WJ, Terry J, Dobson J (2005) In situ characterization and mapping of iron compounds in Alzheimer's disease tissue. J Alzheimers Dis 7, 267-272. Abstract

Nunomura A, Perry G, Aliev G, Hirai K, Takeda A, Balraj EK, Jones PK, Ghanbari H, Wataya T, Shimohama S, Chiba S, Atwood CS, Petersen RB, Smith MA (2001) Oxidative damage is the earliest event in Alzheimer disease. J Neuropathol Exp Neurol 60, 759-767. Abstract

Odetti P, Angelini G, Dapino D, Zaccheo D, Garibaldi S, Dagna-Bricarelli F, Piombo G, Perry G, Smith M, Traverso N, Tabaton M (1998) Early glycoxidation damage in brains from Down's syndrome. Biochem Biophys Res Commun 243, 849-851. Abstract

Pratico D, Clark CM, Liun F, Rokach J, Lee VY, Trojanowski JQ (2002) Increase of brain oxidative stress in mild cognitive impairment: a possible predictor of Alzheimer disease. Arch Neurol 59, 972-976. Abstract

Pratico D, Uryu K, Leight S, Trojanoswki JQ, Lee VM (2001) Increased lipid peroxidation precedes amyloid plaque formation in an animal model of Alzheimer amyloidosis. J Neurosci 21, 4183-4187. Abstract

Sayre LM, Perry G, Harris PL, Liu Y, Schubert KA, Smith MA (2000) In situ oxidative catalysis by neurofibrillary tangles and senile plaques in Alzheimer's disease: a central role for bound transition metals. J Neurochem 74, 270-279. Abstract

Smith MA, Harris PL, Sayre LM, Perry G (1997) Iron accumulation in Alzheimer disease is a source of redox-generated free radicals. Proc Natl Acad Sci U S A 94, 9866-9868. Abstract

View all comments by Hyoung-gon Lee
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View all comments by Xiongwei Zhu

It is well known that the brain contains an intriguing and distinctive pattern of iron deposition (1) and there is a rapidly growing interest in possible roles for iron metabolism in the pathogenesis and possible therapy of neurodegenerative diseases including Alzheimer disease (2). It is generally thought that the involvement of iron in these diseases involves the exacerbation of oxidative stress (3). However, it has been difficult to establish clear-cut mechanisms linking iron storage to disease progression. Many discussions of brain iron have focused on the compound ferrihydrite as the mineral core of the iron-protein complex ferritin. However, these tend to be based not on direct studies of brain iron, but on extrapolations from other tissues (both normal and iron-overloaded), particularly liver and spleen (4-7). The authors of the papers I discuss here (8,9) provide evidence for magnetite as one of the components of brain iron in Alzheimer disease (AD), and they point out that "the oxidative chemistry cannot be understood unless we first understand which species are...  Read more

It is well known that the brain contains an intriguing and distinctive pattern of iron deposition (1) and there is a rapidly growing interest in possible roles for iron metabolism in the pathogenesis and possible therapy of neurodegenerative diseases including Alzheimer disease (2). It is generally thought that the involvement of iron in these diseases involves the exacerbation of oxidative stress (3). However, it has been difficult to establish clear-cut mechanisms linking iron storage to disease progression. Many discussions of brain iron have focused on the compound ferrihydrite as the mineral core of the iron-protein complex ferritin. However, these tend to be based not on direct studies of brain iron, but on extrapolations from other tissues (both normal and iron-overloaded), particularly liver and spleen (4-7). The authors of the papers I discuss here (8,9) provide evidence for magnetite as one of the components of brain iron in Alzheimer disease (AD), and they point out that "the oxidative chemistry cannot be understood unless we first understand which species are present." Studies such as theirs are welcome, as they contribute to an understanding of how iron metabolism in the central nervous system may exhibit features not present in the rest of the body.

In the first paper under review (8), the authors provide important new information on the relation of brain iron to AD derived from x-ray analyses; in the second paper (9) these findings are compared with those from a number of previous studies. The first paper reports the modification of a powerful synchrotron radiation source at the Argonne National Laboratory to permit the focusing of a nominally 400 µm x-ray beam to the order of 3 μm while maintaining the majority of the beam intensity. Using 50 μm brain slices (superior frontal gyrus) from an AD autopsy, the researchers scanned the samples for iron by x-ray fluorescence, first at low resolution (100 μm pixels) to study iron signals from a relatively large area (6 mm x 4 mm), and then at high resolution (~5 μm) to study regions where this signal was found to be exceptionally high. Steps were taken during tissue fixation to avoid changes in the chemical and charge state of the iron and to prevent contamination with exogenous magnetic particles. A K-edge XANES (x-ray absorption near edge spectroscopy) profile was obtained and compared with previously obtained spectra from a number of iron-containing compounds (hemoglobin, ferritin, magnetite, etc.). This analysis suggested that the regions of high iron signal contained predominantly magnetite or a combination of magnetite and ferritin. Slices from brain adjacent to the sections studied by x-ray analysis were subjected to conventional histopathology analysis to demonstrate the presence of AD.

At least 15 different forms of iron oxide have been characterized (10), and several of these have biological implications. For the most part, based on studies of normal and iron-overloaded liver and spleen, it has usually been assumed that the predominant form of brain iron is some version of ferrihydrite present as mineralized cores associated with the protein ferritin or the related compound, hemosiderin. Hemosiderin is usually considered to be an insoluble ferritin degradation product, and it is difficult to characterize precisely (11). The details of its structure may vary from tissue to tissue. Although hemosiderin is often considered to be an important form of brain iron storage, it is seldom directly demonstrated in brain. Within the last few weeks, a new book devoted to iron and neurodegenerative diseases has appeared which questions whether this compound is even present in brain (12). Recent studies have applied new technologies based on Mossbauer spectroscopy, SQUID magnetometry, and electron microscopy to brain hemosiderin and related compounds (13-18). In addition to the current studies, these have raised a number of alternatives to the chemistry of brain iron storage as being predominantly a matter of ferritin, hemosiderin, and ferrihydrite. These alternatives include iron in neuromelanin (19) and bound to lipofuscin (20). The x-ray techniques used in the Collingwood et al. studies do not provide information concerning the possible association of the magnetite detected with proteins or other macromolecules.

New Iron Detection Technique
A growing number of AD researchers would love to find out just what role iron plays in neurodegenerative diseases including Alzheimer's. However, they've been hampered to date by a lack of techniques for defining which forms of iron occur in human and AD brain, and exactly where. [Figure courtesy of Joanna Collingwood]

The question of the extent to which iron storage in the brain involves ferrihydrite, magnetite, neuromelanin, and other compounds is important to the use of magnetic resonance imaging to follow disease-related changes in this storage (21,22). In our own work, we have used T2-mapping of high-field MRI to produce evidence of increased iron deposition in the hippocampus and other brain regions of AD patients compared to age-matched controls (23).

Iron affects the MR image by producing microscopic variations in magnetic field strength, which lead to a shortening of the transverse relaxation time (known as T2 or T2*) of water molecules in the vicinity of the iron deposits. On T2-weighted images, this results in a distinctive loss of signal (hypointensity) in the vicinity of iron deposits. The extent of this effect depends on the amount of iron present, but it is also very dependent on the magnetic properties of the compound in which the iron is contained. For example, magnetite is ferromagnetic, and this implies that, on an atom-by-atom basis, iron in this compound is expected to be more effective in producing endogenous contrast in MRI than a similar amount of iron in ferrihydrite. Theoretically, at least, conversion of iron from one chemical compound to another could be as effective in modifying MRI results as an increase in the total iron present (24). This underscores the importance of the Collingwood et al. studies in investigating the form of iron actually present in the diseased brain. A number of studies have found evidence for increased iron deposition in a wide range of neurodegenerative diseases.

The smallest practical voxel elements in brain MRI of living subjects are on the order of 1 mm,3 although much smaller voxels can be imaged in postmortem tissue. These limitations on the resolution of MRI suggest that very small regions of magnetite accumulation, such as seen in the present studies (diameter ~5 μm), may not be detected in MRI unless their density is such that several of them occur within a single voxel. New MRI techniques are being developed to supplement traditional T2- and T2*-weighting and these may extend the ability of MRI to detect and quantify small regions of iron storage (25,26).

Collingwood et al. have provided new technology and information on the chemical and physical properties of brain iron in AD. Such information, I hope, will eventually lead to a deeper understanding of the role of iron in this important disease and illuminate its potential in the areas of diagnosis and treatment.

The work of our group is sponsored in part by the Department of the Army, U.S. Army Medical Research Acquisition Activity, 820 Chandler Street, Fort Detrick, MD 21701-5014 under contract number W81XWH-05-0331. It does not represent the official government position or policy and no official endorsement should be inferred.

References:
1. Koeppen AH. A brief history of brain iron research. J Neurol Sci. 2003;207:95-97. Abstract

2. Zecca L, Youdim MB, Riederer P, Connor JR, Crichton RR. Iron, brain ageing and neurodegenerative disorders. Nat Rev Neurosci. 2004;5:863-873. Abstract

3. Smith MA, Harris PL, Sayre LM, Perry G. Iron accumulation in Alzheimer disease is a source of redox-generated free radicals. Proc Natl Acad Sci U S A. 1997;94:9866-9868. Abstract

4. St Pierre TG, Webb J, Mann S. Ferritin and hemosiderin: structural and magnetic properties of the iron core. In: Mann S, Webb J, Williams RJP, eds. Biomineralization: chemical and biochemical perspectives. Weinheim; New York: VCH, 1989:295-344.

5. Artymiuk PJ, Bauminger ER, Harrison PM, et al. Ferritin: a model system for iron biomineralization. In: Frankel RB, Blakemore RP, eds. Iron biominerals. New York: Plenum Press, 1991.

6. Powell AK. Ferritin. Its mineralization. In: Sigel A, Sigel H, eds. Iron transport and storage in microorganisms, plants, and animals. New York: Marcel Dekker, 1998:515-561.

7. Crichton RR. Inorganic biochemistry of iron metabolism: from molecular mechanisms to clinical consequences. 2nd ed. Chichester; New York: Wiley, 2001.

8. Collingwood JF, Mikhaylova A, Davidson M, Batich C, Streit WJ, Terry J, Dobson J. In situ characterization and mapping of iron compounds in Alzheimer's disease tissue. J Alzheimer’s Dis. 2005;7:267-272. Abstract

9. Collingwood JF, Dobson J. Mapping and characterization of iron compounds in Alzheimer’s tissue. J Alzheimer’s Dis. 2006 (in press).

10. Cornell RM, Schwertmann U. The iron oxides: structure, properties, reactions, occurrences, and uses. 2nd, ed. Weinheim: Wiley-VCH, 2003, p. 11.

11. Wixom RL, Prutkin L, Munro HN. Hemosiderin: nature, formation, and significance. Int Rev Exp Pathol. 1980;22:193-225. Abstract

12. Crichton RR, Ward RJ. Metal-based neurodegeneration: from molecular mechanisms to therapeutic strategies. Chichester; Hoboken, NJ: J. Wiley & Sons, 2006, p. 5.

13. Dubiel SM, Zablotna-Rypien B, Mackey JB. Magnetic properties of human liver and brain ferritin. Eur Biophys J. 1999;28:263-267. Abstract

14. Cowley JM, Janney DE, Gerkin RC, Buseck PR. The structure of ferritin cores determined by electron nanodiffraction. J Struct Biol. 2000;131:210-216. Abstract

15. Quintana C, Cowley JM, Marhic C. Electron nanodiffraction and high-resolution electron microscopy studies of the structure and composition of physiological and pathological ferritin. J Struct Biol. 2004;147:166-178. Abstract

16. Gossuin Y, Hautot D, Muller RN, Pankhurst Q, Dobson J, Morris C, Gillis P, Collingwood J. Looking for biogenic magnetite in brain ferritin using NMR relaxometry. NMR Biomed. 2005;18:469-472. Abstract

17. Zhang P, Land W, Lee S, Juliani J, Lefman J, Smith SR, Germain D, Kessel M, Leapman R, Rouault TA, Subramaniam S. Electron tomography of degenerating neurons in mice with abnormal regulation of iron metabolism. J Struct Biol. 2005;150:144-153. Abstract

18. Quintana C, Bellefqih S, Laval JY, Guerquin-Kern JL, Wu TD, Avila J, Ferrer I, Arranz R, Patino C. Study of the localization of iron, ferritin, and hemosiderin in Alzheimer's disease hippocampus by analytical microscopy at the subcellular level. J Struct Biol. 2006;153:42-54. Abstract

19. Zecca L, Zucca FA, Wilms H, Sulzer D. Neuromelanin of the substantia nigra: a neuronal black hole with protective and toxic characteristics. Trends Neurosci. 2003;26:578-580. Abstract

20. Castelnau PA, Garrett RS, Palinski W, Witztum JL, Campbell IL, Powell HC. Abnormal iron deposition associated with lipid peroxidation in transgenic mice expressing interleukin-6 in the brain. J Neuropathol Exp Neurol. 1998;57:268-282. Abstract

21. Schenck JF, Zimmerman EA. High-field magnetic resonance imaging of brain iron: birth of a biomarker? NMR Biomed. 2004;17:433-445. Abstract

22. Haacke EM, Cheng NY, House MJ, Liu Q, Neelavalli J, Ogg RJ, Khan A, Ayaz M, Kirsch W, Obenaus A. Imaging iron stores in the brain using magnetic resonance imaging. Magn Reson Imaging. 2005;23:1-25. Abstract

23. Zimmerman EA, Li Z, O'Keefe T, Schenck JF. High field strength (3T) magnetic resonance imaging in Alzheimer's disease. Ann Neurol. 2003;54 (suppl 7):S68.

24. Brooks RA, Vymazal J, Goldfarb RB, Bulte JW, Aisen P. Relaxometry and magnetometry of ferritin. Magn Reson Med. 1998;40:227-235. Abstract

25. Haacke EM, Xu Y, Cheng YC, Reichenbach JR. Susceptibility weighted imaging (SWI). Magn Reson Med. 2004;52:612-618. Abstract

26. Jensen JH, Chandra R, Ramani A, et al. Magnetic field correlation imaging. Magn Reson Imaging. 2006 (in press).

View all comments by John Schenck

A number of studies demonstrate that iron and aluminum are co-deposited in the brains of Alzheimer patients (1) and that the metals interact in enhancement of oxidation. Walton has developed a method of staining aluminum in hippocampal neurons in humans with and without AD (2). Higher levels of the metal were associated with sufficient density of neurofibrillary tangles to kill brain cells by enucleation. One looks forward to a future multi-metal study that compares the location of iron and aluminum in the brain, and compares their interaction.

References:
1. Bouras C, Giannakopoulos P, Good PF, Hsu A, Hof PR, Perl DP. A laser microprobe mass analysis of brain aluminum and iron in dementia pugilistica: comparison with Alzheimer's disease. Eur Neurol. 1997;38(1):53-8. Abstract

2. Walton JR. Aluminum in hippocampal neurons from humans with Alzheimer's disease. Neurotoxicology. 2006 Feb 2; [Epub ahead of print] Abstract

View all comments by Erik Jansson

This is significant in that it provides a new analytical tool for spatially assessing the presence of iron in cells including neurons. Although iron has been reported to be increased in the substantia nigra, the brain region impacted in the disorder, information on cell-specific and/or subcellular localization has been lacking. That is where this group’s finding that iron is present in dopaminergic neurons is most significant for Parkinson disease. When dopamine synthesis was reduced, this resulted in release of iron-dopamine complexes from neurovesicles into the cytoplasm. Release of dopamine from the vesicles could result in increased oxidation, leading to enhanced cell death. This study was performed in the dopaminergic cell line PC12 neuronally differentiated via nerve growth factor. I am not familiar enough with the technique to assess its possible clinical applications (it appears it might only be useful for analyses of postmortem samples), but it does appear as if it would be useful in terms of assessing subcellular iron localization at least in animal models of the disease.

View all comments by Julie Andersen

Comment by Joanna Collingwood, Jon Dobson, Mark Davidson, Chris Batich, Albina Mikhaylova, and Jeff Terry
This interesting study by Ortega et al. uses a high-resolution synchrotron X-ray probe in a newly developed configuration at the European Synchrotron Radiation Facility (ESRF). The technique they describe has, in recent years, been embraced by researchers from the medical community. It is good to see facility expansion and development in this area as synchrotron X-ray chemical imaging is adopted as a valuable technique to study biological samples (e.g., Mikhailova et al., 2000; Mikhaylova et al., 2005; Wang et al., 2005; Collingwood et al., 2005; Palmer et al., 2006; McRae et al., 2006; Chwiej et al., 2007; Arora et al., 2007).

Ortega et al. have achieved an excellent in-plane resolution of 90 nm to map iron, zinc, and potassium. Although subcellular chemical mapping studies are conducted at below 50 nm at facilities such as the Advanced Photon Source (APS) 2-ID beamline, mapping in the hard energy range used by Ortega et al. has usually been performed at...  Read more

Comment by Joanna Collingwood, Jon Dobson, Mark Davidson, Chris Batich, Albina Mikhaylova, and Jeff Terry
This interesting study by Ortega et al. uses a high-resolution synchrotron X-ray probe in a newly developed configuration at the European Synchrotron Radiation Facility (ESRF). The technique they describe has, in recent years, been embraced by researchers from the medical community. It is good to see facility expansion and development in this area as synchrotron X-ray chemical imaging is adopted as a valuable technique to study biological samples (e.g., Mikhailova et al., 2000; Mikhaylova et al., 2005; Wang et al., 2005; Collingwood et al., 2005; Palmer et al., 2006; McRae et al., 2006; Chwiej et al., 2007; Arora et al., 2007).

Ortega et al. have achieved an excellent in-plane resolution of 90 nm to map iron, zinc, and potassium. Although subcellular chemical mapping studies are conducted at below 50 nm at facilities such as the Advanced Photon Source (APS) 2-ID beamline, mapping in the hard energy range used by Ortega et al. has usually been performed at pixel resolutions of 150 nm and above (Kemner et al., 2004; Palmer et al., 2006; Finney et al., 2007). The X-ray subcellular imaging technique itself is an established tool in the synchrotron community (Paunesku et al., 2006), and development of a multilayer Laue lens promises resolutions of just a few nanometers in the hard energy X-ray range (Kang et al., 2006).

Synchrotron X-rays are increasingly being used to map metal ion distributions in human tissues (e.g., Collingwood et al., 2005; Chwiej et al., 2007; Miller et al., 2006) and cell cultures (e.g., Palmer et al., 2006; Ortega et al., 2007). Given that these studies require access to the high-flux, high-energy focused X-ray beams available at third-generation synchrotron facilities such as the APS (Chicago, USA), ESRF (Grenoble, France), and DIAMOND (Oxford, UK), one might reasonably ask why researchers are dedicating such effort towards these studies. With access to a modestly equipped histology lab, it is possible to obtain beautifully stained tissue sections revealing the distribution of specific metals (e.g., iron) and of associated proteins (e.g., ferritin) with relatively little trouble. Likewise, accurate quantification of metal ion concentrations in tissue can be undertaken with readily available techniques such as ICP-MS. Ortega’s latest paper (Ortega et al., 2007) provides a good illustration of some of the benefits the synchrotron approach can bring when compared to more conventional methods.

If we compare a conventional iron stain with a synchrotron X-ray fluorescence map of iron, there are several significant differences. The staining process cannot reveal iron in all its bound forms, so the spatial distribution of iron concentration may be better revealed by a synchrotron map. The chemical element detection sensitivity available at third-generation synchrotrons with focused X-ray beams is outstanding (a few ppm). There is great flexibility in selecting mapping resolution (from tens of nanometers to hundreds of microns). Moreover, the synchrotron techniques are ideal for imaging iron in unstained, unfixed sections, avoiding the interference/alteration of the iron’s chemical state and distribution in the tissue frequently brought about by standard processing and staining techniques (Dobson et al., 1996; Mikhaylova et al., 2004). This approach enables retrospective staining and/or imaging, allowing image correlation with structures of interest (Mikhaylova et al., 2004). The synchrotron approach also routinely allows for the simultaneous collection of metal maps for a wide range of chemical elements (e.g., Collingwood et al., 2005a; Miller et al., 2006).

Ortega et al. map the intracellular Fe, Zn, and K concentrations in cultured PC12 rat neurons, and extend the investigation to explore how iron concentration and distribution in isolated neurons is affected by exposure to iron (with FeSO4) and by inhibition of dopamine synthesis (with AMT). This is a good example of how synchrotron radiation can contribute to multi-modal imaging approaches, where multi-metal maps of unstained cells or tissue sections are correlated with light microscopy of the structures, and—in this case—with dopamine fluorescence, allowing colocalization of iron and dopamine to be observed.

The subject matter of this study is of high importance. While iron accumulation has been quantified in individual unstained unfixed dopaminergic neurons (Oakley et al., 2007), and alterations in regional brain iron are observed in a host of neurodegenerative disorders, including Alzheimer disease, the precise mechanisms by which iron is involved in neurodegeneration are not well understood. The field has gained recent momentum as the application of evolving techniques provides insight into colocalization of metals, regional distributions at cellular and subcellular resolution, and altered chemical and mineral states—all of which are important if we are to understand the contribution of metals to oxidative stress in neurodegeneration.

One particular advantage to synchrotron studies of iron in cells and neurodegenerative tissues is that the mapping can be combined with absorption spectroscopy containing information about the chemical state of the element (Collingwood et al., 2005; 2005a; 2006; Chwiej et al., 2007). In a single experiment, it is possible to use the X-ray beam to map iron concentrations in tissue, and to then look at the absorption profile of individual localized iron accumulations to determine their form (including oxidation state and local chemical environment—e.g., ferrihydrite from ferritin, or deposits of hemosiderin). An application of this combined mapping and characterization technique is to look in situ at altered forms of iron storage in neurodegenerative disease (Collingwood et al., 2005; Mikhaylova et al., 2005; Chwiej et al., 2007)—an important factor in exploring the underlying chemistry in diseases such as Alzheimer’s and Parkinson’s.

The excellent sensitivity of synchrotron X-rays to metal ions, whilst being one of the fluorescence mapping technique’s greatest advantages, also necessitates careful preparation of samples. Conventional fixation and staining techniques can alter valence stages and lead to leaching of iron, so ideally tissue should be fresh frozen and sectioned with a non-metallic blade to prevent particulate contamination. Choice of mounting substrate is also important. Many glass and plastic slides contain trace metal levels that are irrelevant to routine microscopy work, but that will be evident in micro- or nano-focus synchrotron fluorescence mapping (Mikhaylova et al., 2004).

While the synchrotron fluorescence technique is excellent for obtaining relative concentrations over an area of uniform sample thickness, it is more challenging to attempt absolute quantification of elemental concentrations. This is primarily because although the pixel area is defined, it is very hard to define the actual volume sampled by the X-ray beam. For the purpose of interpreting the chemical element maps in a study such as Ortega 2007, it must be recognized that the variation in sample thickness across an isolated cell, even within the area of the beam spot, will affect measured concentrations. The signal intensity is proportional to the absolute number of atoms within the excited volume. Since the X-rays pass through the samples essentially unimpeded, the entire thickness is excited. If the thickness of the sample varies in an unmeasured way, as for example, it does across the isolated cells in these samples, then the actual concentration of the metal cannot be determined.

Another challenge, particularly when working with intact tissue sections, is location of nanoscale regions in unstained sections to enable retrospective image correlation. Following on from our initial use of zinc wire grids (Mikhailova et al., 2000; Mikhaylova et al., 2005) in avian tissue studies, we have proposed the use of numbered lithographic grids deposited onto the slides to facilitate co-registration of images (Collingwood et al., 2005a) and in fact have used lithographically formed un-numbered grids in studies in 2000 and 2001. Where iron is the primary metal of interest, chrome is an ideal choice for the grid, providing a metal signal in the vicinity of, but not overlapping with, the iron. For samples with clearly identifiable metal-bearing features that can be collocated with visual landmarks such as tissue section edges, fissures, or iron rich blood vessels, for example, such grids are unnecessary.

It should be noted that other methods are also available to obtain nano-resolution multi metal maps of cells and tissues. This is illustrated in Quintana 2007, where detailed high-resolution (50-100 nm) studies of metals and their relation to cellular and subcellular structures have been performed on Alzheimer’s tissue using nano secondary iron mass spectrometry (nanoSIMS). One of the significant differences between this technique and the synchrotron X-ray approach is that the latter is non-destructive and the nanoSIMS technique cannot yield chemical state information. There is also some issue with the SIMS technique with matrix effects that can cause very large shifts in sensitivity with slight changes in composition of the matrix.

The synchrotron experiments discussed here are not appropriate for clinical imaging, being directed toward autopsy tissue and in-vitro experiments. Synchrotron radiation has been used for non-invasive coronary imaging since 1987 (Rubenstein et al., 1987; Bertrand et al., 2005), but it is not currently viable for high-resolution mapping of trace metals. Amongst many practical difficulties, perhaps the primary one is the multi-hour imaging times and unacceptable resulting dose to which a patient would be exposed.

However, altered regional brain iron accumulations hold potential for non-invasive clinical detection and diagnosis in neurodegenerative disorders. In particular, the impact of iron on T2 shortening in MRI is being investigated for diseases including Alzheimer’s and Parkinson’s disease (House et al., 2007; Bartzokis et al., 2004; Schenk et al., 2006; Graham et al., 2000).

Meanwhile, it is important that metal-related mechanisms of neurodegeneration continue to be investigated, particularly where results may influence therapies such as metal chelation (Youdim et al., 2004). Synchrotron X-ray fluorescence imaging is a standard tool for scientists in a variety of disciplines, but its recognition as a tool to study the role of metals in neurodegenerative tissues is comparatively recent. There is great potential for creative synchrotron experiments to enable progress in this fascinating area of neuroscience.

References
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