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Stroke. 1995;26:2145-2148

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(Stroke. 1995;26:2145-2148.)
© 1995 American Heart Association, Inc.

Articles

Widespread Appearance of Alz-50 Immunoreactive Neurons in the Human Brain With Cerebral Infarction

Toshiki Uchihara, MD, PhD; Kuniaki Tsuchiya, MD, PhD; Hiromi Kondo; Tadayoshi Hayama, MD, PhD Kenji Ikeda, MD, PhD

From the Department of Neuropathology, Tokyo Institute of Psychiatry (T.U., H.K., K.I.); Department of Neurology, Tokyo Medical and Dental University (T.U., K.T.); Department of Pathology, Tokyo Medical College (K.T.); and Department of Pathology, Tokyo Metropolitan Aoyama Hospital (T.H.) (Japan).

Correspondence to T. Uchihara, MD, PhD, Laboratoire Raymond Escourolle, Service de Neuropathologie, Groupe hospitalier, Pitié-Salpêtrière, 47-83 Boulevard de l'Hôpital, 75651 Paris, Cedex 13, France.


*    Abstract
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Background and Purpose Tau-like immunoreactivity is known to develop in neurons under some experimental conditions simulating ischemia. The purpose of this study is to investigate the expression of tau-like immunoreactivity in the human brain after ischemic insult.

Methods A series of autopsied human brains with or without ischemic lesion were investigated with immunohistochemistry (Alz-50, anti-tau, and anti-ubiquitin) and with silver-staining methods (Gallyas and Bodian methods).

Results Punctate immunoreactivity to Alz-50 was visualized in the cytoplasm not only of the neurons in and around the ischemic lesion but also of the neurons free from classic ischemic changes around the necrosis. Some of the neurons around the ischemic lesion were stained by the Gallyas method. Immunostaining with anti-tau and anti-ubiquitin antibodies and the conventional Bodian method failed to visualize these neurons.

Conclusions The widespread appearance of Alz-50 immunoreactive neurons during the ischemic process signifies that tau-related proteins may be related to ischemic necrosis, but the lack of neurofibrillary tangles morphologically distinguishes ischemic development of tau-related proteins from the neurofibrillary degeneration in Alzheimer's disease.


Key Words: cerebral infarction • neurofibrillary tangles • tau proteins


*    Introduction
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Tau and its related proteins are major components of PHF. They are deposited in the form of A-NFTs in the neurons of the AD brain.1 2 3

However, these proteins are known to also appear in cultured neurons after exposure to glutamate4 5 or in neurons of the animal brain after experimentally induced ischemia.6 7 We therefore examined autopsied human brains with an ischemic lesion to clarify its relation to tau-like IR.


*    Methods
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 *Methods
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Autopsied brains from patients with cerebral infarction were used in this study. The time of occurrence of the cerebrovascular accident was determined from the clinical record and/or according to histological findings8 listed in the TableDown. Two other brains without neurological disorders were used as control. Formalin-fixed, paraffin-embedded blocks including both the area of ischemic necrosis and the surrounding nonnecrotic area were obtained. We stained 10-µm-thick neighboring sections with the following: (1) cresyl violet stain; (2) Bodian stain; (3) Gallyas stain pretreated (to enhance the sensitivity of the staining) with 0.3% potassium permanganate for 10 minutes followed by 1% oxalic acid for 1 minute9 10 ; and (4) tau (tau 2,11 mouse monoclonal IgG, 1:1000, Sigma, and pool 2,12 rabbit IgG, 1:20 000, generously provided by Dr H. Mori), ubiquitin (rabbit IgG, 1:1000, DAKO), and Alz-5013 (mouse monoclonal IgM, 1:200, generously provided by Dr P. Davies) immunohistochemistry.


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  		Table 1. Autopsied Cases With Ischemic Lesion Examined

Sections were treated with 0.2% hydrogen peroxide for 30 minutes and were then incubated for 2 days at 4°C with the primary antibody diluted with PBS containing 0.3% Triton-X and the corresponding blocking serum. The sections were then incubated with one of the biotinylated secondary antibodies corresponding to the primary antibody (1:1000, biotinylated anti-mouse IgG, biotinylated anti-mouse IgM, or biotinylated anti-rabbit IgG, Vector) for 2 hours. After incubation with avidin-biotin-peroxidase complex (1:1000, ABC-Elite, Vector) for 1 hour at room temperature, peroxidase labeling was visualized by incubating sections with 0.05% Tris-buffered saline (pH 7.6) containing 0.03% 3,3'-diaminobenzidine, 0.00015% hydrogen peroxide, 0.05 mol/L imidazole, and 1.0% nickel sulfate. A deep purple reaction product became visible after 15 to 30 minutes.


*    Results
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In the ischemic lesion, where glial reaction, proliferation of the capillaries, and neuronal depletion are apparent on sections stained by cresyl violet (Fig 1ADown), some of the pyramidal neurons were immunostained by Alz-50 (Fig 1BDown). These neurons were scarcely identifiable on sections stained by cresyl violet. Some of the neurons were weakly stained by a modification of the Gallyas method (Fig 1CDown).



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  		Figure 1. Photomicrographs of ischemic lesion 13 days after the ischemic attack in a 68-year-old woman (magnification x140, bar=100 µm). A, Cresyl violet; B, Alz-50 immunostain; and C, modified Gallyas stain. Neuronal depletion and glial reaction are apparent (A). Alz-50 immunolabeled (B) an unexpectedly large number of neurons, which are faintly stained on the Gallyas stain (C).

In the nonnecrotic area without glial reaction around the ischemic lesion, the number and the morphology of neurons were normal (Fig 2ADown). In this area also, pyramidal cells were stained by Alz-50 (Fig 2BDown). The Alz-50 IR was restricted to the neuronal perikarya and was punctate in fashion; it was abolished when the primary antibody was replaced by PBS containing 0.3% Triton-X or by mouse IgM (mouse standard, Tago Diffu-gen). These neurons were not stained by the modified Gallyas method.



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  		Figure 2. Photomicrographs of an apparently intact area around ischemic necrosis in the same case as in Fig 1Up (magnification x240, bar=50 µm). A, Cresyl violet; B, Alz-50 immunostain. No apparent morphological changes are detectable. These neurons were immunolabeled by Alz-50 in punctate fashion.

Anti-ubiquitin antibody, anti-human tau antibodies (tau 2, pool 2), and the conventional Bodian method failed to visualize these neurons. In the areas far from the ischemic lesion as well as in control cases, no neurons were stained by these antibodies or by silver-staining methods.


*    Discussion
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 *Discussion
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After experimentally induced ischemia, some neurons are reported to accumulate tau-like or Alz-50 IR6 7 in their cytoplasm in punctate fashion. This is the first report confirming that a similar process takes place in the human brain during ischemia.

Neurons, not containing NFTs, with accumulated tau-related epitopes were described in some restricted parts of the normal human and animal brain,14 15 16 in the nervous system during its development,17 18 and in the AD brain as "pretangle neurons."1 2 3 19 Kato and colleagues20 reported a case of cerebral infarction with abundant NFTs in the nucleus basalis of Meynert in the affected hemisphere. However, it is unusual for A-NFTs to appear in and around the ischemic focus. These examples indicate that tau-related proteins rather frequently accumulate in neurons, such as the increased Alz-50 IR in the ischemic process, but this does not necessarily lead to the formation of NFTs.

As demonstrated by immunoblotting, Alz-50 or anti-tau antibodies (tau 2, pool 2) bind to a recombinant tau molecule or its extracted form from the normal brain,12 21 22 23 while the positive immunohistochemical staining is at most limited to the axon in the fixed normal brain tissue.24 In the AD brain, on the other hand, these antibodies easily immunostain pathological structures such as A-NFTs and neurite reactions.13 19 25 This discrepancy can be partly explained if some of these epitopes in the tissues remain active after fixation only when tau-related proteins are modified into a pathological state (not yet fully specified), as seen in the AD brain.26 In this study the appearance of Alz-50 IR in the fixed brain tissue was accompanied by a positive staining by the Gallyas method, which labels tau, preferentially pathological (PHF) tau.27 These data suggest that tau molecules are pathologically modified during the ischemic process rather than a mere cross-reaction of Alz-50 to molecules other than tau.28

Alz-50 preferentially immunolabels pretangle neurons or intracellular NFTs but not extracellular NFTs which, on the contrary, are labeled by other anti-PHF tau antibodies, Bodian stain, and thioflavine S.1 2 3 19 25 29 SDS-soluble fraction from AD brain containing fewer aggregated fibrils contains more prominent Alz-50 IR than the SDS-insoluble fraction containing more aggregated fibrils such as PHF.30 The Alz-50 immunoreactive epitope on purified bovine tau became selectively active after treatment with transglutaminase without developing other tau epitopes.31 These data are compatible with the hypothesis that Alz-50 immunoreactive epitope becomes more active than other tau epitopes in the early phase of NFT development.13

Although pretangle neurons and some intracellular NFTs stained by Alz-50 in the AD brain are not always immunolabeled by other anti-tau antibodies,1 2 19 29 the absence of IR to anti-tau (tau 2, pool 2) and anti-ubiquitin antibodies in Alz-50 immunoreactive neurons during the ischemic process suggests that the posttranslational modification of tau in these neurons is different from that in AD (for example, phosphorylation, truncation, or ubiquitination).32 33 34 35 In addition, the punctate IR in these ischemic neurons looks like the Alz-50 immunoreactive structures, which are reported to be nonfilamentous, in normal brains14 and is totally different from the diffuse cytoplasmic staining seen in pretangle neurons1 3 19 in the AD brain.

Alz-50 immunoreactive neurons in the ischemic process do not develop NFTs. On the other hand, pretangle neurons in the AD brain may develop A-NFTs, probably after hyperphosphorylation of tau protein.36 It is essential to know what factor or environment is responsible for tau-related proteins being woven into NFTs. Further in vivo or autopsy studies combined with biochemical analyses will be needed to clarify the state of modification of tau-related proteins in various conditions and its relation to the fibrillary structure at the electron microscopic level. Such a study will provide a clue to understanding the key step in the formation of NFTs after tau-related proteins are expressed.


*    Selected Abbreviations and Acronyms
 
AD = Alzheimer's disease
A-NFTs = Alzheimer's neurofibrillary tangles
IR = immunoreactivity
PHF = paired helical filaments
SDS = sodium dodecyl sulfate

Received April 3, 1995; revision received August 1, 1995; accepted August 3, 1995.


*    References
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 up arrowAbstract
 up arrowIntroduction
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 up arrowResults
 up arrowDiscussion
 *References
 

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