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(American Journal of Pathology. 2005;167:505-515.)
© 2005 American Society for Investigative Pathology

Cerebral Microvascular Amyloid ß Protein Deposition Induces Vascular Degeneration and Neuroinflammation in Transgenic Mice Expressing Human Vasculotropic Mutant Amyloid ß Precursor Protein

Jianting Miao*, Feng Xu*, Judianne Davis*, Irene Otte-Höller{dagger}, Marcel M. Verbeek{ddagger} and William E. Van Nostrand*

From the Department of Medicine,* Health Sciences Center, Stony Brook University, Stony Brook, New York; and the Departments of Pathology{dagger} and Neurology,{ddagger} Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Cerebral vascular amyloid ß-protein (Aß) deposition, also known as cerebral amyloid angiopathy, is a common pathological feature of Alzheimer’s disease. Additionally, several familial forms of cerebral amyloid angiopathy exist including the Dutch (E22Q) and Iowa (D23N) mutations of Aß. Increasing evidence has associated cerebral microvascular amyloid deposition with neuroinflammation and dementia in these disorders. We recently established a transgenic mouse model (Tg-SwDI) that expresses human vasculotropic Dutch/Iowa mutant amyloid ß-protein precursor in brain. Tg-SwDI mice were shown to develop early-onset deposition of Aß exhibiting high association with cerebral microvessels. Here we present quantitative temporal analysis showing robust and progressive accumulation of cerebral microvascular fibrillar Aß accompanied by decreased cerebral vascular densities, the presence of apoptotic cerebral vascular cells, and cerebral vascular cell loss in Tg-SwDI mice. Abundant neuroinflammatory reactive astrocytes and activated microglia strongly associated with the cerebral microvascular fibrillar Aß deposits. In addition, Tg-SwDI mouse brain exhibited elevated levels of the inflammatory cytokines interleukin-1ß and -6. Together, these studies identify the Tg-SwDI mouse as a unique model to investigate selective accumulation of cerebral microvascular amyloid and the associated neuroinflammation.

The progressive cerebral accumulation of amyloid ß-protein (Aß) in parenchymal senile plaques and in the cerebral vasculature is a primary pathological feature of Alzheimer’s disease (AD) and several related disorders.1-3 The Aß peptide is derived from the Aß precursor protein (AßPP), a type I integral membrane protein, through sequential proteolytic processing mediated by ß- and {gamma}-secretase activities.1,4 Several mutations in the AßPP gene have been identified that reside within mid-region residues of Aß, including the Dutch E22Q and Iowa D23N variants, which result in familial forms of early-onset and severe cerebral amyloid angiopathy (CAA).5-9 Recent studies have suggested that cerebral microvascular amyloid accumulation is a better correlate with dementia than parenchymal amyloid plaques.10,11 Moreover, cerebral microvascular amyloid, especially in familial CAA disorders, is associated with a localized neuroinflammatory reaction.9,12-17 Together, these findings underscore the increasing recognition of the importance of cerebral microvascular amyloid-induced neuroinflammation and dementia.

Recently, we generated transgenic mice (Tg-SwDI) that express human Swedish/Dutch/Iowa mutant AßPP in brain.18 The human AßPP transgene contained the double Swedish mutations to enhance ß-secretase processing and the production of Aß peptide.19,20 The Dutch and Iowa mutations were included to yield Aß peptides with highly vasculotropic properties.5,6,9 Our initial characterization of these mice showed that they expressed low levels of transgene-encoded human AßPP but developed early-onset and robust accumulation of Aß in brain, particularly in the cerebral microvasculature.18 Subsequent analysis showed that Dutch/Iowa mutant Aß peptides are poorly cleared from brain, across the blood-brain barrier, into the circulation and that this deficiency was primarily due to diminished low-density lipoprotein-1 (LRP-1)-mediated transport across the blood-brain barrier in the cerebral microvasculature.18,21

In the present study, we investigated the temporal development of cerebral vascular amyloid and its associated pathology in Tg-SwDI mice. We show that with increasing age there is extensive accumulation of fibrillar vascular amyloid, particularly in cerebral microvessels but with lesser involvement of larger meningeal vessels. Progressive accumulation of cerebral vascular amyloid was associated with reduced microvessel densities, vascular cell apoptosis, and vascular cell loss. Notably, there was a significant presence of neuroinflammatory cells strongly associated with the cerebral microvascular amyloid. Tg-SwDI mice also exhibited elevated levels of the inflammatory cytokines interleukin (IL)-1ß and IL-6. These findings support a role for cerebral microvascular amyloid in promoting localized neuroinflammation and suggest that Tg-SwDI are a unique model to investigate these pathological processes associated with microvascular CAA in AD and related disorders.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Animals

Generation of Tg-SwDI transgenic mice on a pure C57BL/6 background was recently described.18 These mice express low levels of human Swedish/Dutch/Iowa mutant AßPP in neurons under control of the mouse Thy1.2 promoter. Heterozygous line B Tg-SwDI and nontransgenic, littermate control C57BL/6 mice ranging from 3 to 24 months of age were used in this study. All work with animals followed National Institutes of Health guidelines and was approved by Stony Brook University Institutional Animal Care and Use Committee.

Histology

Mice were sacrificed at specific ages and the brains were removed and bisected through the mid-sagittal plane. Cerebral hemispheres were immersion-fixed with 70% ethanol overnight and subjected to increasing sequential dehydration in ethanol, followed by xylene treatment and embedding in paraffin. Sagittal sections were cut at 10-µm thickness using a microtome, placed in a flotation water bath at 45°C, and then mounted on Colorfrost/Plus slides (Fisher Scientific, Houston, TX). For quantitative analysis of vessel density and CAA frequency, mouse brain hemispheres were embedded in O.C.T. compound (Sakura Finetek Inc., Torrance, CA) and snap-frozen at –70°C. Sagittal sections were cut at 14-µm thickness using a cryostat, mounted on Colorfrost/Plus slides, fixed in acetone, and stored at –70°C.

Immunohistochemistry

Immunostainings were performed on paraffin sections according to recently published protocols.18 In brief, sections were deparaffinated and rehydrated. Antigen retrieval was performed by treatment with proteinase K (0.2 mg/ml) for 5 minutes at 22°C for Aß, collagen type IV, and astrocyte immunostaining, and by 10 mmol/L sodium citrate solution (pH 9.0) for 30 minutes at 90°C in a water-bath for activated microglia immunostaining. Primary antibodies were detected with horseradish peroxidase-conjugated or alkaline phosphatase-conjugated secondary antibodies and visualized either with a stable diaminobenzidine solution (Invitrogen, Carlsbad, CA) or with the fast red substrate system (Spring Bioscience, Fremont, CA), respectively, as substrate. Sections were counterstained with hematoxylin. The following antibodies were used for immunostaining: monoclonal antibody 66.1 (1:300), which recognizes residues 1 to 5 of human Aß;22 rabbit polyclonal antibodies specific for Aß40 or Aß42 (1:200; Chemicon, Temecula, CA); rabbit polyclonal antibody to collagen type IV (1:100; Research Diagnostics Inc., Flanders, NJ); monoclonal antibody to glial fibrillary acidic protein (GFAP) for identification of astrocytes (1:300, Chemicon); monoclonal antibody 5D4 to keratan sulfate antibody for identification of activated microglia23,24 (1:200; Seikagaku Corporation, Japan); and biotinylated goat anti-mouse IgG (1:200) and ABC reagent (Vector Laboratories, Burlingame, CA) according to the manufacturer’s recommendations.

Confocal Image Analysis

Paraffin sections were deparaffinated, rehydrated, washed in 0.01 mol/L phosphate-buffered saline (PBS), and then antigen retrieval was performed by treatment with proteinase K (0.2 mg/ml) for 10 minutes at 22°C for Aß and smooth muscle cell {alpha}-actin immunolabeling. Nonspecific binding was prevented by incubating with Superblocking buffer in PBS (Pierce, Rockford, IL) containing 0.3% Triton X-100 for 1 hour at 22°C. Primary antibodies were incubated with the brain sections overnight at 4°C. Sections were washed in 0.01 mol/L PBS for three times (5 minutes each), then co-incubated with goat anti-rabbit IgG (Alex 594, 1:2500; Molecular Probes Inc., Eugene, OR) or/and donkey anti-mouse IgG (Alex 488, 1:2500; Molecular Probes, Inc.) for 2 hours in the dark at 22°C. After the first immunofluorescence labeling with primary antibody, detection of fibrillar amyloid was performed by incubating the sections with 1% thioflavin-S in 0.01 mol/L PBS in the dark for 5 minutes at 22°C, followed by washing in 50% ethanol for three times (3 minutes each). Sections were washed, Vectashield (Vector Laboratories) was applied, and a coverslip was placed and sealed with nail polish. The labeled brain sections were examined using an Eclipse E600 confocal laser-scanning microscope (LSM, Nikon, Japan). The primary antibodies used for immunofluorescence labeling were as follows: rabbit polyclonal antibody to Aß1-28 recognizing residues 1 to 28 of human Aß (1:200),25 rabbit polyclonal antibody to collagen type IV (1:100, Research Diagnostics Inc.), mouse monoclonal antibody to smooth muscle cell {alpha}-actin (1:500; Sigma, St. Louis, MO).

Cerebral Vascular Apoptotic Cell Detection

Mice were anesthetized with an intraperitoneal injection of 2.5% avertin and transcardially perfused with cold PBS (0.01 mol/L). Brains were immediately removed and fixed in 10% neutral formalin for 24 hours, followed by xylene treatment and embedding in paraffin. Sections were cut in the sagittal plane at 10-µm thickness using a microtome, deparaffinated, and rehydrated. Terminal dUTP nick-end labeling (TUNEL) was performed on paraffin sections using the ApopTag fluorescein in situ apoptosis detection kit (Chemicon Int. Inc.). In brief, brain sections were rinsed in PBS for 10 minutes, pretreated with proteinase K (20 µg/ml) for 15 minutes at 22°C, and then incubated with equilibration buffer for 10 seconds at 22°C followed by incubating with working strength TdT enzyme at 37°C for 1 hour. Working strength stop/wash buffer was applied for 10 minutes at 22°C. Sections were incubated with working strength anti-digoxigenin-conjugated fluorescein isothiocyanate in the dark for 30 minutes at 22°C. TUNEL-treated sections were incubated with either rabbit polyclonal antibody to Aß1-28 (1:200) or rabbit polyclonal antibody to collagen type IV (1:100, Research Diagnostics Inc.) for 2 hours, followed by incubating with goat anti-rabbit IgG (Alex 594, 1:2500; Molecular Probes, Inc.) in the dark for 1 hour at 22°C. Brain sections were washed, Vectashield (Vector Laboratories) was applied, and a coverslip was placed and sealed with nail polish. The labeled brain sections were examined using a Eclipse E600 confocal laser-scanning microscope (LSM).

Quantitative Analysis of CAA Frequency and Vessel Density

Groups (n = 4) of 3-, 6-, 12-, and 24-month-old Tg-SwDI mice and age-matched nontransgenic littermate C57BL/6 mice were used. Vessel densities and CAA frequency were quantified on systematically sampled serial sections immunostained for collagen type IV or Aß and collagen type IV double-immunostained sections throughout the regions of interest (every 25th section through the cortex or hippocampus; every 10th section through the thalamus; yielding 8 to 10 sections per region). The Stereologer software system (Systems Planning and Analysis, Alexandria, VA) was used to direct an automated optical fractionator for unbiased vessel counting using a motorized x-, y-, and z-axes microscope stage. A x4 objective was used to define the reference space. The automated random systematic sampling within the reference space was performed using a x40 objective. Unbiased estimation of vessel count per mm3 was determined in the fronto-temporal cortex, hippocampus, and thalamus. CAA frequency was calculated by counting the percentage of Aß-associated vessels in the fronto-temporal cortex, thalamic, and subiculum regions.

Electron Microscopic Analysis

Samples were fixed with 2.5% glutaraldehyde in cacodylate buffer for 3 days and postfixed with 1% OsO4 for 30 minutes. After dehydration with ethanol and subsequent treatment with propylene oxide, the samples were incubated with mixtures of propylene oxide and increasing concentrations of epon until 100% epon resin and embedded in Epon 812 (Electron Microscopy Sciences, Hatfield, PA). Ultrathin sections were cut on a Leica Ultracut and stained with uranyl acetate and lead citrate. Photographs were taken on a Jeol 1200 EX/II electron microscope at 60 kV.

Quantitative Analysis of Astrocytes and Activated Microglia

Total numbers of astrocytes and activated microglia in the fronto-temporal cortex, thalamus, and subiculum regions were estimated using the Stereologer software system (Systems Planning and Analysis). Every 10th section was selected and generated 10 to 15 sections per reference space in a systematic-random manner. Immunopositive cells were counted using the optical fractionator method with the dissector principle and unbiased counting rules.26 Criteria for counting cells required that cells exhibited positive immunostaining (GFAP for astrocytes and mAb 5D4 to keratan sulfate for activated microglia23,24 ) and morphological features consistent with each cell type. Cells were counted on either the right or the left hemispheres selected at random, and the numbers in each hemisphere doubled to estimate the total cell number for each brain.

Enzyme-Linked Immunosorbent Assay (ELISA) Measurements

Cerebral microvessels and microvascular-depleted brain fractions were isolated from 12-month-old Tg-SwDI mouse forebrains as described.27 The levels of total Aß40 and Aß42 in guanidine extracts prepared from the isolated microvessels and microvascular-depleted parenchymal fractions were determined by sandwich ELISA analysis as described.28

For measurement of IL-1ß and IL-6 levels, forebrains were isolated from 12-month-old wild-type and Tg-SwDI mice and homogenized in 10 vol of 50 mmol/L Tris-HCl, 150 mmol/L NaCl, pH 7.5, containing protease inhibitor cocktail (SM Biotech, Huntington Station, NY) at 4°C. The samples were centrifuged at 14,000 x g for 50 minutes at 4°C. The supernatants were collected and the protein concentrations were determined using the BCA kit (Pierce). The levels of IL-1ß and IL-6 in the samples were determined using mouse IL-1ß and IL-6 ELISA kits as described by the manufacturer (Biosource International Inc., Camarillo, CA).

Statistical Analysis

Data were analyzed by Student’s t-test at P < 0.05 significance level.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Regional Extensive Accumulation of Fibrillar Microvascular Amyloid in Tg-SwDI Mice

Tg-SwDI mice express vasculotropic mutant human AßPP in brain and were shown to develop early-onset and robust accumulation of cerebral Aß.18 In the fronto-temporal cortex the Aß accumulates primarily as diffuse parenchymal deposits that do not stain with the fibrillar amyloid dye thioflavin-S (Figure 1; A to C) . In contrast, certain regions, such as the thalamus, accumulate large amounts of fibrillar microvascular amyloid (Figure 1; D to F) . The co-localization of fibrillar amyloid with the cerebral microvasculature in Tg-SwDI mice was confirmed by double-fluorescent labeling for collagen type IV and thioflavin-S (Figure 1; G to I) . To determine which isoforms of Aß are present in the diffuse parenchymal and fibrillar microvascular Aß deposits brain tissue sections from 12-month-old Tg-SwDI mice were immunostained with antibodies specific for Aß40 or Aß42. Diffuse parenchymal deposits in the fronto-temporal cortex were strongly labeled with antibodies to Aß40 but only weakly stained with antibodies to Aß42 (Figure 2, A and B , respectively). However, the fibrillar microvascular amyloid deposits in the thalamic region exhibited prominent immunostaining for both Aß40 and Aß42 (Figure 2, C and D , respectively). ELISA analysis of extracts from isolated cerebral microvessels and microvascular-depleted parenchymal tissues showed higher levels of Aß40 than Aß42 in each fraction (Figure 2E) . Although the ratio of Aß40:Aß42 was similar in each fraction ({approx}10:1), the overall amount of each Aß isoform was significantly higher in the cerebral microvessels.



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  		Figure 1. Fibrillar Aß deposits develop exclusively in the cerebral microvasculature of Tg-SwDI mice. Immunolabeling for Aß (A) and negative thioflavin S staining (B and C) in the neocortex of 12-month-old Tg-SwDI mice indicates that the abundant diffuse parenchymal deposits in this region are not fibrillar. Immunolabeling for Aß (D) and thioflavin S staining (E) in the thalamus co-localize (F) identifying abundant microvascular fibrillar amyloid deposits in this region of Tg-SwDI mice. Immunolabeling for collagen IV (G) and thioflavin S staining (H) in the thalamus co-localize (I) confirming the microvascular localization of the fibrillar amyloid in the thalamus of Tg-SwDI mice. Scale bars, 50 µm.

 


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  		Figure 2. Distribution of Aß isoforms in parenchymal diffuse and cerebral vascular fibrillar deposits in Tg-SwDI mice. Immunostaining of 12-month-old Tg-SwDI mouse brain sections for collagen IV (red) and Aß40 or Aß42 (brown) in the fronto-temporal cortex or thalamus. The diffuse parenchymal deposits showed stronger immunostaining for Aß40 than Aß42 (A and B, respectively) whereas the fibrillar microvascular deposits in the thalamus exhibited strong staining for Aß40 and Aß42 (C and D, respectively). E: ELISA analysis for total Aß40 and Aß42 in microvessel and nonvascular parenchymal fractions isolated from 12-month-old Tg-SwDI mice. Data shown are the mean ± SD (n = 4 mice). *P < 0.0002 and **P < 0.0001.

 
The regional and temporal development of microvascular CAA was determined using stereological methods to quantitate the numbers of Aß-immunopositive cerebral microvessels in Tg-SwDI mouse brain sections (Figure 3A) . Notable levels of microvascular CAA were observed starting at ~6 months of age in the thalamic and subiculum regions which markedly increased to 85 to 90% affected microvessels at 24 months of age (Figure 3B) . The extent of microvascular CAA was substantially lower in the fronto-temporal cortex of Tg-SwDI mice although they exhibited progressive accumulation in this region to a level of {approx}15% in 24-month-old animals. Tg-SwDI mice also developed fibrillar amyloid deposits in meningeal vessels showing strong co-localization of Aß immunoreactivity with thioflavin-S staining (Figure 4; A to C) . However, the presence of meningeal vessel amyloid did not appreciably appear until ~12 months of age and was markedly lower than the levels of microvascular amyloid affecting only {approx}8% of vessels at 24 months (Figure 4D) . Together, these findings show that Tg-SwDI mice exclusively accumulate fibrillar amyloid in the cerebral vasculature with the most extensive presence in the microvasculature of the thalamic and subiculum regions of brain.



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  		Figure 3. Progressive cerebral microvascular Aß accumulation in Tg-SwDI mice. A: Representative thalamic region from a 12-month-old Tg-SwDI mouse was immunostained for Aß (brown) and collagen IV (red). B: Quantitative stereological measurement of microvascular Aß deposition in different brain regions of increasing aged Tg-SwDI mice. Data shown are the mean ± SD (n = 4 mice for each age group). Scale bar, 100 µm.

 


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  		Figure 4. Meningeal vessel Aß accumulation in Tg-SwDI mice. Immunostaining for Aß (A) and thioflavin S staining (B) co-localize (C) demonstrating fibrillar amyloid deposition in a meningeal vessel of Tg-SwDI mice. D: Quantitative stereological measurement of meningeal vessel Aß deposition of increasing aged Tg-SwDI mice. Data shown are the mean ± SD (n = 4 mice for each age group). Scale bars, 50 µm.

 
Cerebral Vascular Cellular Pathology in Tg-SwDI Mice

We next examined the effect of cerebral microvascular amyloid accumulation on regional cerebral microvessel densities in Tg-SwDI mice. At a young age of 3 months, before accumulation of microvascular amyloid, there was no difference in the microvessel densities between Tg-SwDI and wild-type mice in any of the brain regions examined (data not shown). Mice were then examined at later ages when regional microvascular amyloid accumulates. In the fronto-temporal cortex, a region with substantial diffuse parenchymal Aß deposits but only low amounts of cerebral microvascular amyloid (Figures 1 and 3) , there was no difference in microvessel densities between Tg-SwDI and wild-type mice (Figure 5) . In contrast, in the hippocampal and thalamic regions, with high microvascular amyloid load, there was a 16% and 17%, respectively, lowering of microvessel densities at 1 year, which increased to 27% and 29%, respectively, at 2 years in Tg-SwDI mice (P < 0.01) (Figure 5) . In the hippocampus and thalamus a {approx}15% decrease (P < 0.02) in microvessel densities was observed in wild-type mice as they aged from 1 year to 2 years reflecting a small normal age-related decline in these regions.



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  		Figure 5. Decreased regional vascular densities in Tg-SwDI mice. Vascular densities were determined in different brain regions of 1- and 2-year-old wild-type mice (gray bars) and Tg-SwDI mice (black bars) as described in Materials and Methods. Data shown are the mean ± SD (n = 4 mice for each group). *P < 0.01 and **P < 0.02.

 
Double-labeling experiments, combining immunolabeling for either Aß or collagen IV and TUNEL staining for apoptotic cells, were performed to identify cerebral vascular cell death in cerebral vessels with amyloid deposition. TUNEL-positive apoptotic cells could be found in the walls of meningeal vessels and co-localized with Aß or collagen immunostaining in Tg-SwDI mice (Figure 6, A and B , respectively). Similarly, apoptotic cells were identified in thalamic microvessels, a region rich in microvascular amyloid (Figure 6C) . Furthermore, the accumulation of amyloid in meningeal vessels was accompanied by a loss of smooth muscle cells in the vessel wall as shown by the depletion of vascular smooth muscle cell {alpha}-actin (Figure 7) . In contrast, no TUNEL-positive apoptotic cells or smooth muscle cell loss was observed in any cerebral vessels of nontransgenic wild-type mice (data not shown).



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  		Figure 6. Apoptotic cerebral vascular cells in Tg-SwDI mice. Immunolabeling for Aß (A) or collagen IV (B and C) in red and TUNEL labeling in green identified apoptotic vascular cells in meningeal vessels (A and B) and thalamic microvessels (C) in 2-year-old Tg-SwDI mice. Scale bar, 50 µm.

 


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  		Figure 7. Loss of smooth muscle cells in amyloid-laden meningeal vessels of Tg-SwDI mice. Immunolabeling for Aß (A) and vascular smooth muscle cell {alpha}-actin (B) in a meningeal vessel of a 2-year-old Tg-SwDI mouse. The arrowheads reveal loss of smooth muscle cells in the amyloid-rich regions of the vessel as shown in the merge image (C). Scale bar, 50 µm.

 
Electron microscopy analysis was performed for the ultrastructural characterization of cerebral microvascular amyloid deposition in Tg-SwDI mice. Figure 8A shows a normal thalamic microvessel in a 12-month-old wild-type mouse unaffected by amyloid deposition. In contrast, Figure 8B depicts a thalamic microvessel from a similarly aged Tg-SwDI mouse presenting a fibrillar amyloid deposit adhering to the microvessel. Although the endothelial cells appear unperturbed pericyte degeneration is observed with less electron-dense cytoplasm containing large vacuoles. In another example, a microvessel-associated amyloid deposit is seen with initial signs of penetrating the vascular basement membrane (Figure 8C) . In this same image a microglial cell can be seen engulfing the amyloid deposit. Another thalamic microvessel in the same Tg-SwDI mouse showed more advanced pathology with multiple fibrillar amyloid deposits extending into the adventitia, reduced vessel lumen, several areas of amyloid penetrating the basement membrane, and multiple microglia interacting with the amyloid deposits (Figure 8D) .



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  		Figure 8. Ultrastructural analysis of cerebral microvascular amyloid deposition in Tg-SwDI mice. A: Normal thalamic microvessel in a wild-type mouse. Vessel lumen (L) and vascular basement membrane (*) are identified. B: Thalamic microvessel in a Tg-SwDI mouse with an amyloid deposit (A) and degenerating pericytes with swollen vacuoles (white arrowheads). C: Tg-SwDI cerebral microvessel with amyloid deposition and an engaged microglial cell (M). Initial penetrance of amyloid into the vascular basement membrane is identified by the open arrowhead. D: Another thalamic microvessel in a Tg-SwDI mouse with more extensive amyloid deposition extending into the surrounding parenchyma, several sites of amyloid breaching the vascular basement membrane, and several engaged microglial cells. Scale bar, 2.5 µm.

 
Progressive Accumulation of Cerebral Microvascular Amyloid-Associated Neuroinflammatory Cells in Tg-SwDI Mice

The ultrastructural analysis indicated that neuroinflammatory cells may be associated with cerebral microvascular amyloid deposition in Tg-SwDI mice. Therefore, we performed double-immunolabeling experiments to identify cerebral microvessels and astrocytes in different brain regions of 12-month-old wild-type and Tg-SwDI mice. Modest astrocyte staining was observed in all examined brain regions of the wild-type mice (Figure 9; A to C) . However, Tg-SwDI mice exhibited pronounced numbers of strongly GFAP-positive reactive astrocytes that were particularly associated with amyloid-laden microvessels (Figure 9; D to F) . Quantitative, unbiased stereological analysis was performed to determine the numbers of astrocytes in the different brain regions of 6- to 24-month-old wild-type and Tg-SwDI mice (Figure 9G) . Wild-type mice exhibited similar numbers of astrocytes in all of the examined brain regions and these numbers did not change with increasing age. In contrast, Tg-SwDI showed a pronounced elevation in the numbers of astrocytes that dramatically increased with age. Moreover, larger numbers of reactive astrocytes were found in the brain regions with the highest amounts of cerebral microvascular fibrillar amyloid (ie, thalamus and subiculum).



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  		Figure 9. Increased cerebral microvascular-associated reactive astrocytes in Tg-SwDI mice. Immunostaining for GFAP (brown) and collagen IV (red) for identifying astrocytes and microvessels, respectively, in different brain regions of 1-year-old wild-type mice (A–C) and Tg-SwDI mice (D–F). G: Quantitative stereological measurement of astrocyte densities in brain regions of increasing aged wild-type mice (gray bars) and Tg-SwDI mice (black bars). Data shown are mean ± SD (n = 4 mice for each group). Scale bar, 50 µm.

 
Similarly, we next performed double-immunolabeling experiments to identify cerebral microvessels and activated microglia in different brain regions of 12-month-old wild-type and Tg-SwDI mice. Wild-type mice showed no activated microglia in any of the brain regions (Figure 10; A to C) . On the other hand, Tg-SwDI mice presented numerous activated microglia in the different brain regions, again strongly associated with the amyloid-laden microvessels (Figure 10; D to F) . This finding is consistent with the ultrastructural co-localization of activated microglia with the cerebral microvascular fibrillar amyloid deposits (Figure 8, C and D) . We next performed quantitative, unbiased stereological analysis to determine the numbers of activated microglia in the different brain regions of 6- to 24-month-old wild-type and Tg-SwDI mice (Figure 10G) . No activated microglia were found in any brain region of wild-type mice of any age (data not shown). In contrast, Tg-SwDI showed robust numbers of activated microglia that markedly increased with age. Again, larger numbers of activated microglia were found in the brain regions with the highest amounts of cere-bral microvascular fibrillar amyloid (ie, thalamus and subiculum).



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  		Figure 10. Increased cerebral microvascular-associated activated microglia in Tg-SwDI mice. Immunostaining for mAb 5D4 (brown) and collagen IV (red) for identifying activated microglia and microvessels, respectively, in different brain regions of 1-year-old wild-type mice (A–C) and Tg-SwDI mice (D–F). G: Quantitative stereological measurement of activated microglial densities in brain regions of increasing aged Tg-SwDI mice. Data shown are mean ± SD (n = 4 mice for each group). Scale bar, 50 µm.

 
Because increased numbers of neuroinflammatory cells were found in the presence of microvascular amyloid in Tg-SwDI mice we next measured the levels of IL-1ß and IL-6, two cerebral microvascular-associated inflammatory cytokines (Figure 11) .29,30 Twelve-month-old Tg-SwDI mice exhibited ~5- and 10-fold increased levels of IL-1ß and IL-6, respectively, compared with same aged wild-type mice. Together, these findings indicate that neuroinflammatory reactive astrocytes and activated microglia are strongly associated with cerebral microvascular fibrillar amyloid and certain neuroinflammatory cytokines are elevated in Tg-SwDI mice.



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  		Figure 11. Elevated levels of inflammatory cytokines in Tg-SwDI mouse brain. The levels of IL-1ß (gray bars) and IL-6 (black bars) in soluble brain extracts prepared from 12-month-old wild-type and Tg-SwDI mice were determined by ELISA analysis as described in Materials and Methods. Data shown are mean ± SD (n = 5 mice for each group). *P < 0.0001.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
The present findings show that Tg-SwDI mice develop early-onset fibrillar Aß deposits exclusively in the cerebral vasculature with only diffuse Aß deposition in the brain parenchyma. In certain brain regions, such as the thalamus and subiculum, microvascular Aß deposition becomes very extensive affecting {approx}90% of the microvessels by 2 years of age. Although fibrillar amyloid is also found in some larger meningeal vessels, the frequency of these affected vessels is much lower. The vascular amyloid is composed of both Aß40 and Aß42 human Dutch/Iowa mutant peptides. In brain regions with extensive vascular amyloid accumulation there are significant decreases in vascular densities, microvascular pericyte degeneration, presence of apoptotic vascular cells, and loss of vessel wall smooth muscle cells indicating the degenerative effects of vascular amyloid in Tg-SwDI mice. The deposition of cerebral microvascular amyloid was accompanied by large increases in the numbers of neuroinflammatory reactive astrocytes and activated microglia as well as elevated cerebral levels of the neuroinflammatory cytokines IL-1ß and IL-6. Together, these results suggest that the Tg-SwDI model is a unique and invaluable paradigm for investigating cerebral vascular Aß deposition and its effects on vessel wall degeneration and vascular amyloid-induced neuroinflammation.

Previously, several different human AßPP transgenic mice have been generated that produce wild-type Aß peptides in brain.31-35 Most of these transgenic mouse lines have been shown to develop diffuse and fibrillar parenchymal plaque deposits with varying degrees of subsequent vascular amyloid formation. Recently, Herzig and colleagues36 reported the generation of transgenic mice that express Dutch mutant human AßPP and produce E22Q Dutch mutant Aß peptides in brain. In this model, Dutch mutant Aß was found to accumulate only in the cerebral vasculature of aged (>24 months) mice indicating the vasculotropic nature of the Dutch mutation. In our recently generated Tg-SwDI mice, the presence of the dual Dutch and Iowa vasculotropic mutations (E22Q,D23N) within Aß substantially accelerates the accumulation of microvascular amyloid with appreciable amounts observed at ~6 months.18 Throughout time, larger meningeal vessels of older Tg-SwDI mice also accumulate amyloid, but to a much lesser extent than in cerebral microvessels. In addition to abundant fibrillar vascular amyloid, numerous diffuse parenchymal Aß deposits develop in Tg-SwDI mice consistent with the Aß pathology found in patients with either the Dutch or Iowa familial CAA disorders.9,37,38 The early-onset and robust accumulation of cerebral microvascular amyloid in Tg-SwDI mice developed despite the low, subendogenous expression of transgene-encoded human SwDI mutant AßPP and low production of Dutch/Iowa mutant Aß peptides in brain.18 Because microvascular deposited Aß peptides are derived from a neuronal source in Tg-SwDI mice this supports the concept of Aß drainage from parenchymal interstitial fluid to the capillary walls.39,40 This process, in conjunction with the ineffective clearance of Dutch/Iowa mutant Aß from brain across the capillary blood-brain barrier into the circulation,18,21 likely leads to the extensive formation of cerebral microvascular Aß deposition that penetrates the immediate surrounding parenchyma, also known as "dyshoric angiopathy," in Tg-SwDI mice.

Subtle, but significant, decreased cerebral microvessel densities were observed in aged wild-type mice. Although the precise mechanism responsible for this small decline is unclear it appears to reflect normal cerebral vascular declines previously reported in the aged rodent and human brain.41-47 However, more robust decreases in cerebral microvessel densities were noted in brain regions of Tg-SwDI mice with high amounts of microvascular amyloid. Similar findings have been reported in another human AßPP transgenic mouse model as well as in AD.42,47 In the presence of vascular amyloid these decreases likely result from the cerebral vascular degenerative effects, and possible anti-angiogenic properties, of deposited Aß peptides.47 Indeed, the accumulation of cerebral vascular amyloid in Tg-SwDI mice led to the decreased integrity of the basement membrane, degeneration of microvascular pericytes, presence of apoptotic cerebral vascular cells, and loss of smooth muscle cells in some larger meningeal vessels. These pathological consequences of cerebral vascular amyloid observed in Tg-SwDI mice are in strong agreement with the pathology found in amyloidotic cerebral vessels of patients afflicted with the familial Dutch-type or Iowa-type CAA disorders.9,37,38,48-50 Moreover, the present findings are highly consistent with numerous previous reports describing the pathogenic effects of deposited fibrillar Aß in human cerebral vascular cell cultures. For example, deposited fibrillar Aß, but not soluble Aß, promotes cell degeneration and apoptosis in primary cultures of cerebrovascular smooth muscle cells and brain microvascular pericytes.51-53 Similarly, deposited fibrillar Aß was shown to stimulate the degradation of vascular smooth muscle cell {alpha}-actin in cultured human cerebrovascular smooth muscle cells.53

Our findings provide significant evidence demonstrating that cerebral microvascular fibrillar amyloid deposition can exclusively induce local neuroinflammation in Tg-SwDI mice in the absence of parenchymal plaque fibrillar amyloid deposition. For example, the levels of IL-1ß and IL-6, two inflammatory cytokines reported to be increased in AD amyloid-containing cerebral microvessels,29,30 were strongly elevated in Tg-SwDI mouse brain. More specifically, reactive astrocytes and activated microglia were found to be tightly associated with the cerebral microvascular amyloid deposition. As shown in Tg-SwDI mice, the numbers of these neuroinflammatory cells were highest in the regions with the most extensive microvascular amyloid (ie, thalamus and subiculum) and increased in conjunction with the increase in cerebral microvascular Aß load in all brain regions. The intimate association of reactive astrocytes and activated microglia with cerebral microvascular amyloid in Tg-SwDI mice is highly consistent with the cerebral vascular localization of these neuroinflammatory cells in patients with the Dutch- or Iowa-type familial CAA disorders.12,14-17 In these disorders, as in the Tg-SwDI mice, the induction of a neuroinflammatory reaction appears to correlate with amyloid extending from the vessel wall into the surrounding parenchymal tissues.

Recent studies have suggested that treatments designed to reduce cerebral vascular amyloid-induced neuroinflammation in afflicted individuals have improved the dementia associated with this particular pathology.54-56 Because cerebral vascular amyloid pathology is also commonly observed in AD, this target may have further implications in combined treatment strategies for this neurodegenerative condition and its related disorders. The present results indicate that Tg-SwDI mice provide a unique model to further investigate the role of cerebral microvascular amyloidosis and its accompanying neuroinflammatory response.


   Footnotes
 
Address reprint requests to Dr. W.E. Van Nostrand, Department of Medicine, HSC T-15/083, Stony Brook University, Stony Brook, NY 11794-8153. E-mail: william.vannostrand{at}stonybrook.edu

Supported by the National Institutes of Health (grant NS36645), the Alzheimer’s Association (grant IIRG-02-3995), and ZonMW (Vidi-Vernieuwingsimpuls, 917.46.331).

J.M. and F.X. contributed equally to this work.

Accepted for publication April 18, 2005.


   References
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 Abstract
 Materials and Methods
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 References
 

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