Yonago Acta medica 2003;46:9–16

An Immunohistochemical Study of Perivascular Plaque inAlzheimer’s Disease and Cerebral Amyloid Angiopathy
Chun Hui Yang, Makoto Otsuka and Eisaku Ohama
Department of Neuropathology, Institute of Neurological Sciences, Tottori University Faculty ofMedicine, Yonago 683-8504 Japan
Immunohistochemical study of perivascular plaques (PPs) in 7 patients with Alzheimer’s
disease (AD) and 4 with cerebral amyloid angiopathy (CAA)(CAA with dementia in 3 patients and CAA with massive cerebral hemorrhage in 1 patient) demonstrated that PPs
were also a common form of amyloid β (Aβ) deposits, like SPs and CAAs, in both AD andCAA, and that they had similar immunostaining characteristics to mature senile plaques(SPs), manifesting as always positive for both Aβ42 and Aβ40 but predominant for Aβ42.
In addition, PPs were always associated with AT8-positive degenerated neurites and GFAPpositive astrocytes and fibers. These findings suggest that PPs as mature plaques contributeto the development of dementia, especially in CAA with dementia which lack AD pathology.
Moreover, semiquantitative analysis revealed no correlation between the number of PPs
and that of SPs, but a good correlation between the number of PPs and that of CAAs,
suggesting that there was a close relationship of the formation of PPs and the developmentof CAAs. PPs were also found around non-CAA arteries, although they were frequentaround varying degrees of CAAs, suggesting that the initial Aβ42 deposits in PPs contributeto the development of CAAs.
Key words: Alzheimer’s disease; cerebral amyloid angiopathy; immunohistochemistry; perivascularplaque; senile plaque

It has been established that amyloid β (Aβ) deposits
in the brain, as senile plaques (SPs) and cerebral
amyloid angiopathies (CAAs), are histopathological hallmarks of Alzheimer's disease (AD)(Mandybur, 1975; Griffiths et al., 1982; Miyakawa
et al., 1982; Vinters et al., 1996; Jellinger, 2002),
and that CAA alone may give rise to dementia in theelderly without AD pathology (Cohen et al., 1997;
Yamada et al., 1997; Vidal et al., 2000; Kalaria, 2002).
In the pathological investigations of AD and CAA,
a great number of studies have focused on SPs andCAAs (Alonzo et al., 1998; Iwatsubo et al., 1994,1995), and elucidated that Aβ42 deposition is predominant in SPs and Aβ40 in CAAs. However, another form of Aβ deposition, perivascular plaques(PPs) have not yet been studied thoroughly. PPs
were first described by Uematsu (1923) as perivascular form of SPs and then by Scholz (1938) as drusige
Entartung (in German). Morel and Wildi (1952) used
the term dyshoric angiopathy which meant a breakdown of the blood-brain barrier. Previous researchers thought that amyloid in PPs extended from the vas-Abbreviations: Aβ, amyloid β protein; ABC, avidin-biotin-peroxidase complex; AD, Alzheimer’s disease; ApoE,apolipoprotein E; CAA, cerebral amyloid angiopathy; CAA-CH, CAA with massive cerebral hemorrhage; CAA-D, CAA
with dementia; GFAP, glial fibrillary acidic protein; NFT, neurofibrillary tangle; PBS, phosphate-buffered saline; PP,perivascular plaque; SP, senile plaque9C. H. Yang et al.

cular wall of CAAs to the surrounding parenchyma
in the cerebral cortex (Neumann, 1960; Mandybur,1986; Plant et al., 1990). Some recent studies haveraised the hypothesis that Aβ is eliminated along
perivascular interstitial fluid drainage pathways ofthe brain and progressively accumulates to form
PPs, and further contributes to CAAs (Weller et al.,1998, 2000; Kalaria, 2002; Kumar-Singh, 2002;
Yow and Weller, 2002). However, immunohistochemical studies focusing on PPs have not everbeen reported. For the purpose of clarifying theneuropathological significance of PPs, we examined the brains of patients with AD and CAA by immunohistochemistry, and investigated the relationship of PPs to SPs and CAAs.

al, DAKO A/S, Copenhagen, Denmark; dilution1:50); anti-glial fibrillary acidic protein (GFAP)(polyclonal, DAKO A/S; dilution 1:1000); and antihuman phosphorylated tau (AT8) (monoclonal,Innogenetics, NV, Ghent, Belgium; dilution 1:1000).
Immunostainings were performed by the avidinbiotin-peroxidase complex (ABC) method. Sections mounted on poly-L-lysine-coated glass slides
were deparaffinized, rehydrated in a graded ethanol
series, washed in distilled water for 10 min, andtreated with 3% hydrogen peroxide diluted in distilled water for 30 min to block endogenous peroxidase activity in the tissue. After washing in 0.01 M
phosphate-buffered saline (PBS) pH 7.4 for 10 min,the sections were resubjected to blocking with 10%
normal serum (Nichirei, Tokyo, Japan) for 30 min
to avoid nonspecific binding of secondary antibodies. The sections were then incubated overnightwith the primary antibodies in a moist chamber at4˚C. After washing 3 times with PBS (5 min each)the sections were incubated with appropriate secondary antibodies for 60 min at room temperature,
treated with the ABC reagent for 60 min, exposed to
0.5% 3,3'-diaminobenzidine-0.005% hydrogen peroxide. For enhancement of Aβ and ApoE4 immunostaining, deparaffinized tissue sections were pretreatedwith 96% fomic acid for 5 min. The sections wereheated up to 110˚C for 10 min to improve ApoE4 immunostaining. Finally, sections were counterstainedwith hematoxylin.
Neurofibrillary tangles (NFTs) were counted
in 5 nonselected × 100 fields in the Ammon’s hornand subiculum in Bielschowsky stain, and wererated as follows: +, 1–10; ++, 11–50 and +++, ≥ 51.
Quantitative analysis of SPs was performed basedon the number of mature and diffuse plaques in thetemporal cortices in Aβ42 and Aβ40 immunostainings in 10 nonselected × 100 fields. The abundance of SPs was rated as follows: +, 1–10; ++, 11–50 and +++, ≥ 51. The rating of CAA severity was
made based on the number of pan Aβ-positive vessels in the occipital cortices with 10 nonselected ×40 fields: +, 1–5 positive vessels; ++, 6–10 positive
vessels and +++, ≥ 11 positive vessels and at least 1
vessel showing complete replacement of the media

Subjects and Methods
Brain tissues were obtained from 7 patients withAD, 3 patients with CAA with dementia (CAA-D)
and 1 patient with CAA with massive cerebral hemorrhage (CAA-CH). All patients were brought toautopsy and neuropathological examination in theDepartment of Neuropathology, Institute of Neurological Sciences, Tottori University Faculty of Medicine, Japan. All tissue specimens were fixed in 10%
formalin for 2 weeks, embedded in paraffin and cut
into 6-µm-thick sections. Routine neuropathological examinations were carried out with hematoxylin and eosin, Klüver-Barrera, Bielshowsky,
Gallyas-Braak and Holzer stains. Summary of clinical and routine neuropathological features areshown in Table 1.
For immunohistochemistry in the present
study, samples were selected from the occipital andtemporal lobes including the Ammon’s horn and serial sections were made. The sections were immunostained with the following primary antibodies: antipan Aβ (Aβ-1) (polyclonal, Wako, Osaka, Japan;
dilution 1:1000); anti-Aβ42 (BC05) and -Aβ40(BA27) (monoclonal, Wako; Aβ ImmunohistostainKit); anti-human apolipoprotein E4 (ApoE4) (5B5)(monoclonal, IBL, Fujioka, Japan; dilution 1:10);
anti-human smooth muscle actin (1A4) (monoclon-10Immunohistochemistry of perivascular plaqueTable 1. Summary of clinical and pathological features of 11 cases examined
Age at death
Duration of Brain weight(year)
Gender illness (year)(g)

Patient

NFT

Rating*SPCAA

Alzheimer’s disease

12
3
4
5
6
757
67
75
56
89
83
79

F
F
FFF
M
F

5
7
2
4
6
4
8—
—
1180
—
1060
1400
860

++
++
++
++
++
++
++

+++
+++
+++
+++
+++
+++
+++++++++++++CAA with dementia (D)

12376
6862

M
MF5167120513601360+++++++++
+++
+++

F

0.25860++++

CAA with massive cerebral hemorrhage (CH)
175

CAA, cerebral amyloid angiopathy; F, female; M, male; NFT, neurofibrillary tangle; SP, senile plaque; —, notweighed.
*NFT: +, 1–10; ++, 11–50; +++, ≥51 in 5 × 100 fields in the Ammon’s horn and subiculum.
SP: +, 1–10; ++, 11–50; +++, ≥51 in 10 × 100 fields in the temporal cortices.
CAA: +, 1– 5; ++, 6–10; +++, ≥11 in 10 × 40 fields in the occipital cortex.

with Aβ. The rating of PPs severity was determined
as follows: +, 1–3; ++, 4–6 and +++, ≥ 7 in 10 nonselected × 40 fields in the occipital cortices.

and fibers within or around them as well (Fig. 1D).
These features are again similar to mature plaques.
PPs were ApoE4-positive in AD patients 1 and 2and CAA-D patient 1, and in these 3 patients SPsand CAAs were also positive for ApoE4 (Fig. 1E).
Thus, PPs resembled mature plaques in that they
were always positive for both Aβ42 and Aβ40, andthat they were always associated with degenerated
neurites and GFAP-positive cells and fibers. Theirincidence was not proportional to that of mature anddiffuse plaques but proportional to CAAs (Table 2).
Although they were more frequent around varying
degrees of CAAs (Figs. 1A, B and F), they werealso found around non-CAA vessels (Fig. 1G), suggesting that PPs were formed earlier than CAAs.

Results

Immunohistochemistry of PPs
PPs were found in all 11 patients. In AD, they weresmall in number compared with SPs, but were numerous in CAA-D and CAA-CH in which matureplaques were absent and only a small number ofdiffuse plaques were found in 3 of the 4 patients(Table 2).
PPs were always immunostained for bothAβ42 and Aβ40, but their number and positivestaining areas were always greater in Aβ42 staining(Table 2, Figs. 1A and B), suggesting earlier deposition of Aβ42 than Aβ40 similar to mature plaques.
All PPs were always associated with varying degrees of neuritic degeneration evidenced by AT8
immunostaining (Fig. 1C) as well as Gallyas-Braakand Bielshowsky stains and with GFAP-positive cells

Immunohistochemistry of CAAs
Different numbers of CAAs were found in all 7 AD
patients: moderate in 3 and mild in 4 patients. They
were marked in 3 CAA-D patients and moderate in
1 CAA-CH patient (Table 1). There was a good correlation in the severity of CAAs between the leptomengeal and parenchymal blood vessels. CAA-11

C. H. Yang et al.

Table 2. Immunohistochemical features of senile plaque (SP), cerebral amyloid angiopathy (CAA)
and perivascular plaque (PP)
Rating*SPPatient

Mature plaqueAβ42+
Aβ40+AT8Diffuse plaque
Aβ42+
Aβ40–
AT8CAAAβ42 Aβ40PP
Aβ42 Aβ40 AT8Alzheimer’s disease1234
567+++
+++
+++
++
++
+++

+++++++++
+++
+++
+++
+++
+++
+++

–––––––+++
++
+++++

+++
++
+++++

+++
++
+++++

+++++++++++++++CAA with dementia (D)

1
2
3

–
–––––

++
++–––

+++
+++
+++

+++
+++
+++

+++
+++
+++

+++
+++
+++

+++

CAA with massive cerebral hemorrhage (CH)
1––+–++

++

++

++

+

Aβ40, amyloid β40 protein; Aβ42, amyloid β42 protein; AT8, anti-human phosphorylate α tau.
* SP: +, 1–10; ++, 11–50; +++, ≥51 in 10 × 100 fields in the temporal cortices.
CAA: +, 1– 5; ++, 6–10; +++, ≥11 in 10 × 40 fields in the occipital cortices.
PP: +, 1– 3; ++, 4– 6; +++, ≥7 in 10 × 40 fields in the occipital cortices.
AT8: +, positive; –, negative.

associated vasculopathies such as double barrelingand clusters of multiple arteriolar lumina were seenin the 3 CAA-D patients.
CAAs were always labeled with both Aβ42and Aβ40 (Table 2). Their staining intensity andpositive-staining areas were, however, greater with
Aβ40 in larger cortical and leptomeningeal arteries(Figs. 1A and B; Figs. 2A and B), but were greaterwith Aβ42 in smaller cortical arteries (Figs. 2A andB; arrows). In the same vessel wall, the 2 Aβ species were sometimes detected in different areas. In
larger leptomeningeal arteries, early small depositsof Aβ42 were always observed at the media adjacent to the adventitia (Fig. 2A) or sometimes at theFigs. 1A–G (p. 13). Immunohistochemistry of perivascular plaques (PPs).
A: Immunostaining for amyloid β42 (Aβ42), showing positive PPs. The occipital cortex in Alzheimer’s disease (AD)(Patient 2).
B: An adjacent section to A immunostained with Aβ40. Positive areas are smaller in the PPs but larger in the arterycompared with A.
C: An adjacent section to A immunostained with AT8, showing degenerated neurites within or around the PPs.
D: GFAP-immunostaining of another serial section, showing numerous positive cells and fibers within and aroundthe PPs.
E: A serial section of A immunostained with ApoE4. The staining pattern of the PPs is similar to B but the artery isintensely positive.
F: Immunostaining for Aβ42, showing positive PPs and artery. The occipital cortex of AD (Patient 7).
G: Immunostaining for pan Aβ (Aβ42 and Aβ40), showing PP around non-CAA artery.
A–G: original magnification, × 100. AT8, anti-human phosphorylated tau; ApoE4, apolipoprotein E4; CAA,
cerebral amyloid angiopathy; GFAP, glial fibrillary acidic protein.

12

Immunohistochemistry of perivascular plaque13ABCDEFGFigs. 1A–G