Assessment of Metal Concentrations in ARich Regions of the Hippocampus, Entorhinal Cortex and Periva

1
Assessment of Metal Concentrations in Aß-Rich
Regions of the Hippocampus, Entorhinal Cortex
and Perivascular Areas in Alzheimer Diseased
Brains using Synchrotron X-Ray Fluorescence
(µSXRF) Flinn, J.M.1, Linkous, D.H.1,
Kesslak, J.P.2, Head, E.2, Lanzirotti, A.3, 4,
Rao, W.3, Jones, B.F.5, Bertsch, P.6,
Frederickson, C.J.7 1.George Mason University,
Fairfax, VA 2.Institute for Brain Aging
Dementia, University of California, Irvine
3.National Synchrotron Light Source, Brookhaven
National Laboratory, Upton, NY 4.University of
Chicago, CARS, Chicago, IL 5.United States
Geological Survey, Reston, VA 6.Savannah River
Ecology Lab, University of Georgia, Aiken, SC
7.NeuroBioTex, Galveston, TX
Results___________________________________________
_________________________________________
Introduction__________________________
Elevated levels of Fe, Cu, and Zn have been
observed in plaques and adjacent tissue from
Alzheimers diseased brains (Lovell, et al,
1998). It is thought that these metals increase
in the aging brain and have a causative role in
the formation of plaques (Bush, 2003). Microprobe
synchrotron x-ray fluorescence (µSXRF) allows the
detection and mapping of trace metals in
untreated, fresh-frozen sections. Ten cases were
examined that included 6 AD cases (Mean age
85.8 years, Braak Braak stage V or VI) and 4
control cases (Mean age 88.8 years, Braak
Braak stage 0-III). 20µm-thick brain sections
were assessed at 0.01 steps for X-ray intensities
of P, S, Cl, K, Ca, Ti, V, Cr, Mn, Fe, Ni, Cu,
and Zn. Adjacent sections were immunostained
with antibodies directed against ß-amyloid (Aß)
1-42 or Aß 1-40. Additional sections were labeled
with Thioflavin-S. In Aß-rich perivascular
regions, K, Fe, and Zn are all elevated.
Concentrations of metals in other Aß-rich
deposits vary, however, Fe delineates Aß-rich
areas most consistently.
Methods______________________________ Tissue
was obtained from the Institute for Brain Aging
Dementia, Department of Neurology, University of
CA, Irvine. 6 AD cases (Mean age 85.8 yrs,
Braak Braak stage V or VI) and 4 control cases
(Mean age 88.8 yrs, Braak Braak stage 0-III)
were examined. Fresh frozen blocks including the
hippocampus were sectioned at 20µm on a cryostat.
Sections for x-ray fluorescence studies were
mounted on silica (metal-free) slides. Adjacent
sections were immunostained with antibodies
directed against either ?-amyloid (A?) 1-42 or
A?1-40. Additional sections were stained with
Thioflavin-S. Double labeled sections were also
used to compare the distribution of both species
of A?.These sections were used to identify
individual plaque types for synchrotron studies
Analyses were conducted at the X26a
Beamline at the National Synchrotron Light
Source, Brookhaven National Laboratory (figure
1). The incident x-ray beam was tuned to 10 keV
using a Si (111) channel-cut monochromator (beam
is collimated to 350µm in diameter with a
tantalum slit set and focused to 10µm in diameter
using Rh-coated microfocusing mirrors in a
Kirkpatrick-Baez type geometry). Energy
dispersive x-ray fluorescence data were collected
using a Canberra SL30165 Si (Li) detector.
Detection limits for most of the elements
analyzed here vary between 0.1 to 5ppm. For
two-dimensional composition mapping, counting
times of 7sec/pixel were used, with step sizes of
10µm between each point. Data for all metals
were collected at each step (multi-channel
analysis) (figure 2).

References and Acknowledgements__________ The
authors would like to thank Avi Friedlich and
Ashley Bush for their help and assistance.     Bus
h, A.I. (2003). The metallobiology of Alzheimer's
disease. Trends in Neuroscience, 26(4),
207-214. Lovell, M.A., Robertson, J.D.,
Teesdale, W.J., Campbell, J.L., Markesbery,
W.R. (1998). Copper, iron and zinc in
Alzheimer's disease senile plaques. J. Neurol.
Science, 158(1), 47-52. This study was supported
by a BNL general user grant for NSLS beamline
access (J.M.F.) a George Mason University
Research Assistantship (D.H.L) ADRC P50 AG16573
(J.P.K., E.H.) the United States Geological
Survey (B.F.J.) and the Savannah River Ecology
Lab (P.B.). The National Synchrotron Light
Source (NSLS) is supported by the U.S. Department
of Energy under Contract No. DE-AC02-76CH00016.
Conclusions________________________ µSXRF
is a useful tool for assessing brain tissue which
allows for the simultaneous measurement of a
suite of metals. In agreement with previous data,
it is demonstrated here that levels of Fe and Zn
increase in A?-rich regions and adjacent tissue
in brains of Alzheimer disease patients. Moreover,
Fe depositions are the most discrete markers of
these areas, and are in most agreement with the
histological sections.  Levels of potassium also
increase, while in contrast with previous
findings, copper elevations were not detected.
Further investigation is needed to fully
understand the localization patterns of
transition metals in different types of A?
plaques (e.g. diffuse, cored) and brain regions,
and how these concentrations may vary with
Alzheimer disease pathogenesis. Nevertheless,
the this data confirms the important role played
by metals in disease morphology.