(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
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 |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
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 |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
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 |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
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 |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
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.
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
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 debris4 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 |
|---|
|
Received April 7, 1997; revision received June 24, 1997; accepted July 9, 1997.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Probst A, Brunnschweiler H, Lautenschlager C, Ulrich J. A special type of senile plaque, possibly an initial stage. Acta Neuropathol (Berl). 1987;74:133-141.[Medline] [Order article via Infotrieve]
2. Dickson DW, Farlo J, Davies P, Crystal H, Fuld P, Yen SC. Alzheimer's disease: a double-labeling immunohistochemical study of senile plaques. Am J Pathol. 1988;132:86-101.[Abstract]
3. Haga S, Akai K, Ishii T. Demonstration of microglial cells in and around senile (neuritic) plaques in Alzheimer brain: an immunohistochemical study using a novel monoclonal antibody. Acta Neuropatho (Berl). 1989;77:569-575.[Medline] [Order article via Infotrieve]
4. Itagaki S, McGeer PL, Akiyama H, Zhu S, Selkoe D. Relationship of microglia and astrocytes to amyloid deposits of Alzheimer disease. J Neuroimmunol. 1989;24:173-182.[Medline] [Order article via Infotrieve]
5. Cras P, Kawai M, Siedlak S, Mulvihill P, Gambetti P, Lowery D, Gonzalez-DeWhitt P, Greenberg B, Perry G. Neuronal and microglial involvement in beta-amyloid protein deposition in Alzheimer's disease. Am J Pathol. 1990;137:241-246.[Abstract]
6. Perlmutter LS, Barron E, Chui HC. Morphologic association between microglia and senile plaque amyloid in Alzheimer's disease. Neurosci Lett. 1990;119:32-36.[Medline] [Order article via Infotrieve]
7. Styren SD, Civin WH, Rogers J. Molecular, cellular, and pathologic characterization of HLA-DR immunoreactivity in normal elderly and Alzheimer's disease brain. Exp Neurol. 1990;110:93-104.[Medline] [Order article via Infotrieve]
8. Wegiel J, Wisniewski HM. The complex of microglial cells and amyloid star in three- dimensional reconstruction. Acta Neuropathol (Berl). 1990;81:116-124.[Medline] [Order article via Infotrieve]
9. Miyazono M, Iwaki T, Kitamoto T, Kaneko Y, Doh-ura K, Tateishi J. A comparative immunohistochemical study of Kuru and senile plaques with a special reference to glial reactions at various stages of amyloid plaque formation. Am J Pathol. 1991;139:589-598.[Abstract]
10. Uchihara T, Kondo H, Akiyama H, Ikeda K. White matter amyloid in Alzheimer's disease brain. Acta Neuropathol (Berl). 1995;90:51-56.[Medline] [Order article via Infotrieve]
11. Miyakawa T, Shimoji A, Kumamoto R, Higuchi Y. The relationship between senile plaques and cerebral blood vessels in Alzheimer's disease and senile dementia: morphological mechanism of senile plaque production. Virchows Arch [Cell Pathol]. 1982;40:121-129.
12. Mountjoy CQ. Amyloid and senile plaques and cerebral blood vessels: a semi-quantitative investigation of a possible relationship. J Neurol Sci. 1982;57:89-103.[Medline] [Order article via Infotrieve]
13. Kawai M, Kalaria RN, Harik SI, Perry G. The relationship of amyloid plaques to cerebral capillaries in Alzheimer's disease. Am J Pathol. 1990;137:1435-1446.[Abstract]
14. Uchihara T, Kondo H, Akiyama H, Ikeda K. Single laser, three color immunolabeling of a histological section with laser scanning microscopy: application to senile plaque related structures. J Histochem Cytochem. 1995;43:103-106.[Abstract]
15. Akiyama H, Schwab C, Kondo H, Mori H, Kametani H, Ikeda K, McGeer PL. Granules in glial cells of patients with Alzheimer's disease are immunopositive for C-terminal sequences of beta-amyloid protein. Neurosci Lett. 1996;206:169-172.[Medline] [Order article via Infotrieve]
16. Paresce DM, Ghosh RN, Maxfield FR. Microglial cells internalize aggregates of the Alzheimer's disease amyloid beta-protein via a scavenger receptor. Neuron. 1996;17:553-565.[Medline] [Order article via Infotrieve]
17. Frackowiak J, Wisniewski HM, Wegiel J, Merz GS, Iqbal K, Wang C. Ultrastructure of the microglia that phagocytose amyloid and the microglia that produce beta-amyloid fibrils. Acta Neuropathol (Berl).. 1992;84:225-233.[Medline] [Order article via Infotrieve]
18. Davis JB, McMurray HF, Schubert D. The amyloid beta-protein of Alzheimer's disease is chemotactic for mononuclear phagocytes. Biochem Biophys Res Commun. 1992;189:1096-1100.[Medline] [Order article via Infotrieve]
19. Akiyama H, Ikeda K, Katoh M, McGeer EG, McGeer PL. Expression of MRP14, 27E10, interferon-alpha and leukocyte common antigen by reactive microglia in postmortem human brain tissue. J Neuroimmunol. 1994;50:195-201.[Medline] [Order article via Infotrieve]
20. Meda L, Cassatella MA, Szendrei GI, Otvos LJ, Baron P, Villalba M, Ferrari D, Rossi F. Activation of microglial cells by beta-amyloid protein and interferon-gamma. Nature. 1995;374:647-650.[Medline] [Order article via Infotrieve]
21. McGeer PL, McGeer EG. The inflammatory response system of brain: implications for therapy of Alzheimer and other neurodegenerative diseases. Brain Res Rev. 1995;21:195-218.[Medline] [Order article via Infotrieve]
22. Uchihara T, Duyckaerts C, He Y, Kobayashi K, Seilhean D, Amouyel P, Hauw J-J. ApoE immunoreactivity and microglial cells in Alzheimer's disease brain. Neurosci Lett. 1995;195:5-8.[Medline] [Order article via Infotrieve]
23. Nakai M, Kawamata T, Taniguchi T, Maeda K, Tanaka C. Expression of apolipoprotein E mRNA in rat microglia. Neurosci Lett. 1996;211:41-44.[Medline] [Order article via Infotrieve]
24.
McGeer PL, Rogers J. Anti-inflammatory agents as
a therapeutic approach to Alzheimer's disease.
Neurology. 1992;42:447-449.
25.
Rogers J, Kirby LC, Hempelman SR. Clinical trial
of indomethacin in Alzheimer's
disease. Neurology. 1993;43:1609-1611.
This article has been cited by other articles:
 |
|
 |
|
  B. Liu, S. Rasool, Z. Yang, C. G. Glabe, S. S. Schreiber, J. Ge, and Z. Tan Amyloid-Peptide Vaccinations Reduce {beta}-Amyloid Plaques but Exacerbate Vascular Deposition and Inflammation in the Retina of Alzheimer's Transgenic Mice Am. J. Pathol., November 1, 2009; 175(5): 2099 - 2110. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. K. Giri, V. Rajagopal, S. Shahi, B. V. Zlokovic, and V. K. Kalra Mechanism of amyloid peptide induced CCR5 expression in monocytes and its inhibition by siRNA for Egr-1 Am J Physiol Cell Physiol, August 1, 2005; 289(2): C264 - C276. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. J. Scolding, F. Joseph, P. A. Kirby, I. Mazanti, F. Gray, J. Mikol, D. Ellison, D. A. Hilton, T. L. Williams, J. M. MacKenzie, et al. A{beta}-related angiitis: primary angiitis of the central nervous system associated with cerebral amyloid angiopathy Brain, March 1, 2005; 128(3): 500 - 515. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. K. Giri, S. K. Selvaraj, and V. K. Kalra Amyloid Peptide-Induced Cytokine and Chemokine Expression in THP-1 Monocytes Is Blocked by Small Inhibitory RNA Duplexes for Early Growth Response-1 Messenger RNA J. Immunol., May 15, 2003; 170(10): 5281 - 5294. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Giri, S. Selvaraj, C. A. Miller, F. Hofman, S. D. Yan, D. Stern, B. V. Zlokovic, and V. K. Kalra Effect of endothelial cell polarity on beta -amyloid-induced migration of monocytes across normal and AD endothelium Am J Physiol Cell Physiol, September 1, 2002; 283(3): C895 - C904. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Giri, Y. Shen, M. Stins, S. Du Yan, A. M. Schmidt, D. Stern, K.-S. Kim, B. Zlokovic, and V. K. Kalra beta -Amyloid-induced migration of monocytes across human brain endothelial cells involves RAGE and PECAM-1 Am J Physiol Cell Physiol, December 1, 2000; 279(6): C1772 - C1781. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Nourhashemi, S. Gillette-Guyonnet, S. Andrieu, A. Ghisolfi, P. J. Ousset, H. Grandjean, A. Grand, J. Pous, B. Vellas, and J.-L. Albarede Alzheimer disease: protective factors1 Am. J. Clinical Nutrition, February 1, 2000; 71(2): 643s - 649s. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Tan, T. Town, D. Paris, T. Mori, Z. Suo, F. Crawford, M. P. Mattson, R. A. Flavell, and M. Mullan Microglial Activation Resulting from CD40-CD40L Interaction After -Amyloid Stimulation Science, December 17, 1999; 286(5448): 2352 - 2355. [Abstract] [Full Text]
|
|
|
|
 |
|
 J. Robertson, J.-M. Beaulieu, M. M. Doroudchi, H. D. Durham, J.-P. Julien, and W. E. Mushynski Apoptotic death of neurons exhibiting peripherin aggregates is mediated by the proinflammatory cytokine tumor necrosis factor-{alpha} J. Cell Biol., October 15, 2001; 155(2): 217 - 226. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||