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Regular Articles |
From the Departments of Neurology*
and
Neurosurgery,
Massachusetts General Hospital,
Charlestown, Massachusetts
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mice transgenic for the amyloid precursor protein (APP), originally developed as a model of AD senile plaque formation, also serve as an excellent model of CAA.4-5 Tg2576 mice express human APP carrying the Swedish mutation (HuAPP695.K670N-M671L), a double mutation in the sequence of APP that favors the formation of the 40-amino acid Aß peptide.6 Amyloid deposition starts at 10 to 12 months of age4 in both the neuropil, as senile plaques, and in cerebral vessels, as CAA. This vessel-associated deposition of amyloid, like that in the parenchyma, seems to be affected by the presence of ApoE in the extracellular environment.7
The present work tests the hypothesis that vessel-associated amyloid in these animals interferes with the anatomical integrity and physiological responses of affected vessels. We report, using double-labeling of SMCs and vessel-associated amyloid and three-dimensional reconstruction of the vessel wall with multiphoton laser-scanning microscopy, an age-dependent disruption of SMCs in the wall of affected leptomeningeal vessels, initially without SMC loss. We then demonstrate that this disruption of SMC organization interferes with the ability of the vessel to respond to both an endothelial-dependent vasodilator (ACh) and an endothelial-independent vasodilator [sodium nitroprusside (SNP)], suggesting an underlying loss of coordinated SMC function in affected vessels.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Tg2576 mice expressing hAPP(Sw) under the hamster prion protein
promoter were obtained from a colony started with a breeding pair from
K. Hsiao (University of Minnesota). These animals have been shown to
develop age-dependent amyloid angiopathy as well as cortical and
hippocampal amyloid plaques similar to those seen in AD.4
Eight animals carrying the transgene and eight nontransgenic
littermates were used for the anatomical measurement of SMCs in pial
vessels. Mice of each genotype were of two age groups, a young group at
6 months of age, and an older group at 14 months of age (Table 1)
. These same groups of animals were
used for the vessel reactivity experiment. Additionally, amyloid
deposition and SMCs were imaged in an older group of three
transgene-positive animals, aged 24.7 ± 2.3 months.
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After in vivo vascular reactivity measurements (see below), animals were sacrificed by an overdose of anesthetic (halothane). Intact crania were removed and fixed in paraformaldehyde [4% in Tris-buffered saline (TBS)] for several days. The presence of the intact skull and craniotomy identified the same population of vessels whose dilation was measured in vivo. The brain was then removed, washed with TBS, treated with 0.5% Triton-X in TBS for 20 minutes, washed again, then incubated in 1% bovine serum albumin in TBS for 20 minutes to minimize nonspecific background staining. Vessels were stained with a combination of Alexa-568 phalloidin (50 µl stock solution/2 ml; Molecular Probes, Eugene, OR) and thioflavine S (thioS) (0.005%; Sigma Chemical Co., St. Louis, MO) in 1% bovine serum albumin in TBS. After 20 minutes in the staining solution, the brains were washed with TBS, and stabilized within a plastic dish with molten bone wax. The brains were covered in TBS, into which a dipping microscope objective was lowered, for imaging.
A BioRad 1024MP multiphoton imaging system (BioRad, Hercules, CA) with a Ti:Sapphire laser (Spectra Physics) operating at 750 nm, with an output power of 25 mW at the back aperture of the objective, was used for imaging. The system was mounted on an upright Olympus BX-50 microscope, equipped with long working distance dipping objectives (x10, NA 0.5; x60, NA 0.8). Custom designed external detectors (W. Zipfel, Cornell University) were used to enhance detection of emitted light. The filter set used separated emitted light into three channels: 360 to 430 nm, 485 to 505 nm, and 525 to 650 nm. The thioS and Alexa-568 signals fell clearly into the first two and third channels, respectively.
SMC Density Measurement
Series of x60 optical sections spaced 2 µm apart were taken through branches of the anterior and middle cerebral arteries on the dorsal aspect of the intact brain. Each optical section was acquired at slow scan speed, with Kalman filtering of two successive scans for noise reduction. Vessel structure was then reconstructed by a maximum intensity projection of the stack of optical sections. A computer-generated index line of random length was drawn perpendicular to the vessel diameter (Scion Image). Linear SMC density was calculated as the number of SMCs along this line divided by the length of the line in µm. Three vessels were measured from each animal; to minimize distortion of the measured SMC density by a sloping vessel, the selected vessels were those whose longitudinal axis most nearly matched the imaging plane. Although the obvious presence of amyloid on the vessels made blind selection with respect to genotype impossible, the measurement of phalloidin-stained SMCs was blind with respect to the age of the animal imaged.
Animal Preparation for Vascular Reactivity Measurement
All experiments were conducted in accordance with National
Institutes of Health and Massachusetts General Hospital Institutional
guidelines. Animals were allowed food and water ad libitum.
Anesthesia was induced with 2.5% halothane and maintained in 1.0%
halothane in 67% N2O and 33%
O2. Mice were intubated transorally, placed in a
stereotaxic frame, and ventilated artificially (SAR-830/P; CWE,
Ardmore, PA). End-tidal CO2 was continuously
monitored by a microcapnometer (Columbus Instruments, Columbus, OH).
The femoral artery and vein were cannulated with a polyethylene
catheter (PE-10, Intramedic; Becton Dickinson, Mountain View, CA) for
continuous arterial blood pressure and heart rate monitoring and for
drug infusion. 
-chloralose (80 mg/kg i.v.) was injected and
halothane was withdrawn gradually for deepening of anesthesia.
Supplemental doses of -chloralose were given as needed to maintain a
stable level of anesthesia, which was periodically tested by arterial
blood pressure and heart rate response to tail pinch. Arterial blood
gas and pH were analyzed before drug superfusion. Rectal temperature
was maintained at 37°C with a thermostatically controlled mat
(temperature control; FHC, Brunswick, ME).
Closed Cranial Window Preparation
Techniques used for measurement of vessel diameter changes in mice
were similar to those described. The head was fixed in a
stereotaxic frame, and the skull exposed by a longitudinal skin
incision. A stainless steel cranial window ring (7.0-mm inner diameter,
1.7 mm in height) containing three flow ports was adhered to the skull
in a loop of bone wax. A craniotomy (2 x 1.5 mm) was made in the
left parietal bone within the ring of the window. The dura was then
opened while the brain surface was superfused with artificial
cerebrospinal fluid (ACSF). A cover glass was placed on the ring and
affixed with dental acrylic. The ports were attached to inflow and
outflow connections, allowing for superfusion of solutions directly
onto the exposed brain; the volume under the window was 
0.1 ml. ACSF
was as follows (in mmol/L): Na+, 156.5;
K+, 2.95; Ca++, 1.25;
Mg++, 0.67; Cl-, 138.7;
HCO3-, 24.6; dextrose, 3.7; and
urea, 0.67. ACSF was kept at pH 7.35 to 7.45 by equilibration with
6.5% CO2, 10% O2, and
83.5% N2. ACSF was circulated by an infusion
pump (0.4 ml/min) via PE-50 tubing connected to the inlet port.
Intracranial pressure was maintained at 5 to 8 mmHg by adjusting the
outlet tubing to an appropriate height above the level of the window;
ACSF temperature within the window was maintained at 36.5 to 37.0°C.
Vessel Diameter Measurement
Pial vessels were visualized with a video microscope system comprised of an intravital microscope (Leitz, Germany), CCD video camera (model 1300; Cohu Inc., San Diego, CA), a camera controller (C2400; Hamamatsu Photonics, Hamamatsu, Japan), video monitor (Sony), and a video recorder (Panasonic). The images were continuously recorded on videotape. The diameter of a single pial arteriole (20 to 30 µm) was measured per experiment by a video width analyzer (C3161, Hamamatsu Photonics) and recorded using the MacLab data acquisition and analysis system. ACSF was superfused for 20 to 30 minutes until a stable baseline diameter was obtained. Acetylcholine (ACh) (10 and 25 µmol/L) and SNP (0.5 µmol/L; Sigma) dissolved in ACSF were then applied to assess vessel dilation. Drugs were superfused for 10 minutes, followed by ACSF superfusion for an additional 20 minutes for washout and return to baseline vessel diameter. The order of application of the two drugs was chosen at random. In a subset of animals (n = 6), two cumulative concentrations of ACh were superfused, without return to baseline between the low and high doses. For each application of drug, the maximum diameter change from baseline was compared. Vessel imaging and data analysis were performed without experimenter knowledge of the genotype or age of animal. Animals that exhibited significant hypotension (n = 2) or hypercapnia (n = 1) during the procedure were eliminateda priori from analysis.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Figure 1
shows the pattern of amyloid
angiopathy on leptomeningeal vessels of a 16-month-old Tg2576 mouse.
This montage of 32 images illustrates how the involvement of the middle
cerebral artery varies along its length, and is typical of all vessels
examined. The larger caliber portion of the vessel seems to be the
earliest and most severely affected, with the amyloid forming complete
rings around the circumference of the vessel. The classic segmental
appearance of the amyloid is evident, and in the most severely affected
portions of the vessel, the amyloid continues uninterrupted for
stretches of several hundred µm. Smaller size vessels have less
amyloid, with sparser deposits, sometimes amounting to isolated slivers
of amyloid on the vessel wall. Amyloid deposition appeared exclusively
on the walls of arterioles, whereas venules, whose silhouettes appear
in the figure background, remained unaffected.
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. Regions of sparse amyloid were
characterized by shards of thioS-positive material between neighboring
SMCs. More heavily affected portions of vessels showed amyloid encasing
individual SMCs, distancing and eventually completely isolating them
from neighboring cells. Although the organization of SMCs in affected
vessels of the 14-month-old group was clearly abnormal (Figure 3, a and b)
, SMCs seemed to have accommodated encroaching amyloid by contracting
along their lateral dimension; SMC loss was not apparent along the
length of the vessel. In accord with these qualitative observations,
quantitative analysis (Figure 4)
showed
that there was no significant change in the linear density of SMCs
within thioS-positive portions of vessels compared to thioS-negative
portions of vessels in 14-month Tg2576 animals or compared to
measurements in nontransgenic littermate controls.
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. The density of SMCs along the length of
the vessel was calculated for portions of vessels affected and
unaffected by amyloid; by 24 months of age, amyloid-laden vessels lost
over half the SMCs relative to unaffected vessels from the same
animals. The SMC density in amyloid-free vessels or portions of vessels
was not significantly different between the 14-month-old and
24-month-old age groups (Figure 4)
, nor did it differ significantly
from SMC density in vessels from nontransgenic 14-month-old,
nontransgenic 6-month-old, or transgenic 6-month-old animals.
Subsequent examination of Nissl-stained histological sections revealed
preservation of endothelial cells even in severely affected portions of
the vessels. No evidence for hemorrhagic stroke was observed.
Loss of SMCs in the vessel wall, as seen in the 24-month-old animals,
is certain to alter dilation in response to physiological or
pharmacological stimulation; the consequence of disruption of SMCs, as
seen in the 14-month-old animals, is unknown. We hypothesized that the
presence of amyloid in the vessel wall would impair vessel function
even before SMC loss. We therefore directly examined the physiology of
pial vessels, using a closed cranial window preparation in the young (6
months) and older (14 months) transgenic and nontransgenic littermate
control animals. We measured the change in vessel diameter to
application of either acetylcholine (ACh), which causes
endothelial-dependent vasodilatation through a nitric oxide-dependent
mechanism,10
or SNP, a nitric oxide donor that acts
directly on SMCs (Figure 4)
. To test the possibility that
overexpression of the APP gene and overproduction of Aß peptide has
an effect on vessel function independent of amyloid deposition, we also
measured vessel response in the 6-month-old transgenic and
nontransgenic animals. The physiological parameters of the four
measured groups are shown in Table 1
. No significant differences were
observed for arterial blood pH, CO2,
O2, or baseline vessel diameter for the four
groups (P > 0.05, analysis of variance). A
significant difference was seen between the arterial blood pressure
measurements for the young nontransgenic group and those for the other
three groups (P < 0.05, analysis of variance),
with the blood pressure both before and after vessel reactivity
measurement being substantially higher in this younger group. No
difference was seen, however, between the mean blood pressure before
the experiment and the blood pressure after the experiment for any of
the experimental groups.
The percentage dilation to application of ACh and SNP for the four
groups is shown in Figure 5
. No
difference in the response to either ACh or SNP was observed between
the 6-month-old Tg+ and Tg-groups. By contrast, in the 14-month-old
animals, vessel dilation was markedly attenuated in response to both
doses of ACh and to SNP in four out of five transgenic animals as
compared to the nontransgenic group. Vessel dilation in these animals,
in fact, was reduced to 25% that of control animals. One outlier in
the transgene-positive group showed essentially normal responses to
both ACh and SNP, values that were six standard deviations away from
the mean of the remaining members of the transgene-positive group. No
difference in any of the physiological parameters of this individual
animal could account for this remarkable discrepancy from other group
members.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The data indicate that amyloid-associated disruption of SMCs impairs response to both endothelial-dependent and endothelial-independent vasodilators at an age predating loss of SMCs in these vessels. Several possibilities for the mechanism of this interference of amyloid with vessel function exist. Amyloid may present a mechanical obstacle to vessel dilation, rendering the vessel wall relatively rigid. This possibility is supported by the long-standing observation that vessels with amyloid angiopathy fail to collapse in postmortem tissue, giving them the classic "stove-pipe" appearance.2 The same inflexibility that maintains vessel diameter postmortem may physically restrict dilation in vivo. Alternatively, physical separation of adjacent SMCs by amyloid may disrupt contraction dependent on their coordinated action. A third possibility, given that the accumulation of amyloid around these vessels ultimately results in significant death of SMC, may be a low-grade toxicity of amyloid on the SMC that interferes with their ability to dilate appropriately, perhaps by altering expression of channel proteins (eg, Ca++-dependent K+ channels11 ) mediating nitric oxide-dependent relaxation in cerebral vessels.12 Amyloid is toxic to endothelial cells in culture,13 and a mutant form of the Aß peptide is toxic to SMCs in culture, although the 1-40 form that predominates in the Tg2576 mouse did not demonstrate direct toxicity to SMCs.14,15 The clear in vivo loss of SMC function, then, may reflect a preliminary stage in a cascade of events that leads to cell loss. The SMC loss seen in the Tg2576 mouse model of amyloid deposition parallels that previously described at the ultrastructural level in postmortem human AD cases.16 Intriguingly, such a model of amyloid-induced SMC dysfunction presents the possibility of restoring vessel function, if the amyloid can be cleared before SMC loss in affected vessels. The development of therapeutic approaches for amyloid clearance17 should enable the testing of this hypothesis in these animals.
Cerebral vessel function has been previously studied in mice overexpressing APP(Sw) on an FVB background (Tg1130H). These mice do not develop amyloid deposits, and die at a relatively young age. In contrast to the present results, the Tg1130H mice showed impaired endothelial-dependent, but not endothelial-independent, changes in cerebral blood flow.18 Differences in the age, background strain (the Tg2576 are on a C57 B/J1 F1 background), or the exact measurement protocols (blood flow versus vessel diameter) might also contribute to observed differences. Taken together, however, the data demonstrate profound impairment of the functional integrity of cerebrovascular responses because of overexpression of mutant APP and Aß deposition, and imply that functional alterations are also likely to occur in CAA and AD. These in vivo results, together with observations that Aß has a positive ionotropic effect on aortic rings studied ex vivo,19,20 support the hypothesis that vessel-associated Aß causes a physiologically relevant impairment of cerebrovascular vessel structure and function.
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Acknowledgements |
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Footnotes |
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Supported by National Institutes of Health grants AG 08487, P01 AG 15453, and P50 NS 10828, and by the Walters Family Foundation.
Accepted for publication November 17, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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