Identification and analysis of senile plaques using nuclear microscopy

180

Nuclear

Instruments

and Methods

in Physics

Research

B54 (1991) 180-185 North-Holland

Identification

and analysis of senile plaques using nuclear microscopy

J.P. Landsberg, B. McDonald

a, J.M. Roberts, G.W. Grime and F. Watt

Nuclear Physics Laboratory Keble Road, Oxford OXI 3RH, UK a Department of Neuropathology, Radcliffe Infirmary Woodcitock Road, Oxford, UK, Nuffield Department of Pathology, John Radcliffe Hospital, Oxford, UK, and MRC Neuroanatomical Unit, Department of Pharmacology, South Parks Road, Oxforrl, UK

The senile plaques and neuro-fibrillary tangles which form part of the pathology of Alzheimer’s disease have come under increasing scrutiny over the last decade. In particular, much work has been done investigating their elemental composition. The suggestion that 75-100% of senile plaques with mature cores contain aluminium and silicon, probably in the form of alum&-silicates, has led to increasing speculation about the role of these elements in the disease. SPM preliminary studies suggest that aluminium and silicon are not present in as great a proportion of senile plaques as presented in the literature. The situation is complicated by the fact that airborne and solubilised salts of aluminium and silicon may be encountered as contamination. They have been found, for example, in granular or crystalline form in the Aristar grade organic laboratory reagents used for staining the tissue, and in the pure pioloform used to back the samples. The latest results from scans of stained and unstained Alzheimer tissue are presented.

1. Introduction Alzheimer’s disease (AD) is a progressive neurodegenerative disorder with no known cause. Pathologically it is characterized by large numbers of neurofibrillary tangles and silver staining “senile” plaques mainly concentrated in the neocortex and the hippocampus [l]. The suggestion that 70-1008 of senile plaques with mature cores contain aluminium and silicon, probably in the form of alumino-silicates [2] has led to increasing speculation about the role of these elements in the disease [3-61. A scanning proton microprobe @PM) has great potential for the investigation of elemental distributions in biological tissue. Accurate elemental analysis is possible down to the parts per million level, an increase in sensitivity of two to three orders of magnitude over an electron probe. By combining nuclear backscattering with proton induced X-ray emission (PIXE) the major elements, carbon, nitrogen and oxygen, together with the trace and minor elements, from sodium and above in the periodic table, can be measured. A topographical picture of the sample can be obtained by detecting the secondary electrons (SEM) scattered from the target 171. The SPM has been used previously in the study of the distribution of inorganic elements in AD [8-lo]. In preliminary studies using both silver stained sections [8] and antibody stained sections [9] aluminium and silicon focal deposits have been observed, in some cases associated with other inorganic elements such as titanium, calcium and iron. During these studies it was found that 0168-583X/91/$03.50

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there are a number of problems which must be overcome before analysis of this type of tissue becomes routine. These problems are investigated here using tissue from a single well characterized case of AD.

2. Experimental details 2.1. The Oxford scanning proton microprobe The SPM unit based in the Nuclear Physics Laboratory, University of Oxford, was set up primarily for the nuclear microscopy of medical and biological specimens. The unit has the facility for elemental imaging using the techniques of PIXE, nuclear backscattering and secondary electron mapping simultaneously [ll]. Samples for analysis are mounted in a sample holder, placed in the target chamber, and the focused proton beam (routinely I 1 pm at 100 pA current) is scanned over the specimen. 2.2. Sample preparation In order to provide analytical consistency all the tissue analyzed came from a single source, an 86 yr old female (C3001) with histologically proven AD. The tissue was taken from the temporal cortex and the hippocampus at autopsy, mounted on cork discs and snap frozen. 10 pm frozen sections were cut using a cryostat microtome at temperatures of about - 30 o C and picked

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and analysis of senile plaques

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Fig. 1. (a) A photomicrograph of an immunohistochemically stained plaque. The area outlined corresponds to the region scanned in the SPM studies. (b) Phosphorus, sulphur, nitrogen and SEM maps of the 100 nrn x 100 pm region shown in (a). The area of higher intensity on the maps (black) correlates with the darkly stained central core of the plaque. No aluminium or silicon was found within the stained area. V. MEDICINE

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up onto pioloform coated slides, the sections were stored at - 20°C prior to being acetone vapour fixed for 10 min. The senile plaques are largely composed of amyloid protein, and this can be immunohistochemically stained using antisera raised against complement component C3d [12]. Double distilled deionized water (mini-Q quality) and Aristar grade reagents were used throughout. C3d antibody (Dakopatts), at a dilution of 1 in 200 in 50 mM tris-HCl pH 7.4, containing 200 mM sodium chloride, was incubated for 60 min at room temperature with the sections, followed by three 5 min washes in tris buffer. The sections were then overlaid with peroxidase conjugated goat anti-rabbit antiserum (Dakopatts) at a dilution of 1 in 100 in tris buffer for 60 min at room temperature, washed three times with tris buffer and the bound antibody complex visualized using diaminobenzidine (Sigma 60 mg/lOO ml in tris, containing 40 ul 30 ~01% hydrogen peroxide). The substrate was applied to the sections and incubated for 3-5 min, resulting in a brown reaction product. The reaction was terminated by washing the sections in milli-Q water.

Fig. 2a. A photomicrograph

and analysis of

senileplaques

The pioloform membrane was floated off the slides by immersion in a tank containing milli-Q water and then laid down across the 8 mm diameter hole in the target holders. The immunohistochemical stain is more sensitive than toluidine blue or silver stain which have also been used to visualize the plaques [13,2,10]. It clearly shows the core and surrounding neuritic processes of the plaque (see fig. 1). It is organic and therefore does not interfere with the analysis of the inorganic components, and is unaffected by scanning with the proton beam, although some tissue discolouration does occur.

3. Results 3.1. Stained

tissue

Fig. la shows two immunohistochemically stained plaques, In the lower plaque, the densely stained core and the lighter neuritic halo can be seen. It was found

of an immunohistochemically stained plaque with a shattered core. The region of discoloured tissue outlined corresponds to the area scanned by the proton beam.

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J. P. Landsberg et al. / Identification and analysis of senile plaques

stained area, as were other focal inorganics, including titanium, iron and chromium. Twenty-six plaques with well defined cores, and three with less well defined cores were scanned in stained tissue. In three cases co-localized aluminium and silicon were associated with the densely stained area, and three cores contained focal silicon without aluminium (although one was co-localized with iron). The plaque cores with the co-localized deposits of aluminium and silicon also contained other inorganics such as titanium, calcium and iron. In four of the areas scanned particulate deposits with roughly the same elemental distributions were found which were not associated with the stained plaque core. This led to an investigation into possible sources of this background. 3.2. Unstained

Fig. 2b. 50X 50 urn elemental maps of the plaque in fig. 2a. The phosphorus, sulphur, nitrogen and SEM maps again clearly show the core The silicon and aluminium maps show regions of high intensity which correlate with the stained core and with the other elemental maps. There were also co-localized deposits of titanium, iron, chromium, copper and zinc in this core.

that plaque cores in stained tissue sections had a characteristic elemental composition; they consistently showed high levels of nitrogen and phosphorus (see fig. lb), elements indicating increased protein, and also of sulphur, an element frequently co-localized with amyloid protein. In addition the dense plaque cores were slightly raised in the dehydrated tissue, producing an outline in the secondary electron map. The regions of higher intensity correlate well with the stained area shown in the photomicrograph (fig. la). Fig. 2a shows an immunohistochemically stained plaque with a shattered “burnt-out” core. The densely stained areas correspond well with the regions of high phosphorus, sulphur and nitrogen levels in fig. 2b, and also with the raised areas shown on the SEM. Co-localized aluminium and silicon were associated with the

tissue

It is possible that the staining procedure could wash out any alumino-silicates in the tissue, and could introduce contamination. This could be avoided by scanning plaques in unstained tissue. The major problem with this is in determining the pathology associated with any inorganic deposits found. It was thought that the characteristic elemental distribution described in section 3.1, combined with the secondary electron picture may allow plaques to be visualized. Work on this approach is in progress. Alternatively, analysis of an unstained tissue section with a known number of plaques would provide an upper limit on the presence of aluminosilicates in the plaque cores. Therefore a section of tissue was taken from the hippocampus of C3001 and freeze dried without staining. The density of plaques was determined from neighbouring stained sections and an area of the grey matter, 4.5 mm by 1 mm was scanned in areas of 250 X 250 urn. This area was expected to contain about 20 plaque cores; no co-localized deposits of aluminium and silicon were found. Four particulate deposits of silicon associated with calcium, iron or titanium were found; these were not associated with aluminium, or with areas of increased phosphorus, sulphur and nitrogen (see section 3.1) and so were not thought to be in plaque cores. It is possible that these deposits were associated with structures in the brain other than senile plaques, or were caused by contamination.

4. Contamination 4.1. Contributions

from

airborne

dust

It was found that particles containing silicon and or aluminium, titanium, calcium and iron occurred in the freshly made pioloform backing. The elemental comV. MEDICINE

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position of these particles is characterisic of atmospheric soil dust, containing no phosphorus or nitrogen [14,15]. The pioloform films were then exposed to a normal laboratory environment for periods of up to two weeks, and re-scanned. No significant increase in background was noted which suggests that the contamination was present in the Aristar grade reagents and double distilled deionized water used to make the films. Our results suggested that phosphorus and nitrogen are invariably associated with the stained plaque core and inorganic deposits not co-localized with phosphorus and nitrogen probably did not originate in the tissue. 4.2. Contamination

from

the staining procedure

The Aristar grade reagents used in staining were filtered through 0.2 ym millipore filters and then evaporated onto pioloform films. Scans of the filtrate showed crystalline accumulations of inorganic elements up to 4 urn across. It is thought that the reagents may contain inorganic contaminants in very low concentrations (sub-ppm) which crystallize out as the solvents evaporate. As the antibodies are of biological origin, this contamination is associated with nitrogen and phosphorus and represents a source of background which it is not possible to eliminate in stained tissue. 4.3. Biogenic

contamination

The third source of contamination found is desquamated skin epithelial cells which are incident on the targets during preparation. It was found that this biological contamination is associated with nitrogen and phosphorus, and usually includes aluminium, silicon, titanium and other inorganics. However, this occurs very infrequently and is randomly distributed across the tissue (unlike the stain which is specifically identified with the plaques). It is usually easy to recognize under the light microscope.

5. Conclusions and discussion Scans of well characterized plaques in stained tissue section should provide clear evidence of the presence or otherwise of alumino-silicates in plaque cores. Preliminary results on stained tissue from a single well characterized case of AD contradict previous findings that 70-100% of senile plaque cores contain aluminosilicates [13] and suggest that between 0 and 20% of plaque cores containing particulate aluminium or silicon. This finding was supported by the scans of unstained tissue. A much larger area was scanned in the unstained tissue (4.5 mm2) than the stained tissue (- 0.2 mm2) and approximately half the number of particulate in-

organic deposits were found. This represents a significantly lower level of background than in the stained tissue, and is evidence that the stain may be introducing contamination. Three major sources of contamination were identified; dust in the pioloform backing, biogenic contamination, and contaminants in the reagents used in preparing the tissue. The presence of aluminium silicon and other organic elements in the staining reagents presents the greatest problem as the other two sources of contamination can be discriminated against by their elemental fingerprint or by light microscopy. Much of the background could be eliminated by scanning unstained plaques, however, plaques are extremely difficult to visualize without staining. It is hoped that they will be identifiable by their elemental composition. An alternative approach is to stain the tissue after scanning, however, this can only be done if the sample remains hydrated during scanning. These methods are still being investigated. It may be possible to scan a plaque core in adjacent sections and determine if the aluminium and silicon occurs in both sections, this would eliminate random particulate contamination. Drying artefact and shrinkage produce distortion in the tissue, however, by locating land-marks such as blood vessels it is possible to identify the same plaque in adjacent sections. The work described in this paper was not intended to produce general conclusions about the presence or effect of aluminium and silicon in the brains of people with AD, but instead attempted to find solutions to some of the many problems with scanning biological tissue in general, and AD tissue in particular.

Acknowledgements The nuclear microscopy described above was carried out by the SPM Unit at the University of Oxford. The unit was set up with the aid of a generous grant from the Wellcome Trust, to whom we are indebted. We also acknowledge the valuable assistance of Michael Marsh and Michael Dawson, members of the SPM unit. Two of the authors, JPL and BM, acknowledge the financial support of the Wellwme Trust.

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J.P. Landsberg et al. / Identification and analysis of senile plaques [3] D.R. Crapper. S.S. Krishnan and S. Quittkat, Brain 99 (1976) 67. [4] S. Duckett and P. Galle, J. Neuropath. Exp. Neur. 39 (1980) 350. [5] P.H. Evans. J. Klinowski, Eiji Yano and Naoko Urano, Free Rad. Res. Comms. 6 (1989) 317. [6] D.P. Per1 and P.F. Good, Lancet (May 2, 1987) 1028. [7] F. Watt and G.W. Grime, eds., Principles and Applications of High-Energy Ion Microbeams (Adam Hilger, Bristol, 1987). [8] F. Watt, G.W. Grime, G.M. Gadd, J.M. Candy, A.E. Oakley and J.A. Edwardson, Proc. 11th Int. Congress on X-ray Optics and Microanalysis, eds. J.D. Brown and R.H. Packwood (1986). [9] F. Watt, G.W. Grime, D.N. Jamieson and B. McDonald, Internal report, OUNP-89-17 (1989). [lo] N.P.-0. Larsson, U.A.S. Tapper, K. Sturesson, R. Odselius and A. Brun, Nucl. Instr. and Meth. B49 (1990) 472.

[ll]

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[15]

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G.W. Grime, F. Watt, M. Dawson, M. Marsh and I.C. McArthur, these Proceedings (2nd Int. Conf. on Nuclear Microprobe Technology and Applications, Melbourne, Australia, 1990) Nucl. Instr. and Meth. B54 (1991) 52. M.M. Esiri and B.M. McDonald, unpublished observations (1990). J.M. Candy, J.A. Edwardson, J. Klinowski, A.E. Oakley, E.K. Perry and R.H. Perry, in: Senile Dementia of the Alzheimer’s Type, eds. J. Traber and W.H. Gispen (Springer, Berlin, Heidelberg, 1985) p. 183. P. Artaxo and W. Maenhaut, Proc. 5th Int. Conf. on Particle Induced X-ray Emission and its Analytical Applications, Amsterdam, the Netherlands, 1989, Nucl. Instr. and Meth. B49 (1990) 366. P. Artaxo and W. Maenhaut, ibid., p. 383.

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