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The Journal of Neuroscience, February 1, 2001, 21(3):858-864
Growth Arrest of Individual Senile Plaques in a Model of
Alzheimer's Disease Observed by In Vivo Multiphoton
Microscopy
R. H.
Christie1,
B. J.
Bacskai1,
W. R.
Zipfel2,
R. M.
Williams2,
S. T.
Kajdasz1,
W. W.
Webb2, and
B. T.
Hyman1
1 Alzheimer's Disease Research Unit,
Massachusetts General Hospital, Charlestown, Massachusetts 02129, and
2 School of Applied and Engineering Physics, Cornell
University, Ithaca, New York 14853
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ABSTRACT |
In Alzheimer's disease, amyloid- peptide aggregates in the
extracellular space to form senile plaques. The process of plaque deposition and growth has been modeled on the basis of in
vitro experiments in ways that lead to divergent predictions:
either a diffusion-limited growth model in which plaques grow by
first-order kinetics, or a dynamic model of continual deposition and
asymmetrical clearance in which plaques reach a stable size and stop
growing but evolve morphologically over time. The models have not been tested in vivo because plaques are too small (by several
orders of magnitude) for conventional imaging modalities. We now report in vivo multiphoton laser scanning imaging of
thioflavine S-stained senile plaques in the Tg2576 transgenic mouse
model of Alzheimer's disease to test these biophysical models and show
that there is no detectable change in plaque size over extended periods
of time. Qualitatively, geometric features remain unchanged over time
in the vast majority of the 349 plaques imaged and re-imaged. Intervals as long as 5 months were obtained. Nonetheless, rare examples of growth
or shrinkage of individual plaques do occur, and new plaques appear
between imaging sessions. These results indicate that thioflavine
S-positive plaques appear and then are stable, supporting a dynamic
feedback model of plaque growth.
Key words:
amyloid; transgenic; Alzheimer; two-photon; in
vivo imaging; senile plaque; microglia
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INTRODUCTION |
Aggregates of the amyloid-
peptide (A) form senile plaques, one of the classic
neuropathological lesions of Alzheimer's disease. In vitro
observations show that A is extremely insoluble and precipitates to
form aggregates (Hilbich et al., 1991 ), and that exogenous A
decorates existing plaques and enlarges them according to first-order
kinetics (Jarrett and Lansbury, 1993; Esler et al., 1996). These
observations led to a model in which a nidus is formed and plaques then
grow in a time- and concentration-dependent manner (Jarrett and
Lansbury, 1993; Esler et al., 1996).
Paradoxically, however, the size distribution of senile plaques appears
to remain constant rather than increasing with increasing duration of
illness (Hyman et al., 1993). Together with observations on the fine
structure of plaques, these data led to a statistical physics-based
dynamic feedback model in which a feedback process was postulated to
limit plaque growth, leading to stable plaque size and, in principle,
the shrinkage of existing plaques (Hyman et al., 1995; Cruz et al.,
1997; Urbanc et al., 1999a,b). Distinguishing between these
possibilities is important for understanding the life history and
pathophysiology of amyloid deposition in Alzheimer's disease, but it
has not been possible because direct measurements of plaques in
vivo have not been achieved.
Tg2576 transgenic mice overexpress a mutant form of the human amyloid
precursor protein and develop senile plaques in an age-related fashion, with amyloid- deposits occurring first at ~8-10 months of age (Hsiao et al., 1996; Irizarry et al., 1997). The number of
plaques increases dramatically with age. Here we develop the successful
imaging of senile plaques in living animals using in vivo
multiphoton laser scanning microscopy through an intact skull window,
which allows us to image and re-image the same brain region many times
over a period of days to months. In this study, we also introduce the
use of multiphoton imaging for chronic, in vivo brain
imaging. Multiphoton excitation is based on the simultaneous absorption
of multiple low-energy photons; the sum of the energies of these
photons is sufficient to excite fluorescence in an appropriate fluorophore. Relatively benign long-wavelength light generates fluorescence that would otherwise require potentially damaging levels
of ultraviolet radiation. Another important benefit is that the
infrared wavelengths penetrate tissue without absorption by blood and
with less scattering than visible light, allowing imaging deeper into
the brain. Multiphoton excitation is achieved by focusing a
sub-picosecond pulsed laser into the sample through a microscope
objective. The higher-order power dependence of multiphoton excitation
results in a restricted excitation volume enabling spatial resolution
of ~1 µm, essentially equivalent to that of confocal microscopy
even in turbid tissue (Denk et al., 1990). The resulting elimination of
out-of-plane free radical generation and photobleaching are additional
advantages for in vivo applications.
For the first application of this new technology, we examined the
natural history of individual senile plaques in the brains of living
transgenic mice. We conclude that individual plaques achieve and
maintain a stable size in accord with the predictions of the dynamic
feedback model.
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MATERIALS AND METHODS |
In vivo imaging of amyloid deposition. Nine male
Tg2576 mice (mean age 18.6 months) (Hsiao et al., 1996) were used for
the in vivo imaging of plaques. These mice express the human
amyloid precursor protein carrying the Swedish mutation under the
hamster prion protein promoter. The skull was prepared 2-6 d before
imaging. Mice were anesthetized with avertin (tribromoethanol; 250 mg/kg, i.p.). A high-speed drill (Fine Science Tools, Foster City, CA) was used to thin each skull in a circular region ~1-1.2 mm in diameter (see Fig. 2a), and a dissecting microscope (Leica,
Wetzlar, Germany) was used for gross visualization of the site.
Heat and vibration artifacts were minimized during drilling by frequent application of artificial CSF (ACSF) containing (in
mM): 125 NaCl, 26 NaHCO3,
1.25 NaH2PO4, 2.5 KCl, 1 MgCl2, 1 CaCl2, and 25 glucose. Skull thickness was repeatedly assessed with a surgical probe (Roboz, Rockville MD), and drilling stopped when the bone displayed flexibility in a central region ~0.6 mm in diameter. Clear
visualization of pial vasculature was an additional indication of skull
thinness. The scalp was then sutured, and the animal allowed to
recover. On the day of imaging, the animal was re-anesthetized, the
scalp was reflected, and the small amount of connective tissue that had
grown in the interim was removed by scraping. The tip of a 22 gauge
needle was used to make a small break in the lateral wall of the skull
preparation to facilitate thioflavine S diffusion into the brain.
Thioflavine S (0.005% in ACSF) (Sigma, St. Louis, MO) was then applied
for 20 min to the site. A small ring of molten bone wax was applied to
the skull surrounding the site, and this well was filled with ACSF to
create an aqueous reservoir for the long working distance, water
immersion dipping objectives (Olympus, Tokyo, Japan). The thin-skull
preparation also eliminates the need for application of a coverslip
(Svoboda et al., 1997) to the imaging site because preservation of this
thin layer of bone is sufficient to stabilize the cardiac and
respiratory motion of the brain inherent in in vivo imaging.
The animal was immobilized in custom-built stage-mounted ear bars and a
nosepiece, similar in design to a stereotaxic apparatus. The thin-skull
site was then placed directly under the objective lens of the
microscope (Olympus BX-50) for imaging (see Fig. 1b).
Two-photon fluorescence was generated with 750 nm excitation from a
mode-locked Ti:Sapphire laser [Tsunami (Spectra-Physics, Mountain
View, CA) , 5.45 W Millenium V pump laser (Spectra-Physics), power at
back aperture of objective 10 mW, pulse 60-100 fs] mounted on
a commercially available multiphoton imaging system (Bio-Rad 1024ES;
Bio-Rad, Hercules, CA). Custom-built external detectors containing
three photomultiplier tubes (Hamamatsu Photonics, Bridgewater, NJ)
collected emitted light in the range of 380-480, 500-540, and
560-650 nm; all thioflavine S figures are from the 380-480 nm
channel. Imaging was performed using the normal scan speed of the
scanhead, dwell time = 1.5 µsec per pixel. Up to four thin-skull preparations were made per animal to maximize the number of plaques available for measurement. Thioflavine S (0.005% in ACSF) was applied
to the preparation at each imaging session. The site was first imaged
with a 10× objective (1230 mm square field; NA = 0.5) to map the
surface of the thin-skull preparation. This low-power map, in
conjunction with orienting markings on the microscope stage and
stereotaxic frame, allowed for precise repositioning of the site under
the microscope objective during subsequent imaging sessions. The
x-y stage encoders (Boeckeler, Tucson, AZ) were calibrated with their origin at the center of the thin-skull site and
were used to preserve the relative coordinates of higher-magnification images within the site. Nine z-series using a 60× objective
(205 × 205 µm; NA = 0.8) were then collected in a 3 × 3 array covering the thinnest portion of the site by moving the
stage exactly 205 µm in the x or y direction.
The incremental z-step was 2 µm, and the series was
collected from the skull surface to a depth of ~150 µm into the
brain. The starting position of the z-axis motor relative to
skull position was recorded for later z-axis alignment during montage generation. After completion of image collection, the animal was removed from the stage, the ring of bone wax was removed, the skull was washed with sterile saline, and the scalp was
sutured. The animal was warmed to 37°C during recovery from anesthesia. Total time of anesthesia was limited to 2 hr.
Image analysis. Montages were reconstructed into a single
stack of images using Scion Image (Scion, Frederick, MD). The area of
individual plaque cross sections was measured in each optical section
by thresholding at 2 SD above the mean of an adjacent background
region. Plaques that did not satisfy the criteria of imaging were
eliminated from the measurement set. Plaques on the edge of the imaging
area or on one of the montage lines were rejected because of the
potential imprecision of moving the animal on the stage. Plaques for
which the intensity was not sufficiently above the background for
appropriate thresholding were also eliminated. This rejected many
plaques, typically deep in the preparation, that appeared to be present
but were too faint to measure using the automatic threshold technique.
Finally, plaques with images that contained any appreciable motion
artifact were rejected. Of the 448 plaques imaged, 349 met these
criteria. Maximal plaque diameter was then calculated from the cross
section of the largest area for each plaque. Volume rendering was
performed using VoxBlast (VayTek, Fairfield, IA) on a Windows NT-based
workstation (Precision 610; Dell Computer, Round Rock, TX).
Angiography. The tail of the animal was warmed on a heating
pad to dilate the blood vessels, and ~0.05 ml of fluorescein (25 mg/ml) in sterile PBS was injected into a tail vein of the mouse at
least 20 min before imaging. The dye did not cross the blood-brain barrier and permitted concurrent visualization of blood vessels throughout the imaging volume in the brain.
Histology. Two groups of animals (n = 3 per
group; mean ages 12.6 and 22.6 months) were used for the histological
measurement of amyloid deposition, measuring amyloid burden and size
distribution as previously described (Hyman et al., 1993). Images of
thioflavine S-stained sections were collected using two-photon
excitation with 750 nm light. All fields of the cortex containing
thioflavine S-positive amyloid deposits were imaged in a given section
until ~80 plaques were imaged per animal. Images were then
transferred to Scion Image (Scion), where a threshold was applied, the
image was filtered slightly to remove noise, and the plaques were
automatically outlined by the particle analysis protocol of the
software. Images were manually edited to remove thioflavine S-positive
blood vessels and edge-effect artifacts. Sections containing few
thioflavine S-positive plaques were exhaustively sampled, and all
plaques within this cortical area were counted. Random systematic
sampling of ~10 fields per section was applied to those sections
containing heavier amyloid burdens, and a 400 × 400 µm counting
frame was used with automatic selection and measurement to count
thioflavine S-positive plaques. Plaques were counted in three sections
per animal in this way; the adequacy of the sampling strategy was reflected in coefficients of error  10%. Results were expressed as
the density of thioflavine S-positive plaques per square millimeter. Statistical significance of the observed difference in plaque number
between the groups was assessed by t test.
For immunostaining of intact brain to detect A, monoclonal anti-A
antibody 10D5 (Elan Pharmaceuticals, South San Francisco, CA) was
directly conjugated to Cy3 using a commercially available kit (CyDye;
Amersham Pharmacia, Piscataway, NJ) and applied in conjunction with
thioflavine S (0.005% in ACSF) to the surface of a fixed,
unsectioned brain for 20 min.
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RESULTS |
We adapted multiphoton microscopy for these studies because it has
unique advantages for in vivo imaging and its resolution is
on the order of 1 µm (Denk et al., 1990). Because only acute in
vivo imaging has been reported to date in any system, we developed a new approach for long-term repeat imaging. A thin, transparent bone
window ~1 mm in diameter and ~20 µm thick is formed with a
high-speed burr in the skull of an anesthetized Tg2576 mouse (Fig.
1a). A small break is made in
the lateral wall of the site to allow for delivery of fluorophore to
the brain, but the bone remains otherwise intact within the thinned
region. An upright Olympus BX-50 fixed-stage microscope containing a
modified stage insert was used for in vivo imaging (Fig.
1b).
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Figure 1.
Preparation of skull for in vivo
imaging. a, Gross appearance of skull through dissecting
microscope before imaging. The pial vasculature is visible through the
intact but thinned region of skull. Anterior midline sutures are also
visible in the image. Scale marks are spaced 1 mm apart.
b, Schematic diagram of the microscope objective during
imaging. The thinned area of skull is bathed in a pool of ACSF
(light gray) that is retained by a ring of bone wax
(dark gray). A small break is made in the lateral wall
of the thinned area to allow for thioflavine S entry. c,
In vivo visualization of thioflavine S-positive amyloid
in a 15-month-old Tg2576 mouse. A single optical section near the
surface of the skull is shown. Thioflavine S-positive amyloid
angiopathy is visible ringing the pial arteriole in this image. The
fainter autofluorescence of the skull bone is visible in the
bottom right corner; the
fibrous autofluorescence of the dura is visible as a
band at bottom right. d,
Another optical section from the same z-series as
c, but 50 µm deeper into the brain, showing a
thioflavine S-positive amyloid deposit in layer 1 of the mouse cortex.
e, Perpendicular volume rendering of the entire stack of
images, with the skull visible at the top, the
amyloid-encrusted pial vessel just beneath, and the thioflavine
S-positive plaque deep in the living brain. The autofluorescent dura
can also be seen as a faint layer between the vessel and the skull. The
approximate levels of optical sections shown in c and
d are represented by dotted lines. Scale
bars, 25 µm.
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Thioflavine S is a standard amyloid-binding fluorophore that excites in
the ultraviolet range and has an emission maximum of ~450 nm. It has
been extensively used to label amyloid deposits in human Alzheimer's
disease tissue (Kelenyi, 1967) as well as in transgenic mouse models of
amyloid deposition. It is among the stains recommended by the
Consortium to Establish a Registry for Alzheimer's Disease for
the neuropathological diagnosis of Alzheimer's disease in postmortem
tissue (Mirra et al., 1991). A dilute solution of thioflavine S was
applied to the brain of a living 18-month-old Tg2576 transgenic mouse
for in vivo visualization of amyloid deposits using
multiphoton microscopy. Optical sections were obtained every 2.0 µm,
from the bone window surface to ~150 µm beneath the surface,
using 750 nm light for two-photon excitation of the fluorophore.
Reconstruction of these thin optical sections revealed thioflavine
S-positive amyloid surrounding pial arterioles with the classic
segmental appearance of amyloid angiopathy (Vonsattel et al., 1991) in
superficial sections (Fig. 1c). Deeper optical sections
(Fig. 1d) revealed parenchymal thioflavine S-positive amyloid plaques. Plaques were visualized in this way up to 150 µm
beneath the surface of the cortex.
The imaged plaques share the morphology of classic thioflavine
S-positive senile plaques seen in tissue from transgenic animals and
from Alzheimer's disease cases, and no such structures were seen in
nontransgenic control littermates. That these structures are indeed
senile plaques was further confirmed by incubation of the postmortem
fixed brain from the transgenic mouse with a fluorescently labeled
antibody to A (10D5; Elan Pharmaceuticals) (Hyman et al., 1992)
directly labeled with Cy3 (Amersham). This double stain revealed
colocalization of thioflavine S with surrounding amyloid-
immunoreactivity (Fig.
2a,b). As expected,
plaques that have a dense core are stained by thioflavine S and are a
subset of all A immunoreactive structures (Schmidt et al.,
1995). Moreover, histological analysis 2-7 d after such imaging
reveals no overt damage, neuronal loss, or increase in reactive
astrocytes (Fig. 2c) as assessed by glial fibrillary acidic
protein staining, suggesting that the thin-skull preparation and
imaging protocol are well tolerated by the living brain.
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Figure 2.
To confirm that the thioflavine S-positive
structures were indeed senile plaques, thioflavine S and an
anti-amyloid- monoclonal antibody, cy3-labeled 10D5 (Elan
Pharmaceuticals), were applied to the surface of a fixed but intact
Tg2576 brain. a, Fluorescence emission in the range
380-480 nm shows thioflavine S staining the amyloid core of a plaque
~40 µm deep into the brain. Scale bar, 10 µm.
b, Emission in the 560-650 nm range shows the Cy3-10D5
staining of the same A surrounding the thioflavine S-positive core.
c, Glial fibrillary acidic protein immunoreactivity in a
section through the area imaged by multiphoton microscopy 2 d
previously. Sparse immunoreactive astrocytes, not substantially
different from adjacent (nonimaged) cortex, suggest minimal tissue
response to imaging. Scale bar, 100 µm.
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The potential of multiphoton microscopy for nondestructive in
vivo imaging opens the possibility of repeated visualization of
plaques over time within a living animal. Figure
3a is an example of the
imaging approach in a live mouse. The skull was prepared, thioflavine S
was applied, and a 3 × 3 matrix of a 615 × 615 µm region
of the site was imaged using a 60× water immersion objective. On
recovery from anesthesia, the animal was returned to its cage where it
showed no sign of impairment or discomfort after imaging. Representative images from one animal collected at an interval of
2 d are shown in Figure 3b-e. Examples of a
plaque (Fig. 3b) and amyloid angiopathy (Fig. 3d)
are shown at the initial imaging session. Two days later, the animal
was re-anesthetized, and thioflavine S was reapplied to the thinned
region of the skull. Imaging was performed under the same conditions as
the initial session. Plaques (Fig. 3c) and amyloid
angiopathy (Fig. 3e) were both clearly revisualized after
2 d and appear to have been unaltered since the initial imaging
session.
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Figure 3.
In vivo imaging of thioflavine
S-positive amyloid deposition in a Tg2576 mouse. a, A
3 × 3 montage of 60× fields acquired on initial imaging day.
Optical sections were obtained every 2 µm for a distance of 200 µm
from the inner surface of the skull; images were aligned in the
x, y, and z axes, then
projected onto a single image revealing amyloid angiopathy and senile
plaques. b, In vivo imaging of a
thioflavine S-positive plaque ~40 µm beneath the skull
surface. This image is a single optical section through the body of the
plaque. Scale bar, 10 µm. c, The same plaque as in
b, re-imaged 2 d later under identical imaging
conditions. d, Single optical section showing
thioflavine S-positive amyloid angiopathy associated with a pial
arteriole. e, The same arteriole as d,
imaged after 2 d.
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To study the natural history of thioflavine S-positive amyloid
deposits, plaques in eight additional animals (mean age 18.6 months)
were imaged over progressively longer time periods. Variation in the
thin-skull preparation prevented revisualization of the entire imaging
volume from one imaging session to the next; the number of plaques
within the imaging site could therefore not be counted in a
statistically unbiased manner. A total of 41 imaging sessions yielded
29 data sets containing plaques that were successfully imaged more than
once. The data sets contained 349 aligned pairs of plaques over
extended time intervals; one animal was re-imaged 150 d
after initial imaging. As many as five separate imaging sessions of the
same volume were obtained in each animal. Qualitatively, the structure
and size of the vast majority of plaques remain remarkably stable over
these extended periods of observation. Fine details of the morphology
of individual plaques are recognizable in subsequent images obtained
months later, including finger-like appendages and small clusters of
dense thioflavine S-positive amyloid (Fig.
4).
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Figure 4.
Sequential imaging of a plaque over time. Images
of a single, identified plaque were obtained at the initial imaging
session and again 45 and 110 d later in a live mouse. Fine details
of the plaque are clearly recognizable over these ranges of time. Scale
bar, 20 µm.
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Quantitative analysis of plaque diameter over time, measured in the
optical section with the greatest diameter, confirms this qualitative
judgment. Analysis of changes in plaque diameter for the entire set of
measurements is presented in Figure 5.
The amount of variability in the two measures, taken as a population,
is essentially the same, regardless of whether the measures were obtained 2 or 150 d apart (Fig. 5a). The initial
measurement of the size of an individual plaque is an excellent
predictor of a subsequent measurement of that same plaque, whether over
an interval of days or months. The slope of the linear regression graph
plotting the size of a plaque at initial imaging to its size at a later
time, taking all measurements for periods over an interval of 2-150 d,
was nearly unity (slope = 0.98;
R2 = 0.89). These data are
consistent with plaques being extraordinarily stable in vivo
objects over an extended period of time, and they suggest that
individual plaques do not continue to grow.
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Figure 5.
Analysis of variability of plaque
measurements. Top, Percentage change
(average ± SD) for all plaque measurements binned into 0.5 month
groups shows no trend in either the average measure or the variability
of measurement over the time interval examined. N values
for each measurement are noted above the SD bars.
Bottom, Linear regression plot of initial measurement
and subsequent measurement for all time intervals, showing tight
correlation for all plaque sizes. The slope of the line approaches
unity (0.98) with a correlation coefficient
R2 = 0.89.
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Close examination of the data sets, however, reveals a small number of
plaques that appear to have either grown or shrunk substantially
between imaging sessions. We were concerned that this apparent change
could be caused by technical factors, and so we systematically applied
working criteria to eliminate known potential sources of measurement
error. For example, we rejected data from plaques that fell on the
border of one of the 205 × 205 µm fields comprising the montage
and from the deepest plaque imaged in a given session. In
several animals, fluorescein angiography was performed at the same time
as thioflavine S imaging to create additional internal landmarks to
facilitate lining up the plaques from one imaging session to another.
After carefully evaluating >300 pairs of plaques, we were able to find
only 14 clear examples of marked growth or resolution (i.e., a change
in size by 40%). Figure 6 shows examples
of plaques from a volume-rendered stack of images of the same region of
cortex, obtained 104 d apart, showing the same four plaques (in
red) as well as the fluorescein angiogram
(green). Qualitative and quantitative analyses show that two of the plaques have grown substantially (~50%), one has become substantially smaller (by >40%), and one has not changed size
at all. These data show that, within the same region and during the
same imaging sessions, some plaques appear to grow and others shrink.
No tendency for large plaques to shrink or small plaques to grow (i.e.,
a correlation between initial diameter and percentage change) was
evident in the data. Technical issues such as thioflavine S
concentration or power at the focal plane cannot account for some
getting larger and others getting smaller within a region as small as a
single three-dimensional field; the most parsimonious explanation is
that, in these instances, plaque size is changing.
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Figure 6.
A subpopulation of plaques change size over time.
The images are two-channel volume-rendered stacks of thioflavine S
plaques (red) and fluorescein angiograms
(green) taken from the same animal at the initial
imaging session (left images) and 104 d later
(right images). Four clearly imaged plaques can be seen
in these volumes, labeled A-D. The
autofluorescence of the dura appears at the upper edge of the volume
stacks and appears slightly different in the images here and in Figure
6 because the image stacks are not exactly coincident at their initial
depth. The graph below represents the percentage change
in diameter for each plaque. The plaques labeled A and
B increase in size by ~50%, plaque C
remains the same size, and plaque D decreases by 40%.
Scale bar, 20 µm.
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With increasing age, the number of thioflavine S-positive plaques in
the cortex is known to increase (Hsiao et al., 1996; Irizarry et al.,
1997). Thus, when re-imaging a volume of the cortex, one would expect
to occasionally find new plaques within the imaging volume. Again, we
used rigorous criteria to ensure that the appearance of a new plaque
did not simply reflect slightly better signal-to-noise characteristics
in a second imaging session than in the first, or a greater depth of
imaging, or a slightly different imaging volume. Compelling examples,
in which a new plaque appeared in a volume that had been previously
imaged, occurred nonetheless. Figure 7
shows a dramatic example of a field with three well defined,
characteristic plaques at the first imaging session and four at the
second imaging session 64 d later.
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Figure 7.
Appearance of a novel plaque in the imaged region.
a, Volume rendering of a set of three plaques during an
initial imaging session. b, Volume rendering of the same
region imaged 64 d later, showing the initial plaques joined by a
novel thioflavine S-positive plaque. The fibrous autofluorescence at
bottom left is dura mater. Scale bar, 50 µm.
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The in vivo longitudinal data noted above suggest that the
average plaque diameter does not vary with age. To examine this conclusion using traditional histological analyses in Tg2576 mice, we
examined thioflavine S-stained sections from mice either 12 months
(n = 3) or 22 months (n = 3) of age
using a BIOQUANT">Bioquant image analysis system (Hyman et al., 1993). The
average number of plaques in the cortex increases nearly sixfold over
this 10 month period, from 2.3 ± 1.4 to 13.7 ± 4.3 plaques/mm2 (mean ± SD,
p < 0.05). The size distribution of plaque diameters does not change appreciably between 12 and 22 months, from 18.1 ± 17.8 to 21.4 ± 16.2 µm (p > 0.05, not
significant). These cross-sectional data are consistent with in
vivo measurements, suggesting that plaque size is stable over an
extended period of time. Together with our in vivo measures,
these data are consistent with a model in which plaques are formed,
reach their maximal size rather quickly, and then stop growing.
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DISCUSSION |
In this report, we demonstrate the successful imaging of senile
plaques in living transgenic animals using in vivo
multiphoton laser scanning microscopy. No other imaging approach has
the resolution necessary to observe these Alzheimer's disease-like
lesions. Thus, very little is known about the natural history of these
deposits in the living brain. Multiphoton microscopy permits
high-resolution imaging of living tissue with minimal photodamage or
toxicity. Imaging through an intact skull window allows chronic,
in vivo brain imaging over periods of days to months. Using
thioflavine S, a sensitive and specific fluorescent reporter for the
dense-core subset of senile plaques, we were able to follow a
population of identified plaques in living transgenic mice over time.
Over periods ranging from days to as long as 5 months, the size and morphology of individual thioflavine S-positive plaques remain remarkably stable. Our results suggest that these plaques, once formed,
are quickly stabilized. If continual deposition of insoluble A is
assumed, this size constancy confirms the predictions of the dynamic
feedback hypothesis (Hyman et al., 1995; Cruz et al., 1997; Urbanc et
al., 1999a,b). Stability of plaque size over time is also in
accord with the predictions of a rapid time-limited aggregation of
peptide, reflecting a transient increase in local peptide concentration
above the threshold for nucleation-dependent growth (Jarrett and
Lansbury, 1993; Esler et al., 1996). An intriguing avenue of further
research is to understand the mechanism and time course of the initial
plaque formation. This imaging technique may provide the means for
addressing this issue in the future. We were also able to observe
shrinkage of individual plaques for the first time, confirming the
hypothesis that, to some extent, plaques are in a dynamic equilibrium
with their environment. This raises the possibility that clearance of
plaques that have heretofore been considered insoluble may be possible
with appropriately targeted therapeutics.
The current observations also raise the new question of why plaques
stop growing. We speculate that glia promptly respond to the presence
of an abnormal deposit in the neuropil either by surrounding it or by
phagocytosis. In Alzheimer's disease and in the transgenic models that
we have studied, glia may play an active role in halting plaque growth
primarily by surrounding the deposits (Frautschy et al., 1998). Recent
experiments using immunization with amyloid- in another transgenic
model of Alzheimer's disease suggest that microglia can
phagocytose antibody-bound amyloid deposits (Schenk et al., 1999). We
hypothesize that glial interaction with amyloid deposits may be the
biological mechanism responsible for the "dynamic feedback"
postulated in the theoretical model (Hyman et al., 1995; Cruz et al.,
1997; Urbanc et al., 1999a,b), stabilizing the size of plaques
and preventing continued growth. Further experiments that specifically
inhibit the activity of microglia should address this hypothesis directly.
Our current experiments do not directly answer questions about
the portion of amyloid- deposits that are not thioflavine S
positive. Insofar as thioflavine S-negative amyloid- deposits are
generally less compact and may not be cross-linked, one might expect
them to be more dynamic. Similarly, our data suggest that the vast
majority of thioflavine S plaques, once formed, do not grow in size and
also do not resolve over the timespan that we studied. Nonetheless, we
cannot rule out the possibility that rare plaques do resolve
spontaneously. We did fail to re-image some plaques, but after using
conservative criteria regarding technical issues that might have
contributed to failure to re-image, we were unable to identify any
unequivocal examples of clearance.
This study demonstrates the ability to observe amyloid plaques
chronically in a living brain using in vivo multiphoton
microscopy that provides resolution on the order of confocal microscopy
at unprecedented depths with negligible tissue damage. This technique will allow longitudinal studies of individual animals subjected to
experimental manipulations and should be particularly powerful for the
investigation of therapeutics targeted at clearing amyloid. Likewise,
development of fluorophores that identify other pathological features,
including the surrounding non-thioflavine S-staining amyloid-, holds
the promise for substantial advances in understanding brain
pathophysiology in transgenic models of disease. In principle, this
same approach could be used to diagnose and follow amyloid- deposition in the human brain in Alzheimer's disease.
| |
FOOTNOTES |
Received Aug. 14, 2000; revised Nov. 3, 2000; accepted Nov. 13, 2000.
This work was supported by National Institute on Aging Grants AG08487,
P01 AG15453, and T32GM07753, as well as generous support from the
Walters Family Foundation.
Correspondence should be addressed to Dr. Bradley Hyman, Alzheimer's
Disease Research Unit, CNY 6405, Massachusetts General Hospital, 149 13th Street, Charlestown, MA 02129. E-mail:
B_Hyman{at}helix.mgh.harvard.edu.
| |
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TOP
ABSTRACT
INTRODUCTION
