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(Stroke. 1997;28:1948-1950.)
© 1997 American Heart Association, Inc.

Articles

Activated Microglial Cells Are Colocalized With Perivascular Deposits of Amyloid-ß Protein in Alzheimer's Disease Brain

Toshiki Uchihara, MD, PhD; Haruhiko Akiyama, MD, PhD; Hiromi Kondo; Kenji Ikeda, MD, PhD

From the Department of Neurology, Tokyo (Japan) Medical and Dental University (T.U.), and Department of Neuropathology, Tokyo Institute of Psychiatry (T.U., H.A., H.K., K.I.).

Correspondence to T. Uchihara, MD, PhD, Department of Neurology, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113 Japan. E-mail t-uchihara.nuro{at}med.tmd.ac.jp

	Abstract

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Background and Purpose Microglial cells are present in the center of senile plaques (SPs) in Alzheimer's disease (AD) brain. Such a localization of microglial cells suggests that they are involved in the deposition or the clearance of amyloid-ß protein (Aß) in the brain. We examined their association with another type of parenchymal Aß deposit, which is termed the perivascular deposits of Aß (PAß).

Methods Thick sections from AD brain were stained with a three-color immunofluorescence method that labeled Aß, activated microglial cells, and vascular endothelial cells simultaneously.

Results Three-dimensional observation under a laser scanning microscope confirmed that perivascular aggregates of activated microglial cells were colocalized with PAß.

Conclusions Microglia occur in association with both SPs and PAß, suggesting that they play important roles in the metabolism of Aß in AD brain.

Key Words: Alzheimer's disease • microscopy, confocal • microglia

	Introduction

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Deposition of amyloid-ß protein (Aß) is one of the most salient pathological features in the AD brain. In addition to Aß deposits within the vessel walls, which is referred to as AA, two types of parenchymal Aß deposits are observed in AD brains: SPs and PAß. SPs are seen as spherical structures in tissue sections. Although SPs are sometimes penetrated in part by the blood vessels, PAß are distinguished on a morphological basis as the cylindrical deposits of Aß that evenly surround the blood vessel. It has been reported that microglial cells are consistently associated with SPs,^{1 2 3 4 5 6 7 8 9 10} while other components such as astrocytes, abnormal neurites, and synapses are not always present in SPs.^{2 4 10} On the other hand, evidence suggested that Aß in AA is derived from the blood vessels.^{11 12 13} The aim of this study is to determine which components are spatially related to PAß. We used a confocal laser scanning microscope to observe thick sections stained simultaneously with three different antibodies, which labeled Aß, microglial cells, and vascular endothelial cells.¹⁴ We report here that on the basis of three-dimensional observation, perivascular aggregates of microglial cells are colocalized with PAß as well as with SPs.

	Methods

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Six brains from patients with clinicopathologically verified AD (age, 67 to 88 years; five men and one woman) were used in this study. Ischemic lesions caused by atherosclerotic vascular changes were absent or, at most, minimal in these cases. Brain blocks from the temporal lobe were fixed in 4% paraformaldehyde for 2 days. After cryoprotection with 15% sucrose, 60-µm-thick floating sections were cut on a freezing microtome. The three-color immunolabeling was performed as described previously.¹⁴ Briefly, the affinity purified rabbit polyclonal IgG (E-50) against a synthetic peptide corresponding to residues 17 to 31 of Aß was conjugated with biotin (Sp-1200, Vector). Before they were stained, sections were treated in formic acid (>99%) for 1 minute. After a preincubation with the blocking serum (5%, goat) in phosphate-buffered saline containing 0.3% Triton X-100, sections were incubated with the anti–von Willebrand factor (1:500, polyclonal IgG made in rabbit, DAKO) to label vascular endothelial cells at 4°C for 2 days. Sections were then incubated with the goat anti-rabbit IgG conjugated with FITC (1:200, Cappel) at 4°C for 6 hours, followed by incubation in normal rabbit serum (50%) for 1 hour. Sections were then incubated with a mixture of anti-HLA-DR (1:300, CR3/43 mouse monoclonal IgG, DAKO) to label microglia and biotinylated anti-Aß (1:200 described above) at 4°C for 2 days. The sections were then incubated with a mixture of anti-mouse IgG F(ab')² conjugated with R-phycoerythrin (1:100, Tago Immunochemicals) and Cy-Chorme (tandem conjugate of R-PE+Cy5 coupled with streptavidin, 1:100, PharMingen) at 4°C for 6 hours.

The mounted sections were observed under a confocal laser scanning microscope (LSM 310, Carl Zeiss). Under excitation with a single 488-nm beam, emission from FITC labeling von Willebrand factor–like immunoreactivity was detected through a long-path filter (<520 nm) and displayed as red. Emission from R-PE labeling HLA-DR–like immunoreactivity was detected through band-path filter (576±10 nm, custom-made at Vacuum Optics Corporation of Japan) and was displayed as green. Emission from Cy-Chrome labeling Aß-like immunoreactivity was detected through a short-path filter (>690 nm) and was displayed as blue. Every 1-µm-thick plane of these triple-labeled sections was scanned serially so that the entire depth of each PAß was visualized. This enabled us to observe the spatial relationship of Aß deposits with microglia and blood vessels on a three-dimensional basis. AA, which was identified as Aß staining of the vessel wall itself, was carefully excluded. After AA was excluded, the parenchymal subsets of Aß deposits were then classified into two categories: (1) SPs, which represent spherical deposits regardless of their type, such as classic SP, neuritic SP, and diffuse Aß deposits; and (2) PAß, which represent the cylindrical deposits surrounding the blood vessels. On a three-dimensional basis, one can easily distinguish these two structures.

	Results

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Fig 1 demonstrates simultaneous labeling of three epitopes on the same visual field of a thin optical plane. An aggregate of activated microglia is associated with PAß. Another aggregate is also seen in the center of a senile plaque (arrow). We observed more than 30 PAß in this study. All PAß were present in the gray matter. In the white matter, no vessels were surrounded either by PAß or by aggregates of microglial cells. In all 1-µm-thick planes optically scanned, PAß were always accompanied by aggregates of microglial cells. Fig 2 illustrates, from top to the bottom, nine consecutive optical planes of the same section as Fig 1 scanned at every 3 µm. These vessel-associated microglial aggregates were restricted within the perivascular parenchyma where PAß were present.

View larger version (189K): [in this window] [in a new window]   Figure 1. Three-color immunostaining (top left) of von Willebrand factor for blood vessel (red, top right), Aß (blue, bottom left), and HLA-DR for microglia (green, bottom right). Microglia are seen in the center of the Aß deposits (arrow), which have no relation to blood vessels. Original magnification x150 (bar=100 µm). In the vessel wall, pericytes or vascular endothelial cells expressing HLA-DR are also stained green. Red color for von Willebrand factor epitope, if superimposed on green HLA-DR staining, gives yellow fluorescence in the vessel wall.

View larger version (169K): [in this window] [in a new window]   Figure 2. The same area as Fig 1, optically sectioned every 3 µm (from top left to bottom right) under a confocal microscope. Note the colocalization of activated microglia with the perivascular Aß deposits on three-dimensional basis. Original magnification x100 (bar=100 µm).

	Discussion

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Aß in PAß has been considered to be derived from the blood vessels because of its close spatial relationship.^{11 12 13} In this study we demonstrated that microglial aggregates, in addition to the vessels, are a component that is always associated with PAß. Microglial aggregates were not associated with vessels that lacked PAß. Association of microglial cells with SPs has been reported repeatedly by many investigators.^{1 2 3 4 5 6 7 8 9 10} Previous studies using the combination of multilabeled thick sections and confocal microscopy confirmed that aggregates of microglial cells are the only cellular component that is consistently found in SPs in both gray and white matter of AD brain.^{10 14} Together with the results of this study, we speculate that microglial cells play an essential role in the metabolism of Aß in both PAß and SPs.

At present, it remains unknown whether these Aß-associated microglial cells are involved in the deposition or the clearance of Aß. In AD brains, microglial cells sometimes contain Aß-immunoreactive granules.^{15 16} Some investigators claim that microglial cells generate amyloid fibrils,¹⁷ while others consider that they only scavenge debris^{4 5} after being stimulated at least in part by the chemotactic effect of Aß.¹⁸ Activation of microglial cells in the Aß deposits may also have a deleterious effect by secreting a variety of biologically active molecules such as cytokines,^{19 20} complement proteins (for review, see Reference 21²¹ ), and apolipoprotein E.^{22 23} This appears to be relevant to the recently reported effect of anti-inflammatory agents that retard the progression of dementia of AD patients.^{24 25} These drugs are supposed to suppress microglial activity directly or indirectly. Microglial cells may be key cells in the Aß deposition of AD brain.

	Selected Abbreviations and Acronyms

 

Aß = amyloid-ß protein AA = amyloid angiopathy AD = Alzheimer's disease PAß = perivscular deposits of amyloid-ß protein SPs = senile plaques

Received April 7, 1997; revision received June 24, 1997; accepted July 9, 1997.

	References

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