Ž .Brain Research 791 1998 63–66

Research report

Cerebrovascular muscle atrophy is a feature of Alzheimer’s disease

George Perry a,), Mark A. Smith a, Catherine E. McCann a, S. L. Siedlak a, Paul K. Jones b,Robert P. Friedland c

a Institute of Pathology, 2085 Adelbert Road, Case Western ReserÕe UniÕersity, CleÕeland, OH 44106, USAb Department of Epidemiology and Biostatistics, Case Western ReserÕe UniÕersity, CleÕeland, OH 44106, USA

c Department of Neurology, Case Western ReserÕe UniÕersity, CleÕeland, OH 44106, USA

Accepted 23 December 1997

Abstract

We examined vascular amyloid-b deposition and other abnormalities in the posterior cerebral artery of consecutive cases ofŽ . Ž .Alzheimer’s disease AD compared to controls. Smooth muscle atrophy was a consistent feature in the cases of AD examined p-0.01

and was surprisingly independent of adjacent amyloid-b deposition. These findings suggest that vascular abnormalities are a consistentfeature in AD and may be an important contributor to the pathogenesis and complications of AD. q 1998 Elsevier Science B.V.

Keywords: Amyloid-b ; b protein precursor; Hypoperfusion; Oxidative stress; Vasculature atrophy

1. Introduction

Degeneration of the smooth muscle layer and replace-ment by amyloid-b deposits is found in cases of

Ž .Alzheimer’s disease AD with a clinical history of cere-w xbral bleeding 1,8,17,23 . This could be an important fea-

ture of even a larger proportion of AD than those withdocumented cerebral bleeding since over ninety percent ofcases of AD show amyloid-b deposition in at least one

w xbrain vessel 3,6,7,12 . Given the reported cytotoxicity ofw xamyloid-b 16 , one hypothesis is that amyloid-b deposi-

tion initiates muscle cell death and leads to vascularw xdegeneration 8,24 . Nonetheless, the relationship between

muscle cell degeneration, amyloid-b deposition and AD isincompletely understood.

The present study was designed to determine for a largeŽ .blood vessel entering the brain i the extent of amyloid-b

deposition and b-protein precursor expression in relationŽ .to distance from entry of a vessel into the brain; ii

whether vascular abnormalities were a common feature ofŽ .large blood vessels in AD and iii whether such abnormal-

ities were dependent on amyloid-b deposition.

) Corresponding author. Fax: q1-216-368-8964; E-mail:gxp7@po.cwru.edu

To accomplish these goals, we selected the posteriorcerebral artery, a vessel with an anatomy conducive tocollection of its full length at autopsy and that enters aregion of the brain, occipital cortex, with extensive AD-re-lated pathology.

2. Materials and methods

2.1. Tissue and fixation

Ž .The artery 4–9 cm long from the occipital cortex andextending toward the circle of Willis was collected fromconsecutive AD and aged and young non-demented cases.

ŽCases were classified as either control i.e., non-dementedwith no clinical or pathological diagnosis of neurological

. Ždisease or Alzheimer’s disease based on clinical andpathological criteria established by CERAD and an NIA

w x. Ž .consensus panel 9 Table 1 . Tissue samples were fixedŽ .in methacarn methanol:chloroform:acetic acid; 60:30:10

at 48C overnight, dehydrated in ascending ethanol andembedded in paraffin. During embedment, the vessel wasplaced so that longitudinal sections would include the fulllength of the vessel and attached occipital cortex. A longi-tudinal section was taken to directly evaluate the distanceof amyloid-b deposition and b-protein precursor expres-sion from the cerebral cortex.

0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved.Ž .PII S0006-8993 98 00006-7

( )G. Perry et al.rBrain Research 791 1998 63–6664

Table 1Summary of the findings on the posterior cerebral artery for cases of ADand controls

a 2Case Diagnosis Age Musclertotal SPrmm

1 Control 93 0.530"0.011 02 Control 32 0.540"0.011 03 Control 74 0.575"0.011 04 Control 43 0.595"0.017 05 Control 86 0.629"0.022 06 AD 91 0.355"0.013 1067 AD 90 0.387"0.016 1908 AD 57 0.411"0.014 2529 AD 79 0.425"0.019 3410 AD 85 0.427"0.018 20211 AD 85 0.490"0.015 10512 AD 89 0.493"0.017 29013 AD 73 0.510"0.011 8314 AD 83 0.522"0.018 1115 AD 83 0.522"0.016 8516 ADrCAA 79 0.421"0.012 13517 ADrCAA 90 0.441"0.025 14018 ADrCAA 81 0.456"0.014 15519 ADrCAA 69 0.512"0.021 5620 ADrCAA 79 0.527"0.015 46

There is a consistent reduction in the ratio the circumferential muscleŽ .layer to the total vessel thickness musclertotal in cases of AD whether

or not there was amyloid-b deposition within that vessel, congophilicŽ .angiopathy CAA .

a Mean"standard error.

2.2. Immunocytochemistry

Paraffin-embedded blocks were cut at 6 mm and im-munocytochemistry performed using the peroxidase–anti-

Ž . Xperoxidase PAP method with 3 3-diaminobenzidineŽ .DAB as chromogen. Mouse monoclonal antibody to

Ž . w xamyloid-b 4G8 10 or rabbit antiserum to amyloid-b1-42w x2 were used to stain for amyloid-b following a 5 minincubation of the tissue section with 70% formic acid toenhance labeling. b-protein precursor was localized with arabbit antiserum to recombinant b PP produced in bac-

w xulovirus 2 . Smooth muscle cells were marked with anti-w xbodies to tropomyosin or actin 5,17 .

2.3. Data collection

The relative thickness of the circumferential smoothŽ .muscle layer tropomyosin-positive was determined usingŽ .an ocular scale Bausch and Lomb micrometer disc 31-16

in a Zeiss Axioskop 20 microscope, and this was comparedas a ratio to the total thickness of the vessel wall. Measure-ments were taken along the entire length of the posteriorcerebral artery at 1 mm intervals. The measurements oftotal vessel thickness, circumferential muscle thicknessand, by subtracting, the remaining portion, were made as aratio for each interval; the ratio as well as the other twomeasurements were averaged for each case. All measure-ments were taken where the vessel wall included the full

thickness as evidenced by an intact endothelial and adven-tial layer.

Senile plaque density was determined for an area adja-cent to the arterial entry. In sections immunostained for

Ž .amyloid-b 4G8 , senile plaques were counted in 5 fields2 Ž 2 .of 0.2 mm each 1.0 mm total in layers III–IV, at the

point where the artery enters the occipital cortex.

2.4. Statistics

Parametric and non parametric analysis was used toassess the significance of differences between cases of AD

Ž .with and without vascular amyloid CAA and controlsw x15 .

3. Results

Immunostaining of the posterior cerebral artery of ADand control cases for amyloid-b revealed that 0r5 of thecontrol and 5r15 AD cases contained amyloid-b in thatartery, while in the remaining 10 AD cases, 7 showedamyloid-b in at least one microvessel within the brainparenchyma. In the 5 cases with amyloid-b in the poste-rior cerebral artery, there was focal deposition along thetotal length of the vessel, i.e., there was no relationship ofamyloid-b deposition and distance from the cortex overthe region examined. Smooth muscle cells, identified bytropomyosin and actin, were the site of b-protein precursorwhich was present along the full length of the vessel inboth AD and control cases. Muscle cells in cases of ADwere often disorganized, and the basal lamina and tunicaadventia contributed a greater proportion to the thicknessŽ . Ž .Fig. 1A when compared to the control cases Fig. 1B .

The proportion of vessel wall thickness occupied by thecircumferential smooth muscle-layer showed a significant

Žreduction in all cases of AD ps0.002 by parametricŽ .analysis of variance ANOVA and ps0.004 by non-

.parametric analysis by the Mann–Whitney–Wilcoxon testŽ .compared to the controls Table 1 . The high significance

of the difference is not surprising since neither aged oryoung controls overlapped the values seen in AD. Further-more, the same vascular changes were not noted in twocases of diffuse Lewy body disease and one case of Pick’sdisease. No trend was noted in ratio along the length of the

Ž .vessel data not shown . The difference was still signifi-cant for the 10 cases of AD without amyloid-b in the

Žposterior cerebral artery compared to controls ps0.002.by the Mann–Whitney–Wilcoxon test as well as for the 5

Žcases which had amyloid-b deposits in that artery ps.0.024 by the Mann–Whitney–Wilcoxon test . Interest-

ingly, there was no difference in the ratio when comparingvessels from AD cases with or without amyloid-b deposi-

Žtion in that artery ps0.510 by the Mann–Whitney–.Wilcoxon test . Further, the reduced muscle ratio for AD

( )G. Perry et al.rBrain Research 791 1998 63–66 65

ŽFig. 1. The proportion of the vessel wall thickness occupied by the circumferential smooth muscle layer recognized by antisera to tropomyosin between. Ž . Ž . Ž .arrows is reduced in cases of Alzheimer’s disease A compared to controls B . While the tunica adventia adv appears to be increased in the AD case,

instead it is an apparent increase since only the relative contribution of the adventia increases due to atrophy of the muscle layer. Scale bar s100 mm.

cases is a result of muscle abnormalities rather than anincrease in the adventia or intima because direct measure-ments of the circumferential muscle layer showed a signifi-

Ž .cant decrease ps0.02 by the Student’s t-test while theŽadventia and intima did not significantly increase ps

.0.264 by the Student’s t-test . While direct measurements,rather then ratios, can suffer distortion from the plane ofsection, particularly in longitudinal sections, there is noreason to think cases of AD would differ from controls inthis regard. When we examined the relationship of amy-loid-b deposition as defined by the number of senileplaques in the cases of AD only to the vessel muscle ratioby regression analysis, we found, at best, a weak but

Ž .insignificant correlation rs0.399 p)0.05 .

4. Discussion

In this study, we found that the proportion of thecircumferential smooth muscle layer that contributes to thevessel wall thickness of the posterior cerebral artery is

Ž .significantly reduced in Alzheimer’s disease AD com-pared to controls. However, there is no significant differ-ence between AD cases with or without amyloid-b in thatartery. These results are surprising since they suggestvascular changes may be a common feature of AD that isnot dependent on amyloid-b. Although there are reportsthat the cells adjacent to focal amyloid-b deposits showdegenerative changes during the early stages of amyloid-b

w xdeposition 1,8,17,23,24 , our findings instead suggest thatmuscle cell degeneration related to amyloid-b deposits isnot significant enough to cause change in the vascularmuscle layer in most cases of AD. Instead, we find thatmuscle atrophy is a common finding that occurs indepen-

dently of, rather than only as a response to, adjacentamyloid-b deposition.

If vascular abnormalities, such as those shown here,occur early in the course of AD, changes in vascularperfusion may play an important role in neurodegenera-tion. It may mean that the consistent finding of hypoperfu-

w xsion in AD 4,11 is not only the result of lower energydemand from a damaged brain but a reflection of inabilityof the cerebral vasculature to respond to metabolic needs.Of interest in this regard is the recent finding of Nagata et

w xal. 14 , that although both cerebral blood flow andmetabolism are decreased in AD, the oxygen extractionfraction is increased, suggesting that the reduction in cere-bral blood flow is more than could be accounted for by thelowered state of metabolic demand. Significantly, imbal-ances in energy metabolism can lead to oxidative stress, a

w xpervasive feature of AD 13,18–21,25 . Furthermore, mus-cle degeneration in arteries might explain the higher thanexpected incidence of cerebrovascular infarcts in demented

w xpatients 22 . Clearly more studies that investigate thevasculature of other brain and systemic sites are essentialto clarify the primary or secondary role played by vascularatrophy in AD.

Acknowledgements

We thank Dr. Kim for providing the monoclonal anti-body, 4G8, and Dr. B. Greenberg for the antiserum tob-protein precursor. Supported by grants from the National

Ž .Institutes of Health AG09287 , the American Health As-sistance Foundation and the American Federation of AgingResearch.

( )G. Perry et al.rBrain Research 791 1998 63–6666

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