Early Senile Plaques in Alzheimer's Disease Demonstrated by Histochemistry, Immunocytochemistry, and Electron Microscopy

SHU-ICHI IKEDA, MD, NOBUO YANAGISAWA, MD, DAVID ALLSOP, PHD, AND GEORGE G. GLENNER, MD

To clarify early pathologic changes in Alzheimer's disease, the brains from two cases from a single family with this disease were examined. A mother who died at age 75 with severe dementia showed an abundance of typical senile plaques, neurofibrillary tangles, and cerebrovascular amyloidosis. The senile plaque and cerebrovascular amyloid were strongly immunoreactive to anti-13 protein antibody. Her son manifested erratic and bizarre behav- ior, and was suspected of having committed suicide at age 52. His brain weight and macroscopic observations were normal; how- ever, Bielschowsky's silver impregnation and methenamine sil- ver stains showed numerous argyrophilic plaque-like lesions in the neocortex. They were weakly immunolabeled by anti-13 pro- tein antibody, but lacked any abnormal neurites of Congophilic amyloid deposits. These lesions resembled the "type 3" immu- noreactive lesions (previously reported by us in Alzheimer's dis- ease and Down's syndrome) which seem to be an early stage of senile plaque formation. These putative early plaque lesions were also examined by methenamine silver electron microscopy, and were seen to consist of loose aggregations of irregular spindle- shaped structures with a heavy deposition of silver grains, with genuine amyloid fibrils not being apparent. It is believed that the accumulation of 13-protein immunoreactive material without amyloid fibril formation might be an initial step in the develop- ment of the senile plaque, and that the son, having extensive cortical involvement with type 3 plaque lesions, demonstrated clinical manifestations of less completely developed Alzheimer's disease. HUM PATHOL 211:1221--1226. �9 1990 by W.B. Saunders Company.

Alzheimer's disease is a degenerat ive brain dis- o rde r with a progressive dement ia which develops in middle or late life. T h e pathologic findings of this disease are characterized by neurofibri l lary tangles, senile plaques, and cerebrovascular amyloidosis, and

similar lesions are commonly seen in adult Down's syndrome brains. 1,2 Amylo id fibrils isolated f rom cerebrovascular 3,4 and senile plaque amyloid 5 in both Alzheimer's disease and Down's syndrome are com- posed o f a un ique 4.2 kd po lypept ide t e rmed "[3 protein". This amyloid pro te in is thought to be de- rived by proteolysis f rom a much larger cell mem- brane glycoprotein precursor , 6,7 the gene for which is located on ch romosome 21.8,9 Polyclonal and mono- clonal antibodies for [3 pro te in have been repor ted to react specifically with amylo id deposi ts o f senile plaques and ce r eb ro v ascu l a r walls at bo th light- microscopic 1~ and electron-microscopic 12 levels.

Some recent his tochemical and immunohis to- chemical studies 13-a6 have revealed impor tan t new in- format ion on the pathogenesis o f the senile plaque: a special type o f senile plaque with weak 13 protein im- munoreac t iv i ty called a "d i f fuse p laque" , 13 "pre- amyloid deposit", 14 or " type 3 immunoreac t ive le- sion ''z5 has been noted. This type o f plaque lesion lacks any his tochemical ev idence o f degene ra t i ng neurites or amyloid deposits, and many similar le- sions in the absence o f typical senile plaques are com- monly seen in the brains o f individuals between 30 and 40 years of age with Down's syndrome. 17-21 It has been suggested, therefore , that these lesions are se- nile plaques at a very early stage of development . In this paper we repor t a probable case with early path- ologic changes o f Alzheimer 's disease, with a special focus on the histochemical , immunocytochemica l , and ul trastructural f indings of these putative early senile plaques.

From the Department of Medicine (Neurology), Shinshu Uni- versity School of Medicine, Matsumoto, Japan; the Department of Molecular Biology, Psychiatric Research Institute of Tokyo, Tokyo, Japan; and the Department of Pathology, University of California at San Diego, School of Medicine, La Jolla, CA. Accepted for pub- lication January 24, 1990.

Supported by Grant No. 01770541 from the Ministry of Ed- ucation, Science and Culture, Japan; Grant No. AG-05683 from the National Institutes of Health, Bethesda, MD; and Grant No. 84-84559 from the California State Department of Health Services.

Key words: Alzheimer 's disease, senile plaque, amyloid, [3-protein, methenamine-silver stain.

Address correspondence and reprint requests to Shu-ichi Ikeda, MD, Department of Medicine (Neurology), Shinshu Uni- versity School of Medicine, Matsumoto 390, Japan.

�9 1990 by W.B. Saunders Company. 0046-8177/90/2112-000755.00/0

MATERIALS AND METHODS

Only limited clinical information was available on the following two cases. Case no. 1 was a white woman who developed certain adverse personality changes around age 65, and soon afterwards an impairment of her cognitive ability became evident. These symptoms were slowly pro- gressive, degenerating into severe memory deficits and con- fusion at about age 70. At the age of 73 she was bedridden with inability to communicate, and she died at age 75. Her medical history revealed that her mother had suffered from an apparently similar disorder. Case no. 2 was the son of case no. 1; he was observed to have had some de- terioration in memory and cognitive impairment at age 50, and subsequently erratic and bizarre behavior was evident

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for about 1 year prior to his death at age 52 by drowning, possibly self-inflicted. To demonstrate familial occurrence of Alzheimer's disease, we tried to contact other family members; however, we were unable to obtain any addi- tional information.

Brain tissue blocks were routinely taken from case no. 1. Tissue samples prepared from three different neocorti- ces, diencephalon through thalamus and cerebellum, were available from case no. 2. Serial sections from formalin- fixed and paraffin-embedded blocks were stained with he- matoxlin-eosin, alkaline Congo red, modif ied Biel- schowsky's silver impregnation, 22 methenamine silver (MS) stain combined with 0.1% cresyl violet Nissl counter- stain, 23,24 and by immunocytochemical methods with an anti-[3 protein antibody. Amyloid was identified by its char- acteristic apple-green birefringence under the polarizing microscope after Congo red staining.

Immunoperoxidase staining was performed using the avidin-biotin peroxidase technique described previously? 5 The primary reagent was monoclonal antibody 4D 12/2/611 raised against a synthetic peptide consisting of residues 8- 17 of [3 protein; all immunostained sections were pretreated with 98% formic acid 25 to enhance the immunoreactivity of amyloid-related proteins. Using an eyepiece graticule at a magnification of • 100 the number of immunoreactive plaque-like lesions seen in case no. 2 was counted in 10 randomly selected fields fi-om sections of the midfrontal gyrus. A preembedding staining technique was used for

electron-microscopic examination of MS-positive plaque le- sions: dewaxed sections were stained with MS and then em- bedded in Epoxy resin using an inverted gelatin capsule method. Thin sections without fur ther electron-dense stains were observed under a Hitachi HS-9 electron micro- scope (Tokyo, Japan).

RESU LTS

T h e brain weight o f case no. 1 was 952 g, and gross examina t ion o f the bra in showed general ized m a r k e d a t rophy, especially in the f ronta l lobes; coro- nal sections revealed severe cortical th inning with di- lated lateral ventricles. T h e a p p e a r a n c e of the brain in case no. 2, however , was normal , and it weighed 1480 g.

Light M i c roscop i c Findings

Microscopic examina t ion o f the cor tex in case no. 1 revealed a decreased n u m b e r o f neurons with a diffuse distr ibution o f senile plaques (Fig 1, left) and neurofibr i l lary tangles. Extensive and f requen t cere- brovascular amyloid deposits were also seen (Fig 1

FIGURE 1. Neocoffical findings of case no. 1. Closely adjacent sections from the same frontal lobe block. [Left] Bielshowsky's silver stain reveals many typical core-containing senile plaques. [Bar = 100 i~m.] [Insert] A polarized view of a Congo red-stained section showing extensive Congophilic angiopathy in the subarachnoidal vessels, [Bar = 50 I~m.] [Right] Immunostaining. Many discrete plaques with positive immunoreactivity and ill-defined immunoreactive areas with a granular appearance are visible. [insert] Methenamine silver-positive typical senile plaques. [Bar = 100 i~m.]

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EARLY SENILE PLAQUES IN ALZHEIMER'S DISEASE [Ikeda et all

left, insert). The plaques were easily observed in all Congo red-, Bielschowsky's silver impregnation-, and MS-stained sections, and the latter two staining meth- ods revealed that many plaques had a central core surrounded by abnormal neurites (classical or mature plaques, 26 Fig 1, left and Fig 1, right, insert). The subcortical gray matter was less involved, and the cer- ebellum contained a few scattered compact plaques with a small amount of amyloid deposition in the sub- arachnoid vessels. Immunostaining showed numer- ous immunoreactive discrete plaques and plaque-like lesions with faint immunoreactivity (Fig 1, right), to- gether with positively stained cerebrovascular walls.

The specimens from case no. 2 showed well- preserved cortical neurons, but neither senile plaques with amyloid deposition nor cerebrovascular amy- |oidosis were seen after careful examination of the Congo red-stained sections from all five tissue blocks. However, both Bielschowsky's silver impregnation and MS staining disclosed a large number of plaque- like lesions distributed widely throughout the neocor- tical sections examined; they were identified as ill- defined argyrophilic areas by the former method, the vast majority of which lacked abnormal neurites (Fig 2, left). A few primitive plaques 26 were also observed. The MS stain clearly showed that some of the larger plaque-like lesions contained one or more neuronal perikarya (Fig 2, center). In immunostained serial

sections numerous ill-defined immunoreactive lesions with a coarse granular appearance (ranging from 20 to 150 p~m in a diameter) were seen throughout the entire neocortex (Fig 2, right), and these appeared to correspond to the plaque-like lesions revealed by the two silver staining methods. The mean number of immunoreactive plaque-like lesions per mm 2 was 32.0, ranging from 15 to 62. Additionally, a very small number of neurofibrillary tangles were also seen in the neocortical sections, but the diencephalon and cerebellum did not show any significant lesions.

Electron Microscopic Findings

This examination was restricted to the brain sec- tions of case no. 2, and specimens were prepared from selected areas containing many MS-positive plaque-like lesions as seen under light-microscopic examination. Most of these lesions were without a dis- crete core, and consisted of sparse aggregations of frequently ramified spindle-shaped structures with a heavy deposition of silver particles (Fig 3, left). Since deparaffinized tissues were used, the cerebral ultra- structure was not sufficiently well-preserved to clearly dist inguish all tissue componen t s of the senile plaques. However, after an extensive search, no ap- parent amyloid fibrils were seen within or around the concentrated areas of silver particles (Fig 3, right).

FIGURE 2. Neocortical findings of case no, 2. Closely adshows numerous argyrophiUc areas without abnormal neularger ones indicated by arrowheads contain neuronal c[Bars = 100 t~m,] [Insert] A magnif ied picture of the immu

jacent sections from the same frontal lobe block [Left] Bielshowsky's silver stain rites. [Center] Methenamine silver stain discloses similar plaque-like lesions and

ells bodies. [Right] Immunostaining reveals many type 3 immunoreactive lesions. noreactive lesion, Note that there is no discrete central core, [Bar = 50 ~m,]

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FIGURE 3. Electron-microscopic findings of MS-positive early plaque lesions. [Left] Low magnification electronmicrograph shows sparse aggregations of irregular spindle-shaped structures selectively labeled by silver particles. [Bar = 5 ixm.] [Right] Under higher magnification, no obvious amyloid fibrils are visible in the areas with heavy deposition of silver grains. [Bar = 1 ~m.]

DISCUSSION

The abundance of neurofibrillary tangles and se- nile plaques with amyloid angiopathy in the brain of case no. 1 is consistent with a diagnosis of Alzheimer's disease, and many senile plaques in this patient con- tained an amyloid-rich core accompanied by degen- erating neurites in the periphery (mature plaques). Amyloid deposits in both cerebrovascular walls and senile plaques were strongly immunoreactive to the anti-[3 protein antibody. On the basis of all the clinical and pathologic information, this case is considered to be Alzheimer's disease at an advanced stage.

Case no. 2 was suspected of having early clinical manifestations of (probable familial) Alzheimer's dis- ease, but routine histopathologic examination of the brain failed to disclose any pathognomonic findings for this disorder. However, using sensitive histochem- ical and immunocytochemical staining methods, many plaque-like lesions were found to be distributed extensively throughout all areas of the neocortex. These were seen as ill-defined argyrophilic areas with weak 13-protein immunoreactivity that were not asso- ciated with either degenerating neurites or Congo red-positive amyloid deposits. This type of plaque re- sembled the "type 3" immunoreactive lesions 15 that were previously reported by us in Alzheimer's dis- ease. Subsequent studies 17-21 on a series of cases of Down's syndrome have suggested that senile plaques

may undergo sequential structural changes, and that the type 3 lesions (sometimes t e r m e d "di f fuse plaques ''13 or "pre-amyloid deposits") 14 are an early stage of senile plaque formation.

It is well-known that a small number of typical senile plaques occur in the brains of aged nonde- mented individuals, 27,2s and the age-related brain amyloid is also composed of [3 protein? 5,29-32 How- ever, the number of [3 pro te in- immunoreac t ive plaque lesions seen in case no. 2 is much larger than that reported by us x5 or others 3a in age-matched con- trols, and it also satisfies the widely accepted criteria 3a for the pathologic diagnosis of Alzheimer's disease. These findings suggest that case no. 2 might be an example of Alzheimer's disease at a very early stage, with pathologic changes that would eventually de- velop into a picture similar to that of case no. 1 with advanced Alzheimer's disease having many typical or classical senile plaques.

The detailed pathogenic mechanisms involved in the formation of senile plaques remain unclear, and it has been controversial whether or not the 13 protein- immunoreact ive early plaque lesions de- scribed above contain genuine amyloid fibrils or

13 17 I n [3-protein precursors. - The MS sta' ing used in this study is a very sensitive method for detecting se- nile plaques, especially in demonstrating early plaque lesions. 13,2~,24 This is a simplification of the Yajima- modified periodic acid methenamine silver method 34

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i

A B C 0

FIGURE 4. Schematic illustration of possible temporal structural changes in the developing senile plaque. [A] Type 3 lesion showing 13- protein immunoreactive material without amyloid fibril structures or abnormal neurites. [B] Primitive plaque accompanied by the deposition of 13-protein amyloid fibrils and dystrophic neurites, [C] Classical or mature plaque with a discrete amyloid core sur- rounded by dystrophic neurites. [D] Compact or burned-out plaque consisting solely of amyloid without degenerating neurites.

of Jones, 35 and has proven to be useful for the present electron-microscopic study. Although the present case no. 2 showed numerous MS-positive plaque lesions in the neocortex under light micros- copy, the ultrastructural examination did not reveal the presence of any amyloid fibrils in those lesions with either heavy or light deposition of silver parti- cles. Similar electron-microscopic findings 2~ have been reported in two cases with Alzheimer's disease, and it has been also suggested that the areas with accumulation of silver grains contain an electron- dense amorphous substance with a partially fibrous structure. 23 All of these ultrastructural observations support the concept 14,15,x7 that the type 3 plaque le- sions may be composed of [3 protein or [3-protein precursors 36 that are not in the antiparallel [3-pleated sheet configuration characteristic of amyloid fibril structure? 7 Further ultrastructural studies, including immunoelectron microscopy, are required to confirm this.

On the basis of current histochemical, immuno- cytochemical, and electron-microscopic findings of plaque lesions, 13-16,23 including those reported in cases with Down's syndrome, 17-21 we consider that the senile plaque undergoes stages as outlined in Fig 4. Suggestions as to the origin of the [3 protein com- prising the senile plaques have included a serum derivation, t,3s resulting from compromise of the blood-brain barrier in amyloid-afflicted vessels, and a neuronal der iva t ion9 ,4~ The origin of this protein, however, still remains uncertain.

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