Acta Neuropathol (1999) 98 : 461464

Springer-Verlag 1999

S H O RT O R I G I N A L C O M M U N I C AT I O N

Daniele Sandmann-Keil Heiko Braak Masayasu Okochi Christian Haass Eva Braak

Alpha-synuclein immunoreactive Lewy bodies and Lewy neurites in Parkinsons disease are detectable by an advanced silver-staining technique
Received: 2 December 1998 / Revised, accepted: 25 March 1999

Abstract Immunostaining with anti--synuclein is used to detect Lewy bodies and Lewy neurites in cases of Parkinsons disease and related disorders. To prove that the result of a modern silver method is equivalent to that achieved with immunoreactions for -synuclein, individual sections were successively processed using both methods. The silver-stained sections showed all of the immunoreactive Lewy bodies, and thin Lewy neurites were detected equally well by both techniques. The present study, therefore, points to the capabilities of a modern silverstaining method which is less time consuming and less expensive than immunocytochemical techniques. Key words Lewy body Parkinsons disease Immunocytochemistry -Synuclein Silver-staining

Introduction
Parkinsons disease (PD) and related disorders are the result of severe cytoskeletal alterations in a few susceptible types of nerve cells. A major criterion of these illnesses are pathological changes in the form of Lewy bodies (LBs) in perikarya and Lewy neurites (LNs) in cellular processes. LBs and LNs develop at predisposed sites in the peripheral and enteric nervous system, as well as in the central nervous system [3, 8, 12, 14, 16]. A sizeable proportion of PD cases are still incorrectly diagnosed clinically [9, 13] because of damage superimposed by other neurodegenerative illnesses. Accordingly, it is necessary to identify the main illness by means of postmortem demonstration of LBs and LNs. Although
D. Sandmann-Keil H. Braak () E. Braak Department of Anatomy, J. W. Goethe University, Theodor Stern Kai 7, D-60590 Frankfurt/Main, Germany e-mail: Braak@em.uni-frankfurt.de Tel.: +49-69-6301-6900, Fax: +49-69-6301-6425 M. Okochi C. Haass Department of Molecular Biology, Zentralinstitut fr Seelische Gesundheit, J5, D-68159 Mannheim, Germany

large LBs can be detected with acidic dyes, routine stains (H & E) are not sufficiently sensitive to detect all of the PD-related alterations. Particularly the small, inconspicuous cortical LBs and fine networks of LNs (occurring, e.g., in the CA2 sector of the Ammons horn and in select nuclei of the amygdala; [6]) escape recognition. To detect the less noticeable pathological changes with certainty, other methods must be applied. The main options are immunoreactions and progressive, effective silver-staining methods. Recently, the presynaptic protein -synuclein has been reported to be present in all types of LBs and LNs [1, 11, 1821]. Immunocytochemical demonstration of -synuclein presently is regarded as the gold standard for reliable recognition of the entire spectrum of PD-associated cytoskeletal alterations. There clearly is a need for a simple and less-expensive method, but one which equals immunoreactions for synuclein in detecting all of the LBs and LNs present in a section. Such a method not only would be beneficial for routine diagnostic; it could also be implemented in various ways in research settings. There are significant knowledge gaps, for example, regarding the lesional patterns evolving in PD and related disorders, and astoundingly little information is available regarding the location of subcortical and cortical induction sites, the manner of disease progression, the symmetry of involvement, and the range of individual variability. The present study, therefore, is aimed at drawing neuropathologists attention to the advantages provided by a modern silver-staining method which fulfills these requirements, yet is not limited by the inconsistencies of conventional silver techniques. It can be applied without reservation to routinely fixed autopsy material, even if the material has been stored for decades in formaldehyde solutions. It is possible to counterstain sections for Nissl material and/or other structures for easy identification of architectonic units. Use of this technique does not require any particular skill and is considerably less time consuming and less expensive than application of immunocytochemical methods.

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Fig. 1 AF Comparison between -synuclein immunoreaction (left column) versus Campbell-Switzer silver pyridine technique (right column) carried out consecutively in one and the same section. Both methods demonstrate Lewy bodies and Lewy neurites in Parkinsons disease equally well. Note the presence of -synuclein-immunoreactive Corpora amylacea (arrows in C) and the absence of the Corpora amylacea in Campbell-Switzer staining (arrows in D). A, B Small cortical Lewy bodies and Lewy neurite; C, D large Lewy body; and E, F Lewy neurites in the dorsal vagal area of brain stem (bv blood vessel)

Materials and methods

To demonstrate that the quality of the pictures resulting from the recommended silver method is equivalent to that achieved with immunoreactions for -synuclein, individual sections were successively processed using both methods. For this study brain tissue was obtained at autopsy and included three cases of clinically diagnosed, neuropathologically confirmed PD and one control case. Brains were fixed by immersion in a 4% aqueous solution of form-

aldehyde. Tissue blocks encompassing typical predilection sites for LBs and LNs (the anterior cingulate region of the telencephalic cortex, the basal ganglia including portions of the thalamus and the insular cortex, and the dorsal vagal area of the brain stem) were embedded in paraffin to achieve thin sections (410 m) and in polyethylene glycol (PEG) for 50- to 150-m-thick sections [2, 17]. The sections were first subjected to immunoreaction using polyclonal antiserum against -synuclein (dilution 1:1000) [15], following a standard protocol including prevention of nonspecific binding and inhibition of endogenous peroxidase. Bound antibodies were visualized with the ABC reagent (Vectastain Elite Kit, Vector) and the chromogen 4-chloro-1-naphthol (Sigma C 8890). The sections were cleared in a water-soluble medium (Karion Merck 2993) and were cover-slipped transiently. Structures immunopositive for -synuclein were photographed (Fig. 1A, C, E), and their positions were documented with the aid of the vernier scale. Subsequently, the cover slips were removed and the chromogen was decolorized with 70% ethanol. The same sections then were silverstained according to a method originally proposed by Campbell et al. [4] for Alzheimer-related alterations [2, 10]. Silver nucleation sites were induced by placing sections in a pyridine silver solution and subsequently were visualized by physical development per-

463 mitting tight control of the entire staining procedure [7]. Optimally processed sections appeared in a deep brownish to purple shade, with the white matter more intensely stained than the gray matter. Sections were cleared and mounted in a synthetic resin (Permount Fisher). All of the LBs and LNs formerly shown to be immunopositive for -synuclein were then photographed a second time.

Results
Sections processed with the silver method showed LBs and LNs in clear contrast against the background (Fig. 1B, D, F). All of the immunoreactive LBs and LNs could be located again in the silver-stained sections. The size and shape of -synuclein-positive structures closely matched the respective features of the silver-stained elements, so that even fine filigree elements could be detected equally well with both techniques. Occasionally, for thick sections, the silver technique revealed some additional LBs and LNs which could not be detected by the immunoreaction owing to imperfect penetration. In contrast to the immunoreaction, -synuclein-positive Corpora amylacea remained unstained by the silver-method (Fig. 1, see arrows). In addition to reliable demonstration of LBs and LNs, the silver method displayed some of the pathological changes related to Alzheimers disease, such as -amyloid deposits and extraneuronal ghost or tombstone tangles [4]. All of the structures referred to were easily distinguishable from LBs. To a variable extent there was staining of normal axons, preferentially axons with a large diameter and myelin sheath. The trained eye will be able to distinguish between normal axons and LNs. Normal axons are lengthy, straight structures with a constant diameter, whereas LNs usually are shorter and characterized by varicosities and end-swellings. Moreover, from 20 additional clinically diagnosed and neuropathologically confirmed PD cases, we stained two consecutive sections of the above mentioned regions with anti--synuclein and the method according to Campbell et al. [4]. In all of these pairs of sections LBs and LNs were stained in about the same distribution pattern and density.

mogeneous staining throughout the entire thickness of a section, which is a prerequisite for semiquantitative evaluation. The silver technique under consideration can be used for large numbers of sections. It is not limited to thin paraffin sections, but also can be applied to sections cut at greater thickness. The homogeneity of staining throughout the entire thickness of a section facilitates evaluation. A section thickness (50150 m) perfectly meets the requirements of low-power stereomicroscopy, while permitting conventional light microscopy. This is advantageous because the large number of pathological changes superimposed on each other enables more accurate recognition of the key pathoarchitectonic features, such as the thick plexus of LNs in the CA2 sector of the Ammons horn or layer-specific accumulations of LBs in certain cortical areas which can even be evaluated with the naked eye. The technique is particularly useful for the economical processing of numerous large sections through the entire human brain (double-hemisphere sections), a prerequisite for any attempt to enhance our knowledge of the manner of disease progression, symmetry of affection, and range of individual variability in PD.
Acknowledgements This work was supported by the Bundesministerium fr Bildung, Wissenschaft, Forschung und Technologie. The authors would like to thank Dr. K. Del Tredici for revising the English manuscript stylistically.

References
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Discussion
A major weakness of immunoreactions for demonstration of -synuclein is the troublesome nonspecific co-staining of structures that bear a likeness to LBs, such as the Corpora amylacea (Fig. 1; [5]). Moreover, application of immunoreactions usually requires some skill, is time consuming, and expensive (the costs for the -synuclein immunoreaction are roughly 40 times higher than those for the Campbell-Switzer technique). Accordingly, immunocytochemical approaches are usually not employed for routine diagnostic purposes, or are restricted to only a moderate number of small paraffin sections in a few select cases. In addition, immunoreactions do not guarantee ho-

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