Three-Dimensional Atlas of Iron, Copper, and Zinc ... - ACS Publications

We constructed a 46-plate reference atlas by aligning quantified images of metal distribution with corresponding coronal sections from the Allen Mouse...
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Three-Dimensional Atlas of Iron, Copper, and Zinc in the Mouse Cerebrum and Brainstem Dominic J. Hare,*,† Jason K. Lee,† Alison D. Beavis,† Amanda van Gramberg,† Jessica George,‡ Paul A. Adlard,‡ David I. Finkelstein,‡ and Philip A. Doble† †

Elemental Bio-imaging Facility, University of Technology, Sydney, New South Wales, Australia The Mental Health Research Institute, Royal Parade, University of Melbourne, Victoria, Australia

‡

S Supporting Information *

ABSTRACT: Atlases depicting molecular and functional features of the brain are becoming an integral part of modern neuroscience. In this study we used laser ablation-inductively coupled plasma-mass spectrometry (LA-ICPMS) to quantitatively measure iron (Fe), copper (Cu), and zinc (Zn) levels in a serially sectioned C57BL/6 mouse brain (cerebrum and brainstem). Forty-six sections were analyzed in a single experiment of approximately 158 h in duration. We constructed a 46-plate reference atlas by aligning quantified images of metal distribution with corresponding coronal sections from the Allen Mouse Brain Reference Atlas. The 46 plates were also used to construct three-dimensional models of Fe, Cu, and Zn distribution. This atlas represents the first reconstruction of quantitative trace metal distribution through the brain by LA-ICPMS and will facilitate the study of trace metals in the brain and help to elucidate their role in neurobiology.

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reduces the potential for contamination, while greatly improving the precision of analysis of regional concentration data when compared to bulk excision and analysis. Our laboratory employs laser ablation-inductively coupled plasmamass spectrometer (LA-ICPMS) instruments for this purpose, which allow for routine analysis of tissue sections with image resolution in the low-to-mid micrometer range.7 Previous efforts to recreate models of trace element distribution across numerous serial tissue sections have focused on narrow segments of the brain specific to particular applications.8 Development of a reference atlas depicting trace metal distribution throughout a larger area of the brain is a priority, particularly now that therapeutic agents targeting redox-active metals are showing promise for the treatment of Parkinson’s disease and Alzheimer’s disease.6,9−12 In this study, we have generated the first atlas of Fe, Cu, and Zn in the cerebrum and brainstem of the C57BL/6 mouse, one of the most common mouse strains used in neuroscience research. In

nimal models are essential tools for studying human disease, and in recent years the ability to interrogate the molecular basis of disease, particularly in regard to neurodegenerative disorders, has been enhanced through the development of functional brain atlases. This includes the Allen Brain Atlas1 and the Rodent Brain Workbench,2,3 which catalogue genetic and molecular features specific to brain regions. Recent advances in microscopy have allowed for rapid, high-resolution fluorescence imaging of mouse neuroanatomical features by serial two-photon tomography.4 In this regard, there is currently no atlas that details the distribution of other critical features of the brain, such as metal content. Metal ions have a significant role within the brain, where they are involved both in normal physiological processes. Metal ions are both precipitating and potentiating factors in several neurodegenerative diseases.5 Consequently, metals are frequently studied in animal models of neurodegeneration.6 Accurate analysis of trace levels of metals in discrete brain regions is not without its difficulties; the ubiquitous nature of redox-active metals in the brain (including Fe, Cu, and Zn) increase the potential for contamination during dissection and bulk analysis. Imaging of metals in tissue sections significantly © 2012 American Chemical Society

Received: December 12, 2011 Accepted: March 30, 2012 Published: March 30, 2012 3990

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Figure 1. Experimental setup and workflow for atlas and 3D image construction. (a) 30 μm sections were taken at 150 μm intervals and mounted on gelatinized microscope slides. (b) 80 μm diameter vertical line scans moving at 160 μm s−1 were drawn over the slides. Lines were positioned with no space between each other to ensure total tissue coverage. (c) Each line of ablation produced a single data file. Images were produced using Interactive Spectral Imaging Data Analysis Software, where images were also separated into single files for each section. (d) Image registration was performed using Atlas3D, where stereotaxic coordinates and structural features were aligned. Here, the iron image for Plate 36 (bregma −2.980 mm) is aligned according to Fe-rich substantia nigra (circled). (e) Three dimensional imaging was performed using registered images in ISIDAS, which were then imported into 3D imaging software, such as MayaVi2 (shown) and ParaView 3.10.

obtained from Monash Animal Services (Monash University, Australia). All experiments were carried out in accordance with the local animal ethics committee requirements (Howard Florey Animal Ethics Committee) and National Health and Medical Research Council standards of animal care. The 4month old male C57BL/6 mouse was anaesthetized with sodium pentobarbitone (Lethobarb; 100 mg kg−1) and perfused with 30 mL 0.1 M phosphate buffered saline (PBS), pH 7.4. The brain was removed, placed in 4% (w/v) paraformaldehyde (Sigma Aldrich) and 0.1 M PBS (4 °C), pH 7.4 overnight, and then transferred into a sucrose solution (30% in PBS) for three days to cryoprotect the tissue. The

producing this atlas, we aimed to demonstrate that regional metal concentrations were consistent with more labor intensive and potentially contaminating bulk dissection and digestion methods and that relative metal distributions can be associated with specific brain structures. We present our results so that they may be used as a complementary data set for functional neuroscientists.

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EXPERIMENTAL SECTION

Animal Preparation. The mice were housed according to standard animal care protocols and fed standard laboratory chow and tap water ad libitum. C57BL/6 male mice were 3991

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brain was then rapidly frozen at −80 °C before sectioning through the cerebrum and brainstem, collecting 30 μm thick sections at 150 μm intervals (Figure 1a). A total of 46 sections were analyzed. Sections were taken up to the level of the cerebellum at approximately −5.00 mm from bregma. Sections were mounted on gelatinized glass microscope slides. LA-ICPMS Analysis. Analysis was performed using a New Wave Research UP-213 laser ablation instrument (Kennelec Technologies, Mitcham, Victoria, Australia) fitted with a Large Format Cell (LFC). The LFC has a 25 × 25 cm chamber with a low-volume roving sampling cup. The standard insert was modified to fit 8 microscope slides on which samples and tissue standards were mounted. Argon was used as a carrier gas. The laser unit was hyphenated to an Agilent Technologies 7500cx ICPMS instrument (Forrest Hill, Victoria, Australia) fitted with a ‘cs’ lens system and platinum sampler and skimmer cones. Prior to analysis the system was tuned for sensitivity using NIST 612 Trace element in glass and in-house produced tissue standards. Oxide formation arising from trace O2 in the Ar carrier gas was controlled by limiting 232Th16O+/232Th+ to 6% of gross signal. Atlas Construction. Images were positioned in Atlas3D according to sectioning intervals (distance from bregma), identification of previously characterized metal-rich regions (including substantia nigra, hippocampus, and periaqueductal region) and alignment to structural features (including cortical edges and ventricles). Figure 1d shows alignment of an iron image with the identified iron-rich substantia nigra (compacta 3993

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Figure 3. Reference key and left hemisphere Fe, Cu, and Zn images for Plate 24 (bregma −2.48 mm, Allen Atlas Coronal Level 79). Glossary for reference key can be found in Supplementary Figure 1 and is adapted from the Allen Brain Reference Atlas.16

Figure 1). A representative plate (Plate 34, bregma −2.48 mm) is shown as Figure 3. Regions of Interest. Regional metal concentration ranges in specified brain regions are given Table 2. Concentrations

disrupted during sectioning, and the right hemisphere was used with the image mirrored.15 The completed trace element reference atlas for a C57BL/6 mouse brain is available for download with the Supporting Information (Supplementary 3994

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Table 2. Interquartile Ranges ± %RSD in Measurement for Fe, Cu, and Zn at the Deep Supragranular Pyramidal Layer (ISO3) Fe (μg g−1) Cerebrum (CH) cerebral cortex (CTX) cortical plate (CTXpl) cortical subplate (CTXsp) cerebral nuclei (CNU) striatum (STR) pallidum (PAL) Brainstem (BS) interbrain (IS) thalamus (TH) hypothalamus (HY) midbrain (MB) sensory related (MBsen) motor related (MBmot) behavioral state related (MBsta) hindbrain (HB) pons (P) medulla (MY)

Cu (μg g−1)

Zn (μg g−1)

12.0 22.5 12.5 18.2

± ± ± ±

0.52 0.98 0.54 0.79

1.67 2.88 1.54 2.47

± ± ± ±

0.05 0.09 0.05 0.08

7.77 12.4 7.49 11.7

± ± ± ±

0.22 0.35 0.21 0.33

19.3 34.0 22.3 41.3

± ± ± ±

0.84 1.48 0.97 1.80

2.02 3.50 1.59 3.13

± ± ± ±

0.06 0.11 0.05 0.11

7.44 11.3 6.61 10.7

± ± ± ±

0.21 0.32 0.19 0.30

15.1 23.6 17.4 26.6

± ± ± ±

0.66 1.03 0.76 1.16

1.92 3.07 2.43 4.43

± ± ± ±

0.06 0.10 0.08 0.14

5.18 7.49 6.39 9.48

± ± ± ±

0.15 0.21 0.18 0.27

15.5 19.8 13.4 18.7 17.6 42.2

± ± ± ± ± ±

0.67 0.86 0.58 0.81 0.77 1.84

1.69 2.31 1.60 2.62 1.47 2.45

± ± ± ± ± ±

0.05 0.07 0.05 0.08 0.04 0.08

4.90 7.07 4.21 6.52 4.34 7.21

± ± ± ± ± ±

0.14 0.20 0.12 0.19 0.12 0.21

10.5 15.3 13.0 16.7

± ± ± ±

0.46 0.67 0.570.73

1.08 1.70 1.26 7.24

± ± ± ±

0.03 0.05 0.04 0.23

3.46 5.22 3.79 5.22

± ± ± ±

0.10 0.15 0.11 0.15

Figure 4. (a) Hippocampal region (HIP) (including dentate gyrus and Ammon’s horn), periaqueductal gray (PAG) and substantia nigra, reticulate part (SNr) from ARA Brain Explorer application. (b) Combined three-dimensional zinc (HIP, > 14 μg g−1), copper (PAG, > 5 μg g−1) and iron (SNr, > 20 μg g−1) content for corresponding regions. See Supplementary Movie 2 for animation.

ranges we observed for Fe, Cu, and Zn were consistent with data previously reported by two-dimensional imaging of equivalent animal specimens.7,21 We found that whole brain metal levels were comparable to those previously reported for Fe,22 Cu,23 and Zn24 in C57BL/6 mice using bulk dissection and digestion methods. The regional metal concentration ranges shown in Table 2 were also comparable with values obtained through dissection and digestion of discrete regions of fresh frozen Wistar rat brains,25,26 suggesting possible loss of metals during fixation was insignificant.27 It should be noted, however, that extended chemical fixation may result in metal redistribution,28 thus the brain was subjected to paraformaldehyde fixation for less than 24 h. Fe, Cu, and Zn distribution was interpolated for each 150 μm space between sections. True representation of total metal content and distribution would require analysis of serial sections with no interval spacing, which would require significantly longer analysis time and larger ablation cell dimensions. Development of rapid imaging protocols will aid in analyzing these larger sample sets.13 The regional distribution of metals was consistent with traditional histopathological staining techniques, including Timm’s stain for unbound Zn(II) and Cu(II) and Perls Prussian blue for bound and unbound Fe(II).29−31 Fine features of the main olfactory bulb nuclei were clearly visible in the iron images; with comparatively lower iron levels in the mitral cell layer demarcating the glomerular and granular cell layers. High concentrations of iron were in the midbrain (specifically the hippocampus, striatum, substantia nigra, and in the interpeduncular nuclei (Supplementary Movie 1), possibly arising from the presence of iron-rich tyrosine hydroxylasepositive cells,32 among other iron-containing proteins. Copper concentrations were higher around the choroid plexus, which was likely due to the high level of Cu-containing ceruloplasmin protein.33 Cu levels were also higher in hypothalamic nuclei and along the length of the internal capsule. Supplementary Movie 1 shows areas of maximal

whole braina 17.2, 15.7 a

3.31, 2.26

8.65, 8.05

Reported as average and median concentration.

extracted from regions of interest are reported as the interquartile range (IQR), which is a robust statistical measure of distribution in a large, nonparametric data set.20 Structures were identified according to registration of metal images with corresponding reference plates in the ARA. Regions of interest were extracted at the third layer (deep supragranular pyramidal layer) of the six-layered hierarchical scheme used by the ARA. This level of detail was consistent with the 80 μm lateral resolution of the metal images. Three-Dimensional Image Construction. We constructed three-dimensional models of iron, copper, and zinc distribution using both hemispheres. For areas of high metal concentration we used three-dimensional imaging of data points determined to represent concentrations above designated values for each metal (Fe ≥ 20 μg g−1; Cu ≥ 5 μg g−1; Zn ≥ 14 μg g−1) (Supplementary Movie 1−2). Figure 4 and Supplementary Movie 2 show how three-dimensional imaging can be used to isolate and compare Fe, Cu, and Zn concentrations in three discrete brain regions (substantia nigra reticulata part, periaqueductal gray, and hippocampus, respectively).

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DISCUSSION In generating this atlas it was important to validate the quantitative data provided. In this respect, the concentration 3995

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copper concentration located along the ventricles. The most prominent Zn concentrations were in the cerebral cortex and hippocampal regions, consistent with the elevated density of synaptic Zn29,34 in these areas. Zn2+, which rises to high micromolar concentrations at the synapse during activity,35 is believed to play a key role in learning and memory via its function as a neuronal messenger and a modulator of synaptic transmission and plasticity.36−38 Specifically, Zn2+ may achieve this through targeted interactions with numerous proteins, including ZnR (GPR39),39 TrkB,40 NMDAR2b,37 and p75(NTR).41 Contributions to both brain Cu and Zn concentrations are also expected from highly abundant antioxidant enzymes, such as the superoxide dismutase family. The 80 μm spatial resolution used was insufficient to discern metal distribution below the third of six layers of the isocortex used by the ARA, including Zn variation within granular layer of the hippocampus and dentate gyrus.35 Smaller laser beam diameters can be employed to improve spatial resolution, the trade-off being extended sample runtimes. Alternately, highresolution atlases of specific brain regions can be produced using the methods outlined here. Concentration data were obtained for a single animal sample and should be viewed as indicative of relative and potential absolute concentration ranges. Further developments to the method we describe in terms of practical applications as a quantitative tool will be made through the analysis and statistical scrutiny of a larger cohort of animals. Regardless, like structural atlases that use single animals to produce images, this atlas provides new insights into the relationship between Fe, Cu, and Zn in specific brain regions. The integration of the ARA into LA-ICPMS imaging demonstrates the potential of the technique to be used in a wider range of applications related to neuroscience and provides researchers with an additional resource complementary to the structural and genomic features provided by the ARA. Data acquisition and a range of commercially available large volume ablation chambers allow for automated sampling. Integration of generated images with commercial life science image processing software suites has the potential for high-throughput analysis of larger sample sets and rapid screening of animal models of disease.

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ASSOCIATED CONTENT

S Supporting Information *

Supplementary Figure 1 − Portable document file (.pdf) of 46-plate atlas of iron, copper, and zinc in the C57BL/6 mouse cerebrum and brainstem with glossary of brain regions. Supplementary Movie 1 − mp4 video of reconstructed three-dimensional models of iron, copper, and zinc distribution in C57BL/6 mouse cerebrum and brainstem. Supplementary Movie 2 − mp4 video of iron, copper, and zinc in substantia nigra reticulata, periaqueductal gray, and hippocampus, respectively; with corresponding Allen Mouse Brain Atlas reference. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Corresponding author address: P.O. Box 123, Broadway, NSW, 2007, Australia. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The development of the ISIDAS program by Grigory Dorokhov and Michael Lake from the Computational Research Centre of Expertise, UTS is gratefully acknowledged. D.H. would like to acknowledge the Australian Research Council for financial support through the Linkage Projects grant program.

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CONCLUSIONS We have generated the first brain atlas depicting the threedimensional rendering of metal concentrations throughout the entire cerebrum and brainstem of the laboratory mouse. These data provide a fundamental resource that will facilitate interrogation of the basic mechanisms underlying both normal and pathological brain states, including neurodegenerative disorders such as Alzheimer’s and Parkinson’s diseases. The technology underpinning this resource, and indeed the atlas itself, represents a significant advance over more traditional methods of metal analysis such as histological stains and bulk tissue digests as it provides a high-resolution spatial profile of the distribution of multiple metal ions. Not only is bulk dissection and digestion a complex procedure prone to contamination, but spatial information is lost in the process. While trace metals have become a clear target for novel therapeutic approaches to treating neurodegeneration,11 a complete picture of metal distribution throughout the brain of common animal models is lacking. The development of an atlas of metal distribution in the C57BL/6 provides neuroscientists with a resource to further aid in deducing the role of metals in neurological disorders. 3996

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