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Abstract Immunostaining with anti- -synuclein is usedto detect Lewy bodies and Lewy neurites in cases of Parkinsons disease and related disorders. To prove thatthe result of a modern silver method is equivalent to thatachieved with immunoreactions for -synuclein, individ-ual sections were successively processed using both meth-ods. The silver-stained sections showed all of the immu-noreactive Lewy bodies, and thin Lewy neurites were de-tected equally well by both techniques. The present study,therefore, points to the capabilities of a modern silver-staining method which is less time consuming and less ex-pensive than immunocytochemical techniques.

Key words Lewy body Parkinsons disease Immunocytochemistry -Synuclein Silver-staining

Introduction

Parkinsons disease (PD) and related disorders are the re-sult of severe cytoskeletal alterations in a few susceptibletypes of nerve cells. A major criterion of these illnessesare pathological changes in the form of Lewy bodies(LBs) in perikarya and Lewy neurites (LNs) in cellularprocesses. LBs and LNs develop at predisposed sites inthe peripheral and enteric nervous system, as well as inthe central nervous system [3, 8, 12, 14, 16].

A sizeable proportion of PD cases are still incorrectlydiagnosed clinically [9, 13] because of damage superim-posed by other neurodegenerative illnesses. Accordingly,

it is necessary to identify the main illness by means of postmortem demonstration of LBs and LNs. Although

large LBs can be detected with acidic dyes, routine stains(H &E) are not sufficiently sensitive to detect all of thePD-related alterations. Particularly the small, inconspicu-ous cortical LBs and fine networks of LNs (occurring,e.g., in the CA2 sector of the Ammons horn and in selectnuclei of the amygdala; [6]) escape recognition. To detectthe less noticeable pathological changes with certainty,other methods must be applied. The main options are im-munoreactions and progressive, effective silver-stainingmethods.

Recently, the presynaptic protein -synuclein has beenreported to be present in all types of LBs and LNs [1, 11,1821]. Immunocytochemical demonstration of -synu-clein presently is regarded as the gold standard for reliablerecognition of the entire spectrum of PD-associated cyto-skeletal alterations.

There clearly is a need for a simple and less-expensivemethod, but one which equals immunoreactions for -synuclein in detecting all of the LBs and LNs present in asection. Such a method not only would be beneficial forroutine diagnostic; it could also be implemented in vari-ous ways in research settings. There are significant knowl-edge gaps, for example, regarding the lesional patternsevolving in PD and related disorders, and astoundinglylittle information is available regarding the location of subcortical and cortical induction sites, the manner of dis-ease progression, the symmetry of involvement, and therange of individual variability.

The present study, therefore, is aimed at drawing neu-ropathologists attention to the advantages provided by amodern silver-staining method which fulfills these re-quirements, yet is not limited by the inconsistencies of conventional silver techniques. It can be applied withoutreservation to routinely fixed autopsy material, even if thematerial has been stored for decades in formaldehyde so-lutions. It is possible to counterstain sections for Nisslmaterial and/or other structures for easy identification of architectonic units. Use of this technique does not requireany particular skill and is considerably less time consum-ing and less expensive than application of immunocyto-chemical methods.

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

Alpha-synuclein immunoreactive Lewy bodies and Lewy neuritesin Parkinsons disease are detectableby an advanced silver-staining technique

Acta Neuropathol (1999) 98 : 461464 Springer-Verlag 1999

Received: 2 December 1998 / Revised, accepted: 25 March 1999

SHORT ORIGINAL COMMUNICATION

D. Sandmann-Keil H. Braak ( ) E. Braak Department of Anatomy, J. W. Goethe University,Theodor Stern Kai 7, D-60590 Frankfurt/Main, Germanye-mail: Braak@em.uni-frankfurt.deTel.: +49-69-6301-6900, Fax: +49-69-6301-6425

M. Okochi C. HaassDepartment of Molecular Biology,Zentralinstitut fr Seelische Gesundheit, J5,D-68159 Mannheim, Germany

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Materials and methods

To demonstrate that the quality of the pictures resulting from therecommended silver method is equivalent to that achieved withimmunoreactions for -synuclein, individual sections were succes-sively processed using both methods. For this study brain tissuewas obtained at autopsy and included three cases of clinically di-agnosed, 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 sitesfor LBs and LNs (the anterior cingulate region of the telencephaliccortex, the basal ganglia including portions of the thalamus and theinsular cortex, and the dorsal vagal area of the brain stem) wereembedded in paraffin to achieve thin sections (410 m) and inpolyethylene glycol (PEG) for 50- to 150- m-thick sections [2,17]. The sections were first subjected to immunoreaction usingpolyclonal antiserum against -synuclein (dilution 1:1000) [15],

following a standard protocol including prevention of nonspecificbinding and inhibition of endogenous peroxidase. Bound antibodieswere visualized with the ABC reagent (Vectastain Elite Kit, Vec-tor) and the chromogen 4-chloro-1-naphthol (Sigma C 8890). Thesections were cleared in a water-soluble medium (Karion Merck 2993) and were cover-slipped transiently. Structures immunoposi-tive for -synuclein were photographed (Fig. 1A, C, E), and theirpositions were documented with the aid of the vernier scale. Sub-sequently, the cover slips were removed and the chromogen wasdecolorized with 70% ethanol. The same sections then were silver-stained according to a method originally proposed by Campbell etal. [4] for Alzheimer-related alterations [2, 10]. Silver nucleationsites were induced by placing sections in a pyridine silver solutionand subsequently were visualized by physical development per-

462

Fig. 1AF 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 inParkinsons disease equally well. Note the presence of -synu-clein-immunoreactive Corpora amylacea ( arrows in C ) and the ab-sence of the Corpora amylacea in Campbell-Switzer staining ( ar-rows in D ). A, B Small cortical Lewy bodies and Lewy neurite;

C, D large Lewy body; and E, F Lewy neurites in the dorsal vagalarea of brain stem ( bv blood vessel)

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mitting tight control of the entire staining procedure [7]. Optimallyprocessed 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 (PermountFisher). All of the LBs and LNs formerly shown to be im-munopositive for -synuclein were then photographed a secondtime.

ResultsSections processed with the silver method showed LBsand LNs in clear contrast against the background (Fig. 1B,D, F). All of the immunoreactive LBs and LNs could belocated again in the silver-stained sections. The size andshape of -synuclein-positive structures closely matchedthe respective features of the silver-stained elements, sothat even fine filigree elements could be detected equallywell with both techniques. Occasionally, for thick sec-tions, the silver technique revealed some additional LBsand LNs which could not be detected by the immunoreac-tion owing to imperfect penetration. In contrast to the im-

munoreaction, -synuclein-positive Corpora amylacea re-mained unstained by the silver-method (Fig. 1, see ar-rows).

In addition to reliable demonstration of LBs and LNs,the silver method displayed some of the pathologicalchanges related to Alzheimers disease, such as -amyloiddeposits and extraneuronal ghost or tombstone tan-gles [4]. All of the structures referred to were easily dis-tinguishable from LBs. To a variable extent there wasstaining of normal axons, preferentially axons with a largediameter and myelin sheath. The trained eye will be ableto distinguish between normal axons and LNs. Normalaxons are lengthy, straight structures with a constantdiameter, whereas LNs usually are shorter and characterizedby varicosities and end-swellings.

Moreover, from 20 additional clinically diagnosed andneuropathologically confirmed PD cases, we stained twoconsecutive sections of the above mentioned regions withanti- -synuclein and the method according to Campbell etal. [4]. In all of these pairs of sections LBs and LNs werestained in about the same distribution pattern and density.

Discussion

A major weakness of immunoreactions for demonstrationof -synuclein is the troublesome nonspecific co-stainingof structures that bear a likeness to LBs, such as the Cor-pora amylacea (Fig. 1; [5]). Moreover, application of im-munoreactions usually requires some skill, is time con-suming, and expensive (the costs for the -synuclein im-munoreaction are roughly 40 times higher than those forthe Campbell-Switzer technique). Accordingly, immuno-cytochemical approaches are usually not employed forroutine diagnostic purposes, or are restricted to only amoderate number of small paraffin sections in a few selectcases. In addition, immunoreactions do not guarantee ho-

mogeneous staining throughout the entire thickness of asection, which is a prerequisite for semiquantitative eval-uation.

The silver technique under consideration can be usedfor large numbers of sections. It is not limited to thinparaffin sections, but also can be applied to sections cut atgreater thickness. The homogeneity of staining through-out the entire thickness of a section facilitates evaluation.

A section thickness (50150 m) perfectly meets the re-quirements of low-power stereomicroscopy, while permit-ting conventional light microscopy. This is advantageousbecause the large number of pathological changes super-imposed on each other enables more accurate recognitionof the key pathoarchitectonic features, such as the thick plexus of LNs in the CA2 sector of the Ammons horn orlayer-specific accumulations of LBs in certain cortical ar-eas which can even be evaluated with the naked eye. Thetechnique is particularly useful for the economical pro-cessing of numerous large sections through the entire hu-man brain (double-hemisphere sections), a prerequisitefor any attempt to enhance our knowledge of the manner

of disease progression, symmetry of affection, and rangeof individual variability in PD.

Acknowledgements This work was supported by the Bundes-ministerium fr Bildung, Wissenschaft, Forschung und Technolo-gie. The authors would like to thank Dr. K. Del Tredici for revis-ing the English manuscript stylistically.

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