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The accumulation of the ƒ-amyloid protein (Aƒ)
in brain is central to the pathogenesis of Alzheimer's disease.
A very promising therapeutic strategy is to stimulate removal of
the protein by immunization with Aƒ itself. However, the recent
termination of Alzheimer's disease immunotherapy Phase II testing
due to patients developing encephalitis has made imperative further
study of the efficacy and safety of Aƒ immunization in animal
models. The squirrel monkey (Saimiri sciureus) is an ideal model
for such studies as these New World primates naturally develop Alzheimer-like
cerebral amyloid lesions with age, especially in the walls of blood
vessels. As a basis for assessing potential side-effects of immunization,
this study sought to determine if amyloid-bearing vessels in the
brains of non-immunized monkeys are associated with perivascular
inflammation or microhemorrhage. We show that squirrel monkeys develop
cerebral amyloid in the form of both parenchymal and vascular deposits.
These unevenly distributed lesions primarily are found in cortical
gray matter and are immunoreactive for both Aƒ40 and Aƒ42.
Larger amyloidotic vessels, where a preference for Aƒ40 is
found, are less common than affected capillaries. Significantly,
hemorrhagic events in aged, non-immunized animals are very rare,
and we saw no evidence of encephalitis. These data show that aged
squirrel monkeys have significant brain amyloidosis but a low incidence
of Aƒ-linked vascular lesions, and therefore are an optimal
model for testing Alzheimer immunotherapies.
Alzheimer's disease (AD) is a progressive, incurable
disorder of mental function. Existing treatments only modestly improve
cognition, in a subset of patients, and the effects are transient.
AD is characterized by the accumulation of specific proteins in
the brain, particularly two proteins called amyloid and tau. Aƒ
occurs in two forms, 40 and 42 amino acids long. Aggregated forms
the cores of senile plaques (protein complexes with abnormal or
reactive brain cells) and deposits in the walls of blood vessels,
known as cerebral amyloid angiopathy (CAA). In 1999, a landmark
study showed that senile plaques can be cleared from the brains
of transgenic mice following immunization with the Aƒ peptide2
(AN 1792; Elan Pharmaceuticals). Elan began human clinical trials
of immunization, but in Phase II studies in AD patients it was discovered
that a subset of treated patients developed brain inflammation that
resulted in the immediate termination of the trials3. It was later
reported that the brains of two immunized patients showed large
areas appearing to have been cleared of senile plaques4,5. Significantly,
these patients also showed evidence of CAA-linked inflammation,
and one of them had multiple microhemorrhages in brain areas with
CAA5, suggesting that CAA might contribute to microhemorrhage and
inflammation in vaccinated patients6,7. Further testing is necessary
to determine specific interactions between Aƒ immunization
and CAA, and thereby develop a safer method for treating AD6. Squirrel
monkeys (Saimiri sciureus) naturally develop deposits in the form
of senile plaques and CAA at around 13 years of age 8,9. Humans
and squirrel monkeys have the same amino acid sequence and develop
similar neuropathologies6. Hence, squirrel monkeys are well-suited
for testing therapies for AD that are designed to remove abnormal
Aƒ deposits from the brain. However, to date nothing is known
about the relationship of CAA to inflammation in the normal aged
squirrel monkey.
Coronally sectioned tissue along the rostro-caudal
plane from two aged (20 and 21 years), one young (8 year) male squirrel
monkeys for systematic, comprehensive sampling. Immunohistochemistry.
Brains were fixed, blocked coronally into seven slabs, sectioned
at 25-40 ?M thickness using a cryostat, and mounted. Sections were
stained systematically with 1) hematoxylin and eosin; 2) immunostain
for Aƒ using a general antibody and one each specific to Aƒ40
and Aƒ42; 3) Perls stain for iron. A series of sections was
doubly stained for Aƒ and iron. Histological analysis. Amyloid
deposits and inflammatory change were mapped in selected sections
representing all levels of the forebrain. Cortical area was determined
by stereological point counting methods. The numbers of senile plaques
and amyloidotic or cuffed vessels were quantitated. The relative
abundance of Aƒ40 and Aƒ42 immunoreactivity was determined
and the relationship of CAA to microhemorrhage. Data analysis. The
young squirrel monkey showed no evidence of amyloid deposition in
any part of the brain. The number of Aƒ-immunoreactive blood
vessels was determined in defined anatomical regions of identical
areas in each animal to determine overall CAA load. Microhemorrhage
was assessed. Data were analyzed by analysis of variance (ANOVA).
Total numbers and cortical densities of amyloid deposits were calculated.
1. Aged squirrel monkeys develop cerebral amyloid
angiopathy and senile plaques. These lesions mostly occur in cortical
gray matter.
2. CAA and senile plaques are not evenly distributed in the neocortex:
The density of deposition decreases caudally.
3. Hemorrhagic vessels occur, but are rare.
4. CAA is more common than senile plaques and is mostly in capillaries.
5. There is no significant difference in immunoreactivity for Aƒ40
and Aƒ42 in the microvasculature and plaques. However, in larger
vessels Aƒ40 reactivity predominates. In this experiment, we
determined the anatomical distribution of Aƒ deposits in the
forebrain, and tested the hypothesis that vascular amyloid in affected
regions is associated with augmented inflammation and microhemorrhage
in normal aged squirrel monkeys. We found that the Aƒ protein
does not accumulate evenly in brain; rostral regions develop a greater
burden than caudal regions. Similarly, Aƒ deposition is more
prevalent ventrally than dorsally (data not shown). Amyloid deposition
in the white matter is rare, as most deposits are found in cortical
gray matter. In humans, most CAA is in larger brain vessels, and
the 40 amino acid strain of amyloid predominates. In squirrel monkeys,
most CAA is in small capillaries, where the 40- and 42-amino acid
forms are equally represented. However, as in humans, Aƒ40
predominates in the relatively few larger amyloid-bearing vessels.
While hemorrhagic vessels do occur in aged squirrel monkeys, their
incidence is very low. Only one ruptured vessel was present in 43
sampled brain sections of one aged animal, and no hemorrhages were
found in other subjects. Furthermore, there was no evidence of cerebral
inflammation in any animal.
Monkeys develop significant brain amyloidosis that varies in density
across brain regions; 2) Most vascular amyloid in squirrel monkeys
is in capillaries, and is a mixture of Aƒ40 and Aƒ42;
3) In the absence of Aƒ immunization, cerebral microhemorrhage
and inflammation in aged squirrel monkeys are unusual, even when
heavy CAA is present. Therefore, hemorrhagic or encephalitic side-effects
due to Aƒ immunization will be detectable with high sensitivity
in this model. This preliminary study of normal aged squirrel monkeys
lays the foundation for subsequent testing of the safety and efficacy
of active ƒ-amyloid immunization in an animal model of Alzheimer-like
cerebral amyloid angiopathy. We have recently begun immunization
treatment of 3 aged monkeys with Aƒ, and our preliminary data
show a circulating T-cell response and increased antibody titers
in 2 animals. Our next step will be to determine if Aƒ clearance
takes place, and whether this is accompanied by inflammation or
microhemorrhage.
This work was supported by a SIRE grant from Emory University to
Hanie Elfenbein and Woodruff Foundation grant (256879) to Lary Walker,
PhD. Thanks also to Rebecca Rosen, Dr. Todd Preuss and Dr. John
Redmond of the Yerkes National Primate Research Center and Center
for Behavioral Neuroscience. Thanks also to Dr. Chris Ibegbu, Harriet
Robinson and Jack Orkin of Yerkes, and Harry LeVine of the University
of Kentucky, for help with the immunization study.
People with Alzheimer's disease develop deposits of a sticky protein
called B-Amyloid in their brains and on the blood vessels which
course through their brains. Squirrel monkeys, a species of New
World primates, also develop these clumps of protein. This research
posed questions of the location, density, and distribution of the
build-up in squirrel monkeys to determine its possible relation
to brain inflammation and stroke.
Immunostaining, histology, sterological point counting, microscopy.
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